Fertilitatea

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Principles and Practice of

Fertility Preservation
Principles and Practice of

Fertility Preservation
Edited by
Jacques Donnez
Department of Gynecology and Andrology, Universite Catholique de Louvain, Brussels, Belgium
S. Samuel Kim
Division of Reproductive Endocrinology, University of Kansas, Kansas City, KS, USA
CAMBRID GE UNIVERSIT Y PRESS

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Library of Congress Cataloging in Publication data
Principles and practice of fertility preservation / edited by
Jacques Donnez, S. Samuel Kim.
p. cm.
Includes index.
ISBN 978-0-521-19695-6 (hardback)
1. Human reproduction. 2. Cancer Treatment
Complications. I. Donnez, J. II. Kim, S. Samuel.
QP251.P77 2011
612.6 dc22
2010041958
ISBN 978-0-521-19695-6 Hardback
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Every effort has been made in preparing this book to provide
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Although case histories are drawn from actual cases, every
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authors, editors and publishers therefore disclaim all liability
for direct or consequential damages resulting from the use of
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manufacturer of any drugs or equipment that they plan to use.
This book is dedicated to our courageous patients fighting cancer
Contents
List of contributors x
Foreword by Roger G. Gosden xv
Foreword by Edward E. Wallach xvi
Preface xvii
Acknowledgements xviii
Section 1: Introduction
Section 3: Reproductive biology

and cryobiology
The evolution of ART

Peter R. Brinsden
The effect of chemotherapy and

radiotherapy on the human
reproductive system 11
W. H. B. Wallace, R. A. Anderson and D. Meirow
Fertility preservation in non-cancer

patients 23
Javier Domingo, Ana Cobo, Mara
Sanchez and Antonio Pellicer
Basic cancer biology and immunology

Roy A. Jensen, Lisa M. Harlan-Williams,
Wenjia Wang and Shane R. Stecklein
Breast cancer and fertility

preservation: a view from oncology
Carol Fabian and Jennifer Klemp
Breast cancer therapy and

reproduction 62
Larissa A. Korde and Julie R. Gralow
Pediatric cancer therapy and fertility

Pinki K. Prasad, Jill Simmons and
Debra Friedman
Cancer epidemiology and

environmental factors in children,
adolescents and young adults 83
Karina Braga Ribeiro and Paolo Boffetta
Life and death in the germ line:

apoptosis and the origins of DNA
damage in human spermatozoa 101
R. J. Aitken and B. J. Curry
10 Molecular aspects of follicular

development 114
Zhongwei Huang and Dagan Wells
11 Fundamental cryobiology of
reproductive cells and tissues:
concepts and misconceptions 129
Erik Woods, Sreedhar Thirumala, Xu Han
and John K. Critser
Section 2: Cancer biology,

epidemiology and treatment
4
35
12 Fundamental aspects of vitrification

as a method of reproductive cell,
tissue and organ cryopreservation 145
Steven F. Mullen and Gregory M. Fahy
49
Section 4: Fertility preservation

strategies in the male
73
13 Hormonal suppression for fertility

preservation in the male 164
Gunapala Shetty
14 Cryopreservation of spermatozoa: old
routine and new perspectives 176
E. Isachenko, V. Isachenko, R. Sanchez,
I. I. Katkov and R. Kreienberg
vii
List of contents
15 Transplantation of cryopreserved
spermatogonia 199
Jill P. Ginsberg and Ralph L. Brinster
16 Cryopreservation and transplantation
of testicular tissue 209
Christine Wyns
17 Assisted reproductive techniques and
donor sperm in cancer patients 225
Wayland Hsiao, Elizabeth Grill and Peter
N. Schlegel

strategies in the female:
medical/surgical
18 Use of GnRH agonists for prevention
of chemotherapy-induced
gonadotoxicity 239
Susannah C. Copland and Megan Clowse
19 Ovarian transposition 250
Carrie A. Smith, Erin Rohde and
Giuseppe Del Priore
20 Fertility-saving surgery for cervical
cancer 257
P. Mathevet and A. Ciobanu
21 Results of conservative management
of ovarian malignant tumors 266
Philippe Morice, Catherine Uzan and
Sebastien Gouy

strategies in the female: ART
22 Embryo cryopreservation as a fertility
preservation strategy 279
Pedro N. Barri, Anna Veiga, Montserrat
Boada and Miquel Sole
23 Oocyte cryopreservation: slow
freezing 283
Andrea Borini and Veronica Bianchi
viii
24 Cryopreservation of human oocytes

and the evolution of vitrification
technology for this purpose 293
Michael J. Tucker and Juergen Liebermann
25 Cryopreservation and transplantation
of isolated follicles 305
Marie-Madeleine Dolmans and Anne Van
Langendonckt
26 ART and oocyte donation in cancer
survivors 310
Ina N. Cholst, Glenn L. Schattman and
Zev Rosenwaks
Section 7: Ovarian
cryopreservation and transplantation
27 General overview of ovarian
cryobanking 328
S. Samuel Kim
28 Ovarian tissue cryopreservation
Debra A. Gook and David H. Edgar
342
29 Ovarian tissue transplantation 357

Jacques Donnez, Jean Squifflet and
Marie-Madeleine Dolmans
30 Whole ovary freezing 367
J. Ryan Martin, Jason G. Bromer and
Pasquale Patrizio
31 Ovarian transplantation: whole ovary
transplantation 377
Mohamed A. Bedaiwy and Tommaso Falcone
Section 8: In vitro follicle growth

and maturation
32 Molecular and cellular integrity of
cultured follicles 389
David F. Albertini, Gokhan Akkoyunlu
and S. Samuel Kim
33 In vitro growth systems for human
oocytes: from primordial to
maturation 397
Evelyn E. Telfer and Marie McLaughlin
List of contents
34 Contributions of ovarian stromal cells

to follicle culture 409
David J. Tagler, Lonnie D. Shea and Teresa
K. Woodruff
35 In vitro maturation of GV oocytes
M. De Vos and J. Smitz
421
36 Clinical potential of in vitro

maturation 431
Baris Ata, Einat Shalom-Paz, Srinivasan
Krishnamurthy, Ri-Cheng Chian and
Seang Lin Tan
Section 9: Future technologies

37 From pluripotent stem cells to
germ cells 440
Rosita Bergstrom and Outi Hovatta
38 Artificial ovary 448
Christiani A. Amorim
39 Predicting ovarian futures: the
contribution of genetics 459
Elizabeth A. McGee and Jerome F. Strauss, III
Section 10: Ethical, legal

and religious issues with
fertility preservation
40 Psychological issues of cancer
survivors 467
Allison B. Rosen, Kenny A.
Rodriguez-Wallberg and Kutluk Oktay
41 Fertility preservation: ethical
considerations 479
Pasquale Patrizio and A. L. Caplan
42 Legal aspects of fertility preservation
Nanette R. Elster
488
43 Christian ethics in fertility

preservation 497
Brent Waters
Index 507
The color plate section will be found between
pages 366 and 367.
ix
Contributors
R. J. Aitken
Discipline of Biological Sciences and ARC Centre of
Excellence in Biotechnology and Development,
School of Environmental and Life Sciences,
University of Newcastle, Callaghan, NSW,
Australia
Gokhan Akkoyunlu
Department of Histology and Embryology, Faculty of
Medicine, Akdeniz University, Antalya, Turkey
David F. Albertini
Kansas University School of Medicine, Kansas City,
KS, USA
Montserrat Boada
Department of Obstetrics, Gynecology and
Reproduction, Institut Universitari Dexeus,
Barcelona, Spain
Paolo Boffetta
Mount Sinai School of Medicine, New York, NY, USA
and International Prevention Research Institute,
Lyon, France
Universite Catholique de Louvain, Brussels, Belgium
Andrea Borini
Tecnobios Procreazione, Centre for Reproductive
Health, Bologna, Italy
R. A. Anderson
Division of Reproductive and Developmental Science,
University of Edinburgh, Edinburgh, UK
Karina Braga Ribeiro

Faculdade de Ciencias Medicas da Santa Casa de Sao
Paulo, Sao Paulo, Brazil
Baris Ata
McGill Reproductive Centre, Department of
Obstetrics and Gynecology, Division of Reproductive
Endocrinology and Infertility, McGill University,
Montreal, Quebec, Canada
Peter R. Brinsden
Bourn Hall Clinic, Bourn, Cambridge, UK
Pedro N. Barri
Barcelona, Spain
Mohamed A. Bedaiwy
Assiut School of Medicine, Assiut, Egypt and
Department of ObstetricsGynecology, University
Hospitals Case Medical Center, Case Western Reserve
University, Cleveland, OH, USA
Rosita Bergstrom
Karolinska Institute, Stockholm, Sweden
Veronica Bianchi
Tecnobios Procreazione, Centre for Reproductive
Health, Bologna, Italy
Ralph L. Brinster
Department of Animal Biology, University of
Pennsylvania School of Veterinary Medicine,
Philadelphia, PA, USA
Jason G. Bromer
Yale University Fertility Center, New Haven, CT,
USA
A. L. Caplan
Center for Bioethics, University of Pennsylvania,
Philadelphia, PA, USA
Ri-Cheng Chian
Department of Obstetrics and Gynecology McGill
University, Montreal, Quebec, Canada
List of contributors
Ina N. Cholst
Ronald O. Perelman and Claudia Cohen Center for
Reproductive Medicine, Weill Cornell Medical
College, New York, NY, USA
A. Ciobanu
Gynecologic department, Hopital
Femme-Mère-Enfant, Bron, France
Megan Clowse
Division of Rheumatology and Immunology, Duke
University, Durham, NC, USA
Ana Cobo
Instituto Valenciano de Infertilidad (IVI),
Valencia, Spain
Susannah C. Copland
Duke Fertility Center, Durham, NC, USA
John K. Critser
General Biotechnology LLC, Indianapolis, IN and
Comparative Medicine Center, University of
Missouri, Columbia, MO, USA
B. J. Curry
Discipline of Biological Sciences and ARC Centre of
Excellence in Biotechnology and Development,
School of Environmental and Life Sciences,
University of Newcastle, Callaghan, NSW, Australia
Giuseppe Del Priore
Indiana University School of Medicine, Department
of Obstetrics-Gynecology, Indianapolis, IN, USA
M. De Vos
Center for Reproductive Medicine, UZ Brussel
(VUB), Brussels, Belguim
Marie-Madeleine Dolmans
Department of Gynecology, Cliniques Universitaires
Saint Luc, Brussels, Belgium
and Gynaecology, University of Melbourne,

Victoria, Australia
Nanette R. Elster
DePaul University College of Law, Chicago, IL,
USA
Carol Fabian
University of Kansas Cancer Center, Kansas City, KS,
USA
Gregory M. Fahy
21st Century Medicine, Inc., Fontana, CA,
USA
Tommaso Falcone
Department of ObstetricsGynecology, Cleveland
Clinic, Cleveland, OH, USA
Debra Friedman
Cancer Control and Prevention Program, Vanderbilt
Ingram Cancer Center, Nashville, TN, USA
Jill P. Ginsberg
Department of Pediatrics, Division of Oncology,
University of Pennsylvania School of Medicine,
Childrens Hospital of Philadelphia, Philadelphia,
PA, USA
Debra A. Gook
Reproductive Services, Royal Womens Hospital/
Melbourne IVF and Department of Obstetrics
and Gynaecology, University of Melbourne,
Victoria, Australia
Julie R. Gralow
Division of Medical Oncology, University of
Washington/Seattle Cancer Care Alliance, Seattle,
WA, USA
Javier Domingo
IVI Las Palmas, Las Palmas de Gran Canaria, Spain
Elizabeth Grill
Reproductive Medicine at New York-Presbyterian
Hospital/Weill Cornell Medical Center, New York,
NY, USA
Jacques Donnez
Department of Gynecology and Andrology,
Sebastien Gouy
Service de Chirurgie Gynecologique, Institut
Gustave-Roussy, Villejuif Cedex, France
David H. Edgar
Reproductive Services, Royal Womens Hospital/
Melbourne IVF and Department of Obstetrics
Xu Han
Comparative Medicine Center, University of
Missouri, Columbia, MO, USA
xi
Lisa M. Harlan-Williams
University of Kansas Cancer Center, Kansas City,
KS, USA
Juergen Liebermann
Fertility Centers of Illinois, Chicago-River North IVF
Center, Chicago, Illinois, USA
Outi Hovatta MD
Division of Obstetrics and Gynecology, Huddinge
University Hospital, Huddinge, Sweden
J. Ryan Martin
USA
Wayland Hsiao
Weill Cornell Medical College, New York, NY,
USA
Elizabeth A. McGee
Department of Obstetrics and Gynecology, VCU
School of Medicine, Richmond, VA, USA
Zhongwei Huang
Nuffield Department of Obstetrics and Gynaecology,
University of Oxford, Oxford, UK
Marie McLaughlin
Centre for Integrative Physiology, University of
Edinburgh, Edinburgh, UK
E. Isachenko
Department of Obstetrics and Gynecology, University
Womens Hospital, Ulm, Germany
P. Mathevet
Hopital Femme-Mère-Enfant, Bron, France
V. Isachenko
Department of Obstetrics and Gynecology, University
Womens Hospital, Ulm, Germany
Roy A. Jensen
University of Kansas Medical Center, Kansas City, KS,
USA
I. I. Katkov
University of San Diego in La Jolla, San Diego, CA,
USA
S. Samuel Kim
Division of Reproductive Endocrinology, University
of Kansas, Kansas City, KS, USA
Jennifer Klemp
University of Kansas Cancer Center, Kansas City, KS,
USA
Larissa A. Korde
Division of Medical Oncology, University of
Washington/Seattle Cancer Care Alliance, Seattle,
WA, USA
R. Kreienberg
Department of Obstetrics and Gynaecology,
University Womans Hospital, Ulm, Germany
Srinivasan Krishnamurthy
Department of Obstetrics and Gynecology, McGill
University, Montreal, Quebec, Canada
xii
D. Meirow
IVF Unit, Fertility Preservation Laboratory, Sheba
Medical Center, Tel-Aviv University, Israel
Philippe Morice
Gustave-Roussy, Villejuif Cedex, France
Steven F. Mullen
Reproductive Cryobiology, 21st Century Medicine,
Inc., Fontana, CA, USA
Kutluk Oktay
Division of Reproductive Medicine and Infertility and
Laboratory of Molecular Reproduction and Fertility
Preservation, Westchester Medical CenterNew York
Medical College, Valhalla, NY, USA
Pasquale Patrizio
USA
Antonio Pellicer
University of Valencia, Valencia, Spain
Pinki K. Prasad
VanderbiltIngram Cancer Center, Nashville, TN,
USA
Kenny A. Rodriguez-Wallberg
Karolinska Institute and Karolinska University
Hospital Huddinge, Fertility Unit, Department of
Obstetrics and Gynecology, Stockholm, Sweden
Erin Rohde
of ObstetricsGynecology, Indianapolis, IN,
USA
Allison B. Rosen
Fertility Preservation Institute, Department of
Obstetrics and Gynecology, Westchester Medical
CenterNew York Medical College, Valhalla, NY, USA
Zev Rosenwaks
College, New York, NY, USA
Mara Sanchez
Gynaecology and Obstetrics at the University of
Valencia, Valencia, Spain
R. Sanchez
Center of Biotechnology in Reproduction,
Department of Basic Sciences, La Frontera University,
Temuco, Chile
Glenn L. Schattman
Center, New York, NY, USA
Peter N. Schlegel
James Buchanan Brady Foundation, Weill Cornell
Medical College, New York, NY, USA
Einat Shalom-Paz
McGill Reproductive Centre, Department of
Obstetrics and Gynecology, Division of Reproductive
Endocrinology and Infertility, McGill University,
Lonnie D. Shea
Department of Chemical and Biological Engineering,
McCormick School of Engineering and Applied
Science, Northwestern University, Technological
Institute, Evanston, IL, USA
Carrie A. Smith
of Obstetrics-Gynecology, Indianapolis, IN, USA
J. Smitz
Center for Reproductive Medicine, UZ Brussel
(VUB), Brussels, Belgium
Miquel Sole
Barcelona, Spain
Jean Squifflet
Shane R. Stecklein
University of Kansas Medical Center, Kansas City,
KS, USA
Jerome F. Strauss, III
Virginia Commonwealth University Health System,
Sanger Hall, Richmond, VA, USA
David J. Tagler
Department of Chemical and Biological Engineering,
McCormick School of Engineering and Applied
Science, Northwestern University, Technological
Institute, Evanston, IL, USA
Seang Lin Tan
Department of Obstetrics and Gynecology at McGill
University; McGill University Health Centre, McGill
Reproductive Centre, Royal Victoria Hospital,
Evelyn E. Telfer
Centre for Integrative Physiology, University of
Edinburgh, Edinburgh, UK
Sreedhar Thirumala
General Biotechnology LLC, Indianapolis, IN,
USA
Gunapala Shetty
Department of Experimental Radiation Oncology,
University of Texas M. D. Anderson Cancer Center,
Houston, TX, USA
Michael J. Tucker
Shady Grove Fertility RSC, Rockville, MD, USA and
Georgia Reproductive Specialists, Atlanta, GA,
USA
Jill Simmons
Division of Pediatric Endocrinology Vanderbilt
University School of Medicine, Nashville, TN, USA
Catherine Uzan
Gustave-Roussy, Villejuif, France
xiii
Anne Van Langendonckt

Department of Gynecology, Cliniques Universitaires
Saint Luc, Brussels, Belgium
Dagan Wells
Nuffield Department of Obstetrics and Gynaecology,
University of Oxford, Oxford, UK
Anna Veiga
Reproductive Medicine Service, Department of
Obstetrics, Gynecology and Reproduction, Institut
Universitari Dexeus, Barcelona, Spain
Teresa K. Woodruff
Department of Obstetrics and Gynecology, Feinberg
School of Medicine, Northwestern University,
Chicago; Robert H. Lurie Comprehensive Cancer
Center of Northwestern University, Chicago,
IL, USA
W. H. B. Wallace
Division of Reproductive and Developmental Science,
University of Edinburgh, Edinburgh, UK
Wenjia Wang
University of Kansas Medical Center, Kansas City,
KS, USA
Erik Woods
General Biotechnology LLC and Indiana University
School of Medicine, Department of Microbiology and
Immunology, Indianapolis, IN, USA
Brent Waters
Stead Center for Ethics and Values,
GarrettEvangelical Theological Seminary, Evanston,
IL, USA
Christine Wyns
Department of Gynecology, Universite Catholique de
Louvain, Brussels, Belgium
xiv
Foreword
One of the first and most poignant cases that

impressed on me the importance of fertility preservation was that of a young policewoman. She was
already engaged to be married when she was diagnosed
with non-Hodgkins lymphoma. Her eagerness to preserve fertility and avoid premature menopause was so
understandable, although in those days the only technology available for her was experimental. Nevertheless, she grasped the straw, saying, It was worse to hear
the treatment would sterilize me than when my doctor
told me I had cancer. . . . She died.
The prospects for survival of young cancer patients
are much better now than 20 years ago and continue to improve, but progress has often depended
on more aggressive, and hence gonadotoxic, treatment regimens. Recognition of the damaging effects of
chemotherapy and pelvic irradiation, and sometimes
of the disease process itself, in patients of reproductive
age both women and men as well as children is now
common knowledge and has encouraged innovative
technology and surgery, giving hope of preserving
the potential for biological parenthood after cancer.

Indeed, although the priority of medical care is to maximize the chances that patients will overcome their disease, to overlook the late effects is now regarded as
neglect of a vital duty. The new developments may also
help non-cancer patients, including those wishing to
forestall ovarian aging, something that ought to be less
controversial than often depicted in the media.
Recent growth in the field has been signaled by the
launch of an international society for specialists in fertility preservation, biomedical symposia, review articles and a few books, but none as comprehensive as
this volume compiled by Professors Jacques Donnez
and S. Samuel Kim. The topics authored by foremost
researchers and practitioners in the 43 chapters range
from biology and oncology to technology and surgery,
including bioethics and law. Such a large work confirms that this field has moved beyond the pioneering
stage towards maturity, and it will likely be an important reference for some years to come.
Roger G. Gosden
xv
Foreword
Three decades have elapsed since the historic birth of

Louise Brown in England. This momentous event, the
introduction of human in vitro fertilization (IVF) as an
approach to treating infertility, set the stage for hundreds of thousands of infertile couples who were ultimately to benefit from this exciting new technology. As
with many medical/scientific breakthroughs, the initial process has evolved significantly from its prototype
which involved a natural cycle, less than ideal monitoring and laparoscopic egg recovery. During the 30
years since IVF became a reality, new drugs and techniques have developed for follicle stimulation. Ultrasonography has come of age and is now used for follicle monitoring, egg retrieval, embryo transfer and
even detection of early pregnancy. New cryopreservation techniques have enabled embryo, egg and ovarian
tissue storage. Further refinements include specialized
growth media for embryos, intracytoplasmic sperm
injection, transdermal surgical sperm retrieval, preimplantation genetic diagnosis, egg donation and use
of surrogate or gestational carriers. Couples seeking
correction of infertility problems have not been the
only beneficiaries of our new reproductive technologies.
The term fertility preservation refers to a
totally new direction for these exciting technological
advances. Fertility preservation applies to individuals
who have deferred pregnancy for a variety of reasons,
such as single women who choose to have their eggs
preserved for fertility insurance and cancer patients
about to embark on extirpative surgery, radiation
or chemotherapy to cure their disease. In many
instances, these women and men are neither married
nor have specific plans for having offspring at the time
xvi
of their impending cancer therapy. Such individuals

now have a variety of options available to them which
could be applied to heightening their opportunites for
parenthood in the future.
Unintended consequences of our new reproductive technologies encompass profound social and ethical implications. For the cancer victim, awareness of
the consequences of treatment on her future fertility
must be stimulated by the oncologist during indoctrination to what may lie ahead. For young individuals, under age for providing informed consent, assent
is required through guidance by both physician and
parents. Requiring an invasive procedure for obtaining gametes to place in storage has its drawbacks, and
the long-term storage of gametes becomes even more
complex for families of individuals who succumb to
their disease.
Professors Donnez and Kims book clearly points
out that the social and ethical complexities of fertility preservation for cancer patients require the collaboration of oncologists and reproductive endocrine and
infertility specialists to provide the best possible information and strategic plan for each patient. The textbook unites multiple disciplines while covering basic
reproductive physiology, principles of cancer therapy,
age-associated issues and ethical dilemmas. It intertwines the bittersweet combination of passion for procreation and the hazards of advancing age and lethal
disease. The substance and structure of this text should
advance the missions of both infertility specialist and
oncologist.
Edward E. Wallach
Preface
Fertility preservation has become a very prominent

area of interest in reproductive medicine and oncology. In the twenty-first century, fertility preservation
is no longer a theoretical concept but an essential clinical discipline in medicine. Increased long-term cancer
survival has intensified the need for fertility preservation strategies, as fertility is the leading quality of life
issue for young cancer survivors. Although the focus of
fertility preservation has mainly been limited to cancer patients in their reproductive years, its clinical relevance may well be expanded to non-cancer patients,
and much broader clinical applications are expected in
the future.
In the past few years, we have witnessed huge scientific and technological advances in fertility preservation methods, as well as accumulation of an enormous
amount of related information and knowledge. As
pioneers who have actively participated in the development of emerging technologies in fertility preservation, we felt the need to publish a comprehensive book
that would reflect all aspects of this exciting new field.
This book covers the full range of scientific concepts
and emerging techniques, including the latest developments in oocyte cryopreservation, in vitro follicle
culture and ovarian cryopreservation and transplantation.

The first section (three chapters) serves as a general introduction to the field of fertility preservation,
followed by two sections (nine chapters) dedicated to
cancer biology, epidemiology and treatment, as well
as reproductive biology and cryobiology. In section
four, fertility preservation in the male is discussed
(five chapters). The following sections (19 chapters)
are devoted to fertility preservation strategies in the
female, divided into four categories: medical/surgical;
assisted reproductive technology (ART); ovarian cryopreservation and transplantation; and in vitro follicle growth and maturation. The last two sections
(seven chapters) address future technologies and ethical, legal, moral and religious issues related to fertility
preservation.
We are confident that this book will provide a
theoretical and practical guide for scientists, embryologists, nurses and clinicians working in reproductive medicine and oncology. In addition, it will
be a valuable resource for anyone wishing to learn
more about this field for patient care or research
purposes.
xvii
Acknowledgements
We, Jacques Donnez and S. Samuel Kim, would like to

express our gratitude to Cambridge University Press
and its editors, Nisha Doshi, Alice Nelson, Joanna
Endell-Cooper and, in particular, Nick Dunton, who
supported the idea of publishing this book. We greatly
appreciate the time and effort of all contributing
authors. Indeed, publication of this book would not
have been possible without their expertise, knowledge
and enthusiasm. We would also like to acknowledge
the devoted members of the International Society for
Fertility Preservation (ISFP), who are the future in
this field. Sincere appreciation is extended to Norma
Turner and Marie-Madeleine Dolmans for their dedication to the ISFP.
S. Samuel Kim is grateful to each member of
the IVF team at the Center for Advanced Reproduc-
xviii
tive Medicine, the University of Kansas Medical Center, for their deep and enduring dedication, and to
Carl Weiner, my department chair, for his advice and
encouragement. I am indebted to Roger Gosden, my
mentor and best friend, for his keen insight, relentless support and wisdom. My heartfelt thanks go to my
beloved family, Kris, Jean, Melissa, Derek, Monica and
my mother.
Jacques Donnez would like to thank all the
members of his clinical and research teams at the Universite Catholique de Louvain, who work tirelessly in
pursuit of scientific advancement and clinical excellence and understand that there is no life without
pressure. I am also eternally grateful to my wife, children and grandchildren for their unwavering love and
support.
Section 1
Chapter
Introduction
The evolution of ART

Peter R. Brinsden
To understand science, it is necessary to know its history.

Auguste Compte (17981857)
It could be said that the first instance of assisted reproductive technology (ART) was when an eminent surgeon, John Hunter (172893) of London (Figure 1.1),
assisted a woman in becoming pregnant by taking a
semen sample produced by her husband, who had
Figure 1.1 John Hunter (172893). The first reported person to

successfully perform artificial insemination in a human. See plate
section for color version.
hypospadias, and inseminated her with that specimen.

This was an assisted conception, although it is not
strictly within the definition of the present-day ARTs,
which involve the manipulation of sperm, oocytes and
embryos in vitro and include:
r in vitro fertilization (IVF)
r intracytoplasmic sperm injection (ICSI)
r gamete intrafallopian transfer (GIFT) now
rarely practiced
r zygote intrafallopian transfer (ZIFT) now rarely
practiced
r oocyte and embryo donation
r cryopreservation of sperm, oocytes and embryos
r gestational surrogacy
r in vitro maturation of oocytes
r pre-implantation genetic diagnosis.
However, although John Hunters treatment of his
patient was one of the first instances of outside interference with the human reproductive process, mans
interest in fertility and conception in both animal
species and in humans goes back thousands of years.
As early as the fifth century BC, Hippocrates
(c. 460370 BC), who is commonly thought of as the
father of medicine, believed that both males and
females produced the liquor which blended within
the womans body and created babies. Some 100 years
later, Aristotle (384422 BC) proposed the theory that
children are the product of the mingling of male and
female seed. This firmly opposed the then prevailing theory that children were from the male seed
and women were merely the receptacle for the child.
This latter idea prevailed until the sixteenth century,
when William Harvey (15781657) (Figure 1.2), having studied the behavior and fertility of the King of
Englands herd of deer, wrote De Generatione Animalium in 1651, which described the egg as being
Principles and Practice of Fertility Preservation, eds. Jacques Donnez and S. Samuel Kim.
C Cambridge University Press 2011.
Published by Cambridge University Press.
Figure 1.3 Anton van Leeuwenhoek (16321723). The first person

to study animal and human sperm under microscopes, which he
constructed himself. See plate section for color version.
Figure 1.2 William Harvey (15781657). The first person to
describe the egg as responsible for the production of all creatures.
See plate section for color version.
responsible for the production of all creatures. It was

from this research that his famous expression ex
ovo omnia [from the egg everything] arose. It was
from this time onwards that the science of animal and
human reproduction really began to develop. However, it was not really until the development of the optical microscope that researchers were able, for the first
time, to study sperm, oocytes and, later, fertilization.
Anton van Leeuwenhoek (16321723), a Dutch
draper and amateur scientist (Figure 1.3), was fascinated by the potential of the new science of
microscopy. He built his own microscopes and, among
many other specimens, he studied sperm of different
animal species; in 1677 he reported his findings to the
Royal Society in London. He believed that each sperm
was the beginnings of an individual animal or human
and, if it was nourished in the womb, it would produce the next generation. This went against the prevailing opinion at the time that the woman produced
the seed and the male merely produced the fertilizing
power to produce offspring.
Dalenpatius in 1699 stated that he could see a

miniature human within a single sperm, and the idea
that humans were pre-formed within a sperm prevailed for more than a century, even though this report
was later found to be an hoax and Dalenpatius the
fictitious name of the perpetrator of the hoax. It was
Reinier de Graaf (164173) who first described the
development of ovarian follicles later to become
known as Graafian follicles in his honor but he never
discovered oocytes within the follicles. He also supported the work of fellow Dutchman, van Leeuwenhoek and was aware of the importance of his work on
microscopes. De Graaf died at the early age of 32 years.
Lazzaro Spallanzani (172999) (Figure 1.4), an
Italian scientist, studied the behavior of semen microscopically and performed the first known attempts at
insemination of a dog. He is also credited with the very
earliest attempts at IVF in experiments with frogs; he
is also said to have been the first to freeze and thaw
sperm in 1776.
In 1826, Karl Ernst von Baer (17921876) (Figure
1.5) first identified oocytes in the ovaries of a bitch.
He also finally established that mammals develop
from oocytes and reported on organogenesis of early
Chapter 1: The evolution of ART
Figure 1.4
Lazzaro Spallanzani
(172999).
Performed the first
known
insemination of a
bitch, first in vitro
fertilization with
frogs and was the
first to successfully
freeze and thaw
sperm.
mammalian embryos. Von Baer is credited with being

the founding father of modern embryology.
In the mid nineteenth century, extensive research
was carried out on reproduction by a number of
researchers who reported their observations on the
process of fertilization in primitive organisms; in particular, Henry Nelson (1852), Newport (1853), van
Beneden (1854) and Hertwig (1876). Nelson observed
the penetration of ascaris oocytes by spermatozoa;
Newport made similar observations in amphibians,
while both van Beneden and Hertwig are credited with
the first observations on fertilization in mammals.
It is probable that Walter Heape (18551929), a
physician and professor at the University of Cambridge, England, was the first scientist to successfully
transfer embryos into rabbits in the early 1890s. Only
one pregnancy and delivery was reported, but this
experiment showed for the first time that it was possible to remove embryos from one animal and transfer
them to another, without interfering with their development.
In their reviews on the early history of IVF, both
Bavister [1] and Clark [2] give the opinion that 1951
was probably the critical boundary defining the
beginning of the modern era of IVF. Both Colin
Bunny Austin (19142004) and M. C. Chang (1908
91) discovered the need for spermatozoa to undergo
capacitation and the acrosome reaction before they
are able to penetrate the zona pellucida of the oocyte.
Later, in 1963, Yanagimachi and Chang, were able to
achieve the first live births after transfer of hamster
Figure 1.5 Karl

Ernst von Baer
(17921876). The
first person to
identify oocytes in
the ovaries of a
bitch and to
identify that
mammals develop
from oocytes.
Credited as being
The founding
father of modern
embryology.
oocytes fertilized in vitro using spermatozoa capacitated in vitro [3]. Work continued, more or less successfully, over the next decade attempting to achieve
fertilization in vitro and live births of various other
mammalian species.
Some of the earliest observations on fertilization of
human oocytes were made by Robert Edwards (1925)
and published in 1965 in a landmark paper: Maturation in vitro of human ovarian oocytes [4]. It was not
possible for Edwards to progress further with efforts
to achieve IVF of human oocytes for clinical use without close collaboration with clinical colleagues, who
were able to provide a supply of human oocytes usually from patients having ovarian wedge resections for
polycystic ovary disease. It was the need for Edwards
to be able to obtain these supplies of pre-ovulatory
human oocytes that brought him and gynecologist
Patrick Steptoe (191388) together in 1968.
Patrick Steptoe became known to Robert Edwards
because he had brought laparoscopy, whereby the
female pelvic organs could be visualized by a relatively
minor operation, to England from Europe where Steptoe had studied it under both Raoul Palmer (1940
95) and Hans Frangenheim (19202001). On his
return to England, he further developed the technique
of laparoscopy, and shortly afterwards he wrote his
famous short textbook Laparoscopy in Gynaecology in
1967 [5]. His first major paper: Laparoscopy and ovulation followed in 1968 [6]. Shortly afterwards, he further developed the laparoscopic technique to enable
aspiration of oocytes from follicles under direct vision.
Robert Edwards started his career in reproductive
biology at the Institute of Animal Genetics and Embryology, Edinburgh, in 1951, having just been demobbed
Figure 1.6 Patrick Steptoe and Robert

Edwards at the birth of the worlds first
in vitro fertilization conceived baby
Louise Brown on July 25, 1978. Courtesy
of Bourn Hall Clinic. See plate section for
color version.
from the British Army. There, under the supervision

of Professor Alan Parkes, he did his PhD on reproductive genetics. He moved on to the Institute of Medical Research, London, and then to the University of
Cambridge in 1963 to join two well-known researchers
in reproductive physiology: Professors Alan Parks and
Bunny Austin. There Edwards continued his work on
immunology and oocyte maturation. He then spent a
short time in the USA at Johns Hopkins University,
where he collaborated with two other greats in the
field of human ART, Drs. Howard and Georgeanna
Jones. On his return to Cambridge, he continued his
work on human oocytes. In 1968, Edwards, who had
heard about Steptoes work in retrieving pre-ovulatory
human oocytes laparoscopically, contacted him and
met with him at a Royal Society of Medicine meeting
in London. They quickly struck up a working relationship and friendship which, between 1968 and 1978, led
them to further develop the techniques of human IVF
and applied it to the clinical treatment of intractably
infertile women.
Steptoe and Edwards soon started to produce landmark papers together in 1969: Early stages of fertilisation in vitro of human oocytes and matured in
vitro [7]; and also in 1969: Identification of the midpiece and tail of the spermatozoon during fertilisation
of human eggs in vitro [8]; and in 1970: Laparoscopic
recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins [9]. They also carried out the first treatment cycles of oocyte recovery
with tubal insemination (ORTI), as they called the procedure, which was later to become known as gamete
intrafallopian transfer (GIFT).
Between the years 1968 and 1978, while they were
working closely together, Steptoe was in Oldham, Lancashire, England, working in a National Health Service hospital, and Edwards was at the University of
Cambridge. When the clinical treatment of infertile
women started, there followed a number of very difficult years in which none of the first 40 patients they
treated by IVF and embryo transfer (ET) became pregnant. In 1976 they did achieve their first pregnancy following transfer of a single blastocyst, but this subsequently turned out to be an ectopic pregnancy [10].
After 102 failed embryo transfers, including the one
ectopic pregnancy, Leslie Brown was treated and subsequently became pregnant following her first embryo
transfer. This was achieved in a natural IVF cycle,
with no stimulation; one oocyte was collected and a
single eight-cell embryo was transferred. There followed a difficult pregnancy for Mrs Brown, but her
baby, Louise Brown, was delivered by cesarian section on July 25, 1978 (Figure 1.6). Much to everyones
relief, baby Louise was found to be a perfectly normal,
fit and healthy infant. This momentous achievement
was announced with a simple publication as a letter
in the Lancet: Birth after reimplantation of a human
embryo [11]. The arrival of Louise Brown was heralded as The baby of the century. This was in spite of
considerable criticism and opposition by clinical and
Figure 1.7 Worlds first in vitro

fertilization conference, held at Bourn Hall
in 1981. Courtesy of Bourn Hall Clinic.
scientific colleagues, the lay press and by many representatives of different religious faiths.
Work on human IVF was also being conducted
elsewhere in the world, particularly in Melbourne,
Australia, where the team of Professor Carl Wood and
Dr. Alan Trounson achieved the birth of the worlds
fourth IVF baby, Candice Reed, in June 1980 [12]. At
the same time, Drs. Howard and Georgeanna Jones
had been working in Norfolk, Virginia, USA, and Elizabeth Carr, the first US in-vitro conceived baby, was
born on December 28, 1981 [13].
In England, meanwhile, Patrick Steptoe and Robert
Edwards were unable to continue their work on human
IVF, since neither the UKs National Health Service nor
any of the Universities or the Medical Research Council were willing to provide funding to help them to continue their work. They eventually found Bourn Hall,
an old Jacobean manor house in the Cambridgeshire
countryside, where they founded the Worlds first IVF
treatment and research center Bourn Hall Clinic
which opened in September 1980 (Figure 1.7). There,
Steptoe and Edwards continued their research and,
by 1986, they had achieved 500 live births [14]. Steptoe and Edwards achieved a number of distinguished
national and international awards over the next 3
years, but, in 1988, Patrick Steptoe fell seriously ill with
prostate cancer and died on March 21, 1988. Robert
Edwards continued to work as Scientific Director of
Bourn Hall and as Editor of the newly formed journal
Human Reproduction, which he cofounded. In 1994, he
retired from working at Bourn Hall.
In the early years following the first IVF births in

England, Australia and the USA, other teams were successful in achieving births: in 1982 in France from the
group of Professors Frydman and Testart; and in Sweden from the group of Professor Lars Hamberger. In
1982, in England, both Dr. Brian Liebermans group
and Professor Ian Crafts group also achieved live
births, followed by, in Austria, Professors Feichtinger
and Kemeter, and later that year births also occurred
in Finland, Germany and the Netherlands.
An interesting historical point is the suggestion
that the worlds second IVF baby was achieved in India,
following work by Dr. Subhash Mukhopadhyay. A baby
was born on October 3, 1978 following IVF and ET,
but his achievement was never officially recognized;
indeed, he was derided by his colleagues and officials
in India at the time. He eventually committed suicide
in 1981. However, following a close investigation of his
claim some 27 years later, he was officially accepted
as being the first Indian and second in the world to
achieve a live birth following IVF.
In 1983, the first IVF baby was born following the
transfer of frozenthawed embryos in Australia [15].
Oocyte donation, as a treatment option in IVF programs, also developed from about the mid 1980s, with
the first successful live birth reported from Australia
[16]. Originally developed to treat women with premature menopause, this was extended to treat women
with inherited diseases and, increasingly now, is being
used in the treatment of women in their mid to late
40s, or even older, to help them to have children
late in their lives. In a few countries, treatment using

donated embryos has been permitted and, in some
countries, treatment using gestational surrogates has
become available to treat women without a uterus or
with other reasons meaning that they are unable to
carry a child. Utian et al. published the first report of
an IVF birth in the USA through gestational surrogacy
in 1985 [17].
The need to develop more user friendly techniques to obtain oocytes, other than by the relatively invasive technique of laparoscopy, was developed by Lenz and Lauritsen in 1982, who described
the technique of abdominal ultrasound-guided needle oocyte recovery [18]. Gleicher et al. further developed this technique in 1983, approaching the ovaries
transvaginally but using an abdominal probe [19].
Later, in 1985, Mats Wikland in Sweden developed the
now almost universally used transvaginal ultrasound
probe-guided needle aspiration of pre-ovulatory follicles [20]. The techniques of intrauterine insemination (IUI) and GIFT also developed over the following
years as a more simplified variant of standard IVF.
Over the years since the beginning of human
IVF, many changes have occurred in ovarian stimulation protocols for IVF. The major developments have
been:
r 1970s
natural cycle IVF
clomiphene alone
r 1980s
clomiphene + urinary human menopausal
gonadotropin (HMG)
gonadotropin-releasing hormone (GnRH)
agonists + urinary HMG
flare protocol
ultra-short and short protocols
long luteal phase or follicular-phase start long
protocols
r 1990s
GnRH agonist + urinary-follicle stimulating
hormone (FSH) intramuscularly (im)
GnRH agonist + high purity FSH
subcutaneously (sc)
GnRH agonist + high purity HMG (sc)
GnRH agonist + recombinant human follicle
stimulating hormone (rhFSH) (sc)
r Late 1990s2000+
GnRH antagonists + rhFSH recombinant
human luteinizing hormone (rhLH)
recombinant LH
recombinant human chorionic gonadotropin
(hCG)
fill by mass versus IU recombinant FSH
patient friendly sc injections and use of pens
for injection
As can be seen above, stimulation protocols have
undergone many changes. In the early days, IVF was
conducted in natural cycles or with clomiphene-only
stimulation. It was in 1984 that Porter et al. in London first developed the use of GnRH agonists in IVF
stimulation protocols to prevent premature LH surges
[21], which, over the following years, became the gold
standard for use in IVF stimulation protocols; indeed,
it remains so for many practitioners. Introduction of
the GnRH antagonists in stimulation protocols, first
reported by Frydman et al. in 1991 [22], increasingly
has become used, allowing a more natural cycle and
being more patient friendly, since treatment does
not last as long as do GnRH-agonist protocols. Also
from the mid 1990s, there were major developments
in the production and use of gonadotrophins. These
were produced originally from human menopausal
urine and injected intramuscularly. High purity FSH
and HMG were a great improvement and could be
injected subcutaneously by patients themselves after
training. Also from about the mid 1990s, recombinant gonadotrophins were developed, producing the
purest FSH, LH and hCG. It was in 1992 that Germond et al. [23] and Devroey et al. [24] reported
the first pregnancies using the new recombinant
FSH (rFSH).
More recently still, there has been an enthusiasm
for the concept of IVF lite. This has introduced the
concept of a milder stimulation strategy for IVF in
order to reduce the risk of complications of stimulation, particularly of ovarian hyperstimulation syndrome. It is also thought to improve the chance of
implantation by reducing interference of the development of the endometrium that may occur in some high
dose gonadotrophin stimulation protocols.
In vitro fertilization was never really successful
in the treatment of severe male factor infertility, and
techniques were developed to try to improve the outcome for men with this diagnosis, for whom the
only real option then was the use of donor sperm.

In 1987, Laws-King et al., in Australia, first reported
the microinjection of spermatozoa under the zona
pellucida of oocytes [25]. However, it was not until
1992 that the first pregnancy after ICSI of oocytes with
single spermatozoa was reported by Palermo et al. of
the Free University of Brussels [26]. For the first time,
this technique allowed men with the most intractable
infertility problems to achieve pregnancies with their
partners. This treatment option has been the most
important development in ART since human IVF first
started. Most ART units worldwide now treat 4050%
or more of their ART cycles by ICSI, and some even
advocate using it for all ART cycles.
Other firsts include, in 1989, Handyside and colleagues from London who first showed it is possible
to take a single blastomere from an embryo, perform
pre-implantation genetic diagnosis (PGD) and to sex
the embryo by DNA amplification [27]. This technology has led to the development of a whole new subspecialty of techniques used to diagnose not only the
sex of an embryo but to detect a multitude of genetic
abnormalities, including single gene defects, and also
to perform screening for aneuploidy. In 1990, Verlinsky et al. reported the first polar body biopsy, with a
subsequent embryo transfer and pregnancy [28]. This
has proved to be most useful in countries which do not
allow embryos to be manipulated or biopsied, such as
Germany and Switzerland.
In 1991, Cha et al. developed the technique of in
vitro maturation (IVM) of oocytes and reported their
first pregnancy using this technology [29].
Silber et al. in 1994 reported the first cases of
testicular sperm extraction (TESE) combined with
intracytoplasmic injection [30] for men with obstructive and non-obstructive azoospermia. Other landmark developments were made in the diagnosis of
male factor infertility, particularly in 1996 when Reijo
et al. showed that some men with severe oligoasthenozoospermia had deletions on the Y chromosome [31]. The whole understanding of male factor
infertility has developed dramatically in the last 12
15 years.
The first autologous transplantation of frozen
thawed ovarian tissue was conducted by Oktay et al. in
2001 [32] and, in 2004, Donnez et al. reported the first
live birth after orthotopic transfer of frozenthawed
ovarian tissue [33]. These advances will do much in
the future to improve the reproductive outcomes for
young women who require chemotherapy or radiotherapy for malignancies.

One of the major hurdles still to be overcome
by clinicians and scientists practicing the ARTs is to
reduce the number of multiple pregnancies created. It
is considered now to be unacceptable that some 40
50% of children born as a result of IVF and related procedures are from multiple births, with the consequent
major increase in complications, both for the babies
and for the mothers. There is a very positive move now
towards making the majority of ETs in an IVF program
single embryo transfers be it at day 2, 3 or blastocyst stages. This change in practice is largely being led
by the Northern European countries, where multiple
rates have been reduced to 10% and even, in some
practices, to 5% [34]. However, in certain countries,
transfer of four or more embryos is occurring in some
2535% of cycles, producing twins in 2535% of cycles
and triplets in anywhere between 2.7 and 5.7% of deliveries. This is in spite of large numbers of fetal reductions being performed. However, these alarming figures are slowly reducing, year on year.
One of the most important developments in ART
worldwide during the last 32 years has been the evolution of guidelines or regulatory systems to govern
the practice of the ARTs. The state of Victoria, in Australia, was the first state to pass legislation on IVF in
1984; this became known as the Infertility (Medical
Procedures) Act 1984. The UK was the first country to develop a full regulatory process and regulatory
body. This started with an initial review by a Government appointed body, which produced a report in 1984
known as the Warnock Report. This proposed a UK
regulatory system which would cover clinical and scientific practices of:
r all treatment involving the creation of human
embryos outside the body
r all treatment involving donated gametes
r all storage of human gametes and embryos
r all research on human embryos
The report also recommended that all clinics
providing ART services should be licensed by
a regulatory authority. Following publication of
this report in 1984, voluntary and then interim
licensing authorities were set up to monitor ART
practice in the UK. The Human Fertilisation and
Embryology Act finally passed through Parliament in
1990, which led to the establishment of the Human
Fertilisation and Embryology Authority (HFEA)

in 1991. This body is responsible for the licensing,
regulation and monitoring all units practicing ART
in the UK. The rules and regulations are set out in a
Code of Practice, which is reviewed regularly, and
in 2009 the eighth edition of the Code of Practice was
produced, following passage of an updated Human
Fertilisation and Embryology Act in 2008 [35].
Most countries in 2010 have some form of regulation, more or less strict, while other countries have
guidelines, but there are still countries that have no
regulation or guidelines at all. The state of regulation
and practice worldwide is summarized in a 3-yearly
publication produced by the International Federation of Fertility Societies (IFFS) and published by the
American Society for Reproductive Medicine (ASRM);
the last edition was published in 2007 [36].
The study of fertility, both animal and human, has
fascinated clinicians and scientists for more than two
millennia. Research into fertility and infertility led us,
via many important milestones, to being able to treat
women and men with hitherto untreatable infertility by IVF and related techniques. These treatments
the ARTs are now very well established as mainstream treatments, almost universally accepted and
practiced. Worldwide, there are now an estimated 4
5 million babies who have been born since human
IVF was first successful in1978. Although the early
pioneering days of IVF are over, there is still a limitless amount of research to be done in the field of
ART, particularly in genetics and stem cell research.
It is also to be hoped that IVF and related ARTs will
become still more simple and patient friendly and,
in particular that they may become much cheaper, so
that ART can be provided in the less developed countries, where presently infertile couples are unable to
obtain treatment because of cost. It has been impossible to cover the whole story of the development of
ART over so many years in one short chapter, but the
present status of ART worldwide is built upon the fundamental achievements of the early scientific and clinical pioneers of our specialty. Their story bears more
in-depth study and understanding, to better appreciate what we all struggle to achieve for our patients
families.
A thorough comprehension of the history of IVF would improve the
depth of appreciation of challenges we are facing today, hopefully
resulting in improved outcomes of future treatments [37].
Just as this book was going to press, the Nobel Prize

Committee made the following announcement on the
4th of October 2010:
Robert G. Edwards, the 2010 Nobel Laureate in Physiology or
Medicine, battled societal and establishment resistance to his development of the in vitro fertilization procedure, which has so far led
to the birth of around 4 million people.
The many friends and colleagues of Robert Bob

Edwards are delighted at this very happy and hugely
well-deserved accolade and send him our most sincere
congratulations.
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Further recommended reading

Cohen J, Trounson A, Dawson J et al. The early days of IVF
outside the UK. Hum Rep Update 2005; 11: 43959.
Edwards RG. Introduction: the beginnings of human
in-vitro fertilization. In: Gardner DK, Weissman A,
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Reproductive Techniques. Colchester UK: Informa UK,
2009: pp. 1730.
26. Palermo G, Joris H, Devroey P and Van Steirteghem

A. Pregnancies after intracytoplasmic sperm injection
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Edwards RG and Sharpe DJ. Social values and research in

human embryology. Nature 1971; 231: 8791.
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Pregnancies from biopsied human pre-implantation
Edwards RG and Steptoe PC. A Matter of Life. London:

Hutchinson, 1980.
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Edwards RG and Purdy JM (eds.), Human Conception
in Vitro. London: Academic Press, 1981: pp. 3517.
Jones HW, Jones GS, Andrews MC et al. The programme
for in vitro fertilization at Norfolk. Fertil Steril 1982; 38:
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10
Steptoe PC and Edwards RG. Birth after reimplantation of

a human embryo. Lancet 1978; 2: 366.
Wood C and Trounson AO. Historical perspectives of IVF.
In: Trounson AO and Gardner DK (eds.), Handbook of
In Vitro Fertilization, 2nd edn, Boca Raton FL: CRC
Press, 2000: pp. 114.
Section 1
Chapter
Introduction
The effect of chemotherapy and

radiotherapy on the human
reproductive system
W. H. B. Wallace, R. A. Anderson and D. Meirow
Cancer in childhood is rare, with approximately 1400

new cases per year in the UK, and a cumulative risk
of around 1 in 500 by the age of 15 in resource-rich
countries. With long-term survival rates approaching
73%, it has been estimated that by the year 2010 about
1 in 715 of the adult population will be a long-term
survivor of childhood cancer [1]. Cancer is more common after puberty during the reproductive life span
of men and women [2, 3], and many of these patients
will be cured by combination treatment with surgery,
chemotherapy and radiotherapy. Long-term survivors
are nevertheless at risk of developing a number of
late sequelae [4], including impaired fertility, adverse
pregnancy outcomes and health problems in offspring
[57]. Loss of fertility is one of the most devastating
consequences of radio- or cytotoxic therapy for these
young patients who, having overcome their disease,
have expectations of a normal reproductive life.
Normal ovarian development and

follicular depletion
Current understanding of human ovarian reserve presumes that the ovary establishes several million nongrowing follicles (NGFs) during the second half of
intrauterine life, which is followed by a decline to the
menopause when approximately 1000 remain at an
average age of 5051. With approximately 450 ovulatory monthly cycles in the normal human reproductive life span, this progressive decline in NGF
numbers is attributed to follicle death by apoptosis.
In a recent study, the first model of human ovarian
reserve from conception to menopause that best fits
the combined histological evidence has been described
[8]. This model allows us to estimate the number of

NGFs present in the ovary at any given age, and it
suggests that 81% of the variance in NGF populations is due to age alone (Figure 2.1). Further analysis demonstrated that 95% of the NGF population
variation is due to age alone for ages up to 25. The
remaining 5% is due to factors other than age, e.g.
smoking, body mass index (BMI), parity and stress.
We can speculate that as chronological age increases,
factors other than age become more important in
determining the rate at which NGFs are lost through
apoptosis.
There is speculation that this widely held tenet of
mammalian ovarian function may require revision. A
report in 2004 suggested the presence of germ stem
cells in the adult mouse ovary [9], and two subsequent reports by the same group suggested the ability of bone marrow-derived cells to give rise to new
immature oocytes [10, 11]. Bone marrow transplant
was shown to partially restore the fertility of busulfantreated mice [11] even though all offspring derived
from the host germline. More recently, another group
has identified the presence of proliferative and culturable female germline stem cells in newborn and adult
mouse ovaries [12]. Strikingly, when these cells (transgenically labeled) were injected into a chemotherapytreated ovary, they became enclosed within follicles
and offspring bearing the transgene were produced.
These data provide a basis for re-evaluating the regenerative capacity of the mammalian ovary and new
approaches for overcoming fertility loss. While the
emerging evidence thus appears to provide evidence
in support of the existence of germ stem cells within
the adult mouse ovary, the WallaceKelsey model of
11
10 000 000
NGF population (log10 scale)
1 000 000
n =325
r 2 =0.81
100 000
Observed values
WallaceKelsey model
Lower 95% Cl for model
Upper 95% Cl for model
Lower 95% prediction limit
Upper 95% prediction limit
10 000
1000
100
10
5
0
5
10
15
20
25
30
35
40
45
50
Age (in months from conception to birth; in years from birth to menopause)
55
Figure 2.1 The best model for the establishment of the non-growing follicle (NGF) population after conception and the subsequent decline
until age at menopause is described by an ADC model. The model has a correlation coefficient r2 = 0.81, fit standard error = 0.46 and
F-value = 364. The figure shows the dataset (n = 325), the model, the 95% prediction limits of the model and the 95% confidence interval (CI)
for the model. The horizontal axis denotes age in months up to birth at age 0 and age in years from birth to 51 years. Reproduced with
permission from Wallace and Kelsey [8]. See plate section for color version.
ovarian follicle decline provides no supporting evidence of neo-oogenesis in normal human physiological aging.
Clinical detection of
chemotherapy-induced
ovarian damage
Many studies use amenorrhea as a surrogate for ovarian failure, with biochemical confirmation (i.e. elevated follicle stimulating hormone [FSH] concentration) in some. While amenorrhea may of course have
other causes than ovarian failure, it would seem a reasonable surrogate for population-based studies. However, an important drawback of the use of amenorrhea or elevated FSH measurements is that these only
detect the endpoint of the decline of ovarian function. It would be of considerable utility to have a biochemical or biophysical marker of the number of follicles in the ovary, i.e. the ovarian reserve (Table 2.1
[13]) [14] to allow detection of lesser degrees of damage and earlier changes during the progress to ovarian failure. This would allow improved analysis of the
12
Table 2.1 Qualitative assessment of ovarian reserve

r Early follicle phase, follicle-stimulating hormone (FSH)
r Early follicular phase, inhibin B
r Serum anti-Mullerian
hormone (AMH)
r Sonography: total antral follicle count (AFC)
r Sonography: ovarian volume
r Ovarian biopsy
r Response to ovarian stimulation
Adapted from van Rooij et al. [13].
effects of chemotherapeutic agents on the ovary and

clinically allow individualized advice based on pre and
postchemotherapy analysis of the ovarian reserve. Follicle stimulating hormone remains the most widely
used marker of incipient ovarian failure, but it shows
low sensitivity and considerable inter-cycle variability.
Inhibin B is a product of the granulosa cells of growing
follicles and shows a fair prediction of oocyte recovery following superovulation (the standard in vitro
fertilization [IVF] -based outcome measure of the
ovarian reserve). However, it is produced by the granulosa cells of large as well as small antral follicles, and
thus while its physiological importance in limiting the
inter-cyclic rise in FSH is undoubted, its concentration
Chapter 2: Effect of chemotherapy and radiotherapy
declines only late in reproductive life and thus its

value as a marker of loss of the ovarian reserve is
limited.
Anti-Mullerian hormone (AMH) is best characterized as a product of the fetal Sertoli cells, causing regression of the Mullerian structures in the male.
It was subsequently recognized that AMH is also an
important product of the adult ovary, produced by the
granulosa cells of smaller growing follicles [15]. Crucially, AMH secretion is only initiated at the start of
follicle development and declines abruptly in the early
antral stages. It is thus not a product of the dominant follicle, and only to a limited extent of FSHrecruited follicles. It would therefore be expected to
decline with age and show much less variation across
and between menstrual cycles than FSH or inhibin
B. These characteristics have been confirmed adding
to the convenience of its measurement [16]. Thus,
AMH is the best currently available marker of the
number of small growing follicles in the ovary [13].
It is not a direct marker of the true ovarian reserve,
i.e. the number of primordial follicles, but primordial follicle number is directly related to the number
of early growing follicles (although with quite wide
variability).
Data are increasingly available on AMHs utility to
detect chemotherapy-induced loss of ovarian reserve
in survivors of childhood [1719] and adult [2022]
cancer, and limited data from prospective studies illustrate its ability to reflect acute gonadotoxicity [20, 23].
Anti-Mullerian hormones utility to predict long-term
ovarian function and, more importantly, fertility when
measured before or after chemotherapy remains to be
determined.
Ultrasound can also be used to assess the ovarian reserve. As with AMH, most data derive from the
context of IVF/superovulation, with the number of
oocytes recovered being the primary outcome rather
than short or long-term fertility. Nevertheless, both
antral follicle count (AFC; the number of follicles of
210 mm diameter) and, to a lesser extent, ovarian volume have been explored as markers of ovarian damage during chemotherapy [20, 21]. In a prospective
study of women undergoing chemotherapy for breast
cancer, both AFC and ovarian volume decreased during treatment [20]. However inter-investigator variability is more important than with biochemical variables and, as these tests are also less convenient, it is
likely that AMH and future biochemical developments
will take precedence.
Chemotherapy and the ovary

The ovary is susceptible to chemotherapy-induced
damage, particularly following treatment with alkylating agents such as cyclophosphamide [17, 24]. Ovarian
damage is drug and dose-dependent and is related to
age at the time of treatment, with progressively smaller
doses required to produce ovarian failure with increasing age [25, 26]. Alkylating agent dose is related to subsequent fertility in childhood cancer survivors [27].
The stockpile of primordial follicles found in the
cortex of the ovaries represents the ovarian reserve.
Histological studies of human ovaries have shown
chemotherapy to cause ovarian atrophy and global loss
of primordial follicles [28, 29]. However, these studies of human ovarian biopsies do not provide any
information on the mechanism of injury. The effect
of chemotherapy on the ovary is not an all or nothing phenomenon, and the number of surviving primordial follicles following exposure to chemotherapy
correlates inversely with the dose of chemotherapy and
the nature of the agent [30].
The mechanism involved in the loss of primordial
follicles in response to anti-cancer therapy is not well
understood. A few human and animal studies have
demonstrated that chemotherapy induces damage to
ovarian pre-granulosa cells [31] and that apoptosis
occurs during oocyte and follicle loss [32]. In addition,
injury to blood vessels and focal fibrosis of the ovarian
cortex are further patterns of ovarian damage caused
by chemotherapy, evidenced in ovaries of patients
previously exposed to non-sterilizing chemotherapy
[33]. Fibrosis and vascular changes have also been
reported by others [31, 34], who examined ovarian tissue from girls treated for acute lymphoblastic leukemia
(ALL). As dividing/proliferating cells are, in general,
the major targets of chemotherapeutic agents, it would
seem likely that the granulosa cells of growing follicles
would be the most chemotherapy-sensitive cell type in
the ovary. This may be the reason for the abrupt decline
in serum AMH during chemotherapy [20, 23] and the
cessation of menstrual bleeding for a few months after
chemotherapy, due to loss of growing larger follicles.
Loss of the inhibitory influence of small growing follicles on initiation of primordial follicle growth (as in
some animal models [35]) will result in increased activation of the resting pool of primordial follicles and
thus premature ovarian failure: it is possible that a similar mechanism contributes at least in part to the effect
of chemotherapy in the human.
13
A reduced follicular reserve may result in premature ovarian failure (POF) and menopause many
years post-treatment, even in patients undergoing
chemotherapy at a very young age [24]. Significant
depletion of the primordial follicle stockpile postchemotherapy in a normally ovulating female has been
demonstrated in an animal model [30].
The risk of ovarian failure in several commonly
encountered malignancies and other disorders requiring chemotherapy and/or radiotherapy is presented
in Table 2.2. Cyclophosphamide is widely used in
combination chemotherapy regimens, and high dose
cyclophosphamide (200 mg/kg) is frequently utilized
as conditioning therapy before bone marrow transplantation (BMT), either alone, where recovery of
ovarian function is more likely, or in combination with
other chemotherapeutic agents or total body irradiation (TBI) [36].
Treatment of Hodgkins lymphoma with MOPP
(mechlorethamine, vincristine, procarbazine and
prednisolone) or ChlVPP (chlorambucil, vinblastine,
procarbazine and prednisolone) is associated with
ovarian dysfunction in 1963% of cases [37]. Amenorrhea is more commonly observed in older women,
but long-term follow-up is necessary, as a number of
young women also develop premature menopause.
The BEACOPP regimen (bleomycin, etoposide,
doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone) results in amenorrhea in
approximately 20% of women overall, but this rises to
67% in women treated with 8 cycles of dose-escalated
BEACOPP [38] with, as discussed above, age being
an important factor. Amenorrhea was reported by
95% of women aged over 30 years compared to
51% in younger women. Treatment with an ABVD
regimen (adriamycin, bleomycin, vinblastine and
dacarbazine), which contains no alkylating agents or
procarbazine, results in significantly less gonadotoxicity, especially in patients under 25 years [39]. In a
recent cohort study [40] of 518 female 5-year survivors
of Hodgkins lymphoma aged 1440 (median age: 25
years) at treatment, the Amsterdam group explored
the incidence of POF before age 40. Alkylating agents,
especially procarbazine (hazard ratio [HR]: 8.1) and
cyclophosphamide (HR: 3.5), showed the strongest
associations. Ten years after treatment, the actuarial
risk of premature menopause was 64% after high
cumulative doses (8.4 g/m2 ), and 15% after low
doses (4.2g/m2 ), of procarbazine [29]. A small
study demonstrated normal fecundity in women
14
treated with ABVD [41]. The risk of POF in Hodgkins

lymphoma and breast cancer is summarized in Tables
2.2 and 2.3[42].
In cases of germ cell tumors, fertility-sparing
surgery is possible in a large proportion of patients.
For patients with advanced stage disease, maximum
cytoreductive surgery appears to be beneficial. For
patients who require postoperative chemotherapy,
standard therapy involves a combination of bleomycin,
etoposide and cisplatin. Although POF may occur
in a small proportion of patients, 8099% of those
who undergo fertility-sparing surgery and chemotherapy can expect to maintain reproductive function
[43]. In a group of young women (median age: 25.5
years) who were treated with the VAC protocol (vincristine, actinomycin, cyclophosphamide) for germ
cell tumors, 13% were found to have irregular menses,
15% oligomenorrhea or amenorrhea and 8% persistent
amenorrhea, although 11 of 16 who had attempted to
become pregnant had been successful [44].
Chemotherapy is also used in patients with nonmalignant diseases such as systemic lupus erythematosus (SLE). Pulse cyclophosphamide therapy is
frequently used for active lupus nephritis or neuropsychiatric lupus. The major determinants for the development of ovarian failure in patients with SLE are age
at the start of therapy and the cumulative cyclophosphamide dose (number of cycles and doses) (Table 2.2)
[45]. Women with SLE and related diseases provide
additional challenges for fertility preservation because
of the uncertain course of their disease, and thus the
poor predictability of the total dose of cyclophosphamide that will be required.
Radiation and the

hypothalamicpituitaryovarian axis
The ovaries may be damaged following total body,
abdominal or pelvic irradiation, and the extent of the
damage is related to the radiation dose, fractionation
schedule and age at the time of treatment [25, 46]. The
human oocyte is sensitive to radiation, with an estimated LD50 of 2 Gy [47]. This is the lethal dose to
destroy 50% of NGF present in the ovary. The number of NGF present at the time of treatment, together
with the dose received by the ovaries, will determine
the fertile window and influence the age of POF.
Long-term ovarian failure has been reported in 90%
of patients after TBI (10.0015.75 Gy) and in 97%
of females treated with total abdominal irradiation
Table 2.2 Ovarian failure rates
Study (year)
Treatment
Age (years)
Ovarian failure (%)
Breast cancer
Lower (1999)
Premenopausal
45
35
28
Bines (1996)
Premenopausal
68
Meirow (1999)
44
50
Goodwin (1999)
Burstein (2000)
CMF
CMF
43.7 5.2
65
30
19
3039
30
CAF
3039
AC
30
Jonat (2001)
3040
0
1025
3039
13
Premenopausal
60
Hodgkins lymphoma
Howell and Shalet (1998)
Aggressive treatment
Meirow and Dor (2004)
Second-line therapy (not ABVD)
Bokemeyer (1994)
Infradiaphragmatic Rx
Brusamolino (2000)
Ovarian-sparing protocol
Behringer (2005)
Bone marrow transplantation
Dose-escalated BEACOPP
3857
32
50
25
45
30
30
95
30
51
No. of patients
Sanders (1996)
73
38 (mean)
99
Teinturier (1998)
21
217
72
Thibaud (1998)
31
3.217
80
Meirow (1999)
63
29 (mean)
79
Grigg (2000)
19
30 (mean)
100
Systemic lupus erythematosus

Mok (1998)
70
Boumpas (1993)
39 (0.51.0 g/m2 )
26
7 pulses
12
15 pulses
39
30
Blumenfeld (1996)
2030
20
Appenzeller (2007)
57, 0.75 mg/m2

63 patients
15
100
17.5 (12.3*)
50, 0.5 mg/m2

Manger (2006)
13
0 (20*)
30
39
3040
59
Adapted and updated from Meirow and Dor [3].

AC, doxorubicin, cyclophosphamide; BEACOPP regimen, bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone; CAF, cyclophosphamide, doxorubicin, fluorouracil; CMF, cyclophosphamide, methotrexate, fluorouracil; POF,
premature ovarian failure; SLE, systemic lupus erythematosus.
15
Effective (
) and mean (
Figure 2.2 From our understanding of

the radiosensitivity of the human oocyte
and our knowledge of the natural decline
in primordial follicles with increasing age,
we can provide effective and mean
estimates of the dose required to sterilize a
patient at a known age of treatment.
Adapted from Wallace et al. [51].
) sterilizing doses
22
20
18
Dose (Gy)
16
14
12
10
8
6
4
2
0
10
15
20
25
30
Age at treatment
35
Table 2.3 Risk of permanent amenorrhea in women

High risk
Stem cell transplantation, external beam

irradiation to fields including the ovaries,
breast cancer adjuvant combination
chemotherapy regimens containing
cyclophosphamide, methotrexate,
fluorouracil, doxorubicin and epirubicin in
women 40 years
Intermediate
risk
Breast cancer adjuvant chemotherapy

regimens containing cyclophosphamide in
women 3039 years or
doxorubicin/cyclophosphamide in women
40 years
Low risk
( 20%)
Combination chemotherapy regimens for

NHL, ALL or AML breast cancer adjuvant
chemotherapy regimens containing
cyclophosphamide in women 30 years or
doxorubicin/cyclophosphamide in women
40 years
Very low risk

or no risk
Vincristine, methotrexate, fluorouracil
Unknown
risk
Paclitaxel, taxotere, oxaliplatin, irinotecan,

trastuzumab, bevacizumab, cetuximab,
erlotinib, imatinib
Adapted from Lee et al. [42].

Risk assessment is based on amenorrhea rate. Because some
therapies compromise the follicular reserve without causing
amenorrhea, fertility may be compromised before the cessation
of menses.
ALL, acute lymphoblastic leukemia; AML, acute myeloid
leukemia; NHL, non-Hodgkins lymphoma.
(2030 Gy) during childhood [36, 48, 49]. Our understanding of the LD50 of the human oocyte has made it
possible to estimate the age at which premature ovarian failure may occur. Furthermore, we have estimated
16
40
45
50
the sterilizing dose following any given dose of radiotherapy at any given age, based upon the application of
a mathematical solution to the FaddyGosden model
for natural oocyte decline (Figure 2.2) [50, 51]. This
will help clinicians provide accurate information when
counseling women about fertility following treatment
for childhood cancer.
Gonadotropin deficiency following high dose cranial irradiation (24 Gy in the treatment of brain
tumors) manifests as delayed puberty or absent menses
and can be treated by hormone replacement therapy.
Interestingly, early puberty is often reported in females
with cranial radiation doses of 24 Gy [52]. However, we have shown a subtle decline in hypothalamic
pituitaryovarian function following low dose cranial radiotherapy (1824 Gy). This is characterized
by decreased luteinizing hormone (LH) secretion
throughout the cycle, an attenuated LH surge and short
luteal phases [53], which may compromise reproductive function. Recent data confirm lower fertility in childhood cancer survivors treated with either
hypothalamic/pituitary iradiation 30 Gy or ovarian/uterine irradiation 5 Gy [27].
Nowadays, the most commonly used fertility
preservation measure is the surgical transposition of
the ovaries outside the irradiation field before the initiation of pelvic radiation in adults with gynecological
malignancies [54]. This approach may be considered
for patients not planning to receive high dose systemic chemotherapy. Surgery is effective at protecting
the ovaries from direct irradiation damage, but fertility may be affected by scatter radiation, damage to the
Figure 2.3 Surgical transposition of the

ovaries outside the irradiation field prior
to initiation of pelvic radiation for
Hodgkins disease. Adapted from Meirow
and Dor [3].
Irradiation field
Right
transposed ovary
ovarian vasculature during surgery or torsion of the

transposed ovary (Figure 2.3). To avoid this, tissue
cryopreservation may be considered at the same time
as oophoropexy.
Radiation and the uterus

The uterus is at significant risk of damage following
abdominal, pelvic or TBI in a dose and age-dependent
manner [55]. Uterine function may be impaired following radiation doses of 1430 Gy as a consequence
of disruption to uterine vasculature and musculature
elasticity [48]. Even lower doses of irradiation, as in
TBI, have been reported to cause impaired growth
and blood flow [56]. The clinical consequences are
increased risk of miscarriage and premature delivery
[36]. A uterine contribution to an inability to conceive
following radiotherapy is not clear but seems likely.
A small number of studies have been reported
that attempted to improve uterine function in survivors of cancer with POF. In young adult women
previously treated with TBI, physiological sex steroid
replacement therapy improves uterine function (blood
flow and endometrial thickness) and may potentially
allow them to benefit from assisted reproductive technologies [56]. Larsen et al. studied uterine volume in
100 childhood cancer survivors and assessed uterine
response to high dose estrogen replacement therapy in
three patients with ovarian failure and reduced uterine volume following abdominal and/or pelvic irradiation [57]. There was no significant difference in uterine volume, endometrial thickness or uterine artery
Left
transposed ovary
blood flow following sex steroid treatment, suggesting that higher doses of pelvic radiation cause greater
damage than lower doses (as in TBI), and that this
damage may be irreversible.
Testicular function
In males, testicular damage can involve the somatic
cells of the testis (Sertoli, peritubular myoid and Leydig cells) or the germ cells. Sertoli cells are responsible for nurturing developing germ cells, and Leydig
cells produce testosterone. Recent data have revealed
the essential contribution of peritubular myoid cells in
mediating the effect of testosterone on spermatogenesis. Gonadal damage in males treated for cancer can
result from either systemic chemotherapy or radiotherapy to a field that includes the testes. Cytotoxic
treatment targets rapidly dividing cells and it is therefore not surprising that spermatogenesis is impaired
after treatment for cancer. The exact mechanism of
this damage is uncertain, but it appears to be linked
to depletion of the proliferating germ cell pool and
associated stem spermatogonial cells. Although the
pre-pubertal testis does not complete spermatogenesis and produce mature spermatozoa, cytotoxic treatment given to pre-pubertal boys may impair future fertility. Importantly, the pre-pubertal testis is susceptible
to cytotoxic damage.
Chemotherapy and the testis

As with radiotherapy, the germinal epithelium of the
testis is very sensitive to the detrimental effects of
17
chemotherapy, irrespective of pubertal status at the

time of treatment. Therefore, after receiving gonadotoxic agents, patients may be rendered oligospermic
or azoospermic. Testosterone production by Leydig
cells is usually unaffected, however, and thus secondary sexual characteristics develop normally or are
maintained [58]. Following higher cumulative doses
of gonadotoxic chemotherapy, Leydig cell dysfunction may also become apparent [59]. Recent data
indicate that the proportion of men with mild Leydig cell dysfunction and biochemical, if not clinical, hypogonadism following chemotherapy may be
higher than previously recognized [60]. Low testosterone concentrations are associated with a number of important clinical conditions, including osteoporosis, frailty, metabolic syndrome, cardiovascular
disease and erectile dysfunction, and thus replacement testosterone should be initiated with appropriate
monitoring [61].
Treatment of Hodgkins lymphoma has involved
the use of procarbazine, together with alkylating
agents such as chlorambucil, mustine and cyclophosphamide. While these drug combinations have yielded
excellent survival rates, the majority of male patients
subsequently developed permanent azoospermia [62,
63]. Mackie et al. studied patients with a mean age of
12.2 years at diagnosis who were treated with ChlVPP,
a regimen containing both chlorambucil and procarbazine [37]. On follow-up, 89% of these patients
showed severe damage to the seminiferous epithelium
up to 10 years following therapy. Consequently, the
treatment of Hodgkins lymphoma was modified in an
attempt to reduce the gonadotoxicity, whilst maintaining long-term survival [64]. Treatment with the ABVD
regimen, which contains no alkylating agents or procarbazine, results in significantly less gonadotoxicity,
with no patients demonstrating permanent azoospermia [62]. However, anthracycline exposure in this regimen renders it potentially cardiotoxic in the long term.
The BEACOPP regimen has high gonadal toxicity in
men as in women, with azoospermia reported in 89%
of men and low testosterone concentrations in over
50% [65].
Radiotherapy to the testis

In males, radiation doses as low as 0.11.2 Gy can
impair spermatogenesis, with doses over 4 Gy causing
permanent azoospermia. The somatic cells of the testis
are more resistant than the germ cells, and Leydig cell
18
dysfunction is not observed until 20 Gy in pre-pubertal

boys and 30 Gy in sexually mature males [66].
Within the pediatric and adolescent age group, testicular damage occurs with direct irradiation to the
testes, for example in the management of leukemia
[67]. In patients with leukemic infiltration of their
testes, radiation doses of 24 Gy are used, and this
results in permanent azoospermia [68]. Total body
irradiation applied as conditioning treatment before
BMT also irradiates the testes. However, the effects
of this can be difficult to elucidate as it is usually
given concurrently with alkylating agents, but doses of
910 Gy have produced gonadal dysfunction [69].
Chemoprotection
Preventing chemotherapy-induced damage to the
ovary or testes remains an elusive ideal in the field
of fertility preservation. Most attention has focused
on the potential for protecting the ovaries using the
gonadotrophin-suppressing gonadotropin-releasing
hormone (GnRH) analogues, although a clear biological basis for this approach is unclear as only
later stages of follicular growth are gonadotrophindependent. There may however be other, indirect
mechanisms on smaller follicles, or direct effects of
the GnRH analogues themselves, and the approach
is supported by animal studies in both rodents [70]
and non-human primates [71]. Initial small studies
failed to show any benefit [72] but were substantially
underpowered, and while later studies showed large
apparent benefits [73, 74], lack of randomization and
differences between treated and control groups preclude reliable interpretation. A recent randomized
controlled trial in women under the age of 40 with
breast cancer appears to support a benefit of GnRH
analogue treatment [75], but further studies are necessary to substantiate this finding as there are a number
of potential confounding methodological issues with
study design. Alternative approaches have addressed
the mechanisms of chemotherapy-induced oocyte
apoptosis using sphingolipids [32] and more recently
the c-Abl kinase inhibitor imatinib [76]. In the male,
the immunomodulator AS101 has been demonstrated
to protect against cyclophosphamide-induced sperm
damage and low fertility [77].
Summary
Whilst many children and adults diagnosed with
cancer can now realistically hope for long-term
survival, they must often live with the consequences

of their treatment. Infertility is one of the most devastating adverse effects of cancer treatment in this
patient group. Both chemotherapy and radiotherapy
can impair future fertility, and treatments for certain
cancers can be sterilizing [78]. Although predicting
individual fertility following treatment is extremely
difficult, further epidemiological studies and investigation of markers indicating gonadal damage may be
of use to our patients.
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21
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Section 1
Chapter
Introduction
Fertility preservation in non-cancer patients

Javier Domingo, Ana Cobo, Mara Sanchez and Antonio Pellicer
Introduction
The increasing survival rates of cancer patients [13]
have encouraged many specialists to focus on the irreversible consequences of chemotherapy and radiotherapy. Chemotherapy and radiotherapy treatment for
cancer or other pathologies has resulted in improved
survival rates, but these treatments may also lead
to sterility [4]. The increasing success of oncological
treatments means it is now even more crucial to implement procedures aimed at preserving fertility.
Similarly to cancer patients, there are some nononcological conditions currently treated with gonadotoxic agents, such as patients with autoimmune disorders or some chromosomal abnormalities that can
lead to ovarian failure. There are also other situations
where a woman may benefit from fertility preservation procedures, such as a woman with severe or recurrent endometriosis or a woman who wants to postpone
conception until her late reproductive years [5].
In addition, there are other gynecological situations regularly found in our clinical practice that were
previously difficult to deal with but which now can
be solved. Thus, oocyte or embryo vitrification can
be performed when, for any reason, we should prefer
to transfer embryos in a different cycle to the stimulated one. People with a high risk of hyperstimulation syndrome, the presence of a hydrosalpinx or
polyps during the stimulation, the absence of sperm
in the sample the day of the ovum pick-up or any
bleeding previously to embryo transfer are some situations where vitrification should be considered. As
a large number of embryos are needed, vitrification
may also be helpful for the low-responder patient with
the aim of accumulating oocytes or embryos, especially if pre-implantation genetic screening (PGS) is
planned [6].
Fertility preservation is already well established in

males. It may simply be solved by freezing sperm samples [7], and should be considered in all cases a specialist may suspect any prospective damage to sperm or to
testicular function. More recently, it has become possible to preserve young females gonadal function and
fertility. In this chapter we will focus on female fertility preservation procedures because of their complexity and peculiarities.
Ovarian failure leads to the impossibility of childbearing apart from other problems related to the
menopause, such as vasomotor, skeletal or cardiovascular alterations. Early menopause and infertility are
two of the main consequences for patients treated with
gonadotoxic agents. Interest in fertility preservation
has grown due to the effect of infertility on a womans
quality of life and self-esteem.
Lately, oocyte vitrification and ovarian tissue cryopreservation procedures have been modified with
excellent clinical outcomes [8, 9]. But these are not the
only methods with which to preserve fertility. The use
of gonadotropin-releasing hormone agonists (GnRHa)
is also of interest. In addition, although it is still
considered experimental, immature oocyte retrieval
for the in vitro maturation (IVM) process provides
great expectations for future fertility preservation
[10, 11].
Since fertility preservation procedures show a benefit for patients to be treated with gonadotoxic agents,
a new field in assisted reproductive technology (ART)
has appeared. As users of testicular and ovarian preservation techniques, patients undergoing treatment with
gonadotoxic agents are a new population in fertility clinics. However, fertility preservation procedures
should not be limited to patients undergoing cancer therapy but also applied to any situation where
23
reproductive function is threatened. If fertility may

be diminished, specialists, patients and their families
should be aware that fertility preservation is an option
and that reproductive function may be preserved.
Although some fertility preservation methods
need to be improved, they still must be disseminated
among medical professionals and patients with the aim
of preventing infertility. Future fertility or a pregnancy
is not guaranteed by fertility preservation procedures
but, if done, these procedures may help people face any
treatment with a high risk of ovarian failure, offering
them future hope [12].
Ovarian damage and

decreased activity
Gonadotoxicity a decrease in ovarian activity
depends on several factors, including the age of the
patient; the initial status of the ovaries (referred to as
the antral follicular account); the treatment applied
(chemotherapy, radiotherapy or surgery) and cumulative doses; and the type of agent used.
As many factors may contribute, it is difficult
to establish the exact incidence of premature ovarian failure after systemic chemotherapy. Most ovarian failure data are referenced to cancer patients, who
receive higher doses of chemotherapy and, consequently, have an increased incidence of ovarian failure.
People with autoimmune disorders may also be treated
with chemotherapy but usually at lower doses than
cancer patients. So, chemotherapys consequences may
not be so dreadful but determined by the cumulative
dose.
Although many of the patients treated with
chemotherapy recover their ovarian function once
chemotherapy is completed, there is an increased risk
of premature ovarian failure, especially when related to
age [13] and the use of alkylating agents [14, 15].
Age
The ovarian cortex has a limited number of primordial
follicles that decrease with age. Previous chemotherapy
or surgery can also affect the initial status of gonads
before treatment starts. This will determine the final
impact on ovarian function.
Cumulative doses of cyclophosphamide can cause
infertility in young women [16]. Gonadotoxicity is
directly related to age: the cumulative dose needed
to cause premature ovarian failure decreases as age
24
increases. Its effect can be acute or cumulative, and the

ovarian capacity for recovering is limited. Chemotherapy and radiotherapy frequently induce a reduction in
the number of germ cells, with a loss of steroid hormones, the possibility of mutation or teratogenic consequences [17].
The implications of chemotherapy treatment on
fertility and future pregnancy has a higher relevance
for younger women as most are childless or havent
completed their family. But, as gonadotoxicity is an
age-related process, their younger age will have a protective effect. Many younger patients will naturally
recover their ovarian function and fertility, especially
if the applied chemotherapy doses are low.
Chemotherapy
It is known that chemotherapeutic agents can cause
mutations, DNA adducts and structural breaks, as well
as oxidative damage in somatic and germ cells. Alkylating agents such as cyclophosphamide or ifosfamide
are the most gonadotoxic agents, but also gonadotoxic
are chlorambucil, busulfan, cisplatin, melphalan, carboplatin or procarbazine [14, 15, 18].
The effect of chemotherapy on the ovary is not an
all-or-nothing phenomenon, so the number of surviving primordial follicles following chemotherapy will
depend on several factors such as age, type of agent and
doses received [16].
Chemotherapys alkylating agents join with DNA,
avoiding its replication and transcription [18]. They
are extremely gonadotoxic by acting at any phase of
the cellular cycle (cellular cycle phase independent),
causing damage to the primary follicles. Pathological
examinations of ovarian biopsies in patients treated
with cyclophosphamide show either a total absence
or a significant reduction in the number of inactive follicles, with fibrosis and no signs of follicular
maturation [19].
The mechanism of chemotherapy causing premature ovarian failure is not well known, but granulosa
cells appear to be the crucially affected cells [20]. Cellular edema of pre-granulosa cells is observed, with
queratin deposits and edema of the nucleus of the cell,
which damages the oocyte morphology [21]. Additional factors, such as vascular alterations and fibrosis
of the ovarian cortex, may contribute to the reduction
of follicles [22].
What is clear is that both the antral follicle count
and ovarian volume decrease after chemotherapy.
Chapter 3: Fertility preservation in non-cancer patients
A fast fall in anti-Mullerian hormone (AMH)

and inhibin B concentrations is observed during
chemotherapy, although estradiol concentrations are
maintained [23].
Radiotherapy
Similar to chemotherapy, the effect of radiotherapy on
the gonads depends on age, cumulative doses, fractioned doses and irradiation area. The average doses
needed to destroy oocytes in humans is 2 Gy [24].
Ninety-seven percent of women receiving 5.010.5 Gy
will subsequently undergo ovarian failure [25].
Especially if radiotherapy is applied during childhood, the irradiation area has been associated with
alterations of the uterine function due to the reduction of vascular flow and endometrial thickness [26].
Cranial irradiation with 3545 Gy doses can damage the hypothalamuspituitarygonadal axis but, as
gonads are not affected, they recover their function
with gonadotropin replacement.
Surgery
Repeat ovarian surgery due to endometriosis or
another benign pathology may diminish the ovarian
reserve and lead to premature ovarian failure. Furthermore, in recurrent endometriosis, normal residual
ovarian tissue may be compromised.
Tubal sterilization through electrocoagulation,
when compared to the application of mechanical clips,
also seems to have an adverse effect on ovarian reserve
in the postoperative period. Significant differences
have been detected in ovarian volumes at day 3 and
antral follicle counts at 10 months after the tubal
occlusion [27].
Treatment for rheumatic diseases

There are four main categories of drugs for the treatment of rheumatic diseases: (1) anti-inflammatory
drugs; (2) corticosteroids; (3) immunosuppressive
drugs; and (4) biological agents. These treatments are
split into two main groups: the disease modifying antirheumatic drugs (DMARDs) and the non-steroidal
anti-inflammatory drugs (NSAIDs).
Disease modifying anti-rheumatic drugs

Disease modifying anti-rheumatic drugs are a host
of new drugs. Although most of the patients diagnosed with rheumatic diseases are treated with non-
biological DMARDs, the rate of biological DMARDs

is increasing. The gonadotoxic effects of the antiinflammatory and immunosuppressive drugs have not
been studied with the exception of salazopyrine and
some cytotoxic drugs as described below.
r Salazopyrine impairs fertility in males, although
not females, with a higher incidence of
oligospermia, decreased sperm motility and
higher rates of abnormal forms. Men with
inflammatory bowel disease treated with
salazopyrine showed a higher incidence of fetal
abnormalities among offspring. Folate deficiency
may have a role, as salazopyrine inhibits the
gastrointestinal and cellular uptake of folate [28],
but salazopyrine also has its own role as fetal
abnormalities werent avoided with folate
supplementation. An oxidative stress mechanism
of male-induced infertility has also been described
[29]. Usually, spermatogenesis recovers at about 2
months after withdrawal of the drug [30].
r Cyclophosphamide and chlorambucil are rarely
used in the treatment of rheumatoid arthritis, but
these drugs are very important for patients with
systemic lupus erythematosus. Cyclophosphamide
is gonadotoxic in both sexes. It is not possible to
predict which patients will become infertile and
which will recover reproductive function, this
depending fundamentally on age and the
cumulative dose [30].
Non-steroidal anti-inflammatory drugs

Inhibitors of cyclooxygenases (COX-1 and COX-2)
are involved in ovulation and implantation. Transient
infertility has been described after treatment with
NSAIDs, such as indomethacin, diclofenac, piroxicam
and naproxen. Non-steroidal anti-inflammatory drugs
can inhibit the rupture of the luteinized follicle and,
thereby, cause transient infertility [30].
A decreased sperm count has been found in
chronic male users of NSAIDs at low or moderate
doses [31].
Fertility preservation
procedures
Several strategies have been proposed over the last few
years to protect and preserve the ovarian function in
patients with cancer or suffering from other pathologies with a high risk of premature ovarian failure. Some
25
have demonstrated their efficiency and are now part of

the daily routine of clinical practice, while others are
still under evaluation.
These options include embryo and oocyte cryopreservation, cortical or whole ovary cryopreservation
and GnRHa protection. In vitro maturation of imature
oocytes still needs improvement, but there is no doubt
that it will become an important part of these procedures in the future as the trend in fertility preservation
techniques is directed towards ovarian tissue cryopreservation and further retrieval of immature oocytes
followed by IVM and vitrification [32].
Unlike with cancer patients for whom chemotherapy needs to be started immediately, other patients
usually have no problem with the time frame of the
23 weeks needed to obtain the oocytes, as there is
no hurry to complete the ovarian stimulation. Neither
patients with endometriosis nor young people who
wish to postpone childbearing are inconvenienced by
this time frame. Indeed, some stimulations cycles can
even be performed to increase the number of oocytes
when oocyte vitrification is intended.
26
increased due to a better understanding of oocyte

physiology, the use of improved media and the implementation of new techniques [35]. The number of
pregnancies resulting from oocyte cryopreservation is
constantly increasing, with no apparent increase in
adverse postnatal outcome such as low birth weight or
congenital abnormalities [36].
Embryo cryopreservation
Embryo cryopreservation is a widely accepted method
that is now considered the standard practice for fertility preservation [37]. Previously, oocytes had to
be fertilized to be preserved, as thawed embryos
were considered to achieve higher survival rates than
oocytes. The female patient needed to have a partner
or sperm donor to fertilize the retrieved oocytes, creating embryos that may not have been used in the future,
which had various ethical considerations. Oocyte
cryopreservation avoids the need for sperm at the time
of oocyte retrieval and the results have been similar to
embryo cryopreservation, and thus should be considered an option.
Oocyte vitrification
Ovarian tissue cryopreservation
Oocyte vitrification is a method of cryopreserving

human oocytes which provides an excellent clinical
outcome [33]. Vitrification is solidification of a solution by an extreme elevation of viscosity using high
cooling rates, from 15 000 to 30 000 C/min, which
avoids ice crystal formation and, thus, the damage and
the osmotic effects caused by intracellular ice formation. One of the problems of vitrification is the toxicity
of cryoprotectants. This can be reduced by the use of
an adequate combination of cryoprotectants (ethylene
glycol + dimethylsulfoxide [DMSO] + sucrose) or by
using very low volumes, which increases the speed of
the vitrification process and consequently reduces the
use of cryoprotectant in the vitrification solution [34].
The Cryotop method is a minimal volume device
where oocytes are vitrified in volumes 0.1 l, which
preserves their capacity for fertilization and further
development after warming. Survival rates of 97% have
been referred, with no differences in fecundation and
implantation rates, embryo quality or pregnancy rates
when compared to fresh oocytes [35].
Historically, the slow cooling method for oocyte
cryopreservation has been hampered by its low efficiency and because it did not guarantee reproducible results. However, recently the success rate has
Ovarian tissue freezing for later autotransplantation is

another alternative for fertility preservation in women
with oncological or non-oncological diseases [38].
Immature oocytes in primordial follicles of the ovarian
cortex are less sensitive to cryopreservation damage
[39]. Thus, ovarian tissue freezing is an alternative to
ovarian stimulation and oocyte cryopreservation for
preserving fertility. Orthotopic transplantation of the
frozenthawed ovarian cortex would allow natural fertility and, in the case of failure, in vitro fertilization
would still remain an option. Another advantage of
this approach, apart from future childbearing, is that
patients would be able to restore ovarian function.
Ovarian cryopreservation and transplantation procedures have so far been almost exclusively limited to
avascular cortical fragments. Transplantation of an
intact ovary with vascular anastomosis has been proposed as a way to reduce the ischemic interval between
transplantation and revascularization. To date, there
are only a few newborns from this technique [40].
Recently, 2 newborns, one miscarriage at 7 weeks and
a biochemical pregnancy were related following 6 reimplants [41]. This publication was interesting because
in all 6 patients the whole ovary was cryopreserved
prior to treatment, and tissue from 3 of the patients was
transported 45 h on ice prior to freezing, demonstrating that hospitals may offer cryopreservation without
having the necessary local infrastructure.
The main disadvantage of this technique is that it
requires surgery laparoscopy to obtain the ovarian
tissue and a further re-implant with appropriate incorporation of the cryoprotectant to the tissue. Ischemic
damage and reduced follicular pool usually appear
after transplantation. The active life of the transplanted
tissue will depend on the neoangiogenesis and new
vascularization.
This approach offers great possibilities to patients
in the future, since portions of healthy tissue can be
preserved for a further use when an oophorectomy is
performed for a benign indication. The ethical basis for
performing this surgery for elective cryopreservation
has been discussed [42, 43], but a patients request for
cryopreserving small portions of ovary at the time of
any other gynecologic surgery should not be denied on
ethical grounds.
Currently, ovarian tissue cryopreservation is
only recommended as an experimental treatment in
selected patients. Research is needed to investigate the
revascularization process with the aim of reducing the
follicular loss that occurs after tissue grafting.
Ovarian tissue cryopreservation can serve as a
source of follicle for IVM. Thus, though still experimental, future fertility preservation techniques will
tend towards combining ovarian cryopreservation and
immature egg retrieval for further in-vitro oocyte maturation and vitrification. To become widely accepted,
these procedures need to be safe, easy to perform
and with favorable results. Meanwhile, a combination of ovarian cryopreserved transplantation, further
ovarian stimulation and vitrification of the retrieved
oocytes to be accumulated for an in vitro fertilization
(IVF) cycle can be used. A twin pregnancy was recently
obtained through this method [44].
antral follicles and their evolution to antral follicles

was demonstrated. Thus, GnRHa could avoid follicles
reaching their sensitivity threshold to chemotherapy
by supression of the granulosa cells. But it is not yet
known if the effect would be similar in humans, as
the GnRHa protective effect would not be sufficient
enough for the regimens used in humans, which are
usually longer and with higher doses of chemotherapy than those protocols used in animals. A reduction in the response of breast cancer to chemotherapy can be observed, which is due to the GnRHa effect
blocking the cells in G0 phase of the cellular cycle. This
causes cells to become resistant to chemotherapy as it
is considered that 50% of tumoral cells in breast cancer have receptors for GnRH and its analogues [51].
Experimental studies have demonstrated that both
GnRH agonists and antagonists directly inhibit ovarian cancer proliferation through GnRH receptors overexpressed on 80% of these tumors [49].
Patients with cyclophosphamide-treated systemic
lupus erythematosus or other autoimmune diseases
urgently need ovarian protection as, although doses of
chemotherapy are usually not as high as those given
for cancer treatments, there is a high premature ovarian failure rate [52]. Because of this, GnRHa protection should be considered, as well as other ART fertility preservation procedures.
Gonadal medical protection
Although of great interest for the future, immature egg

retrieval for further in-vitro oocyte maturation and
vitrification is currently not a feasible option.
Most of the follicles in human ovaries remain
primordial. Thus, they would be the most abundant
source of oocytes but, due to their immaturity, IVM is
needed. Primordial follicles can be isolated from either
fresh or cryopreserved ovarian tissues and matured in
vitro for further vitrification.
Many healthy children with normal outcomes have
been born after using this method [54]. The main
Gonadotropin-releasing hormone agonists

Although there is controversy about the use of
GnRHa preventing the ovaries from being damaged
after chemotherapy, the latest prospective randomized
studies do show a benefit [18, 48, 49]. Following the
administration of GnRHa, a reduction in the mitotic
activity of the granulosa cells has been described
[50]. In some studies performed on mice treated with
GnRHa, inhibition of the recruitment process of pre-
Imatinib
Imatinib acts by blocking the apoptotic pathway activated by cisplatin in ovarian germ cells. Cisplatin
induces DNA damage by activating the c-AblTAp63
pathway, leading to cell death. In cell lines, c-Abl phosphorylates TAp63, which induces the activation of
proapoptotic cells. Treatment with the c-Abl kinase
inhibitor imatinib blocks these effects [53].
In-vitro maturation
27
benefit is the absence of stimulation and its low cost,

but results are not consistent enough and still need
to be improved [11, 54]. Pregnancy and implantation
rates are lower than those obtained with standard IVF
cycles [55], and a higher clinical miscarriage rate has
been observed [56]. So, more controlled studies are
needed of the possible long-term effects of IVM on
babies.
This method may be considered for patients in
whom hormonal ovarian stimulation is not recommended due to high estradiol levels, such as breast cancer patients or those suffering from systemic lupus erythematosus. It may also be suitable for patients with
polycystic ovary syndrome (PCOS) or when there is
an urgent need to start cytotoxic therapy.
Transposition of ovaries
Scatter radiotherapy can cause considerable damage
even if the gonads are outside the radiation field. The
purpose of this approach is to avoid the direct exposure of the ovaries to radiotherapy, although the indirect exposure can also cause gonadotoxicity. Thus, it
should be indicated for any pathology that requires
pelvic radiotherapy treatment. When this approach is
performed, 1690% of the patients show the ovarian
function preserved [4547].
Ovarian transposition is not suitable for nononcological patients as radiotherapy is uncommonly
used, although it is very useful for cancer patients
when they are going to receive local radiotherapy.
Indications for fertility preservation in

non-oncological patients
Fertility preservation is not limited to cancer patients,
and can be considered and offered to patients with
many other ordinary conditions. Any patient with a
high risk of premature ovarian failure is a possible
candidate for fertility preservation. The indications
include those listed in Tables 3.1 and 3.2.
Autoimmune diseases
The effect of autoimmune disorders on fertility
depends most of all on a womans reproductive age.
Fertility preservation methods should be considered
in young women with severe systemic autoimmune
rheumatic diseases requiring imminent gonadotoxic
treatment.
28
Table 3.1 Ovarian tissue cryopreservation: the Valencia

program for fertility preservation
Malignant
Breast cancer
Hodgkins lymphoma
Other tumors
Non-malignant
(n = 284)
170
62
52
(n = 17)
Systemic lupus erythematosus
Crohn disease
Endometriosis
Nephropathies
Multiple sclerosis
Wegeners granulomatosis
The availability of new medications for the treatment of rheumatoid arthritis has dramatically changed
the prognosis for these patients, but limited data about
their reproductive or developmental toxicity are available, particularly when the medications are used for
the treatment of rheumatoid arthritis.
Cyclophosphamide is only used for refractory
rheumatoid arthritis. It seems to have a significant
beneficial effect on the clinical evolution of rheumatoid arthritis, but its administration has been limited due to its secondary aggressive effects and the
similar benefits and low risk of other anti-rheumatic
agents. Furthermore, cyclophosphamide treatment for
rheumatoid arthritis has been related to the appearance of some hematological malignant neoplasms and
bladder cancer [57, 58].
Cyclophosphamide is the elective drug of choice
in the treatment of severe manifestations of systemic
lupus erythematosus, such as proliferative nephritis,
affection of the central nervous system, pneumonitis
or severe thromobocytopenia [59].
Cyclophosphamide-treated lupus patients have
premature ovarian failure rates of up to 50% in women
below 30 years and 60% in women between 30 and 40
years of age [60]. In a manner similar to onocological
patients, as survival rates have increased ovarian failure and infertility have become important issues for
these patients.
The use of intermittent cyclophosphamide has
been proposed due to its successful application in
systemic lupus erythematosus and the observed reduction in toxicity. However, there is continued controversy over its results, and it remains unclear if secondary effects of cyclophosphamide are influenced
Table 3.2 Oocyte vitrification with non-oncological indication: Instituto Valenciano de Infertilidad (IVI), Valencia, Spain
No. patients
Age
No. vitrified oocytes
33 3.2
20 (10 4)
Postponing childbearing
65
34 6.1
890 (7.7 5.3)
Gynecological disorders in ART (bleeding, hydrosalpinx, hydrometra)
67
35.8 4.2
532 (7.9 5.6)
Gynecological disorders non-ART
65
36 4.2
346 (5.8 4.8)
Lack of partners sperm sample
18
35 4.1
184 (10.8 5.5)
OHSS
12
32 4.3
286 (19.0 4.5)
Autoimmune diseases
Accumulation previous to PGS-FISH
348
39.5 3.5
2050 (2.5 2.6)
Accumulation previous to PGS-PCR
28
35.5 3.3
264 (5.1 3.3)
543
36.7 3.2
2163 (2.6 1.6)
Low response
ART, assisted reproductive technology; PGS, pre-implantation genetic screening; FISH, fluorescence in-situ hybridization; PCR, polymerase
chain reaction; OHSS, ovarian hyperstimulation syndrome.
by the frequency of administration or the cumulative

dose taken by a patient [58].
There is a concern that exogenous female hormones may worsen disease activity in women with
systemic lupus erythematosus. Due to this, rheumatologists have traditionally discouraged the use of
estrogens in lupus patients. Recent investigations have
shown that estrogens can cause light cutaneous eruptions, but do not decrease disease activity. But these
findings cannot be applied to women with high levels
of anti-cardiolipin antibodies, lupus anticoagulant or
previous thrombosis with low activity lupus, in whom
the use of estrogens may increase the risk of severe
lupus crises [61].
Estrogen may have some use when oocyte cryopreservation is desired, because ovarian stimulation
is needed. Thus, high estradiol levels can result in
patients with severe lupus being stimulated with
gonadotrophins. In these cases, letrozole can be used
for the stimulation in a manner similar to estrogen sensitive cancers. If not, IVM could be an
option.
Letrozole is an aromatase inhibitor that has been
shown to be effective as an ovulation inductor because
it reduces the negative feedback that estradiol exerts in
the hypothalamus and pituitary. This results in an
increased follicle stimulating hormone (FSH) sensitivity in ovarian granulosa cell receptors. Furthermore,
letrozole induces suppression of circulating estradiol
levels [62]. It can be used alone or in combination with
FSH. Letrozole with FSH results in a higher number of
oocytes that can be vitrified with lower estradiol levels,
in a manner similar to physiologicals [63].
As described by Oktay, letrozole stimulation consists of oral administration of 5 mg of letrozole beginning on the 2nd or 3rd day of the cycle and continuing until the day of human chorionic gonadotropin
(hCG) administration. After 2 days of letrozole administration, 150 U of gonadotropins are added. A GnRH
antagonist is administered when a follicle reaches 14
15 mm or the E2 level exceeds 250 pg/ml, and hCG
is given when the leader follicles are 20 mm. Letrozole administration is continued until the appearance
of menses [63].
Postponement of childbearing (age)

Social and economic factors associated with modern
lifestyles have resulted in many women choosing to
delay having children, and so the number of infertile
couples has risen over the last few years. Population
data reveal an increase the age at which both men and
women attempt to conceive their first child.
Over the past 10 years the mean age at which a
woman conceived her first child rose by more than
2 years. The negative effect of age on fertility is well
established, especially for women over 35 years of age
for whom poorer quality oocytes and decreasing ovarian reserve are of greater concern that with younger
women [47, 64]. Reproductive trends and the physiological factors associated with conception in older
women point to growing problems with infertility in
the future.
Cryobanking their own healthy young oocytes is
a good option for women who plan to conceive children late in their reproductive life. This option is even
29
more attractive for women who have a family history of

premature menopause or factors that predispose them
to premature ovarian failure.
benign indication, portions of healthy tissue could be

preserved for future use.
Bone marrow transplantation

Recurrent ovarian surgery (endometriosis)
Repetitive surgery on the ovary due to endometriosis
or any other benign pathology can diminish ovarian
reserve and lead to premature ovarian failure.
Endometriosis is one of the most frequent pathologies in gynecological surgery. Although there is no evidence that surgery can cure infertility, cyst excision is
considered one of the best options to avoid recurrence
of endometriosis and improve fertility. However, excision of endometriotic cysts is also associated with a
significant reduction in ovarian reserve [65], and so
electrosurgical coagulation plays an important role.
Ovarian damage and premature ovarian failure incidence may increase in the case of bilateral endometriotic cysts.
Endometriomas should be removed only in case of
pain, infertility or if it is an asymptomatic endometrioma over 4 cm in size. It is still unclear which is the
best approach when assisted reproduction techniques
are used, but convincing evidence has emerged showing that responsiveness to gonadotrophins is reduced
after ovarian cystectomy and there are no deleterious
effect of surgery on pregnancy rates. So, proceeding
directly to IVF is recommended to reduce the time to
pregnancy and to avoid potential surgical complications. Surgery should be considered only for large cysts
and smaller ones that can obstruct the ovum pick-up
because of their location in the ovary [66].
Excessive surgery often leads to destruction of normal ovarian tissue, which is usually excised along
with the endometrioma wall. Analysis of excised tissue reveals that most of the follicies were close to the
ovarian hilus. Incomplete surgery is associated with
an increased risk of recurrence [67], but there is no
clear evidence as to whether the damage is related to
the surgical procedure, to the previous presence of
endometriomas or both [68]. Following surgery, an
important reduction of the ovarian response to stimulation is observed, but this does not seem to be related
to the dimension of the excised ovarian cyst [65].
In patients with a high risk of ovarian failure,
oocyte vitrification or preservation of ovarian tissue
should be considered before surgery. When a cystectomy or an oophorectomy is performed due to a
30
Bone marrow transplantation for the treatment of both

oncological and non-oncological hematological diseases leads to ovarian failure due to the aggressive
chemotherapy and radiotherapy used to destroy preexisting bone marrow [69].
Chromosomal abnormalities that

can lead to premature ovarian
failure: Turners syndrome
Premature ovarian failure is a common condition of
Turners syndrome. Fertility preservation may not be
feasible for most patients with Turners syndrome.
After careful consideration of increased pregnancy
associated risks, it can be recommended for young age
patients with mosaic Turners syndrome [70].
Spontaneous puberty occurs in 2030% of Turners
syndrome patients and their fertility rates are about 5
10% [71]. This indicates the presence and maturation
of follicles when young, and so this is probably the best
time to attempt fertility preservation procedures.
Ovarian borderline tumors

An ovarian borderline tumor is an epithelial tumor
with a low potential to invade or metastasize. This
low malignant potential tumor accounts for 1015% of
ovarian epithelial tumors. Nearly 80% are stage I at the
time of diagnosis. Prognosis will depend on the histological type and stage at surgery, serous and mucinous being the most frequent histological types. The 5year survival rate for women with stage-I borderline
tumors is about 9597%, but the 10-year survival rate
is only between 70 and 95% because of late recurrence.
The survival rate for advanced stage serous borderline
tumors with non-invasive implants is 85%, while the
rate for tumors with invasive implants is 60%. Mucinous borderline tumors are usually gastrointestinal.
Survival rates are good, except when they are associated with peritoneal pseudomyxoma, which has bad
prognosis due to its extraovarian origin [72].
Treatment for borderline ovarian tumors is similar to that for ovarian cancer and includes hysterectomy with bilateral adnexectomy. However, patients
with borderline ovarian tumors tend to be younger
than women with invasive ovarian cancer, with many

of them nuliparous, and so conservative surgery (cystectomy or unilateral oophorectomy) must be considered for patients with early stage borderline ovarian
tumors [73]. Lately, this concern has been expanded to
women with advanced stage disease [74]. Conservative
management increases the risk of recurrence but does
not affect survival. Recurrence rates are higher when
a cystectomy is performed (58%) instead of an adnexectomy (23%) [73]. Following this treatment, fertility,
pregnancy outcome and survival rates remain excellent.
A biopsy must be performed when any macroscopic implant can be observed on the contralateral ovarian surface. Thirty percent of serous borderline tumors are bilateral and, because these tumors
often are diagnosed after surgery, fertility preservation
should be recommended before the procedure. If ovarian preservation is impossible, oocyte or ovarian cryopreservation before the surgical procedure must be
considered [74].
In any case, fertility preservation can also be done
as preventative measure in case of recurrence and the
need for an adnexectomy.
Similar to borderline ovarian tumors, patient carriers of the BRCA-1 or BRCA-2 mutations have an
increased risk of breast and ovarian cancer, and so a
patient may undergo adnexectomy due to cancer or
just as prevention. Fertility preservation techniques
may also be recommended in such circumstances.
Other routine gynecological situations

There are some other clinical gynecological situations
in the routine clinical practice of ART that can be
solved by using cryopreserved oocytes. These situations are as follows:
r Patients with a high risk of ovarian
r
r
r
r
hyperstimulation syndrome to whom embryo

transfer would not be desirable.
To accumulate oocytes in low-responder patients,
for a further IVF or pre-implantation genetic
screening (PGS) cycle [75].
To postpone transfer to another cycle due to
finding a hydrosalpinx or polyp, or any bleeding
or hydrometra that may appear previous to
transfer [75].
Semen or pathological samples are not available
the day of the oocyte retrieval.
Inappropriate endometrium for transfer [75].
r Establishment of egg-banking for ovum donation

programs. This would simplify the donation
process, since no synchronization between donor
and recipient is needed. Oocyte cryopreservation
would also ensure safer oocyte donations in a
manner similar to semen banks because it would
allow more accurate screening and quarantine for
viral infections.
Ethical concerns about

embryo freezing
Previously oocytes needed to be fertilized to be
preserved because embryo thawing was thought to
achieve higher survival rates than oocytes cryopreservation. Oocytes were fertilized with sperm samples
from the patients partner or a sperm donor, with different ethical considerations for each.
Oocyte and ovarian tissue cryopreservation are
useful as they overcome some of the disadvantages,
ethical concerns and legal restrictions related to
embryo cryopreservation.
Conclusions
For fertility clinics, advances in fertility preservation
procedures have created a new group of patients who
seek to use testicular and ovarian preservation techniques in order to prevent infertility. Specialists should
inform patients about the new approaches that will
allow them to attempt a pregnancy in the future with
their own oocytes, regardless of whether they experience ovarian failure.
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926.
66. Garca-Velasco JA and Somigliana E. Management of

endometriomas in women requiring IVF: to touch or
not to touch. Hum Reprod 2009; 24: 496501.
74. Koskas M, Madelenat P and Yazbeck C. Ovarian low

malignant potential tumor: how to preserve fertility?
Gynecol Obstet Fertil 2009; 37: 94250.
67. Muzii L, Bellati F, Bianchi A et al. Laparoscopic

stripping of endometriomas: a randomized trial on
different surgical techniques. Hum Reprod 2005; 20:
198792.
75. Cobo A, Bellver J, Domingo J et al. New options in

assisted reproduction technology: the Cryotop method
of oocyte vitrification. Reprod Biomed Online 2008; 17:
6872.
Section 2
Chapter
Cancer biology, epidemiology and treatment
Basic cancer biology and immunology

Roy A. Jensen, Lisa M. Harlan-Williams, Wenjia Wang
and Shane R. Stecklein
Introduction
Cancer describes a group of well over 200 diseases,
each with distinct and heterogeneous molecular aberrations that result in a breakdown of cellular mechanisms that govern cell growth, death and differentiation. From the early observation of chromosomal
abnormalities in cancer cells, then the identification of
the first proto-oncogene, and now the detailed analyses of specific genes and signaling networks, we have
come to recognize cancer as an astoundingly complex
acquired genetic disease.
The origin of cancer

Transformation of normal cells
In its infancy, the science of histopathology revealed
that like normal tissues, tumors are composed of cells.
This rudimentary observation proved fundamental in
our understanding that tumors were not the manifestation of some external entity, but rather were aberrant progeny of our own cells. More detailed analyses coupled with improvements in technology allowed
for the categorization of tumors with respect to their
tissue and cell of origin and the microscopic behavior
of the mass. Malignancies which arise from epithelial,
mesenchymal and primitive tissues were termed carcinomas, sarcomas and blastomas, respectively. Furthermore, those neoplasms that retained some semblance of the normal tissue and grew in a confined
manner were termed benign, while those that grew
and invaded the surrounding tissue and/or gave rise
to distant metastases were termed malignant. Today,
the cellular origin of a diverse array of human cancers
is well-known. The remainder of this chapter discusses
our current understanding of the fundamental molecular abnormalities that contribute to malignant trans-
formation of normal cells, the role of the normal and

cancer-associated immune system and the biological
manifestations of cancer.
Oncogenes and tumor suppressor genes

In the 1970s, tumor viruses were believed to be the
cause of human cancers and efforts were made to identify the mechanism(s) by which these viruses could
redirect the cellular machinery of their host to proliferate uncontrollably. However, attempts to isolate
these viruses from human tumors proved unsuccessful, which resulted in a paradigm shift towards the
role of carcinogens as mutagens that mutate normal cellular genes, or proto-oncogenes, to oncogenes
[1]. This was made possible using a novel experimental procedure of transfection to demonstrate that
DNA from chemically transformed cells could induce
cellular transformation in recipient cells, suggesting
that the donor cells carried genes that could function as oncogenes [2]. Mechanisms that have been
implicated for oncogene activation include mutation
(e.g. point mutation in H-ras), gene amplification (e.g.
eRB1B2/neu/HER2) and chromosomal translocation
(e.g. Bcr-Abl) [3].
The technique of cell fusion between a normal cell
and a cancer cell derived from a non-virus-induced
tumor often resulted in non-tumorigenic hybrid cells,
suggesting that genes from the normal cell can substitute for those in the cancer cell and that these
genes antagonize the cancer cell phenotype [4]. But
this approach did not identify specific tumor suppressor genes. Studies involving the RB1 gene provided a genetic explanation. Alfred Knudson Jr. postulated from the kinetics of the appearance of unilateral
(sporadic) and bilateral (familial) retinoblastomas that
the familial form, having already inherited one RB1
gene mutation, only needs to sustain a single somatic
35
Section 2: Cancer biology, epidemiology and treatment
Figure 4.1 Overview of the stepwise

accumulation of mutations in colorectal
cancer.
Time
Normal
Inactivation
of APC
Dysplasia
Activation
of KRAS
Inactivation
of SMAD2/4
In situ
malignancy
Invasive
carcinoma
Inactivation
of TP53
Genetic abnormalities
mutation whereas the sporadic form requires two

somatic mutations [5]. The inactivation of the two
copies of a tumor suppressor gene can occur by mutation, by loss of heterozygosity due to mitotic recombination or gene conversion or by promoter methylation
[6, 7].
Stepwise accumulation of mutations

The identification and early functional characterization of oncogenes and tumor suppressor genes illuminated the role of genetic mutation in the pathogenesis of cancer. While mutations in these genes set the
stage for our understanding of tumorigenesis, mounting evidence suggested that inactivating a tumor suppressor gene or promoting the activity of an oncogene
alone is not sufficient to induce malignant behavior.
We now know that a series of mutations in the lifetime of a cell are required in order to overcome the
superbly effective regulatory mechanisms intrinsic to a
cell that regulate orderly cellular division, differentiation and death. While there is evidence supporting this
stepwise accumulation of mutations in the vast majority of human malignancies, this process was first elegantly demonstrated in the case of colorectal cancer
[8].
Mutational inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene is observed
in approximately 85% of all early stage adenomatous
polyps and dysplastic crypt foci, which are considered
to be premalignant neoplasms [9]. This suggests that
loss of APC is among the earliest genetic lesions in the
progression of colorectal cancer. However, additional
sequential mutations have been identified in KRAS,
SMAD2/4 and TP53 and are thought to be critical for
36
the progression of premalignant polyp to invasive adenocarcinoma (Figure 4.1) [8, 9].
The cancer stem cell theory of cancer

Despite our growing knowledge of the molecular
events that lead to malignancy, a number of fundamental questions remain concerning the cellular etiology of cancer. A growing body of evidence suggests
that only a small number of cells within most human
cancers truly possess tumorigenic properties. This was
first demonstrated definitively in human acute myelogenous leukemia, where transplantation of a single
human leukemic cell into an immunocompromised
mouse was capable of recapitulating the phenotypic
heterogeneity and malignant features observed in the
human patient [10]. Thus the paradigm of a cancer
stem cell (CSC) was born. Accumulating evidence now
supports the existence of CSCs in a number of solid
cancers, including those of the breast, brain, colon and
prostate [1115].
Tissues which undergo regeneration, remodeling
and renewal during an organisms lifetime are thought
to possess a small population of quiescent stem cells
which respond to injury and microenvironmental cues
in order to maintain tissue homeostasis. It is this longlived population of cells, seated at the apex of a hierarchical differentiation pathway that establishes the
various cellular components of adult tissues through
asymmetric division (Figure 4.2). Given that most
human epithelial cancers arise in tissues with relatively rapid cellular turnover rates, it can be implied
that most of the cells within these tissues do not
live long enough to accumulate the requisite number
of mutations required for malignant transformation.
Chapter 4: Basic cancer biology and immunology
Adult
stem cell
Anomalous
self-renewal
Lineage
comitted
progenitor cells
Terminally
differentiated
cells
Tumor
subtype 2
Tumor
subtype 3
Differentiation
atio
or m
nsf
Tra
Multipotent
progenitor
cell
Anaplasia
Tumor
subtype 1
Physiological
self-renewal
Tumor
subtype 4
Figure 4.2 Schematic representation of a normal and a cancer-associated cellular hierarchy.
The long-lived resident tissue stem cell, however, provides an elegant target for mutational transformation. By slowly amassing mutations throughout its
lifetime (i.e. the lifetime of the organism), this cell
may reach the threshold for tumorigenic conversion.
Once transformed, this cell is thought to continue to
undergo symmetric and asymmetric division and generate lineage-committed progeny, albeit the hierarchical nature of this differentiation is perturbed (Figure
4.2). The retention of self-renewal capacity only in the
CSC is responsible for the limited tumorigenic potential of the cells which comprise the bulk of the tumor.
Recent evidence now suggests that lineage-committed
progenitors which have gained self-renewal capacity
may also be potential sources of CSCs.
Molecular pathogenesis of cancer

The heterogeneity between and within distinct types
of human cancers highlights the diversity in mechanisms responsible for transformation. Indeed, aberrations in hundreds of molecules have been implicated
in the causation of various cancers. Despite this variation, most cancer cells exhibit similar aberrations in
general cellular processes.
Anomalous growth factor and growth factor

receptor signaling
During development and in physiological contexts,
such as wound healing and maintenance of tissue
homeostasis, proliferation is regulated by exposure of
cells to soluble growth factors within their environment. These factors bind to and activate integral membrane receptors which transduce the mitogenic signal
across the plasma membrane and then, through the
action of cytoplasmic messengers, into the nucleus.
Ultimately, this cascade results in changes in gene
expression that promote entry into the cell cycle.
Under normal conditions, entry into the cell cycle is
tightly regulated by controlling the release of soluble
growth factors in a spatially and temporally controlled
manner, selectively expressing and localizing growth
factor receptors and modulating the activity of intracellular signaling molecules.
Cancer cells commonly induce autonomous mitogenic signaling by hijacking the growth factor signaling machinery. This occurs by: (1) autocrine production of growth factors; and/or (2) enabling activation
of the growth factor receptor in the absence of ligand.
For instance, in a variety of human epithelial cancers,
37
overexpression of the epidermal growth factor receptor tyrosine kinases EGFR and ERBB2 enables receptor dimerization and constitutive mitogenic signaling
in the absence of ligand [16]. Alternatively, tumor cells
commonly secrete soluble growth factors that act in a
paracrine manner to induce activation of membranebound growth receptors and promote entry into the
cell cycle [17].
Abnormalities in signal transduction molecules

Cells use a number of distinct signaling pathways to
control their proliferation, including Ras, Jak-STAT,
Wnt, nuclear factor-B, Notch, Hedgehog and transforming growth factor (TGF) [1824]. These pathways enable cells to receive extracellular signals that
then pass through a series of cytoplasmic signaltransducing proteins to activate transcription factors
and subsequent gene expression. In cancer cells, the
intrinsic activity, concentration and localization of signaling molecules can be affected. For example, the
inactivation of NF1, a GTPase-activating protein, prevents hydrolysis of GTP to GDP by Ras, resulting in a
constitutively active Ras signaling pathway and uncontrolled proliferation [25]. Additionally, the inactivation of APC prevents formation of the destruction
complex containing GSK-3 and its phosphorylation and subsequent ubiquitylation and degradation of
-catenin, which migrates to the nucleus and associates with Tcf/Lef transcription factors to drive proliferation and prevent differentiation [26].
Cell cycle and checkpoint abnormalities

A cells decision to divide or to become quiescent is
influenced by mitogenic signals in the cells surroundings. Cells are responsive to extracellular signals from
the onset of G1 up to the restriction point. Once a
cell has committed to divide, there are several checkpoints within the cell cycle to ensure genomic integrity
as well as proper replication of DNA and assembly
of the mitotic spindle before the cell can progress
through the cell cycle. The pairings of various cyclins
and cyclin-dependent kinases (CDKs) enable cells to
progress through the cell cycle. Cell cycle regulation
depends on cyclin levels and availability during the
different cell cycle phases, which fluctuate from one
phase to the next. Cyclin D1 levels, however, depend
on the input from a variety of mitogenic growth factors. Cyclin-dependent kinase inhibitors also regulate cyclinCDK complexes and include p15INK4B ,
38
which increases in response to TGF signaling to

inhibit cyclin D-CDK4/6 [27], as well as p21Cip1 and
p27Kip1 , which increase in response to mitogens acting
through Akt/PKB to inhibit cyclin-CDK complexes
that form at later stages of the cell cycle [28, 29]. pRb
phosphorylation by cyclinCDK complexes regulate
its growth-inhibitory activity. In early/mid G1, when
pRb is unphosphorylated or weakly phosphorylated
by cyclin D-CDK4/6, it binds E2Fs and prevents transcription. In late G1, pRb becomes hyperphosphorylated by cyclin ECDK2, which results in its complete
functional inactivation [30, 31]. Various mechanisms
can deregulate cell cycle progression and converge on
pRb, including inactivation of the RB1 gene by mutation or promoter methylation, by gene amplification
of cyclin D1, by point mutations in CDK4, or by loss
of CDK inhibitors [32]. Additionally, the Myc oncoprotein, acting with Max, induces expression of cyclin
D2 and CDK4 in early/mid G1 and of CUL1 and E2Fs
in late G1. By associating with Miz-1, Myc represses
expression of CDK inhibitors to overcome the growthinhibitory effects of TGF (Figure 4.3) [33, 34].
Defective cell death

In rapidly dividing tissues, parenchymal cells are
born from asymmetric division of a stem/progenitor
cell, differentiate and then undergo programmed cell
death. The timely death of these cells ensures normal
tissue homeostasis by limiting the accumulation of terminally differentiated cells and eliminating senescent
and damaged cells. Defects in apoptosis, a form of programmed cell death, are widely observed in various
human cancers. It is now readily apparent that cancer
results not only from exaggerated cellular division, but
also from a failure of normal cells to undergo physiological elimination. The molecular anomalies that are
responsible for defects in cell death are diverse, but
generally represent alterations that interfere with the
activators and/or effectors of apoptosis molecules or
those that provide survival signals in the context of
pro-death signaling.
Human follicular lymphoma presents an elegant example by which human cancer cells interrupt normal cellular turnover. In this malignancy,
the t(14;18)(q32;q21) chromosomal translocation is
widely observed and is the causative genetic lesion that
induces overexpression of Bcl-2 [35]. This protein, an
anti-apoptotic member of the Bcl-2 family of proteins,
inhibits the classical mitochondrial apoptosis pathway
B; CD
C
M
DNA
damage
p27Kip1
p53
TGF
p16INK4A
Mitogens
p21Cip1
G1
MDM2
R point
p15INK4B
D;
CDK4/6
p57Kip2
A; CDC
G2
p18INK4C
p19INK4D
pRb
p14ARF
A;
CD
K2
E; CDK2
E2F1/2/3
Myc
Figure 4.3 Schematic representation of the cell cycle and checkpoint regulatory system.
[36]. This genomic alteration results in accumulation

of B lymphocytes that are unable to proceed through
programmed cell death. Additional genetic aberrations that prevent apoptosis and/or promote survival
have been described in a number of human cancers
[37].
Aberrant differentiation
Mammalian organogenesis proceeds through an elegant hierarchical establishment of cellular and functional components. Each step of this process entails
an increasing degree of cellular commitment that is
driven by changes in transcriptional and/or epigenetic
programs. The end-product of this process is the formation of terminally differentiated cellular components that are specialized to perform specific biological functions. In normal tissue morphogenesis, this
process is tightly regulated by growth factors, morphogens and signals derived from cellular and noncellular components of the microenvironment. The
CSC theory of cancer operates under the premise
that cells within this hierarchical arrangement are targets of transforming mutations and that these different cellular origins account for the heterogeneity observed in human cancers. Well before the CSC
theory gained popularity, dysregulated growth pat-
terns and impaired differentiation, termed dysplasia

and anaplasia, repectively, were widely recognized
in human cancers. Emerging evidence suggests that
defects in cellular fate commitment and the existence
of aberrant cellular hierarchies within human cancers
contribute to malignancy.
Acute promyelocytic leukemia (APL) is an earlyonset myeloid leukemia whose pathogenesis is entirely
related to failed differentiation. The t(15;17)(q22;q12)
translocation is observed in over 95% of APL cases
and results in a reciprocal translocation of the promyelocytic leukemia gene (PML) with the retinoic acid
receptor-alpha gene (RARA) [38, 39]. Retinoid signaling plays a critical role in differentiation of promyelocytes into mature granulocytes. Aberrations in RARA
cause a maturation arrest of promyelocytes and result
in the accumulation of these immature cells within
the peripheral blood. Administration of all-trans
retinoic acid (ATRA) causes the terminal maturation
of promyelocytes and has transformed this rapidly
fatal and incurable malignancy into a disease in which
clinical remission is almost universally achieved [40].
Failure of DNA damage repair systems

Intact DNA damage repair systems are critical to
maintain genomic stability and prevent tumorigenesis,
39
as demonstrated by studies of patients with inherited defects in DNA-repair genes, such as xeroderma
pigmentosum (defect in nucleotide excision repair
[NER]), Nijmegen breakage syndrome (defect in NBS
and processing of double-stranded DNA [dsDNA]
breaks), ataxia telangiectasia (defect in ATM and
homology-directed repair [HDR] of dsDNA breaks)
and familial breast and ovarian cancers (defect in
BRCA1 or BRCA2 and HDR of dsDNA breaks).
DNA damage can result from endogenous biochemical processes (e.g. depurination, deamination, oxidation) and exogenous carcinogens (e.g. radiation, alkylating agents, heterocyclic amines). Cells employ a
variety of mechanisms to repair DNA damage, including mismatch repair (MMR) enzymes that recognize
normal but misincorporated nucleotides and other
enzymes that detoxify (e.g. glutathione-S-transferase)
or remove DNA adducts (e.g. O6 -methylguanine DNA
methyltransferase). Additionally, base excision repair
(BER) recognizes chemically altered bases caused by
endogenous sources, NER recognizes helix-distorting
adducts from exogenous sources, HDR of dsDNA
breaks utilizes the undamaged, homologous DNA
sequence to direct repair and the more error-prone
non-homologous endjoining (NHEJ) results in fusion
of two dsDNA ends [41]. One anti-cancer treatment
approach takes advantage of the failure of DNA damage repair. Poly (ADP-ribose) polymerase (PARP) is
an enzyme that participates in BER, a mechanism
that cancer cells with mutations in BRCA1 or BRCA2
depend on to repair their DNA damage. These cancer
cells become hypersensitive to chemotherapy and radiation when PARP inhibitors are given to prevent repair
by this mechanism as well [42].
Hallmarks of cancer
Despite the tremendous molecular heterogeneity
observed in human cancers, malignant cells exhibit a
common set of behaviors regardless of their tissue of
origin or the specific genomic insults that they have
sustained. These hallmarks of cancer [43] (Figure 4.4)
are the biological manifestations of the molecular
anomalies that exist within cancer cells.
Self-sufficiency in growth signaling

Normal growth factor receptor signaling begins with
cognate ligand-receptor binding followed by cytoplasmic transduction of the signal into the nucleus and
transcription of genes that stimulate cell prolifera-
40
Insensitivity to
anti-growth signals
Limitless
replicative
potential
Self-sufficiency
in growth signals
Genomic
instability
CANCER
Immune
evasion
Evasion
of apoptosis
Sustained
angiogenesis
Invasion and
metastasis
Figure 4.4 The hallmarks of cancer.
tion and cell growth. By the mechanisms described

in the Anomalous growth factor and growth factor
receptor signaling section above, cancer cells have
evolved mechanisms that deregulate growth signaling
and enable them to become self-sufficient [43].
Insensitivity to anti-growth signals

Just as cells respond to growth-promoting cues within
their microenvironment, they also sense and react
to molecular signals which antagonize proliferation.
When grown in vitro, normal cells proliferate until a
confluent monolayer is formed and then become quiescent. This phenomenon of contact inhibition implies
that high cell density and/or extensive contact with
neighboring cells restrains further cell growth. Cells
that ectopically express certain viral or cellular oncogenes and cells derived from established tumors lack
contact inhibition. After these transformed cells reach
confluence, they appear to ignore the anti-growth signals that constrain normal cells and begin piling upon
one another in a disorganized fashion.
We now understand many of the molecular signaling cues that are ignored or otherwise misinterpreted in malignant cells. In normal epithelial cells,
TGF family members appear to be largely responsible for conferring anti-growth signals [44]. Exposure of cells to TGF results in increased expression
of p15INK4B and p21Cip1 (Figure 4.3) [27, 45]. In addition, activation of Smad3 by a ligand-bound TGFR
results in formation of a Smad3-E2F4/5-p107 trimer
that potently inhibits expression of Myc [46]. Cancer cells almost universally find ways to evade the
anti-growth signals mediated by TGF. Mutational
inactivation of the RB1 gene is extremely common
in human cancers and largely compromises the antigrowth effects of TGF because release of E2F1/2/3
is no longer dependent upon formation of a cyclin
D-CDK4/6 complex (Figure 4.3). Alternatively, cancer
cells have been demonstrated to mutate the negative
regulatory sequences in the Myc promoter, resulting
in constitutive expression of this gene, and to inactivate the genes encoding the Smad transcription factors
or the TGF receptors, both of which are necessary to
transduce anti-growth signals into the nucleus [43].
Limitless replicative potential

Mortality is an intrinsic property of normal cells that
is mandated by the unique structure of the ends of our
linear chromosomes. Molecular limitations in eukaryotic DNA polymerases result in the progressive shortening of chromosomes, which when unopposed, leads
to a state of senescence. In this state, cells remain alive
and metabolically active, but no longer enter into the
cell cycle, even in the presence of growth factors. Serial
passaging of cells in vitro revealed that normal cells
undergo a finite number of divisions and then stop
dividing. Cancer cells, however, have the capacity to
proliferate indefinitely in vitro. This fundamental difference implies that the acquisition of immortality is
critical to the success of an incipient cancer.
Landmark discoveries by Barbara McClintock
revealed that specialized structures at the ends of linear chromosomes termed telomeres protect against
chromosomal fusion events [47]. A more evolved
understanding of DNA replication in eukaryotes also
revealed that these repetitive structures prevent loss of
genomic coding sequence through DNA replication.
Because normal somatic cells largely lack the ability to
combat the gradual loss of telomeric sequence caused
by DNA replication, they are fundamentally restricted
in the number of cell divisions through which they
can proceed. Certain normal cell types, such as germ
cells, express the enzyme telomerase. This ribonucleoprotein enzyme complex catalyzes the addition of
repetitive telomeric sequence to the ends of linear chromosomes, thus increasing cellular replicative
potential. The finding that most normal cells lack
appreciable expression of telomerase while 8590% of
all human cancers aberrantly express this gene highlights the importance of evading replicative senescence
and achieving immortality [48]. The remaining 10
15% of human tumors that do not express telomerase achieve immortalization by lengthening their
chromosomes using the repetitive telomeric sequence

on other chromosomes as a template in a telomeraseindependent pathway termed alternative lengthening
of telomeres (ALT) [43, 49].
Evasion of apoptosis
Inactivation of the apoptotic machinery permits the
survival of cells with accumulating mutations and promotes evolution of premalignant to malignant cells.
One strategy that is frequently encountered is inactivation of the p53 pathway. This can result from
mutations in the TP53 gene itself (which normally
induces pro-apoptotic genes), by deletion or promoter
methylation of the ARF gene (which normally blocks
MDM2 action) or by overexpression of MDM2 (which
normally blocks p53 action) (Figure 4.3). Additionally, other components of the apoptotic machinery are
altered, including promoter methylation of the APAF1
gene (which normally assembles with cytochrome c
to form the apoptosome and activate caspase 9), inactivation by mutation of the pro-apoptotic BAX gene
and increased expression of the pro-survival BCL2
gene. Finally, hyperactivation of the PI3KAKT/PKB
pathway by PTEN inactivation (phosphatase which
removes the 3 phosphate group from PIP3 that was
added by the kinase PI3K), or IGF-1/2 overexpression (which activates PI3K) also allows cancer cells to
acquire resistance to apoptosis [43].
Sustained angiogenesis
A tumors ability to attract blood vessels is essential
for its continued growth, otherwise tumor cells located
further from the vasculature experience hypoxia and
may enter apoptosis or become necrotic. The Rip-Tag
transgenic mouse model of islet cell tumor progression
has provided researchers a way to study this angiogenic switch, which involves the heterotypic interactions among the premalignant islet cells, inflammatory cells and endothelial cells [50]. The islet cells produce vascular endothelial growth factor (VEGF) that
is then sequestered by the surrounding extracellular
matrix (ECM) as well as still-unidentified signals that
recruit mast cells and macrophages. The inflammatory cells produce matrix metalloproteinase (MMP) 9
that cleaves specific components of the ECM to release
VEGF for signaling. The endothelial cells then proliferate and form the tumor vasculature in response to activated VEGF. However, the tumor-associated endothelial cells tend to form a poorly organized vasculature
41
with gaps in the walls of the capillaries, leading to

leaky vessels that contribute to high hydrostatic pressure within tumors. Normally, the lymphatic vessels
would drain the fluid, but any lymphatic vessels that
form near the tumor vasculature subsequently collapse
from the pressure. This high fluid pressure poses a
problem when administering anti-cancer therapy [43,
51].
Invasion and metastasis

The effects of primary tumors are responsible for
only 10% of cancer-related deaths. The most insidious aspect of cancer is its ability to spread through
the vascular and/or lymphatic systems and seed distant sites. These metastases disrupt normal tissue and
organ function and are responsible for the remaining
90% of cancer mortalities.
The movement of individual malignant cells from
the site of primary disease to a distant site entails a
series of complex events. Since the vast majority of lifethreatening cancers arise in epithelial tissues, this section will focus specifically on the processes that are
involved in the invasion and metastasis of carcinomas, though many of these events are involved in the
malignant progression of non-epithelial cancers. At
the cellular level, a reprogramming event known as the
epithelial-mesenchymal transition (EMT) is thought
to play a fundamental role in allowing epithelial cells
to acquire motile behavior. This process is associated
with repression of epithelial adhesion markers (especially E-cadherin), reorganization of the cytoskeleton, increased migratory and invasive potential and
resistance to anoikis [52]. Interestingly, TGF signaling appears to play a critical role in induction of the
EMT [53]. Cancer cells appear to thwart the growthinhibitory effects of TGF, while preserving their
responsiveness to the pro-tumorigenic activities of
TGF [54]. Acquisition of a mesenchymal phenotype
also results in expression of MMPs which enable localized invasion beyond the basement membrane into
the underlying stroma. The stromal environment contains both blood and lymphatic vessels that offer the
incipient metastatic cell access to the systemic circulation. The process by which cells enter either blood or
lymphatic vessels is commonly termed intravasation.
After gaining access to the systemic circulation, cancer cells may very well be eliminated before they reach
their final destination. Without the ability to attach to
a basement membrane and without exposure to mito-
42
genic and/or trophic factors provided by the stroma,

cells may die by anoikis. Furthermore, epithelial cells
that enter the blood stream will encounter substantial physical shear forces that may destroy them. Those
few cells that resist death and withstand the unwelcoming environment of the systemic circulation will
lodge in small capillary beds. Through various mechanisms, including those that enabled the cell to move
through the stroma and invade the vasculature, these
cells can leave the vascular lumen through a process
termed extravasation. The last step of the metastatic
cascade, termed colonization, represents the most difficult step in the incipient metastatic cells journey. A
variety of growth and trophic factors dictate the sites in
which disseminated cancer cells will ultimately establish new tumors. Cells which leave the vascular system
and arrive in a location without appropriate stromal
support will likely fail to establish clinically significant
metastases. Those cells which arrive at an anatomic
location that is amenable to their growth will take hold
and establish metastatic growths [55]. While a primary
tumor in an organ like the mammary gland presents
little intrinsic danger, these tumors commonly seed
metastases in the brain, liver and lungs. Additionally, a number of epithelial cancers commonly
metastasize to bone and result in significant pain.
Clinically detectable metastases very commonly compromise structure and/or function of these vital organs
and ultimately cause much of the morbidity and mortality observed in clinical oncology.
Genomic instability
A cell has a variety of mechanisms to ensure the structural integrity of its DNA, including enzymes that
detoxify mutagenic molecules and proteins that recognize and repair the damage. Cancer susceptibility
increases when these repair processes are affected. This
can occur by inherited or somatic mutation of key
proteins or epigenetic alterations such as promoter
methylation that leads to functional inactivation of key
proteins. For example, MMR defects prevent the detection and repair of sequence mismatches in genes, such
as the type II TGF receptor that have microsatellite
repeats in their sequences. In this case, it introduces
a nonsense mutation, which results in a truncated
receptor that can no longer function in its growthinhibitory signaling. Changes in chromosome structure as a result of translocations or fusions of unrelated chromosomes as well as changes in chromosome
number as seen with altered MAD1 or MAD2 (both

involved in the spindle assembly checkpoint that leads
to aneuploidy) also appear to drive tumor progression
[56].
Basic immunology
The immune system has two major features: immune
recognition and immune response. Immune recognition is the ability to distinguish foreign invaders from
self-components, which then leads to the immune
response in which the foreign invader (or non-self
component) is eliminated. The immune response
to a foreign invader involves both innate immunity and adaptive immunity. Innate immunity is the
bodys first line of defense against a foreign invader.
The innate immune response is rapid and fixed and
includes anatomic barriers (e.g. skin), inflammation
and soluble molecules (e.g. interferons [IFNs]). The
major cellular components of an innate immune
response include neutrophils, macrophages, dendritic
cells and natural killer (NK) cells. The adaptive
immune response is the bodys second line of defense
that develops in response to a foreign invader. The
adaptive immune response is a delayed response that
demonstrates antigen specificity, diversity, immunological memory and self/non-self recognition. The
adaptive immune response can be further divided into
humoral and cell-mediated responses. In an adaptive humoral immune response, B lymphocytes interact with their specific antigen (pieces of a foreign
invader); differentiate into plasma cells, which then
secrete antigen-specific antibodies. The secreted antibodies bind to their specific antigen and facilitate
clearance of that antigen. In an adaptive cell-mediated
immune response, T lymphocytes recognize their specific antigen, resulting in the secretion of various
cytokines. Cytokines direct numerous cellular activities, including activation of T helper (TH ) or T cytotoxic (TC ) lymphocytes, leading to cell-mediated toxicity. The major cellular components of the adaptive
immune response are T lymphocytes, B lymphocytes,
NK cells and antigen-presenting cells (e.g. dendritic
cells).
Tumor immunology
The immune systems ability to distinguish self- from
non-self components and react only to non-self components is crucial to an appropriate immune response.
Failure in the ability to distinguish self from non-self
can result in autoimmune reactions or facilitate the

development of a tumor [57]. The immune systems
role in tumor development can be defined in three
parts [58]:
1. Protect the body from viral infections thereby
reducing any virus-induced tumors.
2. Eliminate foreign invaders and resolve
inflammation thereby eliminating an environment
that can be conducive to tumorigenesis.
3. Identify and eliminate tumor cells on the basis of
tumor-specific antigens or molecules induced by
stress.
There is both clinical and epidemiological evidence
that suggests a strong association between chronic
infection, inflammation and cancer [59]. For example,
infection with Helicobacter pylori is associated with
gastric carcinoma and chronic viral hepatitis is associated with hepatocellular carcinoma [6062]. In animals, if certain components of the immune system are
knocked out, there is an enhanced susceptibility to
tumor formation [63]. The tumor microenvironment
would also indicate there is a role for the immune system in tumor progression. Tumor-infiltrating lymphocytes (TIL), NK cells and NK T cells are found to be
associated with tumors. Early on, an increased number of immune cells associated with a tumor is usually
correlated with improved prognosis for numerous different tumor types [64]. Therefore, understanding how
the immune system recognizes and eliminates transformed cells is crucial to the development of effective
anti-tumor therapies.
In 1891, William Coley was the first to attempt to
harness the immune system to treat a patient with cancer [65]. Coley noted that some of his patients with
sarcoma had spontaneous regression of their tumors
and this correlated with a bacterial infection. Coley
then used the bacteria to infect cancer patients and, in
some cases, complete tumor regression was achieved.
In 1909, Paul Ehrlich proposed the idea that the
immune system scans for and eradicates transformed
cells before they manifest clinically [66]. This hypothesis was extended in the 1950s by Lewis Thomas and
Frank MacFarlane Burnet, who proposed that T lymphocytes are the sentinels in the immune response to
cancer and coined the term immune surveillance [67,
68]. Immune surveillance suggests the immune system
is on constant alert against transformed cells. In 2002,
Robert Schreiber and Lloyd Old extended the immune
surveillance theory to indicate that the immune system
43
does play a role in cancer development and updated

the hypothesis to immunoediting [6971]. Immunoediting includes the three Es elimination, equilibrium and escape (Figure 4.5). Elimination is the phase
in which nascent tumor cells are destroyed by elements
of the innate and adaptive immune response. Equilibrium is the phase in which tumor cells are able to persist, but are equally destroyed by the immune response.
However, in the escape phase, due to a number of
mechanisms, the tumor disables the immune response
and tips the balance in its favor in order to grow and
invade. Tumor immune evasion mechanisms include
repression of tumor antigens or major histocompatibility complex (MHC) Class I molecules in order
to hide their identity, expression of T-cell inhibitory
costimulation molecules, induction of T-regulatory
cells (Tregs), repression of NK-cell activation ligands
(e.g. NKG2D) and induction of apoptosis in immune
cells through the release of soluble Fas ligand (FasL)
or inhibitory cytokines like interleukin-10 (IL-10)
[7274].
During the elimination and equilibrium phases,
TILs may be responding to tumor-specific antigens
(TSAs) or tumor-associated antigens (TAAs). The
TSAs are specific to a tumor or type of tumor. These
antigens are encoded by genes exclusively expressed
by the tumor. For example, mutations in the p14ARF
and p16INK4A genes result in an epitope found to activate T cells in melanoma [75]. An immune response
to a TSA is typically effective. In contrast, TAAs are
either expressed only at certain stages of differentiation or are normal gene products that are overexpressed in particular tumors. For example, the oncofetal protein carcinoembryonic antigen (CEA) is typically only expressed early in embryonic development,
but is expressed by 90% of colorectal cancers and contributes to the malignant attributes of a colon cancer
[76]. The immune response to TAAs is typically low
because these proteins still look like self to the immune
system.
However, the immune system can also be a detriment in tumor development. Tregs are understood to
inhibit autoimmune reactions, prevent further expansion of activated T cells and impede anti-tumor immunity [77]. In cancer patients, Tregs are present in
increased numbers in peripheral blood, malignant
effusions and tumor tissues [78]. Tumor cells have the
ability to secrete CCL22 and mediate the trafficking
of Tregs to the tumor via CCR4 [79]. Recruitment of
Tregs results in the inhibition of effector T cell activa-
44
tion and function, immune escape and tumor progression.
Immunotherapy
Manipulation of the immune system is an attractive
approach for treatment of many cancers. There are
numerous immune mechanisms to exploit, including passive cellular, passive humoral, active specific
and non-specific mechanisms. The goals would be
to stimulate anti-tumor response, decrease suppressor mechanisms and/or increase tumor immunogenicity [80].
Adoptive T-cell therapy is an example of passive cellular immunotherapy. Adoptive T-cell therapy
involves the infusion of T cells derived from autologous or allogeneic sources with the goal of eliminating a tumor and preventing its recurrence [81]. T cells
can be stimulated and expanded ex vivo prior to infusion in order to enhance their ability to react to a
tumor. T cells can also be genetically engineered to
express chemokine receptors to promote trafficking to
the tumor [82].
Passive humoral immunotherapy includes administration of exogenous monoclonal antibodies. These
antibodies can block function, enhance function or be
conjugated with toxins or radioisotopes. For example,
antibodies to immune inhibitory signals such as PDL1 or CTLA4 prevent the repression of the immune
response and have been shown to enhance tumor
regression in mice [83].
Active specific immunotherapy involves infusion
of autologous or allogeneic tumor cells with the goal
to enhance anti-tumor response. This mechanism also
includes ex vivo loading of dendritic cells with tumor
antigen followed by infusion into a cancer patient
[84]. Further studies have demonstrated that dendritic
cells which have been engineered to express transgenic
tumor antigens or chemokines are more potent inducers of anti-tumor immunity when compared to loaded
dendritic cells alone [85].
Non-specific immunotherapy includes administration of cytokines such as interferon- (IFN ), IL-2
or IL-12 in order to activate macrophages and NK cells
and enhance anti-tumor activity [86]. Synthetic CpG
oligodeoxynucleotides (CpG ODNs) are agents that
have been demonstrated to stimulate both innate and
adaptive immunity by enhancing antibody dependent
cellular cytotoxicity (ADCC) or serving as an adjuvant
to elicit an anti-tumor immune response [87].
Figure 4.5 The three Es of immunoediting.
A major frustration in effective immunotherapy regimens are Tregs. However, there are a number of mechanisms that have been demonstrated to
decrease Treg activity, clearing the way for an effective anti-tumor immune response. For example, low
dose cyclophosphamide treatment selectively depletes
Tregs, denileukin diftitox (an IL-2-diptheria toxin
fusion protein) reduces the percentage of Tregs in the
peripheral blood of cancer patients and daclizumab
(a CD25 monoclonal antibody) inhibits Tregs in
metastatic breast cancer patients who also received a
multipeptide cancer vaccine [88].
Harnessing the immune system to generate an
effective, long-lasting anti-tumor response is a major
challenge. Continued efforts will make attempts to tip
the balance towards the elimination or equilibrium
phases of immune surveillance (Figure 4.5).
stay anti-neoplastic therapies that are currently in use

are of little benefit in terms of actually curing cancer. Oncologists and cancer biologists may need to reevaluate the ultimate goal of cancer treatment, as many
forms of human malignancy may in fact be incurable.
For these diseases, perhaps a more realistic and reasonable objective is to reduce tumor burden and prevent progression such that cancer becomes a chronic
disease. This very real possibility makes advances in
cancer patient care and survivorship all the more necessary. Towards this end, major advances in reproductive endocrinology, reproductive biology and antineoplastic therapies are needed to ensure that those
diagnosed with malignancy before or during their
reproductive years have the opportunity to reproduce
successfully.
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Section 2
Chapter
Breast cancer and fertility preservation

A view from oncology
Carol Fabian and Jennifer Klemp
Introduction and scope of the problem

It is estimated that 57% of cases of invasive breast cancer (11 000/year) occur in women who are under age
40 at diagnosis [13]. The majority of these cases occur
between the ages of 30 and 40 [13]. As 22% of the first
live births in this country occur in women between
the ages of 30 and 40 [4], many young women with
newly diagnosed breast cancer will not have yet had the
opportunity to have a first child. Other women, who
may have previously successfully conceived, may not
yet have had the opportunity to complete their family. In a survey of women under age 40 at the time
of their breast cancer diagnosis, 56% indicated that
they wanted to have 1 or more children in the future
[5]; however, 10% of women have children after a
diagnosis of invasive breast cancer [68]. While this
low rate of births is likely due to a number of factors,
receipt of adjuvant systemic therapy plays an important role for the majority of premenopausal women
with breast cancer.
Breast cancer is likely to be self-detected in women
under the age of 40 [5, 9], and two thirds of these young
women have a stage II or higher tumor [5, 9]. Even
for those with early stage disease, the prognosis for
women under 40 years with breast cancer is worse than
for older women [10, 11]. Achievement of amenorrhea
is known to reduce recurrence and improve survival
[1214]. The likelihood of later stage disease, worse
prognosis and the combined effects of anti-hormonal
and chemotherapy make it likely that most women
under 40 years will receive treatment, resulting in
depletion of ovarian follicles and a reduced ability to
conceive. Treatment-induced preclusion of later childbirth is likely to foster grief and impact later quality
of life, particularly if there was little discussion regarding alternatives [1518]. Women under 40 years of age
are likely to be offered genetic counseling and testing,
and this is increasingly performed prior to definitive
surgery. The emergence of neoadjuvant chemotherapy shortly after biopsy and before definitive surgery
for women with stage II or higher tumors increases
the complexity and reduces time for standard fertility preservation procedures such as controlled ovarian
stimulation (COS) and oocyte retrieval. How can decisions regarding fertility preservation be incorporated
into the already crowded and emotionally charged
interval immediately following diagnosis?
In this chapter we will cover those issues most likely
to be raised by young women who have been recently
diagnosed with breast cancer or those at high risk of
the disease contemplating assisted fertility procedures.
Questions likely to be posed by women with a
recent cancer diagnosis are: (1) Are the benefits from
chemotherapy worth the possible loss of fertility? (2)
What are the chances that the planned treatment will
result in menopause or the loss of ability to become
pregnant? (3) If I am still able to conceive will a subsequent pregnancy alter my prognosis? (4) If you alter
my treatment to help preserve fertility will it change
my prognosis? (5) How much do fertility preservation
procedures cost and are they covered by insurance?
(6) How is fertility preservation orchestrated with the
rest of my treatment? Young women at increased risk
of breast cancer want to know if ovarian stimulation
is likely to further increase their already elevated risk
of breast cancer. All members of the treatment team
should be able to provide at least a general response to
these questions.
49
Risk of recurrence and reduction with

adjuvant therapy
Although many young women are interested in fertility preservation, breast cancer cure is generally their
number one priority.
The risk of recurrence and death is determined by
tumor size, number of involved nodes, biological characteristics of the tumor and length of follow-up. The
15-year risk of recurrence and death for women under
age 50 who do not receive adjuvant systemic therapy
is 53 and 42%, respectively [19]. Mortality rates at 5
years are about half of that at 15 years. This is primarily because breast cancers (particularly those which are
estrogen receptor positive) continue to recur after 5
years from the date of diagnosis and the often prolonged interval between the onset of clinical metastatic
disease and death.
The type of adjuvant regimen selected for an individual woman is determined by menopause status,
biological characteristics of the tumor and risk of
relapse. Biological characteristics with strong impact
on treatment selection include estrogen and progesterone receptors (ER and PR), proliferation (usually
measured by Ki-67) and presence of the growth factor receptor Her-2 neu. Approximately two thirds of
younger women will have hormone receptor positive
cancers and one third hormone receptor negative and,
in addition, one fifth will have Her-2 neu positive
tumors. Approximately one sixth of newly diagnosed
women will have a so-called triple negative tumor,
which means the cancer does not express ER, PR or
Her-2 neu. All women with hormone receptor negative tumors 1 cm in size will be advised to take
chemotherapy. Even women with hormone receptor
negative tumors 1 cm will often be offered adjuvant
chemotherapy if their tumors are high grade and/or
Her-2 neu positive. The hormone receptor positive
group is very heterogeneous and includes women at
one end of the spectrum with very well differentiated
tumors which are strongly ER and PR positive with
little growth factor receptor expression or proliferation (luminal A). This group is generally offered antihormonal therapy alone in Europe and Canada but in
the USA they are also likely to be offered chemotherapy, particularly if they are under the age of 40. At the
other end of the hormone receptor positive spectrum
are tumors that have a low level of ER or PR and/or
have a high proliferative rate and/or express growth
factor receptors (luminal B). The latter type of hor-
50
mone receptor positive tumor has a worse prognosis

with anti-hormone therapy alone and chemotherapy
in addition is generally advised.
Large meta-analyses of multiple trials with longterm follow-up have been used to assess the effects
of systemic therapy on breast cancer outcomes. The
most widely referenced is that of the Early Breast
Cancer Trialists Collaborative Group (EBCTCG) with
the latest major published outcomes in 2005 [19].
Without adjuvant therapy, EBCTCG analyses suggest
a 12.5% breast cancer mortality rate at 15 years for
women under 50 years with low-risk node negative
tumors, 25% for women with high-risk node negative tumors and 50% for node positive tumors. These
long-term reported outcomes are reflective of early
generation adjuvant regimens such as 5 years of tamoxifen for anti-hormonal therapy and for chemotherapy 612 months of cyclophosphamide, methotrexate
and fluorouracil (CMF); or 46 months of an anthracycline and cyclophosphamide (AC if the anthracycline is doxorubicin); or 6 months of an anthracycline,
cyclophosphamide and flurouracil combination (FAC
or FEC) (Table 5.1). These early generation regimens
with predominately 6 months of treatment reduce 15year breast cancer recurrence by 39% and 44% in
premenopausal estrogen receptor positive (ER+) and
estrogen receptor negative (ER) breast cancer, respectively, and breast cancer mortality by a little over one
third. Six months of FAC or FEC is associated with a
relative reduction of 44% in 15-year mortality. [19].
Tamoxifen alone reduces mortality by 30% for ER+
women under age 50 and 40% for those under age
40. For women under age 50 with an ER+ tumor, the
addition of 5 years of tamoxifen to an anthracyclinecontaining regimen was estimated to reduce mortality
by a relative factor of 57% [19].
Amenorrhea and survival

Achievement of amenorrhea appears to be associated
with a reduction in relapse and improvement in survival in premenopausal women with ER+ tumors [12,
13, 20]. Amenorrhea need not be permanent to achieve
therapeutic benefit. Approximately 2 years of amenorrhea appears to provide the same benefits as permanent amenorrhea [21]. This is an important concept for
women and their healthcare providers to understand
if they are contemplating fertility preservation. Suppression of ovarian function in women under age 40
with ER+ tumors by adding a gonadotropin-releasing
Chapter 5: Breast cancer and fertility: oncology
Table 5.1 Chemotherapy regimens
Regimen
AC
FAC
FEC
CEF
ACT
TAC
CMF
Cycles
Dose
Adriamycin
60 mg/m2
Cyclophosphamide
600 mg/m2
Every 3 weeks Dose dense every 2 weeks

Every 3 weeks
4 and 4
6 or 12
Frequency
5 Fluorouracil
500 mg/m2
Adriamycin
50 mg/m2
Cyclophosphamide
500 mg/m2
5 Fluorouracil
500 mg/m2
Epirubicin
100 mg/m2
Cyclophosphamide
500 mg/m2
Cyclophosphamide
75 mg/m2
D1D14
Epirubicin
60 mg/m2
D1 and D8
5 Fluorouracil
500 mg/m2
D1 and D8
Adriamycin
60 mg/m2
Every 3 weeks
Cyclophosphamide
600 mg/m2
Dose dense every 2 weeks
Docetaxel (Taxotere)
100 mg/m2
Every 3 weeks
or Paclitaxel
175 mg/m2
Dose dense every 2 weeks
Docetaxel (Taxotere)
75 mg/m2
Every 3 weeks
Adriamycin
50 mg/m2
Cyclophosphamide
500 mg/m2
Every 3 weeks
Cyclophosphamide
600 mg
D1 and D8
Methotrexate
40 mg/m2
D1 and D8
5 Flunoorouracil
600 mg/m2
D1 and D8 every 4 weeks
D, day.
hormone (GnRH) agonist to chemotherapy tamoxifen improves disease-free survival by a relative factor
of 25% and may also improve overall survival [19,
2224]. Use of a GnRH agonist or removal of the
ovaries is probably most effective in women who are
still menstruating or who have premenopausal levels
of estradiol following chemotherapy [25].
Newer adjuvant treatments and

additional benefits
The 2005 EBCTCG analyses probably underestimated benefits from adjuvant chemotherapy and antihormonal treatments as long-term data from some of
the newer successful systemic therapy regimens are
not included. These newer regimens include those
incorporating: (1) taxanes; (2) intravenous bisphosphonates for women with hormone receptor posi-
tive cancer; (3) trastuzamab for women with Her-2

neu positive cancer; and (4) dose dense regimens
in which the interval between chemotherapy cycles
is shortened. Overall, without regard to stage, hormone receptor positivity or menopause status, taxanecontaining regimens appear to result in an absolute 3%
increase in survival [26]. Taxanes appear to provide
incremental benefit when given with anthracyline and
cyclophosophamide in women with luminal B, triple
negative or Her-2 neu positive cancers. There appears
to be no incremental benefit for women with luminal
A tumors compared to older regimens [27, 28]. Intravenous bisphosphonates appear to reduce recurrence
by about one third in premenopausal women with
ER+ tumors given anti-hormonal therapy with tamoxifen and ovarian suppression with a GnRH agonist (i.e.
goserelin) [29]. Trastuzamab given with chemotherapy or chemo-hormonal therapy improves relapse free
and overall survival by about 50% in women with
51
Her-2 neu positive tumors compared to chemotherapy

or chemo-endocrine therapy alone[30]. Taxane and
carbopalatinum regimens appear to be as efficacious
as anthracycline cyclphosphamide and taxane combinations when given with trastuzamab to women with
Her-2 neu positive tumors [31] and are probably not
as likely to result in sterility.
Women often want to know which components
of the treatment plan are giving them the greatest
benefit in terms of reduction of risk of relapse and
death. Understanding the risk/benefit of each component allows for a more critical review of treatment
recommendations. Providers generally discuss the
likelihood of relapse following local therapy and then
estimate the incremental benefit likely to accrue with
the addition of each agent. For example, a 35-year-old
woman with a 2 cm, node negative, strongly hormone
receptor positive tumor, with little proliferation (luminal A) might have a 30% risk of relapse at 10 years. She
can expect a 40% relative reduction in risk of recurrence with tamoxifen alone and another 25% relative
reduction with the addition of a GnRH agonist, bringing her absolute risk down to 1314%. An addition of
2 years of an intravenous bisphosphonate could reduce
her risk by an additional one third to an absolute value
of 9%. Adding polychemotherapy with an alkylating
agent might reduce the risk by an additional 20% to an
absolute value of 7%. If she does not use a GnRH agonist, however, the incremental benefit of chemotherapy with the subsequent ovarian hormonal suppression would be higher. On the other hand, a woman
with a weakly hormone receptor positive tumor or one
with a high proliferation rate or presence of growth
factor receptors (luminal B) is likely to have less relative reduction of risk of recurrence with anti-hormonal
agents and more from cytotoxic chemotherapy. Calculation of the relative risk reduction with an individual intervention is much simpler in women with
hormone receptor negative tumors. A woman with a
2 cm, node negative, hormone receptor negative tumor
is likely to have a baseline risk of relapse of 40%. This
risk can be reduced by 40% with chemotherapy if
the woman has a triple negative tumor and by 50%
with chemotherapy plus trastuzamab if the woman has
a Her-2 neu positive tumor. A number of tools have
been developed to facilitate this process including gene
expression panels for ER+ women [3234] and models
incorporating age, stage and biomarker characteristics
such as Adjuvant Online (http://www.adjuvantonline.
com/index.jsp) [35, 36].
52
Standard regimens and how

they might be altered to help
preserve fertility
Women with ER+ tumors (Figure 5.1)
Premenopausal women with an ER+ or PR+ invasive cancer of any size will receive at minimum 5 years
of anti-hormonal treatment with tamoxifen with or
without ovarian suppression or ablation, in addition to
the addition of 23 years of intravenous bisphosphonates. Women with hormone receptor positive tumors
with less favorable biological characteristics, such as
a high proliferation rate, low expression of ER, low
or absent expression of PR or expression of growth
factor receptors, are generally offered chemotherapy
as well. Women continue to relapse between 5 and
10 years after diagnosis even with 5 years of antihormonal therapy adjuvant chemotherapy [37]. This
late relapse rate for women under 35 years is 1.5%
per year and is higher for women who had positive
nodes and/or who are ER+ [37]. Emerging data suggests reduced recurrence when 5 years of an aromatase
inhibitor is added to 5 years of tamoxifen for women
who were premenopausal at the time of initiation of
tamoxifen [38]. Therefore, it has become increasing
more common for oncologists to consider giving adjuvant endocrine therapy for a total of 10 years to women
with node positive ER+ tumors.
Good prognosis ER+ premenopausal women may
not need chemotherapy or may take only four cycles
of a cyclophosphamide-containing regimen. Doxorubicin (adriamycin) combined with a taxane and
omitting cyclophosphamide appears equivalent to an
anthracycline plus cyclophophamide but is probably
less efficacious than all three drugs and should be
reserved for women with fairly good prognosis [26].
The GnRH agonists or analogues are often used
concomitantly with chemotherapy and/or antihormonal therapy in young premenopausal women
with ER+ tumors to improve relapse free survival [22,
39, 40]. These agents stimulate release of luteinizing
hormone (LH) and follicle stimulating hormone
(FSH) from the anterior pituitary and in the first few
weeks may increase ovarian hormone output [41].
With prolonged use ovarian hormone suppression
ensues.
Preclinical studies [4244] and small observational as well as small randomized trials [45, 46]
ER+ BrCa
Good ovarian
reserve
Less favorable
prognosis
Unfavorable risk
Ovary harvest
with cryopreservation
chemotherapy followed by
510 years anti-hormone
therapy + GnRha
Intermediate risk
Consider COS with
letrozole + GnRHa +
oocyte retrieval 5 years
tamoxifen + GnRHa
Good prognosis
> 35 years old

Consider COS
letrozole + GnRHa +
oocyte retrieval 5 years
tamoxifen + GnRHa
< 35 years old

5 years tamoxifen +
GnRHa
Figure 5.1 Fertility preservation in women with estrogen receptor positive (ER+) tumors. BrCa, breast cancer; COS, controlled ovarian
stimulation; GnRHa, gonadotropin-releasing hormone agonist.
indicate GnRH agonist (goserelin) when given during

chemotherapy may also help preserve ovarian function. The recently reported Zoladex (goserelin) in premenopausal patients (ZIPP) study randomized premenopausal women taking six cycles of CMF or no
CMF (women with no positive nodes and a small
tumor could be randomized to no CMF as well) to
concomitant anti-hormonal therapy with tamoxifen
alone, goserelin alone, goserelin + tamoxifen or no
hormonal therapy. The mean age at diagnosis was 45
years. At 1 year after completed endocrine therapy
(36 months from randomization) the proportion of
women with amenorrhea was 90% for controls, 87%
for tamoxifen, 93% for goserelin + tamoxifen and 64%
for goserelin alone (P = 0.006) [46]. The mechanisms
of action of GnRH/luteinizing hormone-releasing hormone (LHRH) agonists in preserving ovarian function are not understood, but may involve reduced
FSH, reduced ovarian perfusion and activation of
GnRH receptors with upregulation of intragonadal
anti-apoptotic molecules [47]. Many reproductive
specialists remain unconvinced that GnRH agonists
improve the ability to conceive, although it is possible that they reduce permanent amenorrhea [48, 49].
A large intergroup randomized trial of a GnRH agonist
versus placebo in ER women undergoing chemotherapy is ongoing and will hopefully answer this question. In the meantime for women with ER+ cancers
desiring fertility preservation, particularly those who

do not undergo oocyte retrieval, use of the GnRH
agonist seems reasonable as it is likely to have a therapeutic effect on the tumor even though there may be
no protective effect for fertility.
Women with ER tumors (Figure 5.2)

Premenopausal women with an ER and PR negative
high-grade tumor of 1 cm or more in size, or those with
positive nodes, will likely undergo 68 cycles of adjuvant chemotherapy in addition to definitive surgery
local radiation. The chance of permanent amenorrhea is 40%. Although there is little concern about
increasing tumor proliferation with drugs for ovarian stimulation and oocyte/egg retrieval, these tumors
are generally rapidly growing, fueling concerns about
treatment delay for fertility preservation, especially
for women requiring neoadjuvant chemotherapy. The
combination of a taxane and carboplatinum or cisplatinum with trastumab appears to be as effective
as cyclophosphamide and an anthracycline for most
women, resulting in less ovarian toxicity [31, 50]. For
women desiring fertility preservation, six cycles of carboplatin and a taxane with trastuzamab can be substituted for chemotherapy with an anthracycline and
cyclophosphamide and taxane. Excellent results are
also being observed with cisplatin as a single agent
53
ERBrCa
Good ovarian
reserve
Less favorable
prognosis
Immediate chemotherapy
with cyclophosphamide
containing regimen
pretreatment ovarian
harvest with
cryopreservation
Good prognosis
* Neoadjuvant chemotherapy
Two cycles of non-cyclophosphamide
regimen
oocyte retrieval, followed by
adjuvant chemotherapy and
surgery
Definitive surgery
COS with letrozole +
GnRHa and oocyte retrieval
adjuvant chemotherapy
* Alternative treatment plan

Figure 5.2 Fertility preservation in women with estrogen receptor negative (ER) tumors. BrCa, breast cancer; COS, controlled ovarian
stimulation; GnRHa, gonadotropin-releasing hormone agonist. Alternative treatment plan.
in neoadjuvant trials of women with triple negative

tumors [51]. Data are very preliminary at this point
and use of cisplatin alone in a woman with a triple negative tumor should only be used in the neoadjuvant
trial setting with follow-up adjuvant chemotherapy
with a standard adjuvant regimen if complete pathological response is not obtained.
In summary, regimens which omit or deliver four
or fewer cycles of cyclophosphamide have the greatest
chance for fertility preservation. For women planning
to take six cycles of cyclophosphamide, flurouracil
and an anthracycline (FEC or FAC) or four cycles
of cyclophosphamide and an anthracycline followed
by four cycles of taxane, pre-chemotherapy oocyte
retrieval in vitro fertilization may enhance later
chance of pregnancy. When this is not possible, ovarian harvest and cryopreservation may be considered. For women with ER+ tumors, use of a GnRH
agonist beginning 2 weeks before chemotherapy and
continuing during and post-chemotherapy, is advised
to reduce the chance of recurrence and enhance
prospects of later successful pregnancy.
54
Chances that treatment will induce

amenorrhea or loss of fertility
The median age at menopause in the USA is 51 years,
but the ability to become pregnant is markedly reduced
beginning about 10 years before cessation of menses
due to depletion of primordial follicles. It is not clear
how many premenopausal women wish to become
pregnant after a diagnosis of breast cancer, but it
is clear that a woman is unlikely to become pregnant if she stops menstruating with treatment and
then fails to resume menses. The main determinant
of chemotherapy-induced amenorrhea is the age of
the woman at the time of diagnosis and the number of cycles of alkylating agent chemotherapy she
has received [52, 53]. The alkylating agent cyclophosphamide is one of the oldest and most effective drugs
in breast cancer. In general, each cycle of cyclophosphamide chemotherapy is associated with an increase
in ovarian age of about 1.53.0 years depending on
dose and frequency [5255]. A woman who takes
the equivalent of 2.43.0 g/m2 of cyclophosphamide
Table 5.2 Agents associated with amenorrhea
Cyclophosphamide
Worst
Carboplatin
Cisplantin
Anthracyclines
Intermediate
Taxanes
Vincas
Antimetabolites
Least
over 1216 weeks can count on adding an approximate 10 years to her ovarian reproductive age, such
that if she is aged 30 at the time her chemotherapy
is initiated, her ovarian reproductive age will be the
equivalent of a 40-year-old woman when her treatment
is completed. While she is likely to resume menses,
her capacity for a birth without assisted reproductive techniques will be marginal. The mechanism of
ovarian toxicity resulting from chemotherapy is not
completely understood, but it is likely to result from
apoptotic changes in pre-granulosa cells that subsequently develop into follicles [56, 57]. Recent investigations suggest that women with greater levels of
neutropenia (often associated with higher doses and
more cycles) are more likely to have permanent amenorrhea as are women with certain single nucleotide
polymorphisms in enzymes responsible for cyclophosphamide metabolism [58, 59]. Drugs more and less
likely to be associated with amenorrhea are given in
Table 5.2.
Rates of amenorrhea that have been observed with
regimens given to good-prognosis node negative premenopausal women are 33% for 6 cycles of CMF and
4 cycles of AC either dose dense or non-dose dense
(there are no published rates for the 4 cycle taxotere
and cyclophosphamide combination) [6065].
Rates of amenorrhea for chemotherapy regimens
often given to high-risk node negative or node positive women such as 6 cycles of FEC or FAC, 6 cycles
of AC or 4 cycles of AC followed by 4 cycles of taxotere are generally double that of the good prognosis
regimens ranging from 5065% [6670]. However, it
appears that giving only three cycles of FEC followed
by thre cycles of taxotere may have less ovarian toxicity
than six cycles of FEC [20]. Fifteen to fifty percent of
women younger than age 40 at diagnosis will eventually resume menses. Recovery rates are higher for regimens with less total cyclophosphamide. Amenorrhea
is likely to be permanent in 90% of women aged over 40
and in 95% of women aged over 45 [68, 71]. A number

of studies indicate a higher rate of prolonged amenorrhea with tamoxifen use after chemotherapy [14, 68].
There is little available data on fertility with platinum
alone or a taxane and carboplatin. However, approximately 50% of women receiving adjuvant platinum and
fertility-sparing surgery for ovarian cancer were able to
successfully bear children [72].
Pregnancy following a breast cancer

diagnosis and risk of relapse
Becoming pregnant after a diagnosis of breast cancer does not appear to result in worse outcomes in
case control studies or cohort studies [3, 5, 6, 74]. In
fact, in several series pregnancy after a diagnosis of
breast cancer appeared to result in a reduced risk of
relapse [7375], particularly for women who waited
for 2 years after diagnosis to conceive [75]. At least for
some series, this may be due to better prognostic features in women who subsequently had a pregnancy [5].
In only 1 series was a 77% reduction in risk of death
observed [74]. For women at high risk of relapse at
diagnosis, most relapses appeared to occur within 5
years of diagnosis [74].
Barriers to fertility preservation

for premenopausal women with
breast cancer
There are three main barriers to implementing fertility preservation in women with breast cancer: cost;
concern about treatment delays; and concern that
increasing sex hormones as a result of COS protocols will stimulate proliferation in ER+ tumors. The
recent discovery of LHRH receptors even in triple
negative tumors [76] and the paracrine interactions
between growth factors and estrogen have made many
clinicians reluctant to sanction COS in women with
recently diagnosed ER breast cancer, especially if they
are to undergo neoadjuvant treatment and the tumor
is still in place.
Cost of fertility preservation

procedures
The cost of fertility preservation procedures is covered
elsewhere in this volume and will vary by institution,
but at our institution the cost of oocyte retrieval, fertilization and cryopreservation is $7000 (medication
55
cost not included) and ovarian harvest and cryopreservation is $6000. Other procedures are available but are
rarely utilized at present [77]. Costs for fertility preservation are not covered by most insurance carriers and
so it may be difficult for young women to come up with
funds on short notice. In addition, in order for embryo
cryopreservation to occur, there must be both egg and
sperm available and for many single women this adds
an additional short-term challenge.
Fertility preservation and adjuvant

therapy delay
For women undergoing surgery first, fertility preservation should not cause significant delays as long as
the surgeon sends the woman for a fertility preservation consultation at the time she is initially seen.
This is most likely to occur in a multidisciplinary
treatment environment where all members of the
team have been educated about fertility preservation
and a Breast Cancer Survivorship Program or similar
facility is readily available to the woman and family
to expedite necessary consultations. Controlled ovarian stimulation and oocyte retrieval requires about
a month for the typical long-form regimen utilizing gonadotrophins (FSH or human menopausal
gonadotropin [HMG]) for ovulation induction; GnRH
agonists to prevent a premature LH surge and oocyte
damage; and human chorionic gonadotropin (hCG)
for ovulation induction oocyte maturation prior to
oocyte retrieval. The GnRH agonists along with
gonadotropins may result in estradiol levels that are
1020-fold higher than are observed in the nonstimulated cycle. For the long form of COS, the GnRH
agonists typically begins at least 14 days before the
anticipated start of gonadotropins which in turn run
10 days in conjunction with the LHRH agonist. A
shorter form of COS which is less likely to result in
high levels of estrogen is the use of the GnRH antagonist Cetrorelix on the 7th day of gonadotropin administration followed by recombinant LH or an LHRH
agonist on day 10 to induce ovulation. Other regimens including one popularized by Oktay et al. uses
letrozole beginning on day 2, FSH days 410 and
the GnRH agonist, leuprolide acetate, days 810 with
oocyte retrieval day 13 [78].
Even with a well-organized team, women undergoing fertility preservation begin their adjuvant
chemotherapy about 12 days earlier than those who
56
are not undergoing fertility preservation proceedures

[79].
For the woman with a large ER tumor who needs
to start neoadjuvant treatment before surgery, COS
and egg retrieval can be attempted between the second and third cycle of chemotherapy, particularly if a
regimen is not employing cyclophosphamide. There is
little experience with this approach, however.
COS, hormone levels and

risk of recurrence
The newer regimens using letrozole and a GnRH
agonist to prevent the premature LH surge and an
LHRH (GnRH) agonist to induce final oocyte maturation do not appear to result in higher hormone levels than a natural menstrual cycle in premenopausal
women [79]. A recent series by Oktays group using
this regimen shows no difference in recurrence for
premenopausal women undergoing fertility preservation with a median follow-up of 2.5 years, and over
two thirds of women in both the control and fertility
preservation groups had ER+ breast cancers [79].
Options other than COS and

oocyte retrieval
Ovarian harvest and cryopreservation may be the only
reasonable option if pregnancy is strongly desired,
COS is not possible prior to full course chemotherapy and donor eggs are not an option. Healthy babies
have been borne from orthotopic or heterotopic ovarian transplantation after chemotherapy [80]. Although
breast cancer can metastasize to the ovary, to date there
is little evidence that tumor cells will be re-implanted
in women with early stage disease [81].
COS and high-risk women

There is an ongoing debate as to whether a possible
short-term increase in hormones as a result of assisted
ovulation results in an increase in the risk of breast
cancer. This question is difficult to answer as many
women undergoing fertility assistance may already be
at increased risk relative to the population because of
null-parity or late age at first live birth. In a recent large
cohort study, a 13% higher risk of breast cancer was
noted in women with ovulation disorders but there
was no significant difference in women undergoing
COS versus those who had not [82]. We are currently

conducting a pilot trial of premenopausal women
receiving letrozole to induce ovulation to determine
whether there is a long-term change in breast tissue proliferation and/or expression of other key genes
in women who undergo fertility assistance procedures but do not become pregnant. Although there
is no current evidence that assisted ovulation significantly increases breast cancer risk, further research is
necessary.
Logistics and summary

Treatment of young women with breast cancer is
increasingly complex and requires a number of consultations and multiple decisions shortly after diagnosis. Adjuvant chemotherapy will be recommended
for the majority of these women. Chemotherapy
anti-hormonal therapy will significantly reduce the
chances of later successful childbearing and commonly used regimens are likely to add 10 years to a
womans reproductive age. Most young women will
receive adjuvant chemotherapy and can increase their
chances of conception by undergoing egg retrieval, fertilization and cryopreservation prior to chemotherapy. Historically, this has been performed in the 4
6 week interval after definitive surgery prior to initiation of systemic therapy Women with a new diagnosis of breast cancer under age 40 are likely to
have larger tumors and, currently, are more likely to
have a recommendation for neoadjuvant chemotherapy prior to definitive surgery than their older counterparts. They are also more likely to have genetic testing and want results prior to a decision about breast
conservation versus mastectomy, which increases the
likelihood of use of chemotherapy prior to definitive surgery. Use of neoadjuvant therapy decreases the
likelihood of egg retrieval prior to chemotherapy. A
variety of methods are being evaluated to increase
the likelihood of pregnancy after chemotherapy other
than pre-chemotherapy egg harvest and in vitro fertilization including pre-chemotherapy ovarian harvest
and cryopreservation; use of an LHRH agonist during chemotherapy; and use of chemotherapy regimens
that do not contain the alkylating agent cyclophosphamide. Decisions about optimal therapy requires
input from the entire treatment team, but the earlier in
the process the woman receives fertility counseling the
greater the number of fertility options that are likely
to be available without compromise in prognosis. If
all members of the treatment team are well-informed

and organized to provide an immediate consultation
for fertility preservation, the patient will begin to talk
with the fertility specialist immediately after diagnosis while she is undergoing staging and meeting with
medical, surgical and radiation oncologists, improving the chance of COS without inducing treatment
delays. We have found that immediate access to a fertility specialist via an intermediary in our Breast Cancer Survivorship Center in the University of Kansas
works well. Regional and national education programs
are ongoing, but Breast Diagnostic Centers should
strive to have logistical information on how women
can access information and/or fertility preservation
programs available in their waiting rooms. National
organizations devoted to this process include Fertile
Hope (http://www.fertilehope.org) and the International Society for Fertility Preservation (http://www.
isfp-fertility.org).
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76. Buchholz S, Seitz S, Schally AV et al. Triple-negative
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78. Oktay K, Turkcuoglu I and Rodriguez-Wallenber K.
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79. Azim AA, Costantini-Ferrando M and Oktay K.

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81. Sanchez-Serrano M, Novella-Maestre E,

Rosello-Sastre E et al. Malignant cells are not found in
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61
Section 2
Chapter
Breast cancer therapy and reproduction

Larissa A. Korde and Julie R. Gralow
Breast cancer is the most common malignancy in

women in developed countries, excluding cancers of
the skin. The American Cancer Society projects that in
2010, 207 090 cases of invasive breast cancer and 54 010
cases of non-invasive breast cancer will be diagnosed
in the USA [1]. The National Cancer Institute estimates
that 2.5 million women with a history of breast cancer
are alive in the USA. While the mean age at diagnosis
of breast cancer in the USA is 61 years of age, approximately 10% of women with breast cancer are diagnosed
at 45 years of age [1, 2]. Younger women with breast
cancer have unique concerns and management issues,
including the effects of treatment on fertility and the
safety and feasibility of pregnancy following diagnosis
and treatment of breast cancer. In addition, the likelihood of an inherited predisposition to breast cancer
increases with younger age at diagnosis. Women with
BRCA1 and BRCA2 mutations have greatly increased
lifetime risks of early onset breast cancer, second primary breast cancers and ovarian cancer, and therefore
also present a number of complex management issues
related to conception and childbearing.
Breast cancer in young women

Breast cancer risk increases with age, with the highest incidence occurring after the sixth decade of life
(Table 6.1 [2]). Incidence is slightly higher among
white women in older age groups, but breast cancer in
women under the age of 45 is more prevalent among
black women. Younger women with breast cancer are
more likely to have poor prognostic features, such
as larger tumor size, regional lymph node positivity,
high nuclear grade, estrogen receptor negativity and
inflammatory disease [3, 4]. A recent registry-based
cohort study in Sweden found that 5-year survival
was poorest among women aged 35 years, despite
Table 6.1 Breast cancer incidence rates per 100 000,

age-adjusted to the 2000 US-standard population; 17
Surveillance Epidemiology and End Results (SEER) Cancer
Registries, 20026
Age at
diagnosis (years)
1519
0.2
White
0.2
Black
2024
1.4
1.3
1.9
2529
8.1
7.8
11.2
3034
25.6
25.3
30.4
3539
58.5
58.0
64.4
4044
118.4
119.3
119.9
4549
185.3
188.1
178.7
5054
229.0
234.7
224.1
5559
288.5
296.1
282.7
6064
351.9
366.8
321.4
6569
394.4
416.5
351.2
7074
415.4
435.9
382.3
7579
441.9
465.7
387.6
8084
428.7
447.9
376.7
85+
342.1
350.5
335.8
From Horner et al. [2], based on November 2008 SEER data submission, posted to the SEER website, 2009.
more aggressive treatment in younger women, and

improved with increasing age. The authors concluded
that these differences in survival could be attributed
to both later stage at diagnosis and to a more aggressive intrinsic biology in tumors diagnosed in younger
women.
Women at genetic risk of breast cancer have an
increased incidence of early onset breast cancer and
a markedly higher cumulative lifetime risk of disease.
It is estimated that 510% of breast cancers occur in
62
All races
Chapter 6: Breast cancer therapy and reproduction
women with an inherited susceptibility to cancer [5].

The majority of these are women with hereditary breast
ovarian cancer syndrome (HBOC), which is explained
by deleterious mutations in the BRCA1 and BRCA2
genes, although a number of less common genetic disorders, such as LiFraumeni syndrome, Cowdens syndrome and PeutzJeghers also include a predisposition to breast cancer [6]. At least half of the diagnoses
of breast cancer in women with inherited BRCA1/2
mutations occur under the age of 50 [7]. Women with
BRCA1 mutations have a 4090% lifetime risk of breast
cancer and a 1040% lifetime risk of ovarian cancer. Women with BRCA2 mutations have an estimated
4050% lifetime risk of breast cancer and a 1020%
lifetime risk of ovarian cancer. In addition, women
with BRCA mutations have a 4060% lifetime risk of
a second breast cancer [6].
Pregnancy-associated breast cancer

Women with an early age at first birth and multiparity
have a decreased risk of breast cancer compared with
nulliparous women and those with late parity. Women
with a full-term pregnancy before age 20 have a 50%
reduced risk of developing breast cancer compared to
those with a first pregnancy after age 35. However,
studies of breast cancer incidence in young women
demonstrate a transient increase in breast cancer risk
in the years following pregnancy [8]. Data suggest that
the transient increase in risk peaks at 35 years after
first delivery and levels off 15 years after delivery. Additionally, the increase in risk appears to be varied in
both length and magnitude based on the number of
pregnancies (i.e. the magnitude of increase in risk is
less in biparous than in uniparous women) [8, 9]. There
may also be a synergistic effect between pregnancy
and family history of breast cancer. One study suggested that in women with no family history, the transient increase in risk was mainly seen in women with
a late age at first live birth, while women with a family history also experienced an adverse effect of pregnancy at younger ages [10]. The mechanism behind
these effects is not well understood, though several
hypotheses have been investigated. Possible explanations include an effect of increased pregnancy-related
hormones such as estrogen, progesterone and growth
hormone on previously initiated cells, the immunosuppressive effect of pregnancy or a pro-tumerogenic
effect of postpartum and post-lactation breast tissue
involution [11].
Concurrent breast cancer is estimated to occur

about 1 in 1500 to 1 in 4000 pregnancies [12]. Since
more women in the USA are delaying childbearing
into their 30s and early 40s, it is likely that this number will increase. The diagnosis of breast cancer during pregnancy is usually prompted by palpation of
a breast mass. Appropriately shielded mammography
and ultrasound are safe during pregnancy. Breast MRI
has not been evaluated due to concern for fetal risk
of gadolinium toxicity and heating/cavitation effects
[13]. Breast biopsy is indicated for definitive diagnosis.
A recent international collaborative study investigated cancer diagnoses, treatment and obstetric and
neonatal outcomes of women with invasive cancer
diagnosed during pregnancy [14]. The study included
216 pregnant women diagnosed with cancer between
1998 and 2008; 46% of study participants were breast
cancer patients. The mean age at diagnosis of all
patients was 33 years. Twenty-four percent of patients
were in their first trimester of pregnancy and 43% and
33% were in the second and third trimesters, respectively. More than half of the patients included in this
study (55%) received chemotherapy, either alone or
in combination with surgery. Among patients receiving chemotherapy during pregnancy, there was a significant increase in preterm labor. In addition, compared with patients that received no treatment, treated
patients had a significantly higher risk of delivering
a small-for-gestational-age child. This was primarily
seen among women treated for hematological malignancy and not seen in women with breast cancer, suggesting that the specific chemotherapeutic agents used
for treatment of certain tumors might have a more substantial impact on fetal growth. There did not appear
to be an increased risk for physical malformations at
birth among children of patients receiving chemotherapy.
As in the non-pregnant state, optimal treatment
of breast cancer during pregnancy generally involves
both local and systemic treatment. Modified radical
mastectomy is the surgical treatment of choice. Sentinel lymph node biopsy during pregnancy has not
been well studied. The dose of radiation delivered to
the fetus with technetium is estimated to be low, but
the use of blue dye mapping during pregnancy is not
recommended. Thus, if only one mapping technique
is used, the sensitivity of sentinel lymph node mapping may be decreased [15]. Breast-conserving therapy should only be considered in the third trimester as
63
radiation therapy is contraindicated during pregnancy,

and thus can only be safely administered postpartum.
Radiation risks to the fetus include a high rate of spontaneous abortion in the first few weeks after conception, an increased risk of congenital malformations
between the 2nd and 8th weeks of gestation and an
increased risk of mental retardation after the 8th week
of gestation. In addition, children exposed to radiation
in utero have an increased risk of childhood cancer
[16].
Few drugs have been studied in the setting of
pregnancy, and thus there are limited safety data on
chemotherapeutic agents in pregnant women. Cytotoxic chemotherapy should be avoided during the
first trimester of pregnancy due to a high risk of
teratogenicity during organogenesis [17]. Two small
studies have reported on the use of combination
chemotherapy for the treatment of breast cancer during the second and third trimesters of pregnancy.
Berry et al. reported no birth defects or serious peripartum complications among women treated with 5flourouracil, doxorubicin and cyclophosphamide at
standard doses [18]. Ring et al. reported on a series
of 27 women treated during pregnancy with either
cyclophosphamide, methotrexate and flourouracil or
an anthracycline-based regimen at five London hospitals; no birth defects were seen in this study, but
one child had intrauterine growth restriction and two
had respiratory problems requiring short-term stays in
the Neonatal Intensive Care Unit [19]. Taxanes have
been even less well studied in pregnancy, with only
case reports suggesting safety of the use of docetaxel
and paclitaxel during the second and third trimesters
[2022]. The delivery of cytotoxic therapy within 3
weeks prior to delivery is associated with both maternal and infant leucopenia. As a result, chemotherapy should be held beyond 35 weeks of gestation
to minimize risk of maternal and infant infection
and hemorrhage [15]. Anti-emetics (including 5HT3
serotonin antagonists) are generally considered safe
during pregnancy and should be used as necessary [15]. Granulocyte-colony stimulating factors have
been used in pregnancy in limited settings [23, 24], and
are considered category C during pregnancy (should
be given only if the potential benefit justifies the potential risk to the fetus). Trastuzumab use in pregnancy
has been associated with oligohydramnios and anhydramnios [2527]; thus, delay of trastuzumab until
after delivery, when feasible, should be considered.
Tamoxifen use has been associated with neonatal mal-
64
formations of the genital tract and craniofacial defects

[15]. Although there are reports of patients receiving tamoxifen during pregnancy without damage to
child [28, 29], its use in the adjuvant setting should be
delayed until after delivery. Teratogenic effects of aromatase inhibitors have been described in animal models [30], but there are no data on their use in pregnancy in humans. The use of gonadotropin-releasing
hormone (GnRH) agonists is not advised during pregnancy, although their use in a series of five patients was
not associated with teratogenicity [31].
Effects of breast cancer treatment on

reproductive function
While the diagnosis of breast cancer during pregnancy itself is rare, about 1015% of breast cancers
are diagnosed in reproductive-aged women, and up to
3% of breast cancers occur in women of peak reproductive age (2535 years) [32], many of whom desire
maintenance of fertility and post-treatment conception. Young age at diagnosis appears to be an adverse
prognostic factor [4], and thus young women are likely
to undergo adjuvant systemic therapy, with attendant
consequences on fertility. Treatment for breast cancer
can impact fertility for a variety of reasons, including a toxic effect of chemotherapy on ovarian follicles,
advice to delay pregnancy due to concern for recurrence of disease and the recommendation for 5 years of
adjuvant endocrine therapy for hormone-responsive
disease, after which age-related decline in fertility is
more likely to be an issue. In addition, ovarian ablation or bilateral oophorectomy may be advised for
women with hormone-responsive cancer or those with
a BRCA1/2 mutation. This is considered particularly
important for the latter group, who also have a significant lifetime risk of ovarian cancer.
Ovarian function following chemotherapy

for breast cancer
The effect of chemotherapy on ovarian function is
related to patient age, and to the specific agent and
dose used [33]. It is important to note a number of
methodological issues arise when assessing fertility
potential following breast cancer. While many studies
report rates of amenorrhea following chemotherapy,
the time point at which menstrual function is assessed
various widely among studies. Furthermore, the presence or absence of menses is an imperfect surrogate for
Table 6.2 Reported rates of amenorrhea with common breast cancer regimens
Regimen
No. of cycles
Younger a women with

amenorrhea (%)
Oldera women with

amenorrhea (%)
CMF (cyclophosphamide,
methotrexate, fluorouracil)
312
1865
7497
Anthracycline plus alkylating agent

(most commonly doxorubicin and
cyclophosphamide)
Variable
3246
73100
Anthracycline followed by taxane

(doxorubicin or epirubicin +
cyclophosphamide followed by
paclitaxel or docetaxel)
4 AC/EC 4 taxane
646
3586
Adapted from Walshe et al. [36].

In most studies, 40 years of age was used as the cut-point to differentiate younger from older premenopausal women, although women
who were 40 years at diagnosis were defined as younger in some studies and older in others.
a
fertility, as some women may maintain menstrual

function but still have impaired fertility. Pregnancy
after cancer treatment is necessarily affected by social
factors and patient preference, and thus is an impractical outcome measure and seldom reported; thus, the
data described here will focus on amenorrhea. Amenorrhea following chemotherapy may be temporary or
permanent, and results from interference of follicular
maturation, with or without depletion of primordial
follicles [34]. For those who do resume menstruation,
data suggest a continued impairment of fertility and
an earlier mean age at menopause [35]. A summary
of studies presenting effects of specific chemotherapy
regimens on menstrual function is shown in Table 6.2
[36].
Chemotherapy regimens that include an alkylating
agent (predominantly cyclophosphamide in the treatment of breast cancer) induce high rates of amenorrhea, ranging from 61 to 97% in women over 40
years and from 18 to 61% in women under 40 years
[33]. Amenorrhea occurs sooner in older women and
is more likely to be irreversible. Higher doses of
cyclophosphamide appear to have more of an effect on
menstrual function; in one study comparing 12 cycles
of single-agent cyclophosphamide (130 mg/m2 ) to 12
cycles of oral cyclophosphamide, methotrexate, fluorouracil (CMF; cyclophosphamide dose 80 mg/m2 ) in
premenopausal women, the rates of amenorrhea were
70 and 63%, respectively [36].
Anthracyclines have been increasingly incorporated into adjuvant treatment for breast cancer, as studies suggest that 36 months of anthracycline-based
therapy is equivalent or superior to CMF [37]. While
many studies suggest that rates of amenorrhea are
lower with anthracycline-based chemotherapy than

with CMF, direct comparisons are difficult due to differences among trials in individual agent dosing and
the number of cycles given. In one trial comparing
six cycles of CMF to six cycles of CEF (cyclophosphamide, epirubicin and fluorouracil), a higher rate
of amenorrhea was seen in CEF-treated patients [38].
Conversely, other studies suggest a lesser impact on
ovarian function with anthracyclines [3941]. These
differences may be partially attributable to a higher
cumulative dose of cyclophosphamide used in the
CMF regimen. Patient age is consistently related to
both risk and duration of amenorrhea due to anthracyclines. In a joint analysis of three prospective trials
using doxorubicin at M. D. Anderson, rates of amenorrhea among women aged 4049, 3039 and 30 years
were 96, 33 and 0%, respectively. A majority of those
40 years of age at diagnosis experienced permanent
menopause, while 50% of patients under age 40 had
resumption of menses [42]. Similar rates of amenorrhea were seen in a study of 249 women receiving
epirubicin [43].
Taxanes have been shown to improve survival in
the adjuvant setting in patients with node-positive
breast cancer and are increasingly used in high risk
node-negative patients, particularly young women, as
young age appears to be a significant risk factor for
recurrent disease. The true impact of taxanes on menstrual function is difficult to determine, as they are
most often given in sequence or combination with
anthracycline-based therapy. Although several small
trials have reported similar or decreased rates of amenorrhea with the addition of taxane to anthracylinebased chemotherapy, a number of trials report higher
65
rates of amenorrhea in patients who receive both

classes of drugs [33]. For example, in the BCIRG001 study, in which 1491 patients were randomized to
either docetaxel, doxorubicin and cyclophosphamide
(TAC) or fluorouracil, doxorubicin and cyclophosphamide (FAC), the rate of amenorrhea was 10%
higher in patients receiving the taxane. Similar results
were seen in a smaller survey study comparing rates
of amenorrhea in women who received four cycles of
doxorubicin and cyclophosphamide (AC) and those
who received AC followed by taxane [44]. Although
there are little data comparing the effect of docetaxel
with that of paclitaxel in terms of menstrual function, one observational study suggested higher initial
rates of amenorrhea with docetaxel versus paclitaxel,
but rates were equivalent at 3 years after treatment [40].
Interestingly, in that study, women receiving AC alone
or with taxane had initially higher rates of amenorrhea
than those receiving CMF, but there was significant
recovery of menstrual function after anthracyclinebased therapy, while CMF resulted in a continued
steady decline in the proportion of patients with menstrual bleeding, suggesting that the effect of CMF on
the ovaries is more likely to be permanent.
Endocrine therapy and reproductive

function
About 60% of premenopausal patients have hormone
receptor-positive breast cancer, and most will be
offered endocrine therapy, either alone or in combination with chemotherapy. The mainstay of hormonal
therapy in premenopausal women is treatment with
tamoxifen for a period of 5 years, which has been
shown to improve both recurrence-free and overall
survival in this population [37]. In one study in which
women were randomized to receive or not receive
tamoxifen along with chemotherapy, the use of tamoxifen decreased the likelihood of menstrual cycling at
1 and 2 years, regardless of chemotherapy regimen
[40], and some women developed irregular menses on
tamoxifen. As noted above, tamoxifen may have teratogenic effects and thus should not be used during
pregnancy. In premenopausal women, treatment with
a GnRH may also be considered, as some data suggest
that these agents may improve outcomes when used in
addition to, or in lieu of, tamoxifen and/or chemotherapy [37]. While it is commonly assumed that only
chemotherapy affects fertility, it is important to note
that hormonal therapy itself and the delay of concep-
66
tion in order to receive several years of hormonal therapy may also affect a womans chance of successful conception. To date, no randomized studies have examined the effect of hormonal therapy on pregnancy outcome [45]. Given the lack of data, many patients with
hormone receptor-positive breast cancer who desire
pregnancy, and their treating physicians, struggle with
whether to shorten the duration of endocrine therapy
or to temporarily interrupt endocrine therapy in order
to achieve pregnancy.
Fertility preservation in breast

cancer patients
As a result of higher survival rates among women
treated for breast cancer, there is an increasing emphasis on quality of life among survivors, and fertility preservation is a key issue among young women
undergoing therapy for breast cancer. The American Society of Clinical Oncology guidelines recommend that all patients interested in future fertility
should be referred for consideration of fertility preservation [46]. Data suggest that although cancer survivors can become parents through third-party reproduction (such as gamete donation and adoption), most
would prefer to have biological offspring [47]. Fertility preservation options in women with breast cancer
depend on the patients age, type of treatment planned,
whether or not she has a partner and the time available
prior to starting therapy.
Embryo cryopreservation
To date, embryo cryopreservation is considered the
more effective approach to fertility preservation. It has
been routinely used for storage of surplus embryos
after in vitro fertilization treatment for infertility. The
post-thaw survival rate of embryos is in the range of
3590%. Successful implantation rates range from 35
to 90%, and if multiple embryos are stored, cumulative pregnancy rates can be 60% [48]. This approach
requires either a male partner or sperm donor. Typically, the patient undergoes 2 weeks of ovarian stimulation with daily injections of follicle stimulating hormone (FSH) beginning at the onset of menses. Follicle
development is monitored by serial ultrasound imaging and blood tests. At the appropriate time, human
chorionic gonadotropin is administered to stimulate
ovulation and oocytes are collected using transvaginal aspiration under ultrasound guidance. Oocytes are
then fertilized in vitro and cryopreserved. In women

with hormone receptor-positive breast cancer (the
majority of women diagnosed with breast cancer),
concerns exist about possible detrimental effects of
the use of endocrine agents to stimulate ovulation
on breast cancer outcome. Oocyte collection without
ovarian stimulation can be attempted, but the embryo
yield is very low. Alternative hormonal stimulation
approaches, such as the use of letrozole or tamoxifen
concurrent with FSH, have been attempted, and do not
appear to increase cancer recurrence rates [49]. Even
in women with hormone receptor-negative breast cancer, concerns about delays in initiation of cancer treatment may limit the feasibility of this approach. Because
the process of ovarian stimulation must begin at the
time of menses and then takes 2 weeks, chemotherapy and other life-saving treatments may be delayed.
While data suggest that the hormonal milieu of natural
pregnancy does not adversely affect breast cancer outcomes, there are no data regarding safety of hormones
to support pregnancy (such as high dose progesterone)
after embryo implantation. In studies published to date
addressing survival of breast cancer patients who have
become pregnant after diagnosis and treatment, only
a small percentage of included patients conceived via
assisted reproductive technologies [50].
Oocyte preservation
Cryopreservation of unfertilized oocytes is an option
for women for whom a partner is unavailable. The
ovarian stimulation and harvesting process is identical to that used for embryo cryopreservation, but
the oocytes are stored unfertilized and then subsequently thawed and fertilized in vitro. Thus, this technique is associated with the same issues regarding timing, potential delay of chemotherapy and exposure to
hormones. Unfortunately, oocyte freezing is technically more complex than embryo cryopreservation and
unfertilized oocytes are more prone to damage during cryopreservation; thus, the overall successful pregnancy rate is likely lower with this procedure [51].

The process of ovarian tissue cryopreservation involves
freezing thin slices of the ovarian cortex, which contains a rich reserve of primordial follicles. This investigational method of fertility preservation requires neither a sperm donor/partner nor ovarian stimulation.
The first ovarian transplant procedure was reported in
2000 [52], and since then there have been several case
reports of successful pregnancies resulting from these
procedures [5355]. Ovarian tissue is removed laparoscopically (this procedure requires general anesthesia)
and frozen, and can be thawed and re-implanted at a
later date, either orthotopically (in the pelvis) or heterotopically (in subcutaneous tissue in the forearm or
abdomen); studies have reported restoration of ovarian function with both methods. Although the cryopreservation technique is highly effective, a large number of follicles may be lost due to ischemia at the
time of re-implantation, so typically the cortex from
an entire ovary is used.
One hypothethical concern with this method is the
potential for re-introduction of metastatic cancer cells.
However, in breast cancer patients without evidence
of systemic disease, the likelihood of occult ovarian
metastases appears to be extremely low, and in the limited reported literature, no cases of cancer recurrence
after ovarian transplantation have been noted [46].
Donor eggs and surrogacy

In patients in whom oocyte or embryo cryopreservation prior to treatment was not possible, particularly those with low ovarian reserve or premature
menopause, in vitro fertilization using donor ova may
be an option. One advantage to this method is a higher
success rate using fresh rather than frozen ova. With
appropriate egg donors, success rates may exceed 60%
per embryo transfer [56]. For those with a high risk
of recurrence, or those on long-term therapy with
tamoxifen or aromatase inhibitors, gestational surrogacy may be a viable alternative to pregnancy.
Attempts to preserve fertility

during chemotherapy: suppression of
ovarian function
Ovarian suppression with a GnRH agonist during
chemotherapy treatment has been suggested as a
means to preserve long-term menstrual function,
though this strategy is controversial. A small study
comparing 54 patients with retrospective controls
suggested a benefit in preservation of ovarian function among women undergoing chemotherapy for
Hodgkins and non-Hodgkins lymphoma [57], with
93.7% of those receiving GnRH agonists resuming
menses vs 37% of historical controls. In a second
study examining the use of GnRH agents for ovarian
67
function preservation in patients receiving chemotherapy (n = 62) compared with retrospective controls
(n = 55), the percentage of patients resuming ovulation and menses was significantly higher in the GnRH
group [58]. In the first prospective randomized trial
of this strategy, 17 women were randomized to either
buserelin or control prior to and during chemotherapy; at 3 years, 4 of 8 women receiving buserelin
and 6 of 9 controls were amenorrheic. In a more
recent randomized study that included 80 women
receiving chemotherapy for breast cancer, the addition of GnRH agonist prior to and during chemotherapy significantly improved the rate of resumption
of menses (89.6% versus 33.3%) and spontaneous
ovulation (69.2% versus 29.6%) [59]. Though compelling, these data require confirmation; the Southwest Oncology Group (SWOG) is currently conducting SWOG S0230 a randomized trial with an accrual
goal of 458 patients evaluating GnRH agonists for
ovarian function preservation in women with hormone receptor-negative breast cancer who receive
chemotherapy [46].
Contraception following breast cancer

As many chemotherapeutic and hormonal agents used
for treatment of breast cancer are known or suspected
to be teratogenic, pregnancy should be avoided during active treatment of breast cancer. Hormonal contraceptives are generally felt to be contraindicated,
particularly in women with estrogen and progesterone receptor-positive tumors, although there is little
established evidence addressing their use [60]. Recent
small studies have addressed the use of an intrauterine levonorgestrelreleasing system (Mirena), which
delivers high local but low systemic doses of progesterone, and suggest that in addition to providing effective contraception, this device may lower the risk of
endometrial pathology in tamoxifen users (61]. However, there is limited data on the effect of Mirena
on cancer incidence and recurrence. Subgroup analyses from one recent cohort study suggested a trend
toward increased risk of recurrence among women
using Mirena at the time of diagnosis who continued with the device in situ, but this effect was not
seen among women with insertion of the device after
a breast cancer diagnosis [62]. In the absence of solid
data, non-hormonal contraceptive methods remain
the standard for women with a previous diagnosis of
breast cancer.
68
Pregnancy following breast cancer

Based on limited retrospective data, pregnancy does
not appear to compromise the survival of women with
a history of breast cancer, and no deleterious effects
have been demonstrated in the fetus [63]. The rate
of pregnancy among women of reproductive age with
a diagnosis of cancer is estimated to be about 50%
lower that of age-matched peers [64]. One retrospective study performed by the International Breast Cancer Study Group described a series of 94 patients who
became pregnant after a diagnosis of breast cancer
[65]. Seventeen percent of study participants had their
first subsequent pregnancy within 1 year of their breast
cancer diagnosis, 24% between 1 and 2 years, 23%
between 2 and 3 years and 31% achieved pregnancy
3 or more years after diagnosis. This study also compared the prognosis among these women to that of 188
matched controls, and found that those women who
attained pregnancy had superior survival compared
with controls, although this difference may represent a
bias toward women with good prognosis tumors and
better overall health being more likely to seek and
achieve childbearing. In any case, this and other studies do not indicate a detrimental effect of subsequent
pregnancy on survival, especially if some interval
occurs between the cancer and the pregnancy [50, 65
68]. Some physicians recommend that patients wait a
couple of years after diagnosis before attempting conception. This allows both time for the body to heal and
time for early recurrences of cancer to manifest, which
may influence the decision to become a parent [50, 69].
In one recent study evaluating pregnancy and disease
outcomes based on time from diagnosis to conception, women who conceived 24 or more months after
diagnosis had significantly improved survival compared with those who did not conceive; women who
conceived 624 months after diagnosis also showed a
trend toward improved survival, though this was not
statistically significant [50]. Given these results, coupled with the fact that fertility rates decline significantly with age, the authors suggested that it may not
be necessary to advise waiting more than 6 months to
attempt conception in patients with localized disease.
The risk of relapse and time to recurrence of
breast cancer is associated with many factors, including stage at diagnosis, lymph node status, tumor grade
and hormone receptor status. Notably, recurrence
risk for hormone receptor-negative tumors is greatest in the first 2 years after diagnosis, and then drops
significantly [70]. Annual risk of recurrence for hormone receptor-positive tumors is more stable, and
is relatively constant from years 15 and then drops
slightly but stays constant from years 512. As younger
women are more likely to develop hormone receptornegative disease, these risks must be considered when
making decisions regarding pregnancy. With regard
to hormone-receptor positive disease, it is important to note that the currently recommended duration of adjuvant endocrine therapy for premenopausal
women is 5 years, and the decision to attempt conception necessitates discontinuation of endocrine therapy.
Thus, the decision regarding when to attempt conception should be individualized.
Lactation following breast cancer

Observational data suggest that breastfeeding after a
diagnosis of breast cancer does not affect prognosis, and there are no data to suggest that breastfeeding poses any health risk to the child [71]. Women
who have been treated with breast-conserving surgery
will require radiation to the affected breast, which
reduces the success of breastfeeding on that side. In
one small study of 11 patients with 13 pregnancies,
successful lactation in the treated breast occurred in
four instances, and one patient was able to breastfeed
from the treated breast for 4 months [72]. Time from
treatment to lactation did not appear to have an effect
on lactation success. However, circumareolar incisions
did appear to adversely affect ability to breastfeed.
Summary and conclusions

Breast cancer is a common disease among women and
frequently affects premenopausal women. Breast cancer during pregnancy is rare and poses unique issues
relating to ensuring adequate treatment of the patient
while not endangering the fetus. Additionally, as the
mean age of childbearing increases, fertility issues after
diagnosis and treatment of breast cancer are increasingly relevant, and present significant challenges to
patients and their healthcare providers. Treatment for
breast cancer, particularly chemotherapy, can have
permanent effects on reproductive capability. While
embryo cryopreservation, oocyte preservation and
ovarian tissue cryopreservation are among the options
available to increase future fertility in young women
at the time of diagnosis of breast cancer, the hormonal
manipulation and timing requirements of these procedures currently limit the feasibility for many breast
cancer patients. Further research is needed to deter-
mine which modalities can successfully aid in protecting and aiding reproductive potential in women
undergoing breast cancer therapy who desire future
fertility.
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Section 2
Chapter
Pediatric cancer therapy and fertility

Pinki K. Prasad, Jill Simmons and Debra Friedman
Background
There are currently an estimated 270 000 survivors
of childhood cancer in the USA [1]. The 5-year survival rate for all childhood cancers is approximately
80% [2], but survival is often associated with a cost.
Treatment with past and contemporary regimens of
chemotherapy and radiation can affect future fertility. Sterility, infertility or subfertility can result from
gonadal removal or damage to germ cells from adjuvant therapy. Damage to the gonads by irradiation
or chemotherapy depends upon the cancer survivors
gender, age at time of treatment, dose of radiation and
fractionation schedule, and total dose and nature of
chemotherapy given [3, 4]. This chapter reviews pediatric cancer therapy and its consequences on fertility.
Effects of chemotherapy and

radiation on ovaries
Normal physiology and potential for fertility
The outer cortex of the ovary contains oocytes and
is the site of hormone production in females. Female
oocyte production ceases during fetal development,
and girls are therefore born with a finite number of
oocytes (2 million at birth with approximately 300 000
left at puberty) [5]. The normal physiology of oocyte
maturation begins in utero, occurs continuously and
is initially gonadotropin-independent. At puberty, the
gonadotropin-dependent phase begins, and follicles
are primed by follicle stimulating hormone resulting in
granulosa cell proliferation. Luteinizing hormone then
triggers ovulation with a potential for fertilization [6].
The normal premenopausal ovary contains degenerating ova and follicles in varying stages of maturity. A
typical female will release 300500 mature eggs during
her reproductive life span.
Due to the non-renewable nature of a females

oocytes, the oocytes are quite susceptible to damage [7]. With the depletion of oocytes by radiotherapy, chemotherapy or normal senescence, the
ovaries undergo atresia [6]. Alkylating agents are
the most common chemotherapeutic agents associated with gonadal damage; these agents are not cellcycle-specific and thus do not require cell proliferation for their cytotoxic actions; however, dividing
cells are more susceptible to damage [8, 9]. As a
result, menstruation and estrogen production ceases
and menopause occurs. Ovarian hormones have critical physiological effects on other organs and bodily
processes including the stimulation of libido, the maturation and function of breasts and vagina, bone mineralization and the integrity of the cardiovascular system.
Effects of chemotherapy
Chemotherapy can cause infertility, premature ovarian failure, menstrual irregularity and delayed puberty.
The effects of chemotherapy on ovarian function are
both agent and dose-dependent, and this effect may be
additive to that resulting from abdominopelvic radiotherapy. Alkylating agents such as cyclophosphamide
affect the resting oocyte in a dose-dependent, cell
cycle-independent manner [10] by affecting undeveloped oocytes and possibly pre-granulosa cells of primordial follicles [11]. The results of alkylating agents
are more pronounced in post-pubertal as compared
to pre-pubertal females, due to the fact that postpubertal females have fewer remaining oocytes. Risks
of menstrual irregularity, ovarian failure and infertility increase with age at treatment, as the normal
aging process is accompanied by an ongoing depletion of oocytes. Amenorrhea and premature ovarian
failure occur more commonly in adult women treated
73
Table 7.1 Gonadotoxic chemotherapeutic agents

Procarbazine
Cyclophosphamide
Chlorambucil
Mustine
Melphalan
Busulfan
Nitrosoureas (BCNU and CCNU)
Ifosfamide
Cytosine arabinoside
Cisplatin
BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; CCNU, 1-(2chloroethyl)-3-cyclohexyl-1-nitrosourea.
with cyclophosphamide than in adolescents [12, 13].

Younger females can tolerate higher doses of alkylating
agents without impairment of fertility when compared
to adult females [10, 1416].
In a study examining the effects of adjuvant
chemotherapy on women undergoing treatment for
soft tissue sarcoma, authors noted that chemotherapy
with doxorubicin, cyclophosphamide and high dose
methotrexate produced irregular menses in 20% of
women and persistent amenorrhea in 20% of women
[17]. Other chemotherapy agents that have been associated with ovarian damage include procarbazine [18].
Table 7.1 lists common chemotherapy agents that are
known to be gonadotoxic.
Frequently, there may initial evidence of ovarian
failure, but recovery often occurs. In a study by Sanders
et al., women who received high dose cyclophosphamide (50 mg/kg/day for 4 days) prior to bone
marrow transplantation for aplastic anemia all developed amenorrhea following transplantation. Fortunately, 80% of the survivors had recovery of normal ovarian function between 3 and 42 months after
transplantation [19]. Ovarian function was evaluated
in women treated with different drug combinations
by Green et al; in women who received a low dose
cyclophosphamide-containing drug combination for
non-Hodgkins lymphoma, ovarian function was normal in all women; however, women who received
a combination of whole abdominal radiation with a
combination of low dose alkylating agents had ovarian
failure [20].
Chemotherapy regimens used as conditioning for
stem cell transplantation are highly gonadotoxic.
Recovery of ovarian function is rare following reg-
74
imens that include busulfan and cyclophosphamide,

though melphalan-based regimens show less reproductive toxicity [21].
In addition to the risk of infertility, female survivors of childhood cancer are also at risk for premature ovarian failure (early menopause) [22]. Byrne
et al. reported a ninefold increase in the incidence
of menopause during their early 20s in female survivors of childhood cancer when compared to the
general population. This study also examined women
who reported having normal menses after the completion of chemotherapy and noted that 42% of women
reported premature menopause (ovarian failure by the
age of 31) compared to 5% of the general population
[22]. Thus, the presence of apparently normal ovarian
function at the completion of chemotherapy should
not be interpreted as evidence of lack of injury to ovarian tissue. Studies have also reported disturbances in
pubertal progression in approximately 30% of patients
treated with alkylating agents during puberty [10, 23,
24]. The Childhood Cancer Survivor Study reported
that 8% of female childhood cancer survivors experienced a non-surgical premature menopause compared
to 0.8% of sibling controls [25].
Effects of radiation
Abdominal, pelvic and total body irradiation (TBI)
may result in ovarian and uterine damage. Damage
induced by radiotherapy results in progressive and
irreversible damage in the ovary, causing amenorrhea
and infertility. The oocyte is very sensitive to radiation
and undergoes irreversible damage at 2 Gy in 50% of
patients [26]. Radiation causes a decrease in the number of ovarian follicles, impaired follicular maturation,
cortical fibrosis and atrophy, generalized hypoplasia
and hyalinization of the capsule.
The frequency of ovarian failure following abdominal radiotherapy is related to the age at time of irradiation and the radiation therapy dose received by the
ovaries. Females treated prior to the onset of puberty
have a greater number of ova than older females;
thus ovarian function is more likely to be preserved
after radiotherapy in pre-pubertal females compared
to post-pubertal females [27, 28]. Whole abdominal radiotherapy produces severe ovarian damage.
Wallace et al. demonstrated that 25% of female
childhood cancer survivors who received abdominal
irradiation therapy doses of 20003000 cGy had premature menopause before the age of 16 years [29].
Chapter 7: Pediatric cancer therapy and fertility
Table 7.2 Effect of fractionated ovarian X-irradiation on

ovarian function in women of reproductive age irradiated for
malignant or non-malignant disease
Effects of chemotherapy and

radiation on testes
Minimum ovarian
dose (Gy)
Effect
Normal physiology and potential for fertility
0.6
None
1.5
No deleterious effect in most young

women. Some risk of sterilization
especially in women aged 40 years
2.55.0
Variable. Aged 1540 years: about

60% sterilized permanently, some
with temporary amenorrhea. Aged
40 years; usually 100% permanent
sterilization
58
Variable. Aged 1540 years: about

70% sterilized permanently; of the
remainder, some with temporary
amenorrhea
100% permanently sterilized
Adapted from Ash [36].
Other reports of childhood survivors treated with

whole abdomen radiotherapy and craniospinal radiotherapy reveal similar results [3032].
All women who receive TBI prior to stem cell
transplantation develop amenorrhea [33] immediately
after treatment. Fortunately, some may have recovery of normal ovarian function. Sanders et al. examined ovarian function following bone marrow transplantation for aplastic anemia or leukemia and noted
that 6% of survivors had recovery of normal ovarian function; these results also indicated that return
of normal ovarian function was highly correlated
with age 25 years [19]. Recovery of ovarian function among female childhood cancer survivors who
received TBI is more favorable if the TBI was given in
fractions and if the child was pre-pubertal [21].
Ovarian failure following radiotherapy is also correlated with the treatment volume. In a study looking
at ovarian failure in long-term survivors of childhood
malignancy [34], ovarian failure occurred in almost
70% of women who received radiation therapy that
included their ovaries in contrast to 0% of women
whose irradiation field did not include their ovaries.
It is also important to consider the risk of ovarian
failure related to radiotherapy when fields outside the
abdomen and pelvis have been irradiated. Direct or
scattered irradiation from craniospinal radiotherapy
has been shown to affect ovarian function [33, 35].
Table 7.2 shows the effect of differing minimum doses
of radiation on the ovaries [36].
Male germinal stem cells are present from the time of

birth, but they do not develop into gametes capable of
fertilizing an oocyte until a male goes through puberty.
The testis is composed of cells that aid in the development of sperm; Sertoli cells support and nurture developing germ cells and are also the site of production of
the glycoprotein hormone, inhibin; and Leydig cells
are responsible for testosterone synthesis [3]. Spermatogenesis is a process that begins at puberty and
continues throughout life. There is a steady turnover
of germ cells in the pre-pubertal testis that undergo
spontaneous degeneration before a haploid state of
maturation is reached. Studies have hypothesized that
during this steady state of turnover, cytotoxic therapy
affects fertility. Testicular function may be damaged by
surgery, irradiation and/or chemotherapy.
Effects of chemotherapy
Testicular dysfunction is among the most common
long-term side effect of chemotherapy in men. The
germinal epithelium is very susceptible to injury by
cytotoxic drugs secondary to its high mitotic rate.
Compared with the germinal epithelium, Leydig cells
are relatively resistant to the effects of chemotherapy
[3739]. However, with more intensive gonadotoxic
regimens, reductions in testosterone concentrations
have been noted that may be clinically relevant.
Similar to females, regimens that include high
doses of alkylating agents are the most toxic to gonadal
tissue. Total dose of cyclophosphamide is one factor that influences the severity of testicular damage. In survivors of Ewings and soft tissue sarcoma,
who received a cumulative dose of cyclophosphamide
7.5 g/m2 , there was an increased risk of persistent oligo- or azoospermia [40]. In studies of male
childhood survivors of acute lymphoblastic leukemia
(ALL), Blatt et al. reported normal testicular function in boys treated for ALL with therapy that did
not include cyclophosphamide or intravenous cytosine arabinoside [41]. Drugs such as procarbazine,
cyclophosphamide and chlorambucil have been shown
to produce prolonged azoospermia in 90% of men
[4244]. Combination chemotherapy that includes an
75
alkylating agent and procarbazine causes severe damage to the testicular germinal epithelium [42, 4446].
Studies of adolescent and young adult male survivors of Hodgkins lymphoma have shown that both
the chemotherapeutic regimen and dose intensity are
important variables affecting reproductive potential
[4, 44, 47]. Those treated with 6 cycles of chemotherapy that included nitrogen mustard, vincristine, prednisone and procarbazine had 90% of infertility due
to azoospermia [44, 47]. However, the adolescent and
young adult males who received 3 or fewer cycles of
identical therapy had only a 50% rate of infertility
[47]. In a study examining more contemporary therapy
in Hodgkins lymphoma, authors found that patients
treated with a regimen of adriamycin, bleomycin, vinblastine and dacarbazine had a 33% risk of infertility [4]. In a recent cohort study conducted by the
European Organization for Research and Treatment
of Cancer, exposure to alkylating chemotherapy was
associated with a significantly higher risk of gonadal
dysfunction among male patients and longer recovery
time of gonadal function [48].
Chemotherapy that includes platinum compounds
can cause prolonged azoospermia in up to 50% of men
[49]. Conditioning regimens that include high doses of
alkylating agents and TBI used for stem cell transplantation cause prolonged azoospermia in more than 50%
of survivors [50, 51].
Chemotherapy appears to lower healthy sperm
counts in cancer survivors but, after an adequate time
off of therapy, some studies suggest that DNA integrity
of sperm is re-established similar to age-matched controls; the amount of time off therapy required for this
recovery has not been adequately quantified [52].
Effects of radiation
When testes are exposed to radiation, sperm count
begins to decrease. Dependent upon dosage, temporary or permanent sterility may result [53]. The degree
and permenancy of radiotherapy-induced testicular
damage also depends upon the treatment field and
fractionation schedule. Table 7.3 shows the effects of
fractionated radiation on spermatogenesis and Leydig
cell function [54]. Males who receive radiation to the
abdominal or pelvic region may still regain partial or
full sperm production depending upon the amount of
injury to the testes. Unlike the germinal epithelium,
Leydig cell function may be more prone to damage
from irradiation in pre-pubertal life than adulthood
76
[55]. Testicular radiation with doses 20 Gy is associated with Leydig cell dysfunction in pre-pubertal boys,
while Leydig cell function is usually preserved with
doses of as much as 30 Gy in sexually mature males.
Exposing the testes to ionizing radiation at a dose
6 Gy causes disturbances of spermatogenesis and
altered spermatocytes with recovery periods dependent on dose [54]. Doses 6 Gy cause permanent
infertility by destroying all stem cells [56].
The testes are directly irradiated in situations such
as testicular relapse in ALL, and the high doses of
radiotherapy required (often 2400 cGy) results in both
sterilization and Leydig cell dysfunction [57]. Total
body irradiation used for stem cell transplantation
conditioning can cause permanent gonadal failure in
approximately 80% of males [21]. Craniospinal irradiation produced primary germ cell damage in almost
20% of children with ALL in a study that utilized
the Childhood Cancer Survivor Study [58]. This study
demonstrated the testes sometimes receive radiation
via body scatter. Scatter occurs when X-rays interact
with tissues near the target of interest, resulting in secondary X-rays that then hit the target [59]. The amount
of scattered radiation is a function of the proximity of
the radiation field to the target, the field size and shape,
the X-ray energy and the depth of the target. Of these,
distance from the field edge is the most important factor. Scatter dose to the testes may be an issue when
treating a field that extends into the pelvis.
Risk of infertility after treatment

Studies have demonstrated that when evaluated as a
group, the fertility of childhood cancer survivors is
impaired. A multicenter study examining 5-year childhood and adolescent survivors of solid tumor cancers
and Hodgkins lymphoma demonstrated a 15% incidence in impaired fertility in survivors; males having
more problems than females [60]. Other studies have
found a number of variables associated with decreased
fertility after cancer treatment in childhood and adolescent survivors of cancer and bone marrow transplantations. These variables include: older age at time
of cancer therapy, type of therapy, site of therapy and
gender [6165].
It is important for physicians treating children and
adolescents to recognize the risks associated with radiation and chemotherapy and the effects treatment will
have on future fertility. In 2006, the American Society of Clinical Oncology provided recommendations
Table 7.3 The effects of fractionated radiation on spermatogenesis and Leydig cell function
Testicular dose (cGy)
Effect on spermatogenesis
Effect on Leydig cell function
<10
No effect
No effect
1030
Temporary oligospermia
No effect
3050
Temporary oligospermia 412 months after

radiation: 100% recovery by 48 months
No effect
50100
100% temporary azoospermia 317 months after

radiation. Recovery begins at 826 months
No effect
100200
100% azoospermia 29 months after radiation.

Recovery begins at 1120 months
No change in testosterone
200300
100% azoospermia beginning at 12 months. May

lead to permanent azoospermia. Recovery variable
generally years)
No change in testosterone
1200
Permanent azoospermia
Decreased basal testosterone. Replacement hormone

not needed to ensure initiation of puberty
2400
Permanent azoospermia
Decreased testosterone. Replacement needed to

ensure puberty
Adapted from Schwartz [54].
for oncologists to help address the possibility of infertility with patients treated during their reproductive
years [66]. The guidelines state that oncologists should
be prepared to discuss fertility preservation options or
to refer the patient to reproductive specialists for further information prior to initiation of therapy. However, there is no consensus on when the appropriate age
of reproductive potential actually occurs or at what age
patients should be referred to a reproductive specialist.
Preservation of fertility
The options for fertility preservation before treatment
differ between females and males. Males have more
available options that are less invasive, less expensive
and more effective.
Preventive strategies for females

before treatment
Progress in reproductive endocrinology has resulted in
the availability of several potential options for preserving or permitting fertility in females prior to receipt of
potentially toxic chemotherapy or radiotherapy. These
procedures are described elsewhere in this book; we
will discuss some strategies specific to pediatric cancer
survivors.
In vitro fertilization and subsequent embryo cryopreservation has been successful and is an established
technique [9]; this is the only non-experimental pretreatment fertility preservation option for adolescent
and young adult women. This intervention is technically complex and can delay initiation of cancer therapy by 24 weeks. This intervention is also limited to
females who are either involved in a stable relationship
or willing to identify a known or anonymous sperm
donor. These issues often preclude this intervention
as an option for adolescent females. Also, this technique requires ovarian stimulation, which precludes
it being an option for those females with estrogensensitive tumors.
Ovarian tissue cryopreservation is a process in
which normal, functioning ovarian tissue is excised
from the ovary and stored cryogenically and is the
only option that can be offered to pre-pubertal girls
[67]. Currently this technique is available only in certain parts of the USA as an experimental protocol. This
technique involves obtaining primordial follicles from
a laprascopic biopsy. The immature follicles are smaller
than mature oocytes and more tolerant to freezing and
thawing [6]. Hundreds of immature oocytes are cyropreserved without the necessity of ovarian stimulation
and subsequent delay of cancer treatment. Ideally, the
stored ovarian tissue is thawed and autotransplanted
into the donor once treatment has been completed.
Studies in humans are still in their infancy, though
there are scattered published case reports that demonstrate its efficacy; a recent paper details 8 years of
77
experience in adult female cancer patients with positive results [68, 69]. Kim et al. reported on four young
cancer survivors who had their ovaries removed prior
to the initiation of cancer therapy and then had a
heterotopic autotransplantation of their ovarian tissue
over the course of a few years after completing therapy. Their report concluded that ovarian function was
re-established in all three patients [69].
Preservation of fertility during

treatment for females
Reduction in the dose or use of alkylating agents
and abdominopelvic radiotherapy is the most effective means of preserving ovarian function and promoting positive reproductive outcomes in pediatric cancer
survivors. Studies have shown that movement of the
ovaries out of the field of radiation (ovariopexy), either
laterally, toward the iliac crest or behind the uterus
may help preserve fertility when high doses of radiation therapy are used [70]. By relocating the ovaries
laterally, it is possible to shield them during radiation
of the para-aortic and femoral lymph nodes. This may
also be helpful for young girls or adolescents undergoing craniospinal radiotherapy for brain tumors. The
ovaries should be marked by the surgeon with clips
that can later be identified by simulator film. Central pelvic blocking at the time of inverted Y field
will prevent direct irradiation, though scatter dose and
transmitted dose will be inevitable [53]. Pelvic radiation, however, still provokes an irradiation of the ovary
of 510%, even if the ovaries are transposed outside
the irradiation area [71]. Although ovarian transposition is relatively effective at preserving the endocrine
function of the ovary in approximately 85% [72] of
cases, 1 study demonstrated that only approximately
15% of patients who wish to become pregnant achieve
this goal [70].
Preventive strategies for males

before treatment
Sperm cryopreservation after masturbation is the most
established and effective method of fertility preservation in males [66]. Sperm should be collected before
initiation of chemotherapy or radiotherapy. Studies
have demonstrated that sperm quality may be compromised in males with Hodgkins lymphoma, leukemia
and testicular cancer [73]. These limitations have been
largely overcome by improving in vitro fertilization
78
technology. Intracytoplasmic sperm injection allows

successful fertilization with a single sperm. Collection
of semen through masturbation in adolescents and
young males may be compromised by embarrassment
and issues with informed consent. Alternative methods of obtaining sperm include testicular aspiration or
extraction, electroejaculation under sedation or anesthesia or from a post-masturbation urine sample [74,
75] Pre-pubertal males pose a challenge for fertility
preservation. Currently, testicular tissue cryopreservation is an experimental option. Ginsberg et al. reported
that 76% of 21 families of pre-pubertal boys with newly
diagnosed malignancies consented to testicular biopsy
cryopreservation and that none of these patients had
any postoperative sequelae [76].
Preservation of fertility in males

during treatment
Cryopreservation of sperm has become the standard practice and should be offered to all newly
diagnosed, post-pubertal males at risk for infertility.
Gonad shielding can be used during radiation therapy but is only possible with selected radiation fields
and anatomy [66]. Fraass et al. reported that a gonadal
shield forming a cup around the testes to reduce the
testicular dose [45] led to a 310-fold reduction in the
radiation dose to the testes, depending upon the distance from the proximal edge of the field. In almost
all cases, the measured dose to the testes was 1% of
the prescription dose. Therefore, for a patient receiving 5000 cGy to a pelvic field, the dose to the testes
would be 50 cGy, which would prevent permanent
azoospermia.
Emerging data on fertility after

cancer treatment
Compared to healthy siblings, the fertility of childhood and adolescent cancer survivors is impaired. The
most significant differences in the relative fertility rates
occur in male survivors treated with an alkylating
agent with or without irradiation [51, 60, 64].
Fertility may be impaired by factors other than the
absence of sperm or ova. Anatomic changes due to
surgery and radiation can affect fertility as well. Retrograde ejaculation occurs with a significant number
of men who undergo retroperitoneal lymph node dissection for testicular cancer. Uterine structure may be
affected by abdominal irradiation in females.
Another issue that is important to disclose to

families is the cost of fertility preservation. Patients
and families are usually responsible for the costs of
preservation, as most insurance companies do not
cover these costs. Fallat and Hutter reported that the
cost of sperm cryopreservation after masturbation was
approximately $1500 for 3 samples stored for 3 years in
2006 [77]. This cost was exponentially higher if alternative methods were needed to obtain sperm or for
prolonged storage [66]. Unfortunately, the cost for fertility preservation for females, which involves more
procedures and anesthesia, can easily be more than
$10 000.
Table 7.4 Guidelines for parents and patients about

preservation of fertility
Role of physician
Adapted from Fallat [77].
Patients who will receive therapy with the potential

to limit or abolish fertility need sensitive, informed
management. Important aspects of management have
been discussed in the previous sections and involve
considerations of gonadal protection, germ cell storage and assisted fertilization. It is imperative that
physicians present this information to families and
patients prior to the initiation of chemotherapy or
radiotherapy. A recent study reported that both pediatric and adult oncologists are uncomfortable with
discussing fertility preservation with cancer patients.
Quinn et al. reported that a physicians discomfort
with this topic was due to a number of variables that
included language barriers, lack of knowledge and success rates of fertility preservation techniques available
and perception that the subject of fertility preservation adds more stress [78]. It is important for oncologists to have access to improved training that will
remove these biases and facilitate these discussions.
A physicians encouragement is a strong predictor of
whether an optional intervention will be considered
by a patient [77]. Oncologists have a responsibility
to inform parents and age-appropriate patients about
the likelihood that their cancer treatment may permanently affect their fertility [66]. Parents of minors or
age-appropriate children should be informed of their
prognosis in realistic terms. The success rates, costs
and experimental nature of specific assisted reproduction techniques should be discussed. Suggestions have
been previously published regarding counseling parents and patients about the preservation of fertility in
children and adolescents with cancer. The authors recommended that evaluation of candidacy for fertility
preservation should involve a team of specialists that
Offer cryopreservation of sperm whenever possible to

male patients and families
Discuss current fertility preservation options for female

children and adolescents
When considering actions to preserve a childs fertility,

parents need to consider childs assent, procedures
involved and whether procedures are proven or
experimental
Instructions concerning disposition of stored gametes,

embryos or gonadal tissue in the event of patients
death should be legally outlined
Concerns about welfare of resulting offspring of

childhood cancer survivor should not be cause for
denying reproductive assistance to patient
includes a pediatric oncologist, a radiation oncologist,

a fertility specialist, an ethicist and a mental health
professional [77]. Table 7.4 lists the guidelines [77].
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childhood or adolescent cancer. N Engl J Med 1987;
317(21): 131521.
61. Humpl T, Schramm P and Gutjahr P. Male fertility in
long-term survivors of childhood ALL. Arch Androl
1999; 43(2): 1239.
62. Jaffe N, Sullivan MP, Ried H et al. Male reproductive
function in long-term survivors of childhood cancer.
Med Pediatr Oncol 1988; 16(4): 2417.
63. Sanders JE, Buckner CD, Amos D et al. Ovarian
function following marrow transplantation for aplastic
anemia or leukemia. J Clin Oncol 1988; 6(5): 81318.
81
64. Green DM, Kawahima T, Stovall M et al. Fertility of

male survivors of childhood cancer: a report from the
Childhood Cancer Survivor Study. J Clin Oncol 2010;
28(2): 3329.
65. Green DM, Kawashima T, Stovall M et al. Fertility of
female survivors of childhood cancer: a report from
the childhood cancer survivor study. J Clin Oncol
2009; 27(16): 267785.
2006; 24(18): 291731.
67. Fabbri R, Venturoli S, DErico A et al. Ovarian tissue
banking and fertility preservation in cancer patients:
histological and immunohistochemical evaluation.
Gynecol Oncol 2003; 89(2): 25966.
Engl J Med 2005; 353(3): 31821.
2009; 91(6): 234954.
70. Morice P, Thiam-BA R, Castaigne D et al. Fertility
results after ovarian transposition for pelvic
malignancies treated by external irradiation or
brachytherapy. Hum Reprod 1998; 13(3): 6603.
82
71. Donnez J and Bassil S. Indications for

cryopreservation of ovarian tissue. Hum Reprod
Update 1998; 4(3): 24859.
Obstet Gynecol 2003; 188(2): 36770.
73. Opsahl MS, Fugger EF, Sherins RJ et al. Preservation
of reproductive function before therapy for cancer:
new options involving sperm and ovary
cryopreservation. Cancer J Sci Am 1997; 3(4): 18991.
74. Bahadur G, Ling KL, Hart R et al. Semen quality and
cryopreservation in adolescent cancer patients. Hum
Reprod 2002; 17(12): 315761.
75. Bahadur G, Ling KL, Hart R et al. Semen production
in adolescent cancer patients. Hum Reprod 2002;
17(10): 26546.
76. Ginsberg JP, Carlson CA, Lin K et al. An
experimental protocol for fertility preservation in
prepubertal boys recently diagnosed with cancer: a
report of acceptability and safety. Hum Reprod 2010;
25(1): 3741.
77. Fallat ME and Hutter J. Preservation of fertility in
pediatric and adolescent patients with cancer.
Pediatrics 2008; 121(5): e14619.
78. Quinn GP, Vadaparampil ST, King L et al. Impact of
physicians personal discomfort and patient prognosis
on discussion of fertility preservation with young
cancer patients. Patient Educ Couns 2009; 77(3):
33843.
Section 2
Chapter
Cancer epidemiology and environmental

factors in children, adolescents and
young adults
Karina Braga Ribeiro and Paolo Boffetta
Introduction
Cancer is a rare disease in childhood. In developed
countries, only about 0.5% of all cases occur in children under 15 years of age. The incidence rates range
between 96 and 138/million children per year for
males and between 70 and 116/million children for
females [1]. Contrary to what is observed in adults,
epithelial cancers (carcinomas) are rare among children, and in this age group solid tumors are predominantly embryonal. Therefore, it is more appropriate to classify childhood cancers according to their
histology. According to the International Classification of Childhood Cancer [2], childhood tumors are
classified into 12 major diagnostic groups: leukemias,
lymphomas, central nervous system (CNS) tumors,
sympathetic nervous system tumors, retinoblastomas,
renal tumors, liver tumors, bone tumors, soft tissue sarcomas, germ cell tumors, epithelial tumors
and other and unspecified malignant cancers (Table
8.1 [2]). Leukemias, brain tumors and non-Hodgkins
lymphomas (NHLs) are the most frequent pediatric
cancers in developed countries, representing almost
60% of all cases, while in developing countries NHLs
are more common than brain tumors (Figure 8.1)
[3]. Each year, an estimated 160 000 cancers are
diagnosed in children worldwide, and an additional
240 000 cancers in adolescents and young adults (aged
1524 years) [1].
Descriptive epidemiology
Childhood cancer
According to the last edition of Cancer Incidence in
Five Continents, the highest cancer incidence rates,
for all tumor sites except skin, are observed in
Kuwait (non-Kuwaitis, 418/million), Italy (Brescia and
Naples, 250 and 234 new cases/million, respectively),
Brazil (Sao Paulo and Braslia, 224 and 219/million,
respectively), Switzerland (Vaud, 214/million), Croatia (203/million) and the USA (non-Hispanic white
population, 179/million) for males, while for females
high incidence is noted in Kuwait (non-Kuwaitis, 500
new cases/million), Brazil (Brasilia and Sao Paulo,
195 and 190/million, respectively), Cyprus (190/million), Italy (Salerno, 172/million), Germany (Munster, 168/million), Portugal (southern region, 160/million) and the USA (non-Hispanic white population,
155/million) (Figure 8.2a, b) [1]. On the other hand,
mortality rates are usually higher in medium-income
countries rather than in high-income countries, with
the highest rates (4 deaths/100 000 habitants per
year) observed in Ukraine, Republic of Moldova,
Romania, Cuba, Latvia, Russia, Estonia and Mexico for
both males and females (Figure 8.3a,b) [4].
In Europe, data from the Automated Childhood Cancer Information System (ACCIS) Project
83
Table 8.1 International Classification of Childhood Cancer, third edition
Group
Subgroup
Description
Ia
Ib
Ic
Id
Ie
Leukemias, myeloproliferative diseases and myelodisplastic diseases

Lymphoid leukemias
Acute myeloid leukemias
Chronic myeloproliferative diseases
Myleodisplastic syndrome and other myeloproliferative diseases
Unspecified and other specified leukemias
II
IIa
IIb
IIc
IId
IIe
Lymphomas and reticuloendothelial neoplasms

Hodgkins lymphomas
Non-Hodgkins lymphomas (except Burkitts lymphoma)
Burkitts lymphoma
Miscellaneous lymphoreticular neoplasms
Unspecified lymphomas
III
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
Central nervous system and miscellaneous intracranial and intraspinal neoplasms

Ependymomas and choroid plexus tumor
Astrocytomas
Intracranial and intraspinal embryonal tumors
Other gliomas
Other specified intracranial and intraspinal neoplasms
Unspecified intracranial and intraspinal neoplasms
IV
IVa
IVb
Neuroblastoma and other peripheral nervous cell tumors

Neuroblastoma and ganglioneuroblastoma
Other peripheral nervous cell tumors
Retinoblastoma
VI
VIa
VIb
Vic
Renal tumors
Nephroblastoma and other nonepithelial renal tumors
Renal carcinomas
Unspecified malignant renal tumors
VII
VIIa
VIIb
VIIc
Hepatic tumors
Hepatoblastoma
Hepatic carcinomas
Unspecified malignant hepatic tumors
IX
IXa
IXb
IXc
IXd
IXe
Soft tissue and other extraosseous sarcomas

Rhabdomyosarcomas
Fibrosarcomas, peripheral nerve sheath tumors and other fibrous neoplasms
Kaposis sarcoma
Other specified soft tissue sarcomas
Unspecified soft tissue sarcomas
Xa
Xb
Xc
Xd
Xe
Germ cell tumors, trophoblastic tumors and neoplasms of gonads

Intracranial and intraspinal germ cell tumors
Malignant extracranial and extragonadal germ cell tumors
Malignant gonadal germ cell tumors
Gonadal carcinomas
Other and unspecified malignant gonadal tumors
XI
XIa
XIb
XIb
XIc
XId
XIe
Other malignant epithelial neoplasms and malignant melanomas

Adrenocortical carcinomas
Thyroid carcinomas
Nasopharyngeal carcinomas
Malignant melanomas
Skin carcinomas
Other and unspecified carcinomas
XII
XIIa
XIIb
Other and unspecified malignant neoplasms

Other specified malignant tumors
Other unspecified malignant tumors
With permission from Steliarova-Foucher et al. [2].
84
Chapter 8: Cancer epidemiology and environmental factors
show an increase in overall incidence of childhood

cancer in all ages [5]. Mean age-standardized rates (per
million) were 118 in the 1970s, 124 in the 1980s and
139 in the 1990s, corresponding to an annual increase
of 1.0% during this period. Significant increases were
observed for leukemias (average annual percentage change [AAPC] = 0.7%, P 0.001), lymphomas
(AAPC = 1.3%, P 0.001), neuroblastomas (AAPC =
2.0%, P 0.001), soft tissue sarcomas (AAPC = 1.8%,
P 0.001), germ cell tumors (AAPC = 2.3%, P
0.001), renal tumors (AAPC = 1.1%, P = 0.017),
hepatic tumors (AAPC = 1.0%, P = 0.027) and bone
tumors (AAPC = 0.4%, P = 0.023). For CNS tumors
increases were higher in Eastern Europe (AAPC
= 2.5%, P 0.001) compared to Western Europe
35
30
More-developed
countries
Percentage
25
Less-developed
countries
20
15
10
5
0
Leukemias
CNS tumors Non-Hodgkins Renal

lymphomas tumors
Hodgkins
lymphomas
Figure 8.1 Childhood cancer types distribution in more

developed and less developed countries, 2002. CNS, central nervous
system. With permission from Ferlay et al. [3].
Figure 8.2 Populations with the highest

age-standardized incidence rate of childhood
cancer (014 years), 19982002. (a) All sites but
skin, males. (b) All sites but skin, females. ASR,
age-standardized annual incidence rate. With
permission from Curado et al. [1].
(a)
New Zealand
Cyprus
Brazil, Cuiab
USA, SEERNon-Hispanic White
Latvia
Italy, Veneto
Portugal, South
Colombia, Cali
Serbia
China, Guangzhou
Italy, Milan
Croatia
Brazil, Braslia
Switzerland, Vaud
Italy, Florence and Prato
Brazil, So Paulo
Italy, Umbria
Italy, Naples
Italy, Brescia
Kuwait: non-Kuwaitis
50
100
150
200
250
300
350
400
450
ASR (per million)

(b)
Germany,
France, Herault
Italy, Veneto
Serbia
Latvia
Belgium, Flanders
Spain, Murcia
Colombia, Cali
USA, SEER: Nonnon-Hispanic white
Croatia
Italy, Florence and
Portugal, South
Canada, Nova Scotia
Germany, Munster
China, Guangzhou
Italy, Salerno
Brazil, So Paulo
Brazil, Braslia
Cyprus
Kuwait: non-Kuwaitis
0
100
200
300
400
500
600
ASR (per million)
85
Figure 8.3 Age-standardized cancer

mortality rates in children (014 years) in
selected populations, 20002004. (a)
Males. (b) Females. ASR, age-standardized
annual incidence rate. With permission
from Ferlay [4].
(a)
Germany
Kyrgyzstan
Norway
Canada
USA
Austria
Singapore
South Africa
Japan
Tajikistan
Venezuela
Mexico
Russian
Albania
Latvia
Cuba
Moldova
Ukraine
Romania
Azerbaijan
ASR (per 100 000 persons)
(b)
Germany
Finland
The Netherlands
France
USA
Canada
Tajikistan
Austria
Japan
South Africa
Albania
Panama
Mexico
Cuba
Lithuania
Russian Federation
Moldova
Ukraine
Azerbaijan
Romania
ASR (per 100000 persons)
(AAPC = 0.8%, P 0.001), while for retinoblastoma

increases were observed only for children aged 1
year (AAPC = 1.1%, P = 0.018) [5]. In the USA, from
1992 to 2004, no significant increase was observed for
total childhood cancer incidence (19 years of age)
(AAPC = 0.4%; 95% CI, 0.1 to 0.8%). However, significant increases were observed for hepatoblastomas
(AAPC = 4.3%, 95% CI 0.28.7%) and melanomas
(AAPC = 2.8%, 95% CI 0.55.1%) [6].
86
Cancer in adolescents and young adults

Incidence rates in the 1524 year age group range
from 10.7 (Poona, India) to 47.2 (Queensland, Australia) per 100 000 persons for males and between
9.9 (Harbin, China) and 60.4 (Iceland) per 100 000
persons for females [1]. The populations with the
highest incidence rates for males and females are
reported in Figure 8.4a and 8.4b, respectively. A
Figure 8.4 Populations with the highest

age-standardized incidence rate of cancer in
adolescents and young adults (1524 years),
19982002. (a) All sites but skin, males. (b) All
sites but skin, females. ASR, age-standardized
annual incidence rate. With permission from
Curado et al. [1].
(a)
New Zealand
Cyprus
Brazil, Cuiab
USA, SEERnon-Hispanic white
Latvia
Italy, Veneto
Portugal, South
Colombia, Cali
Serbia
China, Guangzhou
Italy, Milan
Croatia
Brazil, Braslia
Switzerland, Vaud
Brazil, So Paulo
Italy, Umbria
Italy, Naples
Italy, Brescia
Kuwait: nonKuwaitis
50
100
150
200
250
300
350
400
450
ASR (per million)
(b)
Australia, Western
Germany, Munster
Canada, Ontario
Australia, New South Wales
France, Haut-Rhin
New Zealand
Israel
Belarus
Spain, Navarra
Australia, South
USA, SEERnon-Hispanic white
Canada, Manitoba
Italy, Romagna
France, Vendee
Italy, Salerno
Brazil, So Paulo
Australia, Queensland
Italy, Naples
Italy, Brescia
10
20
30
40
50
60
ASR (per million)
classification system for adolescents and young adults

with cancer has recently been developed [7] and is
summarized in Table 8.2. In a recent publication
from the US Surveillance Epidemiology and End
Results (SEER) program, analyzing all invasive cancers that occurred in the age group 1529 between
1975 and 2000, the most frequent tumor types were
lymphoma (20%), invasive skin cancer (15%), cancer
of the male genital system (11%) and cancer of the
endocrine glands (11%) [8]. As in children, high
mortality rates are experienced in medium-income
countries such as Ukraine, Costa Rica, Mexico,
Russia, Romania, Lithuania, Ecuador, Venezuela and
Cuba, with mortality rates 5.5 and 7.5/100 000
persons for males and females, respectively

(Figure 8.5a,b) [4].
Steliarova-Foucher et al. described an increase
in cancer incidence for adolescents (1519 years)
in Europe between 1970 and 1999, with remarkable
increases for carcinomas (AAPC = 3.9%, P 0.001),
lymphomas (AAPC = 2.4%, P 0.001) and germ
cell tumors (AAPC = 3.9%, P 0.001) [5]. Bleyer
et al., based on data from SEER for the period 1975
2001, has described an increase in incidence of cancer in adolescents and young adults in the USA for age
groups 1519 years, 2024 years and 2529 years [8].
Soft tissue sarcomas and lymphomas were responsible for most of the increase observed for males in the
87
Table 8.2 International Classification for Cancers in Adolescents and Young Adults
Group
Subgroup
Description
1.1
1.2
1.3
Leukemias
Acute lymphoid leukemia
Acute myeloid leukemia
Other and unspecified leukemia
2.1
2.2
Lymphomas
Non-Hodgkins lymphoma
Hodgkins disease
3.1
3.2
3.3
3.4
3.5
3.6
Central nervous system and other intracranial and intraspinal neoplasms

Astrocytoma
Other gliomas
Ependymoma
Medulloblastoma and other primitive neuroectodermal tumors
Other and unspecified malignant intracranial neoplasms
Non-malignant intracranial and intraspinal neoplasms
4.1
4.2
4.3
4.4
Osseous and chondromatous neoplasms, Ewing tumors and other neoplasms of bone
Osteosarcoma
Chondrosarcoma
Ewings tumor
Other specified and unspecified bone tumors
5.1
5.2
5.3
Soft tissue sarcoma

Fibromatous neoplasms
Rhabdomyosarcoma
Other soft tissue sarcomas
6.1
6.2
Germ cell and trophoblastic neoplasms

Gonadal germ cell and trophoblastic neoplasms
Germ cell and trophoblastic neoplasms of non-gonadal sites
7.1
7.2
Melanoma and skin carcinoma

Melanoma
Skin carcinoma
8.1
8.2
8.3
8.4
8.5
8.6
8.7
Carcinomas (except of skin)

Carcinoma of thyroid
Other carcinoma of head and neck
Carcinoma of trachea, bronchus, lung and pleura
Carcinoma of breast
Carcinoma of genito-urinary tract
Carcinoma of gastrointestinal tract
Carcinoma of other and ill-defined sites not elsewhere classified
9.1
9.2
Miscellaneous specified neoplasms

Embryonal tumors not elsewhere classified
Other rare miscellaneous specified neoplasms
10
Unspecified malignant neoplasms
With permission from Birch et al. [7].
age group 2529 years, suggesting that this finding can

be attributed to the emergence of acquired immune
deficiency syndrome (AIDS)-related Kaposis sarcoma
and NHLs [8].
Survival
Survival for children with cancer has continued to
improve over the past 20 years. In high-income coun-
88
tries, since the late 1990s, 5-year survival rates are close
to 80% [9].
In Europe, according to data from 83 populationbased cancer registries in 23 countries participating in the EUROCARE-4 project, 5-year survival
rates for all cancers combined was 81% for children and 87% for adolescents and young adults.
Among children under 15 years of age, very high survival rates were observed for retinoblastoma (97.5%),
Figure 8.5 Age-standardized cancer

mortality rates in adolescents and young
adults (1524 years) in selected
populations, 20002004. (a) Males. (b)
Females. ASR, age-standardized annual
incidence rate. With permission from
Ferlay [4].
(a)
Greece
Sweden
The Netherlands
France
USA
Canada
Austria
Germany
China, Hong Kong
Japan
Venezuela
Lithuania
Northern Ireland
Russian
Panama
Romania
Azerbaijan
Costa Rica
Mexico
Ukraine
Guatemala
10
12
ASR (per 100 000 persons)
(b)
Greece
Sweden
The Netherlands
France
USA
Canada
Austria
Germany
China, Hong Kong
Japan
Mexico
Panama
Venezuela
Ecuador
Cuba
Romania
Azerbaijan
Costa Rica
Russian Federation
Ukraine
Guatemala
10
ASR (per 100000 persons)
Hodgkins lymphoma (95.2%), Wilms tumor (89.1%)

and lymphoid leukemia (85.4%), while less favorable figures were observed for CNS nervous system
tumors (66.8%) and acute myeloid leukemias (66.8%)
[10]. In general, survival rates are lower in Central and Eastern Europe than in Western Europe. In
the USA, for children and adolescents (019 years)
diagnosed 19992005, registered into 17 SEER Cancer Registries, the 5-year relative survival rate for
all cancers was 79.3%, ranging from 59.6 (hepatic
tumors) to 97.0% (retinoblastoma) [11]. Contrasting with the finding for adults, in which cancer survival for European adults is lower than that observed
in USA, no systematic differences were observed
between these two regions regarding childhood cancer
survival [12].
Unfortunately, in spite of advances in diagnosis and treatment, survival rates are still poor
in low and middle-income countries, such as
India (Chennai, 40%) [13]. In several low-income
89
countries (Bangladesh, Philippines, Senegal, Tanzania

and Vietnam), 5-year overall survival is as low as
10% [14].
Risk factors: childhood cancer

Ionizing radiation
Intrauterine and postnatal exposure
Ionizing radiation is a known cause of cancer and other
adverse effects. It is one of the most extensively studied
human carcinogens and may account for about 3% of
all cancers [15]. Epidemiological studies have shown
an association between exposure to medical radiation
during pregnancy and risk of childhood cancer in offspring. The first association between exposure of pregnant women to abdominal diagnostic X-rays and pediatric cancer mortality in offspring was reported in
the Oxford Survey of Childhood Cancer (OSCC), a
nationwide case-control study including all children
(16 years), residing in England, Scotland and Wales,
who died from cancer during 195381 and matched
living controls [16]. The causal nature of the association has been discussed, since the evidence in favor
of an association derives almost exclusively from casecontrol studies, whereas practically all cohort studies
failed to replicate it [17]. However, the most recent
analysis of OSCC data confirmed that intrauterine Xray examination is associated with an increase of 40%
in the risk of childhood cancer [16]. Beginning at the
1970s, abdominal X-ray and pelvimetry were progressively replaced by obstetrical sonography and casecontrol studies carried out in UK [18], China [19],
USA and Canada [20] did not show evidence of an
increased risk of childhood cancer overall or leukemia
associated to this exposure.
The relation between exposure to diagnostic radiation during early childhood and the subsequent risk of
developing a pediatric cancer is less obvious [21]. Linet
et al. have recently reviewed the relevant evidence,
based on 22 studies [21]. Only one case-control study
from China reported an increased risk of all childhood cancers [19]. Among the five studies addressing the risk of acute lymphoblastic leukemia (ALL),
only one reported an association with exposure to
two or more diagnostic X-rays [22]. Concerning brain
tumors, an increased risk was observed in one study
for children exposed to diagnostic skull X-rays at least
5 years before diagnosis (OR = 6.7, 95% CI 1.627.3),
although reverse causality, i.e. the appearance of symp-
90
toms linked to the brain tumor led to a skull X-ray

could not be ruled out [23].
Preconception exposure
Linet et al. have called attention to the difficulties
in assessing results from studies evaluating the association between maternal preconception exposure to
diagnostic medical radiation and childhood cancer
[21]. According to these authors, the data are scarce
and difficult to interpret due to lack of standardization
of the time window of exposure, lack of emphasis on
key anatomic sites of exposure, absence of assessment
of the medical indication for the radiological examination and failure to adjust the results for therapeutic and
occupational radiation exposure [21]. The early results
of the OSCC indicated an association between abdominal X-ray exposures prior to marriage and childhood
cancer [24], but a subsequent analysis of data collected during the course of OSCC did not support
this finding (OR of all childhood cancer for preconception X-ray 1.1, 95% CI 0.91.2) [25]. Most of the
further studies also did not detect an association [19,
2630].
Extremely low-frequency electromagnetic

fields
Extremely low-frequency magnetic fields (ELF-MF)
has been classified by the International Agency for
Research on Cancer (IARC) as possibly carcinogenic
to humans mainly based on epidemiological evidence
derived from studies on childhood leukemia. The first
study assessing the association between exposure to
ELF-MF and childhood leukemia was published in
1979 [31], and results from more than 20 epidemiological studies focusing on this topic have been summarized in two distinct pooled analyses [32, 33]. Greenland et al. pooled original individual data from 12
studies, and summary estimates showed no association when comparing 0.10.2 and 0.20.3 microtesla
(T) categories with the 0.00.1 T category, but a
significant association was found for the comparison
between 0.3 T and 0.00.1 T (OR = 1.7, 95% CI
1.22.3) [33]. In the same year, Ahlbom et al. reported
results of an analysis including individual data from 9
studies published between 1993 and 1999, describing
that for residential magnetic field levels 0.4 T no
evidence of an increased risk of childhood leukemia
was found. On the other hand, residential exposures
0.4 T a year before diagnosis, conferred a twofold

higher risk of childhood leukemia [32].
In a recently published report, including data
from a case-control study carried out in Germany, no
increased risk of childhood cancer was associated with
preconception parental occupational exposure to ELFMF [34]. In addition, an exploratory meta-analysis
performed by the same authors, including other four
studies that evaluated the association between paternal occupational exposure to ELF-MF and childhood
leukemia, resulted in a pooled risk estimate of 1.35
(95% CI 0.951.91) [34].
Parental occupational exposure

Parental occupational exposure might be related to
cancer in children. Several mechanisms can be cited
on the explanation about how these exposures might
lead to an increased risk of cancer in the offspring.
These mechanisms comprises carcinogenic or mutagenic damage to either paternal or maternal germ
cells before pregnancy, injury to the fetus through
transplacental exposure during pregnancy and also
direct exposure of the children through contamination
of breast milk or parental clothes [35].
However, results from studies assessing these associations are somewhat non-homogeneous, mainly
because of poor assessment of exposures, low frequency of exposure, failure to assess exposure windows and multiple comparisons [36].
Occupations and corresponding exposures are
largely more studied for fathers than for mothers [36].
For childhood leukemias, positive associations with
paternal exposure to diverse chemical agents have
been found, including paints, solvents, pesticides and
working in motor-vehicle related jobs [37], but the
results are not consistent among studies.
Brain tumors have been linked to paternal exposure to paints and pesticides and employment in the
petroleum and chemical industries [37]. Exposure to
pesticides and organic chemicals during paternal hobbies has been associated with risk of brain tumor in
children [38].
Paternal exposures to hydrocarbons have been
associated with an increased risk of neuroblastoma in
some reports [39, 40]. Spitz and Johnson described an
increased risk of neuroblastoma associated with jobs
linked to moderate exposures to aromatic and aliphatic
hydrocarbons [40]. A large case-control study conducted by the Childrens Cancer Group and the Pedi-
atric Oncology Group has found significant increased

risks of neuroblastoma for fathers working as landscapers and groundskeepers (OR = 2.3, 95% CI 1.0
5.2), as well as for mothers employed as hairdressers
and barbers (OR = 2.8, 95% CI 1.26.3) [41]. In a
recent literature review, including 47 articles that have
evaluated risk factors for neuroblastoma, Heck et al.
concluded that there is suggestive evidence of positive
association with paternal exposure to volatile and nonvolatile hydrocarbons, wood dusts and solders [42].
On the other hand, findings from recent studies carried out in the UK did not support the role of parental
occupational exposures as important risk factor for
neuroblastoma [43], Wilms tumor [44] or retinoblastoma [45]. MacCarthy et al. assessed 32 paternal occupational exposure groups, among which only leather
was associated with neuroblastoma (OR = 5.0, 95%
CI 1.146.9) [43]. The same group of investigators
has conducted another case-control study to assess
the role of these exposures on the risk of retinoblastoma and have found an association only with paternal occupational exposure to oil mists in metal working (OR = 1.85, 95% CI 1.053.36) [45]. Furthermore,
data from the National Registry of Childhood Tumors
have served as a basis for a case-control study, including approximately 2500 pairs of cases and controls, in
which no associations between paternal occupational
exposures and Wilms tumor was found [44]. In conclusion, epidemiological studies linking occupational
parental exposure to childhood cancer suggest several
potential links, but for none the evidence can be considered strong.
The association between pesticides and childhood
cancer was first suggested after the publication of a
few case reports in which leukemias and other childhood tumors were reported following the utilization
of insecticides and herbicides in the home and yard
[4648]. An increased risk of childhood leukemia has
been found for fathers occupationally exposed to pesticides prior to and during pregnancy [49], as well as
for parental pesticide exposures at home or in gardens
[49].
Other types of childhood cancer including neuroblastoma, Wilms tumor, soft tissue sarcoma, Ewings
sarcoma, NHLs, CNS tumors, colorectal and testicular cancer have also been associated with exposure to
pesticides, based on evidence from case reports and
case-control studies. Although these studies are subject to several caveats, including non-specific pesticide exposure information, small numbers of exposed
91
subjects and potential for information bias, it is

remarkable that many of the reported increased risks
are of greater magnitude than those observed in studies with the adult population, suggesting that children
may be particularly susceptible to the carcinogenic
effects of pesticides [50].
A recent literature review about the association
between pesticides and childhood cancers, including 36 studies published between 1998 and 2006, has
concluded that definite conclusions cannot be drawn
from the existing evidence [51]. According to this
author, although several studies suggest associations
between pesticides and childhood cancers, there is
no consistency regarding tumor types and implied
agents, and estimates derived from case-control studies are usually higher than those obtained in cohort
studies, particularly concerning studies on childhood
leukemia.
Infections
It has been long proposed that infections might cause
childhood leukemia and NHLs, based on two nonmutually exclusive mechanisms. Greaves hypothesized
that a delayed exposure to common infections in
infancy and early childhood leads to an increased
risk of ALL [52], while Kinlen suggested that a large
influx of people into relatively isolated areas (population mixing) might facilitate the spread of a viral infection that occurs when infected and susceptible individuals come in contact with each other, leading to an
increased risk of childhood leukemia [53]. Although
isolated studies have suggested varicella [54], influenza
[55] and EpsteinBarr virus (EBV) [56] as possible etiological factors, no single agent has been compellingly
implied as a cause.
EpsteinBarr virus is linked to Burkitts lymphoma (BL), and this association is based on seroepidemiological studies and the identification of EBV
genomes in tumor specimens from individuals living
in endemic areas [57]. Endemic BL is predominantly
found among children living in equatorial Africa and
in Papua New Guinea, where it is strongly associated
with endemic malaria. In these areas, pediatric lymphomas account for up to 80% of all cancers in children. On the other hand, areas with low incidence of
BL have a much lower (30%) proportion of BL associated with EBV [58], while areas with an intermediate
pattern, like South America, show percentages ranging
between 25 and 70% [5962].
92
EpsteinBarr virus has also been also identified as

a causal agent for nasopharyngeal carcinoma (NPC).
The etiological link between NPC and EBV was
first proposed on the basis of serological evidence.
High antibody titres of immunoglobulin G (IgG) and
immunoglobulin A (IgA) against early antigen or viral
capsid antigen are frequently observed in individuals
with NPC, particularly those with the undifferentiated
tumors. This association was later confirmed by the
finding of EBV genomes in tumor cells [63].
Human immunodeficiency virus (HIV) infection
has also been associated with the development of
malignancies in children, although the occurrence
is less frequent than in adults. Non-Hodgkins lymphomas are the most frequent cancer in children with
AIDS, followed by leiomyomas and leiomyosarcomas,
which are also clearly associated with EBV infection
[64]. Kaposis sarcoma is rare in children [64] and its
etiology probably involves infection with human herpesvirus 8. Therefore, it has been suggested that the
risk for this cancer is higher among those HIV-positive
children who were born to mothers in high-risk groups
(heterosexual transmission via a bisexual partner) or
who became infected postnatally through contaminated blood [65, 66].
Lifestyle factors
The effects of maternal lifestyle during pregnancy on
embryonic and fetal development are well known and,
therefore, effects on the subsequent risk of cancer in
the offspring might be expected [57]. Several features
of maternal lifestyle during pregnancy have been studied regarding their association with childhood cancer, including diet, breastfeeding, smoking and alcohol consumption and the use of cosmetics. In addition,
paternal exposures during the preconception period
are also hypothesized as having effects on the risk
of germ cell mutations [57], and habits like tobacco
smoking and alcohol consumption have also been
assessed regarding the risk of childhood cancer.
Diet and breastfeeding

Parker conducted a non-systematic review of published studies on the association between leukemia/
lymphoma and breastfeeding and suggested that 25%
of the 500 annual cases of childhood acute leukemia
or lymphoma registered at the UK could be prevented if prevalence of breastfeeding was increased to
100% [67]. Results of an analysis conducted within the
context of the UK Childhood Cancer Study, including 3500 cases (1637 with leukemia) and 6964 controls,
have demonstrated a small protective effect for breastfed children, both for leukemia (OR = 0.89; 95% CI
0.801.00) and for all cancers combined (OR = 0.92;
95% CI 0.841.00) [68]. A first meta-analysis comprising 14 case-control studies (including 6835 ALL
cases and 1216 acute myeloid leukemia [AML] cases)
have shown a negative association between long-term
breastfeeding (6 months) and both ALL (OR = 0.76;
95% CI 0.680.84) and AML risk (OR = 0.85; 95%
CI 0.730.98) [69]. In 2005, Martin et al. published
another meta-analysis, including 26 publications (92%
were case-control studies), suggesting that breastfeeding was associated with a 9% risk reduction for ALL
(OR = 0.91; 95% CI 0.840.98), 24% for Hodgkins disease (OR = 0.76; 95% CI 0.600.97) and 41% for neuroblastoma (OR = 0.59; 95% CI 0.440.78) [70].
Results from the Northern California Childhood
Leukemia Study, including 282 case-control sets of
children, in which maternal diet (12 months before
pregnancy) was evaluated through a food-frequency
questionnaire, have shown that the consumption of
vegetables (OR = 0.65; 95% CI 0.500.84), fruits
(OR = 0.81; 95% CI 0.651.00), proteins (OR = 0.55;
95% CI 0.320.96) and legumes (OR = 0.75; 95% CI
0.590.95) were associated with a reduced risk of ALL
[71]. Another study from the same group have also
investigated the role of childs diet on leukemia risk
and authors have reported that the regular consumption of oranges/bananas (OR = 0.49; 95% CI 0.26
0.94) and orange juice (OR = 0.54; 95% CI 0.310.94)
during the first 2 years of life were both associated
with a reduction in risk of childhood leukemia [72].
In addition, a recent Taiwanese report has pointed out
the role of the childs consumption of cured/smoked
meat and fish on increasing the risk of acute childhood leukemia (OR = 1.74; 95% CI 1.152.64), while
it also has shown a protective effect linked to a frequent consumption of vegetables (OR = 0.55; 95% CI
0.370.83) and bean-curd food (OR = 0.55; 95% CI
0.340.89) [73].
Several studies have investigated the role of maternal diet during pregnancy as a risk or protective
factor in relation to pediatric CNS tumors, and the
most compelling and studied hypothesis concerns the
high risk of disease in the offspring of those mothers with a high intake of N-nitroso compounds and
precursors. Results from an international collaborative
case-control study have shown specific associations
between cured meat consumption and pilocytic astrocytomas (OR = 2.5; 95% CI 1.15.8) or ependymomas
(OR = 2.0, 95% CI 1.42.9), as well as negative associations between cruciferous vegetables and anaplastic astrocytomas (OR = 0.4; 95% CI 0.30.7) and also
fresh fish consumption and malignant gliomas (OR =
0.5; 95% CI 0.30.6). Oil products intake was associated with an increased risk of medulloblastoma (OR =
1.5; 95% CI 1.02.2) [74].
Parental tobacco smoking

Cigarette smoking has been shown to increase oxidative DNA damage in human sperm cells [75]. The
link between paternal cigarette smoking and childhood cancer has not been sufficiently evaluated, and
the results of the epidemiological studies have been
contradictory. Most of the studies on maternal smoking and childhood leukemia did not find a significant
positive association and some have shown a protective
association. On the other hand, studies assessing paternal smoking and childhood leukemia reported positive
associations more frequently [76].
In the UK, three studies that have analyzed data
from OSCC did not find a statistically significant association between maternal smoking and childhood cancer [7779]. However, significant positive associations
were found for the paternal smoking habit, with all
three studies showing increased risks for fathers who
were moderate or heavy smokers [7779]. A subsequent report from the Inter-Regional Epidemiological Study of Childhood Cancer (IRESCC) confirmed
the findings for paternal smoking, showing a significant positive trend between the risk of childhood cancer and paternal daily consumption of cigarettes before
the pregnancy [80].
A case-control study conducted by the UK Childhood Cancer Study (UKCCS) has found a statistically
significant association between hepatoblastoma and
maternal smoking (OR = 2.7) and also an increased
and stronger risk when both parents had a smoking
habit (OR = 4.7) [81]. This last finding was subsequently confirmed in a publication with data from the
OSCC, in which an increased risk of hepatoblastoma
when both parents were smokers was shown (OR =
2.7, 95% CI 1.26.1) [82].
There is no consistent evidence regarding a possible association between childhood brain tumors
and maternal smoking [57]. However, Preston-Martin
et al. have reported a positive association with mothers living in a household where someone else smoked
93
during the pregnancy of the index child [83]. Recently,

the findings from the ESCALE study provide additional evidence for a role of paternal smoking during the year prior to birth in childhood CNS tumors
(20 cigarettes/day, OR = 1.4, 95%CI 1.02.1) and
more markedly for astrocytomas (20 cigarettes/day,
OR = 3.2, 95% CI 1.29.1) and ependymomas (20
cigarettes/day, OR = 2.6, 95% CI 1.25.9) [84].
Concerning retinoblastoma, Bunin et al. did not
find a significant association between maternal (OR =
1.1, 95% CI 0.62.1) or paternal smoking (OR = 1.2,
95% CI 0.72.3) during pregnancy [85]. However, a
recently published record linkage study including the
New South Wales Central Cancer Registry and the
New South Wales Midwives Data Collection, in which
more than one million babies, born between 1994 and
2005, were matched to 948 cancer cases, has shown
that maternal smoking was significantly associated
with retinoblastoma (OR = 2.2, 95% CI 1.24.1) [86].
Parental alcohol consumption

The association between parental alcohol consumption and childhood cancer, in particular childhood
leukemias, has been assessed in several studies, but the
overall evidence is not conclusive. More recent studies,
however, have shown an increased risk of childhood
leukemia linked to maternal drinking during pregnancy. MacArthur et al. have reported that both
preconceptional (OR = 1.37; 95% CI 0.991.90) and
during pregnancy maternal drinking (OR = 1.39; 95%
CI 1.011.93) were associated with an increased risk
of childhood leukemia, with a dose-response effect
observed for increasing weekly consumption [87]. A
French population-based case-control study has found
an increased risk of ALL in the offspring of mothers
who reported a consumption of more than 1 drink/day
(OR = 2.8; 95% CI 1.85.9) [88], while other studies
have reported no association [89, 90]. Infante-Rivard
et al. have additionally investigated the interaction
between prenatal parental exposure and childrens
polymorphisms in the metabolizing genes GSTM1
and CYP2E1 [91]. Conversely to what was observed
in the French study, the authors have suggested that
alcohol consumption during pregnancy can have a
protective effect, particularly for those mothers who
drank wine (OR = 0.7, 95% CI 0.50.9). However,
exposure was associated with an increased risk of
disease for those mothers with GSTM1 genotype
(drinking any alcohol, second trimester, OR = 2.3,
94
95% CI 1.05.1; third trimester, OR = 2.4, 95% CI 1.1

5.4) [91].
Parental alcohol consumption has also been associated with other tumors like brain tumors, neuroblastomas, Wilms tumors, germ cell tumors, soft
tissue sarcomas, bone tumors, retinoblastomas and
hepatoblastomas [92]. Overall, there was no strong
evidence for a positive association. Ten of the thirtythree studies observed an increase in risk of childhood cancer associated to parental alcohol consumption. Seven studies reported associations with maternal consumption for the following tumors: leukemia
(five studies), brain tumor (two studies) and neuroblastoma (two studies). Conversely, a protective effect
was described in four studies. In addition, three studies reported a positive association with paternal alcohol consumption, whereas no study showed a protective effect for this exposure [92].
Recreational drugs
Parental illicit drugs use has been associated with several types of childhood cancer, including leukemia,
neuroblastoma, brain tumor and rhabdomyosarcoma.
A case-control study conducted by Robison et al.
reported that the maternal use of mind-altering drugs
(mainly marijuana) prior to or during the index pregnancy was associated to an increased risk of acute nonlymphoblastic leukemia [93]. Kuijten et al., using data
from a case-control study conducted in the USA in
the late 1980s, reported that maternal use of any recreational drug, and particularly marijuana, was associated with an increased risk of astrocytoma in the offspring (OR = 2.8, 95% CI 0.99.9) [94].
A case-control study carried out in the USA,
including 322 cases and 322 matched controls, has
shown that maternal use of marijuana during the year
before the child birth conferred a threefold higher risk
of rhabdomyosarcoma (RMS) (95% CI 1.46.5) [95].
Maternal use of cocaine was also associated with RMS
(OR = 5.1, 95% CI 1.025.0). Both paternal marijuana
smoking (OR = 2.0, 95% CI 1.33.3) and cocaine use
(OR = 2.1, 95% CI 0.94.9) were also associated with
an increased risk of this type of tumor [95].
More recently, findings form a study conducted in
the USA showed that maternal use of marijuana in
the first trimester may also increase the risk of their
child developing neuroblastoma (OR = 4.8, 95% CI
1.616.5) [96].
Zahm and Devesa have reiterated the difficulties needed to make an accurate assessment of these
exposures and the need for additional studies [97]. On

the other hand, these authors have emphasized the
need of continuing prevention efforts even in the absence of consistent evidence of association with childhood cancer, due to the other health problems already
definitely associated with the use of illicit drugs [97].
Overall, a role of lifestyle factors in the etiology
of childhood cancer is plausible, and the evidence is
stronger for paternal tobacco smoking and breastfeeding. For other aspects of lifestyle, notably diet, the
available data do not allow a conclusion at present.
Risk factors for the most

frequent cancers in adolescents
and young adults
Lymphoma, melanoma and testicular, cervical and
thyroid cancers account for the vast majority of cancers in this age group (65% of the total). Among
them, melanoma, cervical cancer and lymphoma are
the tumor types more strongly linked to environmental and lifestyle agents (such as ultraviolet light [UV],
human papilloma virus [HPV], HIV and EBV) [8].
Risk factors for lymphoma have been reviewed in the
previous section.
Melanoma
The etiology of melanomas is not fully understood,
although epidemiological studies have pointed out
several risk factors for developing the disease, including high solar exposure in early childhood, sunburns,
light hair and eye color, pale white skin and sunbed use
[98]. However, the major environmental risk factor for
melanoma is undoubtedly the exposure to UV radiation [98]. A meta-analysis published in 2005, including 57 studies from Europe, North America, Australia,
New Zealand, Argentina, Brazil and Israel, has found
a significant increase in the risk of melanoma associated with total sun exposure (RR = 1.34, 95% CI
1.021.77), with studies published after 1990 showing
a stronger effect (RR = 1.75, 95% CI 1.312.35). Intermittent sun exposure (RR = 1.61, 95% CI 1.311.99)
and sunburn history (RR = 2.03, 95% CI 1.732.37)
were also confirmed as significant risk factors for this
cancer [99].
Testicular cancer
Cryptorchidism is the best characterized risk factor
for testicular cancer. This association was first doc-
umented at the beginning of the nineteenth century

and the presence of this congenital abnormality confers an increase in the risk of testicular cancer of
211-fold. Risk is higher if cryptorchidism is bilateral
or if the condition is not treated before the age of 11
years. Other lifestyle and environmental factors have
been investigated but there is no evidence of a consistent increase in the risk of testicular cancer. In an
extensive review, Garner et al. have summarized the
epidemiological evidence, describing a possible etiological role for maternal characteristics and exposures
(endogenous estrogen level prenatal smoking), characteristics of the child (early height and decreased levels
of androgen), occupational exposures (pesticides) and
reproductive factors (age at puberty, age at pregnancy,
infertility) [100].
Cervical cancer
Infection with HPV is the main risk factor for cervical
cancer, and viral DNA is found in almost 100% of the
tumors. Human papilloma virus oncogenic types 16,
18, 31, 33 and 35 are the most associated with invasive
cervical carcinomas, with the types 16 and 18 causing
approximately 70% of the cancers worldwide. Human
papilloma virus is now recognized as the most common sexually transmitted infection in most populations. Infected women usually clear the infection
within 2 years, but those who become chronically
infected with high-risk HPV types are at greatest risk
for developing cervical cancer [101]. Female adolescents are one of the population groups with highest risk of becoming infected by HPV. It is estimated
that 75% of all new HPV infections are diagnosed
among those women aged 1524 years, with oncogenic HPV types accounting for nearly 50% of the
infections among adolescents. Recently, two vaccines
against HPV were developed: a quadrivalent (protecting against HPVs 6, 11, 16 and 18) and a bivalent vaccine (HPVs 16 and 18). Clinical trials have
shown almost 100% of efficacy of the vaccine in preventing high-grade, pre-cancerous cervical lesions,
caused by the HPV types prevented by the vaccination, for women nave to HPV types included in
the vaccine. Both vaccines are now licensed for use
in women more than 90 countries. Since 2006, the
quadrivalent vaccine is approved for use in the USA,
in girls aged 926 years, and the Advisory Committee
on Immunization Practices (ACIP) currently recommends that all 1112 year-old girls receive this vaccine
95
routinely, with a catch up use in women aged 13

26 years [102].
Thyroid cancer
The only established risk factor for thyroid cancer, particular for papillary carcinomas, is ionizing radiation.
Numerous reports have described the epidemiological aspects of thyroid cancer in areas associated with
nuclear plants, accidents and atomic bombs. Studies
that have assessed thyroid cancer incidence after the
explosion of atomic bombs in Hiroshima and Nagasaki
have demonstrated that the risk was highest among
individual aged 10 years at the time of exposure.
Moreover, a striking increase in thyroid cancer incidence among children was registered in areas surrounding Chernobyl, after the power plant accident
in 1986. In Belarus, the incidence rate previous to
the accident was 1 new case/million per year and it
has increased to more than 100 new cases/million per
year in the post-accident period. Diagnostic and therapeutic uses of radiation are also associated with an
increased risk of thyroid cancer, even if low doses are
applied [103].
Conclusions
Cancer is the second commonest cause of death, after
accidents, among children in high-income countries,
while in low and medium-income countries, improvements in the control of infectious diseases and perinatal mortality will lead to the emergence of cancer in
children as an important public health problem [57].
Incidence rates of childhood cancer have been increasing in the last decades in most countries. It is unclear
whether this reflects a real phenomenon or an artifact
due to better diagnosis and reporting, with improvement of the quality of cancer registries [5]. Current
knowledge on etiological factors remains limited, particularly for tumors other than leukemia and CNS neoplasms. Known causes of childhood cancer include
ionizing radiation, several infectious agents and possibly some behavioral factors; however, they explain
only a small proportion of the cases. Future collaborative studies should be conducted, aiming to have
enough power to test specific hypothesis as well as
to integrate knowledge from different areas, including
pediatric oncologists, epidemiologists and investigators working on basic research. Etiological hypotheses would be better tested in prospective studies, but
very large populations of newborn and children need
96
to be followed up to study a sufficiently large number

of cases. Recently, investigators involved in prospective studies of children have formed the International
Childhood Cancer Cohort Consortium (IC4), to promote large-scale collaboration between existing cohort
studies [104]. Incorporation of biomarkers of exposure and early effect and investigation of interactions
between environmental exposure and genetic predisposition factors represent additional avenues for future
studies on the etiology of childhood cancer.
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100
Section 3
Chapter
Reproductive biology and cryobiology
Life and death in the germ line

Apoptosis and the origins of DNA damage
in human spermatozoa
R. J. Aitken and B. J. Curry
Introduction: apoptosis and DNA

damage in human spermatozoa
Apoptosis, a physiological process for the controlled
deletion of cells, is critical for the regulation of cell
numbers, the management of morphogenesis during embryonic development and the orchestration of
many cellular processes in the adult. Spermatogenesis, the production of functional spermatozoa from
spermatogonial stem cells is no exception. It appears
that a functional apoptotic pathway is necessary for
normal spermatogenesis to develop and without it
infertility ensues. Apoptosis also plays a crucial role in
the maintenance of the testis and its response to external toxicants as well as in the programmed senescence
of terminally differentiated spermatozoa. This chapter
will focus specifically on how apoptosis affects sperm
quality and function, and the implications of this process for both embryonic development and the health
and well-being of the offspring.
A great deal of data has accumulated in recent years
suggesting that human spermatozoa can exhibit some
of the hallmarks of apoptosis including activation of
caspases 1, 3, 8 and 9 [1, 2], annexin-V binding [3, 4],
mitochondrial generation of reactive oxygen species
(ROS) [5] and DNA fragmentation [68]. The latter
is potentially extremely important because DNA damage in the male germ line has been associated with
a wide variety of adverse biological and clinical outcomes. Thus DNA damage in human spermatozoa has
been correlated with poor fertilization and impaired
embryonic development to the blastocyst stage [9, 10]
as well as with the incidence of subsequent miscarriage [1113]. Furthermore, even if such pregnancies
do carry to term, the presence of DNA damage in the

spermatozoa at the moment of conception has been
linked with developmental abnormalities in the offspring leading to a wide range of different pathologies,
including childhood cancer [12].
DNA damage in spermatozoa, assisted

reproductive technology and
embryonic development
This apparent association between DNA damage in
spermatozoa and the health and well-being of any
progeny is particularly significant in the context of
assisted reproductive technology (ART), which has
come to dominate the therapeutic landscape for infertile couples. Thus, the developed nations of the world
are currently seeing an exponential increase in the use
of ART to treat human infertility to such an extent
that 1 in 80 children born in the USA, 1 in 50 born
in Sweden, 1 in 40 born in Australia and 1 in 24 born
in Denmark are the product of this form of treatment.
In 2003, more than 100 000 in vitro fertilization (IVF)
cycles were reported from 399 clinics in the USA,
resulting in the birth of more than 48 000 babies [14,
15]. Worldwide, this figure has now exceeded 200 000
births per annum and is continuing to rise. This massive uptake of ART may herald a developing public
health crisis for at least two reasons. Firstly, human
infertility is a complex multifactorial condition that
is strongly impacted by genetic factors that ART will
ensure are passed onto the progeny [16]. As a consequence, it is a biological certainty that the more we use
assisted conception to treat one generation, the more
101
Section 3: Reproductive biology and cryobiology
we shall need it in the next. Secondly, in at least half of

the patients referred for ART, there is a problem with
the male partner. Since male subfertility is frequently
associated with high levels of DNA damage in spermatozoa, the use of ART invariably means that DNAdamaged cells will be used to achieve conceptions in
vitro that could never have occurred in vivo. This raises
genuine concerns over the safety of IVF procedures
should the DNA damage brought into the zygote by
the fertilizing spermatozoon subsequently lead to disordered embryonic growth and development [15].
We already know that the incidence of birth defects
following assisted conception is double that seen in
the naturally conceived population [17], and there is
good evidence for an increase in imprinting disorders, notably the BeckwithWiedemann and Angelmans syndromes, in such children [18, 19]. Infants
produced by ART are also significantly more likely to
be admitted to a neonatal intensive care unit, to be
hospitalized and to stay in hospital longer than their
naturally conceived counterparts [20]. Large studies of
Scandinavian populations using record linkage have
also shown an increase in the hospitalization of ART
offspring in infancy and early childhood compared
with spontaneously conceived children [2123]. Such
increases in morbidity cannot be completely explained
by multiple births because singletons are similarly
affected [23]. Furthermore, recent independent investigations have shown an eightfold increase in the incidence of undescended testicles in boys conceived by
intracytoplasmic sperm injection (ICSI) [24], while
another study has demonstrated abnormal retinal vascularization in such children [25]. Notwithstanding
the concerns raised in these publications, it must also
be stressed that other authors have been more reassuring about the normality of ICSI children, particularly in terms of their motor skills and cognitive development [2628]. Such a mixed picture for the safety
of ART arises from the literature for a number of
reasons:
1. The incidence of definable events such as overt
birth defects, hospitalizations or significant
pathologies such as cancer is low.
2. The study populations are generally small.
3. The number of confounding variables influencing
the health and well-being of children is great.
4. The duration of follow-up is short.
5. The instruments used for assessing defects in the
embryo (incidence of miscarriage, birth defects,
102
overt changes in the health and behavior of the

offspring) are insensitive.
If we assume that the genetic/epigenetic damage associated with ART is randomly distributed throughout
the genome then the chances that a specific gene will be
directly affected is low, given that 95% of the genome
is non-coding. It is even less likely that if a mutation
does occur in a gene, it will be dominant and cause a
detectable phenotypic or behavioral change in the F1
generation. Clinically, the incidence of even the more
common spontaneous dominant mutations, such as
achondroplasia, approximates to 1 in every 10 000
30 000 births in the population at large [29]. Thus, if
phenotypic change is the criterion, then most ART
studies are simply not adequately powered to determine whether this form of therapy has a significant
effect on the normality of the offspring. In this situation, the absence of evidence cannot be taken as evidence of the absence of problems with ART, and we
should proceed on the assumption that this form of
therapy carries significant risks that have to be quantified and addressed. The notion that DNA damage
in human spermatozoa constitutes part of this risk is
supported by clinical evidence generated around two
major paradigms: aging and smoking.
Age, DNA damage and disease in

the progeny
As men age, they do not stop producing spermatozoa. However, the quality of the gametes they do produce deteriorates, leading to a loss of fertility [30] and
increased levels of DNA damage in the spermatozoa
[31]. The latter has been consistently observed to correlate with age across a number of studies, using a
variety of different methods to detect the DNA strand
breaks. Thus Singh et al. employed a Comet assay to
demonstrate a significant correlation between age and
DNA damage in human spermatozoa [31]. In the same
vein, Schmid et al. found older men to have increased
alkali-labile sites or single-strand DNA breaks in their
spermatozoa using the same assay [32]. Others have
employed the sperm chromatin structure assay (SCSA)
to demonstrate a similar relationship between DNA
damage and male age [33]. Furthermore, both human
and rodent data have revealed age-related increases
in the number of spermatozoa with chromosomal
breaks and fragments [34]. A detailed analysis of chromosome 1 also found that the frequency of sperm
Chapter 9: Life and death in the germ line
carrying breaks, segmental duplications and deletions

was significantly higher among older men compared
with their younger counterparts [35]. In particular, the
frequency of spermatozoa carrying breaks within the
1q12 fragile-site region nearly doubled in older men.
In contrast to female gametes, there was no effect of
age on the frequency of sperm with numerical chromosomal abnormalities [35].
This abundant evidence for an effect of paternal
age on DNA damage in the germ line is important
because a fathers age appears to be a major determinant of the health and well-being of his offspring.
Indeed, for many decades we have been aware that
paternal age has a dramatic influence on the incidence
of spontaneous dominant genetic mutations such as
achondroplasia, Aperts syndrome (acrocephalosyndactyly) and multiple endocrine neoplasia. For example, an analysis of 154 consecutive cases of dominant genetic mutations to determine the parent of origin, revealed that in every single case the mutation
could be traced back to the male germ line, never the
female. Moreover, the appearance of these mutations
was found to be an exponential function of the age
of the father. Such mutations appear to be created by
replication error in the spermatogonial stem cell population, followed by clonal expansion of mutant germ
cells as they enter a selfish pathway of proliferation
[36, 37]. Of course, the genetic damage occurring in
human spermatozoa as a function of paternal age is
not only associated with dominant genetic mutations.
Age is also associated with an increase in the incidence
of complex polygenic neurological conditions in the
offspring including epilepsy, spontaneous schizophrenia, bipolar disease and autism [12, 3841] as well
as an increased rate of death in the F1 generation
associated with congenital malformations, injury and
poisoning [42].
It is clear from the foregoing, that paternal age is
associated with high rates of DNA damage in spermatozoa and that such age-dependent genetic damage
to the male gamete is, in turn, associated with a wide
range of clinical pathologies in the offspring, from
dominant genetic mutations to complex neurological disorders. This age-dependent DNA damage is
thought to be caused by oxidative stress originating
from a combination of diminished antioxidant protection and elevated levels of ROS generation by the spermatozoa [43, 44]. Oxidative stress also appears to be
at the heart of another factor known to increase DNA
damage in the male germ line, smoking.
Smoking, DNA damage and disease

in the progeny
Heavy paternal smoking is thought to place the body
under systemic oxidative attack causing a significant depletion of antioxidant vitamins such as vitamin C and E [4547]. One of the consequences of
the resultant stress is thought to be DNA fragmentation and formation of the oxidized base adduct
8-hydroxy-2-deoxyguanosine (8OHdG) in the spermatozoa [46]. In addition, benzo[a]pyrenes derived
from cigarette smoke can form adducts with sperm
DNA, once metabolically activated by the cytochrome
P450 system [48]. The clinical significance of these
sperm DNA adducts can be found in the positive correlations that have been repeatedly observed between
paternal smoking and serious morbidity in the offspring, including childhood cancer [4953].
Is DNA damage related to

apoptosis in the germ line?
In summary, the above clinical data provide circumstantial evidence that DNA fragmentation in human
spermatozoa is correlated with adverse clinical outcomes ranging from miscarriage to cancer. These conclusions are also supported by animal data indicating that the experimental induction of oxidative DNA
damage in spermatozoa with cigarette smoke, alcohol
or the experimental deletion of antioxidant enzymes,
such as glutathione peroxidase 5, can subsequently
lead to disruptions of embryonic growth resulting in
high rates of abortion and the appearance of birth
defects [5456]. Given that these associations between
sperm DNA damage and abnormalities of embryonic
development clearly exist, it is now critical that we gain
an understanding of how this DNA damage is generated in human spermatozoa and, in the context of this
review, the role of apoptosis in the prosecution of this
process.
Nature of the DNA damage

In order to understand the origins of DNA damage in
the male germ line we first have to understand its precise nature. A small number of studies have actually
examined the damaged DNA from human spermatozoa in the search for clues as to its origins. These studies have revealed that the major base adduct present in
human spermatozoa is 8OHdG, a marker of oxidative
103
Figure 9.1 The powerful correlation

observed by De Iuliis et al. [57] between
DNA fragmentation in human
spermatozoa and oxidative DNA base
damage as indicated by expression of
8-hydroxy-2-deoxyguanosine (8OHdG).
25.0
P < 0.001
22.5
20.0
NH2
Oxidation
O
H
N
TUNEL (%)
NH
17.5
15.0
12.5
10.0
NH
7.5
HO
N
NH2
8-hydroxy,2deoxyguanosine
(8OHdG)
5.0
2.5
0.0
10
15
20
25
30
35
8OHdG (%)
stress. The levels of 8OHdG expression in human

spermatozoa are consistently found to be elevated in
the spermatozoa of infertile men [57, 58]. Moreover,
the presence of this oxidized base adduct has been
found to exhibit an extremely strong correlation with
both DNA damage as measured by the TUNEL assay
and chromatin protamination as assessed by chromomycin (CMA3) staining [57]. Indeed, the correlation between TUNEL and 8OHdG formation is so
strong that we have been forced to conclude that a
majority of DNA damage in the male germ line is
oxidatively induced (Figure 9.1 [57]).
Besides 8OHdG, biochemical analyses of DNA
from infertility patients have also revealed the presence of two ethenonucleosides: 1,N6-ethenoadenosine
and 1,N2-ethenoguanosine. These compounds probably arise from a reaction with 4-hydroxy-2-nonenal,
the main aldehyde released during lipid peroxidation
[59]. These findings are again consistent with oxidative stress being a major factor in the etiology of DNA
damage in the male germ line.
In another study, uncharacterized bulky DNA
adducts were found to be significantly more common
in the spermatozoa of male factor infertility patients
than in a cohort of healthy donors [60]. Furthermore, a
significant negative correlation was observed between
these bulky DNA adducts and sperm concentration
and sperm motility, among patients with impaired
104
fertility [60]. In a further study, polycyclic aromatic

hydrocarbonDNA adducts were found to be more
prevalent in infertile versus fertile men [61]. While the
origin of some of the DNA adducts detected in human
spermatozoa are clear, as in the case of smoking [48],
we clearly still have a lot to learn about the causes of
DNA damage in the germ line. The one thing that does
appear to be certain is that a significant proportion of
the spontaneous DNA damage seen in male infertility
patients is oxidative in nature.
The link between DNA oxidation (8OHdG formation) and DNA strand breakage could be interpreted in
one of two ways. First, the link may be causative. That
is, the formation of oxidative base adducts disrupts
DNA integrity by labilizing the glycosyl bond that
attaches the base to the ribose unit with the resultant
generation of an abasic site. Abasic sites have a strong
destabilizing effect on the DNA backbone which can
subsequently result in strand breaks. Alternatively the
relationship may be indirect. Oxidative base damage
and DNA fragmentation may simply be independent
witnesses to the same fundamental underlying process the ability of spermatozoa to undergo apoptosis. Under these circumstances the DNA strand breakage could be linked to endonucleases activated during
an apoptotic cascade that was triggered by oxidative
stress. Such considerations have encouraged us to consider the process of apoptosis in the male germ line as
these cells differentiate from spermatogonial stem cells

to fully formed spermatozoa.
Ability of the germ line to

undergo apoptosis
The ability of germ cells to undergo apoptosis is
expressed very early in life when the testes are differentiating and adjustments have to be made to achieve
the optimal ratio of germ cells to Sertoli cells. During
this developmental process, excess pre-meiotic spermatogonia are removed by an early wave of apoptosis
that accompanies the first round of spermatogenesis
in the testis. Functional deletion of the pro-apoptotic
protein, Bax, or over-expression of anti-apoptotic factors such as BclxL or Bcl2, generates a male infertility phenotype associated with a perturbed germ cell to
Sertoli cell ratio [62, 63]. This phenotype shows disordered seminiferous tubules filled with spermatogonia,
but no mature haploid sperm, while other tubules lack
any germ cells whatsoever. Later in life, p53 and Fas
are involved in the removal of germ cells that are damaged as a result of exposure to environmental toxicants
or chemotherapeutic agents [64]. A role for apoptosis
in the etiology of spontaneous male infertility has also
been suggested by virtue of the excessively high numbers of apoptotic germ cells observed in the testes of
some infertile males [65]. It has also been suggested
that the DNA damage that features so prominently in
human spermatozoa is the result of an abortive apoptotic process that was initiated during spermatogenesis but failed to run to completion because the extensive remodeling of germ cells to produce spermatozoa,
removes the intracellular machinery needed to effect
cell death [66].
Significance of
spermiogenesis in the
etiology of DNA damage
Spermiogenesis, the process by which round spermatids differentiate into spermatozoa, is a key event
in the etiology of DNA damage in the male germ line.
As indicated above, it is possible that some of the
DNA breaks seen in spermatozoa are the result of an
abortive apoptotic process initiated during spermiogenesis in response to some disruptive event. Equally,
DNA fragmentation in spermatozoa may also be
the result of unresolved strand breaks created dur-
ing the normal process of spermiogenesis in order

to relieve the torsional stresses involved in packaging a very large amount of DNA into a very small
sperm head. Normally, these physiological strand
breaks are corrected by a complex process involving
H2Ax expression, formation of poly(ADP-ribose) by
nuclear poly(ADP-ribose) polymerases and topoisomerase [67]. However, if spermiogenesis should be disrupted for some reason then the restoration of these
cleavage sites might be impaired and the spermatozoa,
lacking any capacity for DNA repair in their own right,
will be released from the germinal epithelium still carrying the unresolved strand breaks.
Experimentally, if physiological strand break
repair during spermiogenesis is impaired then spermatozoa are generated that exhibit high levels of
DNA fragmentation. For example, it is known that the
chromatin remodeling steps associated with spermiogenesis trigger poly(ADP-ribose) (PAR) formation, as
an early event in DNA repair. Knockout mice deficient
in enzymes involved in PAR metabolism (PARP1
[poly(ADP-ribose) polymerase]; PARG [poly(ADPribose) glycohydrolase, 110-kDa isoform]; or both
display DNA strand breaks associated with varying
degrees of subfertility [67]. Similarly the transition
proteins that move into the sperm nucleus during
spermiogenesis, between the removal of histones and
the entry of protamines, are thought to play a key
role in maintaining DNA integrity. If these proteins
are functionally deleted then spermatozoa are generated with poor fertilizing potential, poor chromatin
compaction and high levels of DNA fragmentation
[68]. Such studies clearly indicate that the functional
disruption of chromatin repair mechanisms operative
during spermiogenesis can result in the genesis of
spermatozoa carrying high levels of DNA fragmentation. However, even though disruption of DNA repair
mechanisms during spermiogenesis can result in
DNA-damaged spermatozoa, this does not necessarily
mean that such mechanisms are actually operative in
a clinical context.
Using the fluorescent probe CMA3 to monitor
the efficiency of sperm protamination, many independent laboratories have generated data on the excellent
correlations observed between DNA fragmentation
in the one hand and poor chromatin remodeling
during spermiogenesis on the other (Figure 9.2ac).
This proposed link between defective spermiogenesis and DNA damage is further supported by the
fact that several independent studies have recorded
105
(a)
Figure 9.2 The close relationship

between the efficiency of chromatin
remodeling, as monitored by
chromomycin (CMA3) fluorescence and
DNA damage in spermatozoa: (a)
correlation between CMA3 and
8-hydroxy-2-deoxyguanosine (8OHdG);
(b) correlation between CMA3 and DNA
fragmentation as measured by the TUNEL
assay; (c) image of the TUNEL signals
generated by human spermatozoa
possessing damaged DNA (arrowed).
(b)
30
P < 0.001
50
P < 0.001
CMA3 (%)
25
8-OH-dG (%)
40
20
15
30
10
20
0
0
10
10
15
20
25
30
35
TUNEL (%)
(c)
0
TUNE L
0
10
15
20
CMA3 (%)
correlations between DNA damage in human spermatozoa and elements of the conventional semen profile (specifically sperm count and morphology) that,
in turn, reflect the efficiency of the spermatogenic
process.
Not only is the disruption of spermiogenesis correlated with DNA damage; it is specifically correlated
with oxidative DNA damage as reflected by 8OHdG
formation (Figure 9.2). We postulate that this relationship exists because the poorly remodeled chromatin detected by CMA3 is particularly vulnerable to
oxidative attack by ROS originating from a number
of potential sources including infiltrating leukocytes,
depleted antioxidant systems and excessive free radical
generation by the spermatozoas own mitochondria.
We further propose that of all these potential sources,
the sperm mitochondria are the most important [5].
Experimental conditions associated with the induction of high levels of oxidative DNA damage, such as
exposure to radio frequency electromagnetic radiation
(RFEMR) [9] or the triggering of apoptosis through
the suppression of PI3 kinase with wortmannin [R. J.
Aitken, unpublished observations] invariably involve
the release of considerable amounts of ROS from the
sperm mitochondria.
106
Importance of chromatin remodeling

and oxidative stress
In light of the above considerations, DNA damage in
human spermatozoa appears to have its origins in the
testes and is associated with oxidative stress. We might
interpret these data in two different ways:
1. Impaired chromatin compaction and oxidative
DNA damage are independent events. It is possible
that a variety of different factors are primarily
responsible for impairing the quality of
spermiogenesis (for example endocrine
disruption, environmental toxicants, exposure to
electromagnetic radiation or genetic mutations).
The result of this disrupted spermiogenetic
process is the production of spermatozoa with
poorly remodeled chromatin that, because of the
lack of DNA compaction, are particularly
vulnerable to oxidative stress and the induction of
8OHdG formation. Under these circumstances
the oxidative stress could come from the variety of
sources mentioned above, including impaired
antioxidant defenses or the enhanced generation
of free radicals by either the spermatozoa
themselves or leukocytes in the immediate

vicinity of these cells.
2. Impaired chromatin compaction and oxidative
DNA damage have a common origin in oxidative
stress. We have already cited evidence that DNA
damage in spermatozoa is largely oxidatively
induced. Whether spermiogenesis can also be
adversely affected by oxidative stress is still a
matter of debate. In male toxicology models
involving, for example, the administration of
streptozotocin [69], bacterial lipopolysaccharide
[70], deltamethrin [71], methyl-parathion [72],
aroclor 1254 [73], cyclophosphamide [74] or
formaldehyde [75], oxidative stress is associated
with the disruption of spermatogenesis via
mechanisms that can be reversed by the
administration of antioxidants such as lipoic acid
[76], quercetin [69], Satureja khuzestanica
essential oil [74] and melatonin [75]. In some of
these experiments, the administration of an
antioxidant both improved testicular function and
reduced DNA damage in spermatozoa [74],
suggesting that oxidative stress can underpin both
the disruption of spermatogenesis and the
induction of DNA damage in spermatozoa. In
addition, the induction of oxidative stress with
methyl parathion has been shown to specifically
affect chromatin remodeling during
spermiogenesis and induce DNA damage in
spermatozoa [77]. Such results encourage
speculation that oxidative stress is a major
determinant of the efficacy of spermiogenesis
which, when it becomes disrupted, results in the
generation of spermatozoa that are vulnerable to
oxidative stress, 8OHdG formation and,
ultimately, DNA fragmentation (Figure 9.3
[7880]). A powerful demonstration of the
validity of this hypothesis has been provided by
Zubkova and Robaire [81]. These authors induced
systemic oxidative stress through the
administration of the glutathione-depleting drug
l-buthionine-[S,R]-sulphoximine (BSO). In
response to the oxidative stress so generated, the
rats exhibited evidence of both impaired
spermiogenesis, as evidenced by an increase in
CMA3 staining of the sperm chromatin, and
increased DNA fragmentation.
Thus, oxidative stress can impair spermiogenesis
and this, in turn, precipitates DNA fragmen-
tation via a number of potential mechanisms

(Figure 9.3):
1. Physiological DNA strand breaks introduced to
facilitate DNA folding during spermiogenesis are
left unresolved and persist in the mature gamete.
2. The defective spermatozoa generated as a result of
disrupted spermiogenesis possess poorly
protaminated chromatin that is vulnerable to free
radical attack generating 8OHdG adducts, abasic
sites and, ultimately, DNA fragmentation.
3. The defective spermatozoa, generated as a result
of disrupted spermiogenesis, respond to oxidative
stress by entering a default apoptotic pathway that
results in endonuclease-mediated DNA
cleavage.
Importance of apoptosis in spermatozoa

There is no doubt that spermatozoa can exhibit many
of the features of apoptosis and there is good evidence
that oxidative stress can trigger apoptosis in these cells.
Thus, exposure of human spermatozoa to hydrogen
peroxide (H2 O2 ) can readily trigger an apoptotic cascade characterized by the activation of caspase 3 and
the appearance of annexin-V binding positivity [82].
We have also demonstrated that the activation of an
apoptotic cascade following H2 O2 exposure results in
the induction of mitochondrial free radical generation
[A. Koppers and R. J. Aitken, unpublished observations]. The activation of mitochondrial ROS generation, in turn, induces the formation of 8OHdG adducts
in human sperm chromatin. We have observed this
cascade of cause-and-effect involving oxidative stress,
the activation of mitochondrial ROS generation and
the induction of oxidized base adduct formation following: (1) the direct addition of oxidant (H2 O2 ) to
spermatozoa; (2) the indirect creation of oxidative
stress with RFEMR [83]; and (3) the activation of
apoptosis using the PI3 kinase inhibitor, wortmannin [A. Koppers and R. J. Aitken, unpublished observations]. We have also pursued this apoptotic cascade to determine whether the activation of caspases,
mitochondrial ROS generation and phosphatidylserine (PS) externalization is followed by the activation of
endonucleases that then move into the sperm nucleus
to induce DNA fragmentation.
Surprisingly, this analysis revealed that apoptosis in spermatozoa differs in one important respect
from this process in somatic cells, in that even though
endonucleases may be released from the mitochondria
107
RFEMR
PUFA
Infection
Loss of antioxidants
Heat
Oxidative stress
in testes
Testicular torsion
Toxicants
Disrupted
spermiogenesis
DNA strand breaks created

during spermiogenesis are
not resolved
Sperm DNA not adequately

compacted: vulnerable to
oxidative stress
Defective spermatozoa
become senescent in male
tract and undergo
apoptosis
Apoptosis
Mitochondrial ROS
DNA undergoes oxidative attack

Generates 8OHdG
Topoisomerase
Senescent cells activate endogenous nucleases
Cell death
Extensive DNA fragmentation
Figure 9.3 Hypothesis for the creation of DNA damage in human spermatozoa. This hypothesis posits that a variety of different clinical,
genetic and environmental factors can induce oxidative stress in the testis. One of the processes affected by oxidative stress is the
differentiation of spermatozoa during spermiogenesis. As a result of disrupted spermiogenesis DNA damage will occur in the spermatozoa via
at least three potential routes: (1) Physiological, topoisomerase-mediated strand breaks that occur during spermiogenesis to relieve the
torsional stresses associated with DNA packaging are not resolved and persist in the mature gamete. (2) Defective spermiogenesis generates
spermatozoa possessing poorly compacted, inadequately protaminated DNA that is vulnerable to free radical attack originating from a variety
of sources including depleted antioxidant protection, leukocytic infiltration, redox-cycling xenobiotics and ROS generation by the
spermatozoa themselves. (3) Defective human spermatozoa respond to their imperfect state by prematurely engaging in a process of
programmed senescence equivalent to apoptosis. During apoptosis caspases are activated, the spermatozoa lose their motility,
phosphatidylserine (PS) externalization occurs and the mitochondria start to generate ROS. As a consequence of routes (2) and (3) the DNA is
oxidatively attacked, generating the base adduct, 8-hydroxy-2-deoxyguanosine (8OHdG), which ultimately leads to the creation of abasic
sites and DNA strand breakage. In addition, we propose that as cells enter the terminal stages of senescence, topoisomerase, possibly acting
in concert with endogenous endonucleases [7880], completes the final destruction of the DNA.
(endonuclease G, apoptosis inducing factor) or activated in the cytosol (caspase-activated DNase) during the apoptotic cascade, the physical architecture of
these cells prevents these nucleases from translocating
to the sperm nucleus for two reasons:
108
1. The chromatin is so densely packed that proteins

cannot penetrate into its internal structure.
2. The mitochondria and a majority of the cytoplasm
are located in the sperm midpiece while the
chromatin is located in the sperm head.
Thus, these enzymatic mediators of apoptotic DNA

cleavage cannot be directly involved in the cleavage
of sperm DNA. Rather, apoptosis is associated with
mitochondrial ROS and 8OHdG formation which secondarily induces the non-enzymatic fragmentation of
DNA following the creation of abasic sites. The only
other possibility is that, in order to compensate for
the inability of endonucleases to move into the sperm
chromatin during apoptosis, spermatozoa are constructed with an endonuclease already integrated into
the body of the chromatin, which only has to be activated for DNA cleavage to commence. In this context Sotolongo et al. and Shaman et al. have described
endogenous nucleases that would fit these criteria [78,
79]. These authors envisage that this nuclease activity is initiated by topoisomerase IIB which becomes
activated by divalent cations such as calcium or manganese [80]. The result of this topoisomerase activity is
to cut the inter-toroid regions of sperm DNA to yield
50 kb looped fragments. An uncharacterized second
nuclease activity is then activated that further degrades
the DNA in a process known as sperm DNA degradation (SDD). It is possible that these endonuclease
activities are involved in the final stages of apoptosis
when cell viability is being lost and the plasma membrane is no longer in a condition to exclude divalent
cations such as calcium from the cells interior (Figure
9.3). This observation would be in keeping with our
recent observation that a vast majority of TUNEL positive cells in human sperm suspensions are, in fact, dead
[84].
Lives in the balance: apoptosis

and pro-survival factors
In light of the foregoing discussion, we hypothesize
that oxidative stress impairs the progress of spermiogenesis resulting in the generation of spermatozoa
that may not only carry unresolved physiological
DNA strand breaks acquired during early spermiogenesis, but also possess poorly compacted chromatin
that is vulnerable to free radical attack. Such an attack
could come from many quarters including infiltrating
leukocytes, a lack of antioxidant protection or simply very prolonged periods of storage, i.e abstinence.
It is also possible that spermatozoa created as a result
of impaired spermiogenesis are more likely to enter
an intrinsic apoptotic pathway in response to stress
(Figure 9.3).
As far as we are aware, there are no specific

chemical triggers for apoptosis in human spermatozoa. Rather entry into this pathway appears be triggered by cell senescence, which normally occurs in
the female, not the male, reproductive tract. Following insemination, the female tract responds to the
presence of millions of dead or moribund spermatozoa by triggering a massive leukocyte infiltration
into the lower reproductive tract post coitum [85]. The
phagocytic activity exhibited by these cells must be
silent; in other words, the spermatozoa must be efficiently phagocytozed and removed from the tract in
the absence of an oxidative burst or the production of
pro-inflammatory cytokines. There are many examples
of silent phagocytosis in biology, and a common feature of this phenomenon is the expression of apoptotic
markers, such as PS, on the surface of the phagocytozed cell. This apoptotic marker is thought to instruct
the phagocyte that the target cell should be engulfed
in a non-phlogistic manner [86]. We therefore propose that the activation of this apoptotic cascade in
senescent cells is an adaptation that permits the efficient removal of spermatozoa from the female tract by
phagocytic leukocytes without provoking an inflammatory response. Viewed in this context, the DNA
damage we see in the spermatozoa of male infertility
patients with defective spermiogenesis could represent
their premature entry into a pathway of programmed
senescence involving activation of the intrinsic apoptotic cascade that should have occurred in the female
tract. This hypothesis may explain why male infertility is so frequently accompanied by subclinical levels
of leukocyte infiltration [87].
If entry into this truncated intrinsic apoptotic cascade is the default pathway for spermatozoa, we might
ask what normally prevents them from becoming
senescent. The answer to this question is that for the
kind of prolonged survival that these cells exhibit in
vivo, where they may have to spend up to a week in
the epididymis waiting for ejaculation, followed by
another week in the female tract waiting for an egg to
arrive, exposure to pro-survival factors is critical. The
latter are thought to maintain cell viability by driving
a signal transduction pathway, which has PI3 kinase
and Akt at its head. As long as these enzymes are
phosphorylated they can, in turn, phosphorylate a key
constituent of the apoptotic cascade called BAD. Phosphorylated BAD is bound to a regulatory protein
called 14-3-3 and in this state cannot interact with
the cells mitochondria. However, if the cell should
109
become senescent for any reason then a reduction in

PI3 kinase/Akt phosphorylation occurs which leads to
the dephosphorylation of BAD. The latter then escapes
from the embrace of its 14-3-3 regulator and moves
rapidly to the mitochondria where it triggers pore formation, cytochrome C release and the initiation of
apoptosis. If this is the case, then what are the prosurvival factors that normally keep spermatozoa from
entering this default apoptotic pathway? The study of
such factors is still in its infancy but one candidate
to emerge from recent studies is prolactin. Spermatozoa possess several variants of the prolactin receptor
(including their own unique isoform) and respond to
the presence of this hormone with the stimulation of
PI3 kinase/Akt phosphorylation and the prolongation
of sperm survival [88].
Conclusions
DNA damage in human spermatozoa has been correlated with a range of adverse clinical outcomes including subfertility, miscarriage and morbidity in the offspring. A majority of this DNA damage is oxidative
and is correlated with the disruption of spermiogenesis and the generation of spermatozoa with poorly
compacted chromatin. We hypothesize that the disruption of spermiogenesis is the result of oxidative
stress, which can be induced by a wide range of factors including age, exposure to electromagnetic radiation in the form of heat or RFEMR, smoking, alcohol, insecticides, herbicides and heavy metals (Figure
9.3). The poorly protaminated spermatozoa generated
as a consequence of defective spermiogenesis may not
only carry unresolved physiological strand breaks
but are also vulnerable to oxidative attack. We further
hypothesize that this attack originates largely from the
generation of ROS by defective spermatozoa as they
prematurely enter a default pathway of programmed
senescence characterized by a truncated apoptotic cascade featuring ROS generation, PS externalization and
caspase activation. However, the endonucleases that
translocate to the nucleus during somatic cell apoptosis are prevented from doing so in spermatozoa by
virtue of the unique architecture of these cells. As
a result, apoptosis in spermatozoa initially generates
oxidative DNA damage followed by non-enzymatic
DNA fragmentation. Enzymatic DNA cleavage may
subsequently be induced by topoisomerase and other
uncharacterized nucleases that are bound up in the
structure of sperm chromatin. We propose that the
110
activation of these enzymes occurs close to cell death

or even post mortem in order to ensure the complete
destruction of the cell (Figure 9.3). These concepts are
not only novel but highly relevant to the safety of ART
and the diagnosis, treatment and prevention of male
factor infertility.
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Kobayashi Y. Silent cleanup of very early apoptotic
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87. Ricci G, Perticarari S, Fragonas E et al. Apoptosis in
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88. Pujianto DA, Curry BJ and Aitken RJ. Prolactin
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113
Section 3
Chapter
10
Molecular aspects of follicular development

Zhongwei Huang and Dagan Wells
Introduction
The follicle has a fundamental reproductive role in the
ovary. It consists of the oocyte (in various stages of
growth and development) and its surrounding layers
of supportive cells, the granulosa cells (GCs), cumulus
cells (CCs) (during the antral phase of follicular development) and thecal cells (TCs). These somatic cells
perform essential roles in ensuring optimal development and maturation of the oocyte. They receive signals from the external environment and pass them on
to the oocyte to orchestrate its growth and development until ovulation.
It is now widely recognized that there is bidirectional communication between the oocyte and
surrounding somatic cells and that this is essential
for the creation of a favorable follicular microenvironment in which the gamete can develop. Many complex and intricate molecular pathways are likely to be
involved in the production of an optimal follicle and a
viable gamete. This chapter aims to provide an insight
into the molecular mechanisms likely to be involved in
the various stages of follicular development, as determined from animal and human studies.
The follicle develops in stages

During fetal life, the human ovary is populated by
7 106 oogonia as early as the 4th month of pregnancy with the highest mitotic activity seen just before
meiosis commences [1]. These oogonia, present during the early second trimester, ultimately give rise to
approximately 1 million primordial follicles at birth.
The number of follicles continues to decline during
childhood reaching 300 000 at menarche [2].
Meiosis is initiated in the human fetal ovary at
1112 weeks of gestation [3] when the oocytes enter
prophase and go through synapsis with the exchange
of genetic material between paired homologues. On

completion of recombination, the oocyte progresses to
diplotene of prophase I and enters a protracted arrest
stage known as dictyate. At this stage such oocytes are
known as primary oocytes and are surrounded by a
single layer of flattened pre-granulosa cells. These primary oocytes and their associated cells make up the
pool of primordial follicles, which a woman will utilize during the course of her reproductive life span. Primordial follicles remain quiescent for years until they
are recruited to undergo further development during sexual maturation under the influence of pituitary
gonadotrophin, follicle stimulating hormone (FSH)
and luteinizing hormone (LH).
The pre-granulosa cells will continue to undergo
cyto-differentiation and proliferation to support the
oocyte during its early growth through the transition from the primordial to pre-antral follicular phase.
During this transition, the morphology of the GCs
changes from squamous to cuboidal type, as observed
in the murine ovary [4]. The regulation of GC cytodifferentiation requires the actions of a number of hormones and growth factors [5] (discussed below).
Contact between GCs and the oocyte in its early
growth is maintained via cytoplasmic processes penetrating the zona pellucida and forming gap junctions at the oocyte surface [6]. The gap junctions also
form in increasing numbers between adjacent GCs to
form an extensive network of inter-cellular connections. Through this network, substrates of low molecular weight such as amino acids and nucleotides are
passed to the growing oocyte for its own synthesis
of macromolecules as well as ribosomal and messenger RNA. The nutritional support and trafficking of
macromolecules that this system allows may be particularly important for oocytes due to the avascular
nature of the granulosa layer [7].
114
Chapter 10: Molecular aspects of follicular development
As the follicle undergoes pre-antral to antral transition, the GCs continue to proliferate and as they
do so, follicular fluid accumulates and coalesces to
form a single follicular antrum. The follicular antrum
enlarges during the antral phase but the oocyte does
not increase in size during this period and remains suspended in fluid surrounded by the CCs (which differentiates from GCs during antrum formation). The CCs
remain connected to the rim of GCs by a thin stalk
of cells [7].
Follicle growth takes 85 days in humans and most
follicles become atretic at some stage [8] with the
oocyte continuing to play a critical role in follicular
control and the regulation of oogenesis, ovulation rate
and fecundity [911]. Only a minority of the developing follicles reach ovulation and, in most cases, only
one will complete growth each month, reaching about
100 m in diameter just before ovulation [12]. This
oocyte will be ovulated in response to the mid-cycle
LH surge, which effects the meiotic and cytoplasmic
maturation of the oocyte. The oocyte will complete
meiosis I and arrest at metaphase of meiosis II (MII)
[13, 14]. Meiosis II is only resumed when fertilization
occurs. The rest of the follicle collapses after ovulation
and now becomes the corpus luteum. Both GCs and
TCs contribute to the formation of the corpus luteum
with a switch of morphology and endocrine functions.
The CCs continue to be associated with the oocyte
after ovulation, assisting in oocyte transport along
the Fallopian tube. The cumulus matrix and CCs also
participate in fertilization by influencing spermatozoa
binding and penetration of the cumulus oocyte complex (COC) [15, 16]. Subsequently, the intimate contact between the CCs and the oocyte is broken by the
withdrawal of the cytoplasmic processes.
From the formation of primordial

follicles to the pre-antral phase of
follicular development
The first primordial follicle in human foetuses is
formed at 1522 week gestation, at which point
oocytes are enclosed by a single layer of pre-granulosa
cells [17]. This only happens when primordial germ
cells become oocytes upon the initiation of meiosis.
Pre-granulosa cells are then recruited to the primary
oocyte to form primordial follicles [18].
The formation of primordial follicles is controlled
by retinoic acid (RA) [19]. Retinoic acid acts by bind-
ing to nuclear RA receptors which hetero-dimerize

with nuclear retinoid X receptors (RXR, and ).
The heterodimer will bind to RA-response elements
(RAREs) and thereby control the expression of RAresponsive genes [20]. Primordial follicle assembly (i.e.
formation of primordial follicle) is inhibited by both
progesterone and estrogen [21] but primordial follicle
development is hormone independent [5].
The dynamics of the primordial follicle pool are
not completely understood but involve the interplay
between atresia, activation or maintenance of the
primordial follicle by repressive signals like phosphatase and tensin homolog (PTEN), Foxo3 and antiMullerian hormone. Transition of primordial follicles
to primary follicles requires activating signals which
include stem cell factor (SCF) (also known as Kit ligand), basic fibroblast growth factor (bFGF) and bone
morphogenetic proteins 4 and 7 (BMP-4 and -7) [22].
Primordial follicle activation involves changes to both
GCs and the oocyte. Granulosa cells are stimulated to
resume mitosis and assume a cuboidal shape, while
the oocyte enlarges and cytoplasmic organelles start to
proliferate and differentiate [23]. Subsequent recruitment and proliferation of TCs also take place [5] during primordial follicle activation.
Stem cell factor (Kit ligand) has been found to
induce the primordial to primary follicle transition
[24]. It appears to be a critical factor in stimulating follicular progression as GCs produce SCF, which
appears to act on the oocyte, causing it to enlarge
and initiate development [21, 24]. Furthermore, SCF
also stimulates growth of TCs, their production of
androgens and their expression of keratinocyte growth
factor (KGF) and hepatocyte growth factor (HGF).
Keratinocyte growth factor, produced by the TCs,
is able to stimulate the primordial to primary follicle transition [25] and acts on adjacent GCs as
well. Furthermore, SCF expression is, in turn, stimulated by gonadotrophins, KGF and HGF [5], forming a positive feedback loop. Stem cell factor exerts
its effects through the phosphatidyl-inositol-3-kinase
(PI3K) pathway via phosphorylation of Akt, Foxo3a,
glycogen synthase kinase 3 (GSK-3) and GSK-3
[26]. The rapid oocyte growth during follicular activation is in accordance with the growth enhancing functions of the PI3K pathway [27, 28].
Platelet derived growth factor (PDGF) has been
reported to activate the PI3K pathway [29] as well. The
proteins and mRNA transcripts for two PDGF receptors are detected in human oocytes and the presence
115
of these receptors in human GCs suggests that PDGF

might be involved in the activation of primordial follicles [30].
Another factor which, like SCF, appears to be a
primordial follicle inducing factor is bFGF. Treatment
with bFGF dramatically increases the number of primary follicles and decreases primordial follicle numbers [24, 31]. It is localized in the oocytes of primordial and primary follicles [31], acting on GCs, TCs
and stroma. Basic fibroblast growth factor effects GCs
mitosis [32, 33], steroidogenesis [34], differentiation
[35] and apoptosis [36].
Apoptosis and cellular proliferation of follicular
cells are also influenced by leukemia inhibitory factor (LIF). This acts via a specific transducing receptor, GP130, feeding into the JAKSTAT pathway [37]
in GCs and the oocyte [5]. Leukemia inhibitory factor
promotes development of primordial follicles, a capacity which is enhanced by insulin [38].
A member of the transforming growth factor
(TGF) superfamily, bone morphogenetic protein 7
(BMP-7), also appears to have a role in early follicle development and involves a stroma cell interaction
with the primordial follicle [39, 40]. Another member of the same family, BMP-4, is found to significantly increase primordial to primary follicle transition and is a required survival factor for oocytes
[25]. However, anti-Mullerian hormone (AMH), also a
member of the TGF superfamily appears to have the
capacity to block primordial development. In addition,
AMH is produced in the early secondary follicles, the
pre-antral follicles and antral follicles [41] but is not
expressed in the primordial follicle.
Tumor suppressors, tuberous sclerosis complexes
(TSC) 1 and 2 are observed to suppress the activation
of primordial follicles by suppressing mTOR (mammalian serine/threonine kinase mammalian target of
rapamycin) complex 1 [42]. The TSC1TSC2 complex
suppresses the activity of mTOR complex 1 through
a GTPase activating protein domain located in TSC2.
The function of TSC1 is to stabilize TSC2 and protect
it from ubiquitination and degradation [43].
Foxo3a, a downstream transcription target of the
phosphatise and tensin homolog (PTEN)/PI3K pathway and a substrate of Akt, functions in oocytes to suppress follicular activation [44, 45]. Deletion of PTEN
from oocytes of primordial follicles results in the premature activation of the entire primordial follicle pool
by overgrowth of immature oocytes. The accelerated
oocyte growth is found to be mediated by the enhanced
116
activation of Akt and hence elevated phosphorylation (activation) of ribosomal protein S6 (rpS6), which
is dependent on mTOR complex 1 [45]. Therefore,
PTEN, which inhibits the actions of PI3K, governs follicular activation through control in the initiation of
oocyte growth via PI3K, Akt and Foxo3a [46].
FOXL2, a single exon gene of 2.7 kb, belongs to the
family of winged-helix/fork-head transcription factors
[47] and is expressed in somatic cells of the developing
human ovary before and during follicle formation [48].
This gene product is essential in pre-granulosa cells to
mediate the squamous-to-cuboidal transition and proliferation. In the absence of FOXL2, the whole pool of
primordial follicle gets prematurely activated without
synchronizing with the differentiation and division of
surrounding pre-granulosa cells. This results in follicles that lack multiple layers of functional GCs, ultimately leading to atresia of these follicles [49, 50]. It is
likely that FOXL2 in pre-granulosa cells provides an
inhibitory mechanism via gap junction or paracrine
secretion and maintains the primordial follicles in a
dormant state [42].
Upon activation of primordial follicles, progression from the primary (single GC layer) to secondary
(pre-antral) stage of follicular development requires
further oocyte expansion, GC proliferation and investment of an LH-responsive theca cell layer. These vital
steps are not FSH dependent but remain critically
dependent on paracrine signaling by members of the
TGF superfamily [51]. In primary to pre-antral follicles, activin subunit (INHBA and INHBB) and AMH
[41] gene expressions predominate [52].
Activins (homodimers or heterodimers of INHBA
and INHBB) enhance GCs proliferation and promote
their responsiveness to FSH. At the same instance,
LH responsive thecal androgen synthesis will be suppressed. Appropriate stimulation by FSH will then
divert the formation of activins to that of inhibins
which promote androgen synthesis. Androgen synthesis is in turn linked to FSH augmentation of inhibin
synthesis with the primary aim to ensure estrogen synthesis from the aromatizing of androgens [51].
Anti-Mullerian hormone expression peaks in GCs
of secondary, pre-antral and small antral follicles
4 mm in diameter. In larger antral follicles (4
8 mm), AMH expression gradually disappears [53].
Anti-Mullerian hormone has been shown to suppress
early stages of follicular growth and onset of responsiveness to FSH in vitro, exerting a controlling influence on the rate at which follicles become available
for pre-ovulatory development. The inhibitory effects

of AMH on FSH-sensitivity of follicles may play this
important role in the process of selection. A group
of follicles with lower expression of AMH and hence,
more sensitized to FSH, may be selected from the
batch of AMH-producing growing follicles to continue
growth up to the pre-ovulatory stage [41]. It appears
that AMH produced by growing follicles may act as a
negative paracrine feedback signal on neighboring primordial follicles to inhibit their recruitment [26].
Recent studies have shown that Smad2 and Smad3
are important mediators of TGF signaling in ovarian cells [54, 55]. They are expressed in GCs of preantral and small antral follicles but their expression
becomes weak in the GCs of large antral follicles
[54]. Anti-Mullerian hormone is likely to utilize isoforms of different Smads to mediate its effects on target cells [41]. Anti-Mullerian hormone decreases primordial to primary follicle transition by reducing the
expression of stimulatory factors like SCF, bFGF and
KGF [56].
A multitude of players are likely to be involved in
this crucial stage in ovarian follicular development and
once a primordial follicle has been recruited, it will
be destined for further development. The oocyte commences its growth and secretes glycoproteins which
condense around it to form the zona pellucida. This
separates the oocyte from the rapidly proliferating and
differentiating GCs during the pre-antral phase of follicular development.
The oocyte is shown to be a central regulator of follicular cell function, secreting soluble growth factors,
oocyte secreted factors (OSF), which act on neighboring follicular cells to regulate a broad range of GC
and CC functions, including differentiation, proliferation, apoptosis and luteinization [57]. This has been
affirmed by two landmark studies which demonstrated
that absence of two OSFs, growth differentiation factor
9 (GDF-9) and bone morphogenetic protein 15 (BMP15), causes sterility [58, 59]. Both GDF-9 and BMP15 are from the TGF superfamily, are expressed in
an oocyte-specific manner from a very early stage and
play key roles in promoting follicle growth beyond the
primary stage [60].
As mentioned before, bi-directional communication between the oocyte and surrounding somatic
cells is maintained by cytoplasmic processes and
gap junctions which facilitate the transfer of amino
acids, nucleotides, glucose and metabolites essential
for oocyte and follicle growth and maturation [61].
This communication is so crucial that genetic deletion of the oocyte specific gap junctional subunit,
connexin-37, leads to female sterility in mice, resulting from a lack of mature follicles, failure to ovulate
and development of numerous inappropriate corpora
lutea. In addition, oocyte development in connexin-37
deficient mice is arrested before meiotic competence is
achieved [62].
In addition to oocyte growth and GCs proliferation, the pre-antral follicle further increases its size
and complexity through formation of a basal lamina
between the GC and TC layers in addition to multiplication and differentiation of TCs into two theca layers:
the inner vascular theca interna and the fibrous capsule, theca externa (Figure 10.1).
Pre-antral to antral phase transition

The pre-antral follicle, also known as the secondary
follicle (consisting of the oocyte surrounded by several layers of GCs and theca) continues its development and becomes increasingly FSH responsive.
Further growth and continued survival are increasingly gonadotrophin dependent [63]. Activins in concert with other growth factors, such as insulin-like
growth factors, augment multiple actions of FSH [64]
on immature GCs including upregulation of FSH
receptors and increased formation of inhibin- subunit. Increasing inhibin- diverts GCs from activin to
inhibin biosynthesis and creates the potential for thecal androgen production to be stimulated by inhibin
[52, 65]. The androgen produced is able to synergize
with FSH in promoting GC mitosis and carbohydrate
metabolism, including the increased formation of lactate required for energy production by the maturing
oocyte [66].
The oocyte continues to accumulate large amounts
of transcripts during its growth, while transcriptional
activity ceases at the time of meiotic resumption [67].
It does not increase in size over the antral period but
continues to actively synthesize RNA and turnover
protein.
The GCs continue to proliferate resulting in a further increase in the size of the follicle. The GC and
TC layers become gonadotrophin sensitized with the
expression of FSH receptors in GCs and LH receptors
in TCs, allowing these cells to assume steroidogenic
roles. Follicular fluid, which is composed partly of
GC secretions (including muco-polysaccharides) and
partly serum transudate, starts to form as the GCs
117
Figure 10.1 Primordial follicle to secondary follicle formation (gonadotrophin independent phase): The oocyte, under the stimulatory
effects of retinoic acid, recruits squamous type granulosa cells (GCs) to form the primordial follicle. Steroid hormones inhibit the formation of
primordial follicles. Under the activating effects of stem cell factor (SCF)/kit ligand, basic fibroblast growth factor (bFGF), bone morphogenetic
proteins 4 and 7 (BMP-4 and -7), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF) and keratinocyte growth factor (KGF),
the primordial follicle further develops into the primary follicle with accompanying morphological changes of the supportive cells, i.e. from a
monolayer of squamous type GCs to cuboidal type GCs. Thecal cells then surround the now multiple layers of GCs which encloses the
developing oocyte to form the secondary follicle. Repressive signals like phosphatase and tensin homolog (PTEN), Foxo3 and anti-Mullerian
hormone (AMH), tuberous sclerosis complexes (TSC) 1 and 2 and FOXL2 inhibits the development of primordial follicles to secondary follicles.
proliferate. These drops of follicular fluid coalesce to

eventually form the follicular antrum.
Follicular antrum formation and antral expansion
are absolutely dependent on FSH [68, 69]. The commencement of GC differentiation occurs upon follicular antrum formation, which corresponds approximately to the end of the oocyte growth phase. Granulosa cell differentiation produces two anatomically and
functionally distinct lineages mural GCs that line
the wall of the follicle with primarily a steroidogenic
role and the CCs, which encircle the oocyte [57, 70].
Adequately functioning CCs are essential for ensuring
the survival and subsequent maturation of the oocyte,
allowing it to fulfill its reproductive role [71].
As the pre-antral follicle develops into the antral
follicle, its GCs are not only sensitized to FSH but
also respond to LH directly, since they express both
FSH receptors and LH receptors (LHR) at this time
[72, 73]. Luteinizing hormone receptors are known
to be located on the mural GCs but not on the
CCs or the oocyte [7476] during the antral phase.
Oocytes do not possess functional gonadotrophin
118
receptors but depend on follicular somatic cells to relay

cues via the cumulus-derived microvilli projecting
through the zona pellucida into the ooplasm [75]. The
LH-dependent stages of oocyte and CC maturation are
critically affected by paracrine signaling, most notably
involving GDF-9 and BMP-15 [77]. Growth differentiation factor 9 is anti-apoptotic in pre-antral follicle and
protects GCs from undergoing programmed cell death
by activating the PI3KAkt pathway [78]. Insulin-like
growth factor-1 (IGF-1) also activates PI3KAkt and
has been shown to play an anti-apoptotic role in rat
and bovine GCs by sustaining PI3KAkt signaling. In
humans, where IGF-2 is more abundant, it appears to
act similarly to IGF-1 [78].
The WNT/-catenin (CTNNB1) pathway is also
known to have profound effects on the proliferation, differentiation and survival of GCs [79]. It
enhances FSH-mediated induction of an aromatase
gene (CYP19A1) in GCs, and its regulation of this
gene appears to involve direct interaction with the
transcription factor steroidogenic factor 1 (NR5A1).
Decreasing CTNNB1 levels, or disrupting the
interaction with NR5A1, reduces FSH-induced

CYP19A1 promoter activity and mRNA accumulation
[80]. This affects estradiol synthesis and may adversely
affect follicular development.
Expression of other components of the WNT/catenin pathway, including DVL, AXIN, GSK-3 and
-catenin, are also found in human CCs [81]. Betacatenin is a key effector of WNT/-catenin signaling
and is regulated by the cytoplasmic destruction complex formed by AXIN, GSK-3 and APC. In addition, WNT2 and its receptor FDZ9 have also detected
in human CCs. These findings are compatible with a
model in which WNT2 signals through FDZ9 to regulate the -catenin pathway in human CCs, potentially
recruiting -catenin into the plasma membrane and
promoting the formation of adherens junctions CDH1
[81]. This may ensure the establishment of effective communication and proliferation of the cumulus
oophorus in order to support the oocytes development.
Another player which may have a role in follicular maturation is MATER (Maternal Antigen That
Embryos Require). It is expressed in CCs but not in
mural GCs [82]. It is transcribed only during oogenesis and remains stable until the morula and early
blastocyst stage but disappears in the late blastocyst
stage [83]. It is demonstrated to interact with protein kinase C epsilon (PKC ) in human CCs. As
PKC is believed to function as an anti-apoptotic protein, it is possible that MATERPKC collaborates
with other signaling pathways such as PI3K, Akt and
Ras/Raf/ERK to regulate cell survival and cell death.
Therefore, a proper MATER expression during follicular maturation could be crucial for pro-survival
signal transduction such as via the PI3K/Akt/ PKC
pathways [84].
After the acquisition of LH receptors by GCs,
subsequent follicular maturation comes under direct
LH control. Estrogen secretion is sustained through
LH support of GC aromatase activity and thecal
androgen synthesis, augmented by paracrine inhibin
[52, 73]. Luteinizing hormone-driven nutritional and
information signals from CCs promote oocyte quality [85, 86], and the oocyte in turn secretes BMP15 and GDF-9 to influence CC expansion, apoptosis, carbohydrate metabolism and steroidogenesis in
GCs [77, 87].
These molecules presented here are probably part
of the myriad of factors ensuring the survival and
growth of GCs to sustain follicular development.
Without the support of gonadotrophins on somatic

cells, the oocyte will fail to mature and the entire follicle will undergo atresia (Figure 10.2).
Antral phase and resumption

of meiosis
As the follicle enters into the antral phase (which lasts
about 810 days), the antral cavity becomes filled with
follicular fluid and continues to grow reaching a diameter of 1012 mm [7]. The antral follicle with GCs and
TCs, which can bind LH when the LH surge occurs, is
highly likely to be the dominant follicle which persists to become the pre-ovulatory follicle (measuring
up to 25 mm in diameter). During this phase of follicular development, it is imperative for the oocyte,
arrested at diplotene stage (known as the germinal
vesicle), to resume meiosis within the next 3036 h,
allowing a meiotically competent oocyte to be released
at ovulation.
Stimulation of meiotic maturation by LH occurs
via its action on the surrounding somatic cells rather
than on the oocyte itself [12]. The resumption of meiosis is characterized by the disappearance of the nuclear
membrane, a process referred to as germinal vesicle
breakdown (GVBD).
Cyclic adenosine monophosphate (cAMP) is a negative regulator of GVBD [88]. A constitutively activated G protein-coupled receptor, GPR3, is responsible
for generating cAMP required for maintaining meiotic
arrest during oocyte development [89]. Also, cAMPdependent protein kinase A (PKA) has been shown
to be required for meiotic arrest in oocyte, as knocking down of the regulatory I (RI) subunit of PKA
results in resumption of meiosis in mouse oocytes with
abnormal meiotic spindles and cleavage planes leading to extrusion of large polar bodies [90]. The cAMPdependent PKA regulates the activity of maturation
promoting factor (MPF, also known as Cdk1/cyclin
B1) by phosphatase Cdc25 and kinase WEE1/MYT1
[91]. The MPF is inhibited by inactivation of Cdc25
(through phosphorylation by PKA) and phosphorylation of Cdk1 of MPF by WEE1/MYT1 (activated by
PKA) [12].
Gonadotrophins promote an increase in cAMP
levels in GCs and a decrease of cAMP in oocyte,
thus inducing the resumption of meiosis as well as
cumulus expansion [92, 93]. Activation of mitogenactivated protein kinase (MAPK) by a notable surge
of cAMP in the cumulus oocyte complex, caused by
119
AMH
Secondary follicle
FSH, activins,
inhibins
androgens,
estrogens,
GDF9, BMP15
Primary
oocyte
with zona
pellucida
Basement
membrane
Pre-antral follicle
Thecal layer
Antrum formation
Cumulus cell
layer
enclosing the
oocyte
FSH, insulin-like growth

factors, inhibins,
androgens, estrogens
Antral follicle
Figure 10.2 Pre-antral phase to antral phase transition. As the secondary follicle develops, it becomes more gonadotrophin sensitive with
the expression of gonadotrophin receptors. Under the activation of follicle stimulating hormone (FSH) and the effects of activins and inhibins,
androgen synthesis with estrogen production take place, resulting in the development of the secondary follicle into the pre-antral follicle.
Anti-Mullerian
hormone (AMH) has been shown to suppress early stages of follicular growth and onset of responsiveness to FSH in vitro,
exerting a controlling influence on the rate at which follicles become available for pre-ovulatory development. Enhancing effects from
growth differentiation factor (GDF-9), bone morphogenetic protein 15 (BMP-15) and insulin-like growth factors enables the pre-antral follicle
to develop further into the antral follicle with the formation of the antrum. At the same time, the GCs proliferate and differentiate into the
cumulus cell layer which encloses the developing oocyte. With the antral cavity filled with follicular fluid, the pre-antral follicle now becomes
the antral follicle. See plate section for color version.
stimulation with gonadotrophins, triggers the resumption of meiosis as well as cumulus expansion [94].
The MAPK appears to mediate LH-induced oocyte
maturation by interrupting cell-to-cell communication through phosphorylation of connexin-43 within
the ovarian follicle [95]. The breakdown of communication arrests the supply of cAMP from somatic
cells to oocyte, thereby reducing the levels of cAMP
within the oocyte [96]. This is probably mediated
by termination of gap junctions between CCs and
the oocyte [97]. The rapid increase of cAMP in
CCs may activate phosphodiesterase 3A (PDE3A)
and decrease cAMP level in oocytes, possibly by
cAMP-dependent guanine nucleotide exchange factor (GEF)/PI3K/phosphoinositide-independent protein kinase 1 (PDK1)/protein kinase B (PKB) pathway
[98, 99].
Following the LH surge, a cascade of events is initiated that leads to CC proliferation. The competence to
undergo expansion is a unique characteristic of CC differentiation [100], which has been shown to be critical
120
for normal oocyte development, ovulation and fertilization [101103].

Nitric oxide and/or natriuretic peptides-derived
cyclic guanosine monophosphate (cGMP) may be an
oocyte maturation inhibitor. The cGMP maintains
pre-ovulatory oocytes in meiotic arrest via inhibition of oocyte cAMP phosphodiesterases (e.g. PDE3)
to maintain cAMP level and activation of cGMPdependent protein kinase (PKG) to decrease MAPK
activity [94]. It is possible that accumulation of cGMP
by nitric oxide (NO) and/or natriuretic peptides under
FSH stimulation during follicular growth may serve
to prevent untimely oocyte maturation. The inducible
form of nitric oxide synthase (main source of NO in
the ovary) [104] significantly decreases after human
chorionic gonadotropin (hCG) injection (a similar
effect to the LH surge), which induces a decrease of
NO concentrations in pre-ovulatory follicular fluid
[105]. This results in a decrease of cGMP which
may allow the oocyte to undergo maturation and
ovulation.
Luteinizing hormone-binding to mural GCs leads

to the production of epidermal growth factor (EGF)like growth factors, such as amphiregulin, epiregulin
and betacellulin [106]. These factors are known to
be potent stimulators of oocyte maturation and they
also activate genes like Has2, Ptgs2 and Tnfaip6 associated with CC expansion [107]. Epidermal growth
factor receptor (EGFR) activation by EGF or EGFlike growth factors may be a common pathway mediating meiosis-inducing influence of FSH and LH.
The EGFR is expressed in both CCs and the oocyte,
whereby the EGFR in CCs contributes significantly
to the mediation of gonadotrophin-induced meiotic
resumption [94]. The pathway utilized by EGF and
EGF-like growth factors for the stimulation of meiotic
resumption involves binding to EGF receptor subfamily members with subsequent tyrosine kinase activation and downstream activation of MAPK [99]. Possible production of steroids, e.g. progesterone, estradiol by EGFR and the PI3K/PKB pathway, mediates
gonadotrophin-induced MAPK activation [94].
Sterols and steroid hormones have also been shown
to result in the resumption of meiosis. Follicular fluid
meiosis-activating sterol (FF-MAS), an intermediate
in the cholesterol biosynthetic pathway, is a putative oocyte maturation-inducing substance, demonstrated to stimulate resumption of meiosis in isolated
mammalian oocytes including mouse, rat and humans
[108112]. Addition of FF-MAS to culture medium
promotes progression of the oocyte to metaphase II
and dramatically improves the quality of oocytes produced in vitro [113115].
Progesterone is produced in human CCs [116].
The level of progesterone and its receptors in CCs
are increased by stimulation with LH and FSH in
porcine cumulus oocyte complexes [117]. Progesterone biosynthesis depends on the gonadotrophininduced activity of its biosynthetic enzymes such as
CYP51, delta 14-reductase, delta 7-reductase, P450scc
and 3HSD [118, 119]. Progesterone may induce
GVBD by binding to its receptor, which then decreases
connexion-43 in CCs and, hence, reduction of cAMP
level in oocytes [120, 121]. However, the role of estrogen and testosterone in mediating mammalian oocyte
remains to be elucidated, and it seems that steroids are
not necessary for resumption of mammalian meiosis.
Nevertheless, steroids are probably involved in follicular growth, somatic cell-differentiation and the acquisition of developmental competence of mature ova [94,
122] (Figure 10.3).
Figure 10.3 Antral phase to ovulation: The antral follicle

progresses to develop into the pre-ovulatory follicle under the
stimulation of luteinizing hormone (LH). This transition is
augmented by epidermal growth factors (EGF), EGF-like growth
factors and follicular fluid meiosis-activating sterol (FF-MAS). A
critical step in this transition is the completion of meiosis I in the
maturing oocyte with the extrusion of the first polar body. This
ensures that the oocyte achieves meiotic competence prior to
ovulation. Under the LH surge and increased expression of
metalloproteinases and proteolytic enzymes, the pre-ovulatory
follicle subsequently ruptures and ovulation takes place with the
release of the cumulus oocyte complex. The follicle then collapses
and proceeds to form the corpus luteum. See plate section for color
version.
Ovulation and subsequent

luteinization of the follicle
Luteinizing hormone appears to play a major role in
mediating the final phase of follicular development.
Under LH stimulation, there are significant changes
in the pattern of steroid secretion from primarily aromatizing androgens to estrogens to synthesizing progesterone. The somatic cells have reduced capacity to
bind estrogens and FSH, but become sensitized to LH
and respond by secreting more progesterone. This prepares the pre-ovulatory follicle for ovulation, depresses
the growth in other less mature developing follicles
and promotes the transition to the luteal phase of the
cycle [7].
By the end of the pre-ovulatory phase, the follicle
attains a maximum dimension of 25 mm in diameter, mostly due to the rapid expansion of follicular
fluid volume. This results in the oocyte with its associated CCs to be held tenuously by a thinning stalk
of mural GCs to the rest of the follicle. Under the LH
surge, the oocyte resumes meiosis, extrudes the first
polar body and arrests at metaphase II. Activation of
the Mos/MAPK signaling pathway seems to be the
121
Gonadotrophin-independent phase
Ovarian follicular
development
Gonadotrophin-dependent phase
Figure 10.4 In the ovary, follicular development constitutes of a gonadotrophin-independent and a gonadotrophin-dependent phase.
Coordination of multiple growth factors, hormones and biochemical molecules signaling at timely intervals with the activation of several
pathways, e.g. PI3K, JAK/STAT, WNT/-catenin and MAPK between the growing oocyte, surrounding somatic cells and wider endocrine
system ensures optimum follicular development with the release of a meiotically and developmentally competent oocyte. See plate section
for color version.
cytostatic factor responsible for this second meiotic

arrest [123]. At the time of ovulation, the oocyte
becomes basically transcriptionally inactive [124, 125].
From this point until the 48 cell stage (23 days
after fertilization), the oocyte/embryo must survive on
stockpiled mRNAs and proteins.
Prior to ovulation, due to increased vascularization of the follicle, the follicle appears hyperemic.
There is production of large amounts of prostaglandins
(mainly PGE2 ) and synthesis of a hyaluronan (HA)rich matrix. These events are dependent on LHinduced expression of EGF-like factors (amphiregulin,
epiregulin, betacellin) [106, 126], matrix-associated
molecules (Has2, Ptgs2, Tnfaip6, Ptx3, Cspg2), transcription factor genes (Pgr and RUNX1) and protease
genes (Ctsl and Adamts1). Ovulation seems to be reminiscent of inflammatory responses due to the genes
expressed under LH stimulation [127]. The LH surge
also induces a switch in gene expression in surrounding somatic cells and the generation of an extracellular
matrix within the expanded cumulus oocyte complex.
The EGFR transactivation has been demonstrated
to be essential for the regulation of ovulation [126],
122
and the physiological surge of LH requires a local

sustained activity of the EGFR to mediate and maintain its stimulation in the GCs which synchronizes the
many complex events that finally converge to ovulation
[128].
Matrix metalloproteinases (e.g. gelatinase), serine
proteases such as plasmin and plasminogen activator
(which cleaves pro-collagenase to generate active collagenase) are explicitly involved in ovulation as proteolysis is essential to effect the breakdown of the connective tissue of the follicle. The LH surge leads to activation of these proteases to cause follicular rupture and
the release of the cumulus oocyte complex.
After ovulation, the collapsed follicle transforms
into the corpus luteum. The fibrin core within the follicular antrum undergoes fibrosis over a period of few
days and the membrane propria between the granulosa and theca layers break down and blood vessels invade. Luteinizing hormone is pertinent in terminating GC proliferation and mediates the genetic
transition of GCs to luteal cells (LCs) [129]. The GCs
will cease dividing and hypertrophy to become large
lutein cells rich in mitochondria, smooth endoplasmic
reticulum, lipid droplets and, in many species, a

carotenoid pigment, lutein, which may give the corpus luteum a yellowish or orange tint. Thecal cells will
form smaller lutein cells and produce progesterone
and androgens. This transformation of GCs and TCs
to lutein cells is referred as luteinization.
In humans, the luteal phase lasts from 12 to 15
days. Thereafter, luteal regression occurs with ischemia
and the progressive death of lutein cells leading to
the formation of a whitish fibrous scar known as corpus albicans. A PTEN deficiency in LCs seems to
lead to enhanced phosphorylation and activation of
Akt, and expression of a distinct set of PI3K pathway components including FOXO3. This appears to
enhance longevity in LCs [130]. Hence, PTEN appears
to be expressed in a cell-specific manner in the ovary,
as described previously with its involvement in the
oocyte, GCs at various stages of development and in
the regulation of LCs life span.
Due to the regression of the corpus luteum, there
is a fall in progesterone output. A new cycle then reinitiates in the ovary with the recruitment of primordial follicles and progression of subsequent events as
detailed before.
Concluding remarks
Follicular development consists of gonadotrophinindependent and gonadotrophin-dependent phases
(Figure 10.4). These two phases require the fine coordination of multiple growth factors, hormones and
biochemical molecules signaling at timely intervals
between the growing oocyte, surrounding somatic
cells and the wider endocrine system. The precise control and balance of these systems is essential if the ovulation of a meiotically and developmentally competent
oocyte is to be achieved.
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Activation of meiotic maturation in rat oocytes after
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114. Cukurcam S, Hegele-Hartung C and Eichenlaub
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127. Richards JS, Liu Z and Shimada M. Immune-like

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128
Section 3
Chapter
11
Fundamental cryobiology of reproductive

cells and tissues
Concepts and misconceptions
Erik Woods, Sreedhar Thirumala, Xu Han and John K. Critser
Introduction
Cryobiology as a science deals with the effects of
reduced temperatures on living organisms, their constituent parts and their products. Understanding the
basics of cryobiology to develop improved cryopreservation procedures has been a major challenge to scientists all over the world. Since the complexity of cryobiology is deeply rooted in the complexity of living systems, historically this has entailed coordinated
research efforts among biological and physical sciences
and involved the participation of biologists, chemists,
physicists, engineers, mathematicians and others using
carefully designed empirical evaluations, as well as
investigation and utilization of specific cellular characteristics in theoretical models, all in efforts to apply
cryopreservation to a broad range of cells and tissues
[1].
For the most part, reproductive cryobiology has
been developed to gain time, either for flexibility (e.g.
extension of fertility), logistics (e.g. gamete donation),
back-up (for repeat embryo transfer) or some combination of these [2].
Many misconceptions related to the cryopreservation or vitrification methods stem from the use of
potentially confusing terminology that has evolved in
cryobiology literature. At one level, this occurs in the
labels we place on various categories of general methods for cryopreservation. For example it is common
to refer to methods on the basis of cooling rate such
as slow cooling methods or rapid cooling methods
[35]. In other cases cryobiologists refer to methods as
equilibrium methods, non-equilibrium methods
or even quasi-equilibrium methods [6]. Still, in other
cases the methods are described by the type of con-
tainer or device in which the cells or tissues are cryopreserved such as an Open Pulled Straw method [7],
a needle-immersed vitrification method [8], a Cryoloop method [3] and so on.
The key to sorting-through what all this means in
terms of outcomes is to understand how these labels
(methods) relate to the underlying physiochemical
conditions produced that fundamentally determine
whether cells and tissues survive (or fail to survive)
the series of extreme conditions we subject them to
during the cryopreservation process. For the purposes
of organizing this chapter and to potentially clarify
misconceptions and/or misuses of terminology with
respect to cryobiological processes, the subject has
been divided into three parts consisting of: (1) classical cryopreservation; (2) vitrification; and (3) preservation of reproductive cells and tissues using classical
cryopreservation and vitrification procedures.
Classical cryopreservation
Cryopreservation has been developed to store viable
biological systems at ultra-low temperature (196 C)
in a cryogenic medium such as liquid nitrogen for
extended periods of time. At such ultra-low temperatures, all cellular divisions and metabolic activities
are arrested, such that the systems can be revived and
restored to the same living state as before they were
stored. Most currently used methods for cryopreservation of mammalian cells are indeed derived from centuries of low temperature work on reproductive cells
and tissues [9].
Cells and tissues lose viability (are killed) during
classical cryopreservation due to two major causes:
(1) the formation of ice inside the cells (intracellular
129
ice formation or IIF); and (2) exposure to very high

concentrations of solutes that form as liquid water is
removed from the extracellular solution when it crystallizes and forms ice (solute damage) [10]. In general, these factors are managed by following equilibrium cryopreservation methods where equilibrium
means that the water inside and outside the cells is in
equilibrium (or very near equilibrium). It is important
to understand that equilibrium cryopreservation simply means that the procedure is intended to maintain
(more or less) the same amount of water inside the cell
as outside the cell. All the other components of the system (the cryopreservation medium and the cytoplasm)
are typically not in equilibrium.
During a typical cryopreservation process ice tends
to form at different rates. The generally accepted theory of ice development in biological systems is based
on Mazurs work, which states that the probability of
an ice crystal to form at any temperature is a function of volume [11]. As the extracellular space is much
larger than the intracellular space, ice is likely to form
first in the extracellular space whereas intracellular
solution becomes supercooled. Water is removed from
suspending solution due to this ice formation, and
partially frozen extracellular solution becomes rich in
solutes. This creates an osmotic non-equilibrium state
that provides a driving force for the cellular dehydration or loss of intracellular water [12]. This phenomenon is a function of temperature, cooling rate
and initial amount of solutes dissolved in the water. At
low cooling rates cellular dehydration is the dominant
mechanism while at very rapid cooling rates intracellular ice formation (IIF) is the dominant mechanism
[11, 12]. Both excessive cellular dehydration and IIF
have been shown to be deleterious to the post-thaw
survival of biological systems [1013]. At sufficiently
slow cooling rates the cell loses water to concentrate
the intracellular liquid adequately enough to eliminate
supercooling and maintain chemical potential of intracellular water in equilibrium with that of extracellular water. However, the resulting changes in the extracellular space, such as changes in pH, and/or changes
in ionic concentration, can lead to protein configuration changes, modifying their properties and diminishing or eliminating biological activity. This denaturing of cell proteins due to solute toxicity may be lethal
to cell survival [13]. In addition, mechanical interaction between extracellular ice crystals and cells can
also lead to the physical deformation of cells and rupture of cell membranes [14]. On the other hand, if the
130
cell is cooled too rapidly it is not able to lose water

fast enough to reach equilibrium; it becomes increasingly supercooled and eventually attains equilibrium
by freezing intracellularly. Intracellular ice formation
is generally thought to be lethal as it causes injury to
cellular membranes and intracellular structures. So,
cooling rates which are either too slow or too fast
can and do reduce the post-thaw survival of the cells;
therefore, a cooling rate for maximum cell cryosurvival should and does exist between the high and
low rates [15]. This has been confirmed experimentally for a variety of cells and the curve of cell survival,
plotted as a function of the cooling rate, has a characteristic inverted U-shape [10]. The use of cryoprotectants helps to alleviate some of the problems associated
with solute toxicity and IIF during freezing.
Equilibrium and non-equilibrium

slow freezing
Conventional slow freezing protocols involve pretreatment of cells with cryoprotective agents (CPAs)
in order to remove some water from the cells and
to minimize some other harmful effects of freezing.
Broadly speaking, CPAs can be divided into two classes
including permeating (e.g. polyols such as glycerol or
ethylene glycol) and/or non-permeating (e.g. sugars).
Permeating CPAs are thought to protect via their colligative properties [1] while non-permeating CPAs are
generally thought to protect by dehydration as well as
membrane stabilization [1]. While both types of CPAs
result in some cellular dehydration, the majority of cell
water does not exosmose until slow cooling in the presence of ice to a predetermined temperature. During
freezing the fate of any biological cell depends on the
thermal history it experiences, i.e. cooling rate, end
temperature to which the cell is frozen, the time spent
at the end temperature and warming or thawing rate
[16].
During slow freezing, using low concentrations of
CPA, IIF is generally avoided by cooling cells sufficiently slow so that dehydration reduces the intracellular water at near osmotic equilibrium with the outside
partially frozen solution. This type of slow freezing is
referred as equilibrium slow freezing which avoids
the supercooling of intracellular solution and averts
the formation of lethal IIF. In equilibrium, freezing
cells are typically cooled at 0.11.0 C/min using programmable freezer to a temperature of 60 C or below.
The presumption is that after reaching 60 C, the cells
Chapter 11: Cryobiology of reproductive cells and tissues
essentially contain little or no freezable water and consequently IIF cannot occur during a subsequent plunge
at 196 C [17]. The first successful cryopreservation
of mouse embryos using equilibrium slow freezing
was reported by Whittingham et al. and Wilmut and
Rowson [18, 19]. These authors reported a high percentage of embryo survival when frozen at 1 C/min
to 70 C under near-equilibrium conditions. However, extreme dehydration, as would be encountered
during equilibrium slow freezing, can cause volume
excursions (shrinkage) beyond what some cells can
tolerate and produce irreversible damage to cellular components. Additionally, as described above,
unavoidable excessive salt concentrations can also play
a significant role in damaging the cells.
Alternatively, if the cooling rate is not slow enough
to maintain equilibrium, IIF becomes possible due to
supercooling of intracellular solution in relation to the
frozen extracellular solution at a given temperature.
However, there is an alternate slow freezing approach
often referred as non-equilibrium slow freezing or
interrupted slow freezing [20] during which the cells
are initially cooled slowly (slow enough to avoid IIF) to
an intermediate temperature to render some degree of
dehydration. At such an intermediate temperature, it is
expected that the concentration of unfrozen fraction of
the suspending solution and cytosol are significantly
high enough to enable vitrification and form amorphous glass upon rapid cooling by immersing abruptly
in liquid nitrogen. However, high survivals with this
procedure usually require that the cells be warmed
rapidly during thawing. The reason for this is that
after the initial slow cool to the intermediate temperature, the cells still probably contain a small amount of
freezable water and that water undergoes damaging recrystallization unless the subsequent warming is rapid
[17]. Further, if the concentration of unfrozen fraction is not enough an unstable glass may form during
rapid immersion in liquid nitrogen and devitrification
and/or re-crystallization can occur upon re-warming
[9].
Toxicity and osmotic effects of

cryoprotectants
In general, classical cryopreservation (as well as vitrification) involves the addition of permeating CPAs,
and their extensive use is based on the serendipitous observation by Polge et al. that sperm cells were
able to survive freezing in the presence of a chemi-
cal (glycerol) and did not survive the freezing process

in its absence [21]. Another ground-breaking finding was the utility of dimethyl sulfoxide (DMSO) as
a CPA by Lovelock and Bishop in 1959 [22]. Over the
course of some 50 years, several other compounds have
been found that possess some CPA activity and many
cells and tissues have been frozen, mostly through
empirically derived methods. The literature shows that
there is a considerable divergence in classes of CPAs,
varying from low molecular weight permeable solutes
like DMSO, glycerol, ethylene glycol and sucrose [13,
23] to high molecular weight non-permeable polymers like polyvinylpyrrolidone (PVP), dextran and
hydroxyl ethyl starch [23, 24]. While these compounds
have enhanced cryopreservation outcomes and made
widespread use of cryostorage possible, there is a substantial amount of evidence suggesting that CPAs,
albeit with their benefits, can actually play a direct
role in producing cryoinjury [23, 25, 26]. Detrimental effects of cryoprotectants are almost as relevant
to cryobiology as are their cryoprotective effects [25].
For instance, the CPAs can potentially induce osmotic
injury to the cells during their addition and removal,
and the higher the CPA concentration required, the
greater the likelihood of damage [1]. While permeating CPAs do indeed penetrate cells, none to date
have been identified that cross their membranes as
quickly as water. Addition of a permeating CPA therefore causes the cell to undergo extensive initial dehydration due to osmotic efflux of water followed by
rehydration due to influx of CPA and water. During removal of CPA, cells at first swell due to the
osmotic influx of water and then slowly return to
initial isotonic volume as CPA and water leave the
cell. These repeated volumetric changes can result in
significant loss of functional integrity and even cell
death [27].
The rate at which permeable CPA diffuses into
the cells varies between the cryoprotectants and is
also temperature and concentration dependant [28,
29]. For example, most embryos are more permeable
to propylene glycol than to glycerol, which renders
them less sensitive to osmotic shock when propylene
glycol is diluted out compared to glycerol [28].
For this reason, impermeable solutes such as sugars
are often added to dilution media to prevent excessive osmotic swelling during post-thaw CPA removal.
As mentioned earlier, CPA diffusion across the membrane is also a function of concentration and temperature. The higher the concentration, the faster and more
131
extensive the osmotic shock. This may be overcome

either by gradual or stepwise addition and dilution of
CPAs before freezing and after warming [1]. Temperature has a profound effect since the permeation of CPA
is usually rapid at higher temperatures. In reproductive cells such as embryos, CPA permeation essentially
ceases when temperature reaches around 5 C [28].
Cryoprotective agents also reduce the temperature at
which ice crystallization first occurs, thereby extending the dehydration time during freezing. Therefore,
procedures for the addition and removal of CPA must
be optimized according to specific cell characteristics
to ensure successful cryopreservation. The dynamics
of cell volume changes can be maintained within the
tolerable limits by carefully selecting the optimal type
and concentration along with a cooling rate that yields
the optimum time necessary for diffusion of CPA in
and out of the cell during cooling in the presence of ice.
For this, information is required regarding the osmotic
tolerance limits of cells, defined as the extent of volume excursions cells can withstand before irreversible
loss of function occurs [30]. In addition to that, to optimize addition and removal of CPA it is also necessary
to define other osmotic properties such as osmotically
inactive cell volume, hydraulic conductivity and solute
permeability of the cell [31].
Vitrification
As described in many recent research papers and
reviews, vitrification has become an increasingly
accepted method for preserving embryos, oocytes and,
recently, even sperm [2]. However, despite the growing
popularity of this type of preservation, misconceptions
in the literature abound. Vitrification may be simply
defined as the process of converting a supercooled liquid into a glass-like amorphous solid which is free of
any crystalline structure, either by the quick removal
or addition of heat and/or by mixing with an additive. Vitrification occurs at the glass transition temperature (Tg ) which is lower than the melting point
(Tm ) [32].
In the context of cryobiology, the supercooled liquid is almost always water and the additive is a combination of solutes, typically including salts and one or
more permeating or non-permeating CPAs. The relationship between the quick removal of heat (i.e. cooling rate) and total additive (solute) concentration can
be expressed conceptually as:
PV (S P )B ;
132
where PV is the probability of vitrification, S is the total

solute concentration, p is the specific solution parameter (derived empirically), and B is the cooling rate.
Vitrification prevents both intracellular and extracellular ice formation [33]. One approach to vitrifying
cell suspensions is to use a relatively high concentration of CPA in combination with a relatively low cooling rate. In this case, the cooling rate must be higher
than the critical cooling rate (CCR) required to achieve
vitrification [3436].
To determine the CCR, Ren et al. [36, 37], based on
Boutrons [34, 35] semi-empirical crystallization theory, developed a correlation between the cooling time
and the volume ratio (x) of the ice quantity to the maximum crystallizable ice by using the integral method
without considering the finite expansion. This correlation can determine CCRs through investigations on
the time-temperature transformation (TTT) diagram
and its derivative continuous-cooling transformation
(CCT) diagram [3538]. For solutions with a relatively
high concentration of CPAs, such as 45 M glycerol,
L-2,3-butanediol and 1,2-propanediol, CCRs were
determined as 1034 K/min [36]. Currently available
cooling methods, such as the Open Pulled Straw (OPS)
method [3, 7, 39, 40], can indeed achieve cooling rates
that high. However, as previously described, high concentrations of CPAs often have damaging toxic and/or
osmotic effects on cell survival [33]. Another approach
to achieving vitrification is to use an ultra-fast cooling rate (1056 K/min) to improve vitrification tendencies and decrease the CPA concentration requirement [41]. Critical cooling rates for solutions with a
relatively low concentration of CPAs (such as 12 M)
may reach 1056 K/min [34]. Producing such ultrafast cooling rates is important for vitrification of these
solutions. In addition, previous investigations [34, 35]
have also demonstrated that a crystallization peak
exists as a temperature region typically from 240 to
200 K, where the maximum ice formation happens
during freezing. This region is also a dangerous temperature region (DTR) for vitrification of a CPA solution [41]. The ultra-fast cooling rate should also significantly decrease the time for the sample to pass the
DTR during cooling and hence to improve vitrification
tendencies.
A common misperception held by many in assisted
reproductive technology (ART) laboratories is that
plunging samples into liquid nitrogen (LN2 ) results
in cooling rates sufficiently high to produce vitrification. To gain a better understanding of why this is a
q = hA(Ts Ta );
where q is the heat flow, h is the heat transfer coefficient, A is the surface area, Ts is the surface temperature, and Ta is the reference temperature [42]. Using
this approach we can show the relationship between
the heat transfer coefficient (h) of a sample, and how
this is related to cooling rate, the size of the sample
and the required CPA concentration in the sample to
achieve vitrification at various cooling rates. Plunging samples into LN2 is an example of a pool boiling
approach. Flowing LN2 over a sample is an example of
a forced flow boiling approach. Oscillating heat pipe
(OHP) technology [41] is an example of new technologies that are being developed to increase our ability
to apply vitrification approaches to biological samples
(Figure 11.1 [41]).
Equilibrium and non-equilibrium

vitrification methods
As described above, in the context of cryopreservation,
equilibrium refers to the relative amounts of water
inside the cell and outside the cell being the same
(or nearly so). An ideal vitrification method produces
no ice formation and may therefore be an equilibrium method. If a sufficiently high concentration of
CPA could be added at the beginning of freezing, formation of ice would be totally avoided and the system would vitrify with no supercooling no matter how
slowly it was cooled. The lengthy isothermal equilibration in cryoprotectant solution can be represented
by a long isothermal (near isothermal) streak on the
phase change diagram before the system is cooled to
glass transition temperature without ice crystallization (EV-1 on Figure 11.2 [43]). This approach, which
is independent of cooling rate with no ice crystallization, may be called equilibrium vitrification. It
should be noted that, in equilibrium vitrification, postthaw survival is not dependent on the rate of warming
11
Required CPA for vitrification (M)
misperception, it is helpful to consider the relationship between the heat transfer coefficient (h) of a sample, and how this is related to cooling rate, the size of
the sample and the required CPA concentration in the
sample to achieve vitrification.
The heat transfer coefficient comes from Newtons
law of cooling: the dissipation of heat from a solid to
a fluid is proportional to the temperature difference
between the solid and the fluid. This can be expressed
as:
10
Pool boiling
Forced flow
Boiling
New
technology
h = 103 W/m2 K
h = 104 W/m2 K
h = 105 W/m2 K
6
5
h = 106 W/m2 K
4
0.0
0.2
0.4
0.6
0.8
Sample dimension (cm)
1.0
Figure 11.1 The effect of sample size on the cryoprotectant agent

(CPA) concentration required to achieve vitrification at various
cooling rates. Plunging samples into liquid nitrogen (LN2 ) is an
example of a pool boiling approach. Flowing LN2 over a sample is
an example of a forced flow boiling approach. Oscillating heat pipe
(OHP) technology (data from Jiao et al. [41]) is an example of new
technologies that are being developed to increase our ability to
apply vitrification approaches to biological samples. See plate
as there is no ice to re-crystalize. Using this approach,

Song et al. vitrified vascular grafts by exposing them
to 55% cryoprotective solution and then cooling them
rapidly (43 C/min) to 100 C followed by slow cooling (3 C/min) to 135 C [44]. In another study,
Brockbank et al. [45] exposed porcine cardiac tissue to
an 83% v/v cryoprotectant solution followed by similar cooling to 135 C with good structural preservation. However, the concentration of CPA necessary
to achieve equilibrium vitrification is extremely high
(typically 6080%) and it is often a daunting task to
balance the deleterious effects of toxicity associated
with such high CPA concentration with potential benefits. Earlier, Farrant introduced a liquidus tracking
method, in which he gradually increased CPA concentration while lowering the sample temperature during freezing and gradually removed CPA during rewarming in order to minimize the known toxic effects
of the cryoprotectant [46]. By this method, the sample remained slightly above the melting temperature
on the phase change diagram and effectively cooled
below glass transition temperature without any supercooling (EV-2 on Figure 11.2). Farrant explored his
technique using smooth muscle tissue but, despite his
early success in avoiding ice crystal formation, the
133
NEV
Tm
20
Temperature (C)
40
Figure 11.2 Binary phase diagram for aqueous

mixtures of glycerol showing the principal events
and phase changes associated with cooling. Where
Tm is equilibrium melting point curve; Th is
homogeneous nucleation curve; Td is devitrification
curve; Tg is glass transition curve; EV-1 is the
equilibrium vitrification by the liquids tracking
method; EV-2 is the equilibrium vitrification by warm
equilibration in unfreezable solution and NEV is the
non-equilibrium vitrification. Adapted from
Anderson [43].
EV-1
Supercooled
(Metastable)
Th
60
EV-2
80
Unstable
100
Td
120
140
Tg
160
10
20
30
40
50
60
70
80
100
Concentration (w/w%)
rewarmed tissue was severely impaired both functionally and structurally after exposure for 20 h in
unfrozen CPA medium at 79 C [46]. Subsequent
experiments by Elford and Walter determined that the
toxic effects of DMSO are not solely responsible for the
loss of tissue function, and in fact its function was only
improved after adequate steps were taken to optimize
the solute concentration (the ionic composition) of the
CPA medium during freezing [47]. Nevertheless, Farrants eminent procedure has been the basis for most
of the equilibrium vitrification methods that followed
[4851]. Despite some success achieved, this equilibrium approach has not been actively pursued, presumably because the technique requires lengthy periods
of exposure to toxic solutes at high temperatures and,
more importantly, the equilibration at sub-zero temperatures may not be achievable either in a practical
time scale or without exceeding the tolerance limits of
solute toxicity of the tissue [47, 52].
Vitrication is also possible using less concentrated
cryoprotective solutions if sufficiently rapid cooling
rates are employed. This approach requires the best
possible cryoprotective concentration (critical CPA
concentration) that ideally balances the glass formation ability and toxicity at the temperature of addition
[48]. These optimally adjusted cryoprotectants could
be vitrified by supercooling at realistically feasible
cooling rates through the metastable non-equilibrium
zone between the melting temperature and glass transition temperature on the phase change diagram (NEV
on Figure 11.2) [53]. The success of non-equilibrium
134
vitrification depends on how fast the sample is cooled

through the temperature region of potential crystallization to below glass formation temperature. Since
the supercooled water exists in a state of precarious
equilibrium, the cooling rate has to be rapid enough to
extend supercooling all the way down to glass forming temperature; otherwise any minor perturbations
or suspended impurities can trigger ice nucleation and
crystal growth. In concentrated solutions, the probability of ice nucleation becomes high well below the
freezing point, where the actual rate of ice crystal
growth is significantly low [54]. Clearly, if the cooling rate is rapid, the sample can escape both nucleation and ice crystal growth and reach an amorphous
glassy state. However, if the cooling rate is insufficient
the sample may nucleate but with or without ice crystals. In the later case, if the warming rate is not sufficient, freezing reoccurs as the sample traverses the
nucleation zone first and then the zone of ice growth
as larger ice crystals grow at the expense of smaller
crystals [54]. These phenomena are known as devitrification and re-crystallization, respectively, which are
of great concern during non-equilibrium vitrification.
Another factor that influences the critical CPA concentration required to achieve vitrification under moderate cooling conditions is the volume of the sample. Minimizing the volume of the sample decreases
the amount of liquid which has to be cooled and the
likelihood of ice crystal formation and thereby promotes vitrication [55]. Nevertheless, non-equilibrium
vitrication has been developed and shown to provide
effective preservation for a number of cells, including monocytes [56], organized tissues [44, 4851, 57],
mouse ovaries [58] and pancreatic islets [59].
Preservation of reproductive cells

and tissues
Cryobiology of spermatozoa
The benefits of sperm cryopreservation are numerous in human reproductive medicine. For example,
ART using cryopreserved donor spermatozoa is a
widely available treatment for couples with severe male
infertility factor (such as oligospermia, seminoma or
azoospermia) or for patients without male partners
desiring pregnancy [43]. Another established option
for semen cryopreservation is for men and postpubertal boys who are suffering either from malignant
diseases such as cancer or at risk of fertility impairment [60]. For instance, in patients suffering from
cancer, the germinal epithelium of the testis, from
which the spermatozoa develop, is highly sensitive
to drugs, chemotherapy and radiotherapy treatments.
Therefore, the patients are encouraged to cryopreserve
their sperm before cured and sterilized by these treatments to preserve their ability to father a biological
child in the future either using intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI)
[61]. Human sperm cryopreservation can also be used
to preserve fertility in other situations, such as surgical
procedures that can harm fertility and prior to vasectomy.
It is just over 60 years since Polge et al. described
the use of glycerol to freeze spermatozoa, and several attempts have since been made to develop successful cryopreservation protocols for mammalian spermatozoa with varying success [21]. Cryopreservation of
sperm, developed largely by empirical means, generally employs a slow-cooling rate (about 5 C/min)
starting from physiological temperature to a seeding temperature, followed by rapid cooling next to
the onset of ice formation (100200 C/min), in the
presence of glycerol buffered with egg-yolk citrate
medium [1]. The problem of cryoprotectant toxicity
and osmotic stress, as well as biochemical alteration
and possible effects at the genetic level are widely
investigated to determine optimal CPA addition and
dilution, and cooling and warming rates [62, 63]. Various aspects of sperm cryopreservation, such as chemical composition of extenders and their effects on the
sperm plasma membrane, osmotic tolerance limits,

hydraulic conductivity and CPA permeability, seminal
plasma composition and other factors that influence
the quality and life-span of post-thaw spermatozoa
have also been studied [63, 64]. Also, to better assess
the success of cryopreservation, endpoints other than
the percentage of motility recovery or the assessment
of ultrastructural damage, like energy status, damage to the plasma membrane or to subcellular elements, chromatin stability and chromosomal damage
have been proposed [65]. Many of the studies generated acceptable results in various species, but the
procedures used are still relatively complicated and
time consuming and considerable loss of recovery is
still observed [1]. While, in humans this loss of viability may not be an issue with normal ejaculate volumes and sperm counts, they may be highly significant in the case of oligozoospermic or asthenozoospermic samples [62]. Furthermore, with the development
of ICSI and the availability of techniques for surgical sperm retrieval (both epididymal and testicular),
there is an increased need to store low numbers of
sperm and therefore develop improved freezing techniques in order to maximize survival [62, 66]. Efficient
cryopreservation of small number of sperm retrieved
from these techniques reduces the number of surgical
interventions and thus avoids the complications and
expenses associated with repeated surgeries. Moreover, given the unique characteristics of epididymal
and testicular spermatozoa, conventional methods of
sperm cryopreservation may not be optimal [67].
Novel cryopreservation methods have been
recently proposed to improve post-thaw recovery in
highly compromised and low-number sperm samples.
A novel procedure for efficient cryopreservation of
single human spermatozoa in cell-free human or
animal zona pellucida is reported [66]. Other novel
methods include, using mini straw as carrier for
cryopreservation of microquantities of sperm, direct
cryopreservation of individually selected spermatozoa in microdroplets, the Cryoloop method using
conventional slow freezing, the microencapsulation
of sperm in alginate beads, using ICSI as a carrier for
single sperm cryopreservation and agarose microspheres as analogue to zona pellucid for small sample
sperm cryopreservation (refer to a recent review by
AbdelHafez et al. [67]). In all these methods, sperm
was pre-equilibrated in CPA solution before cooled
either using LN2 vapor or controlled rate freezer and
then immersed in LN2 [67]. Despite their novelty and
135
attractiveness, these methods invariably suffer from

several drawbacks such as labor intensive, high cost,
extreme complexity, sperm loss due to adherence to
carrier walls, risk of cross contamination and sperm
loss during washing off the carrier.
Recently, vitrification methods have been gaining
momentum in sperm cryopreservation. Generally vitrification involves a very high concentration of solutes
to ensure total vitrification without any ice crystal
formation. While quite successful for several types
of reproductive cells, this method of vitrification is
inappropriate for the cryopreservation of mammalian
spermatozoa due to extreme sensitivity of spermatozoa to permeating CPA [68]. However, several studies
have shown success in achieving vitrification of sperm
samples at moderate to zero CPA concentrations. For
example, Schuster et al. demonstrated success by using
Cryoloops for ultra-rapid freezing of a small number
of human sperm with 12% glycerol as CPA in test-yolk
solution [69]. In their pioneering work, Dr. Isachenkos
group from Germany have successfully attempted to
vitrify a small volume of sperm without any cryoprotectants by using different carrier systems such as
Cryoloops, droplets, OPS and grids [6873]. They
demonstrated that vitrification of a small sample
of spermatozoa without cryoprotectant resulted in
higher motility after rapid warming in comparison to
conventional freezing with cryoprotectant [68]. Thus,
it appears that intracellular vitrication can be achieved
at relatively low cooling rates without any CPAs and
preliminary dehydration. This is most probably facilitated by the low intracellular water content and the
presence of abundant high molecular weight components such as proteins, polysaccharides and nucleic
acids in spermatozoa, which affect the viscosity and
glass transition temperature of the intracellular cytosol
[71, 72].
The success rate of slow-freezing cryopreservation of spermatozoa has been, for the most part, satisfactory [1]. Nevertheless, because of the damage
associated with freezing, the motility of post-freeze
spermatozoa is statistically reduced with respect to
pre-freeze motility, and success rates vary among
species and even among individuals within species.
This associated loss of viability may not be an issue
where ejaculate volume and sperm counts are normal.
However, in situations where the sperm retrieval yields
extremely smalls samples, conventional slow freezing may not be suitable and other appropriate methods including vitrification may be considered. Finally,
136
although CPA-free vitrication offers a fast and simple

method relative to slow freezing, the current evidence
is not sufficient to support the use of vitrification as
standardized protocols for semen cryopreservation. To
make any rational conclusions on this, well-designed
randomized trials with appropriate sample sizes are
needed to evaluate the effectiveness of various semenfreezing methodologies.
Cryobiology of oocytes
Cryopreservation of mammalian oocytes is one of the
most intensively studied topics in the field of cryobiology. For example, due to their regular spherical
shape and relatively large size, mouse oocytes were
used as model cells for numerous theoretical investigations and the tests of newly developed cryopreservation methods. Human oocyte cryopreservation plays
not only an important clinical role in assisted reproduction as an adjunct to sperm and embryo cryopreservation, but also a representative of the application of both the freezing and vitrification approaches.
Therefore, in this section, the discussions are concentrated on the current status of human oocyte cryopreservation.
Mature human oocytes

In the mature oocytes, the metaphase chromosomes
are lined up by the thin meiotic spindle along the equatorial plate. Such spindle apparatus is fragile and can
be easily damaged by IIF or cell volume change due
to osmotic effects. The first report of a pregnancy and
subsequent delivery of a baby derived from a frozen
and thawed oocyte appeared in 1986 and 1988, respectively [74, 75]. Several other reports appeared in the
late 1980s describing additional attempts to cryopreserve human oocytes [76, 77]. One notable feature of
these reports is the lack of fundamental experiments
designed to understand the cryobiology of human
oocytes. Instead, simple changes to a standard equilibrium protocol were made and outcomes were assessed.
Changes included altering the addition and removal of
the cryoprotectant (stepwise and at room temperature
[77]) and assessing the effects of the polyol propylene
glycol (PG) versus DMSO [76]. In general, the outcomes of these early reports were poor and highly variable. For example, immediate survival of 136 oocytes
recovered after thawing was 32% [77]; 58% of those
that survived underwent fertilization and 2 pregnancies ensued but neither went to term. In the report by
Al-Hasani et al., 28% of the oocytes frozen in dimethyl
sulfoxide (Me2 SO) survived and only half of those fertilized [76]. Oocytes frozen in PG tended to survive
better (32% and 75% of those fertilized). The second
report of a live birth came in 1987 using an equilibrium
method with DMSO as the permeating CPA [78].
Since 2001, at least 10 reports have appeared
describing the results of freezing human oocytes using
a standard equilibrium method with 1.5 M PG and
sucrose. Some of these reports show very high survival
and early development rates, and this has lead some
individuals to emphatically defend human oocyte
cryopreservation and suggest that the clinical results
are nearly equivalent to the use of fresh embryos.
Other investigators have been more cautious in their
interpretation [79], and in two recent reports with a
very large number of cycles, the results were still rather
poor [80, 81], especially when compared to the use of
fresh embryos [81].
Vitrification has also been utilized as a means to
cryopreserve human oocytes in recent years, gaining
more popularity than traditional equilibrium methods in recent years. Kuleshova et al. described a birth
resulting from an oocyte vitrified with a solution containing ethylene glycol and sucrose [82]. For this work,
the investigators utilized an OPS for the procedure.
Since this time, other reports on human oocyte vitrification have been published [83, 84], with all of these
reports utilizing open container systems to achieve socalled ultra-rapid cooling. However, the use of open
containers represents a potential problem for the possibility of disease transmission in the storage vessel
[85]. On average, the results from the vitrification trials
have been better than the trials using slow cooling (as
measured by the number of oocytes to achieve a pregnancy). However, the results are still limited in number and general conclusions are difficult to reach at this
time. Overall, progress on human oocyte cryopreservation has been significant during the past few years,
as evident by the number of reports appearing in the
literature. However, the procedure is still regarded as
suboptimal and experimental in nature.
Immature human oocytes

Due to the absence of a metaphase II spindle in GV
stage oocytes, it has been suggested that immature
oocytes may be more amenable to cryopreservation
[86]. Several reports have been published describing
attempts to cryopreserve GV stage human oocytes.
A low sodium solution proved more effective with
GV oocytes compared to a standard sodium chloride-
based solution, with similar rates of maturation, fertilization, and cleavage compared to controls [87]. In
a different study, the maturation rate and fertilization
rate of cryopreserved and control oocytes was similar, but blastocyst development was lower [88]. Vitrification has also proved successful with GV human
oocytes [89], with rates of maturation, fertilization and
early development similar between frozen and nonfrozen oocytes. Despite these successes, to date, only
one report describing a live birth after cryopreserving a
GV stage human oocyte appeared in the literature [90].
Clearly, more basic research needs to be undertaken to
improve this technology.
Ovarian tissue
Successful ovarian tissue cryopreservation is one of the
effective clinical options for preserving female fertility.
For example, orthotopic re-implantation of cryopreserved ovarian cortical strips is a promising technique
for restoring ovarian function in women treated with
sterilizing chemotherapy for cancer. Both equilibrium
freezing and vitrification methods have been applied
for human ovarian tissue cryopreservation. Hovatta et
al., using histological assessment, showed that a high
proportion of follicles could survive an equilibriumfreezing method with human ovarian tissue [91, 92].
In the first report, they showed very good morphological survival of ovarian follicles after cryopreservation using either DMSO or PG, with no obvious
differences between frozenthawed and non-frozen
tissue. In the second report, using an in-vitro culture system, they present data suggesting that follicle
development occurs in the previously cryopreserved
tissue, with development rates similar between the
frozenthawed and unfrozen tissue samples. Shortly
after these reports, a study undertaken to determine
the diffusion rates of cryoprotectants into human ovarian tissue was published [93]. The results from this
study suggest that ethylene glycol (EG) and DMSO
diffuse into the tissue more rapidly than PG and
glycerol. These results supported the previous findings of these authors which showed EG and DMSO
to be superior cryoprotectants compared to PG and
glycerol [94], suggesting that the rate of cryoprotectant permeation is a critical factor in successful cryopreservation of human ovarian tissue. A similar conclusion was reached in a different report, where the
time of equilibration prior to cooling had a significant
effect on the proportion of intact follicles post-thaw
[95]. Therefore, it is of both practical and theoretical
137
22.0
R 2 = 0.99
22.5
Log (D )
23.0
EG
DMSO
PG
23.5
24.0
24.5
25.0
25.5
0.34
0.36
100/T
0.38
0.40
Figure 11.3 The Arrhenius relationship between the values of D

(unit: m2 /s) and the measurement temperatures (20, 10, 0, 10 and
20 C). DMSO, dimethyl sulfoxide; EG, ethylene glycol; PG,
propylene glycol.
importance to address the issue of optimizing cryoprotectant permeation into human ovarian tissue by
measuring permeability parameters of the relevant
cells and using this information to model the mass
transport of water and cryoprotectant. Various physical models regarding the permeation of cryoprotectants into tissues have been established. A differential scanning calorimetry method to measure the
in ovarcryoprotectantwater mutual diffusivity ( D)
ian tissues at both super and sub-zero temperatures
was also established [96]. Figure 11.3 shows that the
Arrhenius relationship was strictly followed for the
values of D with different cryoprotectants. Due to the
relatively low values of D (106 cm2 /s), it has been
suggested that the optimal cooling rate for equilibrium
freezing procedures for ovarian cortical strip should
be at the order of 0.5 K/min. For the cryoprotectant
perfusion procedure before cooling, a stepwise perfusion is preferred and the perfusion time should be controlled to approximately 1 h.
Follicle loss after transplantation is likely due in
part to ischemic damage [9799]. However, using fresh
or frozen ovarian tissue from cynomolgus monkeys,
Schnorr et al. showed that only two of four recipients had functional frozen and thawed transplants, yet
five of six of the ovarian transplant recipients receiving fresh tissue had functional transplants [100]. In a
more recent study using ovarian tissue from cynomolgus or rhesus macaques, immediate post-thaw viabil-
138
ity of follicles isolated from the thawed tissue showed

a fairly high percentage of viable follicles (6770%)
with the various freezing methods used. This number
was lower, however, when compared to follicles isolated from fresh tissue (76%). Due to the historical lack
of established methods for the in vitro culture of isolated follicles [101], accurate assessments of the developmental potential of follicles after freezing has been
difficult to establish.
In more recent years, several studies have shown
that ovarian follicles can develop in previously frozen
and thawed human ovarian tissue after xenotransplantation into immunodeficient mice. These reports
showed the development of antral follicles [102], MII
stage oocytes [103] and corporea lutea with increased
levels of circulating progesterone [104, 105]. Despite
these positive signs, the developmental potential of
the oocytes from those follicles remained uncertain.
At least 12 reports describing ovarian graft function
after autotransplantation in humans have been published in the past several years (see Donnez et al.
[106] for a recent review). In many of these studies,
follicular development and accompanying endocrine
changes, suggesting restoration of graft function were
noted. However, to date, only two live births have been
described [107, 108] resulting from transplantation of
previously frozen ovarian tissue; in two other reports,
pregnancies ended in miscarriage [109, 110].
Cryobiology of embryos
Because of its central importance, the cryopreservation of human embryos has become an integral part
of almost every ART program. According to International Committee Monitoring ART (ICMART), the
majority of the children (up to 40% worldwide) born
after ART are now born from cryopreserved embryos
[111], and this number is likely to increase in future.
Embryo cryopreservation is also a widely used method
of fertility preservation for cancer patients [112].
Cryopreservation allows the storage of excess viable
embryos for future use in an in vitro fertilization (IVF)
treatment cycle. In addition, cryopreservation makes
feasible the postponement of embryo transfer in the
event of a mother becoming ill or with patients at high
risk of ovarian hyperstimulation syndrome [113].
Over the years, major empirical advances have
been made to develop successful protocols for the
cryopreservation of embryos. Literature shows that
majority of the studies either used glycerol, EG or
DMSO as CPAs. Recently, however, these cryoprotectants have been increasingly replaced by 1,2 propanediol, which is generally used in combination with
other permeable and non-permeable cryoprotectants.
Propanediol is considered to have higher penetration permeability and is less toxic than DMSO. Both
slow cooling and vitrification have been studied for
embryo cryopreservation. The slow cooling technique
was developed independently in 1972 by Wilmut and
Rowson for bovine embryos and Whittingham et al.
for mouse embryos using DMSO as CPA [18, 19].
The same freezing protocol was applied to human
early-cleavage stage embryos and resulted in the first
report of pregnancy in 1983 [114] and live births in
1984 [115]. In 1985, PG along with sucrose was first
introduced in human embryo freezing and proved
to be quite efficient for 1, 2 or 3 day-old embryos
[116]. Therefore, this method was rapidly applied and
has become the widespread application for embryo
preservation in virtually every full scale IVF program
worldwide [117]. In general, slow freezing of embryos
employ progressive pre-equilibration in a CPA solution and then cooling to a seeding temperature at
which deliberate ice nucleation is initiated to prevent
supercooling. After equilibration for about 1015 min,
the cells are cooled slowly (1 C/min) to about
30 C till they are sufficiently dehydrated and then
finally plunged and stored in liquid nitrogen. Normally, frozen embryos are thawed slowly to prevent
excessive osmotic stresses. Finally, the CPA is removed
either by successive dilution or by using a sucrose dilution technique [118].
Slow cooling, however, is a long process and
requires extensive resources like controlled rate freezers and large quantities of liquid nitrogen. Further, the
studies using slow freezing have reported significant
differences in survival and developmental rates after
warming between laboratories, developmental stages
and quality [119121]. Vitrification, on the other hand,
has increased greatly in use in recent years particularly
for freezing reproductive cells, and avoids the multihour long process of slow cooling to 196 C. For vitrification, embryos are usually loaded with high concentrations of CPA and then plunged directly in LN2
using some specialized carrier or supporting device.
For thawing, usually high warming rates are used to
prevent ice crystallization during devitrification. There
are, however, several factors that need to be taken
into consideration while undertaking the vitrification
of embryos. The type of CPA used, its concentration
and exposure time, all create toxicity issues. These can

be alleviated to some extent by using combinations
of permeating and non-permeating CPAs or by loading embryos with CPAs at sub-zero temperatures [1].
There are also some concerns regarding contamination in LN2 tanks as open carrier systems allows direct
contact of embryos with LN2 posing a risk for disease
transmission and rendering their use in human IVF
debatable [122].
Nonetheless, despite some drawbacks, vitrification
has become a viable and promising alternative to traditional slow freezing, and previous published data
appears to indicate that vitrification produce at least
equal or significantly better results than those obtained
from slow cooling for cryopreservation of mammalian
embryos [122, 123]. For example, Kuwayama et al.
compared slow freezing with vitrification by using
human embryos at various stages of development
[122]. They reported that vitrification resulted in 100%
survival of pronuclear stage embryos with 93% cleavage stage rate and 52% blastocyst rate. Conversely,
with slow freezing the pronuclear stage survival rate,
cleavage stage rate and blastocyst rate were only 89,
90 and 41%, respectively. They further demonstrated
that vitrification was superior to slow freezing for cryopreservation of four-cell embryos and blastocysts.
However, clinical pregnancy rates after the transfer of
frozen thawed embryos were not significantly different
between the two methods of cryopreservation [123].
On the other hand, Rama Raju et al. reported significant difference in pregnancy rates of 35% and 17.4%
after vitrified and slow-cooled 8-cell embryo transfer,
respectively [124]. In a study by Li et al., randomized
cryopreservation of 160 day-3 embryos resulted in no
significant difference in post-thaw survival (vitrification: 89% versus slow freezing: 91%) and clinical pregnancy rates (vitrication: 48% and slow freezing: 38%)
[125].
Although it appears that vitrification is superior to
slow freezing in terms of post-thawing survival rates in
different developmental stages of human embryo cryopreservation, it is premature to make any solid conclusions regarding the relative efficacy of vitrification
versus slow freezing. This is mainly due to the fact that,
at present, there are not enough randomized control
test data available [2]. Therefore, additional randomized controlled trials using uniform criteria for defining post-thaw embryo quality are needed to determine if vitrification should be the preferred method of
embryo cryopreservation in IVF laboratories.
139
Conclusion
Within the realm of reproductive cryobiology, a better understanding of the basic features of what we have
described here as classical cryopreservation and vitrification is critical to accelerate advancement and to
allow individual clinical laboratories to make processing choices appropriate to reaching their desired outcomes. This includes clarification of terms, theoretical and technical details, and a balanced, pragmatic
evaluation of possible risks and potential (or definite)
gains [2]. As the survey of preservation methods and
results for gamete, tissue and embryos presented here
indicates, the ability to preserve these cells and tissues
has had a profound effect on humankind with millions
of births around the globe facilitated by these efforts
while truly optimum protocols are yet to be uncovered. Because of the complex nature of the biological
systems as well as the physical phenomenon in question, a scientific approach balanced between theoretical and empirical methods is absolutely necessary to
ensure procedures continue to be further optimized.
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Section 3
Chapter
12
Fundamental aspects of vitrification as a

method of reproductive cell, tissue and
organ cryopreservation
Steven F. Mullen and Gregory M. Fahy
Introduction
In the field of assisted reproductive technology, vitrification is becoming an increasingly popular method of
cryopreserving cells, tissues and even entire organs [1
4]. The National Library of Medicines public database
(PUBMED) lists an exponentially increasing number
of citations on vitrification over the past decade, many
of them pertaining to vitrification in reproductive biology.
Vitrification in the present context is the process
whereby an aqueous solution is transformed into an
amorphous solidified system as a result of a significant
decrease in temperature [5]. The three-dimensional
arrangement of the molecules in such a system resembles that of a liquid (i.e. remains disordered), but
the viscosity and associated shear relaxation time are
more solid-like. This is in contrast to freezing methods, where molecular rearrangements in the form of
extensive ice formation occur in the solution during
the cooling process.
Vitrification as a means of cryopreservation is an
attractive alternative to freezing for several reasons.
The two most obvious are the complete elimination
of ice formation and its consequent damage, and the
ability to avoid chilling injury by rapidly cooling a
sample through the temperature zone known to be
damaging [68]. Other advantages include reduced
labor and equipment costs [9]; the lack of need to
discover optimum cooling and warming rates; and
scalability from cells to tissues and even to whole
organs. Some recent reports suggest that vitrifying
oocytes and embryos is a more effective means of
cryopreservation than is freezing [1013], provid-
ing additional support for its adoption in a clinical

setting.
The formation of ice during a cryopreservation
procedure is often associated with damage to the biomaterial [14, 15]. More than 30 years ago, intracellular
ice formation (IIF) visualized using cryomicroscopy
was shown to be strongly correlated with irreversible
damage to mouse oocytes [16]. While examples of
non-lethal IIF are found in the literature [1719], it is
currently believed that, in most cases, IIF is lethal [14]
and should be avoided. But even extracellular ice formation can cause significant damage, whether by acting to nucleate intracellular ice [20] or by disrupting
the extracellular matrix of organized tissue or compressing packed cells [21]. Perhaps most significantly,
survival after cryopreservation by freezing is the result
of a compromise in which some cells may die due to
IIF and some may die due to excessive shrinkage, often
making it difficult to achieve 100% survival. Vitrification eliminates this dilemma. Hence, it is reasonable
to expect that the complete avoidance of ice formation
during cryopreservation would improve outcomes.
A discussion of the specific methods of vitrification currently utilized in fertility preservation and the
rationale for those methods will be covered by other
chapters in this volume. The purpose of this chapter is
to provide a brief historical overview of vitrification as
a means of cryopreservation; to delineate some of the
principles governing crystallization, vitrification and
storage in the vitreous state; and to discuss strategies
for developing improved vitrification solutions. Given
that the current emphasis in reproductive cryobiology
has been to come as close to freezing as possible without actually allowing ice to form (in order to be able
145
to use the minimum possible concentrations of cryoprotectants), some understanding of the physics of ice
formation is necessary to understand the risks associated with different vitrification methods and, indeed,
to be able to determine whether vitrification is complete or partial. Interested readers are encouraged to
consult a number of authoritative reviews for further
details [35, 9, 2229].
Historical origins of vitrification as a

means of cryopreservation
The possibility that water might be vitrified was first
proposed by Brayley in the mid 1800s [30], but the idea
of cryopreservation by vitrification was apparently not
introduced until Stiles observed, in 1930, that protoplasm is likely, at very high cooling rates, to form a
finely crystalline or even amorphous mass that in
thawing, might be expected to give again the original system without change [31]. It took Father Basile
J. Luyet, however, to independently develop this idea
into a major research proposal in 1937 [32]. Luyets
rationale for vitrification was that life is due to a
special and exceptional arrangement of the atoms, or
other structural elements of living cells [33] and that
death from freezing seems to result from the disruption of the units which constitute living matter when
the molecules of water are torn away from these units
by the forces of crystallization [34]. This problem
would clearly be prevented by vitrification, and Luyet
observed that the essential problem of the vitrification
technique consists in . . . obtaining a cooling velocity
sufficient to prevent the formation of crystals [32].
Luyet and his colleagues focused on this problem
until about 1958 [24]. In that year, perhaps inspired
by criticism of Luyets evidence for vitrification by
Audrey U. Smith in 1954 [35], two fateful articles were
published in Biodynamica, one by Luyet and Rapatz
and the other by Meryman. These papers showed that
gelatin gels previously believed by Luyet to be vitreous in fact contained ice in the form of evanescent
spherulites that were detectable both optically [36]
and by X-ray diffraction [37]. This revelation abruptly
ended Luyets pursuit of vitrification, and in 1969 he
described his results as mostly negative and of academic interest [38]. Thus, Luyet introduced vitrification as a concept, but he did not provide it as a workable cryopreservation method that was widely adopted
by others. He did, however, continue his studies in
other areas beyond 1958, focusing on ice crystal mor-
146
phology and phase diagram relationships rather than

on the pursuit of vitrification. Ironically, it is these later
studies that ultimately provided the physical basis for
vitrification as it would later develop [24].
In 1978, Boutron and Kaufmann recognized that
in the extreme case of a solution which remains
entirely amorphous even at very slow cooling or warming rates, all cells should be protected [39]. However, they also rightly noted that the high concentrations required for vitrification tend to be toxic [40],
but lower concentrations result in the need for astronomical warming rates to escape from freezing during
re-warming [39], a less than encouraging situation.
Although he ultimately obtained survival of putatively
vitrified red blood cells in 1984 [41], the need to warm
these cells at 5000 C/min and their lack of DNA did
not make a particularly inspiring general case for vitrification, and this limited Boutrons early impact on
the practical development of vitrification as a new
and general method of cryopreservation. However,
Boutrons seminal contributions to our understanding
of the cooling and warming rate dependence of ice formation, and therefore of glass formation and stability of the amorphous state during warming, remain
unparalleled (see discussion below and Mehl [27]).
Fahys trajectory toward vitrification began in
1970, when he read of the partial success of Barry
Elford in recovering smooth muscle strips that were
prevented from freezing at temperatures as low as
79 C by using very high concentrations of dimethyl
sulfoxide (Me2 SO) to depress the freezing point [42].
Fahy pursued this concept as an undergraduate student using frog sciatic nerves [43], and found that the
nerves could tolerate 30% but not 40% Me2 SO [G. M.
Fahy, unpublished results]. As a graduate student he
found that he could treat rabbit renal cortical slices
with 40% Me2 SO at 22 C with reasonable recovery,
but not with 50% Me2 SO at 35 C [44]. His inquiries
into this approach continued when, as a postdoctoral
fellow pursuing the goal of organ cryopreservation, he
learned that, according to unpublished data of Rajotte
and McGann, dog kidney cortical slices could survive
exposure to 30% Me2 SO plus 30% sucrose, an impressively high total concentration of cryoprotectant [45].
However, he soon found that solutions of this kind
required total concentrations of 7080% (plus 5% w/v
glucose added to simulate a carrier solution) to prevent freezing for long periods at 79 C, and most solutions either froze or developed ice spheres millimeters
in diameter (Table 12.1).
Chapter 12: Fundamental aspects of vitrification
Table 12.1 Supercooling properties of solutions containing sucrose.a
% Glucose
% Sucrose
% Glycerol
% Me2 SO
% EG
% MeOH
Resultb
40
30
40
30
30
44
30
40
F, NF
30
40
IS
30
40
NF
30
35
10
IS
30
30
10
30
30
10
NF
20
30
10
IS
20
30
20
IS, NF
20
25
25
IS
20
20
20
20
20
20
20
20
20
10
IS
%w/v glucose and sucrose, % v/v glycerol, Me2 SO, EG and MeOH. EG, ethylene glycol; MeOH, methanol. Experiments carried out from
12/21/1979 to 3/17/1980; previously unpublished data.
Results determined after 1119 days of storage in triplicate 15ml samples in test tubes. F, frozen; IS, ice spheres; NF, not frozen.
Although these results were discouraging, Fahy

reasoned that success might still be obtained if the
central problem of prolonging the time required for
ice to form and grow in the deeply supercooled state
could somehow be solved, and he reflected on how
this might be done without using lethal solute concentrations. The idea of lowering the temperature below
79 C immediately led to a sudden inspiration: what
if solutions of tolerable concentrations could be cooled
all the way to the glass transition temperature without
freezing? They would then be stable indefinitely! Thus
was born the idea of achieving vitrification by the reliable deep supercooling of even organ-sized volumes
into the vitreous state.
Fahy had previously seen that Me2 SO solutions and
pure ethanol remained transparent but fractured when
cooled in liquid nitrogen (LN2 ), so he began extensive and still-unpublished studies of fracture avoidance
in 1980. These studies were soon followed by studies
on reducing toxicity by the use of cryoprotectant mixtures [46], including putative toxicity-blocking amides
[4648], studies on the minimum concentration of
cryoprotectant required for vitrification [48, 49], and
the use of 1,2-propanediol [48, 49] and high hydrostatic pressures [48, 49] to reduce the latter. By mid
1981, he had assembled enough pieces of the puzzle
to present the idea in public for the first time [48] and
published confirmation of the utility of high pressures
[50]. More thorough expositions of the idea were provided in 19821984 [22, 49, 51, 52].
By 1984, Fahy had clearly defined the physicochemical requirements for true vitrification; had
demonstrated the possibility of highly concentrated
yet minimally toxic solutions for vitrification; had
explained the idea of combining permeating and nonpermeating cryoprotectants to induce vitrification in
a manner consistent with the vitrification tendency
of the intracellular compartment; had provided efficient methods for adding and removing vitrifiable
concentrations; had described taking advantage of
osmotic shrinkage for facilitating vitrification; had
demonstrated methods for avoiding fracturing even
in objects larger than rabbit kidneys; and had presented his new preservation method as one that should
be applicable not just to organs but to all cells and
tissues [22]. However, few cryobiologists concerned
themselves with organ cryopreservation, and therefore few read about these developments or realized
their broader applicability. In addition, there still
remained no actual demonstration that living cells vitrified according to Fahys methods could actually survive after re-warming.
147
Fortunately, William Rall became aware of Fahys

ideas and recognized the opportunity to make a major
contribution by proving their applicability to mammalian pre-implantation embryos, a system of great
interest to many cryobiologists then and now. Rall
had himself concluded that slowly frozen embryos survive as a result of intracellular vitrification [53, 54],
so he realized that embryos should be able to tolerate
both intracellular and extracellular vitrification, making embryos an excellent system for an initial demonstration of Fahys method. Rall joined Fahys lab at the
American Red Cross, and the two worked together to
show that embryos could survive vitrification both at
cooling rates applicable to small organs (20 C/min,
achieved by cooling at maximum speed in Fahys small
organ high pressure vessel) and at much higher rates.
Fahy provided the vitrification solution, VS1, and Rall
provided the method for adding and removing it.
The result of this collaboration [55] definitively
established, for the first time, vitrification as a viable
and potentially general alternative to freezing. Since
then, the number of papers on biological vitrification
has grown at an exponential rate [3, 56]. The systems that have been successfully preserved by vitrification to date are too numerous and diverse to list here,
but include many examples from within the realm of
reproductive biology, as reflected in the balance of this
book, and finally even include, as of 2009, the survival
and life support ability of the entire rabbit kidney following vitrification, re-warming and transplantation
[57]. For additional details about the history of vitrification, the reader is referred to other partial accounts
[3, 24, 45, 57, 58].
Physical aspects of vitrification

Ice nucleation and growth
Whether water in aqueous solutions is thermodynamically stable in the liquid or solid phase at a specific temperature is determined by the difference in the Gibbs
free energy (G) between each phase at that temperature
[59]. As the temperature of an aqueous solution is lowered below its melting temperature (Tm ), Gice becomes
less than Gliquid water , and ice becomes the more stable
state. G = H TS, where G is a function of both the
enthalpy (H; heat content) of a system and the entropy
(S; degree of order), T being the temperature on the
Kelvin scale. Both H and S decrease as the temperature
is lowered; hence, whether the difference between the
148
G
Ice
Pure water
Aqueous solution
Tm Solution
Tm Pure water
0
Temperature (C)
Figure 12.1 An example of the relative values of G for pure water,
water containing dissolved solutes and ice. A physical state is more
thermodynamically stable than an alternative state if its value for G
is lower than that alternate states value. As can be seen, Gice is
lower than Gpure water at low temperatures; hence ice is the more
favorable state ( G is negative). The temperature where these lines
cross is the melting point of water (Tm pure water; 0 C), and at that
point, both states are equally stable. Adding solutes to water causes
the curve to shift to the left, decreasing the value of G relative to
pure water. This decreases the temperature at which ice becomes
the more stable state (Tm solution). The curve for solutions would
continually be shifted to the left if additional solutes were added,
further depressing the melting point. The relative difference
between the curves for water and ice at a specific temperature (as
indicated by arrows) is an indication of the degree of supercooling
for a solution remaining liquid below Tm , and can be thought of as a
driving force for a phase transition. Hence, as a liquid is lowered
below its melting temperature, the strength of the driving force
continually increases. Note the difference in the length of the
arrows for G for water-ice (single arrowheads) and solution-ice
(double arrowheads) at specific temperatures. Modified from Angell
and Senapati [59].
two states ( G) is positive or negative depends upon

the relative contribution of H and (TS) to the overall
energy of a system. Overall, G increases as temperature decreases for water in an aqueous solution, and
the same is true for ice. However, Gliquid water increases
faster than Gice , and it is this ever increasing difference
in G for the two states that is the driving force for crystallization (Figure 12.1).
Despite G (Gice Gliquid water ) becoming negative as cooling passes below Tm , immediate crystallization rarely occurs. Crystallization is initiated by random and statistically improbable aggregations of water
molecules to form small volumes of the new phase
(known as ice crystal nuclei) [60]. Creation of a stable nucleus entails overcoming an energy barrier associated with the formation of the liquidcrystal interface. The interfacial energy of a nucleus is related to its
The necessity of the vitreous state

Vitrification can be seen as the means by which
an aqueous solution remains within the bounds of
thermodynamic law. As a solution is cooled to
Ice nucleation
Ice growth
Relative rates
radius of curvature, with smaller nuclei having larger

surface free energies per unit area. As a result, at temperatures slightly below Tm , only very large (and therefore statistically very improbable) nuclei are stable. In
fact, in most instances, ice nucleation during cooling
does not occur through the self-aggregation of water
molecules to form a stable nucleus (a process called
homogeneous nucleation), but rather takes place by
water molecules becoming organized on foreign particles that effectively reduce the amount of iceliquid
interface and thus lower the free energy barrier associated with the formation of a stable nucleus. This
process is termed heterogeneous nucleation, and the
temperature at which this occurs is generally substantially higher than the homogeneous nucleation temperature (Th ). It is only at temperatures far below Tm
that homogeneous nucleation becomes energetically
favorable. The homogeneous nucleation temperature
of pure water is near 40 C [60]. It has been estimated
to require 45 000 water molecules to form a stable
nucleus at 5 C, but only 70 molecules at Th [60].
Once a stable nucleus is formed, crystallization
continues through crystal growth. Crystal growth is a
kinetic phenomenon; molecules must diffuse from the
liquid phase to the interface and rotate to be incorporated into the crystal. As a result, the crystal growth
rate is highest at temperatures near Tm where molecular mobility is high. Nucleation is also a kinetic process, and at lower temperatures the rate of molecular
motion slows so much that the nucleus formation rate
is reduced (Figure 12.2 [61]).
Based upon these ideas, it is easy to understand
why the current methods for oocyte vitrification have
evolved. Cooling very quickly minimizes the time
available for nucleation and crystal growth to occur.
Under the right conditions (see below) a solution can
therefore be cooled and warmed rapidly with no apparent ice formation. Vitrification, however, does not
inherently rely upon very high rates of cooling because
ice nucleation and growth rates go down as solute concentration goes up. High cooling rates simply make
vitrification more likely, and also, as discussed below,
diminish the solute concentration necessary to attain a
vitreous state.
Tg
150
125
Tm
100
75
50
Temperature (C)
Figure 12.2 The maximum rates of ice nucleation and ice growth
occur at very different temperatures in effective vitrification
solutions. The curves represent the temperature dependence of
these two rates for a specific vitrification solution (M22) [58].
Whereas the maximum rate for nucleation occurs near Tg , the
maximum rate for crystal growth occurs near Tm . With permission
from Wowk [61].
temperatures below Tm , an interesting relationship

between the entropy of the supercooled solution and
the entropy of an identical solution undergoing freezing causes the entropy to plummet, but below Tm the
rate of reduction of the entropy of the supercooled liquid is greater than that of the corresponding frozen
solution. If the trend were to continue to even lower
temperatures, the supercooled solution would eventually have less entropy than the corresponding mixture of concentrated solution and ice. Such a situation,
however, would be in violation of the known principles of thermodynamics because a crystal has the lowest entropy possible for a given substance. Kauzmann
discussed this paradox and elucidated a rationale for
its resolution [62].
With a sufficient reduction in temperature, thermal energy becomes insufficient to drive rotational
and translational motions. Water molecules become
trapped in local energy wells due to the removal of
internal energy and the entropy therefore stabilizes,
and remains above that of the ice phase. This event
is referred to as the glass transition, and it occurs at
temperatures between about 110 C [63] and about
130 C [39, 40] for low molecular weight cryoprotectants in water. The viscosity of a glass is around
1013 Poise, 15 orders of magnitude greater than the viscosity of water at room temperature!
149
Figure 12.3 Relationships between the values for the melting

temperature (Tm ), homogenous nucleation temperature (Th ),
devitrification temperature (Td ), the glass transition temperature
(Tg ), the concentration needed to vitrify without homogeneous
nucleation (Cv ), the concentration allowing no devitrification (Cndv ),
the eutectic concentration (Ce; glycerol usually does not crystallize
during cooling) and the concentration that is sufficient to prevent
the growth of pre-existing ice (thus, the unfreezable
concentration, Cu ) for glycerol dissolved in water (modified from
previous representations [3, 22, 49]). In Section I, even above Th , ice
nucleation events, indicated by Xs, are generally inevitable due to
heterogeneous nucleation. On the contrary, in Sections IV and V, ice
formation never occurs during slow cooling and warming. In
Section II, sufficiently rapid cooling results in partially crystallized
glass (PCG) due to the presence of at least homogeneous nuclei
even though visual inspection would suggest the absence of ice.
Section III defines the lowest concentration range for true
vitrification. See text for more details.
The concentration dependence of

vitrification and devitrification
A supplemented phase diagram (Figure 12.3, for glycerol in water in this particular example) [22] is often
used to visualize the relationships between key variables pertinent to vitrification. As can be seen by the
curves in Figure 12.3, Tm and Th both decrease with
increasing solute concentration, and the glass transition temperature (Tg ) increases. With enough solute,
Th can be reduced to below Tg , making vitrification possible without contaminating homogeneously
nucleated ice nuclei. Below this threshold concentration, Cv , one forms partially crystallized glass (PCG)
unless cooling is so accelerated as to drive Th to lower
temperatures such that Th again intersects Tg at the
concentration employed. Partially crystallized glass
formed by rapid cooling with homogeneous nucleation is particularly liable to crystallization on warming to Td , the temperature of devitrification (ice formation on warming).
150
Figure 12.3 is partitioned into sections, with each

section bearing a particular relevance to cryopreservation. In section I, solute concentration is relatively
low and therefore nucleation (via homogeneous and
heterogeneous mechanisms) and extensive ice growth
is inevitable [22] except at ultra-rapid cooling rates
(see below). For solutions with concentrations associated with section II, crystallization during cooling
may be minimal, but nucleation still occurs extensively. Such a solution may appear to be vitrified, and
classification as such may be appropriate. However,
such doubly-unstable glasses (unstable both thermodynamically and by virtue of being extensively nucleated) are prone to devitrification, i.e. extensive crystal formation and growth during warming [2, 5]. Such
instances of crystallization may not be damaging if
cooling and warming rates are high, for the size of
individual crystals may remain relatively small, and
the crystals may be in the form of cubic ice, which
is believed to be more innocuous to biological systems than regular hexagonal ice [41]. At slightly higher
concentrations (region III), homogeneous nucleation
is at least nominally precluded due to Th being below
Tg (nominally because holding just below Tg may
allow the sub-Tg extension of the Th curve to be
observed [64]); the lower boundary of region III thus
defines the minimum concentration needed to vitrify at slow cooling rates (CV ) [22, 49]. However,
until the upper bound of region III, devitrification
is still a problem. Beyond region III, even devitrification fails to occur during slow warming, making
this the ideal region for vitrification. Unfortunately,
for most cryoprotectant solutions, such concentrations
are extremely toxic, and thus cannot be used successfully, although this problem is being overcome
[58, 65].
It should be noted that the locations of these
boundaries are not absolute due to the fact that the
values of Th , Td , and Tg are rate dependent. The values shown apply at cooling and warming rates of
10 C/min, but at extreme cooling and warming rates
associated with some of the open systems utilized for
oocyte vitrification, the point of intersection between
Th and Tg will be at lower concentrations. Thus, solutions containing lower concentrations of cryoprotectants can remain amorphous during vitrification with
these systems. However, mapping phase diagrams at
extreme rates is difficult, and to date we lack diagrams
like Figure 12.3 that are applicable under such conditions.
Th
104
Relative time
1
thom
104
thet
108
tmr
1012
Tg
Temperature
Tm
Figure 12.4 Generalized time scales for a specific fraction of a

solution to crystallize during cooling (via heterogeneous or
homogeneous nucleation) as well as the time necessary for
molecular rearrangements to occur within a solution (tmr ) as a
function of temperature. At temperatures just below the melting
temperature Tm , crystallization can occur, but is unfavorable due to
the instability of small nuclei. With continued temperature
reduction, nucleation occurs and the time for crystallization drops
dramatically. However, with further temperature reduction, the time
for crystallization increases. This occurs because the molecular
movements required for crystal development become constrained
kinetically by the lack of available thermal energy, as reflected by
tmr . The different time scales for crystallization via homogeneous
and heterogeneous nucleation reflect the fact that catalyzed ice
nucleation occurs at higher temperatures and requires fewer
molecular rearrangements to occur. Fortunately, in practice solutes
inhibit heterogeneous nucleation particularly well, tending to
eliminate it at Cv . See text for more details. Tg , glass transition
temperature; Th , the homogeneous nucleation temperature; thet ,
crystallization via heterogeneous nucleation; thom , crystallization via
homogeneous nucleation;Tm , melting temperature. Adapted from
Angell and Senapati [59].
Kinetic aspects of ice avoidance

As described by Angell and Senapati, two time scales
are relevant to crystal formation and growth (Figure
12.4) [59]. As the temperature of a solution drops
below Tm , the time required for a given fraction
of a solution to crystallize initially decreases (upper
two curves). This is due to the increase in the driving force for crystallization as the temperature is
reduced coupled with fast ice growth rates at relatively high sub-zero temperatures. On the contrary,
as a solution cools, the time required for molecular
rearrangements within a solution to occur (i.e. viscosity), also increases (lowest curve). The second time
scale begins to dominate as the temperature is continuously lowered. Eventually, the time necessary for
water molecules to arrange themselves into a crystalline structure becomes very long longer than
observable time scales, thus confining ice development
to higher temperatures. Hence, at lower temperatures
crystallization is thermodynamically favorable, but it
does not occur on a normal time scale simply due to
kinetic barriers that preclude the molecules from joining a crystal.
Boutron used a semi-empirical approach to
describe the dependence of the total quantity of ice
crystallized on the cooling rate for given solutions.
Under the assumptions described in his work, and
according to the most accurate model, the quantity
of ice crystallized during cooling is given by equation
12.1:
1
ln(1 x 3 ) + 0.5 ln(1 + x 3 +1 + x 3 )
1
1
+ 3 arctg(( 3x 3 )/(2 + x 3 )) = k4/|V|
[12.1]
where x is the ratio of ice crystallized on cooling to

the maximum crystallizable ice, V is the cooling rate
and k4 is an empirical constant [66]. In the same work,
he also developed analytical expressions for devitrification on re-warming [66], and the predictive nature
of these equations turned out to be quite accurate (see
also [67, 68]). As an example based upon the figures
published in Boutrons report, a binary solution consisting of 45% (w/w) ethylene glycol (EG) in water
cooled at approximately 80 C/min results in approximately 1/3 of the amount of ice formed compared to
the same solution cooled at 20 C/min. Increasing the
cooling rate to 160 C/min causes the total ice formation to decrease by 95%.
In recent times, oocyte and sperm vitrification have
focused on using the highest possible cooling rates
and the minimum possible concentrations of cryoprotectant [6, 69, 70]. Although differential scanning
calorimetry measurements in this cooling rate regime
are not feasible, Toner et al. [71] were able to estimate the cooling rate necessary to achieve vitrification as a function of concentration even at very low
concentrations by solving Boutrons equations. The
results of these calculations, plus some experimental
data for comparison [68], are provided in Figure 12.5
along with estimates for 0% solute [72, 73]. Figure 12.5
applies to solutions of pure cryoprotectants in water,
but provides a reasonably good indication of the cooling rates needed to justify claims of vitrification using
more dilute concentrations of cryoprotectant in carrier
solutions when the effect of the latter as extra solute is
taken into account.
151
Critical cooling rate (C/min)
for the growth of ice crystals to become detectable as

homogeneous nucleation density becomes more and
more astronomical. Fortunately, vcwr depends not only
on the cryoprotectant concentration but also on the
solute concentration of the carrier solution, which in
some cases lowers vcwr as effectively as an equal mass
of the cryoprotectant itself (Figure 12.6 [3]). Most vitrification solutions also contain sugars and other polymers; these compounds also significantly affect these
critical rates [67, 7577]. It is likely that intracellular proteins also affect the intracellular critical cooling
and warming rates, but little information regarding the
magnitude of the effect is available at this time.
The relationship between critical cooling rates and
critical warming rates for several individual cryoprotectants is shown in Figure 12.7 [7880], which highlights the challenge of avoiding devitrification after a
vitreous state is achieved during cooling with lower
concentrations of cryoprotectant. On the other hand,
examples of the cooling and warming rates allowing
survival of erythrocytes with two different cryoprotectants are also shown and indicate that survival is
possible even when warming is orders of magnitude
slower than the warming rate needed to prevent devitrification. Additional examples of this phenomenon
have been tabulated elsewhere [81]. In further support, Seki and Mazur have recently determined the
cooling and warming rates necessary for high survival
CPAs in water
Figure 12.5 Critical cooling rates for solutions of cryoprotectants

in water in relation to their concentrations. Estimates of the vccr for
pure water from Bald [72] (star) and Bruggeller and Mayer [73]
(circles) are shown at the upper left (0% solute). The gray circles for
glycerol and open diamonds for propylene glycol (PG) are estimates
(derived from [71] and used here with permission). (Ethylene glycol
[EG] data and observed PG data (black diamonds) are from [68].)
Modified from Fahy and Rall [3].
The warming rates necessary to avoid devitrification, i.e. the critical warming rates (vcwr ), are generally higher than those needed to vitrify (critical cooling
rates, vccr ) by several orders of magnitude. Baudot and
Odagescu [68] reported, for example, the values of vcwr
for solutions with 50, 45 and 40% (w/w) EG in water as
853, 1.04 106 and 1.08 1010 C/min, respectively.
Such rates reflect the increasingly short times needed
PG in water
(a)
(b)
Figure 12.6 Effect of the carrier (physiological support) solution on vcwr . (a) When vcwr is plotted as a function of cryoprotective agent (CPA)
concentration only, it can be seen that the presence of the sugar-rich EuroCollins solution carrier (EC) can lower vcwr for 30% w/w
2,3-butanediol (2,3-BD) by 4 orders of magnitude, whereas salt-rich carriers (PBS [circles] and St Thomas solution [stars]) have a smaller
effect. (b) If the same data are plotted as a function of total solute concentration (CPA plus carrier solutes), it can be seen that the effect of the
carrier is approximately the same, gram for gram, as that of the cryoprotectant, at least for 2,3-BD solutions. For solutions of propylene glycol
(PG), the presence of salt carrier solutions may actually slightly increase vcwr . These results allow the contribution of the carrier to vitrification
solution stability to be estimated fairly easily as a rough rule of thumb. (Data from [66, 67, 74].) Modified from Fahy and Rall [3].
152
ciable freezing is avoided on the way down to below Tg ,

but not on the way back up to above Tm .
Thermo-mechanical instability in
vitreous materials and the problem of
glass fracturing
Figure 12.7 Warming rates necessary for either survival (dotted

lines) or the prevention of devitrification (solid or dashed lines) as a
function of the critical cooling rate for solutions of cryoprotectants
either in water or salt-based carrier solutions. Note that vcwr is two
or more orders of magnitude greater than the vccr , a point often
overlooked in the reproductive cryobiology literature. The values for
survival are not meant to indicate the lowest warming rates
compatible with survival, but are only known examples showing
survival at far below the actual vcwr of similar solutions. (Physical
data from [40, 67, 68, 78, 79]; survival data from [18, 41, 80].) BD,
butanediol; EG, ethylene glycol; G, glycerol; PG, propylene glycol.
of mouse oocytes when using a more complex vitrification solution (EAFS 10/10, consisting of 10% [v/v]
EG, 10.7% [v/v] acetamide, 24% [w/v] Ficoll-70 and
0.4 mol/l sucrose [in a stock solution of PB1 medium])
[82]. They found that maximum survival was attained
with a cooling rate as low as 500 C/min provided the
warming rate was approximately 3000 C/min (lower
warming rates reduced survival). They report that the
cooling rate needed to avoid ice formation in this solution is 500 C. Although we have not been able to find
any reference to a critical warming rate for EAFS 10/10,
it is likely to be 3000 C/min based on Figure 12.6.
Although Figure 12.7 supports the ability of cells
to survive after devitrification, it and Seki and Mazurs
observations, emphasize that attaining a high warming rate is more critical than attaining a high cooling
rate, a point that is often ignored due to preoccupation only with ensuring vitrification during cooling.
The same observations also make it apparent that survival alone is not unequivocal proof of vitrification
[35]. This point is often overlooked in the reproductive cryobiology literature, and is particularly relevant
to some of the current methodologies utilized for vitrification. Many of these methodologies might be better characterized as nucleated vitrification procedures
or as one-way vitrification methods in which appre-
Up to this point, we have discussed the stability of

a vitreous solution only in terms of its propensity
to crystallize. Thermo-mechanical instability can also
develop during cooling, and is a concern for long-term
storage of biomaterials at cryogenic temperatures [83],
particularly for systems that are adversely affected by
fracturing or deformation.
When forces are applied to a material, the material
responds by changing shape; for example, if both ends
of a wire are pulled in opposite directions (stress, ),
the wire will stretch (strain, ). If the material returns
to its initial size after the forces have been removed
(think of stretching rubber), the material is said to have
undergone elastic strain. Elastic strain is defined as
the size of the change relative to the initial size; in the
example of the rubber, the final length relative to the
initial. Objects will also experience strain in the direction perpendicular to the applied stress (i.e. the rubber
gets thinner as it is being stretched). The ratio of this
transverse strain to the extension strain (in the direction of the applied force) is called the Poisson ratio. The
proportionality between stress and strain for a given
material is the Youngs modulus (also referred to as the
elastic modulus, E = /) and applies only to elastic
strain. Viscous strain occurs when the stress exceeds
a quantity known as the yield stress, and is associated with non-reversible rearrangement of molecular
positions (think of taffy being pulled, which does not
return to its original geometry). Viscous strain relieves
at least some of the stress that builds up within a
material.
Stress and strain issues in cryopreservation are usually associated with cooling and warming of the system. At high sub-zero temperatures, viscous strain
occurs to a greater extent during cooling of a solution
due to its relatively low viscosity. As the system continues to be cooled, the relative contribution of viscous strain decreases and elastic strain becomes more
influential. Rabin et al. have defined the temperature
at which the effects of these two strains are similar as
the set temperature [84]. This temperature is usually
near, but above, Tg for solutions. Well above the set
153
temperature, elastic strain is insignificant and the solution acts like a liquid. Below the set temperature, the
elastic strain dominates and the fluid acts like a solid
(see Steif et al. [85] for more details).
To avoid stress buildup to the point of fracture, it
is important to consider the effect of thermal gradients through the sample. During cooling at a constant
rate, significant thermal gradients inevitably become
established within all but very tiny systems. Above the
set temperature, minimum stress exists within the system as cooling proceeds. As the system approaches the
storage temperature (usually below the set temperature), the thermal gradients established during cooling can result in significant stress build-up within the
system as the system approaches thermal equilibrium
(e.g. as the still-pliant center of the system cools and
pulls against the rigid outer layers). If the stress developed from this process exceeds the yield stress, fracturing results.
Using a continuum mechanics approach to modeling the stresses built up as a result of temperature
gradients during cooling, Steif and colleagues developed an analytical expression to approximate the maximum tensile stress ( max , which occurs at the center
of a sample) associated with various ideal geometries,
given by the following equation:
max = gE T/(1 v)
[12.2]
where E is the Youngs modulus, is the coefficient

of thermal expansion, T is the temperature gradient
during cooling, and v is the Poisson ratio of the system.
The coefficient g depends upon the specific geometry
of the sample, and is equal to 1/3, 1/2 and 2/5 for plate,
cylindrical and spherical geometries, respectively. T
can be computed from the following equation:
T = f(Hd2 /)
[12.3]
with f being the coefficient for maximum temperature

difference (equal to 1/8, 1/16 and 1/24 for plate, cylindrical, and spherical geometries, respectively), H the
cooling rate, d the diameter of the sample, and the
thermal diffusivity [85].
Several important parameters at cryogenic temperatures, such as the viscosity and thermal diffusivity, are
uncertain. This will influence the precision of such predictions. Nonetheless, such an analysis suggested that
the strains that yield fracturing in vitrification solutions (0.23% for VS55 and 0.18% for DP6) are similar
to those of brittle organic materials [84]. Further work
154
along these lines may permit prediction of the exact

conditions needed for fracture avoidance.
Fracturing is an even greater risk during warming
than it is during cooling (e.g. [86]). It has also been
demonstrated that fracturing is a concern with standard Cryo straws, and relatively slow initial warming
of 1/4; cc straws after removal from LN2 can eliminate
fracture damage [87]. This latter observation supports
the assertion that slow warming above the glass transition temperature allows the stress built up during cooling to be relieved as the viscosity decreases and the
vitrified solution softens. This is the basis upon which
a 5-second air-thaw for a straw containing cryopreserved embryos prior to warming in a water bath was
developed.
Vitreous state storage below Tg

As mentioned above, the glass transition temperature
of vitrification solutions is 6686 C above the boiling point of LN2 (196 C). Storage in LN2 is common
not because of the necessity of this temperature per se,
but due to issues of convenience and stability. Many,
perhaps most, of the current methods used to vitrify
human oocytes use so-called open systems, where the
sample comes in direct contact with LN2 . At this time,
the issue of open systems, and the potential for contamination, is contentious [88, 89]. Storage in nitrogen
vapor is seen by many as a superior alternative, and
eventually may be the most common means to store
vitrified samples.
An increasing amount of genetic resources is being
banked at this time, and such an organized collection
requires long-term stability for overall usefulness [90
92]. Clearly, one should choose a storage temperature
that will match the requirements of storage time. The
optimal storage temperature depends on the vitrification solutions Tg , nucleation characteristics, liability to
damage from fracturing and on the biological systems
viability as a function of time and temperature.
As described by Fahy and Rall, maintaining samples even above Tg may still preserve viability for very
long periods of time [3]. By combining the Vogel
TammannFulcher (VTF) equation, which describes
the temperature-dependence of viscosity, the Stokes
Einstein equation relating the diffusion coefficient to
the viscosity and temperature and the relationship
between diffusion distance, diffusion coefficient and
time, one can calculate the time (t) necessary for the
amount of diffusion to occur at a specific temperature
t = t1 (T1 /T) exp[B[(1/(T T0 ))

1/(T1 T0 )]],
where T0 estimates the limiting temperature for structural change [3]. Figure 12.8a provides an example of
the use of curve-fitting of the VTF equation for a specific vitrification solution (M22) [58], and Figure 12.8b
shows how the results can be applied to the estimation
of acceptable storage times. This particular calculation
is based upon the assumption that biological damage
due to the vitrification solution is dependent on diffusion, and that the total accumulated damage is equivalent to damage caused by exposure to a vitrification
solution for 10 s at 0 C. As can be seen, even at temperatures well above Tg , safe storage times are predicted to
be quite long. For storage below Tg , damage resulting
from diffusional processes is likely to be minuscule.
Such calculations do not, however, take into
account the fact that cubic ice nucleation (which evidently requires minimal diffusion) may still occur at
temperatures even below Tg (see Figure 12.2). Thus,
empirical research results that define a critical warming rate for a system when storage near Tg is very brief
may not extrapolate when storage times are extended,
for devitrification may be a greater concern in the later
case [93]. On the other hand, it is known that jugular veins stored at 130 C (just 7 C below Tg ) for
4 months show no increase in damage compared to
storage for only 24 h (both showed 80% recovery versus controls, [94]). Whether chilling-sensitive systems
will show a similar degree of stability remains to be
seen.
Vitrification solutions and vitrification

solution toxicity
The composition of vitrification solutions is an essential consideration given the relatively high concentrations of cryoprotectants required. Many mixtures
of cryoprotective agents have been tested as vitrification solutions (VSs) for reproductive cells, tissues, and
organs over the past two decades [95, 96]. Ali and Shelton examined a large number of mixtures in search
of the most useful combinations for mouse morulae
[97, 98], and have recently published a useful summary
of their searches and conclusions [95]. Other investigators have only compared relatively small numbers
of solutions for vitrification purposes for reproductive
1e + 14
1e + 13
1e + 12
1e + 11
1e + 10
1e + 9
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3
1e + 2
1e + 1
1e + 0
1e 1
1e 2
20
(a)
Time in years
to equal 10 s at 0C
(T) to equal that obtained at time t1 and a reference

temperature (T1 ):
Log M22 viscosity (Poise)
(b)
1e + 8
1e + 7
1e + 6
1e + 5
1e + 4
1e + 3
1e + 2
1e + 1
1e + 0
1e 1
1e 2
1e 3
1e 4
1e 5
1e 6
1e 7
20
40 60 80 100 120
Temperature (C)
10000 years
100 years
10 years
1 year
1 month
1 week
1 day
1 hour
1 minute
20
20
40 60 80 100 120
Temperature (C)
Figure 12.8 In (a), the fit of the VogelTammannFulcher (VTF)

equation to viscosity data for M22 is shown. The point at the upper
right is an estimate of the viscosity at the glass transition
temperature (1013 Poise). The VTF equation is (T) = A
exp[B/(T T0 )] where is the solution viscosity, T is temperature
and T0 is the estimated limiting temperature for structural change,
which approximates the Kauzmann temperature (see [3, 62] for
more details). For this example, A = 0.009, B = 1112, T0 = 155.4 C
and T is in C. (The composition of M22 can be found in Table 2 of
[58].) Part (b) combines the values of B and T0 from (a) with the
StokesEinstein equation [which relates the diffusion coefficient for
a substance (D) to the temperature-dependent viscosity (T):
D = KB T / (6 (T r), where KB is the Boltzmann constant, T the
absolute temperature and r the particle radius], and the equation
for the time required for a given amount of diffusion to take place at
a particular temperature [3], to provide an estimate by means of
Equation 12.4 of the storage times for viability maintenance as a
function of the storage temperature. Even at temperatures nearly
80 C above the temperature of liquid nitrogen (196 C), it is
estimated that storage times exceed several centuries. See text for
more details.
cells [99101]. A recent meta-analysis of the published literature of full reports over the past 10 years
identified fewer than 10 vitrification solutions having
been tested on in vivo matured human oocytes to date
[102]. This is undoubtedly due, at least in part, to the
155
difficulty of using healthy mature human oocytes for

experimental purposes.
After years of experience, the choice of compositions utilized by many investigators still seems nearly
random, and little consensus seems to exist as to which
solutions are optimal for which reproductive cells.
The empirical success of the Kuwayama method for
oocytes [103] seems to be encouraging more use of
15% Me2 SO + 15% EG as the basic permeating component of the VS for human oocytes [2, 104, 105], but
it has not been shown that this solution is actually better than competing formulas when used in the same
protocol [102].
The wide range of empirical solutions in the literature is not surprising. Analytically, the problem
of mapping the full range of available composition
temperaturetime design space is enormous. How
many agents will be used? In what proportions? At
what total concentration? With what balance of permeating and non-permeating agents, as chosen on
the basis of what rationale? With what loading and
unloading protocol, developed on what basis, and with
what accompanying temperatures? Dissolved in what
carrier solution and combined with that solution in
what way? Compared to what controls? And all justified on the basis of what functional endpoints with
what cells? Given the large number of choices available, the differing intuitions of different investigators
and the limited inclination and/or ability of most laboratories to map out compositional and procedural
variables in detail, a broad spectrum of proposed solutions is only to be expected.
However, many guiding principles have been
developed that may help to provide direction to future
studies and that may eventually lead to more of a
consensus concerning how to design, introduce and
remove VSs. We therefore briefly re-examine here the
principles of good VS construction and use as we
presently understand them.
Good VSs require bad glass-forming agents

According to an analysis published in 2004 [65], the
compositional variable upon which toxicity depends
is q, which is defined as MW /MPG , in which MW is
the molarity of water in the solution and MPG is the
molarity of water-bonding groups (polar groups) on
the permeating cryoprotectants of the VS. Good glass
formers vitrify water at low glass former concentrations and therefore MW and q are relatively high at the
156
threshold concentration needed for the solution to vitrify under standard conditions (qv , pronounced cue
vee star). According to analyses of many VSs tested on
rabbit renal cortical slices, viability is high when qv is
low, and vice versa, indicating that weak glass forming agents are less toxic than strong ones. A similar
trend is apparent from the results of Ali and Shelton
[98] when the toxicity of 13 vitrification solutions on
mouse morulae is assessed [S. F. Mullen, unpublished
results].
This is interpreted to mean that it is preferable to
choose cryoprotectants that compete less strongly with
cellular constituents for access to water, and that water
is actually more available to hydrate biomolecules in
VSs that have lower absolute water concentrations at
qv [65]. However, it is often preferable to use mixtures
of cryoprotectants rather than attempting to select
only the weakest available glass former because the
concentrations needed for any one agent to vitrify tend
to be high enough to introduce specific toxic effects for
that agent that defeat its theoretical advantages [65].
Nevertheless, Ralls choice of glycerol + albumin (VS3
[106]) is consistent with qv theory because glycerol is
a particularly ineffective glass former, polar group for
polar group, compared to other agents. Unfortunately,
glycerol tends to be limited by its low permeability, and
in some systems is able to dramatically lower adenosine triphosphate (ATP) levels by being phosphorylated at the expense of ATP [107].
MacFarlane and colleagues have examined the
physicochemical basis of glass formation in aqueous cryoprotectant solutions by nuclear magnetic resonance (NMR) spectroscopy techniques, and were
able to establish that glass-forming efficacy is directly
related to the water-cryoprotectant hydrogen bond
strength (cryoprotectant basicity) [26, 108]. These fundamental observations might in principle be used to
quantitatively relate qv to hydrogen bond strength
and therefore to relate viability directly to hydrogen
bond strength, but this depth of analysis has not to date
been pursued.
Consider employing cryoprotectant

toxicity neutralization
There is one exception to the rule that weak glassforming agents should be used, and that arises from
the phenomenon of cryoprotectant toxicity neutralization (CTN), which has recently been reviewed in detail
[109]. Certain amides have the remarkable property
of having toxicity that can be blocked by the simultaneous presence of Me2 SO, such that, for example, a
50% w/v total concentration resulting from the combination of 20% w/v formamide with 30% w/v Me2 SO
can have no toxicity even though 20% w/v formamide
by itself can lower viability by 60% [110]. The mechanisms involved are unknown, but sufficient clues are
available to enable mechanisms to be elucidated [109].
In the meantime, CTN enables the exceptionally poor
vitrification tendency of amides to be traded off against
the strong glass-forming tendency of Me2 SO and supplemented with the use of other intrinsically weak
glass formers to enable solutions of exceptionally low
toxicity, high concentration and high overall stability
against ice formation [58, 65, 111].
The use of amides in VSs can be controversial when
considered outside the context of CTN. However, the
toxicological effects of amides in vivo are not meaningful in the context of low temperature addition and
removal in the presence of Me2 SO in vitro, and can
be avoided if necessary by ensuring that all amides are
removed prior to re-warming to 37 C or by very rapid
washout at 37 C.
The choice of acetamide in the original VS1
[55] solution may have been suboptimal [65, 109].
Formamide currently appears to be the amide of
choice, combining high permeability [112], exceptionally poor glass-forming ability [24], lack of denaturing character [109], full toxicity neutralization potential [109] and lack of any documented mutagenicity
or carcinogenicity. Thus far, CTN has been demonstrated only in kidney and liver tissue, and seems not to
apply to brain tissue. Nevertheless, solutions based on
CTN have been effective for brain slices [113], mouse
oocytes [65] and many other systems.
Include extracellular agents in moderation

The vitrification tendency of cytoplasm and of
organelles will differ from that of the extracellular
medium due to the presence of intracellular proteins
and membranes. It was shown in 1984 that this enables
some reduction in the level of permeating cryoprotectant to be accomplished, intracellular protein being
balanced by extracellular polymers [22], and it is now
known that total solution toxicity depends more on
permeating cryoprotectants than on impermeants
[58, 65, 109]. What is less clear is the limit beyond
which this principle cannot or should not be extended.
Excessive reliance on impermeants to achieve vitrifi-
cation may in principle reduce cell viability through

excessive cell shrinkage [114, 115], exacerbating
chilling injury as a result of too much cell shrinkage
[58] and allowing IIF if the glass-forming tendency or
anti-nucleating effectiveness of extracellular polymers
such as ice blockers significantly exceeds that of
intracellular proteins. Cell shrinkage secondary to the
use of polymers may exacerbate hypertonic osmotic
injury upon cryoprotectant addition but will also
limit the amount of cryoprotectant that enters cells
and, consequently, will simplify the removal of that
cryoprotectant after storage [22].
Consider special additives

In recent years, special additives, including most
prominently the ice blockers X-1000 [116] and Z1000 [117] but also including ice-growth inhibitors
[111; 21st Century Medicine, unpublished results],
3-methoxy-1,2-propanediol [58, 118, 119] and, if
needed, N-methylformamide [58], have been introduced to eliminate heterogeneous nucleation and
enhance extracellular and intracellular vitrification
without increasing toxicity when used in low concentrations. These additives and solutions based on
them are commercially available and have been efficacious for numerous diverse systems outside the realm
of reproductive cryobiology, and have allowed good
recovery of vitrified mouse oocytes [65].
Choose your carrier solution carefully

The toxicity and glass-forming ability of a vitrification
solution depend to a significant extent on the carrier or
physiological support solution in which the cryoprotective agents of the vitrification solution are dissolved
(see Figure 12.6; [120]). At 21st Century Medicine, we
use an LM5 carrier solution, which enhances the activity of ice-blocking agents and sustains cell viability well
in the presence of high cryoprotectant concentrations
[58, 65, 121].
Define your needs

The toxicity and ice prevention efficacy of a VS both
increase as the total solution concentration increases.
The former effect should be minimized by determining the minimum required size of the latter effect and
limiting total concentration to what is actually needed.
This point is emphasized particularly because vitrification tendency is sometimes assessed in unnecessarily
large increments of total solution concentration, such
157
as increments of 5% or 0.5 mol/l [95], when smaller

increments might both suffice for attaining sufficient
solution stability and result in important reductions in
overall toxicity.
Eliminate osmotic effects

The negative effects of VSs can arise, of course, not only
from true biochemical toxicity but also from osmotic
effects if the latter are not carefully excluded. Although
mass transfer modeling is the best way to estimate
appropriate protocols for adding and removing VSs
while avoiding osmotic injury [122124], empirical
testing is also required and may be sufficient if the
investigator is sufficiently thorough in verifying that
further increases in exposure time at a given concentration, further reductions in concentration step size
or further decreases in the rate of change of concentration do not result in additional protection [125]. In
general, exponential rates of increase and decrease in
concentration will minimize both toxicity and osmotic
effects [109, 126].
Osmotic effects will be maximized at lower temperatures, but lower temperatures are generally necessary
to minimize toxicity unless chilling injury near 0 C is
so severe as to preclude lower temperature cryoprotectant addition [6, 127]. For systems in which chilling
injury is not exacerbated by excessive cell shrinkage,
low temperature addition of the final VS may only need
to allow cytoplasmic concentration by the exosmosis
of water, cryoprotectant uptake by cells being unnecessary [22].
Summary
Vitrification is becoming an increasingly common
means to cryopreserve reproductive cells and tissues.
Efforts to understand the physics of vitrification have
been underway for decades, and the basic principles
of vitrification are now reasonably well understood,
although many important practical details remain to
be investigated. Achieving vitrification in a laboratory
setting is generally believed to require the use of multimolar concentrations of cryoprotectants, which have
the potential to be toxic to living cells. Fortunately,
some general principles underlying the relationship
between molecular structure, vitrifiability, and toxicity have been elucidated in recent years, increasing
our ability to develop vitrification solutions on a rational basis. While the avoidance of the damaging effects
of ice formation is the basis for choosing vitrifica-
158
tion as a means of cryopreservation, mechanical damage due to fracturing of the vitreous material can also
cause serious injury, in some cases negating the benefits of vitrification. However, utilizing interdisciplinary
knowledge of the fundamental principles of vitrification, the fundamental cryobiology of the cells being
preserved and the derived parameters associated with
current technology, continued improvements in vitrification methods for reproductive cells, tissues and
organs should be possible, enabling the achievement
of superior fertility preservation results.
Acknowledgements
We would like to thank Brian Wowk for stimulating
discussions on thermodynamic aspects of vitrification,
and his assistance in the clarification of those concepts.
We also thank Mehmet Toner for his consent to publish
his estimates in Figure 12.5. This work was supported
by 21st Century Medicine, Inc.
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Section 4
Chapter
13
Fertility preservation strategies in the male
Hormonal suppression for fertility

preservation in the male
Gunapala Shetty
Introduction
Radiation and chemotherapeutic regimens required
for life-threatening diseases, such as cancer or exposure to environmental toxicants, may jeopardize the
fertility of men of reproductive age causing permanent or temporary azoospermia. Young pre-pubertal
patients who cannot supply a semen sample for cryopreservation of sperm have particularly poor fertility prognosis. In the USA alone about 17 000 men
aged 1545 years old are diagnosed each year with
Hodgkins disease, lymphoma, bone and soft tissue sarcomas, testicular cancer or leukemia. Of these, over
3000 are treated with doses of alkylating agents, platinum drugs or radiation that are sufficient to induce
prolonged azoospermia. In addition, over 6000 boys
under the age of 15 are diagnosed each year with cancer, including leukemia, nervous system tumors, lymphomas and other solid tumors. About 80% of them
receive chemotherapy or gonadal radiation, and about
550 of the long-term survivors are azoospermic when
they reach adulthood.
Environmental and occupational toxicants can
also produce prolonged azoospermia. This was most
dramatically shown with dibromochloropropane
(DBCP), as highly exposed manufacturing and
agricultural workers had an increased incidence of
azoospermia [1, 2]. In addition, sterility can be developed during development such as due to cryptorchidism or aging.
Methods to prevent these effects on male fertility
and to restore normal testicular function are of great
importance. A variety of biochemical and biological
approaches (thiol radioprotectors, prostaglandin analogues, growth factors, blockers of apoptotic pathways,
and reduction in blood flow) have been tested to protect the testes in experimental animal model systems
against radiation and chemotherapy (reviewed in [3]).
However, utmost research interest in this field, including all clinical trials, has involved hormonal modulation in attempts to prevent or reverse damage to
the germ line from radiotherapy and chemotherapy.
In this chapter, I will discuss the current knowledge
of hormonal suppression as a means to preserve or
restore fertility in males.
History and hypotheses

The use of hormone suppression for protecting
gonadal function after cytotoxic exposure is based on
the observation that non-cycling cells are generally
more resistant to killing by certain toxicants, particularly anti-neoplastic agents, than are rapidly proliferating cells.
The mechanism originally proposed for the protection of spermatogenesis was that the interruption of the pituitarygonadal axis would reduce the
rate of spermatogenesis and render the resting testis
more resistant to the effects of chemotherapy [4]. This
hypothesis was based on the claim that pre-treatment
with gonadotropin-releasing hormone (GnRH) analogue which in rodents produces reversible inhibition of follicle stimulating hormone (FSH) and
luteinizing hormone (LH) secretion and severe reduction in serum testosterone levels protected spermatogenesis in the mouse from cyclophosphamideinduced damage. However, attempts to repeat these
original observations using more quantitative endpoints revealed that there was no protection [5]. Since
suppression of gonadotropins and testosterone only
164
Chapter 13: Hormonal suppression in the male
blocks the completion of spermatogenesis but has

no effect on the kinetics of the early spermatogenic
cells [6], the premise on which the proposed mechanism was based was incorrect and a negative outcome
should be expected. Furthermore, the stem spermatogonia, which are more important targets than the differentiating germ cells for the long-term effects of cytotoxic damage, did not appear to be affected at all by
hormonal suppression.
Despite the failure until now to observe protective effects in mice, it has been convincingly shown
that suppression of gonadotropins and intratesticular
testosterone levels prior to, during or after exposure
of rats to chemotherapy, radiation or other cytotoxic
agents enhances the subsequent recovery of spermatogenesis, as discussed below. Thus other mechanisms
must be involved and careful attention must be given
to the species used.
Effect of cytotoxic agents on

testicular function
The testis consists of the seminiferous (or germinal)
epithelium arranged in tubules and endocrine components (testosterone-producing Leydig cells) in the
interstitial region between the tubules. The seminiferous tubules contain the germ cells, which consist
of stem and differentiating spermatogonia, spermatocytes, spermatids and sperm and the Sertoli cells,
which support and regulate germ cell differentiation.
Among the germ cells, the differentiating spermatogonia proliferate most actively and are extremely susceptible to cytotoxic agents. In contrast, the Leydig and
Sertoli cells, which do not proliferate in adults, survive most cytotoxic therapies. These cells may, however, suffer functional damage. Frequently, following
cytotoxic therapies, germ cells appear to be absent, and
the tubules contain only Sertoli cells. This could be a
result of the killing of the spermatogenic stem cells, the
loss of the ability of the somatic cells to support the differentiation of a few surviving stem cells or a combination of the two.
The eventual recovery of sperm production
depends on the survival of the spermatogonial stem
cells and their ability to differentiate after exposure to
cytotoxic agents. The reduction should be temporary
provided the stem spermatogonia survive. However,
stem spermatogonia are killed by some of these
agents at varying degrees and recover only gradually,
resulting in prolonged reductions of sperm count; in
the mouse this reduction is directly related to stem cell

death [7]. It is rare that surviving stem spermatogonia
fail to differentiate in mouse testes [8]. In contrast,
after exposure of rats to several anti-neoplastic agents
[9] and other toxicants [10], the stem spermatogonia
that survive are blocked from differentiating and
their progeny undergo apoptosis instead [11]. This
block has been shown to be a result of damage to
the somatic environment within the testis, not to the
spermatogonia [12]. There is, however, no evidence
of a similar spermatogonial block in monkeys [13].
In men, surviving stem cells can remain in the testis
but fail to differentiate into sperm for several years
after cytotoxic insult, so delayed recovery is possible
[14]. At lower doses of these agents, recovery to
normospermic levels can occur within 13 years, but
at higher doses, azoospermia can be more prolonged
or even permanent.
The loss of germ cells has secondary effects on the
hypothalamicpituitarygonadal axis. Inhibin secretion by the Sertoli cells declines and, consequently,
serum FSH levels rise. Testicular blood flow is reduced,
resulting in less testosterone being distributed into the
circulation [15]. Therefore LH levels increase to maintain constant serum testosterone levels. The reduction
in the testis size and increased LH levels also contribute
to an increased concentration of testosterone within
the testis.
Hormone suppression studies in

animal models
Several studies support the conclusion that
gonadotropin suppression does not protect spermatogenesis in mice from damage [8, 16, 17]. In
contrast, numerous reports suggest that hormone
suppression protects rat testes from damage due to
irradiation, procarbazine, doxorubicin, an indenopyridine compound and heating [1826] and enhances
future fertility in the face of these toxicants. In
addition to direct suppression of gonadotropins
with GnRH agonists and antagonists, which also
results in both direct and secondary suppression of
intratesticular testosterone, these studies also utilized
combinations of GnRH analogues with antiandrogens,
systemic physiological doses of testosterone (which
suppresses gonadotropins and results in reduced
intratesticular testosterone levels), progestins (which
are very effective at suppressing gonadotropins but
have weak androgenic activity) and estrogens (which
165
Section 4: Fertility preservation strategies in the male
X
X
X
(a)
(b)
*
*Sp
Sp
*Sp
Sp
*
(c)
(d)
Figure 13.1 Photomicrographs of Lewis brownNorway F1 hybrid (LBNF1) rat testes sections showing normal spermatogenesis in control
testis (a), radiation-induced block in the differentiation of spermatogonia (b,c) and the reversal of this block by gonadotropin-releasing
hormone (GnRH) antagonist treatment. The testes were harvested 13 weeks after 5 Gy irradiation with or without GnRH antagonist treatment
during weeks 37 after irradiation. Note that with no GnRH antagonist treatment all tubules are atrophic (X) and contain only Sertoli cells and
type A spermatogonia (b), with normal (half-open arrow) and dividing (filled arrow) spermatogonia shown at higher magnification (c). With
GnRH antagonist treatment all tubules are repopulating ( ) with mature spermatids in many of them (Sp) (d). Bars = 50 m.
both suppress gonadotropins and inhibit testosterone

synthesis). It should be noted that in all these studies
protection was not assessed directly at the time of
cytotoxic exposure, but rather by the enhanced ability
of spermatogenesis to recover from surviving stem
cells [27], which is actually the most relevant endpoint
for future fertility.
Attempts to protect spermatogenesis in other animal species (dog, monkey) have not yielded any
reproducibly positive results. Although one group
reported that GnRH agonist shortened the time for
recovery of spermatogenesis after treatment of dogs
with cyclophosphamide, cisplatin or radiation [28],
another study reported potentiation of the damage
166
[29]. Studies using hormone suppression in monkeys

have not convincingly demonstrated enhanced recovery of spermatogenesis following gonadotoxic injury.
Although one preliminary report based on a total
of only three baboons suggested that hormone suppression might decrease the gonadal damage from
cyclophosphamide [30], two larger studies using an
adequate number of macaques showed neither protection [31] nor stimulation [13] of recovery of spermatogenesis from radiation damage by hormone suppression treatment.
We proposed that cytotoxic exposure of normal
testis (Figure 13.1a) produces a pronounced block in
the differentiation of surviving stem spermatogonia
Normal
rat
Irradiated
rat
Spermatogonium
T independent
+T
Figure 13.2. Diagrammatic representation of

the role of testosterone in regulating
spermatogenesis at various steps during normal
spermatogenesis and in the irradiated rat testes.
In normal rats, spermatogonial differentiation is
qualitatively independent of testosterone, with
the absolute requirement of testosterone during
spermiogenesis. However, after irradiation
testosterone inhibits the differentiation of
spermatogonia.
Spermatocyte
T independent
T independent
R. spermatid
+T
+T
Sperm
in rat testes (Figure 13.1b,c) and prevention of this

block is the mechanism by which hormone suppression appears to protect spermatogenesis from toxicant
exposure [27]. It is important to note that many of
the studies showing protection involved subchronic
exposure to the cytotoxic agent, so the hormonal suppression was given after the initial exposures, and in
some cases even extended beyond the last exposure
[32]. Furthermore, when the hormonal suppression
was administered to the rats only after the cytotoxic
insult, either immediately or after a delay of several
months, the numbers of differentiated germ cells still
dramatically increased [33]. However, because testosterone, which is required for spermatid differentiation, was suppressed, spermatogenesis proceeded only
to the round spermatid stage: no sperm were produced. Nevertheless, when additional time without
further suppressive treatment was allowed before the
rats were euthanized, all tubules showed almost complete spermatogenic recovery (Figure 13.1d), sperm
counts increased and the fertility of the rats significantly increased [27]. This phenomenon appears to
be quite general: post-treatment with GnRH agonists
or antagonists, with or without antiandrogen, low
dose systemic testosterone, estradiol or hypophysectomy are all effective at stimulating recovery [3436],
and recovery has been stimulated following gonadal
toxicity from radiation, procarbazine [37], busulfan
[38], hexanedione [39], dibromochloropropane [40],
an indenopyridine compound [25] or heat treatment
[41]. The endogenous hormone primarily responsible for the inhibition of spermatogonial differentiation in toxicant-treated rats was testosterone, although
FSH also had a minor inhibitory effect [35], and
other exogenously administered androgens were also
inhibitory [34]. It was also observed that hormonal
suppression after irradiation of mouse testes modestly
but significantly increased the percentage of tubules
in which differentiation of surviving spermatogonia
occurred [G. Wang and M. L. Meistrich, personal communication].
Hormonal suppression with GnRH analogues or
hypophysectomy has also been shown to promote the
survival and differentiation of spermatogonia that are
transplanted into testes of animals that were depleted
of endogenous stem cells. Although the effects were
most dramatic when rat testes depleted by cytotoxic
treatments were used as recipients [12, 42], enhanced
proliferation and differentiation of transplanted spermatogonia were also observed when mouse testes
were used as recipients [4345; G. Wang and M. L.
Meistrich, personal communication].
The inhibition of spermatogenesis by testosterone
after cytotoxic exposure such as radiation appears to
be contradictory to its well-studied role in stimulating and maintaining spermatogenesis (Figure 13.2). In
normal unirradiated and irradiated rats, testosterone
has a similar role in spermiogenesis, as shown by the
failure to find cells that have developed past the round
spermatid stage in both these cases when testosterone
167
Table 13.1 Results of hormone suppression treatments given before and during cytotoxic therapy on spermatogenic recovery in men
Recoveryb
Cytotoxic
therapya
Hormone
treatment
Hormonetreated
Controls
References
Hodgkins
MOPP
36 cycles
GnRH agonist
1/5
No controls
Johnson et al.
[48]
Hodgkins
MVPP, ChlVPP
GnRH agonist +
testosterone
0/20
0/10
Waxman et al.
[49]
Hodgkins
MOPP 4 cycles
Testosterone
70%/23c
70%/22c
Redman and
Bajorunas [50]
Testis Ca
PVB + ADR/CY +
radiation
Medroxy-progesterone
0/4 (2/12)d
2/3 (7/13)d
Fossa et al.
[51]
Testis Ca
PVB
GnRH agonist
6/6
8/8
Kreuser et al.
[52]
Testis Ca
(Seminoma)
Radiation
GnRH agonist +
antiandrogen
12/12
8/8
Brennemann et al.
[53]
Nephritis
Cyclophosphamide
Testosterone
5/5
1/5
Masala et al.
[54]
Disease
Ca, cancer; GnRH, gonadotropin-releasing hormone.

a Chemotherapy regimens are as follows: ADR/CY, adriamycin, cyclophosphamide; ChlVPP, chlorambucil, vinblastine, procarbazine and
prednisone; MOPP, mechlorethamine, vincristine, procarbazine and prednisone; MVPP, mechlorethamine, vinblastine, procarbazine and
prednisone; PVB, cisplatin, vinblastine, bleomycin.
b Fraction of men recovering testicular function as assessed by restoration of sperm counts to normospermic levels unless otherwise noted.
c Actuarial recovery calculated by KaplanMeier analysis.
d Recovery assessed by restoration of follicle stimulating hormone (FSH) levels to within the normal control range.
is suppressed. But, unlike normal rats in which spermatogonial survival and differentiation are qualitatively
independent of testosterone, in irradiated rats the survival and differentiation of type A spermatogonia are
inhibited by this hormone.
The mechanism by which somatic cells in rat testis
are protected from damage if testosterone suppression
treatment is given at the time of cytotoxic exposure is
not known. Neither is the mechanism by which testosterone suppression after cytotoxic exposure enhances
the ability of the somatic elements of the testis to
maintain the differentiation of spermatogonia. Previous studies ruled out the possibility that the protective
effect of hormonal suppression given before cytotoxic
drug exposure was due to reduced delivery to the tissue or altered metabolism of the drug [46]. We have
recently shown that the block in spermatogonial differentiation in rats treated with cytotoxic agents is associated with the increased levels of interstitial edema
in the testes [47]. Based on data indicating that more
rapid stimulation of recovery of spermatogonial differentiation in irradiated rats was achieved by elimination of Leydig cells with ethane dimethane sul-
168
fonate than with total androgen ablation [G. Shetty and

M. L. Meistrich, unpublished data], we are analyzing
the role of Leydig cells as targets for the testosteroneinduced inhibition of spermatogonial differentiation
in toxicant-treated rats. Recent studies in rats also suggested a direct stimulation of spermatogonial differentiation by estradiol after irradiation, in addition to
its indirect effect through suppression of testosterone
[36]. It is yet to be determined whether the spermatogonia having estrogen receptor beta (ER-) receptors
are the target for estradiol, unlike androgens, which
have receptors only on somatic cells.
Clinical trials
Seven clinical trials have been performed in attempts
to demonstrate protection of spermatogenesis in
humans by hormone suppression treatment before
and during cytotoxic therapy, but six indicated no
protection (Table 13.1 [4854]). Three of the trials
involved patients treated for Hodgkins lymphoma
and three involved testicular cancer patients. Treatment with GnRH agonist resulted in only 20% of
patients recovering sperm count after cessation of

chemotherapy [48]. However, no concurrent control
group of patients receiving similar regimens of
chemotherapy without GnRH agonist were enrolled
in this study. In another study, hormone suppression with testosterone combined with GnRH agonist prior to and during chemotherapy was randomized with no hormonal suppression [49], but none
of the patients from the control and treated groups
showed evidence of recovery of spermatogenesis at
13 years after completion of therapy. Suppression
of gonadotropins and intratesticular testosterone levels with testosterone injections alone during treatment [50] also did not provide gonadal protection
benefit: 70% of the patients in both the treated and
control group showed spermatogenic recovery at 3
years. Suppression of gonadotropins with medroxyprogesterone acetate during chemotherapy combined
with radiotherapy did not improve the recovery of
sperm count or normalize FSH levels, which was
used as a surrogate for sperm count in patients in
whom sperm counts were unavailable; indeed, they
appeared to be lower in the patients receiving concurrent treatment with hormonal suppression than in
controls [51]. Two more studies used GnRH agonist
[52] or GnRH agonist plus an antiandrogen (cyproterone acetate) [53] prior to and for the duration of
chemotherapy or radiation therapy, respectively. In
these studies the chemotherapeutic regimen was only
2 courses of cisplatin, vinblastin and bleomycin (PVB)
and the gonadal dose of radiation was 0.2 Gy, which
allowed spontaneous recovery of sperm counts in all
the control patients within 2 years. The time course
of recovery of spermatogenesis after chemotherapy
was identical for the groups of patients with or without GnRH-agonist treatment. Although fluctuations in
sperm counts made it difficult to determine whether
the time course of recovery of spermatogenesis was
affected by hormonal treatment, the time course of
reduction of elevated FSH levels back to pre-treatment
values was similar in controls and in the patient groups
treated with GnRH agonist and antiandrogen.
The one study that demonstrated hormonal treatment preservation of sperm production in men
involved testosterone therapy of men who received
cyclophosphamide as an immunosuppressive therapy
for nephrotic syndrome [54]. During the treatment,
the testosterone suppressed gonadotropin levels and
suppressed the completion of spermatogenesis. All
but one of the men who received cyclophosphamide
alone remained azoospermic 6 months after the end of

immunosuppressive therapy, whereas sperm concentrations returned to normal in all 5 men who received
cyclophosphamide in combination with testosterone
therapy.
The one attempt to restore spermatogenesis by
steroid hormone suppression after cytotoxic therapy
was also unsuccessful [55]. Seven men with azoospermia secondary to high dose chemotherapy and/or
radiation therapy for leukemia or lymphoma in childhood were treated with medroxyprogesterone acetate
combined with testosterone to suppress gonadotropin
and likely intratesticular testosterone levels many years
after the anti-cancer treatment. None of the men
recovered any sperm production during the 24-week
follow-up after the end of hormonal treatment.
Even if the hormonal suppressive treatments that
were successful in protecting and stimulating spermatogenic recovery in rats are applicable to human
males, there may be many reasons for the unsuccessful
outcomes of the aforementioned clinical trials. The use
of testosterone or medroxyprogesterone either alone
[50, 51, 55] or combined with a GnRH analogue [49] is
suboptimal given that, in animal studies, both of these
steroids inhibit the effects of GnRH analogues in stimulating recovery of spermatogenesis after cytotoxic
damage [34, 56]. The number of patients and controls
studied was small [48] and the cancer therapies variable. Some treatment regimens were not sufficiently
gonadotoxic to cause sterility [52, 53]; conversely some
regimens may have delivered doses well above that
needed to ablate all spermatogonial stem cells, since no
evidence of spermatogenesis was observed in almost
all patients even after many years [48, 49, 55]. Thus,
the application of these procedures to humans remains
uncertain.
Analysis of interspecies differences

The results of studies of protection of long-term
gonadal function by hormonal suppression in experimental animals and humans are summarized in Table
13.2 [5, 8, 13, 1621, 31, 33, 37, 38, 40, 4855, 5759].
The dramatic stimulation of recovery of spermatogenesis by hormone suppression in toxicant-treated
rats is in contrast to the less marked effects observed
in mice, the absence of stimulation in macaques,
and generally negative but variable results in
human.
169
Table 13.2 Summary of effects of hormone suppression on protection and/or stimulation of recovery of spermatogenesis after cytotoxic
treatment in different species
Effect of timing of hormone suppression

relative to cytotoxic treatment
Species
Treatment
Before
Mouse
Procarbazine,
doxorubicin,
cyclophosphamide
Cisplatin
Radiation
Radiationa
Rat
After a delay
References
n.d.
Da Cunha et al. [5];

Crawford et al. [17]
n.d.
n.d.
Nonomura et al. [16];
n.d.
Kangasniemi et al. [8]; Crawford

et al. [17]; Wang and Meistrich,
unpublished data
++
++
Meistrich and Kangasniemi [33];

Kurdoglu et al. [57]
Procarbazinea
++
++
n.d.
Delic et al. [18]; Morris and Shalet

[19]; Jegou et al. [20]; Parchuri
et al. [21]; Meistrich
et al. [37]
Busulfan
n.d.
Udagawa et al. [38]; Udagawa

et al. [58]
DBCP
Immediately after
++
Aging
Meistrich et al. [40]
Schoenfeld et al. [59]
n.d.
Boekelheide et al. [13];

Kamischke et al. [31]
Monkey
Radiation
Human
Chemotherapeutic drug
combinations
( radiation)
/?
Johnson et al. [48]; Waxman

et al. [49]; Redman and Bajorunas
[50]; Fossa et al. [51]; Kreuser
et al. [52]; Thomson et al. [55]
Radiation
n.d.
Brennemann et al. [53]
Cyclophosphamide
++
n.d.
Masala et al. [54]
DBCP, dibromochloropropane.
a Instances in which the type A spermatogonia showed a block in differentiation.
+, ++, relative effectiveness at protection or stimulation.
, no protection/stimulation.
?, no protection observed but could not be demonstrated or ruled out with experimental design.
n.d., the effect not tested.
Experimental studies, particularly in rodents, are

of great value in that they may be highly controlled,
have larger sample sizes and can be used to optimize treatments and to elucidate mechanisms. Primate studies have greater variability and uncertainties and sample sizes are limited. The main question
is what aspects of the rodent studies are applicable to the human and what aspects are not. Since
primates and rodents diverged 66 million years ago
(Mya) there will be differences. It is also noteworthy that mouse and rat diverged 41 Mya, whereas
humans and macaques diverged 23 Mya. Thus, sig-
170
nificant differences are expected among rodents and

between rodents and different primates. It is important to understand the mechanism of protection or
stimulation of recovery by hormonal suppression in
order to determine which individual steps in the process will be similar or different between rodents and
primates.
Cytotoxic agents which spermatogenesis is sensitive to in both rodents and primates are appropriate to utilize in experimental studies for extrapolation to men. Mouse and human spermatogenesis are both sensitive to certain alkylating agents
(procarbazine, chlorambucil, busulfan) and radiation as measured by stem cell death and prolonged azoospermia, respectively [60]. However, there
are also differences as spermatogonial stem cells in
mice are sensitive to, and killed, by doxorubicin
(Adriamycin) but not cyclophosphamide, whereas
cyclophosphamide, but not doxorubicin, strongly
induces long-term azoospermia in humans. It has to
be noted that although both primate and mouse spermatogenesis are sensitive to radiation, the human [61
63] and monkey [64] appear be more sensitive to radiation than mice [6567]. Thus, one of the directions of
future research should be on elucidating the reasons
for greater sensitivity of primate stem spermatogonia
to radiation and certain chemotherapeutic agents in
order to develop methods to protect them.
One contribution to the difference in the stimulation of recovery by hormone suppression after cytotoxic treatment may be the interspecies differences in
the block in differentiation of spermatogonia. In rats
exposed to moderate doses of cytotoxic agents, the
induction of a block in spermatogonial differentiation
is a much more likely cause of prolonged azoospermia
than is spermatogonial stem cell death. The reversal
of this block in spermatogonial differentiation appears
to be the mechanism by which hormone suppression protects or restores spermatogenesis in toxicantexposed rats [27], and the occurrence of such a block
should indicate whether hormonal suppression might
stimulate spermatogenic recovery in another species.
Although, in many cases, the seminiferous tubules in
testicular biopsies taken from men with chemotherapy
or radiotherapy-induced azoospermia contain only
Sertoli cells and no spermatogonia [68], occasionally the presence of isolated spermatogonia have been
observed at relatively long times after chemotherapy
treatment [69]. In addition, spontaneous recovery of
spermatogenesis in some men more than 1 year after
radiation [70] or chemotherapy [71] also implies a
block in the differentiation of spermatogonia that survive these cytotoxic exposures. These results suggest
that after some cytotoxic therapy regimens, there is a
potentially reversible block to spermatogonial differentiation in men. It should be noted that there is no
evidence of a similar spermatogonial block in monkeys [13]. The human data emphasize that the cytotoxic therapy regimens need to be carefully selected in
these clinical trials from a range of regimens. To have a
chance of success, doses of cytotoxic therapies should
be chosen at which there is an appreciable block to
spermatogonial differentiation but not the killing of all

stem spermatogonia.
Since many chemo and radiotherapeutic regimens
may result in the complete killing of the stem spermatogonia and the hormonal methods do not protect these cells from cytotoxicity, consideration should
be given to the applications of hormonal suppression
in combination with spermatogonial transplantation.
Cryopreservation of spermatogonia and autologous
transplantation is considered a potential method for
restoring spermatogenesis and possibly rescuing fertility after chemotherapy or radiotherapy [72]. Hormonal suppression could restore the somatic environment in human testes sufficiently to promote the ability
of transplanted stem spermatogonia to develop, as was
the case with rat testes [12].
It is also important to consider whether the molecular and cellular effects of hormonal suppression
are similar in humans and in rats. Although the
basic processes by which GnRH analogues suppress
gonadotropin and testosterone levels and induce a
block in the completion of spermatogenesis in normal adult males are similar in rodents and primates,
there are quantitative differences. Whereas in rats
and humans, GnRH antagonist reduced intratesticular testosterone concentrations to about 2% of that
observed in controls [73, 74], in macaques it only
reduced intratesticular testosterone concentrations to
28% of control [75]. Despite the less marked reduction in intratesticular testosterone levels, spermatogenesis was blocked at the B spermatogonial stage by
GnRH antagonist treatment of the primates [75] compared to the round spermatid stage in rats [8]. In
humans, the block in spermatogenesis was also largely
at the B spermatogonial levels, but later germ cells
to the round spermatid stage were still produced at
20% of control levels [74]. The restimulation of spermatogonial differentiation by hormonal suppression
in rats may be dependent upon the ability of germ
cells to develop to the spermatocyte stage during the
testosterone suppression, and hence might occur in
men. Further germ cell differentiation in the presence
of suppressed testosterone can be induced in human
testes by treatment with FSH [76].
Thus, the mechanism by which hormone suppression protects or reverses the damage to the somatic elements of the testis to stimulate spermatogenic recovery
in rodents is not yet clear. Knowledge of the mechanism in appropriate animal models should allow the
identification of treatment targets downstream from
171
the initial action of the hormones. It may be possible to suitably modulate those targets in primates to
release the blockade in spermatogenic differentiation,
while maintaining hormone levels, which should allow
spermatogonial differentiation. In addition, elucidation of mechanisms for greater sensitivity of primate
germ cells to cancer therapeutic agents and development of ideal conditions for germ cell transplantation
in primates may open strategies to recover spermatogenesis and sperm count in toxicant-exposed human
males.
Acknowledgements
The authors research in this review was supported by
Research Grant ES-08075 from the US National Institute of Environmental Health Sciences (National Institutes of Health). I am thankful to Dr. Marvin Meistrich
for his valuable help in preparing this review.
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175
Section 4
Chapter
14
Cryopreservation of spermatozoa
Old routine and new perspectives
E. Isachenko, V. Isachenko, R. Sanchez, I. I. Katkov and R. Kreienberg
Introduction
Cryopreservation of male and female gametes has
been long established, and nowadays low-temperature
storage of human spermatozoa is a routine technique in assisted reproduction. This technique offers
the following advantages over the use of fresh ejaculated spermatozoa: (1) storage of both homologous
or donor sperm for subsequent intrauterine insemination, in vitro fertilization (IVF) and intracytoplasmic
sperm injection; (2) long-term storage of known quality donor semen; (3) the ability to quarantine donor
semen until appropriate testing can be completed;
(4) preservation of epididymal or testicular spermatozoa/tissue for subsequent intracytoplasmic sperm
injection or necessary diagnosis [1, 2]. Recently, fertility preservation has been a hot topic and attracted
much attention because chemotherapy and radiotherapy result in a significant reduction of spermatozoa
quality and, as a consequence, a following indefinite
period of infertility [35]. Human genome banking
is one specific approach that can be used to preserve
male genetic material before sterilization, chemotherapy or radiotherapy. It can also be used for males
with autoimmune diseases [2, 4, 68] or before some
treatments and special kinds of surgical procedures
which may lead to testicular failure or ejaculatory
dysfunction [9].
The empirical methods of cryopreservation developed in the 1950s are still used today [10, 11] and based
on use of a relatively slow cooling rate (1170 C/min)
in a region of critical temperatures (10 to 60 C). At
present the major steps used for cryopreservation of
different kind of cells can be summarized as follows:
(1) Adding cryoprotective agents (CPAs) before
freezing. This substance enhances post-thaw survival
by limiting the crystallization of water. (2) The seeding
of samples at freezing or around freezing point. (3)

The thawing of the cells. (4) Removing the CPAs from
the cells after thawing [12]. Cells can endure storage at
very low temperatures (below 100 C). However, the
cooling and warming processes associated with the
intermediate zone of temperature (10 C to 60 C),
which cells must traverse twice once during cooling
and once during warming can be lethal to cells [11].
The aim of slow cooling rates is to maintain a very
delicate balance between ice crystal formation and
the growing concentration of dissolved substances.
The danger is cell damage due to crystallization of
intracellular water and osmotic and chilling injury;
cytoplasm fracture; or even effects on the cytoskeleton, genome or genome-related structures [13
16].
Despite these dangers, the use of programmable or
non-programmable conventional slow freezing allows
the simultaneous preservation of relatively big volumes of diluted ejaculate or prepared spermatozoa,
from 0.25 to 1.0 ml [1719], with satisfactory results
based on the motility of spermatozoa after thawing
[2022], the integrity of the acrosomal and cytoplasmic membrane [23], the functional activity of mitochondria [24, 25], DNA stability [26, 27] and the
prevention of phospholipids translocation inside the
spermatozoa membranes [28, 29].
Vitrification: a promising
new direction
The success of these current procedures brings up the
reasonable question: Why do we need to develop
other technique and what advantages will it give
us compared to the current system? This new
technique is vitrification and it could be beneficial
176
Chapter 14: Cryopreservation of spermatozoa
compared with the conventional slow freezing

method. The vitrification method uses no specially
developed cooling program; it does not need to apply
permeable cryoprotectants; it is much faster, simpler
and cheaper; and it can also provide a high recovery
of motile spermatozoa after warming as effective protection of spermatozoa against cryodamage [3033].
The method is based on the cooling of the cells by
direct immersion into liquid nitrogen (LN2 ), thereby
avoiding the formation of big intracellular ice crystals
[34]. In routine work, the vitrification method
using high concentrations of permeable cryoprotectants was successfully applied in 1985 for mouse
embryos [35], and at present is successfully used
for the preservation of female gametes [36, 37] and
embryos [38, 39]. However, to apply this protocol to
spermatozoa cryopreservation is impossible because
of the resulting osmotic and cytotoxic effects [13, 40].
The promising results after successful vitrification
of frog [41] and fowl [42] spermatozoa were not
confirmed in subsequent investigations [43, 44], and
work in this direction stopped for 40 years. At present,
as an extremely rapid method of cryopreservation
[45], vitrification is being investigated extensively and
applied to embryos [46] and oocytes [26, 47] but very
seldomly to spermatozoa, with the exception of a few
reports [26, 27, 30, 32, 33, 4853].
At present, preservation techniques for natural
diversity fall into two categories: (1) in-situ preservation the preservation and protecting of individual species in their natural setting; and (2) ex-situ
preservation the preservation of individual species
genomic material in combination with breeding programs [54]. In human medicine, the cryopreservation
of male gametes offers both men and boys the possibility of preserving their fertility. The general creation of
cryobanks, for the low-temperature storage of genetic
materials, came after the discovery of the beneficial
effects of glycerol and non-permeable cryoprotectants
on plant cryostability [55]. Subsequent investigation
of glycerols properties applied to mammalian spermatozoa supports these results [5659]. These empirical methods, which were subsequently developed in
the 1950s for use in many species and still applied
today, have opened a new era of permeable cryoprotectants. However, before now, the technology for sperm
cryopreservation did not provide complete protection
of the motility of cryopreserved/thawed cells; motility
dropped to about 50% of their prefreezing value, with
considerable intersample fluctuation taking place [13,
60]. The question of diminished spermatozoa motility after cryopreservation is crucial since this variable
is known to be the first affected by cryopreservation
[61], although the mechanism of sperm impairment
and its mechanical and/or physicalchemical etiology
remains unclear. The reason for cell damage could be
due to mechanical cell injury, as a consequence of
intracellular or extracellular ice crystal formation, and
osmotic damage due to extensive cell shrinkage during
conventional freezing. The warming process also has a
negative influence on cells, possibly through excessive
osmotic swelling [10, 62]. Both these factors accompany slow freezing due to the chemical and physical damage of the sperm cell membranes, which is
the result of changes in lipid phase transition and/or
increased lipid peroxidation and active production of
reactive oxygen species [63] and the subsequent loss
of sperm motility [24, 64]. The permeable and nonpermeable cryoprotectants, used during conventional
freezing to prevent intracellular ice formation, can be
damaging due to the so-called CPAs toxicity [30]. This
toxicity includes damage to the cells membrane due to
the osmotic and chemical influence of CPAs on cells
during freezing and/or thawing [62, 65], which activates an apoptosis-like mechanism [66] that can also
lead to chromatin damage [67]. All of these findings
suggest that, quite apart from ice crystal formation,
slow cooling, especially the thawing of spermatozoa,
is intrinsically deleterious.
Compared to conventional slow freezing, the vitrification technique, even though it has its own peculiarities, is a real alternative.
How does vitrification work

in principle?
Luyet wrote that crystallization is incompatible with
living systems and should be avoided wherever possible [34]. However, he considered the cooling of small
living systems at ultra-high speeds of freezing possible, and that this could eliminate big crystal formations and create instead a glass-like (vitreous) state
[34]. This constituted the origin of the idea of vitrification but not, however, the beginning of the vitrification of organs, which was unthinkable at the speed
of freezing and thawing demanded by Luyet [34]. It
is known that vitrification is used as a natural form
of cryoprotection in some arctic plants [68]. In contrast to slow-rate freezing protocols, during vitrification the entire solution remains unchanged and the
177
water does not precipitate, so no ice crystals are formed

[69]. The physical definition of vitrification is the solidification of a solution (water is rapidly cooled and
formed into a glassy, vitrified state from the liquid
phase) at low temperature, not by ice crystallization
but by extreme elevation in viscosity during cooling
[70]. Fahy expressed this as follows: . . . the viscosity of the sample becomes greater and greater until the
molecules become immobilized and the sample is no
longer a liquid, but rather has the properties of a solid
[69]. However, vitrification is a result of high cooling
rates associated with high concentrations of cryoprotectant. Inevitably, this is biologically problematic and
technically difficult [71].
There are two ways to achieve the vitrification of
water inside cells:
1 To increase the speed of temperature conduction.
2 To increase the concentration of cryoprotectant.
In addition, by using a small volume of high concentration cryoprotectant (1 l), very rapid cooling rates
of 15 00030 000 C/min can be achieved (e.g. T
from 196 C to 25 C = 221 C/0.5 s = 5). The strategy of vitrification is realized by decreasing the volume of the ice crystals of extracellular water and the
total elimination of the crystallization of intracellular
water.
A practically glass-like solidification of solution is
achieved by vitrification of the bulk solution without any sizable portions of ice during cooling and
thawing (see our previous description [30]). This process is performed substantially faster than with equilibrium freezing (during which ice is formed). It is
achieved by the combination of relatively slow to moderate speeds of cooling (up to 105 C/min) with the
use of high concentrations (3.58.0 mol/l) of permeable CPAs, or by solidification of the bulk solution by abrupt cooling at a very high speed to temperatures below the glass-transition temperature of
the solution by directly plunging the specimen into
LN2 . Thus, the formation of a glassy state (with some
molecule regulation) is accompanied by an extreme
elevation in viscosity (proportional to the cooling rate)
of pure water/solution during cooling. In the glassy
state all physicalchemical processes are completely
arrested. It is known that it is possible to provide cryoprotection of cells by alcohols (including glycols),
amines (including amides), sugars, inorganic salts and
macromolecules (including proteins and polysaccharides) and dimethylsulfoxide (DMSO) (polar aprotic
178
solvent is a solvent that shares ion-dissolving power

with protic solvents but lacks an acidic hydrogen
and has a high dielectric constant and high polarity) [12]. However, it is also well-known that the
fastest of all permeable CPAs (the most used such as
ethylene and propylene glycol, glycerol, DMSO) have
a toxic influence on living cells [7274]. The toxic
effect of highly concentrated permeable CPAs (critical CPA concentration, Cv) are possibly lowered by
including some non-permeable CPAs, such as carbohydrates (saccharides), polymers (polyvinylpyrrolidone), polyols (polyethylene glycol), polysaccharide
(Ficol), amines (acetamide, formamide), inorganic
salts (sodium citrate, ammonium sulfate), proteins
(albumin, antifreeze peptide/glycopeptide), phospholipids (hen egg yolk) or by using the combination
of two or more permeable and non-permeable cryoprotectants [72]. As a rule, carbohydrates are used
for sperm cryopreservation to compensate for the
decrease in osmotic pressure caused by the permeable cryoprotectant glycerol, which works as an additional dissolvent and has the property to decrease
the mediums osmotic pressure [75]. For example, the
combination of sugars with permeable CPAs [76] has a
major influence on the vitrification properties of such a
cryoprotective mixture, resulting in a lowering of permeable CPAs toxicity to embryos and oocytes and a
significant decrease in the concentration of permeable
cryoprotectants needed for efficient cryopreservation
[77]. In general, the incorporation of non-permeating
compounds into the vitrifying solution, and the incubation of the cells in this solution before any vitrifying, helps to withdraw more water from the cells and
to lessen the exposure time of the cells to the toxic
effects of the cryoprotectants. This is not surprising
as the sugars, as non-permeable cryoprotectants, possess unique properties: during the cells dehydration
they compensate for the osmotic pressure drop (acting as an osmotic buffer to reduce the osmotic shock
that might otherwise result from the dilution of the
cryoprotectant after cryostorage), simultaneously stabilizing the cells membrane [48, 7880]. Cells naturally contain high concentrations of protein, which
is helpful in vitrification. Higher concentrations of
cryoprotectants are needed for extracellular than for
intracellular vitrification. It was demonstrated that
in certain circumstances a polymer can reduce the
Cv on average by 7%, and by as much as 24% in
combination with increased hydrostatic pressure [70].
Early studies evaluated the potential beneficial effects
of adding macromolecular solutes to the vitrification

solution to facilitate vitrification [81]. These polymers
can protect embryos against cryo-injury by mitigating the mechanical stresses that occur during cryopreservation [82]. They do this by modifying the vitrification properties of these solutions by significantly
reducing the amount of cryoprotectant required to
achieve vitrification itself [83]. They also influence
the viscosity of the vitrification solution and reduce
the toxicity of the cryoprotectant through lowered
concentrations.
Furthermore, the polymers may be able to build
a viscous matrix for the encapsulation of cells, and
also prevent crystallization during cooling and warming [81]. Indeed, ONeill observed that addition of
polyethylene glycol (PEG) resulted in greatly improved
viability of oocytes following cryopreservation, and
vastly reduced the variability seen with vitrification
solution alone [84]. The possible toxic effect of permeable CPAs was reduced by exposing the cells
to a graded series of pre-cooled concentrated solutions. The combination of these described methods and increasing the rate of cooling and warming reduces both the toxic and osmotic effects of
cryoprotectants.
The higher cooling rate may be provided by use of
an electronmicroscopical grid [85]; or by a nylon loop
allowing an effective reduction in the concentration
of single permeable cryoprotectant; or by substituting
the single permeable cryoprotectant with a mixture of
permeable cryoprotectants [40]. The small amount of
such cryopreservation solution (0.11.0 l) immersed
directly into LN2 assists in achieving the maximal cooling rate. The optimal cooling rate is achieved with the
following specially designed packaging systems: Open
Pulled Straws [86]; the Flexipet denuding pipette [40];
micro-drops [87]; electron microscope copper grids
[88]; the Hemi-straw system [89]; the Cryotop [90];
small nylon coils [91] or nylon meshes [92]; the Cryoloop [93]; the Cryoleaf [94]; the Cryotip [95] and
some others. All of these packaging systems have been
successfully used in routine human and veterinary
reproductive medicine for the vitrification of oocytes
and embryos [47, 96101]. In our experience, similar results can be achieved in the absence of the conventional CPAs provided that the cooling/warming
speed is high enough to ensure both intracellular and
extracellular vitrification [30, 33]. In general, the rate
of cooling/warming and the concentration of CPAs
required to achieve vitrification are inversely related.
In other words, the faster the cooling and warming is undertaken, the lower the critical solute concentration necessary to obtain ice-free vitrification
[34, 41, 77]. Given the biological and physiochemical effects of cryoprotectants and the high concentrations used in vitrification, it is not surprising that
cryoprotectant toxicity has been described as a key
limiting factor in the cryobiology of vitrification [34,
41]. In an attempt to avoid this toxicity, we achieve
ultra-rapid cooling and warming rates, in the range of
0.51.0 106 K/min, using a relatively dilute vitrification medium (around 12% total solute content) of
similar solute concentration to semen or blood; thus,
resurrecting the original vitrification approach of
Luyet.
Spermatozoa vitrification techniques

The era of vitrification began with the famous work
of Luyet [34]. The success of Luyets vitrification technique was supported by Shaffner applying the technique to frog spermatozoa after vitrification of fowl
sperm [42]. However, the subsequent applying of this
technique to the spermatozoa of different kind of
animals, including humans, showed non-promising
results: spermatozoa survival was very low or lacking
[43, 44]. The problem was the low concentration of
CPAs tolerated by sperm at high speed. Active investigation in this direction stopped for 25 years, and it
only began again in 1980 following the fundamental work of Rall and Fahy [35]. They discussed vitrification of mouse embryos by application of high
CPA concentrations and a relatively low speed of cooling and warming. It opened new horizons for applying this technique to different kind of cells, including tissues from different living organisms. The main
approach to vitrification of spermatozoa is the same as
used for other types of mammalian cells [102]. However, it was impossible to completely extrapolate this
technique to male gametes, due to the lethal osmotic
effects and possible chemical alterations following use
of highly concentrated permeable cryoprotectants in
combination with a high mode of cooling (2000
30 000 C/min).
Indeed, applying conventional methods of vitrifying to human spermatozoa resulted in very low or
null spermatozoa survival rates. This prompted the
idea of exploring vitrification methods that would not
require high concentrations of potentially toxic CPAs.
Seventy years of cryobiology experience had showed
179
Table 14.1. The relationship between the size (approximate) of mammalian sperm heads and stability of gametes to cold shock
Boar
Bull
Ram
Rabbit
Cat
Dog
Stallion
Human
Length (m)
9.0
9.1
8.1
7.7
7.7
7.0
6.5
4.6
Maximal width (m)
5.0
4.7
4.0
4.5
3.2
5.0
3.4
3.2
Area of projection (m2 )
37.5
34.2
28.0
Sensitivity of spermatozoa to cold shock
++++
+++
+++
++
15.2
10.8
From Watson and Plummer [107] with permission.
that cryoprotectants are not always needed for successful vitrification. The first investigations in this direction had very contradictory results. Luyet, the pioneer in vitrification investigation, showed that a small
specimen cooled very rapidly could be vitrified without substantial loss of viability [34]. There followed
subsequent investigations by Jahnel [103] and then
Parkes [104] in which they performed cryoprotectantfree cryopreservation of human spermatozoa cooled
in LN2 and liquid helium (269.5 C) using glass or
metal tubes as packaging with a large volume of sperm
suspension (a few milliliters). They did not get such
promising results, seemingly due to the difficulty of
achieving a quick enough mode of warming. All subsequent attempts to vitrify mammalian spermatozoa
using this approach resulted in low or null survival [43,
44], mostly because of the critical speed of freezing and
warming, which is very high for low concentrations of
cryoprotectants. Such situations were why it was considered that the vitrification could only be achieved
using high concentrations of permeable cryoprotectants alone or in combination with non-permeable
cryoprotectants [105]. However, classical vitrification
requires a high percentage of permeable cryoprotectants in medium (3050% compared to 57% with
slow freezing) and is unsuitable for the vitrification of
spermatozoa due to the lethal osmotic effect [65, 70].
It was reported that, by optimizing regimens for the
addition and removal of the CPAs, it may be possible
to significantly reduce the damaged effect of these substances on male gametes during the equilibration time
before freezing [13, 62, 65, 106]. But, based on numerous publications dedicated to this theme, it must be
emphasized that generally, in the majority of species,
sperm cannot tolerate the high concentrations of cryoprotectants conventionally used for vitrification. One
peculiarity of male gametes could be a significant factor: the shape and size of the sperm head, which defines
the cryosensitivity of the cell. Comparative studies on
180
various mammalian species (boar, bull, ram, rabbit,

cat, dog, horse, human) showed a negative correlation between the size of the sperm head and cryostability (Table 14.1 [107]) [108]. Among the abovementioned species, human spermatozoa possessed the
smallest size with maximal cryostability [10]. Taking
all of this into account, we believed that vitrification
of human spermatozoa with no or a low amount of
permeable cryoprotectants, in combination with a relatively small sample size (from a few to hundreds
of microliters), could be successful. Such a combination would achieve a very quick mode of cooling and
warming. This was theoretically predicted in 2000 by
Bischofs group [106]. They wrote that one of the optimal cooling rates for spermatozoa theoretically lies
between 5000 and 7000 C/min. A few years later, we
were able to prove this theoretical prediction. In our
first studies, we demonstrated the possibility for successful vitrification of human spermatozoa without
permeable cryoprotectants, using 1% of human serum
albumin (HSA) as the chosen non-permeable cryoprotectant [30, 33]. The chosen package system was
self-manufactured cryoloops. To investigate the effectiveness of this method of cryopreservation, we compared it with conventional slow freezing. Comparison
of the physiological parameters of spermatozoa following cryopreservation was done using split-sperm samples. For cryopreservation of spermatozoa, suspension
was performed using the vitrification method without permeable cryoprotectants using cryoloops as the
package system. Following this, a sample of spermatozoa suspension (20 l volume of drops) was placed
onto a copper loop (5 mm diameter) and then directly
plunged into LN2 . The warming of the cryoloop was
performed by plunging the cryoloops into a centrifuge
tube with 10 ml of sperm-preparation medium at
37 C under intense agitation. The routine conventional slow freezing was done using French straws
[109] and standard cryoprotective solution (TYBG,
Figure 14.1 Damaged and undamaged DNA after warming.

Fluorescent staining was performed using SYBR green stain
(working concentration 1:200). In healthy cells, the fluorescence was
confined to the nucleoid: undamaged DNA is supercoiled and does
not migrate very far from the nucleoid. In cells that have incurred
damage to the DNA, the alkali treatment unwinds the DNA,
releasing fragments that migrate from the nucleoid and form the
so-called comet-tail (circled). See plate section for color version.
Scandinavian IVF Science, Sweden). The swim-up prepared spermatozoa, conventionally frozen with cryoprotectants immediately after thawing, showed 38%
motility compared to 49% after vitrification without cryoprotectants. The number of morphologically
normal spermatozoa after applying both cryopreservation methods was not significantly different (27%
for slow frozen with permeable cryoprotectants versus 26% for vitrified without permeable cryoprotectants, P 0.01). Programmable freezing in the absence
of cryoprotectants dramatically reduced all sperm
variables.
The negative influence of freezing on cells can lead
to chromatin damage and is strongly correlated with
mutagenic effects [23]. It has been shown that there is a
correlation between fertilization rates in IVF [110] and
intracytoplasmic sperm injection (ICSI) [111] with
fragmented DNA. We performed the comet assay to
investigate the occurrence of apoptosis in spermatozoa DNA after vitrification (Figure 14.1). Our albumin estimates [26] showed that devitrification (especially intracellular) did not occur [112] during cooling
or, especially, during re-warming/resuscitation, due to
the small amount of specimen and cells, the high vis-
cosity of the solution and the high speed of cooling and warming [30]. Our investigation showed that
no significant difference in DNA integrity was found,
independent from the mode of cooling and the presence of cryoprotectants (P 0.05) (Figure 14.2) [26].
We found that the DNA integrity of vitrified spermatozoa was comparable with standard slow frozen
and thawed spermatozoa. The DNA integrity (P
0.05) was found to be unaff1ected by the vitrification method. Our results supported the results of the
Schrader group [113] and Duty [114]. They found no
difference in sperm chromatin structure assay results
for cryopreserved or non-cryopreserved sperm, or for
slowly or flash-frozen specimens [113]. They suggested
that the unique packaging of sperm DNA protects
it from intracellular fluid shifts and the formation
of the nuclei of crystallization during the cooling
warming cycle. The use of spermatozoa vitrified using
this method for IVF of human oocytes resulted in successful fertilization and development to the blastocyst
stage [33].
The next modification of the vitrification method
was the direct dropping of spermatozoa suspension
into LN2 (Figure 14.3 [115]) [32, 49]. Immediately
following vitrification, 30 l of sample was dropped
directly into LN2 . The temperature of the foil was
determined by an electrical thermometer. After 5 min
of cooling, the solution-precursor-solid (SPS) droplets
were placed into pre-cooled LN2 Cryovials and stored
there until the time of use. The warming of the solid
sphere was performed by directly plunging it into
a centrifuge tube with 10 ml of sperm-preparation
medium (SPM) at 37 C under intense agitation.
Based on our previous data and the peculiarities
of carbohydrates, we decided to investigate the contribution of disaccharide sucrose to sperm cryoprotection during cooling/warming using the dropping
method. The investigation focused especially on the
ability of sucrose to support the motility of spermatozoa, prevent the artificial induction of capacitation or
acrosome reaction and support mitochondrial stability during vitrification/warming. The viable, acrosome
reacted and capacitated spermatozoa (Figure 14.4)
were detected using the double-fluorescence Chlortetracycline (CTC)-Hoechst 33258 staining technique
[116].
The results have shown that the medium supplemented with 1% HSA + 0.25 M sucrose allowed the
motility rate to increase after warming to 57%, compared to HSA alone (45%) or pure medium without
181
105
100
DNA integrity (%)
95
90
85
80
75
70
65
Swim up
Swim up
Slowly frozen Slowly frozen
Vitrified
Vitrified
without
with
without
with
without
with
cryoprotectant cryoprotectant cryoprotectant cryoprotectant cryoprotectant cryoprotectant
Method of treatments
Figure 14.2 DNA integrity of spermatozoa according to different treatments and cryopreservation methods. Each bar represents the
medium, 25 and 75 percentile, minimum and maximum values. Bars with different letters inside of each treatment group represent significant
differences (P 0.05). With permission from Isachenko et al. [26].
Figure 14.3 Scheme of the spermatozoa vitrification procedure.

(1) Single channel pipettes with adjustable volume (30 l). (2)
Spermatozoa suspension. (3) Strainer. (4) Foam box. (5) Liquid
nitrogen. (6) Distance between bottom of strainer and surface of
liquid nitrogen (minimum 3 cm). With permission from Isachenko
et al. [115]. See plate section for color version.
cryoprotective substances (19%) (Figure 14.5a) [32].

The medium supplemented only with HSA provided
21% motility of spermatozoa after 24 h of culture
compared to 2.5% motility with non-supplemented
medium. The combination of two HSA and sucrose
non-permeable substances in the cryopreservation
182
medium provides 32% motility of spermatozoa after

24 h of culture. Our data showed that the percentage
increase of capacitated spermatozoa after warming was
not significantly different to controls in both groups
(HSA: 7%; HSA + sucrose: 11%; control: 9%; P 0.1).
The acrosome-reacted spermatozoa in the group with
both supplements (HSA and sucrose) were detected
in 6%, in the group with one supplement (HSA) in
11% and in the control in 10% (P 0.05). The physiological condition of spermatozoa, such as capacitation
and acrosome reaction, was not affected by the composition of cryoprotective medium (Figure 14.6) or by
the vitrification technique (Figure 14.5b). The results
of our investigation corresponded to data of Esteves
et al. [117]. It showed no statistical difference between
numbers of capacitated and acrosome-reacted spermatozoa of control and experimental groups. According to the data, the acrosome reaction after cryopreservation may involve some complex mechanisms rather
than a physiological change induced by capacitation.
However, at present, routine conventional freezing for
human sperm cryopreservation does not induce the
capacitation process or acrosome reaction but also
does not sufficiently preserve mitochondrial integrity
[24, 25].
To evaluate mitochondrial activity, we measured changes in the mitochondrial membrane
(b)
(a)
(c)
(d)
Figure 14.4 Example of non-capacitated (a), capacitated (b), acrosome-reacted (c) and non-viable (d) spermatozoa. At least 200
spermatozoa were observed in each plate and 3 patterns were identified (see Figure 14.2). (a) A uniform fluorescence on the head of the
spermatozoa (non-capacitated spermatozoa). (b) A band of fluorescence diminished in the post-acrosomal region and a relatively shining
fluorescence in the acrosomal region (capacitated spermatozoa). (c) A fluorescence in the complete head of the spermatozoa, except a
tenuous band of fluorescence in the equatorial segment (acrosome-reacted spermatozoa). The slides were viewed using a Zeiss Axiolab
Epifluorescence microscope that was equipped with an excitation/emission filter of 485 nm/520 nm under 400 magnification. The
non-viable spermatozoa were observed with the filter set 09 (450490 nm). The dead spermatozoa displayed a pattern of blue fluorescence in
the whole head (Figure 14.2d). With permission from Isachenko et al. [115]. See plate section for color version.
potential (M ) using a unique fluorescent

cationic dye, 5,5
, 6,6
-tetachloro-11
, 3,3
-tetraethylbenzamidazolocarbocyanin iodide (Figure 14.7),
commonly known as JC-1 [118]. This test was
performed as per manufacturers instructions for
Mitochondrial Permeability Detection Kit AK-116

(MIT-E-TM , BIOMOL International LP, Plymouth
Meeting, PA) and applied for the early detection
of the initiation of cellular apoptosis. However, the
mature healthy human ejaculated spermatozoa are
incapable of undergoing apoptosis [119]; they lack
the necessary assemblage of enzymes and signaling
pathways possessed by cells capable of undergoing
this process, and the lack of orange-red fluorescence
due to binding of this cationic probe by the sperm
mitochondria implies only the loss of energy conservation capacity. The mitochondria are most probably
uncoupled rather than membrane damaged; but
uncoupled mitochondria are evidence of unhealthy
sperm and so this determination is a useful one to
R
document post-thaw sperm survival by determination of mitochondrial activity. It has previously been
demonstrated that mitochondrial activity and viability
are equally susceptible to cryopreservation-induced
damage [25]. It has been suggested, however, that
current conventional freezing techniques for human
sperm cryopreservation are good enough to preserve
all these structures [24, 25]. The non-permeable
cryoprotective substances also showed stable cryoprotective effect on the mitochondrial membrane (Figure
14.5c). It was observed that the medium alone has no
cryoprotective effect (8%). The combination of HSA
and sucrose is more effective then addition of HSA
only (65 and 33%, respectively; P 0.05).
Unfortunately, these methods have a large technological disadvantage: their techniques do not prevent
direct contact with LN2 . At present, numerous publications exist about microbial contamination of the
different kinds of biological material cryopreserved
in LN2 [120123]. Because of this, a special kind of
183
Motility of
spermatozoa
immediately after
warming
Motile/viable spermatozoa (%)
100
90
80
70
Motility of
spermatozoa
after 24 h
60
50
Viability
immediately after
warming
40
30
20
10
0
HTF
HTF + HSA + S
HTF + HSA
Vitrification media
(a)
Capacitated/acrosome
reacted spermatozoa (%)
100
Capacitation
80
Acrosome
reaction
60
40
20
0
HTF
HTF + HSA
HTF + HSA + S
Vitrification media
(b)
Spermatozoa with stable

mitichondrial membrane
potential (%)
100
80
60
40
20
0
HTF
(c)
HTF + HSA
Vitrification media
HTF + HSA + S
Figure 14.5 (a) Cryoprotective effect of human tubal fluid (HTF) alone or in combination with human serum albumin (HSA) or with HSA +
sucrose on motility and viability of vitrified spermatozoa. (b) Cryoprotective effect of HTF alone or in combination with HSA or HSA + sucrose
on activation of capacitation and induction of acrosome reaction of vitrified spermatozoa. (c) Cryoprotective effect of HTF alone or in
combination with HSA or HSA + sucrose on the mitochondrial membrane integrity of vitrified spermatozoa. Asterisks denote statistical
differences between respective values of compared groups (P 0.05). With permission from Isachenko et al. [115].
package system needed to be developed, one that

allowed the biological material to separate from LN2 .
However, caution was needed as the package system
can significantly reduce the speed of cooling, resulting in undesirable results after warming. Therefore,
184
it was necessary to try and develop an aseptic vitrification technique that prevented direct contact with
LN2 . To do this, it was necessary to discover if the
cooling or warming rate of vitrification most affected
the spermatozoa parameters. According to the famous
90
Motility immediately after

dilution
Motility after 24 h
80
Viability
70
Capacitation
60
Acrosome reaction
Percent (%)
100
50
Mitochondrial membrane
potential
40
30
20
10
0
HTF
HTF + HSA HTF + HSA + S

Vitrification media
Figure 14.6 Influence of human tubal fluid (HTF) medium alone or in combination with human serum albumin (HSA) or HSA + sucrose on
motility, viability, activation of capacitation, induction of acrosome reaction and mitochondrial membrane integrity before cryopreservation.
No statistical differences were found between the respective values of compared groups (P 0.1) except for motility 24 h after in vitro culture
(P 0.05). With permission from Isachenko et al. [115].
(a)
(b)
Figure 14.7 Example of undamaged (a) and damaged

mitochondria (b). In undamaged mitochondria the mitochondrial
membrane potential (M ) is intact and the JC-1 reagent
aggregate inside of the non-damaged mitochondria and fluoresces
red. In our case, the midpiece is yellow, as expected for the red
fluorescence from JC-1 aggregates merging with the green
fluorescence of JC-1 monomer dispersed throughout the cell
plasma membrane. In damaged mitochondria, the M is broken
down and the JC-1 reagent disperses though the entire cells and
fluoresces green. The changes in M is measured using an
unique fluorescent cationic dye, 5,5, 6,6-tetachloro-11,
3,3-tetraethyl-benzamidazolocarbocyanin iodide, commonly
known as JC-1. See plate section for color version.
investigations of Luyet, devitrification and the growth

of ice crystals formed during cooling could be a key
factor promoting cell damage during re-warming and
thawing procedures [34, 41]. To accelerate the velocity of warming, we directly placed the specimens in a
warm solution under gentle agitation, ensuring a very
high rate of warming. This procedure prevents the substantial devitrification (re-crystallization) of the vitri-
fied intracellular solution and an uncontrolled increase

in the size of intracellular crystals due to the high
speed and very short time of warming. Our estimations [30, 124] showed that during cooling and, especially, warming devitrification (especially intracellular) would not occur due to the following: (1) the high
viscosity of the freezing medium and the small specimen size; (2) the high viscosity of the intracellular
matrix and the small size of the cells, their low water
content and their high degree of compartmentalization; and (3) the very high speed of warming (higher
than the speed of cooling) [112, 125].
Our results [27] supported the data. The results
showed that during vitrification applying a cooling rate
of around 160250 C/min in combination with very
fast mode of warming (38 C) was enough to preserve
60% motility. The results for the recovery of motile
spermatozoa after slow vitrification was unstatistically different to spermatozoa cryopreserved using
the quick vitrification method (Figure 14.8). DNA
integrity was found to be non-affected by vitrification
mode and was around 90%. The results from IVF show
approximately equal fertilization potential for fresh
human spermatozoa samples compared with swim-up
prepared CPA-free samples vitrified by directly plunging into LN2 or into nitrogen (N2 ) vapor (Table 14.2).
This is not surprising, because human spermatozoa
contain large amounts of proteins, sugars and other
components that make the intracellular matrix highly
viscous and compartmentalized. The additional factor
185
100
Figure 14.8 The motility and DNA

integrity of spermatozoa after
cryoprotectant-free cryopreservation with
quick (vitrification) and slow (freezing)
cooling. There is no difference between
the respective rates of vitrified and frozen
spermatozoa (Pab 0.05). With
permission from Isachenko et al. [27].
90
80
70
Rate (%)
60
b
Motility
50
DNA integrity
40
30
20
10
0
Fresh (control)
Quick
Mode of cooling
Slow
that could be an influence on successful vitrification at

such a low mode of cooling is the small size and high
degree of compartmentalization of the sperm head.
In such conditions, even if small (non-lethal) crystals start to form during this relatively slow cooling,
there would be insufficient time for substantial growth
during cooling. It is known that a major problem for
such metastable systems is the regrowth of crystals and
devitrification during warming. All of these findings
led us to speculate the possibility that we could achieve
intracellular vitrification of the human spermatozoa
even at such a low rate of cooling. Guided by these
results, we have two modifications of the aseptic technique (Figure 14.9) [49]. The first modification, aseptic
cooling [49] in LN2 using an Open Pulled Straw (Figure 14.9a) was performed as follows: 510 l of spermatozoa suspension was drawn inside Open Pulled
Straws by capillary effect [86]. The straw was located
inside of a sterile 0.5 ml insemination straw, hermetically sealed with an ultrasound hand-held sealer and
plunged into LN2 .
The second modification (Figure 14.9b), aseptic
cooling in LN2 using an insemination open straw [49],
was performed as follows: 12 l of sample was located
on the inner wall, not far from the end of an insemination straw. For the same purpose, it is also possible to use the Cut Standard Straw (CSS) (Figure 14.10),
which is made from a standard insemination 0.25 ml
straw cut at an angle 45 [77]. The straw is placed
186
inside a sterile 0.5 ml insemination straw and sealed

the same way as for the Open Pulled Straw method.
The open pulled or insemination straws are rapidly
warmed by immersion in 1.5 ml microcentrifuge tubes
containing 1 ml of SPM at 37 C after being expelled
from their packaging.
Comparative investigation of these four modifications of the vitrification technique showed that all
cryopreservation regimens give about a 40% reduction in spermatozoa motility in comparison with nontreated swim-up control. No statistically significant
difference was found in these parameters between all
the regimens of cryopreservation tested [49].
Recently, the CSS aseptic vitrification technique
was applied to ejaculate containing 13 106 motile
spermatozoa/ml, 50% progressive motile and 30%
morphologically normal spermatozoa [126]. The
described methodology was finalized in Chile and
approved by the University of Temuco de La Frontera,
Ethics Committee. For vitrification, human tubal
fluid (HTF) with 1% HSA and 0.25 M sucrose in
the end-concentration was applied [127]. The 0.5 M
sucrose was prepared in bi-distillate water with 1%
HSA filtrated and frozen until use. After dilution, the
cell suspension was maintained at 37 C in 5% CO2 for
5 min before the cooling procedure was performed.
Then 10 l of spermatozoa suspension was deposited
on the end of the inner part of a CSS, packaged
into sterile 0.5 ml insemination straws, hermetically
10
12
12
13
15
16 h, 2 PN
and 3 PN
(n)
3 transfer
3 transfer
3 transfer
3 transfer
3 transfer
3 transfer
2 transfer
2 transfer
48 h, 46
blastomeres
(n)
56 h, EB and
BL (n)
Oocytes
(n)
16 h, 2PN
and 3PN
(n)
48 h, 46
blastomeres
(n)
56 h, EB
and BL
(n)
After vitrification with quick cooling
(2PN) oocytes with two pronuclei; (3PN) oocytes with three pronuclei; (EB) early blastocysts; (BL) blastocysts.
No differences between spermatozoa vitrified using quick and slow cooling (P 0.01).
With permission from Isachenko et al. [27].
Oocytes
(n)
Total
oocytes
(n)
After fresh spermatozoa (control)
Oocytes
(n)
16 h, 2PN
and 3PN
(n)
48 h, 46
blastomeres
(n)
56 h, EB
and BL (n)
After freezing with slow cooling
Table 14.2. Fertilization properties of spermatozoa after cryoprotectant-free cryopreservation with rapid and slow cooling: results of in vitro fertilization and culture
(a)
(b)
Figure 14.9 Two modifications of aseptic vitrification technique for spermatozoa. (a) Photograph of container and method for Open Pulled
Straw vitrification and warming of spermatozoa. (1) Open pulled straw. (2) Suspension of spermatozoa (510 l). (3) Meniscus of suspension.
(4) 90 mm straw. (5) Heat-sealed end of 90 mm straw. (6) Marked end of Open Pulled Straw. (7) Tube for warming. (8) Warming medium.
White arrow indicates the direction of thawing and swim-up of sperm suspension. (b) Photograph of container and method for open straw
vitrification and warming of spermatozoa. (1) Tip of pipette. (2) Open straw. (3) Drop of spermatozoa (0.52 l). (4) A 90 mm straw. (5)
Heat-sealed end of a 90 mm straw. (6) Marked end of open straw. (7) Tube for warming. (8) Warming medium. White arrow indicates the
direction of thawing and swim-up of sperm suspension. With permission from Isachenko et al. [49].
Figure 14.10 Photographs and scheme of warming using the Cut

Standard Straw (CSS) container for vitrification. (1) A closed 0.5 ml
straw. (2) Cut Standard Straw. (3) Vitrification medium with embryo.
(4) Tube with solution for warming and removal of cryoprotectant.
With permission from Isachenko et al. [77].
sealed from both sides with a hand-held sealer (Medical Technology GmbH, Bruckberg, Germany) and
plunged into LN2 . The warming of the spermatozoa
sample was performed a day earlier than the planned
intracytoplasmic sperm injection procedure as fol-
188
lows: the package system was partly removed from

LN2 and the over part of the 0.5 ml straw was cut
off. Then the CSS was removed from the packaged
straw and quickly immersed into 1.5 ml, pre-warmed
to 37 C, HTF with 1% HSA in a 2 ml Eppendorf tube.
This mode allows very quick warming, with a speed
of approximately 90 000 C/min. Next, the spermatozoa were concentrated by centrifugation at 340 g
for 3 min. The resulting pellet was resuspended in
10 l of the same medium and used for further culture, evaluation and ICSI. The motility of spermatozoa was estimated with a Maklers chamber under
the light microscope at 400 magnification. At the
same time the following parameters were assessed:
progressive motility (categories a and b); viable,
acrosome-reacted, capacitated spermatozoa; and stability of mitochondrial membrane (M ). After 30
min post-warming, the spermatozoa showed the following rate of physiologicalmorphological parameters: 85% motility (a + b); 10% with signs of capacitation; 5% with signs of acrosome reaction; and 70%
spermatozoa with non-damaged mitochondrial membrane.
For induction of superovulation, pituitary suppression was achieved using a long protocol with a
gonadotropin-releasing hormone (GnRH) analogue
microdose, followed by ovarian stimulation with
recombinant follicle stimulating hormone (FSH) and

human menopausal gonadotrophin.
Ten cumulus-oocyte complexes were retrieved 36 h
after the administration of 10 000 IU of human chorionic gonadotropin (hCG). The oocytes were denuded
with 80 IU/ml of hyaluronidase and 8 mature MII
oocytes were microinjected with vitrified/warmed
spermatozoa. Eighteen hours later, six oocytes showed
signs of normal fertilization. The embryos were cultured in vitro for 48 h and transferred to the uterus cavity (Frydman Ultra Soft Catheter with Echo Tip, CCD,
France) under ultrasonographic guidance.
Fifteen days after transfer, a -hCG value of
148 IU/L was registered, a clinical twin pregnancy was
confirmed by ultrasound at 7 weeks of gestation and 2
healthy babies were born at term.
Is vitrification suitable for IVF, ICSI

and insemination?
Compared to conventional slow freezing, all modifications of the vitrification technique only allow the
simultaneous cryopreservation of a small amount of
spermatozoa suspension (from 0.5 to 30.0 l). So, the
advantage of programmable or non-programmable
conventional slow freezing [1719] is the ability to
simultaneously preserve a relatively large volume of
diluted ejaculate or prepared spermatozoa (from 0.25
to 1.0 ml) [2022, 50]. Because of this, our subsequent
study aimed to develop an aseptic vitrification technique for handling a large volume of spermatozoa suspension, which would allow this method to be applied
for purposes such IVF or intrauterine insemination as
they require a large volume of spermatozoa.
We used the results of our previous investigation
concerning slow vitrification and a quick warming
rate [27] to help us develop an aseptic vitrification
technique for handling large volumes of spermatozoa
suspension [128]. This is because the packaging system, when compared with the sample directly making contact with LN2 , considerably slowed down the
speed of cooling. This problem was solved by using
for the warming step a large volume of warm (37 C)
sperm preparation medium (5 ml) with gentle agitation to accelerate the melting, and the removing of
unclosed content from both side straws for the sample. Such a system allows the transition of heat from
the warm sperm preparation medium to the sample
through the plastic wall and, at the same time, provides direct contact between warm solution and the
3
1
4
Figure 14.11 The scheme of the warming process

of a vitrified spermatozoa sample. (1) A sample straw.
(2) A spermatozoa sample. (3) A 15 ml plastic
centrifuge tube. (4) Sperm preparation medium. The
arrow heads shows the direction of the heat moving
from the warm sperm preparation medium through
the plastic wall of straw to the vitrified spermatozoa
sample. The large arrows below the sample straw
shows the direction of movement of the warm
sperm-preparation medium inside of the sample
straw. The direct contact between the warm sperm
preparation medium and the vitrified spermatozoa
sample accelerates its melting.
solid sample (Figure 14.11). Such straws filling and

warming achieves the necessary speed of warming and
protection required for spermatozoas life-important
parameters. To check the before and after results of
vitrification/freezing, we analyzed spermatozoa motility and applied highly sensitive methods to analyze the
cells integrity and function.
Flow cytometry (FACSCalibur, Becton Dickinson)
was applied to analyze the following:
1 Mitochondrial membrane potential integrity. To
evaluate mitochondrial activity, we measured the
changes in the M using a unique fluorescent
cationic dye, 5,5
, 6,6
-tetachloro-11
,
3,3
-tetraethyl-benzamidazolocarbocyanin iodide,
commonly known as JC-1 [118].
2 Cytoplasmic membrane integrity (CMI). The
integrity of the plasma membrane was assessed
with a LIVE/DEAD sperm viability kit, which is
used to stain nucleic acid probe molecular
(SYBR-14 dye) and propidium iodide (IP).
3 Acrosomal membrane integrity (AMI). The
acrosomal membrane integrity was assessed using
the technique described by Mendoza et al. [129].
4 Phosphatidylserine translocation (PST). To
determine of the phosphatidylserine translocation
in the sperm, we applied the anexin V-FITC
staining technique (APOPTESTTM -FITC, Nexins
Research, the Netherlands).
5 Level of DNA fragmentation. For determination of
DNA fragmentation in spermatozoa [130], we
used TUNEL (In Situ Cell Death Detection Kit,
Fluorescein, Roche Applied Science, USA), which
189
*
*
*
(a)
(d)
(b)
(e)
(g)
(h)
(c)
(f)
Figure 14.12 The vitrification procedure for big volume of spermatozoa suspension. (a) The 0.25 ml plastic straws are halved and
dark-marked from one side (dark asterisks) and a 0.5 ml packaging straw (white asterisks). (b) The non-marked side of the half-straw is filled
with spermatozoa suspension. (c) A 0.25 ml half-straw, hermetically closed from both sides, is filled with spermatozoa suspension. (d) Holding
it in a horizontal position, the 0.25 ml half-straw filled with spermatozoa suspension is marked from one side (arrowed) and placed into a
0.5 ml packaging straw, closed from both sides. (e) Using tweezers and keeping it in a horizontal position, this closed packaging system is
directly immersed into liquid nitrogen (LN2 ) and submerged for over 5 s to prevent the flow of spermatozoa suspension spreading into
packaging straw. (f) The vitrified sample is stored in LN2 . Using tweezers, the dark-marked part of the Sealed Pulled Straw (SPS), approximately
1.01.5 cm, is removed from the LN2 and the end of packaging straw is cut. (g) With the help of a 200 l pipette tip (Eppendorf AG, Hamburg,
Germany), the suspension-filled fixed straw is quickly removed from the packaged straw and (h) immersed into a 15 ml plastic tube
containing 6 ml of human tubal fluid (HTF) and human serum albumin (HSA) prewarmed to 37 C with gentle agitation to accelerate the
melting and removing of content. See plate section for color version.
is based on the detection of single and

double-stranded DNA breaks occurring at early
stages in apoptosis.
The results of this technique were compared to the slow

conventional frozen technique. For this purpose all
supernatants from each swim-up-prepared ejaculate
were centrifuged, then diluted with the same medium
to achieve the concentration of 5 106 spermatozoa/ml and then finally divided into three equal parts.
The first part non-frozen swim-up prepared spermatozoa served as a control, the second part was conventionally frozen and third part was vitrified.
190
Conventional freezing/thawing was performed

according to routine procedure in LN2 vapor (80 C
10 cm over the LN2 surface) for 30 min using freezing medium. The frozen samples were stored a minimum of 24 h before evaluation. The thawing of samples was performed in a 37 C water bath, washed
for 5 min at 340 g and pellets resuspended with
HTFHSA.
The vitrification/warming method [128] for a relatively large volume of spermatozoa suspension (100 l,
compared to small volume 130 l) was performed
as follows (Figure 14.12af): The suspension of swimup-prepared spermatozoa was diluted 1:1 with a solution of 0.5 M sucrose (0.25 M end concentration) to
100
90
After warming
80
70
Motility (%)
achieve the final concentration of 5 106 spermatozoa/ml. For vitrification, one half of the 0.25 ml plastic straw was used. For this, the straw was cut into
two parts and a dark mark made from one side. All
subsequent manipulations were done strictly in a horizontal position to prevent the loss of spermatozoa
suspension. The half-straw was filled from the nonmarked side with 100 l of spermatozoa suspension.
Then sticky tape in a horizontal position was put into a
0.5 ml packaging straw, previously hermetically closed
from the other side, and hermetically closed. Then
the straw packaging system (SPS), held by tweezers to
strictly keep it in a horizontal position, was directly
immersed into LN2 and submerged there for over 5 s
to prevent the flow of spermatozoa suspension into the
packaging straw. It was then stored in LN2 for at least
24 h before use.
The warming of spermatozoa suspension was done
as follows (Figure 14.12g,h): Before removing the SPS
from the LN2 it was necessary to find the side of
the dark-marked spermatozoa suspension-filled straw.
Using tweezers, this part of the SPS, approximately
1.01.5 cm, was removed from the LN2 . The end of
the packaging straw was cut and then, with the help
of pipette tip for 200 l, the suspension-filled straw
was fixed, quickly removed from the packaged straw
and immersed into a 15 ml plastic tube containing
10 ml of HTFHSA prewarmed to 37 C with gentle agitation to accelerate the melting and removing of content. After warming, the spermatozoa were
concentrated by centrifugation at 340 g for 5 min
and the sediment was resuspended with HTFHSA.
As a control, before cryopreservation we tested the
influence of basic medium (HTFHSA), vitrification
medium (HTFHSAsucrose) and the TESTyolk
buffer (TYB) medium used for conventional freezing,
on the physiologic parameters of spermatazoa (Figures
14.13, 14.14). The motility of spermatozoa 1 h after
warming, with subsequent incubation at 37 C in a CO2
atmosphere, and after 24 h in vitro culture is shown in
Figure 14.13. The data shows that our newly developed
method of vitrification for relatively large volumes of
spermatozoa suspension (100 l, compared to small
volume 130 l) achieves high spermatozoa motility (62%) after warming and satisfactory motility after
24 h in vitro culture (22%). This is not statistically different from slow frozen spermatozoa at 24 h (24%).
Indeed, all of the data are similar to the slow-freezing
technique.
60
After 24 h
*
*
50
40
30
20
10
0
Native
Vitrification
Slow
Type of treatment
Figure 14.13 Influence of type of sperm treatment on

spermatozoa motility after warming/thawing and after 24 h of in
vitro culture. Different superscripts indicate significant differences
(P 0.05).
The influence of the type of treatment on the stability of mitochondrial membrane potential M ,
the plasmatic and acrosomal membrane, induction of
cryo-capacitation (phosphatidylserine translocation)
and level of DNA fragmentation is shown in Figure
14.14. The data shows that aseptic vitrification had
a significantly stronger protective effect on the mitochondrial membranes (Figure 14.14a) potential stability (72%) compared to conventionally frozen spermatozoa (30% P 0.05).
The integrity of the cytoplasmic membrane (Figure 14.14b) was significantly affected by both cryopreservation methods, but protection of the cytoplasmic membrane by conventional slow freezing was significantly lower (30%, P 0.05) than by vitrification
(54%).
Both aseptic vitrification and conventional freezing negatively influence the acrosomal membrane
integrity (28.0 6.9% versus 41.4 2.5%, respectively; P 0.05; Figure 14.14c). Although the acrosomal membrane integrity was lower after applying aseptic vitrification, the index of spermatozoa integrity was
non-statistically different from the number of conventionally frozen spermatozoa with non-damaged acrosomal membrane.
It was noted that this vitrification technique
strongly prevented the translocation of phosphatidylserine (2%; Figure 14.14d) compared to
phosphatidylserine quantitative indexes of conventionally frozen spermatozoa (20%; P 0.01). Both
191
100
potential (JC-1)
80
103
60
40
101
PSAFITC 2 VII 280607.001
10
10
1
*
Native
Vitrification
Slow
Type of treatment
100
101
102
103
104
10
20
Translocation of
phosphatidylserine (anexin V)
103
15
102
10
Native
Vitrification
Slow
100
101
Type of treatment
8
7
6
5
4
3
2
1
0
ANEX VIT 2 250607.018
104
10
25
(d)
(e)
10
10
10
10
10
Fragmentation of DNA
(TUNEL)
104
Damage of acrosomal
membrane (PSA-FITC)
10
I VII 290607.031
103
104
102
SYBR-14
103
10
102
20
10
101
10
101
10
10
10
30
10
100
40
Slow
10
Vitrification
Type of treatment
Native
Native
Vitrification
Type of treatment
Slow
100
10
4
1
20
50
Acrosomal membrane
damage ( %)
40
(b)
(c)
60
10
Phosphatidylserin
(anexin V) translocation (%)
Integrity of cytoplasmatic
membrane (SYBR-14)
Slow
Type of treatment
10
Cytoplasmic membrane
integrity (%)
80
Vitrification
100
Native
(a)
100
102
20
0
PM VIT 2 250607.076
104
DNA fragmentation (%)
potential (%)
100
101
102
103
104
Figure 14.14 Influence of type of sperm treatment on spermatozoa mitochondrial membrane potential integrity, plasmatic and acrosomal
membrane integrity, stability of phosphatidylserine and level of DNA fragmentation. (a) Mitochondrial membrane potential integrity. (b)
Plasmatic membrane integrity. (c) Acrosomal membrane integrity. (d) Phosphatidylserine (anexin-V) translocation ability. (e) Level of DNA
fragmentation. Different superscripts indicate significant differences (P 0.05).
methods of cryopreservation, aseptic vitrification and

conventional freezing (Figure 14.14e) had no negative
influence on cells DNA integrity (P 0.5).
Future perspectives on the long-term

storage of male gametes
cheap, quick and successful for different types of reproductive (different stages of maturity), somatic, stem
cells and, seemingly, tissues. Due to the lowering of
a solutions glass transition temperature, permeable
cryoprotectants:
r prevents actual freezing and
r maintains some flexibility in a glassy phase.
Long-term storage of frozen cells and tissues remains

elusive in both theoretical and routine cryobiology,
and future investigation applying nanotechnology is
needed. The principle behind future investigations is
that the developmental rate of cells depends on the
concentration of cryoprotectants and the speed of
cooling and warming. Right now, cryobiology investigations are exploring vitrification and freezing-drying
(lyophilization) techniques.
Vitrification is a future perspective in the longterm storage of male gametes because the method is
Vitrification without permeable cryoprotectants also

allows us to:
r avoid cryoprotectants toxicity and osmotic stress
r avoid damage to the plasmatic and mitochondrial
membrane during equilibration with
cryoprotectants
r protect plasmatic and mitochondrial membrane
against lipid peroxidation and the formation of
reactive oxygen species
r avoid DNA damage.
192
The other future perspective in the long-term storage of male gametes is lyophilization. This method
provides a low cost for storage and transport of preserved materials because the biological material can
be stored at 4 C and shipped at ambient temperatures. This technique is very suitable for preservation
of the genome (nucleus), because nuclear viability is
not equivalent to cell viability and is not destroyed by
freezing/drying [131135]. In 2003, Ward, from the
Yanagimachi group, wrote wrote that: (1) the freezedrying procedure itself causes some abnormalities in
spermatozoa but freezing without cryoprotection does
not; and (2) long-term storage of both frozen and
freeze-dried spermatozoa is not deleterious to their
genetic integrity [136]. Freezing without cryoprotection is highly successful, simple and efficient but, like
all routine sperm storage methods, requires LN2 . Liquid nitrogen is also required for freeze-drying but
sperm can then be stored at 4 C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is
the preferred method for preservation of spermatozoa
from mouse strains carrying unique genes and mutations. The genomic integrity of cells can be maintained
after freeze-drying and it is possible to produce offspring from the cells using nuclear transfer techniques
[131, 137, 138]. In this case, the long-term preservation of mouse sperm by desiccation is economically
and logistically attractive [139].
Conclusions
r It is feasible for human spermatozoa to be vitrified

without using permeable cryoprotectants. This is
either by directly plunging into LN2 or freezing in
N2 vapor beforehand, followed in both cases by
rapid thawing.
r The speed of warming plays a decisive role in
vitrification independent from the type of cells.
r The DNA of spermatozoa from normospermic
samples is stable and independent from the type
of treatment.
r A mixture of non-permeable cryoprotectants
(HSA + sucrose) can significantly enhance
mitochondrial integrity and prevent initiation of
capacitation and the acrosome reaction process.
r The vitrification procedure without permeable
cryoprotectants effectively protects the important
physiological parameters of spermatozoa.
r The developed aseptic vitrification technique for

large volume (100 l) spermatozoa suspension
retains the full functionality of spermatozoa.
r In contrast to conventional freezing, the method
is quick and simple and does not require special
cryobiological equipment.
r The evaluation of motility and long-term survival
of spermatozoa allows us to conclude that all four
investigated methods can be used successfully.
However, the method of aseptic vitrification is
recommended because it minimizes the potential
risk of microbial contamination.
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Section 4
Chapter
15
Transplantation of cryopreserved
spermatogonia
Jill P. Ginsberg and Ralph L. Brinster
Background
Over the last several decades, survival rates for childhood cancer have steadily increased. In fact, with
the overall cure rate for pediatric malignancies now
approaching 80%, current estimates indicate that 1
in every 640 young adults in the USA will be a survivor of childhood cancer [1]. Unfortunately, many
survivors struggle with medical late effects of their
treatment including disorders of the endocrine system, cardiac and pulmonary dysfunction, secondary
neoplasms and infertility. Gonadal damage is a relatively common consequence of the treatments used
to cure pediatric cancer. The extent of cytotoxic germ
cell damage depends on the specific chemotherapeutic
agents used and the cumulative doses received. Alkylating agents (particularly cyclophosphamide, ifosfamide, nitrosureas, chlorambucil, melphalan, busulfan and procarbazine) are the most common class
of drugs known to effect gonadal function and their
impact has been studied extensively [2]. Additionally,
the testes have a very low threshold for radiation exposure, and even small doses are known to be gonadotoxic. As treatment regimens for pediatric oncological
malignancies have improved, more and more survivors are entering their reproductive years [3]. Maintenance of fertility is extremely important with regard
to long-term quality of life for these survivors [4, 5].
Consideration must be given to whether a childs fertility is likely to be impacted by his treatment. Ideally, this should occur before the start of therapy, when
a window of opportunity may exist to preserve the
patients future reproductive potential [48]. Pubertal
males can produce a semen sample prior to starting
gonadotoxic therapy and cryopreserve the sperm for
future use. Because current methods for oocyte fertilization can utilize as few as one motile sperm (e.g.
intracytoplasmic sperm injection [ICSI]), this method
has proven to be successful even when the number of
cryopreserved sperm is small [5, 9, 10].
Unfortunately, pre-pubertal males pose a particular challenge for fertility preservation because
these boys cannot produce mature spermatozoa for
cryopreservation. During embryonic development,
primordial germ cells (PGCs) migrate to the genital
ridge and differentiate into gonocytes [11, 12]. In the
mammalian postnatal testis, gonocytes are the first cell
population committed to male germline development.
Before puberty, these cells then give rise directly to
spermatogonial stem cells (SSCs). In the mouse, which
has a short pre-pubertal period (3 weeks), some
gonocytes transition to SSCs and others undergo an
early differentiation directly to type A1 spermatogonia
by day 6 of life. During the first 23 months after birth
in humans, which have a long pre-pubertal period
(12 years), the gonocytes are replaced by adult
dark (Ad) and adult pale (Ap) spermatogonia that
are thought to represent the reserve SSC and active
SSC pool [12, 13]. These Ad and Ap spermatogonia
undergo activation beginning at approximately age
5 years, particularly to type B spermatogonia. By
age 10 years, these SSC represent about 10% of total
spermatogonia. During puberty, the SSCs in all species
provide the foundation for spermatogenesis, through
self-renewal and differentiation to daughter cells.
Although the germ cells of the pre-pubertal testis contain a small number of the self-renewing SSCs they do
not yet have mature spermatozoa. For these at risk prepubertal boys, current practice does not provide any
options for fertility preservation at cancer diagnosis.
199
Culture
Transplantation
Cryopreservation
Sperm
production
Testis
biopsy
Chemotherapy
or irradiation
Figure 15.1 Male germline stem cell preservation. Before treatment for cancer by chemotherapy or irradiation, a boy could undergo a
testicular biopsy to recover stem cells. The stem cells could be cryopreserved or, after development of the necessary techniques, could be
cultured. After treatment, the stem cells would be transplanted to the patients testes for the production of spermatozoa. From Brinster [14]
with permission. See plate section for color version.
A potential approach to this issue is the use of

cryopreserved testicular tissue. Ideally, pre-pubertal
testicular tissue could be acquired and banked prior
to initiating gonadotoxic cancer therapy (Figure 15.1,
[14]). Years later, once the patient is ready to begin
a family, this tissue could then be thawed and the
stored germ cells re-implanted into the patients own
testes [14, 15]. Alternatively, the stored cells could be
matured and expanded in vitro until they can achieve
fertilization by use of ICSI. Cryopreservation of the
testicular tissue has been shown to be feasible in many
species including mouse, rat, pig, baboon and humans,
and the ability to of restore spermatogenesis are equivalent to using fresh cells [11, 16, 17]. Unfortunately, the
initial testicular biopsy, which is small, contains very
few SSCs, and cryopreservation and techniques used to
remove malignant cells results in loss of SSCs. Therefore, there is an insufficient number to result in fertility following transplantation. For this transplantation
approach to be useful clinically, stem cells from the
biopsy sample must be isolated and expanded in vitro
prior to re-introduction [15]. While significant strides
have been made in animal model research in this area,
translational use of testicular tissue cryopreservation
in humans remains experimental [9, 18].
200
Development of spermatogonial
transplantation
Spermatogonial stem cells are responsible for the continual production of spermatozoa throughout adult
life. Both SSCs and the surrounding cells in the seminiferous tubules regulate the biological activity of
these cells. Considerable research has been dedicated
to understanding the interactions between SSCs and
the surrounding somatic cells for proper sperm production. For example, Sertoli cells are thought to be
extremely important for SSC growth and development
by secreting growth factors that regulate these germ
cells [19, 20]. Importantly, a critical breakthrough in
the characterization of SSCs has been the development
of the germ cell transplantation technique [11, 15, 21].
In 1994, Brinster and Avarbock developed the first animal model of SSC transplantation [21]. Injection of
spermatogenic cells into the seminiferous tubules gave
rise to donor cell-derived foci of spermatogenesis in
the recipient testes.
Figure 15.2 presents a schematic overview of spermatogonial transplantation in mice. First, transgenic
mice carrying a LacZ or green fluorescent protein
(GFP) transgene are used as donor mice. The marked
Chapter 15: Transplantation of cryopreserved spermatogonia
Germ
cells
Enzyme
In situ
injection
digestion
Testis with
transgene
35 months
Donor genes
transmitted
X Mate
Progeny
Figure 15.2 Testis cell transplantation method. A single-cell suspension is produced from a fertile donor testis. The cells can be cultured or
microinjected into the lumen of seminiferous tubules of an infertile mouse. Only a spermatogonial stem cell can generate a colony of
spermatogenesis in the recipient testis. When testis cells carry a reporter transgene that allows the cells to be stained blue, colonies of donor
cell-derived spermatogenesis are identified easily in the recipient testes as blue stretches of tubule. Mating the recipient male to a wild-typed
female produces progeny, which carry donor genes. From Brinster [11] with permission. See plate section for color version.
donor testis cells are digested to make a single-cell

population suspension. The cell suspension is introduced into the seminiferous tubules of recipient mice
testis that contain few to no germ cells after treatment
with busulfan that destroys endogenous spermatogenesis. The recipient mice can become fertile to father
progeny. Notably, resultant offspring carry the donor
mouse haplotype [21]. Research utilizing animal models of male infertility (e.g. busulfan-treated nude mice)
has demonstrated that there are several methods to use
germ cells from testicular tissue to obtain mature spermatozoa for fertilization, including autotransplantation, allotransplantation and xenotransplantation [11,
22]. Autotransplantation is considered more acceptable than allotransplantation or xenotransplantation,
although both of the latter have been used successfully
in mouse models [17, 21].
Morphological identification of
spermatogonial stem cells
The number of SSCs is very low in the testis of
an adult mouse. It is estimated that SSCs consti-
tute only about 0.03% of testicular germ cells [23].

Typically, about 106 germ cells are introduced into
the recipient testis depending upon stem cell concentration, among which only a few hundred cells
could be stem cells. About 20 spermatogenic colonies
will develop, depending on the stem cell concentration. Colonization efficiency is estimated to be
about 510%.
The paucity of SSCs in comparison to differentiating germ cells and somatic cells within the testis
has challenged the field to develop reliable markers of
these specialized germ cells, so they can be identified
unequivocally for subsequent isolation and enrichment in vivo or in vitro. Tangible advances include
identification of SSCs or SSC-like spermatogonia by
morphology, chemical phenotype and functional characteristics in relevant animal models. For example, the
study of rodent SSCs was previously hampered by the
lack of techniques for purification and long-term in
vitro maintenance. However, recent methodological
advances have been developed and refined for rodent
germ cell identification and transplantation along with
improved culture conditions for SSC expansion and
growth [20, 2426]. These techniques have led to
201
the characterization of many aspects of SSC biology,

including the identification of growth factors such as
glial cell line-derived neurotrophic factor (GDNF) as
the main regulator of rodent SSC self-renewal [25
27]. Morphologically, gonocytes that give rise to SSCs
are larger in diameter than nearby somatic cells, and
tend to rest loosely on the basement membrane of
the seminiferous tubules. Because of the difference in
size and morphological characteristics between gonocytes and somatic cells and absence of the differentiating stem cells, micromanipulation techniques to
select these two cell types from single cell suspensions
isolated from pre-pubertal human and mouse testes
have been enabled [27]. Selected populations were distinctly homogenous, and virtually pure populations
of germ cells and somatic cells have been obtained
and validated phenotypically by immunological techniques for well-established markers that differentiate
gonocytes/SSCs from somatic cells [27]. For example,
selected gonocytes from mouse testes and spermatogonia from human testes have been identified using
the markers zinc finger and BTB domain containing
16 (ZBTB16), ubiquitin carboxyl-terminal esterase L1
(UCHL1) and deleted in azoospermia-like (DAZL),
along with a lack of labeling for GATA-binding protein 4 (GATA4), which is found in Sertoli cells but
not germ cells [19, 28, 29]. In contrast, selected populations of somatic cells were negative for ZBTB16,
UCHL1 and DAZL and positive for GATA4. Data
indicate that micromanipulator selection of gonocytes
from pre-pubertal human testis and neonatal mouse
testis cell suspensions is an effective technique for the
enrichment of germ cells and provides essentially pure
( 99%) populations of cells for downstream molecular and cellular analyses as well as germ cells for transplantation purposes. These advances also enable future
comparisons of SSCs from humans and rodent species
for planned transplantation interventions. Moreover,
a similarity in self-renewal and survival mechanisms
between human and mouse SSCs may exist, because
transplantation of testis cells from non-rodent species,
including human, into testes of immunodeficient mice
allowed the maintenance and limited replication of
spermatogonia in the recipient seminiferous tubules
for periods of 612 months [11, 17, 27]. Additionally, comparison of molecular and cellular fingerprints
from isolated human spermatogonia and mouse gonocytes could provide details regarding specific gene
expression patterns. The degree and characteristics of
gene expression similarity would allow extrapolation
202
of our knowledge about mouse SSCs to the difficult

study of human germline cells, and ultimately impact
our understanding of human male fertility and infertility.
Isolation, purification and culture of

murine spermatogonial stem cells
As stated above, the number of SSCs in the male
mouse testes is relatively low and identification of
these cells is not straightforward. In 1999, Shinohara et al. demonstrated that 1 and 6 -integrins
are specific surface markers for mouse SSCs [30].
Cells that were positive for these markers were
selected using a magnetic bead procedure on a testicular cell suspension. Using this method with the
transplantation model, there were a greater number
of colonies of spermatogenic cells originating from
donor cells in the recipient when enhancement of the
concentration of the SSCs was used [31]. In 2004,
Kubota et al. demonstrated the essential role of glial
cell line-derived neurotrophic factor (GDNF) for in
vitro proliferation of SSCs, which were enriched from
mouse testes [25]. Using a well-established enrichment
strategy, (Thy-1+ ) SSCs were identified and isolated.
The stem cells were then cultured in a well-defined
serum-free medium, which led to successful expansion, and enabled identification of essential growth
factors for this critical cell type. Importantly, Kubota
et al. demonstrated that these stem cells grew best in
culture with the addition of specific growth factors
and their cognate receptors, including GDNF, basic
fibroblast growth factor (bFGF), and GDNF family
receptor alpha-1 (GFR1) [25]. They cultured murine
SSCs for 4 months, and then in vivo spermatogenesis was restored after transplantation back into the
recipient [25].
Over the last decade, methods have been developed for rodent germ cell transplantation and SSC
culture conditions [11, 20, 2426]. Specifically, GDNF
has been established as the main regulator of rodent
SSC self-renewal [25, 26]. The c-Ret receptor tyrosine kinase (RET) and the cofactor GFR1 bind to
initiate intracellular signaling cascades within SSCs
[25, 27, 32]. By examining GDNF withdrawal in
rodent SSC cultures, several GDNF-dependent genes
have been identified, including B-cell CLL/lymphoma
6, member B (Bcl6b), basic helix-loop-helix family,
member e 40 (Bhlhb2), Ets variant gene 5 (Etv5),
Tight
junction
Sertoli cell
Sertoli
cell
-1
GFR
Human
spermatogonial
stem cell
SFK
PI3K AKT
PI3K/AKT
Etv5, Bcl6b
Self-renewal
Differentiation
Figure 15.3 A proposed model of human spermatogonial stem cell (SSC) self-renewal regulation by glial cell line-derived neurotrophic
factor (GDNF), which has been demonstrated to have an essential role in regulating rodent SSC self-renewal. The model is similar to those
suggested for mouse SSC self-renewal. In this model, GDNF binds to RET and the GFR1 coreceptor with possible intracellular protein kinase
signaling through SFK and PI3K/AKT downstream pathways to regulate the expression of specific genes, such as Etv5 and Bcl6b, which are
involved in SSC self-renewal. However, other genes not regulated by GDNF (e.g. Zbtb16, Taf4b and Lin28), are likely controlled by different
signals and may block differentiation but not be involved directly in self-renewal. Genes for these regulatory molecules have been shown to
be highly expressed in pre-pubertal human spermatogonia, mouse gonocytes and mouse SSCs. The basement membrane (green), on which
the SSC rests, is generated by the peritubular myoid cells (dark brown) and Sertoli cells (tan). From Wu et al. [27] with permission. See plate
homeobox C4 (Hoxc4), LIM homeobox 1 (Lhx1)

and Tec protein tyrosine kinase (Tec) [27, 32, 33].
Notably, Bcl6b and Etv5 have implicated by several
independent research studies to be involved in regulating rodent SSC self-renewal [3236]. Glial cell linederived neurotrophic factor also activates downstream
signaling cascades including phosphatidylinositol 3kinase (PI3K), serine-threonine kinase AKT family
(AKT) and Src family kinase (SFK) that impact rodent
SSC maintenance and self-renewal (Figure 15.3). Thus,
GDNF is considered a factor critical for SSC selfrenewal. Importantly, inclusion of GDNF in culture
media is essential for SSC self-renewal in vitro and
additional supplementation with bFGF or spidermal
growth factor (EGF) augments those effects. Currently,
SSC culture systems that support long-term SSC selfrenewal are available only for mouse, rat and hamster [34]. Additional research is required to determine
which specific cell surface and/or intracellular mark-
ers are expressed on human testicular SSCs to enable

the rapid and reproducible accession of enriched SSC
cell populations for downstream analyses and clinical
utilization.
Culture of human spermatogonial

stem cells
Spermatogenesis in vitro from biopsied germ cells
is considered to be an excellent alternative for
pre-pubertal boys with malignancies, particularly of
hematopoietic origin, who carry a risk of relapse after
transplantation. The ability to mature stem cells to
spermatids in vitro would offer an important option
to pre-pubertal cancer patients. Unfortunately, enormous hurdles remain for bringing the in-vitro maturation processes into the clinical setting. Even for
autotransplantation of SSCs a major challenge is
that human germ cells, similar to murine germ cells
203
described above, likely yield a low number of SSCs, as

104 germ cells may contain only 2 or 3 stem cells [14,
23]. Methods are needed to isolate and increase the
number of human SSCs available to be subsequently
autotransplanted or matured in vitro. Unfortunately,
the initial biopsy contains very few SSCs, and success
of either of these procedures is likely to be equivocal. Therefore, even for autotransplantation, which is
a procedure already established in animals, to be used
clinically, stem cells from the biopsy sample must be
isolated and expanded in vitro prior to re-introduction
[14]. Spermatogonial stem cell isolation from prepubertal human testis biopsy samples by itself is not
likely to be sufficient to restore fertility following autologous transplantation because the number of SSCs
recovered from a biopsy is small. Our recent study
found that testicular biopsies from pre-pubertal boys
(n = 9; range 210 years) weighed 31.5 3.7 mg and
provided 3.9 0.6 105 cells per biopsy [27]. The concentration of spermatogonia is predicted to be about
3% of the cell population (estimated to be approximately 11 700 spermatogonia per biopsy), and the
number of Ad and Ap spermatogonia with stem cell
potential in this population is unknown. For comparative purposes, spermatogenesis restoration to approximately half of the seminiferous tubules of a sterile
mutant mouse testis and resultant fertility to approximately half of sterile mice requires transplantation of
approximately 150 SSCs per testis. As the adult human
testis (about 12 g) is nearly 120 times larger than a
mouse testis (about 0.1 g), approximately 18 000 SSCs
would need to be transplanted to each human testis
for a comparative level of fertility restoration, assuming the same successful response level was obtained
to human SSC transplantation [27]. Furthermore, a
crucial factor is verifying that the biopsied testis cells
do not contain cancerous cells. This process requires
use of cell sorting procedures such as fluorescenceactivated cell sorting (FACS, and see later), effectively
necessitating more SSCs than recovered from a biopsy
[27]. Therefore, simple transplantation of the SSC cells
harvested from a single biopsy is not likely to be sufficient to restore fertility. The culture and expansion
in number of healthy, cancer-free SSCs is essential for
effective clinical use of human SSC transplantation to
restore fertility. A recent report indicates that expansion of human germ cells in vitro is feasible [37]. However, much work yet will be required to routinely culture and increase the number of human SSCs and
assure their quality.
204
Cryopreservation of spermatogonial
stem cells
Once spermatogonial transplantation systems were
established in vivo, it became critical to determine
whether SSCs could be cryopreserved for potential fertility preservation in humans that would otherwise
be infertile due to their gonadotoxic pediatric cancer treatments. To date, these procedures are primarily experimental, and the current success lies within
animal models, not actual human patients. Notably,
the process of SSC transplantation in animal models includes a number of steps in the handling of
SSCs, and preparing the receiving testis for transplantation. Stem cells must be appropriately isolated and
enriched. For practical purposes in human testis banking, SSCs and related germline cells must be cryopreserved. Cryopreservation enables future manipulation and evaluation of SSCs in culture over a period
of time to select cells with the desired properties (i.e.
non-malignant). Investigators have routinely collected
testis cells from many species after enzyme digestion,
frozen these cells and stored them in liquid nitrogen [14, 16, 38, 39]. When ready to be utilized, cells
are thawed and transplanted into sterile recipients.
This procedure has been determined experimentally to
be quite effective, as infertile mice that receive cryopreserved SSC transplants develop donor-cell derived
spermatogenesis [16]. To date, several groups have
demonstrated that SSCs can be cryopreserved and not
lose their ability to restore spermatogenesis in animal models [11, 16, 38]. Transplantation of cryopreserved and fresh testis cells from non-rodent species,
including human, into testes of immunodeficient mice
has been successfully completed and the data demonstrate that cryopreserved donor human spermatogonia colonize mouse testes similar to fresh spermatogonia (Figure 15.4) [17]. Recently, primate testicular
cell suspensions were frozen [38] and, after thawing,
viability was found in 58% of the cryopreserved cells.
Importantly, Keros et al. have documented successful freezing protocols using human testicular tissues
[39]. Specifically, slow programmed freezing with 5%
dimethyl sulfoxide (DMSO) as a cryoprotective agent
is efficient in maintaining spermatogonia, Sertoli cells
and the stromal compartment from testicular biopsies during the freezing, thawing and subsequent tissue
culture procedures required for successful accession
and utilization of human clinical samples. It has been
estimated that 5066% of spermatogonial cells can
(a)
(b)
Figure 15.4 Detection of human germ cells transplanted into seminiferous tubules of recipient mouse testes using a baboon testis specific
antibody that identifies human spermatogonia. (a) Donor human spermatogonia in mouse tubule 4 months after transplantation of
cryopreserved cells. This panel shows that cryopreserved cells also colonize mouse testes as observed with freshly transplanted cells. (b)
Donor human spermatogonia in mouse testis 5 months after transplantation. These donor cells were transplanted without cryopreservation.
C 2002 American Society for
Bar = 100 m (a) and 40 m (b). From Nagano et al. [17] with permission from Elsevier Science, Inc.
Reproductive Medicine. See plate section for color version.
survive after freezing and thawing, making long-term

cryopreservation a feasible means for storing viable
cells for future fertility [39].
Removal of malignant cells

Despite significant advances in spermatogonial cell
biology and subsequent fertility management, malignant contamination remains one of the main concerns
surrounding autologous transplantation. The risk is
substantial with hematological cancers, in which the
testes can act as sanctuary sites for leukemic cells.
Advances in our ability to detect cancer specific chromosomal or molecular abnormalities will be necessary in order to screen gonadal tissue for malignant
cells prior to autotransplantation [40, 41]. Using minimal residual disease detection in hematological diseases might be very useful since one malignant cell
can be detected among 106 normal cells [42, 43].
The most sensitive techniques are polymerase chain
reaction (PCR) based and use break-point regions
of leukemia-specific chromosomal aberrations or the
immunoglobulin or T-cell receptor gene rearrangements [43, 44]. Another methodology for screening
malignant cells in residual tissues is FACS. Specifically, progress in addressing this question has been
made in mouse models, particularly with the use of
FACS technology to negatively sort malignant cells
from cell suspensions [4547]. Fujita et al. restored fertility in sterile mice after transplanting SSCs isolated
from mice with leukemia, without inducing leukemia
in the recipient mice [45]. They used positive selection
of SSCs by flow cytometry for CD45 (a surface marker

for leukemic cells) negative cells. Another alternative
method to screen human testicular tissue for malignant potential might be to inject an aliquot of the
actual clinical sample suspension into immunodeficient mice prior to delivering into the patient. In this
scenario, animal models could be used to test cell suspensions with regard to their malignant potential. If
a rodent develops a malignancy following transplantation, then the suspension that was used to inoculate the mouse would be rejected for use in human
candidates. Regardless of what method is utilized to
remove malignant contamination, the number of SSCs
collected from a testicular biopsy need to be further
expanded to accommodate for these techniques of
removing malignant cells. Indeed, specific cultures of
SSCs could be used to eliminate cancer cells that are
likely to be dependent on different growth factors and
culture conditions.
In summary, the potential for transferring tumor
cells within cryopreserved and subsequently cultured
and/or expanded testicular tissue back into the patient
is of paramount concern. Children most at risk of
transmitting cancer cells include those with a hematological malignancy such as acute leukemia. However,
local invasion from solid abdominal or pelvic malignancies cannot be excluded such as the patient with
rhabdomyosarcoma with testicular involvement. The
potential for in vitro maturation of sperm from prepubertal testicular biopsies could overcome this problem by screening and selecting for SSCs that do not
have malignant potential.
205
Ethical concerns
As testicular cryopreservation is an experimental procedure in humans, efficacy and safety research is governed under the auspices of federal regulations for
clinical trials involving children. These federal regulations mandate that federally funded clinical research
protocols including children that involve greater than
minimal risk, and that present the potential of direct
benefit to these subjects, must be reviewed by an Institutional Review Board (IRB) [48]. The IRB approval
will only be granted when direct benefits of the
experimental treatment outweigh its risks, and that
research is likely to provide important information
that leads to better understanding of the conditions
therein [48]. Strict adherence to these guidelines is
crucial regardless of the funding source for clinical
trials involving children, as they provide a solid ethical cornerstone for clinical and translational research
[48].
Tissue banking centers that accrue gonadal tissue for pediatric cancer patients must inform the parents of the options for disposition of these materials at a future time [49]. These issues are not trivial,
as the legal system upholds the prior wishes from an
individual regarding the disposition of reproductive
material, which is controlling after death. Therefore,
tacit instructions that biological materials must be
destroyed, or alternatively released to research, have to
be honored. Clarity in the handling of biological samples is particularly important in the situation where
pre-pubertal patients have cryopreserved gonadal tissue. Specifically, parents or their legal guardians must
give directions, in writing in advance, for future tissue disposition, and they must be urged to specify
what should be done with banked tissue if their child
dies.
As a young child cannot give his consent for the testicular biopsy cryopreservation, parental consent must
be granted. Testicular biopsy is an invasive procedure
and purely experimental [50]. In practice, a testicular biopsy should be performed under the same general anesthesia that is used to insert a central line for
chemotherapy. Although harvesting gonadal tissue
may be of high risk if taken in isolation, within the
context of the childs illness it may pose minimal additional risk [49]. Therefore, it should be combined with
a procedure that requires anesthesia. Lastly, it is important to remember that testicular tissue cryopreservation is still at a highly experimental stage and careful
206
counseling and consent is essential. To be valid, consent must be informed, voluntarily obtained and given
by a competent person. The consent process must not
raise unrealistic expectations [49, 51].
Conclusions
Little is known about the biology and regulation of
human germline cells, particularly regarding maintenance and regulation of SSCs, which are the foundation of spermatogenesis throughout adult life.
Spermatogonial stem cells reside on the basement
membrane of the testis seminiferous tubule, and their
decision regarding self-renewal versus differentiation
determines the efficiency of spermatozoa production. In mice, approximately 0.03% of testes cells are
stem cells, and they resemble other early differentiating spermatogonia, a morphological characteristic
observed phylogentically across mammalian species
[14, 52]. Spermatogonial stem cells arise directly from
gonocytes within days in mice and over a few months
in humans. The limited knowledge about these critical cells arises from their rarity and relative absence of
distinguishing morphological characteristics. To date,
cryopreservation is feasible in that all species in which
it has been tried; cryopreservation of testicular tissue
has been successful with significant numbers of SSCs
being recovered. Unfortunately, cryopreservation procedures results in some loss of SSCs and the SSCs
from human testicular biopsies are insufficient to reestablish fertility after transplantation. An important
goal of future basic and translational research is to generate methods to expand the SSCs in number. In addition to the basic science issues regarding the accession
and maturation of SSCs for transplantation, a plethora
of practical and ethical issues also need to be addressed
for appropriate application of human SSC usage for
fertility preservation in boys following gonadotoxic
pediatric cancer treatments.
Acknowledgements
Financial support was provided by an Ethel Foerderer
Award (JPG), National Institute of Child Health
and Human Development (NICHD) grant HD061217
(JPG), NICHD grant HD044445 (RLB), NICHD grant
HD 052728 (RLB) and the Robert J. Kleberg, Jr. and
Helen C. Kleberg Foundation (RLB).
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Section 4
Chapter
16
Cryopreservation and transplantation of

testicular tissue
Christine Wyns
Introduction
Due to remarkable advances in the treatment of childhood cancer, we have seen great improvements in life
expectancy with up to 80% of children surviving their
disease, resulting in a growing population of adult
long-term survivors of childhood malignancies [1].
Although oncological treatments are highly effective, a major concern is their adverse impact on fertility [2, 3].
Since rapidly dividing cells are the target of chemoand radiotherapy, these treatments act not only on cancer cells, but also on germ cells. Differentiating spermatogonia proliferate the most actively and are thus
extremely susceptible to cytotoxic agents, although the
less active stem cell pool may also be depleted [4].
Consequently, although the pre-pubertal testis
does not complete spermatogenesis, there is evidence
that cytotoxic treatment given to pre-pubertal boys
affects fertility [5]. In addition, the presence of a steady
turnover of early germ cells that undergo spontaneous
degeneration before the haploid stage is reached [6, 7]
may possibly explain why the pre-pubertal state does
not offer any protection against gonadotoxic treatments.
Recovery of sperm production after a cytotoxic
insult depends on the survival and ability of mitotically quiescent stem spermatogonia (type A dark) to
transform into actively dividing stem and differentiating spermatogonia (type A pale) [8].
The somatic compartment of the testis may be
more resistant to chemotherapeutic treatment, since
these cells have a low or absent mitotic rate. Neverthe-
less, increased concentrations of luteinizing hormone

(LH) and symptomatic reductions in testosterone levels [2], both signs of Leydig cell impairment, have been
described. Evidence of Sertoli cell functional impairment following chemotherapy responsible for germ
cell differentiation inhibition where germ cells have
survived has also been reported [9].
Loss of fertility: who can benefit from

fertility preservation?
Loss of fertility in adult life is a major psychologically
traumatic consequence of cancer treatment. Indeed, in
a quality of life analysis of former oncological patients,
about 80% viewed themselves as potential parents,
and the vast majority of younger cancer survivors saw
their cancer experience as pivotal in preparing them
to be better parents [10]. Therefore, since post-therapy
recovery of spermatogenesis remains unpredictable, it
is important to inform patients facing infertility as a
side effect of their treatment of all the options available
to preserve their fertility [3].
There is also considerable evidence that gonadotoxic treatments like hematopoietic stem cell
transplantation (HSCT) can cure a variety of
non-malignant disorders in children, so fertility
preservation should not be reserved solely for boys
with cancer [11, 12]. It should also be considered for
other benign conditions where seminiferous tubule
degeneration is expected over time, such as Klinefelters syndrome [13]. The indications for immature
testicular cryopreservation in case of malignant and
non-malignant disease are summarized in Table 16.1.
209
Table 16.1 Indications for immature testicular tissue

cryopreservation in case of malignant and non-malignant
disease
Malignant
r
r
r
r
r
r
Leukemia
Hodgkins disease
Non-Hodgkins
lymphoma
Myelodysplastic
syndromes
Solid tumors
Soft tissue sarcoma
Non-malignant
1
HSCT in case of:

r Hematological disorders:
thalassemia major, sickle
cell disease, aplastic
anemia, Fanconis
anemia
r Primary
immunodeficiencies
r Severe autoimmune
diseases unresponsive to
immunosuppressive
therapy: juvenile
idiopathic arthritis,
juvenile systemic lupus
erythematosus, systemic
sclerosis, immune
cytopenias
r Osteopetrosis
r Enzyme deficiency
disease: Hurlers
syndrome
Risk of testicular degeneration
r Klinefelters syndrome
therefore remains the only option for fertility preservation in pre-pubertal males.
Immature testicular tissue

cryopreservation
Since pre-pubertal boys cannot benefit from sperm
banking, a potential alternative strategy for preserving
their fertility involves storage of testicular tissue in the
hope that future technologies will allow its safe utilization. It is important to stress, however, that this strategy is still experimental.
As pre-pubertal testicular tissue contains SSCs
from which haploid spermatozoa are ultimately
derived, these cells can either be cryopreserved as a
cell suspension [19], in the form of tissue fragments
[2022] or even as a whole organ.
It is nevertheless worth noting that, in 20% of Tanner stage II boys, spermiation has already started [23],
allowing cryopreservation of haploid gametes that may
subsequently be considered for in vitro maturation
(IVM), if necessary.
HSCT, hematopoietic stem stell transplantation.
Cell suspensions
Fertility preservation options before
gonadotoxic therapies
In order to reduce the deleterious effects of gonadotoxic therapies, different strategies have been tested.
Improving therapeutic regimens using less
gonadotoxic protocols [14] could enable spontaneous recovery of spermatogenesis, but their use is
not always possible without compromising patient
survival.
Limiting radiation exposure by shielding or removing the testes from the radiation field should be implemented whenever possible [3].
Minimizing testicular damage from cancer treatment or protecting SSCs in vivo is another approach.
Gonadal protection through hormonal manipulation
was reviewed in Chapter 13.
Anti-apoptotic agents such as sphingosine-1phosphate [15] and AS101 [16] and various other
cytoprotective substances [17, 18] have also been used
with partial success in rodents.
Currently available drugs to prevent testicular
damage from cytotoxic therapy have not proved helpful in humans so far. Testicular tissue cryopreservation
210
Cell suspensions have been developed with a view to

facilitating cryopreservation, as cell heterogeneity in
tissue pieces renders tissue freezing more challenging. However, preparation of cell suspensions requires
mechanical and/or enzymatic digestion of tissue, compromising cell survival and cell-to-cell interactions
necessary for cell proliferation and differentiation [19].
Post-thaw viability of 2982% has been reported in
various animal models [24] and up to 60% in human
testicular cell suspensions, regardless of cryoprotective
agent [19, 25].
Tissue pieces
Cryopreservation of testicular tissue pieces may be
considered as an alternative method capable of maintaining cell-to-cell contacts between Sertoli and germinal stem cells, and therefore preserving the stem cell
niche necessary for their survival and subsequent maturation [26]. Other advantages of this method may be
preservation of the Sertoli cells, since there is evidence
of their reversion to a dedifferentiated state as a consequence of chemotherapy [9], and Leydig cells, whose
preservation may be useful to alleviate the hormonal
imbalance caused by cytotoxic therapy [2].
Chapter 16: Cryopreservation and transplantation of testicular tissue
Better survival rates of Leydig cells were obtained

when dimethyl sulfoxide (DMSO) was used (80%
compared to 50% with 1,2propanediol [PROH])
[27]. Structural integrity and functional capacity were
demonstrated after cryopreservation and culture of
pre-pubertal testicular tissue [20, 21].
Because of the complexity of the tissue architecture, cryopreservation protocols must strike a balance between optimal conditions for each cellular
type. In addition, problems can arise when extracellular ice forms, as it can cleave tissues into
fragments. Furthermore, rapid solute penetration of
highly compacted tissue is vital to ensure high
final concentrations of cryoprotectant at temperatures that minimize cytotoxicity. Post-thaw survival
and seminiferous tubule structure are profoundly
affected by both the type of cryoprotectant and freezing rates [28], so optimization of freezethawing
protocols is mandatory. Dimethyl sulfoxide, rather
than ethylene glycol (EG), PROH or glycerol, was
shown to better preserve structures within tissue
[27, 29] and best maintain tissue capacity to initiate spermatogenesis [30], and slow-programmed
freezing to better protect spermatogonial morphology
[21].
Two teams have reported freezing protocols for
pre-pubertal human testicular tissue, both yielding
good structural integrity [20, 21]. Using different cooling and freezing rates, Keros et al. observed a difference mainly in terms of survival of spermatogonia,
with 94% of intact spermatogonia found after freeze
thawing and culture with their best protocol [21]. This
protocol, albeit slightly modified by the addition of
sucrose, was further applied to evaluate the functional
capacity of cryopreserved human immature testicular tissue (ITT) after orthotopic xenografting [22, 31].
Preservation of spermatogonia (able to proliferate) was
demonstrated. An overview of all studies on cryopreservation of ITT is presented in Table 16.2 [2022,
31].
Whole testis
Due to the small number of SSCs contained in a testicular biopsy and the small size of a childs testis, it is
possible that cryopreservation of a whole testis may be
more appropriate, with a view to later organ autografting. Cryopreservation methods for whole testes need
to be developed, however, as has been done for whole
ovaries [32].
Fertility restoration after immature

testicular tissue cryopreservation
In the light of results obtained from animal studies, frozen diploid precursor cells may provide some
hope of fertility restoration in pre-pubertal boys in
the absence of haploid gametes. Three approaches may
be considered: transplantation of purified cell suspensions back to their own testes; autografting of testicular pieces, testicular cell aggregates or whole testes;
or IVM up to a stage at which they are competent for
normal fertilization through intracytoplasmic sperm
injection (ICSI). The latter option, i.e. IVM, is beyond
the scope of this chapter.
None of these approaches have proved efficient or
safe in humans as yet. These potential options have
mainly been studied in animals, and lessons learned
from these studies will be reviewed in detail.
Testicular germ cell transplantation

In this approach, spermatogenesis is reinitiated after
transplantation of isolated testicular stem cells to germ
cell-depleted testes. Spermatogonial stem cells (SSCs)
are recognized by Sertoli cells and relocate from the
lumen onto the basement membrane of seminiferous
tubules. Because stem cells have unlimited potential to
self-renew and produce differentiating daughter cells,
SSC transplantation offers the possibility of long-term
restoration of natural fertility.
The technique was first described in 1994 [33]. Testicular germ cells isolated from pre-pubertal mouse
testes were injected into the seminiferous tubules of
adult mice with Sertoli cell-only syndrome induced by
busulfan treatment. Normal donor spermatogenesis,
recognized by developing germ cells carrying the lacZ
gene encoding -galactosidase, was initiated and sustained.
Although this approach has yielded healthy
progeny displaying the donor haplotype in animals
[34], it has not yet proved successful in humans (see
Progress towards human clinical application section,
below).
Lessons learned from transplantation

of fresh testicular stem cells in animals
Outcome of the technique
Autologous SSC transplantation has been reported in
mice [33], rats [35], pigs [36], goats [37], cattle [38],
211
Table 16.2 Overview of studies on cryopreservation of pre-pubertal human testicular tissue
(Non)
controlled
Outcome
(germ cells)
Outcome
(endocrine
compartment)
Reference
Cryoprotectant
Kvist et al.
[20]
EG 1.5 M Sucrose
0.1 M
Slow controlled
Start: 1 C,
2 C/min to
9 C, hold 5 min
+ seeding,
0.3 C/min to
40 C,
10 C/min to
140 C, LN2
Culture 2
weeks
Well preserved STs

Presence of intact
SG (c-kit+ )
Well-preserved
interstitial cells
Testosterone and
inhibin levels
similar to fresh
tissue
Keros et al.
[21]
DMSO 0.7 M
Slow controlled
Program 1:
Start: 4 C, hold 30
min, 1 C/min to
0 C, hold 5 min,
0.5 C/min to
8 C, seeding,
hold 10 min,
0.5 C/min to
40 C, hold 10
min, 7 C/min to
70 C, LN2
Culture 24 h
70 7% ISTs in
frozen-cultured
tissue (versus
71 7% in fresh
tissue and 77 4%
in fresh-cultured
tissue) 94 1%
intact SG in
frozen-cultured
tissue (versus
93 2% in fresh
tissue and 83 1%
in fresh-cultured
tissue)
Undamaged
stromal structure:
80 29% of
frozen-cultured
samples (versus
99.49 0.88% of
fresh samples and
97 2% of fresh
cultured samples)
Rapid
controlled
Program 2:
Start: 4 C, hold 30
min, 1 C/min to
8 C, seeding,
hold 10 min,
10 C/min to
80 C, LN2
20 14% ISTs in
frozen-cultured
tissue 50 43%
intact SG in
frozen-cultured
tissue
Undamaged
stromal structure:
29 28% of
frozen-cultured
samples
Slow controlled
Start: 0 C, hold 9
min, 0.5 C/min
to 8 C, hold 5
min + seeding,
hold 15 min,
0.5 C/min to
40 C, hold 10
min, 7 C/min to
80 C, LN2
Immediate
post-thaw
evaluation
0.71 0.89 SG/ST in

frozenthawed
tissue (versus
0.45 0.35 SG/ST in
fresh tissue)
Not assessed
Xenografting
3 weeks
82.19 16.46% ISTs

in frozen-grafted
tissue (versus
93.38 6% in fresh
tissue) 14.5% SG
recovery after
freezing and
grafting
Xenografting
6 months
55 42% ISTs in
frozen-grafted
tissue 3.7 5.5% SG
recovery 21%
proliferating SG
Differentiation up to
pachytene stage of
prophase
Wyns et al.
[22]
Wyns et al.
[31]
DMSO 0.7 M
Sucrose 0.1 M
DMSO 0.7 M
Sucrose 0.1 M
Slow controlled
Freezing rate
Type of
evaluation
Start: 0 C, hold 9
min, 0.5 C/min
to 8 C, hold 5
min + seeding,
hold 15 min,
0.5 C/min to
40 C, hold 10
min, 7 C/min to
80 C, LN2
Signs of
steroidogenic
activity by 3-HSD
IHC and TEM
DMSO, dimethyl sulfoxide; EG, ethylene glycol; HSD, hydroxysteroid dehydrogenase; IHC, immunohistochemistry; LN2 , liquid nitrogen; SG,
spermatogonia; (I)ST, (intact) seminiferous tubule; TEM, transmission electron microscopy.
212
monkeys [39] and dogs [40]. Restoration of fertility

from donor stem cells has only been reported in mice
[34], rats [41], goats [37] and chickens [42].
Heterologous transplantation does not appear to
be as successful as autologous transplantation, probably because of the phylogenetic distance between
species. Rat gonocytes produced mature spermatozoa after xenogeneic transplantation to the testes of
mice, but qualitative and quantitative abnormalities
of sperm were observed [43]. Abnormal spermatozoa
were also found when hamster germ cells were transplanted to mice, probably reflecting the limited ability
of mouse Sertoli cells to fully support hamster germ
cells [44].
Spermatogonial stem cells from all other mammalian species examined (i.e. rabbits, dogs, pigs, bulls,
stallions, non-human primates and humans) were able
to colonize the seminiferous tubules of mice and generate colonies of stem cells, but could not differentiate beyond the stage of spermatogonial expansion [45
49]. One study nevertheless demonstrated some early
meiotic spermatocytes after transplantation of male
porcine germ cells to mice [50]. This suggests that the
initial steps of germ cell recognition by Sertoli cells,
migration to the basement membrane, initiation of cell
proliferation and possibly some early steps of differentiation are conserved among evolutionarily divergent
species.
Efficiency of the technique

The extent of spermatogenesis has been shown to
depend on the number of transplanted stem cells, with
an almost linear correlation [51], and on the quantity
and quality of stem cell niches in the recipient testis
[35].
In rodents, the observed colonization rate was no
higher than 1/20 SSCs [51], thus showing low colonization efficiency. The colonization rate of slowly
cycling type A dark spermatogonia in primates was
expected to be much lower, estimated to be as low
as 0.00150.003% in rhesus monkeys [48]. Recipient
age appears to have an impact on colonization efficiency, since more and larger spermatogenic colonies
were generated in preadolescent recipient mouse testes
than in adult testes [52]. Better niche accessibility and
niche proliferation due to Sertoli cell multiplication,
elements facilitating colony formation and an increase
in seminiferous length during testicular enlargement
may be involved. This should be taken into account to
ensure optimal transplantation time in clinical practice.
Techniques for SSC enrichment

and expansion
Because of the small number of SSCs in a testis
(2/10 000 germ cells) [6], the small size of testicular
biopsies recovered for fertility preservation and the
low efficiency of recolonization after transplantation,
increasing the number of SSCs prior to transplantation
is essential. Ideally, isolation of pure stem cells would
be the most effective method to increase the number
of SSCs in a suspension and therefore transplantation
efficiency.
Adequate purification will probably be best
achieved by cell-sorting techniques, such as magneticactivated cell sorting (MACS) or fluorescenceactivated cell sorting (FACS) based on cell characteristics and membrane antigens. These techniques
have already been shown to improve transplantation
efficiency in mice [53]. So far, the highest level of SSC
enrichment has been achieved based on THY1 expression [54]. As conserved expression of some markers of
undifferentiated spermatogonia (PLZF, GFR-1 and
THY-1) exists between mice and non-human primates
[48], there is hope that cell enrichment techniques
may be extended to humans.
While expansion of pure stem cells in culture
appears to be possible, albeit with limited cell proliferation, better results were achieved using culture on
feeder layers with a combination of growth factors or
applying serial transplantation procedures [55, 56].
So far, strategies for in vitro expansion of SSCs
have only proved successful in rodents. Using various
growth factors and hormones, a 2 1014 -fold expansion in total neonatal mouse testicular cell number was
achieved over 160 days [56] and, after 2 years, the
cultured cells showed 1085 -fold logarithmic proliferation, retaining characteristic morphology and yielding fertile offspring after stem cell transplantation [57].

of frozen testicular stem
cells in animals
Since high survival rates do not guarantee preservation of the functionality of frozenthawed cells, it is
important to evaluate their capacity to self-renew and
213
differentiate through transplantation of cell suspensions. Experiments on human germ cell transplantation were not able to achieve this goal since, after
6 months xenotransplantation to immunodeficient
mice, only proliferative activity was observed [49].
Hence, studies in animals will help us elucidate some
important considerations for clinical application.
The potential of frozen murine testicular cells
to resume spermatogenesis after transplantation was
demonstrated for the first time by Avarbock et al. in
1996 [58]. Live birth of offspring achieved after transplantation of frozen testicular cell suspensions provided final proof of successful cryopreservation [59].
Although it appears that the functional capacity of
mouse SSCs may be compromised by cryopreservation
[60], this was not observed by Kanatsu-Shinohara et al.
[59]. Moreover, rhesus SSCs retained normal colonization capacity after freezing and transplantation to mice
[48], suggesting that possible functional impairment
due to cryopreservation involves germ cell differentiation rather than their ability to recolonize stem cell
niches.
Progress towards human

clinical application
In humans, preclinical in vitro studies using cadaver
or surgically removed testes have demonstrated the
feasibility of transplanting germ cell suspensions into
testes. Fifty to seventy percent of seminiferous tubules
were filled by means of intratubular injection [19] or
injection into the rete testis, with needle placement
controlled by ultrasonography [61].
A clinical trial was initiated in Manchester (UK)
in 1999 to evaluate germ cell transplantation in cancer patients but, as far as we know, no information is
available on the fertility of these patients [62]. Drawing
conclusions from this trial will nevertheless be problematic, as endogenous spermatogenesis and spermatogenesis issuing from transplanted cells will not be
distinguishable.
Testicular tissue grafting

Testicular tissue grafting involves transplantation of
SSCs with their intact niches and thus within their
original microenvironment. Since testicular tissue
grafting has not yet been reported in humans, available data will be reviewed on the basis of observations
made in animals.
214
To date, haploid germ cells isolated from mouse

testis homografts and rabbit testis xenografts have
been used with ICSI to generate offspring [63]. Xenogeneic rhesus sperm generated in host mice have also
been shown to be fertilization competent, allowing in
vitro embryo development at a rate similar to that
reported for in situ rhesus testicular sperm [64]. In
view of these encouraging results in animals, there is
every hope that it will be possible, in the near future,
to autograft cryopreserved testicular tissue of patients
rendered sterile after fertility-threatening therapies
and restore their fertility.

of fresh testicular tissue in animals
Grafting of testicular tissue from several mammalian species into immunodeficient mouse hosts has
resulted in varying degrees of donor-derived spermatogenesis. Complete spermatogenesis following testicular grafting has been reported in mice, rabbits,
hamsters, pigs, goats, cats, bovines, horses and sheep
[6370], as well as macaques [64, 71]. By contrast,
germ cell differentiation blockage was observed in
marmosets [30, 65, 72].
The mechanisms underlying these species-specific
differences in spermatogenic differentiation remain
unknown, but some hypotheses can be proposed.
First, differences between host and donor
gonadotropic hormones [73] may lead to inefficient interaction between murine gonadotropins and
grafted donor testicular tissue. Supplementation with
exogenous gonadotropins could therefore be useful.
Indeed, xenografts of ITT from rhesus monkeys to
mice treated with exogenous gonadotropins showed
some degree of sperm differentiation, compared to
blockage at the spermatogonial level observed in
untreated mice [71]. However, observations from
two different studies do not support this hypothesis, since autologous grafts of marmoset tissue
[72] and xenografts of marmoset and horse ITT
to gonadotropin-supplemented recipient mice [69]
showed blockage in germ cell differentiation. These
conflicting results suggest that species-specific differences in gonadotropins are not the only explanation
for differentiation impairment.
Second, as suggested by studies on testicular tissue
xenografts from macaques and marmosets, speciesspecific structural differences in seminiferous tubule
organization [74], resulting in modified paracrine
Figure 16.1 Histological appearance (hematoxylin/eosin sections)

of donor testicular tissue from a 44-year-old man after 3 weeks
orthotopic xenografting at 200 magnification. Most tubules show
degenerative changes, i.e. sclerosis, while the rest contain mainly
Sertoli cells. See plate section for color version.
interactions, might explain differences in germ cell differentiation within grafts.

Third, the initiation and extent of differentiation
may be influenced by the stage of germ cell development and intensity of spermatogenesis at the time of
grafting. Indeed, complete spermatogenesis was not
reported in xenografted tissue when donor testicular
tissue contained post-meiotic germ cells at the time of
grafting in any species, including humans, and most
grafts regressed or contained degenerated tubules (Figure 16.1) [65, 69, 75, 76].
The reasons for the poor outcome of adult testicular tissue xenografts are so far unknown. However, studies in rodents have suggested that adult tissue could be more sensitive to ischemia than immature
tissue, and that hypoxia related to the grafting procedure may be involved [65]. This hypothesis was supported by studies in bovines, showing higher expression of some angiogenic factors in grafts from younger
donors [77]. Furthermore, pre-treatment of testicular
tissue with vascular endothelial growth factor (VEGF),
a potent angiogenic factor, was found to increase the
number of tubules containing elongating spermatids
[78].
Nevertheless, the angiogenesis hypothesis is probably insufficient to explain this poor outcome, since
donor age-dependent variations in germ cell differentiation have also been observed in immature donors
[68, 71]. Variations in Sertoli cell maturation at the
time of grafting, or their developmental susceptibility
to the detrimental influence of endocrine disruption
due to the xenografting environment, may account for

the inability of these cells to support germ cell differentiation, and may thus be involved in the age-dependent
variations found [68, 71]. Donor age-dependent differential gene and subsequent protein expression in
donor tissue prior to grafting may also be implicated
[77].
Besides causing spermatogenic differentiation
impairment, xenografting has been shown to be
inefficient in some species. Indeed, only 510% of
seminiferous tubules in xenografts produced elongated or elongating spermatids in bulls [68], kittens
[67] and horses [69]. Furthermore, in non-human primate testicular tissue grafts, only 2.84.0% of tubules
contained mature sperm [64, 71]. The reasons for this
low spermatogenic efficiency need to be understood
in order to improve the success of this approach.
Initial germ cell loss, as reported in bovine and
monkey xenografts [71, 79], could explain these poor
results. Decreased expression of glial cell line-derived
neurotrophic factor (GDNF), involved in germ cell
self-renewal, has been described in grafts [77], suggesting that the grafting procedure itself could negatively
influence the number of germ cells. However, tissue
culture performed prior to xenografting to increase the
number of SSCs did not result in a higher percentage
of seminiferous tubules with elongating spermatids at
the time of graft removal [78], indicating that other
factors may be responsible for the low spermatogenic
efficiency.

of frozen testicular tissue in animals
An overview of studies on cryopreserved testicular tissue grafting in various animal models was recently
reported by Geens et al. [24]. In rodents, cryopreservation of ITT led to the birth of healthy offspring [63].
There is therefore every hope that this approach can be
extended to humans.
A number of studies in animals designed to evaluate the effect of freezing on the functional capacity of
germ cells have shown that freezing does not appear
to affect the functional capacity of frozen germ cells
on a qualitative basis [29, 30, 63, 65, 66, 80]. Loss
of SSCs after cryopreservation was nevertheless suggested, since Ohta and Wakayama reported lower colonization efficiency after grafting frozenthawed testicular pieces [80].
215
Lessons learned from

xenotransplantation of fresh
human testicular tissue
Very few studies have been published on xenotransplantation of human testicular tissue [75, 81]. Adult
testicular tissue grafting has yielded poor results,
showing mainly sclerotic seminiferous tubules [75, 81]
and some isolated spermatogonia in 21.623.1% of
grafts [75].
Grafting of human ITT from pre-pubertal boys
[82] did not result in complete spermatogenesis,
although graft and germ cell survival were shown to be
more favorable than in mature tissue grafts. Goossens
et al. observed mainly Sertoli cell-only tubules and
just a few surviving spermatogonia 4 and 9 months
after grafting, constituting considerable spermatogonial loss [82].
Lessons learned from

xenotransplantation of frozen
human testicular tissue
No studies have reported xenografting of cryopreserved adult testicular tissue in humans and only two
have been published on cryopreserved ITT xenotransplantation in humans [22, 31]. Grafts were performed
orthotopically to immunodeficient mice. After grafting frozenthawed cryptorchid tissue for 3 weeks,
14.5% of the initial spermatogoniaI population survived, with 32% of these cells showing proliferative
activity, not significantly different from the 17.8% in
fresh tissue. The number of Sertoli cells was unchanged
and 5.1% were proliferative compared to 0% in fresh
tissue. Raised follicle stimulating hormone (FSH)
levels in the castrated mice, the removal of some
inhibitory mechanisms that normally operate in quiescent immature testes and/or other paracrine factors
were suggested to play a role in the Sertoli cell multiplication. In order to study the capacity of frozen SSCs to
self-renew and differentiate, long-term grafts of normal immature tissue were performed. After freeze
thawing and 6 months xenografting, 3.7% of the initial spermatogonial population remained, with 21% of
these cells showing proliferative activity.
Since considerable loss of spermatogonial cells
occurred, it is essential to evaluate to what extent cryopreservation itself is implicated. Freezing does not
appear to have a major impact. Indeed, no decrease
216
in spermatogonial cell numbers between fresh and

frozenthawed testicular pieces [22] and a very high
survival rate (94 1%) of spermatogonia after freezing
and culture [21] were observed. Furthermore, regarding the effect of cryopreservation on the differentiation capacity of human SSCs, the remaining spermatogonia retained the ability to reinitiate spermatogenesis, although normal differentiation beyond the
prophase of the first meiosis could not be proved [31].
Indeed, spermatid-like structures were detected (Figure 16.2ac), albeit slightly smaller than control spermatids (P = 0.045), but these structures did not show
characteristic markers of postmeiotic cells or acrosome development by immunohistochemistry (IHC).
As shown in Figure 16.3, signs of preservation of
the steroidogenic capacity of Leydig cells were also
observed [31].
Testicular cell grafting

Isolated cell grafting was first described in cotransplantation experiments where Sertoli cells were used
for their immunoprotective properties. The capacity
of dissociated Sertoli and myoid cells to reaggregate
and form seminiferous cords after xenotransplantation was first demonstrated by Dufour et al. [83]. This
approach is challenging, however, since the different
cell types, including Leydig cells, have to form functional three-dimensional cell associations after transplantation in order to produce a supportive microenvironment for spermatogenesis, whereas in tissue grafts
these are already formed.
Isolated cells from immature mammelian testes
were nevertheless able to reproduce partial or complete spermatogenesis and initiate steroidogenesis
after ectopic or orthotopic grafting.
Moreover, spermatogenesis in grafts originating
from single-cell pellets was shown to be morphologically identical to spermatogenesis occurring in grafts
of intact testicular tissue in pigs [84]. Offspring were
finally obtained after mouse germ cell transplantation
in ectopically reconstituted tubules created from isolated murine testicular cells [85].
Studies assessing germ cell differentiation and/or
Leydig cell functionality after xenografting of isolated
immature testicular cells in mouse recipients are summarized in Table 16.3 [8688].
In the light of these animal studies, this approach
looks promising from a clinical perspective. Indeed,
besides allowing reconstitution of a functional stem
Figure 16.2 Histological appearance

(hematoxylin/eosin sections) of donor
testicular tissue from a 12-year-old boy (a)
after 6 months orthotopic xenografting
at 200 magnification; (b) showing
pachytene spermatocytes (arrow) and
spermatid-like cells (inset) at 400
magnification; and (c) spermatid-like cells
at 1000 magnification. See plate section
for color version.
(b)
(a)
(c)
Control
3 HSD
N
Nm
50 m
TEM
Fresh
L
M
Cm
Fresh
2.8 m
50 m
SER
F/T/G
L
Nm
Cm
SER
F/T/G
2.8 m
L
cell niche, this technique offers the possibility of using

cell sorting methods before grafting and could therefore be beneficial when testicular tissue is potentially
contaminated with cancer cells.
In addition, it may help to elucidate factors regulating spermatogenic events after grafting and potential reasons for loss of spermatogenic activity after a
gonadotoxic insult [87].
50 m
Figure 16.3 Steroidogenic activity in

Leydig cells evaluated by transmission
electron microscopy (TEM) (left) and
immunohistochemistry (IHC) (right). The
TEM shows fresh and
frozen/thawed/grafted Leydig cells
showing intact structures of nuclear and
cytoplasmic components and activity.
Magnification 12 000. Bm, basement
membrane; Cm, cell membrane; F/T/G,
frozen, thawed and grafted for 6 months;
L, lipid droplets; M, mitochondria; N,
nucleus; Nm, nuclear membrane; SER,
smooth endoplasmic reticulum: site of
conversion of pregnenolone to
testosterone. The IHC shows fresh and
frozen/thawed/grafted Leydig cells that
are stained for 3 -hydroxysteroid
dehydrogenase (3 HSD), converting
pregnenolone to progesterone. See plate
Whole testis transplantation

The first convincing demonstration of human testis
transplantation was reported in 1978 by Silber [89].
An anorchid man was grafted with a testis from his
genetically identical twin brother and, after vessel
microanastomosis and vasovasostomy, serum testosterone levels increased and 15 million spermatozoa per
milliliter of ejaculate were observed.
217
Table 16.3 Studies on immature testicular cell xenografting
Donor
species
Recipient
species
Graft
localization
Tubule
reconstitution
Sperm
differentiation
Gassei et al. [86]
Rat (after
culture)
Nude
mouse
Back skin
Yes
Few putative
spermatogonia
No further
differentiation
IHC identification of
Leydig cells
Production of
bioactive
testosteronea
Kita et al. [85]
Mouse,
rat
Nude/SCID
mouse
Back skin
Yes
Round
spermatids
Offspring from
mouse-testis-cell
transplants
Not assessed
Yes
Not assessed
Honaramooz
et al. [84]
Pig
Nude/SCID
mouse
Back skin
Yes
Complete
spermatogenesis
IHC identification of
Leydig cells
Production of
bioactive
testosteronea
Arregui et al.
[70]
Sheep
Nude
mouse
Back skin
Yes
Complete
spermatogenesis
Production of
bioactive
testosteronea
Zhang et al.
[87]
Bovine
(after
culture
37 days)
Nude
mouse
Back skin
Testis
Yes
Yes
No germ cells
No germ cells
Not assessed
Watanabe et al.
[88]
Neonatal
pig
Nude/SCID/NOGBack skin
mouse
Yes
Complete
spermatogenesis
Not assessed
Reference
Pig
Steroidogenesis
Production of bioactive testosterone assessed by increase in seminal vesicle weight.

IHC, immunohistochemistry.
More recently, transplantation of rat testes showed

active spermatogenesis in 42% of fresh isotransplants but, after transplantation of intact cryopreserved testes, none was found to be functional [90].
Safety issues
Cancer cell contamination
The most important, life-threatening concern of
spermatogonial transplantation is the risk of reintroducing malignant cells. Indeed, the majority of
pediatric malignancies metastasize through the blood,
thus carrying a high risk of malignant contamination
of the testes. The risk is greater with hematological cancers, as the testes can act as sanctuary sites for leukemic
cells. Indeed, it has already been shown that as few as
20 leukemic cells injected into a testis can induce disease relapse [91].
Germ cell isolation and cell-sorting techniques
enabling complete purification of SSCs therefore need
218
to be validated before safe transplantation can be contemplated. While cell-sorting methods have shown
promising results in animal studies, the same cannot
be said of humans [92].
One of the reasons for suboptimal cell sorting
may be that the surface antigens are shared by other
stem cells, namely hematopoietic stem cells involved
in hematological cancers. Immunophenotyping malignant cells from each patient, followed by inclusion of
patient-specific cancer antigens for cell sorting, should
therefore improve the success of the technique. For
this purpose, we strongly advise storing patient blood
and/or tumor samples before therapy.
As no specific marker exclusive to SSCs has yet
been identified, allowing positive selection of these
cells through cell sorting techniques, further research
on surface markers should focus on the complete elimination of cancer cells from cell suspensions before
sorted preparations can be safely transplanted.
Cancer cell contamination is also a major
concern in tissue autografting. Since it has been
reported that leukemic cells can survive cryopreservation/xenotransplantation and increase the incidence
of generalized leukemia in the nude mouse host
[93], testicular tissue autografting after cure can
only be considered for patients in whom there is no
risk of testicular metastases or who have undergone
gonadotoxic therapies for non-malignant disease.
Infectious transmission
Due to the risk of infectious transmission from animals to humans, testicular xenografting should not be
considered for reproductive purposes at present. This
approach is nevertheless useful for the evaluation of
the functional capacity of germ cells and should therefore form part of the assessment of germ-cell cryopreservation protocols, for the understanding of testicular physiology and pathophysiology and for testing
malignant contamination of tissue before autografting.
Birth defect risks

Goossens et al. recently reported smaller litter size,
significantly lower fetal weight and reduced length in
first generation mouse offspring after germ cell transplantation, suggesting imprinting disorders [94]. Further investigation is therefore required to elucidate the
underlying reasons before autotransplantation can be
safely introduced into clinical practice.
Ethical concerns
Learning that a child has cancer is devastating for all
concerned and treatment needs to begin quickly, leaving very little time for the impact of possible future
sterility to sink in. However, the inability to father
ones own genetic children might have a huge impact
on the psychological well-being of patients in adulthood [10], so it is crucial to inform them of the potential consequences of their therapy on future fertility. Ethical concerns have been expressed about ITT
cryopreservation, highlighting the importance of the
risk/benefit balance [95]. Because of the small size of
testes from pre-pubertal children, immature gonadal
tissue sampling may be considered too invasive a procedure, which must therefore be done for good reason. However, in the two available studies on testicular tissue harvesting in young cancer patients [21, 22],
no major surgical complications occurred during testicular biopsy. Mean biopsy volume was about 5% of
testicular volume which, according to morphological
studies [6], should provide enough germ cells for fertility preservation. Furthermore, in a follow-up study of
cryptorchid boys who had undergone testicular biopsy
during orchidopexy, no adverse long-term effects were
reported [96]. Regarding general anesthesia, since this
biopsy is generally performed under the same anesthesia as that used for placement of the central line for
chemotherapy, there is no additional risk involved.
When considering the benefits of tissue harvesting,
the safety and effectiveness of fertility preservation and
restoration procedures are essential issues. Children
and their parents should be informed of the experimental nature of this approach and the fact that there
is no guarantee of fertility restoration. Parental consent
and the childs ascent, meaning he was given the opportunity to discuss the procedure, should be sought. As
obtaining fully informed consent from children is difficult, substituted consent from parents should for now
be limited to the safekeeping of tissue [95, 97].
Conclusion
Providing young people undergoing gonadotoxic
treatment with adequate fertility preservation strategies is a challenging area of reproductive medicine, but
every patient should be given the chance to consider
fertility-sparing options because the detrimental effect
of such therapy on gonadal function remains unpredictable. Hormonal or cytoprotective drug manipulation aimed at enhancing spontaneous recovery of
spermatogenesis remains a possibility for the future.
Preservation of SSCs offers the prospect of several realistic applications, although none is feasible in humans
at this point in time. Future advances in fertility preservation technology rely on improved understanding of
the cryobiology of gonadal tissue and cells.
Before considering fertility restoration options,
patient selection is essential, since risks vary according to disease. No single (or simple) algorithm can
so far summarize all the possible strategies for fertility preservation and restoration in case of gonadotoxic therapy in pre-pubertal boys, but the most appropriate course of action may be selected according to
the scheme shown in Figure 16.4. Over the next few
years, research should focus on how to extend successful experiments in animals to young boys and on the
identification of the ideal microenvironment for SSC
development. Resolving numerous important technical issues discussed in this chapter should lead to safe
and efficient methodologies for fertility restoration
219
Spermatids/
sperm
High risk of
cancer cell
contamination
ITT
cryobanking
ICSI
IVM
Diploid cells
Low risk of
cancer cell
contamination
Cell transplantation after

efficient cell sorting and
xenotransplantation for
exclusion of cancer cell
contamination
Spontaneous
conception
Tissue autografting after detection of

MRD by RT-PCR and xenografting to
exclude tissue contamination
IUI
IVF
ICSI
ICSI
Figure 16.4 Fertility restoration strategy after gonadotoxic therapy in pre-pubertal boys. ICSI, intracytoplasmic sperm injection; ITT,
immature testicular tissue; IVF, in vitro fertilization; IUI, intrauterine insemination; IVM, in vitro maturation; MRD, minimal residual disease;
RT-PCR, real-time polymerase chain reaction.
after storage of preserved gametes, and the development of ethically accepted pilot protocols, which will
then need to be submitted for further ethical approval
before definitive and universal clinical implementation. Until then, samples should at least be banked after
providing careful counseling and obtaining informed
consent, making sure the patient understands there is
no guarantee of success. Preservation of testicular tissue from todays pre-pubertal patients will allow them
to consider various fertility restoration options that
will emerge in the next 2030 years, giving them hope
of fathering children with their own genetic heritage.
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Section 4
Chapter
17
Assisted reproductive techniques and

donor sperm in cancer patients
Wayland Hsiao, Elizabeth Grill and Peter N. Schlegel
Introduction
Recent advances in the treatment of cancer have lead
to greater longevity and an increased recognition
that quality of life including paternity is significant
issues for cancer survivors. We will focus primarily
on patients with testicular cancer and lymphoma that
generally affects younger patients in the reproductive
window with an excellent overall survival. However,
one must realize in our modern society the age of desiring paternity has increased due to postponement of
marriage as well as for other social reasons. Therefore, this chapter will focus not only on those who
completed chemotherapy as children but adult cancer
patients as well.
Chemotherapeutic effects
The effect of chemotherapeutics on spermatogenesis
varies by both the drug administered as well as the
cumulative dosage and will be discussed in other
chapters. However, it is obvious that due to their high
proliferative index, chemotherapeutics will be toxic to
germ cells while Leydig cells seem more resistant to
the effect of systemic chemotherapy. Therefore, serum
levels of testosterone may be within normal limits
and yet impairment in sperm production may lead to
infertility.
There is no diagnostic test to tell whether spermatogenesis will return after chemotherapy. Men with
return of sperm to the ejaculate may even conceive naturally and all reproductive options are open to them.
However, a focus on men who are rendered azoospermic after therapy remains a clinically important subgroup of patients who are candidates for treatment. Up
to 13.8% of men are azoospermic prior to chemother-
apy [1, 2]. What are we to do with this population? This

chapter will discuss the treatment options available
to azoospermic males prior to chemotherapy as well
as post-chemotherapy azoospermia. Though multiple
advanced reproductive techniques are now available
to the post-chemotherapy population, it remains an
obvious recommendation that men bank sperm prior
to any chemotherapy or gonadal radiation treatment,
optimizing options for effective treatment.
Sperm banking before

chemotherapy/radiotherapy
In most cases, sperm collection and cryopreservation of sperm is a simple task prior to the initiation
of chemotherapy or radiation therapy. In men with
normal ejaculation, sperm cryopreservation involved
the collection of a masturbatory sample that is subsequently frozen. Barriers to universal sperm banking are variable but may include inadequate counseling
by physicians, inability to afford sperm banking, limited sperm quality for cryopreservation, pre-pubertal
or young emotional age of the patient, religious restrictions on sexual activity or the sense of urgency to begin
cancer treatment.
Unfortunately, low rates of sperm banking prevail. One study that examined young adult survivors
of childhood cancer found that nearly 60% reported
uncertainty about their fertility status, and only half
recalled a healthcare provider discussing potential
reproductive problems associated with treatment [3].
In 2002, a survey of cancer patients treated at two
major cancer centers revealed that only 51% of respondents had been offered sperm banking and only 24%
of respondents actually banked sperm [4]. A lack of
225
information was the most common reason for not

banking sperm in up to 25% of patients in this study
[4]. Surveys in the USA [5, 6], UK [7], Australia and
New Zealand [8] show that many oncologists do not
routinely provide information on and referrals for
banking sperm to teenagers and young men, despite
claiming that sperm banking should be offered to all
men at risk of infertility from cancer treatment. Sperm
banking seemed to be offered less when the patients
had aggressive disease or poor prognosis [6]. But even
with successful banking, rates of subsequent utilization of cryopreserved sperm appear to be low. Several recent reports from sperm banking facilities concur that 20% of men who store sperm before cancer treatment end up using it to try to conceive [2, 9
11]. Of course, such numbers could be artificially suppressed by: (1) lack of survivorship for some patients
who elect to bank sperm; (2) the long delay between
medical therapy for cancer and subsequent election to
have children; (3) recoverability of sperm production
for some men despite toxic treatment regimens; and
(4) the lack of insurance support for assisted reproductive treatments in many areas of the USA.
Therefore, all men should be offered and the majority should undergo sperm banking. For the minority of
patients who are azoospermic at the time of presentation, more advanced sperm retrieval techniques may
be needed to cryopreserve sperm.
Chance of recovery of spermatogenesis

The chance of recovery of spermatogenesis depends
on the chemotherapeutic regimen as well as the baseline function of the patient. Notably, alkylating agents
such as cyclophosphamide have been the most extensively studied in this group of drugs and seem to
have the most dramatic reproductive effects. However,
sperm will return to the ejaculate in numbers sufficient
for natural conception in many if not most patients
depending on the chemotherapeutic regimen, radiation treatment, surgical treatment and baseline semen
parameters. For patients with return of sperm to the
ejaculate, reproduction may involve spontaneous conception to intrauterine insemination (IUI) to in vitro
fertilization (IVF). However, Schmidt et al., in a review
of 67 cancer survivors, found that 57% of men were
azoospermic after chemotherapy [12]. The rest of this
chapter will focus on the treatment options available to
those who remain persistently azoospermic or severely
oligospermic.
226
At present, only crude measures of predicting

the return of spermatogenesis after chemotherapy are
available. During chemotherapy, follicle stimulating
hormone (FSH) levels invariably increase. Kader and
Rostom [13] found that persistently elevated levels of
FSH at 2 years after chemotherapy is associated with
a higher chance of azoospermia. It is worth noting,
however, that in our micro-dissection testicular sperm
extraction (mTESE) series we have not found FSH to
be a predictor of sperm retrieval (to be discussed later)
[14]. Otherwise, we eagerly await better markers that
may indicate return of spermatogenesis.
Treatment options for azoospermic

men after chemotherapy
Traditionally the patient who presents with azoospermia after chemotherapy has been considered sterile if
they did not bank sperm prior to chemotherapy. However, advances in advanced assisted reproductive techniques have enabled even these men to successfully
father children with newer techniques for testicular
sperm extraction. The realization that the testicle is
not uniform in terms of spermatogenesis, has allowed
retrieval of sperm from those men once deemed sterile. With the introduction of intracytoplasmic sperm
injection (ICSI), we now have the ability to enable conception with very low numbers of sperm. For the man
who is azoospermic after chemotherapy there remains
a number of choices for reproduction, including the
use of previous cryopreserved sperm as well as use
of fresh sperm retrieved either by electroejaculation
or testicular sperm extraction. Finally, if a patient is
unable or unwilling to successfully pursue any of the
previous options the option of donor sperm remains.
Use of cryopreserved sperm

The existence of a previously cryopreserved sperm
greatly simplifies the algorithm for the postchemotherapeutic azoospermic man and, essentially,
the couple can go directly to IVF. The outcomes are
good for cryopreserved sperm. Hourvitz reported
on 118 couples undergoing 169 IVFICSI cycles at
the Weill Cornell [15]. From 1994 to 2005, using
cryopreserved sperm and ICSI, there was a fertilization rate (per injected egg) of 77.6%, a clinical
pregnancy rate of 57% (96/169) and a 50% delivery rate (85/169). As a historical control, a similar
population was evaluated from 1992 to 1994 at the
Chapter 17: ARTs and donor sperm in cancer patients
same institution prior to the routine use of ICSI.

Using conventional IVF, the fertilization rate was 32%
and the delivery rate was 24% (13/54) [15]. Agarwal
et al., in a retrospective study of 29 patients, reported
the outcome of use of cryopreserved sperm from
men with various malignancies [11]. They showed
that with assisted reproductive technology (ART),
couples with cryopreserved sperm prior to cancer
therapy can be successfully treated to achieve pregnancy. Schmidt et al., in a study of patients presenting
to a Danish fertility clinic, noted that 22/35 live births
in their series were due to the use of cryopreserved
sperm [12]. Thus, the use of cryopreserved sperm is
both viable and successful.
In the patient who is azoospermic

prior to chemotherapy
Patients with leukemia, lymphoma and testicular cancer often present with suboptimal semen parameters
[1619]. Whether one type of malignancy presents
with poorer outcomes is controversial [1, 20, 21]. However, it is notable that up to 13.8% of cancer patients
presenting to sperm banks are azoospermic [1, 2].
For the patient azoospermic prior to chemotherapy,
there is an even greater urgency with regard to the
expediency of treatment. Time is critical and these
patients must balance the urgency of getting necessary
chemotherapy with the additional worry of successful
preservation of spermatozoa. Any fertility treatment
must be of minimal morbidity limiting any delay of
the patient from the needed chemotherapeutics. For
these patients, there is the option of cryopreservation
of testicular spermatozoa from normal testis, cryopreservation of spermatozoa from the diseased
testis, or preserving epididymal or vasal sperm (for
obstructed patients). It is likely that in this setting,
yield of spermatozoa from any of these sperm retrieval
techniques may be low. The advent of ICSI has made
treatment of these patients feasible, since only small
numbers of sperm are needed for successful IVF.
Sperm extraction is possible from the contralateral
normal testis or from the affected testis (if retrieval is
done after separation of the testis from the patient) for
testicular cancer at the time of orchiectomy. In patients
with lymphoma, sperm extraction can be done in
either one side or both testes simultaneously prior to
chemotherapy. In 2003, Schrader et al. reported 31
men with either testicular cancer or lymphoma [22].
In testicular cancer patients, the TESE sample came
from the contralateral testicle, which was also evaluated for concomitant carcinoma in situ (CIS). The
lymphoma patients underwent bilateral TESE. The
sperm retrieval rate was 43% (6/14) in testicular cancer and 47% (8/17) in patients with Hodgkins or nonHodgkins lymphoma [22].
There also is the possibility of vasal or epididymal sperm extraction after orchiectomy from the
orchiectomy specimen. Baniel and Sella reported three
azoospermic patients with testicular cancer who at the
time of radical orchiectomy had vasal and epididymal sperm preserved [23]. After the specimen had
been resected, sterile extraction of sperm with cryopreservation of sperm from the vas and epididymis
was done. They reported two pregnancies from three
couples [23].
Preserving tissue or sperm from the tumor containing testis can be done on a separate sterile field
(a backtable) after orchiectomy or, in cases of partial orchiectomy, the surrounding normal testicular
parenchyma can be examined and sent for cryopreservation. A number of groups have reported on these
techniques in a limited number of patients with good
success at sperm retrieval [2426].
Use of spermatozoa from men who have

received chemotherapy or radiation
Men who have received chemotherapy, and even men
who had non-gonadal radiation therapy, will have
increased rates of sperm aneuploidy for 6 months or
more after treatment. A variable increase in sperm
DNA fragmentation has also been observed after
chemotherapy, much of which will return to baseline
within 1 year after treatment. Some studies have suggested an increase in birth defects for offspring of men
treated with chemotherapy or non-gonadal radiation
within the past year. Taken together, these data suggest caution in suggesting that patients attempt to have
children early after chemotherapy or radiation treatment.
In the patient who is anejaculatory after

chemotherapy and is oligospermic or
normospermic
Any retroperitoneal surgery such as retroperitoneal
lymphadenectomy may affect the sympathetic chain
or structurally compromise the bladder neck and may
227
affect antegrade ejaculation. Partial ejaculatory function may be preserved depending on the degree of
nerve sparing. The preservation of ejaculatory function obviously has a tremendous impact on fertility
rates. In Norwegian testicular cancer survivors who
had chemotherapy, those with intact antegrade ejaculation have an 83% paternity rate, while rates for paternity were only 10% in the anejaculatory group [27].
An option for anejaculatory patients is to undergo
electroejaculation. This is a procedure generally performed under general anesthesia in the sensate patient.
We do not routinely catheterize the patient prior to
electroejaculation. The patient is placed in the lateral
decubitus position. Anoscopy is performed to confirm that the rectum is empty and no rectal mucosal
abnormalities are present. The rectal probe is inserted
completely into the rectum with the electrodes oriented anteriorly, over the prostate and seminal vesicles.
Stimulation is carried out with a standard electrical
stimulation system. The pattern of electrical stimulation has been empirically evaluated but appears to
work best with a gradually increasing voltage peaked
sine wave stimulation that is abruptly ceased, with at
least 57 s delays between stimulations. The procedure
is also monitored by observation of penile tumescence
and rectal temperature. Typically, penile tumescence
is noted first, followed by seminal emission. When
seminal emission ceases, rectal temperature of 38 C is
observed, or a maximum of 30 volts is attained, then
electrostimulation is stopped. Anoscopy is performed
again to insure that there is no rectal mucosal injury,
which is a potential complication of this procedure.
The patient is turned supine and urethral catheterization is carried out. An initial retrograde specimen
is diluted in human tubal fluid (HTF) buffered with
HEPES and plasmanate, pH 7.4, and sent for immediate processing, as is the antegrade ejaculate. The bladder is then irrigated with HTF, and this second retrograde specimen is sent for immediate processing as
well.
Ohl et al. performed electroejaculation in 24
testicular cancer patients (23 of which had undergone retroperitoneal lymphadenectomy) and observed
seminal emission in all 24 patients [28]. Greater than
10 million motile and progressive sperm were obtained
in 88% (21/24) of patients. In total, 17 couples underwent IUI and the overall cycle fecundity rate was 9%.
Seven clinical pregnancies were detected and there
were five live births. Electroejaculation has also been
successfully combined with IVF (and obtained a 53%
228
fertilization rate [29]) as well as IVF/ICSI with a

75.5% fertilization rate[30]. At Weill Cornell, we have
reported a fertilization rate of 75.5%, a clinical pregnancy rate of 56% per retrieval and an implantation
rate of 33% per embryo [30].
In 1998, Rosenlund et al. looked at 17 couples treated for testicular cancer where most (14/17)
received chemotherapy and most patients acquired
sperm through electroejaculation [31]. They employed
IVF or ICSI and had a fertilization rate of 5557%
in both groups, and the ongoing pregnancy rate for
the whole cohort was 57% per cycle [31]. The study
demonstrated that treated testicular cancer patients
can successfully undergo ART with electroejaculated
spermatozoa.
In the patient who is azoospermic

after chemotherapy
Azoospermia after chemotherapy can be due to the
patients chemotherapeutic regimen, the use of radiation, the extent of surgery, the disease itself, the baseline function of the patient or any combination of the
aforementioned factors. While these men were once
considered sterile, the use of advanced reproductive
techniques has enabled paternity in a subset of this
population. Specifically, the realization that the testis
is not uniform and that there may be small pockets of spermatogenesis in these patients has enabled
us to retrieve sperm in patients with non-obstructive
azoospermia using mTESE [32].
For men who are azoospermic after chemotherapy, spontaneous recovery may occur in at least a subset of patients within 28 years. For men treated with
alkylating agents, the duration of azoospermia may be
longer, so a period of observation prior to attempted
testicular sperm extraction is recommended. For men
treated with platinum-based regimens, most men who
will have sperm return to the ejaculate can have sperm
detected within 2 years. As in any patients with nonobstructive azoospermia, percutaneous aspirations or
biopsies, while possible, are more likely to yield low
numbers of sperm and require multiple treatments. In
our view, the low yield, uncertainty of sperm retrieval
and intratesticular bleeding/scarring make these procedures less favorable, especially in this patient population with multiple insults to spermatogenesis. The
risk of testicular injury along with low spermatozoa
yields led to the development of mTESE [32]. Our data
Eversion of
testicular
parenchyma
for microdissection
Figure 17.1 A transverse incision is

made in the testis and the testicular tissue
is everted and micro-dissected for
thorough inspection of all tubules.
Centrifugal vessels
and that of others suggest that mTESE yields the highest sperm retrieval rate in this population.
Evaluation prior to mTESE includes a through history, sexual history, chemotherapy history physical
exam and hormonal profile. On physical exam, attention is paid to the fullness of the epididymis as well
as testicular volumes. The technique of mTESE is as
follows. This technique involves placing a wide incision in the tunica albuginea in an avascular region
and eversion of the testicular parenchyma for microdissection (Figure 17.1). With high power magnification, subtunical vessels as well as intratesticular
vessels can be identified and preserved. Microscopic
dissection and direct examination of seminiferous
tubules allow identification of the rare regions that
contain sperm in men with non-obstructive azoospermia (NOA). The tubules with spermatozoa are wider
and more opaque than the fibrotic Sertoli cell-only
tubules (Figure 17.2). Overall, mTESE has been shown
to result in a higher number of sperm harvested,
increased chance of retrieving sperm and decreased
testicular tissue removed [32, 33]. The only predictor of successful treatment is the most advanced stage
seen on biopsy and not the predominant stage [34].
Testicular volume, serum FSH levels and the etiology of NOA appear to have little or no effect on the
chance of sperm retrieval [14, 34, 35]. Postoperative
ultrasound has demonstrated fewer acute and chronic
changes after micro-dissection as compared to conventional TESE [36]. Of course, an increased number of
biopsies is always counterbalanced by a greater risk
of damage to the vascularity of the testis, and so the
Figure 17.2 Intraoperative picture of tubules more likely to harbor

spermatogenesis, as indicated by forceps. See plate section for color
version.
surgeon must be constantly aware of this. For selection of the initial side, we prefer to start on the side
with larger testicular volume or the side with the more
advanced spermatogenic pattern seen on histology if a
prior biopsy was done (with the most advanced being
normal spermatogenesis followed by late maturation
arrest, early maturation arrest and Sertoli-cell only pattern, in that order).
At Weill Cornell Medical College we have
performed 81 mTESEs in 70 post-chemotherapy
patients. These patients presented with a variety
of malignancies, with the most common being
Hodgkins lymphoma, leukemia, testicular cancer and
229
Table 17.1 Patients with underlying medical conditions treated with chemotherapy
Medical condition
No. of patients (n = 81)
Percentage
Hodgkins lymphoma
29
35.8
Testicular cancer
13
16.0
Leukemia (AML, ALL)
13
16.0
12
14.8
Sarcoma
8.6
Neuroblastoma
3.7
Medulloblastoma
1.2
Wilms tumor
1.2
Mediastinal germ cell tumor
1.2
Nephrotic syndrome
1.2
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia.
non-Hodgkins lymphoma (Table 17.1). The mean

number of years since chemotherapy was 18.6 years
(range 134 years). The mean male age at mTESE was
35.3 years (range 2253 years) and the mean female
age was 32.5 years (range 2143 years). Mean baseline
FSH was 24.0 (range 4.262.7) and mean testosterone
was 352 (range 64814). Our sperm retrieval rate was
43.2% (35/81). Fertilization rate was 57.0% 192/337.
Clinical pregnancies were defined as a heartbeat seen
on transvaginal ultrasound 32 days after embryo
transfer. The live birth rate was 42.9% (15/35) with 10
singleton deliveries and 5 twin deliveries. We noted
a lower sperm retrieval rate with lymphoma 34%
(14/41) than for testicular cancers 85% (11/13) [W.
Hsiao et al., unpublished data].
Damani et al. reviewed the University of California, San Francisco (UCSF) and Boston University
experience in 2003 [37]. The series consisted of 23
patients who underwent chemotherapy for a number
of reasons but mostly for testicular cancer. They either
underwent conventional testicular sperm extraction
or fine needle aspiration mapping and subsequent
TESE. Sperm was successfully extracted in 65% (15/23)
and a total of 26 cycles of ICSI were performed.
The mean fertilization rate was 65% with a delivery/ongoing pregnancy rate of 20.8% in 11 couples.
In a study of 12 patients post-chemotherapy, a multibiopsy approach TESE was undertaken and sperm successfully retrieved in 5/17. Eight ICSI cycles were performed with a fertilization rate per injected oocyte of
68%. There was only 1 live birth from 7 embryo transfers. [38].
230
Donor sperm
Third-party reproduction
Additional counseling is recommended for those
patients who choose the assistance of third-party
reproduction. There is universal agreement that the
psychosocial, emotional and ethical complexities of
donor conception require thorough exploration both
for those donating and those receiving gametes [39
41]. In most clinics, a mental health professional
(MHP) meets with prospective donors and recipients to explain the known psychological, social and
legal implications of third party reproduction. It
has been argued that the assistance of a MHP is
essential to promote complete examination of the
many dilemmas faced by those who receive gametes
[4244].
The MHP may need to help a patient address previously unresolved grief regarding the cancer diagnosis and treatment. When donor back-up treatment is
being considered, it should be carefully explored prior
to treatment and should not be a decision by default
at the last minute (e.g. after poor TESE results and
oocyte retrieval during an IVF cycle). Often times, a
patients hopes that viable sperm will be found may
interfere with their ability to fully consider all aspects
of a donor sperm back-up plan. While a patient may
have grieved at the time of the cancer diagnosis, a
diagnosis of infertility may reopen the grieving process by adding another dimension to the illness and
may interfere with the desire to move forward with a
donor.
Couples embarking on the path of third-party

reproduction must mutually agree that their best alternative to genetic parenthood is the use of donated
sperm or embryos. They must think about what it
means to be a parent and how parenting a child who
is not genetically connected to both of them may be
different from parenting a child who is genetically
connected to only one of them. Couples who feel
strongly about their genetic lineage may view donor
sperm/embryo as severing their ancestral ties. The ultimate loss of ones ability to create a child can create a
powerful emotional crisis as well as feelings of sadness,
anger and bereavement. There are several psychological losses to overcome in non-biological parenting: loss
of biological posterity; loss of self-esteem and a sense
of wholeness; loss of the ability to give ones partner a
child; loss of the fantasized child that will embody the
best of both parents; loss of a sense of control, health
and well-being; and loss of the belief in the fairness
of life. Resolution of these losses is best conceived as
a process [45, 46].
In addition to thinking about how a third party,
known or unknown, will affect their feelings about
themselves and their relationship, most importantly,
couples must think about their relationship with their
potential child. Recipients must decide whether or not
they plan to tell the potential child about how he or she
was conceived and how much interaction, if any, they
want the donor to have with the child. Couples often
carry many fears and fantasies about gamete donation,
including concerns that the donor will try to re-claim
the offspring or that the child will wish to seek out
his/her real mother or father. The man who is unable
to use his own gametes may wonder if he is capable
of loving someone elses child. For some couples this
process brings up thoughts and feelings about adultery,
and they must work toward separating the act of sexual
intimacy from the act of procreation. Others fear that
the biological and genetic inequality of donor sperm
may eventually threaten their relationship.
Anonymous versus known sperm donation

There are three main types of gamete donation [47].
In anonymous donation, donors are typically selected
from sperm banks and couples choose their donors
from profiles with non-identifiable information such
as physical characteristics, intelligence, academic history, professional background, hobbies, nationality,
social history, religion and blood type. In other coun-
tries, it is sometimes the doctor that selects the donor

according to phenotypological similarities with the
male partner. The issue of an identified, known or
interfamilial donor is another option for couples.
When the donor is willing to disclose their identity,
sometimes including meeting the parents (and possibly the child) in the future, the donor is referred to
as a known or identified donor. A directed donor is a
friend or relative of the intended parents who chooses
to donate solely to that specific family.
The decision to use a known or unknown donor
is only one of the many choices that affect all parties involved, including the potential offspring. Those
who support anonymous sperm donation insist that
anonymity is beneficial to the donor, the recipients
and the donor offspring. Some couples strongly desire
anonymous donation because they wish to maintain
privacy about the donor decision, while others come
to the process with additional losses because they do
not know anyone who would be appropriate as a donor.
Most couples who choose anonymous donation feel
protected by the anonymity and feel that it creates a
psychological barrier between them and the donor,
enabling them to feel more secure as a family. Couples do not want the child to be confused about who
his or her parents are or reject the non-genetic parent. They may also wish to conceal the donation from
disapproving family members, especially those for cultures less accepting of sperm donation. Many couples
worry that if their family knew about the donation, the
child would not be loved or accepted in the same way
as a full genetic child [4850]. They typically feel that
telling the child of his or her birth by sperm donation
would subject the child to social or psychological disorders, which could be especially unsettling if the child
wanted to find out more information about the donor
but could not.
In recent years, a strong tendency in favor of nonanonymous sperm donation has emerged in Europe
and Australia. Several countries have enacted laws or
are taking into consideration permitting children to
gain access to information about their genetic fathers
once the child has reached maturity [51]. Proponents
of non-anonymous sperm donation argue that human
beings have a fundamental interest, and perhaps even
a legal right, to know their biological origins. Not
telling the child of his or her origins violates that childs
autonomy. Proponents believe that disclosure is a key
part of open and honest communication with children,
which helps to avoid secrets in the family that can
231
damage family relationships and generate possible

strain and anxieties.
Those who support known or non-anonymous
donation feel more comfortable in having control over
the source of the gametes as well as the knowledge of
medical and social histories. Couples feel comforted
by knowing firsthand the donors personality, temperament and physical attributes, and may feel relieved
with not having to deal with the social and relational
confusion inherent in familial donation. Some choose
a close friend, while others fear that sperm donation
could jeopardize their relationship if something went
wrong or if they did not conceive, or if the donor
became attached to the child and viewed it as his.
In some cases, known donors also give the child the
option of knowing his or her genetic parent, which
some donor recipient parents feel may help facilitate
a more secure identity for their children.
Gamete donation has made it possible for participants to cross generational lines and has raised
many complicated ethical issues. In 2003, the American Society for Reproductive Medicine (ASRM) issued
an Ethics Committee Report on family members as
gametes donors and surrogates [52]. While this report
approved of many types of interfamilial gamete donations, it recommended a careful screening to ensure
that the decision to donate gametes to a family member
protected the autonomy of the donor. It also advised
that semen donation should not be carried out in
those situations in which the child would result from
an incestuous (father-to-daughter donation) or consanguineous union (brother-to-sister donation). Furthermore, it recommends that family members (the
extended family of the infertile couple) must be accepting of the resulting child. Interfamilial donations that
the participants plan to keep secret from the larger
family system should be carefully evaluated.
When an identified, known or interfamilial donor
is being used there are specific psychological issues
that need to be systematically addressed by the infertility counselor. It is necessary to assess the relationship between the participants to establish whether the
reproductive plan is in the best interests of all of those
involved, including the potential child. It is also important to obtain the history of the relationship between
the donor and the recipient couples to ensure that there
is no coercion or other hidden agendas. The nature of
the relationship and boundaries between the potential
child and the donor must be carefully examined and
clearly defined for all parties. Should the relationship
232
between the donor and the parents be strained at some

time in the future, there is potential for traumatization
of the potential child as well as other family members
[49, 51]. In some cases, it is the counselor who needs
to help one party say no to an uncomfortable request.
In child-to-parent donations, the counselor must
address the imbalance of power and the inherent
boundary violations that may leave the family system
or the relationships vulnerable and at risk. Because
most children feel indebted to their parents to some
degree, these children are not truly free to say no to
their parents request in the same way they are free
to say no to anyone else. In addition, many experts
feel that the nature of the relationship may be violated as children are providing for their parents while
these parents are still competent. The infertility counselor must also consider a son or daughters relationship with the stepparent to make sure that there are no
sexual overtones. In the case of a father donating to a
son, some professionals feel more supportive, because
the concept of a parent giving to their child is already
built into the parentchild relationship [51, 53, 54].
Oftentimes couples may ask a brother to donate
while others may ask another relative such as a cousin
or a nephew. Brother-to-brother donations may appear
to be ideal on many levels because of their similar
genetic makeup and continuity of the bloodline, but
it is only as good as the health of the relationships
between the two siblings and their respective spouses.
If couples choose a sibling to donate, old patterns of
sibling rivalry may get stirred up. There are also many
social and emotional entanglements that could occur
in the family if their child for example, has an uncle
who is considered his genetic father and a cousin
who is his half-sibling. When third parties are involved
in family building, especially when they are a family
member, recipient parents may fear that their children
are likely to form a stronger attachment to the donor
than to them. When a family gamete cycle fails to result
in pregnancy, all involved are extremely disappointed,
and as family members search for an explanation, old
family dynamics may be reenacted resulting in blame
or feelings of guilt [53, 54].
It is critical for all of the parties involved in any
type of known donation to have a clear understanding
of boundaries and to think through scenarios that
may challenge these arrangements in the future. All
parties should be in agreement regarding disclosure to
others as well as to the potential child. Ultimately, the
donor should feel comfortable allowing the recipients
to make all decisions related to disclosure, the pregnancy and the upbringing of the potential child.
The infertility counselor must help all of the parties
involved explore their motivations, concerns, expectations, wants, hopes and fears regarding the process.
Disclosure to children created with

donor sperm
Whether or not children conceived using donated
gametes should be told about their genetic origins and
be able to access identifying information about the
donor and/or be allowed to contact the donor are some
of the most disputed ethical issues raised by the practice of assisted reproduction. The question of open disclosure versus secrecy is a complicated one, involving profound ethical, legal, religious and psychosocial
issues. Some believe that it is not justifiable to keep
such information secret, either because it is argued that
children have an inherent human right to know about
their genetic/gestational beginnings or because of concern about the effect of secrecy on family [5557]. Others have argued that due to the limited available evidence about the risks and benefits of disclosure versus
openness, neither view should be imposed on couples
using donor gametes [5860].
It is not surprising that a growing number of
infertility counselors, professional organizations and
government regulatory agencies recommend pretreatment counseling prior to donor sperm. Although
the decision to use donated sperm is private, the
issue of disclosure of this decision to others or to the
resulting offspring often creates anxiety, questions and
uncertainty for the recipient couple. Infertility counselors can provide a forum for patients to safely explore
their thoughts and feelings about disclosure. There are
arguments both for and against disclosure, and each
couple should be allowed to decide with the help of
infertility counselors which choice is best for them
and their child(ren) within the context of these issues.
Experts agree that the more the recipient couple feels
comfortable and prepared for this parenting option,
the more likely it is that they will be fulfilled as parents
and will make decisions that are in the best interest of
the child.
Haimes identifies three competing strategies for
the management of genetic origins [61]. The first is
full secrecy, on the grounds that there are no obvious benefits to be derived from disclosure and there
is some risk of stigmatizing the offspring. The second
is telling the truth about the means of conception and

providing some basic information about the donor, but
not revealing the donors identity. The rationale here is
to ensure that the offspring does not suffer from the
effects of the family keeping such information secret.
The third approach is telling the offspring as much as
possible about the gamete donation and the donor, on
the grounds that he or she will need information to
develop a full sense of identity. Pennings proposed a
fourth approach whereby donors may choose between
anonymity or identification and recipients can opt for
either an anonymous or identifiable donor [62].
Historically, parents have generally not been
encouraged to tell others or their children that the
family came to be through non-traditional means.
The debate between disclosure and secrecy has its
historical roots embedded in the traditions of sperm
donation and adoption. The first case of artificial
insemination using a donor was documented by
William Pancoast, who claimed to have performed the
procedure in secret. Thus began a trend of secrecy that
has continued for over a 100 years for most couples
choosing donor sperm. There are many reasons why
donor sperm has been shrouded in secrecy. Male infertility is often associated with impotence or a lack of
virility and sexual functioning, and thus carries with
it a shameful stigma that is seldom discussed openly
among health professionals [51]. Donor insemination
(DI) itself has been associated with masturbation and
the involvement of a second male has been suggestive
of an extramarital affair.
There are many reasons why a couple may choose
not to tell family, friends and/or the child. Shame often
fuels a non-disclosure stance. Unfortunately, shame is
a byproduct of secrecy, which only increases the couples feelings of inadequacy and may decrease their
ability to form close relationships with the child. Couples may hold fast to the illusion that if they do not tell,
they will remain protected from the sadness of not having a family by traditional means. For some religions,
the use of donor sperm is discouraged since it is viewed
as having a third party in marriage and has been compared to sexual infidelity. However, families who protect secrets develop a complicated system of interpersonal relationships with taboo-like undercurrents that
children often pick up on. This system of secrecy can
inadvertently promote family estrangement and create unhealthy alliances between those who know and
those who do not know and may also undermine the
trust that is vital to a healthy parentchild relationship.
233
When parents choose not to tell their children, they

often live in fear that they may find out about their origins and that the bond of trust that children have with
their parents will be threatened.
This trend toward secrecy appears to be slowly
changing as people have come to recognize the hazards of secrecy and the needs and rights of the offspring to access information about their biological origins. The psychological wishes of sperm donors and
their attitudes toward non-anonymity and disclosure
are increasingly given consideration. Recent findings
showed an increase in donor programs that offer openidentity between donors and offspring [63, 64].
In addition, legislation, professional guidelines and
MHPs in several countries have supported open information sharing [51]. In 2004, the ASRM published
recommendations that encouraged disclosing the use
of donated gametes to offspring [65]. Legislators
responses to this debate vary. In some countries, such
as France, Denmark and Spain, gamete donation only
occurs with reciprocal anonymity between donors and
recipients. In others, such as the UK, Austria, New
Zealand, Sweden and some states in Australia, irrespective of whether the donor is anonymous or known,
it is a legal requirement that identifying information
about the donor is recorded to enable a child born
as a result of a donor procedure to access it in the
future [66]. Policy changes in other countries have
varied. Although there is no agreement within the
European Union regarding access to identifiable information about the donor, from 2006 on, all member
countries are required to document information about
gamete donors for a minimum of 30 years [51]. There
are still some countries, such as Italy, where ART legislation was introduced in 2004, where treatment using
donated gametes is banned [67].
Nevertheless, as a result of different values attached
to family, marriage and the childs well-being, most
families do not disclose the use of donor insemination
to children [51, 68]. Reasons cited for non-disclosure
include the desire to protect the child from either the
distress and stigma of finding out that the father is
not the genitor; the impossibility of accessing information about the donor; to protect the father from
being rejected by the child; the belief that disclosure
is unnecessary; concerns that family relationships may
be damaged as a result of disclosure; the lack of educational material/resources; and lack of support and
guidance on how to tell the child [66, 6971]. Couples
also report the desire to normalize the situation, both
234
so that the child does not feel different and the husband
does not feel embarrassed, and to allow the parents to
feel that they are an ordinary family.
Religion and culture often influence attitudes about
the use of donor sperm. Donor insemination remains a
morally questionable treatment option in many countries. Using donor gametes is forbidden in some religions. In Islam, only a married couples eggs and sperm
may be used in procedures to treat infertility because
religious law dictates the preservation of the genetic
line. The Vatican does not endorse any assisted reproductive techniques, even insemination with the husbands own sperm [72]. Some Catholic bioethicists
have supported assisted reproductive techniques if no
other alternatives exist to allow a couple to have children but still do not find use of donor sperm acceptable
[73].
More often, couples are being encouraged to provide genetic disclosure to offspring. This emerging
trend of more openness may reflect legislative and cultural changes as well as the impact of counseling and
the advent of guidance materials parents can use for
talking to their children [51]. The main reasons that
couples choose to disclose include: the children had a
right to this information; the parents wanted to avoid
the burden of secrecy and the risk of disclosure by
somebody else or accidental discovery; and the parents believed that technical advances in genetics could
result in a genetic mismatch discovered by the offspring, again resulting in inadvertent disclosure [74,
75].
If couples choose to disclose the information to
their children, they should also understand the distinctions between openness and privacy. Couples still
have the right to discern who will know, how they will
find out and when they will share the information.
Given the stigmatizing nature of using donor sperm
and male-factor infertility, the couple needs to be in
agreement about with whom they will share information regarding infertility treatment, diagnosis and
their use of donor sperm. If they do not intend to talk
to their future child about his or her donor sperm origins, they must attempt to ensure that their confidants
will not disclose this to the child accidently nor discuss
the issue of donor sperm with others.
Information sharing will not only be an individual decision based on personal preference but will
also depend on cultural and religious factors, such
as the degree of acceptance of donor sperm as well
as legal practices regarding access to information and
donor identity. Research on how parents of donor offspring make decisions about disclosure reveals that
even when couples are initially opposed to disclosing
to their offspring, most ultimately come to a united
disclosure decision either intuitively, or after discussions influenced by the couples local sociopolitical
environment, professional opinion, counseling, religious and cultural background, family relationships
and personal, psychological and ethical beliefs [76].
Moreover, when gamete donation is used because of a
parents history of cancer, telling the child may provide
reassurance about his or her own lifetime cancer risk.
Age of disclosure appears to be important in determining donor offsprings feelings about their donor
conception. It seems that it is less detrimental for children to be told about their donor conception at an
early age [77]. Those told later in life report more negative feelings regarding their donor conception than
those told earlier. Offspring from heterosexual couple families are more likely to feel angry at being lied
to by their mothers than by their fathers. The most
common feeling toward fathers was sympathetic.
While the research in this area is somewhat limited, the
largest prospective follow-up of donor insemination
children to date documents normal social and psychological adjustment of children and families as the
cohort reached 12 years, despite the decision of almost
all parents to maintain secrecy with their offspring
[78]. Golombok et al. compared natural, adopted in
infancy, donor sperm and IVF children up to the age
of 12 years and found that parents who conceived
without difficulty had higher levels of parenting stress
than the other groups [79]. No significant differences
were found between types of families in the childrens
behavior, adjustment at home or school or feelings
toward their mothers or fathers. In assessing the quality of the parentchild relationship, Golombok also
found that IVF, DI and adoptive mothers had higher
levels of warmth and emotional involvement with their
children [79]. In vitro fertilization or DI fathers were
rated as displaying more warmth toward their children
than natural conception or adoptive fathers.
Overall, cancer survivors tend to view parenthood
in a positive manner and feel that their experience with
cancer would make them better parents [4]. Although
cancer survivors can become parents through options
such as adoption and third party reproduction [80],
most prefer to have genetic offspring [5, 81], even
if they have concerns about abnormalities that could
result if the parent conceived before cancer treatment
[82] or anxiety about their own longevity or their

childs risk of cancer [4]. Research is needed to address
the psychological sequelae of a cancer diagnosis and
treatment, and the contribution that fertility preservation may make on quality of life post-treatment.
More information available to oncologists, reproductive endocrinologists, urologists and MHPs working
with cancer patients, could be beneficial in facilitating the psychological adaptation and quality of life of
cancer survivors, and helping them make difficult and
critical decisions regarding future fertility and family
building.
Acknowledgements
Doctor Hsiao is supported by a grant from the Frederick J. and Theresa Dow Wallace Fund of the New York
Community Trust.
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Section 5
Chapter
18
Fertility preservation strategies in the female: medical/surgical
Use of GnRH agonists for prevention of

chemotherapy-induced gonadotoxicity
Susannah C. Copland and Megan Clowse
The use of gonadotropin-releasing hormone agonists

(GnRHa) for prevention of chemotherapy-induced
gonadotoxicity remains controversial [1, 2]. Ovarian
suppression to prevent oocyte loss during chemotherapy was proposed based upon the observation that
non-cycling cells appear more resistant to cytotoxicity
and that pre-pubertal girls resume menstruation after
cancer treatment more often then post-pubertal girls
[3]. Studies in rats and primates provide some support for the use of GnRHa for ovarian protection [4,
5]. Studies in humans that suggest benefit, however,
have been predominantly observational with historic
controls. The efficacy of GnRHa to protect the ovary
during chemotherapy has yet to be proven in adequate
randomized control trials; however, use of this treatment strategy with the goal of preventing premature
ovarian failure is becoming more common. The 2006
American Society of Clinical Oncologists consensus
statement on fertility preservation recommendations
for cancer patients emphasized the need for safety and
effectiveness data and recommended that patients considering the option of GnRHa enroll in clinical trials [6]. Several randomized trials are in progress, both
for patients utilizing gonadotoxic agents for cancer
and for rheumatologic conditions; however, the results
of these trials have yet to be published. This chapter
summarizes the current evidence and debate for and
against the use of GnRHa during chemotherapy.
Natural GnRH and ovarian

steroidogenesis
Natural human GnRH is released from the hypothalamus in a pulsatile fashion to stimulate gonadotropin
release from the pituitary gland. The gonadotropins,

follicle stimulating hormone (FSH) and luteinizing hormone (LH), dictate the ovulatory cycle and
consequent ovarian steroid and peptide hormone
production. The ovarian hormones provide feedback to the hypothalamus and pituitary to modulate gonadotropin release. The complex interplay of
hypothalamic pituitary ovarian signaling facilitates the
three distinct phases of the menstrual cycle: the follicular phase, during which the dominant follicle is
selected, estradiol levels rise and the endometrium
proliferates; ovulation, during which the dominant
follicle releases the oocyte and becomes the corpus
luteum; and the luteal phase, during which the corpus luteum produces estradiol and progesterone, the
endometrium becomes secretory to facilitate implantation of a potential pregnancy, until eventual menstruation with corpus luteum involution if pregnancy
does not occur (Figure 18.1).
Mechanism of GnRHa action

The GnRHa suppress the hypothalamicpituitary
ovarian axis, and thereby suppress ovarian ovulatory follicle development and resulting steroidogenesis. Endogenous GnRH is a 10 amino-acid peptide.
Sustained release synthetic GnRHa alter 1 or 2 of
the 10 amino acids facilitating greater binding affinity and slower degradation. The GnRHa bind to GnRH
receptors on the pituitary gland for longer periods
than natural human GnRH. After exposure to GnRHa,
the pituitary gonadotropes increase the GnRH receptor number, which increases production and release
of gonadotropins, a phenomenon commonly known
as the flare response. With prolonged gonadotrope
239
Section 5: Female fertility preservation: medical/surgical
Hypothalamus
Progesterone
GnRH
+
Pituitary
Estradiol
Inhibin
FSH
+
LH
+
Ovary
E2/P4
+
Uterus
Figure 18.1 Hypothalamic pituitary ovarian signaling. E2, estradiol;

FSH, follicle stimulating hormone; GnRH, gonadotropin-releasing
hormone; LH, luteinizing hormone; P4, progesterone.
exposure to GnRHa, the ligandreceptor complexes

are internalized and degraded; this results in a decrease
in GnRH receptor number, and concordant desensitization of the pituitary to further GnRH stimulation.
Therefore, after the initial release of FSH and LH, further gonadotropin release is prevented. Without pituitary gonadotropin stimulation, the ovulatory ovarian
cycle ceases, resulting in decreased estradiol and progesterone production to postmenopausal levels and
amenorrhea [7].
Clinical use of GnRHa

The flare response
With the initial dose of GnRHa, the pituitary gland
releases endogenous gonadotropins. This initial FSH
release, or flare, can stimulate the ovary. After continued GnRHa exposure, further FSH release is prevented. The amount of FSH released is greatest in the
first few days following the first dose of GnRHa. By
12 weeks, low gonadotropin levels are achieved. The
extent of the flare response to GnRHa depends upon
the time of administration with a patients menstrual
cycle. During the follicular phase of the menstrual
cycle, ovarian steroids are low providing little endogenous suppression of the pituitary and a larger flare
response. During the luteal phase of the menstrual
cycle, ovarian steroids produced by the corpus luteum
provide endogenous suppression of the pituitary and
240
decrease the extent of the flare. Women with polycystic

ovaries and increased ovarian stroma exhibit an exaggerated flare when compared to normal controls [8].
Theoretically, ovarian stimulation from the flare
response may make the ovary more vulnerable to
insult from chemotherapy. Indeed, chemotherapy
itself may stimulate the ovary. Letterie reported a
greater number of medium and large follicles in
cytoxan-exposed rats than control; ovarian suppression with GnRHa or oral contraceptive pills was not
protective [9]. To mitigate any potential harm from the
flare response, administration of GnRHa 2 weeks prior
to chemotherapy start has been proposed.
Others have proposed using a GnRH antagonist
(GnRHant) concordant with the initial dose of GnRHa
to decrease the intensity and duration of the flare.
The GnRHants competitively inhibit GnRH binding
to the pituitary GnRH receptors. A depot formulation is not approved for use in human; however, the
short-acting GnRHant can be administered every 3
days during the initial days of GnRHa-induced flare
to mitigate the gonadotropin release. Coadministration of GnRHant with GnRHa decreased the duration
of the flare response in Roth et al.s pilot study of children with precocious puberty or short stature [10]. For
the added expense of GnRHant to be worthwhile, the
decrease in flare response would need to add sufficient
benefit to outweigh increased cost and not add any
additional harm. Danforth et al.s report of GnRHant
depletion of primordial follicles in mouse raises concern for potential harm [11]. Where GnRHa significantly decreased the primordial follicle loss associated
with cytoxan administration and had no direct effect
on primordial follicle count in the absence of cytoxan,
GnRHant did not protect the ovary from cytoxan.
Indeed, both systemic and direct ovarian GnRHant
administration resulted in significant decreases in primordial follicle count even at doses insufficient to
decrease uterine weight, a surrogate for gonadotropin
suppression [11]. Danforth et al.s surprising report
of a negative effect of GnRHant on primordial follicle number is in direct contrast to Meirow et al.s
prior report of GnRHant protecting against cytoxaninduced follicular decline in mouse [12]. Meirow et al.
using a different strain of mouse and a different dose
and frequency of GnRHant, reported no direct effect
of GnRHant on follicle number compared to saline
and a protective effect of decreased primordial follicle loss when used concurrently with cytoxan [12].
These discrepant findings require further study to
Chapter 18: GnRH agonists for prevention of gonadotoxicity
delineate the effect of GnRHant on primordial follicles

with and without chemotherapy [13].
Dose
Gonadotropin-releasing hormone analogues can be
administered in many formulations with different
durations of action. In human studies, the dose of
GnRHa has been evaluated by the ability to suppress
ovarian and testicular steroid hormone production.
Whether this dose is sufficient for ovarian protection depends on the potential mechanism of protective effect. Some propose that GnRHa may protect
the ovary by inducing a pre-pubertal state. Whether
dose and formulation of GnRHa influences the level
of pituitary suppression at the end of the labeled depot
period has been examined in children using GnRHa
to treat central precocious puberty. With each repeat
dose there is the possibility of an increase in FSH
release if the pituitary suppression from downregulation has decreased. The rise in FSH and LH after repeat
dosing was higher with the lower dose, longer depot
duration formulations (Depotleuprolide 3.75 mg
1 month, 11.25 mg 3 months) than the higher dose,
shorter depot duration formulation tested (Depot
leuprolide 7.5 mg 1 month). While a greater magnitude of pituitary responsiveness was seen with the
lower dose, longer duration formulations, gonadal
steroid production and pubertal stage did not change
[14]. Mohamed et al. also documented a statistically
significant increase in pituitary response to GnRH
administration after 2 months of downregulation with
goserelin 3.6 mg subcutaneous pellet in 21 patients
with endometriosis [15]. Changes in ovarian steroids
were not reported; therefore, it is not known whether
the pituitary responsiveness at the end of the depot
period is clinically significant. Pending additional
studies with biochemical and clinical outcomes, the
relevance of these fluctuations of FSH at the time of
repeat dosing is unknown.
The influence of obesity upon GnRHa effectiveness has been questioned. Obese men using GnRHa
for treatment of prostate cancer exhibit higher testosterone levels than men with normal body mass indexes
[16]. The GnRHa active peptide is hydrophilic and
is therefore most influenced by the constant volume
of the extracellular fluid compartment. The synthetic
polymer linked to the GnRHa, however, is lipophilic.
Agarwal therefore postulated that adiposity may influence the rate of enzymatic breakdown of the lipophilic
polymer; more rapid breakdown would empty the

depot site sooner resulting in a decrease in gonadal
suppression before the labeled period [17]. Further
study is needed to determine whether obese patients
would benefit from higher doses or different formulations of GnRHa.
Side effects
The most common side effects of GnRH analogues are
related to the subsequent estrogen deprivation. Vasomotor symptoms, hot flushes, night sweats, vaginal
dryness and headaches can occur. With longer duration of administration and the subsequent estrogen
deprivation, bone loss can occur. Side effects can be
mitigated by hormone replacement. A combination
estrogenprogestin product such as a birth control pill
will protect bone and prevent vasomotor symptoms. If
estrogen exposure is contraindicated, a progestin-only
formulation can be of benefit.
GnRHa and ovarian reserve

The most established effect of GnRHa is reduction of
gonadal steroid production via cessation of the ovulatory ovarian cycle. The ovulatory cohort of oocytes,
however, has reached a point in development where
they will either ovulate or undergo degeneration [18].
Therefore, despite the established effect of GnRHa
to prevent the ovulatory cohort from ovulating, this
cohort of oocytes will still be lost to atresia. Dormant
primordial follicles are the largest contributor to the
total oocyte pool. For GnRH analogues to protect an
ovary from chemotherapy, they need to exert effects
beyond preventing ovulation of the ovulatory cohort
and protect total oocyte number or ovarian reserve.
Ovarian reserve reflects the number and quality of
oocytes remaining in the ovary. Women are born with
their total oocyte complement. Though women start
life with over 1 million oocytes, egg number declines
with age. Several hundred thousand remain at puberty.
Only 300400 will ever be released through ovulation;
the rest go through atresia.
Before puberty, oocyte number decreases via
atresia, but without the appropriate hypothalamic
pituitary signaling, there is no ovulation. After
puberty, hypothalamicpituitary signaling results
in pituitarygonadotropin stimulation to the ovary.
Each month a group of oocyte-containing follicles gain the ability to compete to ovulate. If the
hypothalamicpituitary signals are present, these
241
follicles will compete, the oocyte from the dominant

follicle will be released through ovulation, and the
rest of the group will undergo atresia [18]. Surgery,
chemotherapy and radiation cause loss in ovarian
reserve that is superimposed upon the loss due to
natural reproductive aging.
The studies of GnRHa during chemotherapy enumerated below have focused on menstrual outcomes.
While menses is a surrogate marker for ovarian function, menstrual function does not completely predict
the clinical outcomes of most interest: steroidogenesis and ovulation resulting in live birth. The average
age of menopause is 51 years; the average age of last
baby born in societies not using contraception is 41
years. Therefore there are 10 years of menses before
menopause that are not on average resulting in birth
[19]. Future studies need to include the clinical endpoints necessary for patient counseling including: live
birth, hormone production, including any changes to
bone density and age of eventual menopause.
Clinical data about GnRHa and

ovarian preservation
Cotherapy of a GnRHa during chemotherapy has been
under investigation since the mid 1990s. Observational studies, without a control group, show some
promising results (Table 18.1 [2026]). In each of these
studies the definition of maintained ovarian function
was resumption of menses in the months following
chemotherapy. Some studies also measured FSH and
estradiol; these were not measured on cycle day 3, but
randomly at the time of follow-up. For women undergoing multiple doses of chemotherapy with or without
radiation, the rate of menses return ranged from 80 to
97%. Several pregnancies were reported in these studies, but none reported the number of women who tried
unsuccessfully to conceive.
Age at the time of chemotherapy appears to be
an important determinant of the success of this therapy. In one study of women with early breast cancer,
97% of women under the age of 40 resumed menstruation compared to 42% over the age of 40. In addition,
menses were slower to return following chemotherapy
with advancing age [20].
The total dose of chemotherapy was also important. In a study of women with lupus nephritis, only
2 out of 25 women had ovarian failure, both were
over the age of 35 and received a second cycle of
cyclophosphamide for relapse [26]. In one small report
242
of 5 women, the only woman without a resumption of menses received a more aggressive course of
chemotherapy over 34 weeks for stage IVA Hodgkins
disease, compared to 1624 weeks for stage IIA or B
Hodgkins disease in the other women [25].
The marrow-ablative regimen required for stem
cell transplant resulted in ovarian failure in all
30 women despite prior treatment with leuprolide
3.75 mg [23]. Most of the women received busulfan
4 mg/kg for 4 days and cyclophosphamide 60 mg/kg
for 2 days; other women received variants that
included anti-thymocyte globulin (ATG) or melphalan. Only one of the women had menstrual bleeding during the time of thrombocytopenia following
chemotherapy. Hormonal status was followed over the
following year with FSH consistently elevated (median
35 mIU/ml, range 3080 mIU/ml) and estradiol low
(median 15 pg/ml, range 1025 pg/ml).
Bone density declined during GnRHa therapy in
several studies. In one study of 18 women, the hip
and spine T scores declined by an estimated 0.4 points
during the 6 months of cyclophosphamide, high dose
prednisone and leuprolide. In the following 6 months,
the BMD increased, though not back to baseline, in
the women who had a resumption of menses. It did
not increase in the women with premature ovarian
failure [21]. Another study showed both decreases
and increases in bone density during therapy with
some women with normal density developing osteopenia, several improving from osteoporosis or osteopenia, and many remaining unchanged [27]. Another
reported a significant decline within just 3 months of
goserelin therapy, with median T scores falling from
1.50 to 2.40. Bone density measurements following
the resumption of menses were not reported in this
study [20].
Some studies included add-back estrogen therapy to diminish the side effects of GnRHa therapy. In
the only study that reported side effects, women did
not receive add-back therapy and 97% had hot flashes,
90% headaches, sweating or mood changes, and 59%
reported vaginal dryness [20]. It is not clear how effectively estrogen therapy might relieve these symptoms.
Some women, particularly those with breast cancer
and lupus, are not candidates for estrogen therapy.
Studies with a control group

While much of the data from observational studies is
encouraging, it does not document the true benefit of
18
45
30
5
25
Perez Pampin et al. [21]

(Spain 2006)
Castelo-Branco et al. [22]

(Spain, 2009)
Chiusolo et al. [23] (Italy 1998)
Potolog-Nahari et al. [24]

(Israel, 2007)
Franke et al. [25] (the

Netherlands, 2005)
Dooley et al. [26] (USA, 2000)

23
40
92
80
89
89
83
72
96
21
24/25
15
97
Percentage with
ovarian function
27
No. with
ovarian function
Menses
Menses
Menses
Menses and FSH
Menses
Menses
Either menses or
FSH40
Menses
FSH 40
Definition of
ovarian function
Lupus nephritis: intravenous pulse CYC Leuprolide 3.75 mg

q 4 weeks
Hodgkin s disease: chemo and XRT

Goserelin 3.6 mg 12 weeks prior then q 4 weeks
3 Hodgkins, 1 non-Hodgkins, 3 B cell lymphoma, 1 breast

cancer, 1 Wegeners granulomatosis
Goserelin 3.6 mg 12 weeks prior then q 4 weeks
Stem cell transplant: 11 AML, 7 Hodgkins, 7 non-Hodgkins,

1 CML, 3 ALL, 1 MM
Leuprolide 3.75 mg 30 days prior to chemo then 1 dose
28 days after 1st dose
30 Hodgkins, 15 non-Hodgkins lymphoma: chemo and XRT

Triptorelin 3.75 mg 12 weeks prechemo then q 4 weeks
plus tibolone or OCP
Lupus: intravenous monthly pulses of CYC Lupron 3.75 mg
Early breast cancer Goserelin 3.6 mg 1 week prior, then

q 4 weeks
Disease, chemotherapy
ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; chemo, chemotherapy; CML, chronic myeloid leukemia; CYC, cyclophosphamide; FSH, follicle stimulating hormone;
MM, multiple myeloma; OCP, oral contraceptive pill; XRT, radiation therapy.
29
Del Mastro et al. [20] (Italy,

2006)
Author (Location of
study, year)
GnRHa
Table 18.1 Clinical data from studies of gonadotropin-releasing hormone agonist (GnRHa) co-therapy without a control group
GnRHa. To do this, randomized controlled trials of

this therapy are needed. At this time, two randomized
trials have been published, in addition to nine other
studies that compared women treated with GnRHa cotherapy to a control group. All but one of these studies
agree that GnRHa therapy increases the likelihood of
resuming menstruation following chemotherapy. We
completed a meta-analysis of the first 9 published studies and found that GnRHa co-therapy is associated
with a 68% increase in the rate of preserved ovarian
function [28]. This study excluded the two most recent
studies, both of which corroborate this finding [29, 30].
The first published study did not demonstrate benefit, with 4 of 8 women with GnRHa co-therapy resuming menses versus 6 of 9 women without this treatment
[31]. This study is the only one to use intranasal buserelin three times per day to maintain ovarian suppression. Unfortunately, this dosing was likely inadequate
to fully suppress the pituitaryovarian axis. A study
of intranasal buserelin for the therapy of breast cancer demonstrated that method of GnRHa therapy was
inadequate for full ovarian suppression [32].
All subsequent studies have shown that co-therapy
with GnRHa helps to retain ovarian function during
chemotherapy.
A study that was conducted in the early 1990s
but reported in 2009 found somewhat contradictory
results [30]. Women with breast cancer were treated
with cyclophosphamide, methotrexate, fluorouracil
(CMF) chemotherapy if lymph nodes were positive
and were divided into 4 groups for 2 years of endocrine
therapy: control, goserelin (3.6 mg q 4 weeks), tamoxifen (40 mg po qd) or goserelin plus tamoxifen. The
women who received goserelin alone during CMF
treatment resumed menstruation 36% (8/22) of the
time. This compares to 10% of the control group, 13%
of the tamoxifen group and 7% of the tamoxifen plus
goserelin group. Why the goserelin group had more
menstruation resumption than the combined tamoxifen plus goserelin group is unclear, but the authors
speculate that the estrogenic properties of the tamoxifen may have diminished the effects of the GnRHa.
It should also be noted that the first dose of goserelin
was given simultaneously with the first chemotherapy
dose, likely leading to a greater level of ovarian damage
as the chemotherapy affected the ovary during a time
of increased activity.
Two recent randomized trials have shown benefit
from GnRHa co-therapy. Badawy et al. randomized 80
women with breast cancer to co-therapy with gosere-
244
100%
80%
60%
40%
20%
0%
With GnRHa
No GnRHa
Figure 18.2 Percentage of women with a return of ovarian

function following chemotherapy. Each study is a different color.
GnRHa, gonadotropin-releasing hormone agonist. See plate section
for color version.
lin 3.6 mg every 4 weeks [29]. Of the women who

received the co-therapy, 90% resumed menstruation
and 69% resumed spontaneous ovulation 8 months
following chemotherapy, compared to 33% menstruating and 26% ovulating after chemotherapy alone.
Guiseppe Loverro and his team treated 29 women with
Hodgkins disease to either triptorelin or no co-therapy
[33]. All of the women with co-therapy resumed menstruation compared to 47% of those without it (Figure
18.2 and Table 18.2 [3439]).
Putative mechanisms of ovarian impact

The mechanisms by which GnRH analogues could
affect ovarian reserve are the subject of much debate.
Putative mechanisms include: changes to ovarian stromal blood flow; direct effect of GnRH; indirect effect
through suppression of FSH; and GnRH-dependent
immunological changes.
Ovarian stromal blood flow

If prolonged GnRHa administration decreases ovarian blood flow, then less chemotherapy may reach
the ovary. Existing studies of ovarian blood flow after
pituitary downregulation, however, report conflicting
results. In Engmann et al.s study of 99 women undergoing pituitary down regulation for in vitro fertilization (IVF), ovarian stromal artery peak systolic velocity significantly decreased after 23 weeks of daily
subcutaneous buserelin [40]. Dada et al. also showed
decreases in utero-ovarian blood flow after pituitary
downregulation with both subcutaneous buserelin
and intranasal nafarelin [41]. In contrast, Jarvela et
al.s 2003 study of 40 women undergoing pituitary
Table 18.2 Clinical data from controlled studies of gonadotropin-releasing hormone agonist (GnRHa) co-therapy
GnRHa
Author (Location of
study, year)
No. with ovarian

function
75
70
No GnRHa
Percentage
with ovarian
function
No. with ovarian

function
Percentage with
ovarian function
93
82
38
46
100
44
30
27
90
26
23
100
83
14
14
100
15
47
100
17
11
65
Somers et al. [38]

(USA, 2005)
20
19
20
14
70
Waxman et al. [31]

(UK, 1987)
50
67
Pereya et al. [39]

(Argentina, 2001)
12
12
100
Sverrisdottir et al. [30]

(Iceland, 2009)
22
36
72
10
Badawy et al. [29]

(Egypt, 2009)
39
35
89
39
18
33
Blumenfeld and Eckman [34]

(Israel, 2005)
Blumenfeld et al. [35]
(Israel, 2000)
Castelo-Branco et al. [22]
(Spain, 2007)
Dann et al. [36]
Israel, 2005)
Loverro et al. [33]
(Italy, 2007)
Petri et al. [37]
(USA, 2004)
downregulation for IVF found no difference in threedimensional power Doppler vascularity color indices
after 2 weeks of daily subcutaneous buserelin [42].
Yu Ng et al.s report of 85 women undergoing luteal
pituitary downregulation for IVF after a median of
10 days of intranasal buserelin also found no difference in three-dimensional power Doppler flow indices
[43]. The goal of GnRHa administration in IVF is to
prevent ovulation without overly suppressing ovarian
response. In the context of these IVF studies using
lower daily GnRHa dosing, GnRHa have not been
reproducibly proven to decrease ovarian blood flow;
the potential effect of higher doses of GnRHa from
depot formulations on ovarian stromal blood flow is
as yet unknown.
Direct and indirect effects of GnRH or FSH

Direct effects of GnRHa or FSH on ovarian tissue may
influence ovarian response to chemotherapy. Direct
95%
effects would assume presence of receptors upon the

primordial follicle, which has not yet been described.
Indirect effects via changes in detoxifying enzymes or
immunological responses would not require receptor
presence upon primordial follicles, and have therefore
also been proposed.
GnRH expression in the ovary

In Choi et al.s immunohistochemical study of premenopausal ovaries, GnRH-I, GnRH-II and GnRH
receptor were identified in the granulosa cell layer
of pre-ovulatory follicles, the corpus luteum and the
ovarian surface epithelium, but not the primordial to
early antral follicles which represent the largest portion of the ovarian reserve [44]. The absence of GnRH
receptors on the primordial follicles makes a direct
GnRHa effect at the primordial follicle level less likely.
Pre-treatment of granulosa cell cultures from
pre-ovulatory follicles with the GnRHa buserelin
245
prior to doxorubicin exposure preserved subsequent

granulosa cell estradiol production in response to
FSH-containing media. In the absence of GnRHa
pre-treatment, doxorubicin induced a concentrationdependent inhibition of estradiol secretion. Similar
results were seen with leuprolide. The granulosa cells
for the study were obtained, however, from aspiration of pre-ovulatory follicles where GnRH receptors
have been identified. This evidence may not reflect the
behavior of granulosa cells from primordial and preantral follicles, which lack GnRH receptors and are the
majority of the follicle pool [45].
Emons et al.s phase II clinical trial of the GnRHant
cetrorelix in the treatment of plantinum-resistant
ovarian or Mullerian cancer, reviews three signaling
mechanisms by which GnRHa and GnRHant may
exhibit dose-dependent inhibition in the majority of
ovarian and endometrial cancers [46]. The ligandbound GnRH receptor couples to G-protein i activating: (1) inhibition of mitogenic signaling, with reduced
epidermal growth factor (EGF) -induced cell proliferation; (2) nuclear factor kappa B anti-apoptotic mechanisms; (3) cell cycle arrest in G0 /G1 phase via activation of Jun kinase pathway and induction of AP1. These inhibitory effects of GnRHa and GnRHant
upon cancer cell proliferation raise concern for prevention of intended chemotherapy-induced apoptosis
and unintentional protection of cancer cells if GnRHa
or GnRHant are co-administered with the chemotherapeutic regimen [46]. Data from the LH-releasing
hormone (LHRH) -agonists in the Early Breast Cancer Overview group is, however, reassuring. In Cuzick et al.s meta-analysis of premenopausal women
with hormone-receptor positive breast cancer enrolled
in randomized controlled trials including a GnRHa,
addition of GnRHa to tamoxifen, chemotherapy or
both (n = 3754) reduced the hazard rate for recurrence by 12.7% (95% CI 2.421.9) and for death after
recurrence by 15.1% (95% CI 1.826.7) [47]. The addition of both GnRHa and tamoxifen to chemotherapy (n = 1210) reduced the hazard rate for recurrence by 26.7% (95% CI 12.338.7) and for death after
recurrence by 24.4% (95% CI 6.439.0) [47]. This survival benefit with addition of GnRHa to chemotherapy in hormone-receptor positive breast cancer suggests that, even if the described inhibitory effects of
GnRHa upon cancer cell proliferation are involved,
the survival benefits in the context of hormonereponsive breast cancer outweigh the theoretical
risks.
246
FSH expression in the ovary

An indirect effect of GnRHa on the ovary via suppression of FSH has also been discussed. While FSH
is clearly involved in cyclic recruitment of the ovulatory cohort, a role for FSH in the dormant cohort
of primordial follicles has yet to be shown. Oktay
et al. performed real-time polymerase chain reaction
(RT-PCR) for FSH-receptor mRNA on ovarian biopsies from 11 women at cesarian: none of the primordial follicles; 33% of the primary and 2 layer follicles; and 100% of multilayer follicle expressed FSH
receptor mRNA [48]. Patsoula et al. identified FSH
receptor mRNA in surplus oocytes from IVF [49].
Pre-ovulatory oocytes collected during IVF, however,
cannot be assumed to reflect the expression of primordial follicles [49].
Indirect effects of FSH suppression

While a direct effect of FSH on primordial follicles
is less likely given their absence of FSH receptors, an
FSH-mediated effect in the ovary is still plausible. Toft
et al. documented a FSH effect on detoxifying enzymes
glutathione transferase in the rat liver and ovary [50].
While the glutathione transferase isoenzymes expression vary in response to rat sexual maturation and
exogenous gonadotropin administration, clinical outcomes were not assessed [50]. Oktay et al. have postulated that changes detoxifying enzymes theoretically
may cause harm to the ovary if the gonadotoxicity of
chemotherapy were subsequently increased [51].
For GnRHa to be of benefit to fertility preservation,
they would likely need to spare both oocyte quantity
and quality. Familiari et al. evaluated follicle number
and structure in ovarian biopsies from women with
Hodgkins disease before multidrug chemotherapy
or depot-medroxyprogesterone acetate (depot-MPA),
before chemotherapy with depot-MPA and after 45
menstrual cycles after chemotherapy with depot-MPA
ovarian suppression [52]. While coadministration of
depot-MPA appeared to spare follicle numbers when
compared with numbers reported from prior studies,
a higher proportion of follicles from biopsies obtained
after chemotherapy were undergoing atresia [52]. The
long-term significance of this increased proportion of
atretic follicles, and whether ovarian suppression with
GnRHa would yield similar ultrastructural findings,
requires further study with longer follow-up of clinical outcomes.
Immunological effect of GnRHa

Whether the immunological effects of GnRHa could
influence the gonadotoxic effect of chemotherapy has
yet to receive much attention. Umathe et al. report
that GnRHa prevents stress-induced immunosuppression in mice [53]. Leuprolide subcutaneously administered 30 min prior to restraint stress prevented
the decrease in thymus weight, leukocyte count and
humoral and cell-mediated immune response markers
seen in controls. Prior intracerebroventricular administration of GnRHant and castration did not change
results. Therefore, this immunological effect appears
peripheral, independent of the hypothalamic effect
[53]. In their report of GnRHa use prior to stem
cell transplant, Sutherland et al. reported that goserelin 3 weeks prior to and after hematopoietic stem
cell transplantation (HSCT) increased neutrophil and
lymphocyte numbers, enhanced T-cell regeneration,
and increased disease-free survival without exacerbating graft-versus-host disease [54]. Clearly a peripheral
effect of GnRHa on the immune system may influence
the effect of chemotherapy both on the primary goal
of cancer treatment, but also on any putative fertility
preservation outcomes.
Hypothetical mechanisms of
ovarian protection
Blumenfeld hypothesized additional potential mechanisms for GnRHa to protect ovarian function: via
decrease in secretion of growth factors by suppression of FSH-dependent follicular turnover; potential
upregulation of sphingosine-1-phosphate, an antagonist of the proapoptotic second messenger ceramide;
or by protection of undifferentiated germ-line stem
cells should they be proven to exist in humans
[2]. Whether these putative mechanisms require further study will be predicated upon whether GnRHa
are proven to protect not only the resumption
of menses, but also the retention of true ovarian
function with both sustained cyclical steroidogenesis and live birth in those women desiring future
parenthood.
Given the many and varied influences on different
organ systems attributed to GnRH both centrally and
peripherally, robust evaluation of a role for GnRHa in
fertility preservation during chemotherapy awaits adequately powered randomized controlled trials powered for the true outcomes of interest: live birth, time
to menopause and survival. The data amassed so far

supports further investigation, but the lack of randomized data on the effect of GnRHa on long-term ovarian
function and survival outcomes leaves concern regarding potential harm.
While the role of GnRHa in fertility preservation
during chemotherapy is being clarified in randomized controlled trials, women facing gonadotoxic treatment should also receive counsel regarding the more
mature fertility preservation options already proven to
result in live birth. These include the well-established
embryo cryopreservation, as well as the newer and still
investigational oocyte and ovarian tissue cryopreservation.
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249
Section 5
Chapter
19
Ovarian transposition
Carrie A. Smith, Erin Rohde and Giuseppe Del Priore
The relocation of ovaries for their protection in women

diagnosed with cancer in the pelvis was mentioned as
early as 1958 by McCall et al. [1, 2]. At that time, the
procedure was termed oophoropexy and considered to
be revolutionary, controversial and cutting edge fertility preservation. Now, over 50 years later, this procedure has another, perhaps more accurate name, of
ovarian transposition. Paradoxically, it is still considered controversial as the next generation of physicians
becomes experienced in its benefits and limitations.
Although the discussion of ovarian transposition
has not changed much in the last 50 years, womens
reproductive behavior has, making transposition a
more important topic. This is because women are, on
average waiting longer to have their first child, desiring subsequent children at later ages, and are becoming more aware of their available fertility options [3
5]. Although cancers of the pelvic region still remain
rare in women of reproductive age, their incidence
increases with age, thus leaving women with delayed
first pregnancy more vulnerable to the possible loss of
fertility.
The increase in the number of potentially affected
women can be estimated based on cancer incidence
and fecundity. For instance, even if the general incidence of primary pelvic malignancies is approximately
1/10 000, then a delay from age 24 to age 25 in the
median age of first conception may expose a significant
number of women to a cancer diagnosis before first
planned or desired pregnancy. Conservatively, there
are at least 10 million reproductive age women in the
USA with a fecundity rate of 20% or more. Using different underlying assumptions will affect the number
in a predictable manner. Ranges of high and low estimates can thus be assumed for planning and policy
issues. Regardless, even the most optimistic assumptions will result in a predicted yearly increase in the
Table 19.1 Suggested eligibility criteria for ovarian

transposition
r Age 40 years
r Cervical cancer 3 cm in diameter
r Cervical cancer confined to the cervix
r No evidence of lymph-vascular space invasion
r No evidence of lower uterine segment involvement
r Early stage (IA) well-differentiated (grade 1) endometrial
cancer
r Any malignancy that requires pelvic radiation therapy with
or without hysterectomy
number of women at risk, all potential candidates for

fertility preservation and, if indicated, ovarian transposition (Table 19.1).
In addition to the change in womens reproductive behavior, their reproductive expectations have
changed as well. Shover et al. found that 75% of women
with cancer expressed a desire to have children in the
future, a majority of whom continued to desire children even if they were to die young [5]. This statistic
has been reproduced and reported by multiple investigators in a variety of disease states. Although estimates
vary considerably mostly due to study design and questionnaire nuances, the results can never be interpreted
as only affecting an insignificant minority. More often,
any conclusion that minimizes the importance of fertility preservation, and therefore a therapeutic intervention such as ovarian transposition, actually reflects
an investigator bias [6].
This is demonstrated clearly in a series of three
papers reporting potentially affected cervical cancer
patients over a span of 20 years in the literature. All
three investigator groups defined the potential number of patients interested in fertility preservation based
solely on age. In the first paper, reported in 1990
by Maddux et al., the fertility interested group was
defined strictly by age 25 [7]. Using this cut off, only
250
Chapter 19: Ovarian transposition
2% of cancer patients would be potential ovarian transposition candidates. A few years later, Mariani et al.
used age 35 which increased the potential number
of affected patients to 10% [8]. Finally, a New York city
group reported in 2004 that more than 40% of their
patients would be interested in fertility preservation
[9]! This group used age 40 as the cut off, reflecting a
significant change in definition and perspective from
the earliest study. Obviously none of these studies had
an objective basis for their methods. Instead the arbitrary cut offs were based on contemporary and fluid
definitions of reproductive age. Because of the uncertain estimates of the size and definition of the affected
population, all patients should be counseled regarding fertility preservation, including the use of ovarian
transposition.
Ovarian function is compromised when damaged during surgery, exposed to radiation, and/or
chemotherapy. Studies have shown that the amount
of radiation to eliminate 50% of a young womans
primordial follicles is 24 Gy, with 20 Gy of radiation exposure resulting in complete and permanent
ovarian failure [1014]. Pelvic radiation to treat gynecological cancers can be as much as 70 Gy planned
prescribed dose to the ovarian fossa. Given the lack of
precision inherent in radiation planning versus delivery, the actual received dose that the ovaries absorb can
be significantly higher.
In addition, after decades of a relatively stable
delivery system using linear accelerators, radiation
delivery has developed many more options designed to
improve cancer care, increase cures and decrease toxicity [1012, 14]. However there is little to no information on the affect of these modalities on in situ or
transposed ovaries. The general impression and hope
is that there will be less toxicity; however, caution must
be used in counseling patients treated with these latest
modalities that transposition is or is not indicated until
data become available.
Chemotherapy has been found to have a highly
variable chance of acute ovarian failure [14, 15].
There is also a highly variable increased long-term
chance of premature ovarian failure [16]. As discussed
below, the variation in reported chemotherapy affects
is due largely to study design and outcomes. Ovarian transposition done to preserve ovarian function
may paradoxically increase the susceptibility to subsequent toxic exposure. This is only a theoretical concern
based on the reports of ovarian failure following other
pelvic surgeries, especially hysterectomies. Disruption
of normal ovarian vasculature through hysterectomy is

associated with changes in serum estradiol and follicle
stimulating hormone (FSH) levels in retained ovaries.
It is possible that ovarian transposition could result in
a thrombosed or otherwise compromised ovarian vascular blood supply. This could, again theoretically, render the ovaries more vulnerable to the toxic effects of
chemotherapy.
It is important to note the limitation on assessing ovarian function and reserve when considering
ovarian transposition. There is no universally accepted
metric to determine current ovarian function or future
potential. Immediate assessments including estradiol,
follicle counts, FSH and similar measures correlate
poorly with reproductive, i.e. fertility, potential. Often,
resumption of menses is used as a very crude assessment of ovarian function. In addition to the poor
correlation between menstruation and fertility potential, this data is often reported at arbitrary followup intervals such as 6 months. When counseling a
patient regarding transposition, the limitation of the
data should be addressed to avoid unrealistic expectations.
Once the decision to perform ovarian transposition has been reached, the surgical method must be
considered [17]. There have been several proposed
methods, including open laparotomy or minimally
invasive. If technical reasons such as body size and
shape allow, minimally invasive approaches are preferred. Each is relatively simple with documented
pregnancies after the transposition.
Another alternative often not considered today is
a whole ovary, vascular anastomosis to an extraperitoneal site [18]. The technique is often referred to as
a transplant or a heterotopic autotransplantation
procedure because it shares characteristics of other
common organ transplantation such as the kidney
transplants. In fact, it can be thought of as a transposition with important differences. The addition of
a vascular anastomosis is not a significant difference.
The anastomosis is an easy routine procedure for transplant, vascular and plastic surgeons. What is significantly different is the microenvironment of the alternative locations. It is possible that the relative zero
gravity of the abdominal peritoneal cavity is an essential, or at least an important, parameter in normal
ovarian function.
Alternative transposition locations including the
upper extremity [18] have not been shown to be successful by resulting in pregnancy, but have shown
251
Figure 19.1 Radiation field. See plate section for color version.
return of ovarian function, including growth of

follicles. Limited function and utility have been
reported only as isolated cases. Alternative sites may
be considered as part of a research protocol. Ovarian transposition to the upper abdominal cavity
using superior epigastric vessels or internal mammary
should be investigated carefully.
Currently the relocation of the ovaries with an
intact and uninterrupted gonadal vascular bundle to
another intraperitoneal location is the only practical
option [12, 13, 17]. This can be accomplished using
minimally invasive (robotic, single site or conventional laparoscopy). The goal is the same, to remove
the ovaries and typically fallopian tube, to outside the
pelvis (Figure 19.1). Typical radiation borders include
the ovarian fossa and extend beyond the true pelvis.
For this reason, there is no absolutely perfect location for every disease and radiation treatment plan.
Surgeons should consult with the requesting radia-
252
tion therapist to understand the target tissue and dose.

Together, surgeon and radiotherapist can select the
best location [19].
In general, for gynecological malignancies, cervical and uterine cancers are the most likely indications for adjuvant or definitive radiation treatment
to the pelvis, but pelvic radiation is also done for
Hodgkins lymphoma, pediatric sarcomas and rectal
cancer [11]. Vulva cancer has limited indications for
whole pelvic radiation and disease is relatively rare
in reproductive age women. Ovary cancer is very
unlikely to require radiation therapy while still being
an indication for transposition. For a unilateral, welldifferentiated, radiation-sensitive dysgerminoma, it is
theoretically possible to consider transposition of the
contralateral ovary but highly improbable.
For the typical cervical and endometrial cancer
diagnoses, the treatment fields follow closely the lymphatic drainage of the primary tumor. For instance,
since obturator lymph nodes are often involved with,

or at risk for, metastatic cancer, the radiation field
will include the obturator fossa and extend laterally
approximately 2 cm (Figure 19.1). The target area then
moves more medially and cephalad. This superior field
area allows the para-colic gutter to be a relative sanctuary for the ovary [1921]. Because of the relatively
common use of pelvic radiation after endometrial or
cervical cancer, ovarian transposition may be considered for all these patients at the time of the definitive
cancer operation.
The ascending and descending colon make up the
medial border of the acceptable location for the transposed ovary. The inferior border is defined by the anterior iliac crest. There is no lateral or cephaled limit to
where the ovaries can be relocated to. This direction is
only limited by the moblization of the gonadal vessels,
i.e. the infundibulopelvic ligament [22]. There may be
an advantage to moving the ovaries as far as possible
from the target tissue to minimize scatter. Moving the
ovaries as far as possible may also be an advantage in
preventing the migration of the transposed ovary back
into the field.
Possibly due to the effect of gravity and lax unsubstantial points of fixation, a significant number of
ovaries are found to be back in the radiation field
on postoperative imaging [1921]. Fixation on the
abdominal wall requires incorporation of more than
just the peritoneum. The underlying fascia and muscle
should be part of the fixation when using suture or any
other method. The ovarian bundle is more limited in
its choices for fixation. Too aggressive a purchase may
occlude vascular supple. Too flimsy a purchase may
slip and become loose.
It is important to attach radio-opaque staples to
the ovaries or perform an MRI to detect this potential movement [23, 24]. There is no question that rigorous multiple sutures may be more likely to retain the
ovaries in the intended location. However, obviously,
the more substantial the sutures the greater the risk
for ovarian ischemia from occluded or kinked blood
vessels. This is a similar concern regarding the tension
used to move the ovary ever further laterally or cephalad. Tension on the vascular pedicle may lead to compromise of ovarian function [20, 21, 25].
It is hard to estimate a true incidence of ovarian
failure after transposition because of the small numbers in the case series reported [20, 26, 27]. Other limitations include the variable case ascertainment used.
Too often menses at 6 months or some other arbi-
Figure 19.2 Surgical anatomy. See plate section for color version.
trary definition of success is used. However, the consensus estimate is that a substantial minority of transposed ovaries will cease to function after transposition.
Techniques have been devised to minimize this complication.
For instance, a report indicated that nearly all ovarian function can be preserved, at least in the short
term, using a retro-peritoneal approach. Briefly in this
technique, the ovary and its distal blood supply are
brought retroperitoneal superior and lateral to the distal incised utero-ovarian ligament. A window is made
in the paracolic gutter peritoneum and the ovary gently pulled through this opening (Figure 19.2).
This technique theoretically improves outcomes by
preventing ovarian peritoneal inclusion cyst as the
ovary retains its normal relationship with the peritoneal surface [21, 28]. In addition, this technique
avoids the creation of a potential internal hernia by
a taunt gonadal pedicle. When the ovary is directly
attached to the abdominal wall, it should be done so
with as little tension as possible on the gonadal vascular bundle. Ideally it should lie flat against the side
wall without tension. There have been no direct comparisons of the two techniques.
How the ovary is attached to the lateral abdominal wall varies by operator preference [22, 24, 25].
Suture has an advantage of being able to precisely place
253
Figure 19.3 Intensity modulated

radiation therapy (IMRT). See plate section
for color version.
the purchase on both the ovary and the abdominal

wall. However, because of the relative difficulty of tying
and suturing laparoscopically, alternatives have also
been reported. These alternatives include hemostatic
clips and hernia-type staplers. The hernia staplers are
very secure and purchase the underlying fascia easily. The hemostatic clips require a secondary incision
be made to allow it to be slipped under the fascia. Any of the above mentioned techniques can be
performed using either open or minimally invasive
techniques.
No location is entirely safe from the internal scatter radiation dose that occurs during all conventional
treatments. There is a theoretical reduction in this
scatter dose using enhanced delivery modalities such
as intensity modulation (Figure 19.3). However, there
have been no reliable reports on ovarian function after
these advances in radiation therapy.
There have been reports of complications after
transposition [28]. These include the usual postoperative issues of infection, bleeding and hernias. Especially disappointing complications include ovarian
failure supposedly due to ovarian vein thrombosis and
torsion. Reoperation for peritoneal or ovarian cyst is
more common than in the general population.
There have also been reports of spontaneous conception after transposition including the intact Fallopian tube [28, 29]. In certain patients, the Fallopian
tube can be stretched sufficiently to allow the con-
254
nection to the uterus to be retained. In most cases, a

tension-free transposition requires transaction of the
utero-ovarian ligament and transaction of the medial
Fallopian tube.
In some of the cases of spontaneous conception,
the ovaries and tubes have been documented to have
returned to their original natural position after completion of the planned radiation therapy. However,
pregnancies have been reported while still in the transposed location. There are even reports of planned
temporary relocation of the ovaries in hopes of promoting spontaneous conception [23, 29]. It must be
remembered that most women is whom transposition
is indicated, either the uterus is removed or it is radiated often destroying the endometrium. The reports of
spontaneous pregnancy usually involve patients with
medial transposition behind the uterus, with central
shielding and lateral radiation therapy.
Ovarian function is almost guaranteed to be
entirely lost without some intervention before pelvic
radiation therapy. Ovarian transposition is a relatively simple option that should be considered with
all patients at risk for ovarian failure due to radiation.
Unfortunately it is not possible to guarantee preservation of ovarian function or future fertility using any
technique [30]. Alternative continue to be developed
including ovary cryopreservation and other cuttingedge options [3134]. Until then, transposition is a
reasonable option.
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22. Mitchell JD, Hitchen C and Vlachaki MT. Role of

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of craniospinal irradiation on the ovaries: a case
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23. Kung FT, Chen HC, Huang CC, Ho JT and Cheng
BH. Preservation of ovarian germinal follicles by
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failure following abdominal irradiation in childhood.
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24. Huang KG, Lee CL, Tsai CS, Han CM and Hwang LL.
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11. Wo J and Viseanathan A. Impact of radiotherapy on

fertility, pregnancy, and neonatal outcomes in female
cancer patients. Int J Radiat Oncol Biol Phys 2009;
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25. Morice P, Thiam-Ba R, Castaigne D et al. Fertility

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26. Van Eijkeren MA, Van Der Wijk I, Al Sharouni SY

et al. Benefits and side effects of lateral ovarian
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28. Terenziani M, Piva L, Meazza C et al. Oophoropexy: a

relevant role in preservation of ovarian function after
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Section 5
Chapter
20
Fertility-saving surgery for cervical cancer

P. Mathevet and A. Ciobanu
Introduction
Patients with early cervical cancer (FIGO stage IB1
or less) are conventionally considered treated with a
surgical approach while those with more advanced
disease are treated by radiotherapy with concurrent
chemotherapy. Surgical treatment usually involves a
radical hysterectomy with pelvic lymphadenectomy
except for those with early microscopic disease (FIGO
stage IA1) where conisation or extrafascial hysterectomy lymph-node dissection would be sufficient.
With the good implementation of cervical screening
programs in most developed countries, more women
are presenting with early cervical invasive disease. For
these women, the long-term prognosis is good, with
survival of over 90%. With good long-term survival,
preservation of fertility potential and quality of life
become more important issues and have been the focus
of recent surgical advances in the treatment of cervical
cancer.
For FIGO stage IA1 without lymph-vascular space
involvement, cold-knife conisation with a complete
lesion resection is recommended as a fertilitypreserving treatment, since the rate of parametrium
and lymph-node involvement is negligible. For larger
tumors, or in case of lymph-vascular space involvement, due to risk of lymphatic extension, resection of
the pelvic lymph nodes and of the proximal part of
the parametrium is recommended. For these patients,
preserving the fertility was a challenging option until
recently.
The recent development in surgical oncological
technologies aims at improving their effectiveness
while decreasing their morbidity. Therefore, the endoscopic and in particular laparoscopic techniques were
introduced in surgical oncology. With these concepts
of optimization of the treatments and reduction of
their consequences, the gynecologists also proposed

fertility-sparing techniques for young women with
cancer and a desire to preserve reproductive function.
Twenty years ago it was completely impossible to have
a child after treatment for cervical cancer from an
oncological and surgical point of view. This context
emphasizes the originality of the step of Professor Dargent, when he created and carried out the first surgical operation aiming at preserving the fertility among
young women presenting with early cancer of the uterine cervix.
While the first intervention was carried out at
the end of the year 1986, the first publications were
made in 1994 [1], allowing us to verify the absence
of oncological risk associated with this modern surgical operation. Thus, in the 1990s, Dargents operation began to be distributed worldwide, with the help
of British, Canadian and American surgical teams in
particular. These different teams [27] have confirmed
our experience of the radical trachelectomy and and
explored the opportunities to achieve a pregnancy and
also the risk of relapse after this type of operation.
Currently, more than 500 women have benefited
from this intervention with relatively reduced morbidity worldwide and a perfectly controlled risk of
relapse. More importantly, they have had the possibility of achieving a pregnancy, and having healthy children, following conservative radical treatment of early
cervical cancer.
Dargents operation is the realization of a laparoscopic pelvic lymph-node dissection associated with
a radical cervical amputation through a vaginal
approach [1, 8]. This radical resection includes a
total amputation of the uterine cervix associated with
the vaginal cuff along with the proximal part of
the parametrium. Concerning the uterine cervix, the
257
resection is comparable to that which can be obtained

from a Wertheims operation type Piver II. However,
the concept of radical cervical amputation is not a
totally new concept since some authors had already
proposed it, for instance, by Aburel and Nowak in the
1940s and 1950s. But no one achieved pregnancy after
the completion of this type of intervention that had
been carried out through an abdominal approach. It
was therefore the development of laparoscopy (which
limits adhesions and reduces the risk of infertility
associated to the radical trachelectomy) which helped
obtain pregnancies and live births following surgical
treatment for early cervical cancer.
Operative technique
In our team, the operation always begins with a laparoscopic pelvic lymph-node dissection, which includes
sentinel node identification. This technique of the
sentinel node identification discovers, in a significant proportion of cases (1520%), lymphatic drainage
channels with unusual locations of sentinel nodes.
Identification of the sentinel node allows the realization of frozen sections, their result being of paramount
importance for the next surgical step. Indeed, if the
frozen-sections evaluation is positive, the planned radical trachelectomy is cancelled and the patient receives
a full laparoscopic pelvic, common iliac and paraaortic lymph-node dissection followed by radiotherapy and chemotherapy. Detection of the sentinel node
is performed with a dual technique of isotopic colloid

particle (Nanocys ) and color (Patent Blue ) injection in the cervix (in the four quadrants) (Figure 20.1),
and then the sentinel nodes are identified through
laparoscopy (Figure 20.2). This identification is followed by a full pelvic lymph-node dissection performed from the obturator membrane until the iliac
bifurcation, with the removal of all external iliac lymph
nodes (below the vein and at the contact of the external iliac vein and artery). In the case of a tumor larger
than 2 cm in maximal diameter, the cellulo-adipose
tissue of the distal parametrium is dissected through
laparoscopy (corresponding to a lymph-node dissection of the parametrium).
The next step is that of the radical trachelectomy performed through the vagina [8]. The steps
of this operation reproduce those of the intervention
of SchautaStoeckel. Dargents operation includes the
following steps. The first step is the realization of a
vaginal cuff. It is rarely necessary to remove more than
R
258
Figure 20.1 Visualization of right pelvic

lymphatic channels

through laparoscopy after Patent Blue injection in the cervix. See
plate section for color version.
Figure 20.2 Dissection of a right pelvic sentinel node. See plate

12 cm of vaginal mucosa. Thus, a rim of vaginal tissue is delineated circumferentially using 68 Kocher
forceps (Figure 20.3). In order to reduce bleeding and
to help for the following dissections, 20 cc of xylocaine
plus epinephrine is injected at regular intervals in the
vaginal fold. A round incision of the vaginal wall is
made just above the Kocher forceps. Once the incision is fully made, the Kocher forceps are removed and
the anterior and posterior edges of the vaginal cuff are
grasped together with 46 Chroback clamps in order
to isolate the cervix and to prevent tumor spillage.
These clamps allow good traction onto the operative
specimen in order to help the following steps. Then
dissection of the vesico-vaginal septum is performed
Chapter 20: Fertility-saving surgery for cervical cancer
Figure 20.3 Performance of the vaginal cuff. See plate section for
color version.
Figure 20.4 Dissection of bladder pillars and identification of the

left ureter. See plate section for color version.
on the midline with scissors. The space is usually avascular and, when fully dissected, the bladder base is
mobilized with a vaginal retractor. The following step
is the opening of the para-vesical space. On the left
side, the opening is obtained while dissecting along
the vaginal wall at 3 oclock level. It should be noticed
that the upper part of this space has been dissected
through laparoscopy helping the vaginal approach. A
retractor is put into the para-vesical space, and palpation of the bladder pillar and the ureter is performed
between the surgeon finger and the retractor. The precise location of the ureter can easily be defined. And
so, the bladder pillars are excised after coagulation.
The goal of this dissection is to evidence the ureter
(Figure 20.4), and then to fully cut the bladder pillars
in order to free the bladder base and to move away
the lower part of the ureter from the operative field.
The same procedure is performed on the patients right
side. The following steps are then performed on the
posterior aspect of the operative specimen. It first consists of opening the Douglas pouch. This opening is
performed with scissors on the midline, as for a simple vaginal hysterectomy. Then a vaginal retractor is
put in place in order to evidence the utero-sacral ligaments. These ligaments are coagulated and divided
leading to opening of the para-rectal space and identification of the posterior aspect of the parametrium.
Then the next steps are modifications of the ones performed during the Schauta operation. Indeed, the division of the parametrium is carried out while preserving the uterine artery. With retractors in place in the
para-vesical and para-rectal spaces, the anterior and
posterior aspects of the parametrium and the ureter
are evidenced. Then the parametrium is divided while

preserving the arch of the uterine artery. The division of the parametrium can be made by clamping
and ligation or with the use of electrosurgery devices.
The ureter should be fully dissected and left under the
view while performing the parametrium division. This
procedure is performed on both sides. The following
step is the ligation of the cervico-vaginal vessels with
preservation of the uterine artery (Figure 20.5). Then
the uterine cervix is cut with a cold knife at the level of
the lower part of the isthmus (Figures 20.6 and 20.7).
The operative specimen includes the entire cervix until
the isthmus, the proximal part of the parametrium
about 2 cm in length and the vaginal cuff (Figure 20.8).
Frozen sections are performed onto the upper margin
of the operative specimen in order to be sure that the
section is in healthy zone. If this margin is not clear, the
radical trachelectomy is transformed into a Schauta
operation. Concerning the operative specimen, a final
pathological analysis will be performed following the
same procedure as the one recommended for cone
specimens.
The Douglas pouch is then closed with a pursestring absorbable suture. In the case of future pregnancy, a permanent cerclage is set around the uterine isthmus. A non-absorbable polyethylene tape is
usually used. The cerclage is made following the Benson technique with the knot lying posteriorly (Figure
20.9). Then the vaginal anastomosis is carried out by
two Sturmdorff sutures and two angle sutures with
absorbable sutures.
In the postoperative period, a bladder catheter is
left in place for 4 days. On the 4th postoperative
259
Figure 20.6 Section of the operative specimen at the level of the

uterine isthmus. See plate section for color version.
Figure 20.5 Drawing of the ligation of cervico-vaginal vessels.
Figure 20.8 Operative specimen of a radical trachelectomy

showing the vaginal cuff and the proximal parametrial resection.
Figure 20.7 Drawing of the section of the operative specimen at

the level of the uterine isthmus.
260
Figure 20.9 Set up of the isthmic cerclage. See plate section for
color version.
day, the ablation of the catheter is performed after

checking the bladder function, allowing the patients
discharge.
Usually, we advise patients to wait 2 years before
considering a pregnancy (as the majority of relapses
occur within 2 years). However, in the event of lesion of
good prognosis and/or patients approaching the limit
of childbearing age, this time can be shortened to 6
months.
Our experience
In our department, from December 1986 to December 2008, Dargents operation was proposed to 171
patients. For 11 of these patients, the final intervention was changed either because of a lymph-node invasion (6 cases) or because of isthmic involvement by
the tumor (5 cases). Thus, 160 radical trachelectomies
were carried out. The average age of the patients was
31.5 years. The proportion of nuliparous patients was
56%.
The initial stages of the tumors were: stage IA1
or (+ lymph vascular space invasion) IA2 in 38 cases
(24%); and stage IB1 in 122 patients (76%). The histological types were: squamous in 123 cases (77%), glandular in 35 cases (22%) and other types (of which 1
was neuroendocrine) in 2 cases (1%). The maximum
tumoral size was 2 cm in 130 cases (81%) and 2 cm
in 30 cases (19%). The presence of lymph vascular
space invasion (LVSI) was found in 51 cases (32%).
The mean operating time was 192 min (including
the associated laparoscopic pelvic lymph-node dissection). The only intraoperative complications observed
(2 cases, 1%) were a ureteral laceration and a bladder breach, which were sutured without further damage. The average hospital stay was 4.6 days. During
the postoperative course a notable complication was
observed in 14 patients (19%). Nine (6%) patients presented a postoperative bleeding requiring a surgical
revision. Two pelvic lymphocysts required a surgical
marsupialization. Two abscesses (pelvic and parietal)
were drained. A case of uretero-vaginal fistula required
a ureteral anastomosis without sequellas. It should be
noted that the rate of complication was significant,
especially in the first-operated cases because of the
experimental character of the surgical technique. Concerning the last 70 patients who went through surgery,
no operative complication was observed and the postoperative course was marked by 3 (2%) of the 14 complications previously described.
In nine patients, adjuvant radiotherapy was recommended either because of a lymph-nodal or parametrial tumor involvement at the final histological examination (seven cases) or because of a massive LVSI
(two cases). The average follow-up of the patients is 92
months. To this date, 8 (5%) relapses were observed,
leading to death in 6 cases. A neuroendocrine case of
cervical cancer presented 2.5 years after surgical treatment with distant metastases leading to the death of
the patient. Four nodal relapses were observed (located
in the latero-pelvic, common iliac and para-aortic
areas). Despite the treatment of these recurrences
(surgery and radiotherapy), three of these patients
died. A patient with a stage IB1 adenocarcinoma presented a central-pelvic relapse on the uterine isthmus. She was operated on and irradiated and is currently doing well. Two patients presented relapses in
the parametrium and unfortunately could not be saved
despite loco-regional treatments.
The prognostic factors of the relapses were evaluated. After exclusion of the case of neuroendocrine
cancer, which represents an inadequate indication of
radical trachelectomy, the only statistically significant
factor for relapse is tumoral size. Thus, in the event of
maximum tumoral size 2 cm, 6 relapses (20%) were
observed against 1 relapse (1%) when the size was
2 cm (P 0.05). The presence of LVSI is associated
with an increased risk of recurrence, but the difference
is not statistically significant: 5 relapses (10%) in the
event of the presence of LVSI, against 2 relapses (3%)
in the absence of LVSI (P = NS). The histological type
has no effect on the risk of recurrence.
The last update of the pregnancies obtained after
radical trachelectomy was carried out in 2005. At that
time, we had observed the birth of 49 live healthy children following radical trachelectomy performed in our
department. All the childbirths were carried out by
cesarian section. The evaluation of the fertility after
Dargents operation made it possible to find infertility in relation to the operation (primarily by isthmic
stenosis) in 16% of the cases. The rate of late miscarriage is relatively important in our practice since it is
15% of all the pregnancies. However, this rate of late
miscarriage was lowered by the introduction of various procedures: the use of isthmic cerclage, which
made it possible to halve the rate of miscarriage, and
then the closing of the uterine cervix according to the
Saling procedure, which made it possible to reduce
the rate of late miscarriage in our experience from 21
to 12%.
261
So, the results of our experience demonstrate that

the radical trachelectomy is a valid response in the
treatment of young women affected by an early cervical
cancer and wishing to preserve their fertility. Dargents
operation does not appear to increase the risk of cancer
relapse and allows, in a significant proportion of cases,
pregnancies and, especially, live births. Our experience
has been also confirmed by data from other teams in
the world.
Experience worldwide [27,

9, 10]
Radical trachelectomy has been performed by numerous surgical teams across the world, and currently
more than 500 patients have benefited from this operation. Recently, a survey of the experience of most
teams practicing this intervention throughout the
world was presented. Thus, in total, 557 patients (mean
age = 31.5 4.4 years) have benefited from radical
trachelectomy. The stages of the cervical cancers were:
stage IA in 34% and stage IB1 in 76% of patients. The
observed recurrence rate was 28 (5%) of cases. In 3
cases the recurrence was only cervical intraepithelial
neoplasia (CIN) or vaginal intraepithelial neoplasia
(VaIN) and therefore 25 (4.5%) women had an invasive recurrence. Twelve (2.1%) women died from this
recurrence and 4 others are living with progressive disease.
Concerning pregnancies after radical trachelectomy, the data are based on 445 patients for which
the information was available. The late miscarriage rate
is 15% (22 cases identified) and 125 live healthy children were born by cesarian section. Thus, the results
of other teams confirm the validity of the technique
in terms of oncological risk, possibility of pregnancies
and live births after completion of a radical trachelectomy.
Discussion
Dargents operation or radical trachelectomy was
invented to enable preservation of fertility among
women with early cervical cancer. The current followup of the first cases over 20 years confirms the hopes of
Professor Dargent: women benefiting from this operation have an excellent opportunity to have a healthy living child after this treatment without increased oncological risk [8]. The benefits of Dargents operation are
linked to the laparoscopic approach, which reduces the
262
risk of adhesions on pelvis organs and the vaginal route

that allows the preservation of uterine body and its
optimal vascularization [6]. The limits of this procedure are mainly related to the vaginal approach, which
may be difficult for some surgeons not trained in this
approach.
Also, following the excellent results achieved by
the Dargents operation, several teams have proposed
operating variants through laparotomy [1116] or
laparoscopy [17, 18]. These modifications are subject
to criticism because they are usually associated with
the section of the uterine arteries and, thus, with a partial devascularization of the uterus. In addition, the
laparotomy approach is associated with an increased
risk of adhesions. Data from the literature also concur that the rate of pregnancy after radical trachelectomy by laparotomy or laparoscopy is lower than by
the vaginal approach [19]. Currently, we believe that
only the original operation described by Daniel Dargent must be promoted in order to preserve the fertility in young women affected by an early cervical
cancer.
The experience of pioneer teams around the world
helps to refine indications of this operation. Preferential indications are represented by cervical cancer stage
IA2 and IB1 of 2 cm maximal diameter. Unusual histological types must be excluded from this treatment.
Moreover, lesions extending high in the endocervix are
a contraindication to this operation, which may be too
close to the upper limit of the tumor when performing
the isthmic section. It is therefore necessary that the
patient planned for a radical trachelectomy must have
a preoperative exploration with colposcopy and pelvic
MRI to exclude significant extension of the lesions in
the parametrium or the endocervix [8]. In particular,
MRI should define the extent of the lesion in relation
with the isthmus, considering that at least 810 mm of
normal cervix above the lesion should be obtained at
surgery.
Currently, we have enough data to say that Dargents operation is a valid surgical alternative from an
oncological and obstetrical point of view, but many
questions remain unanswered:
1. Should a closing hysterectomy be performed after
the patient has obtained the number of children she
wanted? Only the risk of centro-pelvic recurrence
in long-term studies allows us to answer this
question. Our experience enables us to estimate
that this risk is very low, even negligible. In
addition, the realization of a hysterectomy after

radical cervical amputation is not an easy
operation. We believe today that there is no need
to consider a closing hysterectomy among these
patients. However, this data should be modulated
according to the histological type of the initial
lesion. Indeed, it is known that cervical
adenocarcinomas sometimes have a multifocal
character which could potentially foster a possible
isthmic recurrence in these patients.
2. What are the best treatment options in order to
increase the patients chances of obtaining a term
pregnancy after a radical trachelectomy? Indeed,
after a Dargents operation the rate of late
miscarriage as well as premature birth is
significant. Two main mechanisms are involved in
the increased risk of those obstetrical
complications: mechanical incompetence of the
cervix and subclinical membrane infection linked
to the significant reduction in the amount of
cervical mucus. Isthmic competence can be
improved by making a definitive isthmic cerclage
performed at the same time as the trachelectomy.
Concerning the infectious factor, we propose
closure of the cervix following the Saling
technique during pregnancy. This surgical
procedure allows us to isolate the membranes of
the vaginal bacteria and reduces the risk of
membrane contamination and chorio-amniotitis.
Some authors also advocate an antibiotic
treatment throughout the pregnancy [20].
However, the evaluation of the benefits of these
different therapeutic approaches is practically
difficult. Besides, prophylactic approach by
monitoring the residual cervical length by
ultrasound has not yet received appropriate
studies. Also, time off work and bed rest may be
important as the uterus gets heavier.
3. Is the radical trachelectomy too large for small
cervical cancers 2 cm in diameter? I.e. can we
achieve a less radical treatment in the case of very
early cervical cancer: simple amputation of the
uterine cervix or even a large conization? Its a
recurring question in the support of early cervical
cancers. But data from the literature are very poor
in this area. Attempts to reduce surgical morbidity
in the 1970s by Professor Burghardt failed due to
an important and dangerous recurrence rate.
However, radiological exams and histological
evaluation techniques have progressed and we
currently better understand in which cases you

can reduce surgical aggressiveness: invasive tumor
of 2 cm in diameter without the presence of
LVSI. Recent works have been presented in this
area by a Czech team [21, 22] and experiments
have been started in the USA. However, to
demonstrate equivalence in terms of oncological
risk with less extensive surgery compared to
radical surgery, statistically the numbers of
patients must be very important and therefore
make this type of study hardly feasible.
4. Can we consider the realization of conservative
treatment for stage IB tumors larger than 2 cm? At
present, this indication is not accepted by most
teams that practice the radical trachelectomy.
However, initially, a significant proportion of
patients were performed on for tumors of this
size, either because of the intense desire of the
patient or because of inadequate performance of
the MRI. More accurate analysis of women with
tumors between 2 and 4 cm and treated with
radical trachelectomy shows a substantial
recurrence rate (to the order of 15%) but
comparable to that achieved with the performance
of Wertheims operation in a similar group of
patients with cervical cancer. So we do not create a
major oncological risk by performing the radical
trachelectomy in this particular group of patients
having a tumor 24 cm in diameter. However,
several teams are working on experiments of
neoadjuvant treatments in order to reduce tumor
size, and therefore to be able to perform
conservative surgery in patients with stage IB
cervical cancer larger than 2 cm. These
preliminary experiments using neoadjuvant
chemotherapy (usually a polychemotherapy with

Taxotere , Holoxan and Cisplatine ) lead to
excellent response rates and a reduced risk of
recurrence (Figures 20.10 and 20.11) [23].
Moreover, these patients have a good recovery of
ovarian function and their fertility seems
preserved, with already isolated cases of
pregnancy described [24, 25]. This approach is
interesting and probably will lead to therapeutic
developments in the future.
R
Conclusion
Dargents operation was developed during a period
of reduction in aggressive operative techniques in the
263
References
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La trachelectomie elargie: une alternative a`
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2. Plante M, Renaud MC, Hoskins IA et al. Vaginal
radical trachelectomy: a valuable fertility-preserving
option in the management of early-stage cervical
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literature. Gynecol Oncol 2005; 98: 310.
Figure 20.10 Pre-treatment epidermoid cervical carcinoma stage

IB2 (45 mm) in a 25-year-old young woman, neoadjuvant
chemotherapy is planned. See plate section for color version.
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trachelectomy a safe alternative to radical
hysterectomy for patients with stage IAB carcinoma
of the cervix? Cancer 1999; 86: 22739.
4. Sonoda Y, Abu-Rustum NR, Gemignani ML et al. A
fertility-sparing alternative to radical hysterectomy:
how many patients may be eligible? Gynecol Oncol
2004; 95(3): 5348.
5. Schlaerth JB, Spirtos NM and Schlaerth AC.
Radical trachelectomy and pelvic lymphadenectomy
with uterine preservation in the treatment of cervical
cancer. Am J Obstet Gynecol 2003; 188: 29
34.
6. Klemm P, Tozzi R, Kohler C, Hertel H and Schneider
A. Does radical trachelectomy influence uterine blood
supply? Gynecol Oncol 2005; 96: 2836.
7. Burnett AF, Roman LD, OMeara AT and Morrow
CP. Radical vaginal trachelectomy and pelvic
lymphadenectomy for preservation of fertility in early
cervical carcinoma. Gynecol Oncol 2003; 88:
41923.
Figure 20.11 Same patient after four courses of neoadjuvant

chemotherapy: complete regression of the lesion. See plate section
for color version.
field of gynecological cancers. This operation allows

the preservation of the fertility of young women
affected by an early cervical cancer. The worldwide
experience of this surgical technique, and the fact that
this technique has become recognized, allows us to
confirm that after a radical trachelectomy, pregnancies
are possible and there is no increase of oncological risk.
The current indications of Dargents operation are well
defined: invasive cervical cancer 2 cm in maximal
diameter; usual histological types of cervical cancer;
and a young patient wishing to preserve her fertility
[26]. In the future, new indications will be developed
in association with neoadjuvant chemotherapy.
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8. Dargent D, Ansquer Y, Arnould P and Mathevet P.

Laparoscopic vaginal trachelectomy: a treatment to
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trachelectomy. Am J Obstet Gynecol 2003; 189:
137882.
10. Boss E, van Golde R, Beerendonk C et al. Pregnancy
after radical trachelectomy: a real option. Gynecol
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11. Smith JR, Boyle DC, Corless DJ et al. Abdominal
radical trachelectomy: a new surgical technique for the
conservative management of cervical carcinoma. Br J
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12. Rodriguez M, Guimares O, Rose PG. Radical
abdominal trachelectomy and pelvic
lymphadenectomy with uterine conservation and
subsequent pregnancy in the treatment of early
invasive cervical cancer. Am J Obstet Gynecol 2001;

185: 3704.
13. Ungar L, Palfelvi L, Hogg R et al. Abdominal radical
trachelectomy: fertility-preserving options for women
with early cervical cancer. Br J Obstet Gynecol 2005;
112: 3669.
14. Abu-Rustum NR, Sonoda Y, Black D et al.
Fertility-sparing radical abdominal trachelectomy
for cervical carcinoma: technique and review of
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20. Roman LD. Pregnancy after radical vaginal

trachelectomy: maybe not such a risky undertaking
after all. Gynecol Oncol 2005; 98: 12.
21. Rob L, Charvat M, Robova H et al. Less radical
fertility-sparing surgery than radical trachelectomy in
early cervical cancer. Int J Gynecol Cancer 2007; 17:
30410.
22. Rob L, Pluta M, Strnad P et al. A less radical
treatment option to the fertility-sparing radical
trachelectomy in patients with stage I cervical cancer.
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trachelectomy: two new surgical techniques for the
conservation of uterine arteries. Int J Gynecol Cancer
2006; 16: 1698704.
23. Plante M, Lau S, Brydon L et al. Neoadjuvant

chemotherapy followed by vaginal radical
trachelectomy in bulky stage IB1 cervical cancer: case
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abdominal radical trachelectomy. Gynecol Oncol 2008;
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24. Kobayashi Y, Akiyama F and Hasumi K. A

case of successful pregnancy after treatment
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17. Lee CL, Huang KG, Yen CF and Lai CH. Laparoscopic
radical trachelectomy for stage IB1 cervical cancer. J
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18. Cibula D, Ungar L, Palfalvi L, Bitio B and Kuzcl D.
Laparoscopic abdominal radical trachelectomy.
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Rob L. High-dose density chemotherapy
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Gynecol Oncol 2008; 111: S10510.
265
Section 5
Chapter
21
Results of conservative management of

ovarian malignant tumors
Philippe Morice, Catherine Uzan and Sebastien Gouy
Introduction
Conservative and functional surgery is increasingly
used in surgical oncology. Its aim is to preserve organ
functionality and to limit radical resections. The development of new surgical procedures in oncologic gynecological surgery is a perfect example of this evolution.
Although radical surgery remains the gold standard
for the treatment of ovarian and cervical cancer, a conservative approach can be considered in patients with
early stage disease, in order to preserve their ovarian
function and fertility. These procedures are proposed
to selected patients, depending on the histological subtypes and prognostic factors. Ovarian cancers are classified as epithelial (including borderline and malignant
tumors) and non-epithelial cancers.
Borderline ovarian tumors

The definition of borderline ovarian tumors (BOT)
is based on the histological characteristics of the
ovarian tumor and not on peritoneal implants. Four
characteristics are used to define BOT: (1) epithelial
proliferation with the formation of a papillary configuration; (2) a definable demonstration of atypical
epithelial activity; (3) mild or moderate nuclear atypicality (these three characteristics are essential to differentiate BOT from ovarian cystadenoma); and (4) the
absence of stromal invasion (which distinguishes BOT
from invasive carcinoma) [1, 2]. Peritoneal implants
are associated with BOT in 1040% of cases. They are
either non-invasive in 80% of cases (without stromal
invasion) or invasive in 20% of cases [38]. A noninvasive implant was defined as a glandular or papillary proliferation, devoid of stromal invasion. Non-
invasive peritoneal implants can be subdivided into

two types, the epithelial type (with predominantly
epithelial components) and the desmoplastic type (in
which the epithelial components are embedded in a
predominantly inflamed, desmoplastic stroma). Invasive implants are defined as a proliferation in the peritoneum with stromal invasion [3, 4, 6]. If biopsies
or resection of peritoneal implants are too superficial, the degree of invasion cannot be accurately determined, and such implants should be considered as
non-specified implants. In order to avoid this drawback, large biopsies or resection of peritoneal implants
should be performed during the surgical procedure
[8]. The pathological examination is therefore crucial
to confirm: (a) the diagnosis of BOT (and peritoneal
implants); (b) to determine prognostic factors; and (c)
to decide upon the optimal treatment. In order to carry
out adequate sampling, at least one section per cm of
the greatest dimension of the ovarian tumor and the
totality of the peritoneal implants should be examined
[9].
In patients with non-invasive implants, complete
surgical cytoreduction of peritoneal lesions is the only
treatment likely to improve survival. The prognosis of
patients with non-invasive implants is good [8]. However, when peritoneal implants are invasive, lesions
evolve into more aggressive disease in one third of the
cases [5, 6, 8]. In such cases, adjuvant therapy should
be discussed.
A new entity designated micropapillary serous
carcinoma (MP) associating peritoneal implants with
a borderline tumor was described 10 years ago, in
order to identify a subgroup of patients with a poor
prognosis. Tumors with an MP pattern are more commonly associated with invasive implants. In that study,
266
Chapter 21: Conservative management of ovarian tumors
the presence of an MP pattern was an adverse prognostic factor [9]. However, Eichhorn et al. reported that
the evolution of patients with non-invasive implants
associated with an MP pattern was similar to that of
patients with non-invasive implants without an MP
pattern [10]. The real prognostic impact of this entity
continues to fuel debate.
Modalities of conservative surgery and

clinical outcomes
The standard treatment of BOT consisted of a
total abdominal hysterectomy and bilateral salpingooophorectomy, peritoneal cytology, omentectomy and
multiple peritoneal biopsies. These procedures allowed
us to perform adequate staging and to eventually propose adjuvant therapy exclusively to patients with
invasive peritoneal implants. The prognosis of BOT
is excellent. However, late recurrences (after 5 or 10
years) may occur [11]. Conservative surgery is defined
as preservation of the uterus and at least a part of one
ovary, in order to preserve fertility. BOT arise in a
young population, where fertility is a major issue. The
analysis of conservative management data is crucial
in such patients. An analysis of the literature regarding the conservative management of BOT is difficult,
because most of the series are retrospective and the
duration of follow-up is too short (5 years) to accurately evaluate the exact recurrence rate. Furthermore,
the percentage of patients who were adequately staged
varied among the series and depended on the treatment center. These variable numbers could account
for the differences in recurrence rates. Three recent
reviews were published concerning the results of conservative surgery in such tumors [1214]. The risk of
relapse, estimated at between 0 and 25%, is increased
after this type of surgery [1214]. It is also greater
after a cystectomy (between 12 and 58% of cases) [12
14]. Some of these recurrences were observed long
after treatment of the initial BOT (latest recurrences
were 72 months in the series reported by Gotlieb et al.
and 240 months in our series) [15, 16]. It is therefore
highly likely that some of them are in fact new primary tumors, and not real recurrences of the initial
BOT.
Lim-Tan published one of the first series on conservative surgery in BOT [17]. He reported on 35 patients
(33 with stage I disease) treated conservatively [17].
In order to decrease the risk of recurrence after a cystectomy, Lim-Tan recommended a complete patho-
logic analysis of the margins to rule out any microscopic invasion [17]. Nevertheless, in the recent series
reported by Papadimetriou, among 18 patients who
underwent a cystectomy with histologically free margins, 7 relapsed [18].
In order to reduce the rate of relapse on the remaining ovary, some authors propose initial complete staging surgery with routine biopsies of the spared ovary.
In our series, we performed this procedure in 14
patients with a macroscopically normal ovary. We
found no tumor implants. Like Bostwick and Tazelaar, we observed only one relapse on the macroscopically normal ovary, that was routinely biopsied [19,
20]. It is important to note that this is not a harmless procedure, as it may induce infertility because of
postoperative ovarian adhesions. Macroscopic inspection appears to be sufficient. Biopsies should be performed only in case of macroscopically suspicious
lesions.
The high recurrence rate implies that the optimal
treatment in patients with an intraoperative diagnosis
of BOT, is a unilateral adnexectomy, which reduces the
risk of relapse. A cystectomy should be performed only
in cases of bilateral tumors and/or in patients with only
one ovary (a previous history of adnexectomy). In case
of a recurrent BOT on the remaining ovary, further
conservative management (cystectomy) may be proposed to these patients, in order to preserve fertility.
In our series, eight patients with recurrent BOT were
reoperated on conservatively for their recurrent disease [16]. Six pregnancies were obtained in five of these
patients [16, 21]. In the series by Gotlieb et al., three
cases of conservative management of recurrent BOT
were reported [15]. Those 3 patients were alive after 6
months, 2 years and 7 years of follow-up [15].
Survival of patients after

conservative surgery
Does this increased risk of relapse affect the survival
of patients? Zanetta et al. reported on a series of 189
patients who underwent fertility-sparing surgery [22].
Seven cases of progression into invasive ovarian carcinoma were observed, one of them very shortly (9
months) after the initial treatment of the BOT [22].
Six of these patients were alive after treatment of their
recurrence [22]. In the recent series reported on by
Park et al. among 164 conservatively treated patients
with a median follow-up of 70 months, 9 recurrences
were observed (with only one invasive recurrence in
267
the lung and pericardium, 82 months after the initial

treatment) [23]. This latter patient died of her recurrent disease [23].
In the literature, among 1500 cases of conservative surgery in BOT, nearly 10 cases of patients
treated conservatively for early stage disease developed
a recurrence in the form of invasive carcinoma [24].
Invasive recurrent disease is therefore a rare event
in patients with early stage disease. Most recurrent
lesions were BOT with an excellent prognosis. Such
major data explain why this increased recurrence rate
following conservative surgery finally had no impact
on survival [15, 2123, 2528]. Conservative surgery
can therefore be safely performed in young patients
treated for BOT and carefully followed up.
Nevertheless, it is not scientifically possible to
affirm that there is no potential oncological risk after
conservative management of BOT. Several series compared outcomes and survival after conservative and
radical treatment of BOT [15, 2123, 2528]. However, such a comparison is very difficult because, in
most cases, patients treated radically had more adverse
prognostic factors (particularly advanced stage disease). Furthermore, if there is a potential risk for survival, it must be very small and only series including
a very large number of patients treated conservatively
with prolonged survival could explore such an important question. Only two large series of conservative
treatment have actually been published: the Zanetta
et al. series mentioned above and the recent series by
Park et al. involving 189 and 164 cases [22, 23]. This
high number of patients is nevertheless not sufficient
to address the issue of a difference in survival between
radical and conservative treatment of BOT.
have been published. These two series concluded that

conservative management may be proposed to patients
with peritoneal implants, providing these implants are
entirely removed, with a reliable pathologic interpretation [22, 32]. Zanetta et al. reported on 12 patients
with non-invasive implants treated conservatively: 3 of
them developed a recurrent BOT in the spared ovary
and 1 had progression to invasive ovarian carcinoma,
but all 3 were salvaged with surgery and chemotherapy. All these patients were free of disease when this
paper was written [22]. We recently reported on a
series of 41 patients treated conservatively for a BOT
with peritoneal implants [32]. Twenty-two recurrences
occurred most of which were non-invasive. Nonetheless, one patient died of this recurrence [32]. The risk
of lethal outcomes is therefore rare when serous BOT
with non-invasive peritoneal implants are treated conservatively. Complete resection of implants is crucial
in this context.
Yet is it possible to propose this surgical management to patients with invasive peritoneal implants?
Zanetta et al. reported on seven patients with invasive implants treated conservatively [22]. Five BOT
recurrences were observed in the spared ovary, but
all patients were salvaged with surgery and were alive
[22]. In our experience, we performed this treatment
in three patients with invasive implants, one of whom
had progressive peritoneal disease [32]. In the series
reported by Prat and De Nictolis the only patient
treated conservatively for BOT with invasive implants
died of recurrent disease [29]. Considering the aggressiveness and the poor prognosis of BOT with invasive
peritoneal implants, it seems judicious to propose conservative therapy exclusively to patients with BOT and
non-invasive implants [32].
Limits of conservative surgery
Histological subtypes of the ovarian tumor
Conservative management could be safely offered to

most patients with early stage disease. However, in
which subgroup of patients does such a procedure constitute a potential (small) oncological risk? It is very
difficult to answer to this question because we have no
specific data on this topic. Nevertheless, three different
potential limits should be considered:
When we examined in detail the data of patients with

early stage disease who developed a recurrence in the
form of adenocarcinoma, the majority of them develop
a mucinous tumor. Four out of five patients who died
(of recurrent disease) after conservative management
of early stage disease had a mucinous tumor [2225].
This could be explained by the fact that most of these
tumors were large and the histological analysis of such
bulky tumors is difficult.
Disease stage
Data in the literature concerning conservative management of BOT with peritoneal implants are rare
(Table 21.1) [22, 2932]. To date, only two large series
268
Presence of a serous tumor with an MP pattern

The last potential limit concerns the presence of a
serous tumor with an MP pattern. Only one series
Table 21.1 Literature review of recurrence rates in advanced stage serous ovarian borderline tumor (in particular with invasive implants)
after conservative treatment
No. of invasive
implants
Death in patients
with invasive
implants
No.
Zanetta et al. [22]
25
10
Prat and De Nictolis [29]
10
Longacre et al. [30]
21
De Iaco et al. [31]
21a
Uzan et al. [32]
41
22
Total
97
40 (41.2%)
2 (2%)
11
No. of
deaths
Recurrence in
patients with
invasive implants
No. of
recurrences
Mixed histology (mucinous, serous tumor).
was published on the conservative management of

BOT with MP features. It included 15 cases (8 stage
I and 7 stage III disease) [33]. Eleven recurrences
were observed: six of them exclusively on the ovary,
three exclusively on the peritoneum (invasive peritoneal disease in one) and two on the ovary and peritoneum. One of the last two patients succumbed to
the recurrence (in the form of invasive adenocarcinoma). The other patients were actually disease free.
Five patients achieved eight spontaneous pregnancies
[33]. This recurrence rate might appear to be high but
should be correlated with the high rate of bilateral
ovarian involvement (2/3 patients) and with the fact
that half of the patients had peritoneal implants. To
date, while awaiting further publications on this topic,
there is no indication for systematic radical treatment
in patients with an MP pattern (except in cases with
invasive peritoneal implants).
Fertility results after conservative surgery

Pregnancies have been reported in patients with
conservatively treated BOT. Lim-Tan et al. initially
reported on eight patients who conceived [17]. Eight
series (involving 10 patients desiring pregnancy)
specifically reported the obstetrical results of conservative treatment [15, 16, 22, 23, 27, 28, 34, 35]. The
rate of spontaneous pregnancies ranged between 30
and 80%.
Nevertheless, in spite of conservative management
in BOT, some of these patients will experience infertility. Can ovarian stimulation or in vitro fertiliza-
tion (IVF) be proposed to these patients, given that a

number of studies incriminate hyperstimulation in the
onset of BOT and ovarian cancer? In vitro data suggest
that gonadotropins and/or a high dose of estrogens do
not induce the proliferation of cell cultures from BOT
[36].
Clinical data in the literature concerning this concrete case are rare (Table 21.2) [28, 32, 34, 3746]. The
largest multicentric experience in 30 patients previously treated conservatively for BOT and who underwent IVF procedures (or simple ovarian stimulation)
was reported by Fortin et al. [45]. Thirteen pregnancies were reported and four recurrences (all of them
in the form of borderline disease) [45]. It therefore
seems possible to propose hyperstimulation to patients
with stage I BOT without affecting the patients prognosis. However, we think that the number of stimulation cycles should be limited, in order not to increase
the potential risk of recurrence. Data in the literature
on the safety of hyperstimulation in patients with peritoneal implants exclusively concern case reports (Table
21.2). It therefore does not seem possible to propose
guidelines concerning hyperstimulation and IVF in
these patients, even though some successful cases have
been reported.
Bilateral salpingo-oophorectomy should be performed in patients with bilateral massive BOT and/or
a recurrent BOT on the remaining ovary, and in
whom preservation of a part of one ovary is unfeasible. Pregnancies have been reported in patients who
underwent a bilateral salpingo-oophorectomy (with
uterine preservation) for BOT, from donated oocytes
269
Table 21.2 Literature review of cases of ovarian induction or in vitro fertilization (IVF) procedures in patients with a previous history of a
borderline ovarian tumor
Series
Nijman et al. [37]
No. of
patients
Ovarian
induction (no.)
No. of
IVFs
Mantzavinos et al. [38]
Hershkovitz et al. [39]
Hoffman et al. [40]
Morris et al. [34]
Beiner et al. [41]
Attar et al. [42]
Fasouliotis et al. [43]

Fauvet et al. [28]
Marcickiewicz and
Brannstrom [44]
Fortin et al. [45]
No. of
pregnancies
No. of recurrences
(after stimulation)
2 (1 spontaneous
after IVF)
1 (III C
micropapillary)
Rapidly progressive
peritoneal disease
11
30
27
13
Park et al. [46]
1 (IIIC nodal)
Uzan et al. [32]
or the successful transfer of frozen embryos, obtained

before bilateral salpingo-oophorectomy [4749].
Cryoconservation of ovarian tissue could also be
proposed in such cases, but no pregnancies have been
obtained under such conditions [50].
Patient follow-up is based on a clinical examination
and abdomino-pelvic and vaginal ultrasonography. A
paper on this topic by Zanetta et al. failed to demonstrate the value of using blood markers in this context
but, pragmatically, we use them combined with two
other procedures [51].
Should we reoperate patients to remove the
remaining ovary when fertility is no longer an issue for
those who have conceived after conservative treatment
of a BOT? No standard practice exists in the literature
regarding this question. Although the recurrence rate
is between 0 and 25%, routine oophorectomy appears
to be useless in 75100% of cases [14]. Furthermore,
these recurrent lesions (mostly BOT) could easily be
cured, using a simple surgical procedure. In our institution, the systematic removal of the spared ovary is
thus not mandatory provided patients are followed up
regularly. Oophorectomy is then proposed exclusively
for relapses. However, some patients prefer to undergo
an oophorectomy after achieving a pregnancy, for psychological reasons.
270
No. of
stages II/III
Epithelial ovarian cancer

Indications for conservative surgery
The differential criterion between epithelial ovarian cancer (EOC) and BOT is the invasion of the
ovarian stroma. The standard surgical procedure for
EOC is a radical hysterectomy with bilateral salpingooophorectomy. The results concerning conservative
management of EOC are difficult to analyze in the literature, because many of the published series are either
mixed dealing with conservative treatment in epithelial and non-epithelial ovarian cancer, or including
invasive and borderline ovarian tumors and considering them as epithelial lesions. Some studies reported
on the results of conservative management but epithelial, borderline and non-epithelial tumors were all
included. Only a few studies (5 series [10 cases]) have
focused on conservative treatment exclusively in EOC
(Table 21.3) [5257]:
r Colombo in 1994 and Zanetta in 1997 published
the first series specifically devoted to EOC. These
series included 56 patients [52, 53].
r An American multicenter study (8 different
centers in USA) including 52 cases was reported
in 2002 [54].
Table 21.3 Literature review of results conservative management in epithelial ovarian cancer (5 series reported including 10 cases)
Stage IA
grade 1
Stage IA
grade 2
Stage IA
grade 3
Stage IC
grade 1
Stage IC
grade 2
Stage IC
grade 3
1 recurrence out
of 24 patients
3 recurrences
out of 8 patients
1 recurrence out
of 4 patients
No recurrence
out of 10
patients
1 recurrence out
of 6 patients
No recurrence
out of 3 patients
2 recurrences
out of 33
patients
2 recurrences
out of 6 patients
No recurrence
out of 3 patients
No recurrence
out of 5 patients
1 recurrence out
of 3 patients
No recurrence
out of 2 patients
1 recurrence out
of 13 patients
4 recurrences
out of 14
patients
1 recurrence out
of 3 patients
2 recurrences
out of 2 patients
No patient
1 recurrence out
of 1 patient
Park et al. [56]
1 recurrence out
of 29 patients
No recurrence
out of 3 patients
4 recurrences
out of 4 patients
1 recurrence out
of 15 patients
1 recurrence out
of 2 patients
2 recurrences
out of 2 patients
Anchezar et al.
[57]
1 recurrence out
of 10 patients
No patient
1 recurrence out
of 1 patienta
No recurrence
out of 3 patients
No recurrence
out of 1 patient
No recurrence
out of 1 patient
Total
6 recurrences
out of 109
patients
9 recurrences
out of 31
patients
7 recurrences
out of 15
patients
3 recurrences
out of 35
patients
8 recurrences
out of 12
patients
3 recurrences
out of 9
patients
Italian series
Zanetta et al.
/Colombo et al.
[52, 53]
American series
Schilder et al. [54]
French series
Morice et al. [55]
Patient considered as having a stage IA grade 3 tumor after pathological review of the initial tumor at the time of the recurrence.
r A French multicenter study was published on a

series of 34 patents with EOC with strict inclusion
criteria (a systematic review of slides, complete
staging surgery and chemotherapy for patients
with stage IC-55).
r The largest series was recently published by Park
et al. in 2008 with inclusion criteria very close to
those of the French series [56].
r A recent series from Argentina reporting 18
patients [57].
Initially, Di Saia proposed conservative treatment for
EOC, but with selected inclusion criteria (i.e. patients
who desire fertility; who are willing to undergo
close gynecological follow-up and a stage IA; wellencapsulated ovarian cancer without peritumor adhesions and without involvement of the ovarian surface;
and/or no mesovarium infiltration and negative peritoneal washings) [58]. The histological type plays a
major role among inclusion criteria. Thus, only serous,
mucinous and endometrioid EOC can be considered
for conservative management even if a recent Asian
paper suggested that such management could be proposed for clear cell tumors [59]. While awaiting further
studies concerning such histological subtypes, patients
with clear cell and anaplastic EOC should not be con-
sidered for conservative treatment, because of the high

risk of relapse on the remaining ovary.
The results reported in those four studies suggested
that conservative surgery could be safely performed in
patients with stage IA grade 1 (and probably grade 2)
disease (Table 21.3). In 15 patients with stage IA, grade
3 disease, 6 recurrences were observed (Table 21.3).
Consequently, conservative management should not
be performed in such cases.
Stage IC disease was the subject of heated debate
because of conflicting results in the five published
series (Table 21.3). Clarifying the criteria used to classify patients with stage IC disease in those different series could potentially explain the differences
between the series. In the 1988 FIGO classification,
patients are classified as having stage IC disease in case
of a uni- or bilateral tumor with: (a) tumor spread
on the surface of the ovary (excrescences); and/or (b)
ascites containing malignant cells or positive cytology after positive washing; and/or (c) capsular rupture during the morphologic analysis. Thus, patients
included with stage IC disease were probably dissimilar in terms of the criteria used to classify disease
as stage IC. Furthermore, the histological subtype
(mucinous, serous or other) was perhaps somewhat
different in those four series concerning this substage
271
of disease. Such fine differences could explain the

absence of homogeneity in the literature. If we take
into account the recent data from Park et al., conservative management could probably be considered in
stage IC, grade 1 disease but should not be performed
for grade 2 or 3 disease [56].
A recent paper using the analysis of the Surveillance, Epidemiology and End Results (SEER) database
suggests the absence of deleterious impact on survival of preserving the ovary in stage IA or IC disease [60]. As stated by the authors, to detect a 20%
difference in survival for patients with stage IC disease, a cohort of 1282 pts with 52 deaths is required
[60]. Thus, as none of the published series included
such a large number of patients, it is not possible to
draw definitive conclusions about the safety of conservative management in this setting. In the recent paper
from Schlaerth et al., the survival of 20 patients treated
conservatively for a stage I disease, is similar to the
survival of patients (45 years) treated radically [61].
Nevertheless, such comparison is somewhat inadequate because the prognostic factors, even if none of
them reach the level of the statistical significance, had
a trend to be better in the group of patients treated
conservatively [61]. Conservative surgery should not
be proposed to patients with disease exceeding stage I
[5557].
In patients with a limited indication for conservative surgery (stage IA grade 3 disease, stage IB or
IC grade 2 or 3 disease) an alternative option could be
considered: the removal of both ovaries but with uterine conservation (without uterine curettage at the time
of staging surgery) to preserve a possibility of fertility (oocyte donation or another procedure). This
option has never been explored in EOC but should be
evaluated. In the recent SEER database analysis, there
was no impact on survival of uterine preservation in
stage IA or IC disease [60].
The prognosis of patients with recurrent EOC after
conservative surgery remains poor, particularly when
recurrent disease arises outside the preserved ovary
[62].
Surgical procedure for conservative surgery

This conservative surgery should only be considered
after adequate surgical staging. This staging should
include peritoneal washings, excision of any suspicious
peritoneal lesions, multiple peritoneal biopsies, omentectomy and endometrial curettage. A pelvic and para-
272
aortic lymph node dissection is usually discussed in

early stage disease, particularly in the case of mucinous
tumors [63].
Munnell proposed a systematic biopsy of the
remaining ovary [64]. He also considered that contralateral microscopic involvement existed in 12% of
EOC [63]. However, systematic biopsies of contralateral ovarian cancer can induce infertility by provoking postoperative adhesions on the remaining ovary.
Moreover, many authors did not find any microscopic
implants in the macroscopically normal ovary [53, 55].
Yet Benjamin et al. found microscopic disease in the
contralateral ovary which was macroscopically normal in 3 patients (2.5%) in their series of 118 patients
with stage I EOC [65]. However, these 3 patients
had a grade 3 tumor and none of the patients with
stage I, grade 1 or 2 disease had occult metastasis
on the contralateral ovary [65]. Consequently, we do
not recommend routine biopsies of the contralateral
ovary if preoperative vaginal ultrasonography did not
reveal deep parenchymous abnormalities in the initially undiseased contralateral ovary and if it appears
to be macroscopically normal during the surgical
procedure.
Fertility results following conservative

treatment of EOC
Few fertility results are available in the literature.
Zanetta et al. obtained 27 pregnancies in 20 patients
[52]. In the American series, 17 pregnancies were
reported in 24 patients attempting to conceive [54]. In
the French series, only 9 pregnancies were achieved
and Park reported on 15 pregnancies in 19 patients
[55]. In the series reported by Anchezar et al., 7 pregnancies were reported in 6 of the 7 patients who
attempted to conceive [56]. In the case of persistent
infertility, ovarian stimulation or IVF continues to be
contraindicated.
Patient follow-up is based on a clinical examination, blood markers and the use of systematic imaging
(abdomino-pelvic ultrasonography).
Recourse to completion surgery after childbearing
(or after the age of 40 in patients who fail to become
pregnant) is still debated. However, a case of a recurrent EOC 10 years after conservative treatment, could
suggest discussing the removal of the remaining ovary,
in order to reduce the risk of recurrence on the spared
ovary.
?
?
37
No. conservative
management
Menstration
maintained
No. pregnancies
No. conservative
management,
stages II/III/IV
11
24/26
50
14
16
19/20
19
1e
43/45
74
74
Low et al.
2000 [70]
46
72
Tewari et al.
2000 [69]
46
55 in 32
patients
128/130
138
169
Zanetta
et al. 2001
[71]
Papers reporting exclusively on endodermal sinus tumor or non-dysgerminomatous tumors.

Paper reporting only on dysgerminomatous tumors.
c Menstruations considered as similar to those observed before chemotherapy.
d Five patients excluded from assessment of menstruations because of a lethal recurrence.
e Pregnant patient.
108
No. patients
26
129
Series
69
Brewer
Peccatori Mitchell
et al. 1995 et al.a 1999 et al.b
[67]
[66]
1999 [68]
11 (9 pregnancies)
11
11
26
32/40c
38 in 29
patients
39
55
Zanagnolo
et al. 2004
[73]
64
106
Tangir
et al. 2003
[72]
Table 21.4 Literature review of fertility results following conservative management in germ cell tumors (series published after 1995)
10d
15
29
8 (4
?
pregnancies)
6 in 5
patients
19/23c
23
23
Ayhana
Boran
b et al. 2005 et al. 2005
[74]
[75]
15
15
20
Kangb
et al. 2008
[76]
4 pregnancies
19 in
12 patients
39/40
41
52
De La Motte
Rougea
et al. 2008 [77]
Non-epithelial ovarian cancer

Non-epithelial malignant tumors are characterized
(compared to epithelial cancers) by: (1) the occurrence
of disease in younger patients; and (2) an (overall)
good prognosis of this tumor (even in case of extraovarian disease) as in most cases the chemo-curability
of these tumors is excellent. They could be classified
into two main groups: malignant germ cell tumors
(MGCT) and sex cord stromal tumors (SCST).
Malignant germ cell tumors

Most of the papers concerning the results of conservative surgery in non-epithelial cancers, concern
this group of tumors (Table 21.4) [6677]. The most
frequent lesions in this group are dysgerminomas,
endormal sinus tumors (EST), malignant teratoma and
mixed subtypes. The type of chemotherapy administered against such tumors is the BEP regimen
(bleomycin, etoposide and cisplatin). Conservative
surgery is the standard management in young patients.
Staging (nodal or peritoneal) procedures are discussed in this context. In non-dysgerminomas with
a macroscopically normal ovary, biopsies of the contralateral ovary are not recommended. In dysgerminomas, this procedure could be considered because
there is a potential risk of occult disease in 10%
of cases. For example in the recent series by Boran
et al., 2 out of 17 patients (11%) with a macroscopically normal contralateral ovary had occult involvement [74].
The fertility results in the series reported after 1995
are shown in Table 21.4. Menstruation and endocrine
ovarian function were maintained in a very large
majority of these young patients treated with the BEP
regimen (Table 21.4). Conservative management of
a part of one ovary could be considered in patients
with bilateral involvement (in the case of teratomas) or
in patients with peritoneal disease treated with adjuvant chemotherapy (particularly in dysgerminomas or
malignant teratoma) (Table 21.4).
Given the high curability rate among these
patients, no completion surgery is discussed after
childbearing.
Sex cord stromal tumors

The most frequent subtypes of these tumors are granulosa cell, SertoliLeydig and thecal cell tumors. Very
274
few papers are devoted to the conservative management of such tumors (mostly case reports or short
series). In the series by Zhang et al., among the 376
women treated for SCST, 71 young patients underwent
uterine preservation for stage I disease [78]. The survival of patients treated conservatively and radically
was similar [78].
Two important characteristics observed in granulosa tumors exert an impact on conservative surgery:
bilaterality is uncommon (between 2 and 8% of cases
[79] and these tumors are frequently associated with
endometrial disorders (hyperplasia or cancers). Consequently, random biopsies of the contralateral ovary
are not required (if macroscopically normal) but uterine curettage should be systematically performed. The
overall prognosis of granulosa cell tumors is good
in early stage disease (stage IA) and conservative
management could be considered in young patients
with a similar stage. However, conservative management should not be proposed for higher stages
(or in the case of ovarian capsule rupture during
initial surgery) because the prognosis is less clear
cut.
The use of completion surgery after childbearing
continues to be debated in SCST [79].
Conclusions
Conservative treatment yields good fertility results
and does not affect the survival of patients with borderline ovarian tumors. It should be considered for young
women desiring fertility, even if peritoneal implants
are discovered at the time of initial surgery. In case of
infertility, medically-assisted procreation techniques
may be proposed to patients with stage I BOT with a
limited number of stimulation cycles.
In patients with epithelial ovarian cancer, conservative surgery of an ovary and the uterus can only be
considered in adequately staged patients, with a stage
IA, grade 1 (and probably 2) serous, mucinous or an
endometrioid tumor and careful follow-up. Such management could probably also be safely proposed for
stage IC, grade 1 disease.
In patients with non-epithelial ovarian cancer, conservative surgery is highly applicable, particularly in
patients with malignant germ cell tumors.
Acknowledgement
The authors thank Lorna Saint Ange for her editing.
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Section 6
Chapter
22
Fertility preservation strategies in the female: ART
Embryo cryopreservation as a fertility

preservation strategy
Pedro N. Barri, Anna Veiga, Montserrat Boada and Miquel Sole
Introduction
Survival rates after cancer have increased significantly
in recent decades; however, these treatments also have
drawbacks and patients (or parents in the case of children) must be informed of the long-term side effects
of oncological treatments and the possible options for
preserving the fertility of these patients. It is important to set out clearly the possible risks of developing
ovarian failure or azoospermia with oncological treatments. These will depend on the age of the patients and
on the type, dose and duration of chemotherapy, and
on the field, dose and duration of radiotherapy.
The strategy must be individualized in each case
depending on:
r Patients age.
r Type and stage of the cancer.
r Therapeutic plan to be followed.
r Foreseeable long-term effects.
r Possibility or impossibility of delaying the start of
treatment.
r Whether or not the patient has a partner/spouse.
r Biology of the tumor and potential for metastasis
in ovary.
If ovarian stimulation is possible, embryo cryopreservation is the method with the greatest chance of success so far. However, it is only possible if there is a
partner or if the patient accepts donor sperm. It is
very important to clarify which are the safest ovarian stimulation protocols to be used in these patients,
although current data suggest that in certain cancers a
cycle of ovarian stimulation does not increase the risk
of recurrence.
Since the first pregnancy obtained from cryopreserved human embryos [1], the yield and safety
of embryo cryopreservation programs have improved
notably with successful freezing of zygotes and cleaving of embryos and blastocysts, and pregnancies
are regularly obtained after thawing and transfer
of embryos in any of these stages. Our first pregnancy after transferring a previously frozen embryo
was obtained even though only one of the embryos
original four cells survived thawing and was transferred [2].
Outcome of embryo-freezing programs

Embryo cryopreservation is the method of choice and
with the greatest chance of success; however, it can only
be applied if there is a male partner or if the couple
accept donor sperm.
It is accepted that 1520% of the 3 million children
born after in vitro fertilization (IVF) were conceived
following the transfer of embryos that had previously
been frozen and thawed. That means that these 500 000
or so children make up a wide population that proves
the efficacy and the safety of embryo cryopreservation
[3, 4].
Nevertheless, cryopreservation affects the potential for embryo implantation, which is lower than for
fresh embryos. It is important to choose the freezing
protocol among the slow-freezing methods or those
that use vitrification, just as it is also important to
choose the stage at which the embryo is to be frozen.
Zygotes are normally frozen on 2PN, cleaving embryos
or blastocysts, with comparable rates for survival after
thawing and for pregnancy [5].
279
Section 6: Female fertility preservation: ART
Table 22.1 Institut Universitari Dexeus embryo freezing program: 19872004

20 318 embryos thawed
Cycles
Transfers
Thawed embryos
Survival
Pregnancy/transfer
Babies born
6715
5354
20.318
66%
22.5%
807
Table 22.2 Institut Universitari Dexeus Embryo freezing

program (n = 2547 transfers) Results according to frozen
time (years)
Frozen time
Pregnancy rate/transfer
57 years
16/44
36.4%
79 years
5/16
31.3%
9 years
5/8
62.5%
5 years
853/2479
33.68%
5 yearsa
26/68
38.2%
Totals
One baby born after 10 years and 1 baby born after 14 years of
cryopreservation.
At the Instituto Universitario Dexeus, Spain, we

use zygote-freezing only in cases of fertility preservation in oncological patients or in some egg donation cycles where it is not possible to synchronize
the donors cycle with that of the recipient. An analysis of our results for zygote-freezing in the period
20022007 showed that survival rates were 86.5% with
pregnancy rates per transfer of 41.1%. It is important
to check embryo viability after thawing by cultivating the embryos overnight to confirm that they have
started the first embryonic divisions. Freezing cleaving embryos gave rather lower survival rates of around
70%. Similar rates were achieved by freezing blastocysts and in these cases the post-thawing viability was
evaluated at 4 h to check blastocyst expansion [59].
In our experience, with our embryo-freezing program during the period 19872004, and with 20 318
embryos thawed, we had a global survival rate of
68%. A total of 5354 cryoreplacements were performed
leading to the birth of 807 children and with a birthper-cryoreplacement rate of 22.6% (Table 22.1). One
important aspect to bear in mind when this technique
is applied to oncological patients is to analyze the
results according to the time that the embryos have
been frozen. Our data show that this variable is not relevant as the pregnancy rates obtained following cryoreplacement before and after 5 years of freezing do not
differ (Table 22.2).
280
Recently vitrification has been developed for both

oocyte and embryo cryopreservation. The results so
far seem to show higher post-thaw survival rates and
higher rates of development to the blastocyst stage
[10, 11]. Implantation and clinical pregnancy rates are
within the normal range. As far as safety is concerned,
studies on the neonatal outcome after embryo vitrification have shown no increase in the congenital malformation rate with a neonatal outcome comparable to
fresh embryo transfers [3, 12, 13].
Another point to take into account in these cases
is the analysis of the patients preferences regarding
future use of the embryos if the patient should die. In
such cases, Spanish law allows the patients to decide
between anonymous donation of the embryos to other
couples, donation for research or destruction of the
embryos. We feel that the patients freedom of choice
must be respected with explanations of all the possibilities being given before unfavorable circumstances
arise that could condition their decision [14, 15].
Ovarian stimulation protocols for IVF

and embryo cryopreservation in
oncological patients
In oncological patients two special circumstances
often arise: a short time to stimulate ovulation and the
necessity of not reaching high estradiol levels. It is also
important to start the ovarian stimulation before the
chemotherapy as the results of the IVF cycle will be
very poor if a course of chemotherapy has been performed [16].
In these cases, ovarian stimulation can be done
using aromatase inhibitors in combination with
gonadotropins. Tamoxifen was the first drug used in
these protocols but letrozole soon proved to be just
as safe and much more effective [17]. Letrozole is a
powerful third-generation aromatase inhibitor; with a
half-life of 48 h it significantly suppresses plasma estradiol levels. Data from recent publications suggest that
letrozole is more effective than tamoxifen in ovulation
stimulation protocols for oncological patients [18].
Chapter 22: Embryo cryopreservation
With regard to the gonadotropins, it seems to be

better to use recombinant follicle stimulating hormone (FSH) preparations devoid of luteinizing hormone (LH) to limit the estradiol levels that will be
reached during stimulation. To prevent early luteinization or premature ovulation it is fundamental to combine these drugs with a gonadotropin-releasing hormone (GnRH) -antagonist analogue so that we can act
at any point in the menstrual cycle.
There is now evidence that the survival of oncological patients who have followed an ovarian stimulation protocol for an IVF cycle, and the freezing of
any embryos that are obtained, is identical to that of
patients who do not undergo this protocol [17].
If the oncologist authorizes a 23-week delay in the
treatment, we will start treatment with GnRH antagonists at any point in the cycle and will start stimulation when plasma estradiol is below 50 pg/ml. After
this, the patient will begin treatment with 5 mg/day of
letrozole for 5 days, combining this treatment with the
daily administration of 150 IU of recombinant FSH.
The ovulatory discharge will be with a bolus of GnRH
agonist, and letrozole or antagonist treatment will be
maintained following follicular aspiration.
Results of embryo cryoreplacements in

oncological patients
The first cases of embryo freezing in oncology patients
took place more than 10 years ago, with the application of a natural IVF cycle with embryo cryopreservation prior to chemotherapy for carcinoma of the breast
[19]. However, the first pregnancies were published
some years later with a case of embryo cryopreservation after diagnosis of stage IIB endometrial cancer
and subsequent pregnancy in a gestational carrier [20].
Recently several pregnancies have been published
in oncology patients who had frozen their embryos
before starting chemotherapy [1418]. In our experience, we had a birth of a healthy boy 4 years after
embryo cryopreservation was carried out in a patient
with a bilateral borderline ovarian tumor who was
treated initially with an IVF cycle in which 4 embryos
could be frozen. Conservative surgery of the ovarian
tumor was practiced later and at 3 years the embryos
that survived freezing and thawing were cryoreplaced.
A clinical pregnancy was obtained that led to the birth
of a healthy boy. The improved results from vitrification of oocytes means that now some oncology
patients have opted for this strategy and the first births
achieved in this way have been published [21].
In applying embryo-freezing techniques to preserve the fertility of oncology patients, it is very important to know the couples preference for the disposition of any unused embryos. Couples are normally
offered the possibility of choosing, in the event of death
or unforeseen circumstances, between the destruction
of the embryos, donation for research, anonymous
donation to other infertile couples or leaving the decision to the surviving partner. The oncology patients
make similar choices to the infertile patients who
undergo IVF with regard to donation of the embryos
for research or to other infertile couples, but most
oncology patients reject destruction of the embryos
and accept this option in a much lower percentage than
normal IVF patients [14].
Conclusions
Up to now, embryo cryopreservation has been the only
clinically accepted method for preserving the fertility
of oncology patients before they undergo chemotherapy and/or radiotherapy. The post-thawing pregnancy
rates are acceptable and are around 30% per cryoreplacement depending on the number of embryos available and their quality.
However, this option does have some drawbacks
such as:
(a) It is not feasible for pubertal girls.
(b) Ovarian stimulation may be contraindicated and
oocyte retrieval and IVF may cause a delay in the
initiation of oncological treatment that may not
be acceptable in some cases.
(c) Spermatozoa from a male partner or from a donor
is required.
(d) Possible religious or moral objections.
References
cryopreservation, thawing and transfer of an eight-cell
embryo. Nature 1983; 305(5936): 7079.
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3. Wenherholm UB, Sodertrom Anttila V et al.
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Madam K. Neonatal outcome after vitrified day 3
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14. Klock SC, Zhang JX and Kazer RR. Fertility
preservation for female cancer patients: early clinical
experience. Fertil Steril 2010; 94: 14955.
15. Luna M, Boada M, Aran B et al. Couples opinions
regarding the fate of surplus frozen embryos. Reprod
Biomed Online 2009; 19 (Suppl. 2): 1115.
16. Dolmans M Demylle D, Martnez-Madrid B and
Donnez J. Efficacy of in vitro fertilization after
chemotherapy. Fertil Steril 2005; 83: 897901.
17. Oktay K. Further evidence on the safety and success of
ovarian stimulation with letrozole and tamoxifen in
breast cancer patients undergoing in vitro fertilization
to cryopreserve their embryos for fertility
preservation. J Clin Oncol 2005; 23: 38589.
18. Oktay K and Sonmezer M. Fertility preservation in
gynecological cancers. Curr Opin Oncol 2007; 19:
50611.
19. Brown JP, Moden E, Obasaju M. and Ying YK.
Natural cycle in vitro fertilization with embryo
cryopreservation prior to chemotherapy for carcinoma
of the breast. Hum Reprod 1996; 11:1979.
11. Loutradi KE, Kolibianakis EM, Venetis CA et al.

Cryopreservation of human embryos by vitrification
or slow freezing: a systematic review and
meta-analysis. Fertil Steril 2008; 90(1): 18693.
20. Juretzka MJ, OHanlan KA, Katz SL, El-Danasouri I

and Westphal LM. Embryo cryopreservation after
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subsequent pregnancy in a gestational carrier. Fertil
Steril 2005; 83: 10415.
12. Takahashi K, Mukaida T, Goto T and Oka C.

Perinatal outcome of blastocyst transfer with
vitrification using Cryoloop: a 4 year follow-up study.
21. Yang D, Brown SE, Nguyen K et al. Live birth after the
transfer of human embryos developed from
cryopreserved oocytes harvested before cancer
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282
Section 6
Chapter
23
Oocyte cryopreservation
Slow freezing
Andrea Borini and Veronica Bianchi
In the last 20 years significant improvements have been

made in in vitro fertilization (IVF) procedures especially involving gamete cryopreservation. Several factors have driven the research in this field including the
law (in Italy) and need for optimization of particular
applications (like egg donation). Moreover, the possibility to freeze eggs is an important requirement to preserve fertility potential in patients at risk for fertility
loss due to premature ovarian failure or invasive treatments like chemotherapy and/or radiotherapy. Ovarian cryopreservation presents a valid alternative to egg
freezing in some circumstances. The possibility to store
oocytes for a later use is also an important consideration for women who choose to postpone motherhood
for personal or professional reasons.
The ability to freeze eggs as a routine IVF procedure is an important tool to optimize egg donation and
allow for independent synchronization of donor and
recipients, and quarantine of specimens for sexually
transmitted diseases. Another fundamental benefit of
egg cryopreservation is to avoid potential problems
associated with legal status and ownership of cryopreserved embryos in the event of divorce.
Chemical and physical aspects

Although the first human live birth from cryopreserved oocytes was reported more than 20 years ago
[1], success rates in assisted reproductive technologies
using frozen oocytes have lagged behind those using
frozen embryos or blastocysts. Technically speaking,
slow-freezing protocols were the only option available
to cryopreserve oocytes for about 10 years, until development of vitrification over the last 5 years as an alternative to the original methods.
Any newly developed protocol should consider the
biochemical and physical properties of the oocyte. Cell
survival is intimately associated with the composition

and permeability characteristics of the cell membrane,
the surface to volume ratio of the cells and the difference in osmotic pressure between the two sides of
the membranes [2, 3]. Moreover, the cytoplasm of the
oocyte contains a high proportion of water in comparison to other cells; damage due to ice crystal formation in the phase transition of water to ice was an
initial hurdle to overcome during freezing procedures.
Protocols that include dehydration of oocytes before
and/or during the cooling procedure reduce ice crystal formation and lead to improved clinical outcomes.
Consequently, cryobiology studies analyzing the physical and chemical proprieties of cryoprotectant agents
(CPAs) have been an important contribution to this
field. The first fundamental studies were conducted
by Arrhenius et al. and established an important connection between temperature, activation energy and
the rate of chemical reactions, showing that long-term
preservation was possible at very low temperatures due
to decreased rates of all the biological reactions [4].
Later, Mazur et al. formulated a series of equations aimed to explain the multifactorial cellular damage due to freezing procedures [5, 6]. First, it was
important to analyze the membrane properties of the
cell, regarding permeability and internal osmotic pressure. Subsequently, it was necessary to monitor how
these parameters changed during the cooling phase
according to the freezing rate, decreased temperature,
change in vapor pressure and solute concentrations
during ice crystal formation. From these observations,
Mazur established that a low cooling rate maintains an
osmotic balance between the two sides of the membrane; however, the hypertonic milieu that surrounds
the cell may cause irreversible damage to the membrane protein structures. Faster cooling rates, on the
contrary, limit this situation but also prevent all the
283
intracellular water from leaving the cell and, consequently, result in ice crystal formation and cell death.
To partially overcome to this issue, CPAs have been
introduced; in fact one role of CPAs is to protect biological materials against cooling injuries from ice crystals and high concentrations of solute during cryopreservation [7, 8].
Since the introduction of glycerol as a permeable cryoprotective agent in sperm cryopreservation
by Polge et al., and the subsequent discovery and use
of dimethyl sulfoxide (DMSO), many cells and tissues have been frozen but the cooling procedures were
mostly theoretical [9, 10].
All freezing methods developed to date rely on the
presence of one or more CPAs in molar concentrations.
In general cryoprotectants may be divided into two
groups:
(a) Permeating agents such as glycerol, DMSO,
ethylene glycol and 1,2-propanediol (PROH). All
of which are chemically characterized as having a
relatively low molecular weight and can penetrate
the lipid bilayer of the cell with a slower
permeability than water.
(b) Non-permeating agents that remain in the
extracellular solution as a result of their size or
polarity. These CPAs include sugars and
macromolecules such as sucrose, Ficoll and
raffinose, as well as proteins and lipoproteins.
Cryoprotective agents can reduce the toxic effects
of high concentrations of other compounds in the
solution [11], and their protective effects are related to
their hydrophilic ability to create hydrogen bond and
consequently to interact with water and to achieve high
aqueous solubility.
With glycerol and PROH, the hydrogen bonding is
between the hydrogen of the OH groups and the water.
The oxygen on the DMSO molecules bind to the water
protons with the release of heat. During slow cooling,
when the cells are very dehydrated and are surrounded
by concentrated salts, the cryoprotectants appear to
reduce damage caused by the high levels of salt.
These CPAs, however, can have dramatic osmotic
effects upon cells during the freezing/thawing procedures; when exposed to permeating solutes the oocyte
undergoes extensive initial dehydration and shrinkage
followed by a return to near the original volume as the
cryoprotectant enters the cell and replaces the intracellular water. This causes a double flux across the membrane (the water exits the cell while the CPA enters)
284
that influences both the intracellular solute concentration and the cell volume. The extent of shrinkage and
swelling can cause damage or even cell death due to
the osmotic stress acting on the oocyte membrane. To
reduce stress from excessive swelling, non-permeating
molecules are added to the CPA mixture. Sucrose is the
most commonly used, but trehalose and choline have
also been utilized in cooling protocols [1214].
Usually, in the most widely adopted slow cooling
protocols, cryoprotectant concentrations are around
1.5 M for the penetrating agent (mainly PROH or
DMSO) and 0.10.3 M for the non-penetrating agent
(usually sucrose).
The analysis of cell-volume change dynamics is
important to evaluate approaches to improve freezing protocols. Addition of cryoprotectants can cause
cell damage due to volume changes and chemical toxicity; optimal exposure should aim to minimize
osmotic stress while avoiding chemical toxicity and
allow sufficient permeation and dehydration to achieve
protection from freezing injury.
Several authors approached those issues with different ideas: Yang et al. tried to increase cryoprotectant exposure temperature to achieve faster dehydration rates [15].
Quintans et al. adopted a stepwise addition of
the permeating cryoprotectant (PROH) to reduce volume excursion [16], while Boldt et al. tried to use a
sodium-depleted freezing medium combined with a
lower seeding temperature to improve post-thawing
recovery [17].
However, no measurement of the actual response
of the cells to these changes was performed in these
studies. To address this, Paynter et al. tried to improve
the cryopreservation technique by evaluating several
factors involved during the equilibration of oocytes
to cryoprotectant interaction and decreased temperatures [18]. The authors measured the osmotic response
by monitoring the oocytes for 10 min during a twostep addition of the permeating cryoprotectant PROH
(0.75 and 1.5 M PROH). Following this, the oocyte
osmotic response to 1.5 M PROH and 0.2 or 0.3 M
sucrose was measured. Those authors found that each
oocyte shrank during the first exposure to the cryoprotectant (0.75 M PROH) as water left the cell and
then gradually re-expanded as water and cryoprotectant entered. The entire volume equilibration process
took around 10 min to complete.
During the second exposure to an increased cryoprotectant concentration (1.5 M PROH) the oocytes
Chapter 23: Oocyte cryopreservation slow freezing
underwent the same phases of shrinkage and reexpansion, while in the last set of exposure experiments (1.5 M PROH plus sucrose) the shrinkage rate
was faster with an obvious reduction of cell volume
before freezing to avoid intracellular ice crystal formation.
This paper targeted the important concept of a
shrinkage and re-expansion time frame in which
oocytes can recover after PROH exposure.
An equally important consideration for oocyte survival involves the removal of permeating cryoprotectants. When a cell containing cryoprotectant is placed
in medium with a lower concentration of cryoprotectant, water enters the cells to dilute the cryoprotectant at a faster rate than the cryoprotectant can leave
the cells, which causes swell and potential burst. This
problem can be overcome by controlled removal of the
cryoprotectants in a series of decreasing concentration
steps.
This does not prevent the cells from swelling, but
reduces the corresponding magnitude and achieves the
desired outcome of gradual CPA removal and rehydration in a controlled manner. Cells are moved from one
solution to the next after they re-acquired their normal
level of hydration.
An alternative method for removing cryoprotectants from cells is to use a high concentration of a
non-penetrating molecule such as sucrose; this extracellular high concentration serves to balance the
high intracellular cryoprotectant concentration as it
reduces the difference in osmolarity between the intra
and extracellular environments. The oocytes shrink
proving that both the cryoprotectant and the water are
leaving the cell reducing the intracellular volume. The
use of high sucrose concentrations allows a one step
thawing protocol that is simpler and faster than multistep dilution procedures.
Morphological variables
In addition to surviving the cryopreservation/warming process, the oocyte needs to maintain competence
to fertilize and develop in vitro to the appropriate
embryonic stage without any structural alterations.
It has been widely demonstrated that low temperatures and cryopreservation procedures in general may induce critical damage to the cell substructures, especially due to the peculiar characteristics of
the metaphase II (MII) oocyte. At this maturational
stage the chromosomes are aligned on the metaphase
plate in strict relation to the meiotic spindle. This is

a delicate structure responsible of the correct chromosome segregation during the maturational process
that ends with extrusion of the second polar body.
The meiotic spindle is a highly dynamic bipolar structure made of microtubules that have the ability to
disassemble and reassemble under particular conditions. They are dimers composed of and tubulin,
more dispersed at the microtubule organizing center
(MTOC) and more compacted at both ends in contact with the kinetochore; the spindle has a typical barrel shaped structure with the chromosomes suspended
within it.
Meiotic spindle is frequently abnormal in older
woman both with regard to chromosome alignment
and the microtubule structure, showing that the
increase in the genetic abnormalities is often associated with poor structural quality of the egg [19]. This
study was conducted on women undergoing natural
cycles, thus avoiding bias related to ovarian stimulation or in vitro oocyte aging.
Another factor associated with meiotic spindle
damage is temperature. Several studies using the
mouse model showed that low temperature can cause
depolymerization of the tubulin dimers and a transient
or permanent loss of the spindle [20, 21].
In mouse, oocyte incubation at 25, 18 or 4 C can
lead to microtubule disassembly with an increase of the
monomeric tubuline, but within 1 h at 37 C the spindle
is able to significantly reassemble [22].
Conversely, in the human, this structure is more
sensitive to fluctuations and the rate of recovery is
limited. If temperature is lowered from 37 to 27 C
the spindle depolymerized within 5 min and can
repolymerize only if this suboptimal condition is not
extended beyond 20 min [20, 23].
During cryopreservation oocytes undergo more
stressful situations, due to prolonged exposures to
low temperatures leading to spindle alterations and a
potential increase in chromosome segregation errors
resulting in higher aneuploidy rates or even fertilization failure. It is also known that cryoprotectants play
a key role in protecting this structure if the concentration and exposure time is appropriate [11].
Conflicting opinions have been expressed in
which some authors reported that freezing procedures
alter meiotic spindle organization [24] while others
reported no significant damage occurred after freezing [25, 26]. Cytogenetic analysis performed for chromosomes 13, 18, 21 X and Y did not highlight an
285
increase in aneuploidies or chromosomal dispersion in

embryos derived from fresh or frozenthawed oocytes
(28 versus 26%, respectively) [27].
In order to have a better understanding, several studies on recovery of the meiotic spindle postthaw have been conducted using two approaches:
R
the Polscope
and the confocal immunostaining
microscopy, the latter undoubtedly offering the most
detailed data on spindle structure but unfortunately
requiring a fixation step that causes loss of oocyte
R
has been used instead as an
viability. The Polscope
alternative approach. It is a microscopy optical system
that allows the observation of highly ordered subcellular structures, such as the spindle, through polarized
light [28, 29]. It has been showed that microtubules
are responsible for spindle birefringence and that spindle retardance is related to microtubule density [30].
This system is very important for routine application
as it allows visualization of the spindle and the inner
layer of the zona pellucida, and to roughly evaluate the
amount of the microtubules present in the oocyte by
measuring the retardance.
Compared to immunostaining or other microR
offers the advantage of
scopy methods, the Polscope
being non-invasive to preserving oocyte viability while
allowing repeated observations over time [31]. In the
literature there is a general agreement about correlation between the presence of the spindle and fertilization rate during IVF [3234] or embryo development potential [35]. This system has also been applied
to evaluate spindle recovery after human oocyte cryopreservation using different protocols.
Rienzi et al. used a 1.5 M PROH plus 0.1 M
sucrose slow-freezing protocol and showed a spindle recovery in 37% of the oocytes immediately after
thawing [36]. In the subsequent steps, the spindles
disappeared in all the eggs, only to reappear in all
surviving oocytes within 3 h of incubation at 37 C.
Similar data were published by Bianchi et al. using
a comparable freezing protocol with higher sucrose
concentration (0.3 M) [37]. Immediately after thawing, only 22.9% of oocytes showed a weak birefringence signal, while only 1.2% of oocytes displayed a
high signal. Three hours after thawing, the proportion of oocytes exhibiting a weak or high intensity signal was 49.4 and 18.1%, respectively. Finally, after culture for 5 h following thawing, a weak birefringence
signal was detected in 51.8% of oocytes, while 24.1%
showed a high signal. There was a statistically significant increase in signal restoration after 3 h of cul-
286
ture (P 0.001). Those data were partially confirmed

later by confocal microscopy where unfrozen control
oocytes were compared with frozen oocytes fixed at
0, 1, 2 and 3 h after thawing [38]. All the control
oocytes (100.0%) displayed bipolar spindles with constriction evident at both poles. Following cryopreservation, there was a significant reduction of oocytes
with bipolar spindles directly following thawing (T0;
59.1% bipolar), although after 1 h of culture (T1) 85.7%
of oocytes regained bipolar spindles. Oocytes cultured
for 2 (T2) or 3 h (T3) following thawing displayed 73.7
and 72.7% bipolar spindles, respectively. Moreover, at
T2, oocytes from older patients (36 years) showed
a significantly lower rate (11.1%) of chromosome
alignment compared to younger patients (35 years)
where 70.0% of oocytes displayed a normal configuration (P 0.05). A similar trend was evident at T3
where only 41.7% of oocytes from older patients maintain chromosome alignment compared with 60.0% in
oocytes from younger patients.
Confocal microscopy analysis has been used to
generate more data from oocytes frozen using different protocols based on PROH and sucrose cryoprotectants at different concentrations (0.1 and 0.3 M) [40].
A higher sucrose concentration is responsible for a significantly improved survival rate, but this also might
be associated with an increased osmotic stress for
the oocyte [18] and consequent spindle damage. The
confocal analysis on fresh and frozen thawed oocytes
better clarified this hypothesis. The authors showed
that, of the 104 oocytes included in the unfrozen
group, 76 (73.1%) displayed normal bipolar spindles
with equatorially aligned chromosomes. Spindle and
chromatin organizations were significantly affected
(50.8%) after cryopreservation involving lower sucrose
concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using 0.3 mol/l sucrose
(152 oocytes). From those data it is possible to affirm
that cryopreservation procedures induce damage in
oocytes but that this is relatively acceptable using
higher sucrose concentration. This can be explained
by the higher dehydration reached with a 0.3 mol/l
sucrose protocol that limits the amount of intracellular water and prevents ice crystal formation. Consequently, the spindle is better preserved. This is
supported by the clinical data obtained using these
protocols [4143]. Additional ultrastructure investigations were performed comparing PROH with ethylene
glycol (EG) as the permeating cryoprotectant. The frequencies of normal spindle configuration were lower
in frozen EG or PROH oocytes compared with fresh

oocytes (53.8, 50.9 and 66.7%, respectively, P 0.05)
[44].
Besides the meiotic spindle, other subcellular
structures have to be preserved during freezing
thawing procedures. The zona pellucida, a multilaminar structure composed of glycoprotein, has a fundamental role in the normal fertilization process. The
sperm can bind to specific sites inducing the release
of cortical granules that prevent the entrance of other
sperm through glycoprotein inactivation [45]. During freezethaw procedures this structure can be damaged, thus compromising the post-thaw fertilization
potential. The so called zona hardening was first
described in mouse oocytes showing that the fertilization rate dramatically decreased after thawing.
Normally, the entrance of the sperm inside the
egg causes a transient increase in the intracellular calcium concentration [46], causing release of the cortical
granules and consequent sperm binding glycoprotein
inactivation. This might be related to the use of penetrating cryoprotectant in the cryopreservation protocols that induce premature cortical granules release
and is responsible for calcium oscillations inside the
cell [47]. The entrance of the cryoprotectant is probably associated with a calcium flow toward the cell that
causes an increase in the intracellular calcium concentration [48]. Moreover, it has been demonstrated that
DMSO and PROH can also cause a proteolytic modification of the sperm binding protein ZP2. Adding fetal
bovine serum to the freezing mixture can reduce the
zona hardening and the inactivation of the ZP2 even
though it does not prevent cortical granule release. A
protein known as fetuin competes with the enzymes
released from the granules which are responsible for
the zona hardening [49]. In the human there is limited
data on the fertilization rate by conventional IVF after
thawing since the use of intracytoplasmic sperm injection (ICSI) technique could bypass the issues related
to zona hardening. More data are available on the
cortical granules loss. In 1988 Sathananthan et al.
observed a reduction in numbers [50] that has been
confirmed more recently using electron microscopy
[44, 51, 52]. Ghetler et al. analyzed cortical granule
exocytosis by either confocal microscopy or transmission electron microscopy (TEM) [52]. Mature oocytes
exhibited increased fluorescence after cryopreservation, indicating the release of the granules; this was
confirmed by TEM that revealed a drastic reduction
in their amount at the cortex of frozenthawed MII
oocytes. They demonstrated that the use of PROHfreezing protocol for human oocytes resulted in extensive cortical granule exocitosis. Those results were
later confirmed by Nottola et al. who showed that the
amount and density of cortical granules appeared to
be abnormally reduced in some frozenthawed samples, despite the slow-freezing protocol [51]. These
abnormal features were frequently associated with an
increased density of the filamentous texture related to
the occurrence of zona hardening. The same result
was obtained by these authors using EG and sucrose
as cryoprotectants [44]. As previously mentioned, the
MII oocyte has a complex subcellular structure that
includes the meiotic spindle, cortical granules and
other features like mithocondria or smooth endoplasmatic reticulum (SER). These ultrastructural components were not taken into account in the initial studies,
since oocyte survival after thawing was primarily correlated to morphological appearance of the egg. Lately,
several authors focused their attention on the possible consequences related to subcellular injuries. Nottola et al. analyzed fresh and frozenthawed oocytes
using a slow cooling method with PROH and sucrose
(0.1 or 0.3 mol/l) as cryoprotectants [51]. The oocytes
were then processed for electron microscopy observations. All the oocytes showed a homogeneous cytoplasm and an intact zona pellucida (ZP) with abundant and uniformly dispersed organelles (mainly
mitochondriasmooth endoplasmic reticulum aggregates and mitochondriavesicle complexes). Nevertheless, a degree of microvacuolization was detected in
the ooplasm of some frozenthawed oocytes, particularly in those treated with higher sucrose concentration. Another interesting study comparing fresh and
frozen thawed eggs (PROH plus 0.3 mol/l sucrose)
was published by Gualtieri et al. [53]. The authors
showed evidence that in fresh samples mitochondria
had a regular shape with few short cristae, whereas in
the frozenthawed group a high percentage of oocytes
(72%) showed a variable and, in some cases, a very high
fraction of mitochondria with decreased electron density of the matrix or ruptures of the outer and inner
membranes. Moreover in those oocytes, the mitochondrial damage was associated with SER swelling.
Slow cooling protocols and outcomes

As implied by the name, this protocol is characterized
by a slow decreasing temperature rate. Several mathematical models have been used to define an optimal
287
curve applicable to oocytes since the freezing rate is

vital to achieve sufficient and progressive dehydration,
and thereby minimize the potential of intracellular ice
formation.
The slow cryopreservation protocol applied worldwide to oocytes is the same as that normally used for
embryo freezing, and it is based on a slow freezerapid
thaw program. The cryoprotectant agents most frequently used are PROH and sucrose; the combination
of penetrating and non-penetrating agents allow a better outcome in terms of oocyte stability. The oocytes
are maintained for 10 min in a 1.5 M PROH solution with 20% protein supplement for equilibration
phase. During this time the PROH enters the cell as
water exits the oocyte. The second step is performed
in a 1.5 M PROH solution supplemented with various concentrations of sucrose (according to different
protocols) and 20% protein source. During this 5 min
exposure, the oocyte increases its dehydration in a
proportional way to the amount of sucrose contained
in the solution. The oocytes are subsequently loaded
in straws and placed in an automated Kryo 10 series
biological vertical freezer (Planer Kryo GB). Freezing
solutions are cooled from 20 C to 7 C at a rate of
2 C/min. Manual seeding of oocytes within straws is
performed at near 7 C and this temperature is maintained for 10 min in order to allow uniform ice propagation. The temperature is then decreased to 30 C at a
rate of 0.3 C/min and then rapidly lowered to 150 C
at a rate of 50 C/min. Straws are then directly plunged
into liquid nitrogen at 196 C and stored.
Thawing consists of rapid rewarming (air for 30 s
and then 40 s in a 30 C water bath) and subsequent
stepwise dilution of the cryoprotectants; first in 1.0 M
and then in 0.5 M PROH solutions supplemented with
sucrose (depending on the sucrose concentration used
during freezing procedure) for 5 min each, and then in
a sucrose solution for 10 min and in PBS solution for
an additional 10 min. Finally, the oocytes are returned
to culture media at 37 C to support recovery.
The first pregnancy using oocyte cryopreservation was obtained in 1986 with a protocol tested on
mouse eggs [1]. The author used DMSO as permeating cryoprotectant and partially removed cumulus
cells before freezing. The results were not the same as
those obtained in the animal model; the main problem
emerged following the first attempt which showed a
low oocyte survival rate that was totally incomparable
to embryo freezingthawing results. Consequentially,
this procedure was abandoned for many years. How-
288
ever, in the last 10 years, several prompted renewed

interest; higher survival rates in cancer patients or the
need to preserve fertility in general pushed several
groups forward in improving this technique.
The first protocol used was the same Lassalle
et al. applied to embryo freezing and was based on a
1.5 M PROH plus 0.1 M sucrose mixture [54]. Despite
the great results on embryo survival, this protocol
was not the one tailored for egg freezing. One of the
first studies with this protocol was published in 2004
where, out of 737 oocytes the survival rate was just
37%, while the fertilization and cleavage rates were
45.4 and 86.3%, respectively [43]. The ICSI technique
was used to inseminate after the first report of Porcu
et al. but, even so, the rate of zygotes obtained was
not very satisfying [55]. A total of 15 clinical pregnancies were achieved (25.4% per transfer and 22% per
patient) with an implantation rate of 16.4%. The outcome was much lower in terms of success than results
obtained with frozen embryos and analogous conclusions were reached by other groups using the same
protocol.
The need to improve several aspects of oocyte cryopreservation procedures led to a wide set of experiments that allowed this technique to become a routinely used approach in IVF. Paynter et al. pointed
out first the need to increase oocyte dehydration in
order to raise survival rate [56]. The protocol suggested
by Lassalle did not guarantee a sufficient water flow
out of the cell thus causing intracellular ice formation
and subsequent cell death [54]. The importance of a
non-permeating agent (such as sucrose) in the freezing solution has been deeply analyzed; this, in fact,
enhances oocyte dehydration. The higher the sucrose
concentration, the more the cell dehydrates, as water
rapidly leaves the cytoplasm to dilute the high concentration of extracellular solutes. The exposure time to
cryoprotectant should be long enough to enable sufficient cell dehydration, but not so excessive as to damage the cell by altering the intracellular pH.
To this purpose, Fabbri et al. raised the sucrose
concentration from the standard 0.1 to 0.2 and 0.3 M,
improving the survival rates significantly (34, 60 and
82%, respectively) as a result of a more adequate
dehydration [40]. The data were confirmed by several
authors, but the worldwide results were often related to
a small number of patients with restricted indications
and low numbers of eggs [5759].
The most exhaustive data were produced by Italian
groups in the next few years due to introduction of the
Italian law (40/2004) in which oocyte freezing was no

longer an option but rather a main pillar in the IVF
routine.
During fresh cycles only a few oocytes can be
inseminated; therefore, cryopreservation is the only
option to avoid wastage of surplus eggs and consequent repeated ovarian stimulation. During the thawing cycles it is possible to use just a limited number
of oocytes, which can lead to obvious disadvantages
such as the poor or insufficient number of embryos
transferred and consequent unsatisfactory pregnancy
outcomes. This situation brought about a new challenge: the need to find a protocol that could maintain
high survival and fertilization rates and enhance the
implantation rate/oocytes. In this way, even the thawing of few eggs/cycles can lead to a high pregnancy
rate. The first reports were mainly based on a slow
freezing using high sucrose concentration (0.3 M);
Chamayou et al. showed no difference in fertilization
rate between fresh and sibling frozenthawed oocytes
cryopreserved with 0.3 M sucrose protocol [60]. The
cleavage rate and embryo quality were significantly
reduced in the frozenthawed group (P 0.001) confirming, once again, that an excess of dehydration may
compromise the oocyte developmental potential. La
Sala et al. despite the very good data related to fertilization, cleavage and embryo quality did not show
an improvement in pregnancy and implantation rate
(4.2 and 5.8%, respectively) [61]. The same result was
obtained by Borini et al. on 927 oocytes from 146
patients [41]. Again, very good survival (74.1%), fertilization (76%) and cleavage (90.2%) rates were not supported by corresponding results in terms of pregnancy
and implantation rates, which were still disappointing (12.3 and 5.2%, respectively). Another study was
performed by Levi Setti et al. with analogous results
[42]. Oktay in 2006 published a meta-analysis calculating the combined outcomes of 26 reports using slow
freezing, mature oocytes and ICSI, published prior
to June 2005 [62]. These studies included approximately 4000 thawed oocytes. Clinical pregnancy per
thawed oocyte was 2.4%, while the implantation rate
per transferred embryo was 13.1%. Analysis of the 7
studies using slow freezing published between June
2005 and March 2006 evidenced a clinical pregnancy
per thawed oocyte (n = 2409) of around 2.2%, while
implantation rate per transferred embryo was down
to 6.5%.
More interesting was the retrospective study conducted by De Santis et al. who highlighted the differ-
ences between the two protocols applied in the literature at that time (using 0.1 and 0.3 M sucrose) [63]. It
was surprising that, while the survival and fertilization
rates were significantly improved using higher sucrose
concentration, the pregnancy and implantation were
much improved using low sugar-based solutions. This
may be due to the subtle equilibrium between dehydration enhanced by sucrose and possible subcellular
damages caused by the same cryoprotectant. At this
point it was clear that, besides the biological good
outcome, the high sucrose-based protocol was not producing good results in term of pregnancy and implantation.
Starting from this important observation, Bianchi
et al. established a modified cryopreservation protocol
in which the freezing solution contains 1.5 M PROH
and 0.2 M sucrose in order to reduce the impact of
shrinkage during cooling procedures [64]. The higher
sucrose concentration (0.3 M) was used during thawing, after Fabbri et al. [40], in keeping with the original
idea of Lassalle et al. [54], who used a thawing solution in which sucrose concentration was higher than
that employed in the freezing solution. The survival
rate reported was high and comparable to the previous studies (76.0%) as is the rate of fertilization (76.2%)
and embryo cleavage (93.7%) [64]. The significant difference was evidenced in the pregnancy rate, which
was 21.2, 18.9 and 21.8% per embryo transfer, thaw
cycle and patient, respectively, and the implantation
rate was 13.4%. Pregnancy rate per cryopreserved
thawed oocyte was 4.9% and the implantation rate per
oocyte was 6.9%. These results represent an important achievement in the everyday application of oocyte
freezing.
In a multicenter study, Borini et al. showed that,
out of 2046 patients involved in oocyte cryopreservation, the overall survival rate of thawed oocytes was
55.8% [65]. In 940 thaw cycles, the mean numbers
of inseminated oocytes and fertilization rates were
significantly decreased versus fresh cycles outcomes
(2.6 0.7 versus 2.9 0.2 and 72.5 versus 78.3%,
respectively), as were the rates of implantation (10.1
versus 15.4%), pregnancy rates per transfer (17.0 versus 27.9%) and pregnancy rates per cycle (13.7 versus
26.2%). Even though differences in clinical outcome
were found among centers, a pregnancy rate per thawing cycle above 14% was achieved by most clinics.
It is evident that oocyte cryopreservation is able to
add a valuable option to routine IVF procedures, especially in countries with severe law restrictions.
289
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58. Fosas N, Marina F, Torres PJ et al. The births of five
Spanish babies from cryopreserved donated oocytes.
Hum Reprod 2003; 18: 141721.
59. Tjer GC, Chiu TT, Cheung LP et al. Birth of a healthy
baby after transfer of blastocysts derived from
cryopreserved human oocytes fertilized with frozen
spermatozoa. Fertil Steril 2005; 83: 15479.
60. Chamayou S, Alecci C, Ragolia C et al. Comparison
of in vitro outcomes from cryopreserved oocytes and
sibling fresh oocytes. Reprod Biomed Online 2006; 12:
7306.
61. La Sala GB, Nicoli A, Villani MT et al. Outcome of
518 salvage oocyte cryopreservation cycles performed
292
as routine procedure in an in vitro fertilization

program. Fertil Steril 2006; 86: 14237.
62. Oktay K, Cil PA and Bang H. Efficiency of oocyte
86: 7080.
63. De Santis L, Cino I, Rabellotti E et al. Oocyte
cryopreservation: clinical outcome of slow cooling
protocols differing in sucrose concentration. Reprod
64. Bianchi V, Coticchio G, Distratis V et al. Differential
sucrose concentration during dehydration (0.2 mol/l)
and rehydration (0.3 mol/l) increases the implantation
rate of frozen human oocytes. Reprod Biomed Online
2007; 14: 6471.
65. Borini A, Levi Setti PE, Anserini P et al. Multicentric
observational study on slow-cooling oocyte
cryopreservation: clinical outcome. Fertil Steril 2010,
Jan. 2. [Epub ahead of print]
Section 6
Chapter
24
Cryopreservation of human oocytes and

the evolution of vitrification technology
for this purpose
Michael J. Tucker and Juergen Liebermann
Introduction
In principle, the aims we expect from a cryopreservation technique that allows us to store a biological
material at the low temperature of liquid nitrogen
(196 C) with the tissue free of any crystalline structure, and with arrest of all molecular diffusion and
chemical processes which would otherwise precipitate
degradation and aging, are as follows:
1.
2.
3.
4.
To be able to arrest the metabolism reversibly.

To maintain structural and genetic integrity.
To achieve acceptable survival rates after warming.
To maintain developmental competence
post-cryostorage.
5. For such a technique to be reliable, relatively
simple and repeatable.
To accomplish these fundamental goals, two basic
approaches have been developed: controlled slow-rate
freezing [1] and a rapid cooling technique referred to
as vitrification [2]. Specifically, the ability to be able
to routinely cryopreserve human oocytes represents
an important step forward in assisted reproductive
technology (ART), and comes after more than two
decades where studies of oocyte cryopreservation have
long been the focus of unsuccessful efforts to perfect
its clinical application. Very recently, however, vitrification as an alternative to traditional slow-freezing
protocols has been shown to provide high degrees of
success for cryopreservation of mature metaphase-II
(MII) human oocytes.
In this chapter we shall consider the evolution
of the practical application of cryopreservation tech-
nology to achieve consistently acceptable levels of

cryosurvival of this highly cryosensitive gamete while
retaining its inherent competency.
In general, the cryopreservation of any biological
material includes six principal steps:
1. Initial exposure to a cryoprotectant, which
promotes gradual cellular dehydration by partial
removal of intracellular water.
2. Cooling (slow or rapid) to sub-zero temperatures
(196 C).
3. Secure storage at this low temperature.
4. Thawing or warming with gradual cellular
rehydration.
5. Dilution and removal of the cryoprotectant agents
and replacement of the cellular and intracellular
fluid at a critical rate and temperature.
6. Ultimately, recovery and return to a physiological
environment.
Compared to conventional slow-rate freezing where
the concentration of the cryoprotectant is relatively
low, and the cooling rate is very slow to avoid deleterious ice crystallization, vitrification is an ultrarapid cooling technique that requires higher concentrations of cryoprotectant. The physical definition of vitrification is the solidification of a solution
at low temperature, not by ice crystallization (as in
conventional freezing) but by extreme elevation in
viscosity during cooling; in this approach the cells
are placed into the cryoprotectant and then plunged
directly into liquid nitrogen. Water is largely replaced
by the cryoprotectant. For the vitrified state to be
successfully achieved, cooling rates have to be high.
293
Figure 24.1 Vitrification is the solidification of a solution (glass

formation) without ice crystallization. Two droplets of different
solutions plunged directly into liquid nitrogen: the right droplet is
pure Dulbeccos phosphate-buffered saline (DPBS) with ice
crystallization; in contrast ,the left droplet contains an equi-molar
combination of 15% ethylene glycol (EG) and dimethyl sulfoxide
(DMSO) with 0.5 M sucrose in DPBS without ice crystallization (i.e.
glassification in the vitrified state).
For example, with human oocytes the cooling rate has

to be in excess of 15 000 C/min. During vitrification
an aqueous solution is transformed directly from the
liquid phase to a glassy vitrified state (Figure 24.1).
With this method no ice crystals form that can damage the cells or the tissues. While the terms freezing
and thawing are commonly used for conventional
cryopreservation, the terms vitrifying and warming should be used for vitrification procedures. A par-
ticular problem for cells and tissues can be the high

concentrations of cryoprotectants that are required to
effect vitrification, as there exist biological limitations
on the concentration of cryoprotectant that can be tolerated by the cells during vitrification. Thus, a major
consideration during the development of vitrification
protocols has been to attempt to increase the speed of
temperature change, both during cooling and warming, while reducing the concentrations of cryoprotectants as low as possible without endangering the efficacy of the vitrification process [3].
In the clinical context there has been considerably
less experience with vitrification technology in human
ART, and it is still in the process of being accepted as a
standard means to cryopreserve oocytes and embryos,
although this situation is very rapidly changing both
for cryostorage of embryos [416] and oocytes [17
28]. Indeed, the number of publications regarding vitrification in ART has increased exponentially over the
last decade (Figure 24.2).
Why cryopreserve human oocytes?

The last few years have seen a significant resurgence
of interest in the potential benefits of human oocyte
cryostorage [19, 29]. The benefits include the formation of donor egg banks to facilitate and lessen
the cost of oocyte donation for women that are
unable to produce their own oocytes, through greater
ease of coordination and synchronization of donor
and recipient cycles [3032]. Also of major potential
Number of entries for vitrification
250
200
200
161
157
132
122
150
102
100
64
50
12 15 14
1 4 1 6 1 5 6 5 5
21
52
31
22 23 30 29
19
66
19
77
19
79
19
84
19
86
19
88
19
90
19
92
19
94
19
96
19
98
20
00
20
02
20
04
20
06
20
08
48 48
96
73 74
Years
Figure 24.2 The explosion of interest in the use of vitrification is beginning to affect clinical embryo and oocyte storage. (Data derived from
the Scopus Library the worlds largest abstract and citation database of research literature and web sources; it offers access to 245 million
references and 27 million abstracts from over 15 000 peer-reviewed journals.)
294
Chapter 24: Cryopreservation of oocytes vitrification
benefit is the provision of oocyte cryostorage services

for women wishing to delay their reproductive choices
for a variety of reasons. For women about to undergo
therapy potentially harmful to their ovaries that may
threaten their reproductive health, oocyte cryostorage represents the only truly proven clinical recourse
[33]. Additionally, certain countries have restricted
access to cryopreservation of human embryos by limiting the numbers of oocytes that may be inseminated;
therefore, restricted insemination of oocytes has been
undertaken with cryostorage of the surplus oocytes
becoming the standard reproductive protocol, albeit
with variable outcomes [20, 34]. Limited insemination
of oocytes by patient choice (elective limited insemination [ELI]) probably has a burgeoning relevance
for in vitro fertilization (IVF) worldwide as comfort
grows with reduced superovulation, improved oocyte
cryostorage and single embryo transfer. The growth in
ELI will be driven both by womens appreciation of
the legal benefits of ownership of their own gametes
[29] and the improvement in IVF embryo selection
technology [35]. There also exist certain clinical situations where oocyte cryopreservation can be applied
to rescue an ovarian stimulation cycle; for example,
in an IVF attempt where unexpectedly no spermatozoa are available, then the oocytes can be cryostored
until such time as a resolution can be found, and this
may even involve cryopreservation of both gametes
and embryos [36]. Even with an intrauterine insemination cycle, where ovarian stimulation might be too
exuberant, then oocyte retrieval and cryostorage can
be applied, leaving one or two follicles intact to allow
for intrauterine insemination (IUI) to be undertaken
as planned to achieve live births [37] (M. J. Tucker et
al., Unpublished data from Shady Grove Fertility RSC,
20079) (Table 24.1).
Relevance of human oocyte

cryopreservation
The majority of oocyte freezing so far applied clinically
has been based directly on traditional human embryo
cryopreservation protocols. Such protocols utilize a
slow-freeze/rapid-thaw approach necessitating use
of a programmable freezer, and these protocols have
produced to date the majority of the approximately
900 offspring worldwide [26]. This is in comparison to
500 000 offspring born from mostly conventionally
cryopreserved embryos. Fortunately to date, no significant increase in abnormalities has been reported
Table 24.1 Clinical use of donor and autologous oocyte

carrier and
vitrification using the Cryolock (Biodiseno)
M199-based vitrification media (Irvine Scientific, CA) or media as
in Table 24.3
Donor
oocytes
Autologous
oocytes
Warming cycles
12
21
Oocyte survival (%)
53/64 (83%)
140/187 (75%)
Fertilization (%)
48/53 (91%)
101/140 (72%)
Transfers
12
17
Pregnancies/embryo
transfer (%)
9 (75%)
5 (29%)
Implantations (%)
9/24 (37.5%)
7/48 (14.5%)
Unpublished data from Shady Grove Fertility RSC, 20079.
from these cryostored oocyte pregnancies [38], regardless of the historical concerns that cryopreservation
of mature oocytes might disrupt the meiotic spindle and thus increase the potential for aneuploidy in
the embryos arising from such eggs. These concerns
have mostly been allayed by publications that show
no abnormal or stray chromosomes from previously
frozen oocytes [39], and fluorescence in situ hybridization (FISH) comparison of embryos from fresh and
thawed oocytes show no increase in anomalies [40].
There also appears to be adequate recovery of the
meiotic spindle post-cryopreservation whether using
conventional or vitrification technology [4143]. With
respect to cryostorage of donated oocytes, there have
been a number of reports that have described success with this approach [23, 30, 32, 44, 45]. Indeed
this approach has now been formally commercialized.
Cryostorage of womens own oocytes was originally reported with three pregnancies established in
the late 1980s by two centers [46, 47]. Following a hiatus of several years, these early reports were reproduced by others [17, 30, 48]. These pregnancies mostly
arose from the freezing of oocytes that had been
collected for purposes of infertility therapy where
couples may have had religious or ethical concerns
with embryo cryopreservation; or when couples consented to research studies; or even when spermatozoa
were unexpectedly unavailable after oocytes have been
retrieved during an IVF cycle. The latter circumstances
would have occurred following an unsuccessful testicular biopsy or when the partner was unable to produce
a semen sample for a variety of unanticipated reasons.
295
Figure 24.3 (a) Mature metaphase-II (MII) stage oocyte; (b) immature MI stage oocyte; and (c) germinal vesicle (GV) stage oocyte.
All of these pregnancies arose from frozenthawed

mature oocytes, but for one notable exception, where
a pregnancy arose from an immature germinal vesicle (GV) stage oocyte [49]. It has been considered that
this stage of oocyte maturity might prove to be a more
successful approach for cryopreservation because its
oolemma is more permeable to cryoprotectant and its
chromatin is more conveniently and safely packaged in
the nucleus [50]. This has not proven to be the case,
largely because such eggs still have to undergo GV
breakdown and maturation to the MII stage before fertilization, and therefore their developmental competency is not so clearly established as with fully mature
oocytes that are cryostored. The source of GV stage
eggs, their specific derivation, and whether they have
been exposed to any exogenous gonadotropins may
well play a key role in the competency of such immature eggs [51]. Recently, with the increased interest
with in vitro maturation (IVM) as an alternative to
standard IVF with superovulation, oocyte cryopreservation has played an increasing role, although to date
only mature oocytes have been successfully cryostored
using vitrification after IVM [52].
Whether using mature oocytes or not, conventional freezing technologies have suffered from inconsistency in terms of cryosurvival. Consequently, there
have been attempts recently to introduce significant
modifications to overcome this apparent drawback.
One approach has been to replace sodium as the
principal cation in the cryoprotectant with choline
that does not diffuse through the plasmalemma, in
an attempt to reduce the potential for cell toxicity arising from high solute concentrations (solution effects) that occur during cooling as the water
becomes ice, so removing water from the system [53].
This cationic replacement has been shown to be beneficial in a mouse model of conventional freezing, but
296
begs the question why embryos can routinely be safely

and successfully frozen in sodium-based media [54].
Another modification that has been adopted has been
to increase the concentration of the non-permeating
cryoprotectant sucrose in the freezing media [55].
Such modifications appear to have improved clinical outcomes; indeed, higher sucrose concentration
may improve cryosurvival. However, it may not necessarily improve overall clinical outcomes [56]. Intracytoplasmic sperm injection (ICSI) has become the
accepted norm for insemination of oocytes postthaw, to avoid any reduction in sperm penetration of
the zona pellucida due to premature cortical granule release precipitated by the cryopreservation process [39, 57]. However, it has recently been suggested
that the use of calcium-free cryoprotectant during vitrification of mouse oocytes may lessen zona hardening, thus increasing the fertilization of the oocytes
following conventional insemination [58]. In any
event, as cryostored oocytes after thawing/warming
are often in low numbers and represent precious
gametes, it seems only reasonable to apply ICSI, as
an acknowledged successful adjunct to IVF technology, to achieve the highest consistent levels of fertilization.
Oocytes retrieved after controlled ovarian hyperstimulation will be principally mature (metaphase of
meiosis II with extruded first polar body [MII]: Figure 24.3a). Oocytes from unstimulated cycles or following minimal use of gonadotropins may be mostly
immature (intermediate post-germinal vesicle breakdown with no polar body extruded [GVBD] or MI
stage and germinal vesicle stage [GV]: Figure 24.3 b, c).
Oocytes may be cryopreserved at any stage, but mature
or GV stage oocytes are preferred. If GV stage oocytes
are cryostored, IVM of these eggs post-thawing will
need to be accomplished before fertilization can be
attempted. Alternatively, immature oocytes may be left

to mature prior to cryopreservation. A pregnancy from
the former approach cryopreserving GV stage oocytes
with subsequent thaw and IVM resulted in a successful live birth over 10 years ago [49]; however, the latter
approach has proven to be more effective, with vitrification of only those oocytes that reach maturity in
vitro [38, 59].
It has long been understood that unfertilized
oocytes are more difficult to cryopreserve than cleavage stage embryos. This has largely to do with the
oocytes surface to volume ratio, the reduced membrane permeability of its oolemma, the temperaturesensitive metaphase spindle and zona pellucida and its
susceptibility to parthenogenetic activation and chill
injury. Membrane characteristics also vary between
oocytes and embryos and between oocytes of differing
maturities [60].
Outcomes with oocyte

cryopreservation
The scientific literature on oocyte cryopreservation
seems to grow daily. Most reports focus on clinical
pregnancy rates [61, 62] and, as such, while this data
is helpful to increase our confidence with the technology, it does little to research new directions for oocyte
cryopreservation. It can be difficult to establish baseline performance with any technology in human ART;
however, an acceptable level of research can be undertaken in the area with appropriately donated tissue; i.e.
with full Institutional Review Board approved patient
consent. One such source is oocytes that remain
unfertilized or unmatured the day after retrieval in
IVF; or that are immature on the day of retrieval
and not available as such for ICSI, which nevertheless reach maturity overnight and are donated by
the woman undergoing IVF therapy. Table 24.2 is one
such example that shows the oocyte post-warming survival rates after using two different carriers for vitrification: the Cryoloop and the Hemi-straw. A total of
928, aged and donated for research, human oocytes
that failed to fertilize were subjected to vitrification.
(The oocyte survival rate following 24 h of culture
appeared lower in the Cryoloop group than in the
Hemi-straw group [80.6 versus 85.4%], but this difference was not significant [ 2 ; P = 0.061].) Overall the
percentage of surviving oocytes was 83.0% (771/928),
which is a level that has helped promote clinical
Table 24.2 Post-warming survival of human failed-fertilized

oocytes after 24 h culture relative to the different vitrification
carriers used
Vitrification technique
Cryoloop
Hemi-straw system
Number of oocytes
448
480
Oocyte survival
361 (80.6%)a
410 (85.4%)b
Overall survival
a
b
738/890 (82.9%)
From Liebermann and Tucker [63].

P = 0.061, according to 2 test.
introduction of this technology for oocyte cryostorage

(Table 24.2) [63].
Improving current oocyte

cryopreservation protocols
Oocyte cryopreservation is finally entering the mainstream of techniques in assisted reproduction. For
sometime the reticence to apply oocyte cryopreservation was largely due to the perception that there were
no obvious clinical niches requiring its routine use.
As a reproductive technology it has more recently suffered from the stigma of being seen as experimental (ASRM Committee Opinion; http://www.asrm.
org/Media/Practice/Essential elements.pdf). This is
changing due to legislation that has restricted use
of embryo cryopreservation in some countries, and
to a growing awareness on the part of certain infertile women that choose not to inseminate all of their
oocytes either for ethical or practical issues (ELI).
Cryostored oocytes are the property of only the
woman, as opposed to cryostored embryos that are
co-owned [29]. It was predicted over 10 years ago
that the most likely area of early routine adoption of
oocyte cryopreservation was to facilitate oocyte donation, as was illustrated at that time by the first offspring that arose from cryostored donor oocytes [30].
In any event, practice makes perfect is the key here,
so that the more clinical experience we gain, the more
we will hone our skills with oocyte cryopreservation.
Animal models go only so far to enable us to improve
human assisted reproduction; ultimately, it is clinical
application which is the key to improvement, as has
been seen with embryo cryopreservation. With oocyte
donor egg banking, information in terms of clinical success of protocols is generated within months not
years, as would be the case with cryostorage of oocytes
297
Table 24.3 Typical vitrification and warming media
EG (%) Sucrose (M) DMSO (%)

Equilibration solution
7.5
0.0
7.5
Vitrification solution
15
0.5
15
Warming solution
1.0
Dilution solution
0.5
Holding solution
0.0
DMSO, dimethyl sulfoxide; EG, ethylene glycol.

Holding solution: hepes-buffered human tubal fluid (modifiedHTF) plus 20% protein supplement (e.g. SPS; Sage Biopharma,
CA).
for single women concerned with their future reproductive choices (insurance freezing/self donation)
[64]. In accepting that human oocyte cryopreservation is here to stay, it is of great importance that we
research the consequences of this technology carefully,
to ensure that we do no harm, through establishment
of an effective registry of outcomes and births from
oocyte cryopreservation.
Conventional cryopreservation has seen significant improvement in consistency of outcomes the past
few years in the form of the introduction of ICSI
as the routine insemination approach after cryostorage, choline-based (sodium-depleted) cryo-media and
use of higher concentration sugar solutions. Within
the confines of conventional freezing technology there
may be subtle variations in approach that will only be
revealed through ongoing clinical studies. Major leaps
of technology, such as injection of trehalose into the
oocytes before freezing [65], while theoretically attractive, nevertheless impose an increased level of invasiveness that seems to run contrary to the wish to simplify oocyte freezing. However, we should remain open
minded given that little has been done to date to assess
oocyte quality at the cellular level, other than to determine the status of the meiotic spindle [41].
With regard to vitrification as a means to cryopreserve oocytes, increased speed of cooling through use
of better designed carriers and protocols that lessen the
concentration of cryoprotectant used, while hastening
exposure and procedure times, have put this technology on the map [66], and excellent embryo quality can
be obtained from vitrified oocytes [67]. Furthermore,
the most widely used protocol for vitrification of biological material is a two-step partial equilibration in
the cryoprotectant, and a three-step warming procedure in sucrose only (Table 24.3).
298
Establishing and troubleshooting

clinical oocyte cryostorage
In an attempt to help clinics to set up or evaluate oocyte
cryopreservation as a standard clinical procedure, a
series of considerations are made here to assist with
focus on how to achieve consistent success:
r Finding the right carrier for loading and storing
oocytes is essential. A wide variety of carriers is
available, which can be either open or
closed/sealed systems. An important
requirement for a carrier is that it provides safety,
easy handling, high recovery rate as well as high
cooling rates (Figures 24.4 and 24.5). In addition,
achieving consistent success with vitrification
technology requires a distinct learning curve
(e.g. Figure 24.6).
r Selection of oocytes for cryopreservation is critical
for consistently good cryosurvival and acceptable
clinical outcomes from use of such oocytes.
Selection criteria, in addition to noting the precise
maturational stage (see Figure 24.3ac) include:
size (to avoid use of oversized giant oocytes),
morphology and cytoplasmic health (avoidance
of vacuolation, heterogeneity, etc: Figure 24.7).
r If, in spite of good selection practices for the
oocytes to be cryopreserved, cryosurvival is poor,
then the following potential problem areas should
be investigated:
1. Test the cryoprotectant media, vitrification
carrier plus other consumables and the
protocol in general with a mouse oocyte
bioassay.
2. Confirm the appropriate temperatures for
work surfaces pre-cryopreservation and
postcryopreservation.
3. Confirm that production or delivery of the
cryoprotectant media were undertaken
correctly.
4. With respect to vitrification in particular, be
aware of how vitrification is less
protocol-driven with less precise timing of
steps, given that passage of the oocytes both
into and out of the vitrification and warming
solutions is more dependent on what is
actually happening to the cell in terms of
shrinkage and re-expansion (Figures
24.824.11). Thus, a flexible approach is highly
recommended.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
5. Finally the speed of both the final vitrification

step and the warming step out of the liquid
nitrogen is critical, such that if these steps are
delayed, cryosurvival may well be
compromised due to an increased potential for
ice crystal formation.
Note that morphological cryosurvival itself is not the
sole criterion to judge the success of oocyte cryopreser-
Open system
(Direct contact
with LN2
Cooling rates
>20 000C)
Figure 24.4 Vitrification carriers. (af)

Cryoloop; (gi) Hemi-straw
(homemade).
Figure 24.5 Vitrification carriers.(ac)

Cryoleaf; (de) HSV (High Security
Vitrification Kit); (f) Cryotip; (gh) Cryotop;
(i) Fibre Plug.
Closed system
(No direct
contact with LN2
Cooling rates
~1750C)
vation: the oocytes themselves must be able to fertilize normally at an acceptable rate; develop morphologically as would fertilized fresh oocytes; and ultimately to implant at a clinically appropriate rate. Poor
embryonic cleavage is especially indicative of cryptic
cyto-skeletal damage that may have incurred during
the cryopreservation process. It has to be remembered,
however, that oocytes are only as good as they were
299
Vitrification/warm (% survival)
120
100
80
60
40
20
0
% Survival
5
Trials
Figure 24.6 Oocyte vitrification preclinical studies at Reproductive

Specialists of New York for one embryologist using a Cook Flexipet
tip within a sealed Cryo-straw where a distinct learning curve
occurred. Cumulative viability post-warming from all trials was
144/168 (85.7%), establishing clinical acceptability for that
individual.
Figure 24.7 Preselection of oocytes for vitrification. For example,
an oversize (probably diploid), dysmorphic and vacuolated oocyte
after stripping in hyaluronidase (40 IU/ml).
Figure 24.8 Collapsed metaphase-I (MI) oocyte in 7.5% ethylene

glycol (EG)/7.5% dimethyl sulfoxide (DMSO) prior to vitrification.
Figure 24.10 Mature metaphase-II (MII) oocytes post-vitrification.
300
Figure 24.9 Immature metaphase-I (MI) oocytes post-vitrification

(note no polar body present).
Figure 24.11 Human oocytes after vitrification in 15% ethylene

glycol (EG)/15% dimethyl sulfoxide (DMSO) plus 0.5 M sucrose, with
warming in dilutions of sucrose.
at their time of collection, and fundamental compromise of oocyte quality may occur during ovarian stimulation, retrieval and, more problematically, innate
quality issues related to a womans physiology may
make assessing these developmental issues less clear
cut. Indeed, there exists a natural variability between
women, and often between stimulation cycles from the
same woman; such fluctuations in oocyte quality may
have a significant negative impact on innate competency of such eggs. However, it is our mission during
oocyte cryopreservation to minimize any deleterious
conditions that reduce oocyte quality further on a permanent basis.
The continuing evolution of oocyte

cryopreservation
Before the currently established vitrification protocol becomes fossilized as the standard approach for
oocyte cryostorage, it is important to consider the
effort and studies that have been undertaken over the
years to achieve success with this technique. So it
remains important for us to continue to seek improvement in outcomes for all women regardless of their
needs for oocyte cryostorage and the quality of their
oocytes. There are two main ways to efficiently achieve
the vitrification of water inside cells. One is to increase
the temperature difference between the samples and
vitrification medium, and the second is to find materials with fast heat conduction. However, the actual rate
during vitrification procedures may vary extremely
depending on the device used, technical proficiency
and even the specific movement of the carrier at
immersion into the liquid nitrogen. Every oocyte has
its own optimal cooling rate, and oocytes are cells that
are exquisitely prone to chilling injury. To date vitrification as a cryopreservation method has had relatively
little practical impact on human assisted reproduction.
With improving awareness of the technology, plus its
refinement especially in the commercial arena, added
to the growing numbers of reports of successfully completed pregnancies following vitrification makes for an
extremely encouraging future for its wider adoption,
and it bodes well for the wider spread use of vitrification for cryopreservation of both human oocytes and
embryos. Whether there exists a need to improve cooling rates yet further with use of low-pressure slush
liquid nitrogen machines [68], use of high hydrostatic
pressure systems to condition oocytes prior to vitrification [69] or to undertake cooling on supercooled
metal surfaces [70] remains to be seen. Such developments do detract from the simplicity and low-cost benefits of vitrification; however, even higher cooling rates
may reduce even further the need for the relatively
high concentrations of cryoprotectant and so allow vitrification to be an even more low-impact cryopreservation approach for oocytes.
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Section 6
Chapter
25
Cryopreservation and transplantation of

isolated follicles
Marie-Madeleine Dolmans and Anne Van Langendonckt
Concept of minimal residual disease

For pre-pubertal patients and women who cannot delay the start of chemotherapy, cryopreservation of ovarian tissue is the main option available
to preserve their fertility before cancer treatment.
Indeed, the prospect of reversing treatment-related
premature ovarian failure by autotransplantation of
frozenthawed ovarian tissue harvested before chemoradiotherapy is becoming increasingly real, with 11
live births already reported using this technique [1
7]. It is expected that, in the near future, an increasing
number of cancer patients cured of their disease will
request re-implantation of cryopreserved ovarian tissue [8].
However, very little has been published on the
safety of ovarian tissue transplantation in cancer
patients [9]. One major concern raised by the use of
ovarian cortical strips is the potential risk that the
frozenthawed ovarian cortex might harbor malignant
cells. This could induce a recurrence of the disease
after re-implantation if biopsy is carried out before
chemotherapy. Shaw et al. previously reported that
ovarian grafts from AKR mice could transfer lymphoma to recipient animals [10]. More recent studies tested the safety of cryopreserved human ovarian tissue from lymphoma patients for transplantation,
and suggested that ovarian tissue transplantation in
Hodgkins disease is safe [1113]. Selection of patients
for the procedure should therefore take into account
the malignancy type and its activity. In breast cancer,
for example, metastasis may spread to the ovaries [11],
though a recent study by Sanchez-Serrano et al. did
not evidence malignant cells in ovarian cortex from
breast cancer patients by immunohistochemistry [14].
In leukemia, malignant cells may be present in the

bloodstream, with the risk of transferring leukemic
cells [15].
Hematological malignancies are the most frequent
indication for ovarian tissue cryopreservation in our
department, and represent 44% of all malignant indications. Among them, Hodgkins lymphoma accounts
for 22% of cases, followed by leukemia (11.3%) and
non-Hodgkins lymphoma (11%) (Figure 25.1).
Because frozenthawed ovarian tissue may contain malignant cells that could potentially lead to disease recurrence after re-implantation, we decided to
conduct a study to evaluate the presence of leukemic
cells in cryopreserved human ovarian tissue from
patients with chronic myeloid leukemia (CML) and
acute lymphoblastic leukemia (ALL). Detection of
minimal residual disease in ovarian tissue was carried
out by histology, real-time quantitative polymerase
chain reaction (RT-qPCR) and long-term xenografting
(6 months).
Histology did not reveal any malignant cells. By
RT-qPCR, 2 of the 6 CML patients were positive for
BCR-ABL in their ovarian tissue, while among the
12 ALL patients, 7 of the 10 with available molecular
markers showed positive leukemic markers (translocations or rearrangement genes). Four mice grafted with
ovarian tissue from ALL patients developed intraperitoneal leukemic masses.
In conclusion, this study evidences, by RT-qPCR,
ovarian contamination by malignant cells in acute as
well as chronic leukemia, while histology fails to do
so. Moreover, our results also indicate that chemotherapy before ovarian cryopreservation does not exclude
malignant contamination. Finally, re-implantation of
305
3.8
3.2 2.7 2
0.6
Leukemia
11.3
Hematological
malignancies
44.3
9.6
Hodgkin's
lymphoma
22
22.6
Breast cancer
Ovarian cancer
Rhabdomyosarcoma
Cervical cancer
Liposarcoma
Leukemia
Non-Hodgkin's
lymphoma
11
Other cancers (pancreas, stomach,)

Osteosarcoma
Colorectal cancer
Ewing's sarcoma
Hodgkin's lymphoma
Non-Hodgkin's lymphoma
cryopreserved ovarian tissue from leukemic patients

puts them at risk of disease recurrence.
Concept of follicle isolation

To avoid transferring malignant cells together with
grafted ovarian tissue, ovarian follicles may be isolated
from the surrounding tissue and then either cultured
for in vitro follicle maturation or transplanted directly
into the recipient.
Culturing isolated follicles from the primordial
stage is a particularly attractive proposition, since they
represent more than 90% of the total follicular reserve
and show high cryotolerance [16]. It is widely reported
that isolated primordial follicles do not grow properly
in culture [17, 18], because follicle isolation [18, 19]
or partial follicle isolation[17, 18] severely impairs follicular viability in culture, and that primordial follicles develop best in organ culture. Indeed, encouraging results were achieved by Hovattas team [20] and
Telfers team [21] where follicles grown in culture of
human ovarian cortical strips could be isolated at the
secondary stage and grown in vitro to the late preantral/early antral stage. Nevertheless, recent publications show that encapsulation of isolated pre-antral
follicles in alginate beads may be a suitable system
for in vitro culture of small human pre-antral follicles, as they can survive and grow after enzymatic isolation and in vitro culture for 7 days [22]. However,
this in vitro approach is difficult in humans due to
the prolonged duration of folliculogenesis, and obtaining antral follicles from primordial follicles in vitro
remains challenging.
306
Figure 25.1 Indications for ovarian

tissue cryopreservation in case of
malignant disease at Saint Lucs
University Hospital, Brussels, Belgium in
2008. See plate section for color version.
This led us to consider an alternative strategy,

which involves grafting of isolated ovarian follicles.
For human primordial follicles, mechanical isolation
is not possible due to their size (3040 m) and their
fibrous and dense ovarian stroma, so enzymatic tissue digestion with collagenase is generally used. Different types of collagenase (Ia, II, IX, XI) have been
employed for this purpose, either alone [17, 23] or
in combination with DNase or mechanical isolation
[18, 19, 2428]. However, the drawback of collagenase,
which is a crude preparation derived from Clostridium histolyticum, is that it may contain high endotoxin
levels that could severely impair culture and grafting
outcomes. It also shows substantial variations in effectiveness between batches [29], which may explain the
discrepancies in results between different groups. This
appears to indicate that some crude collagenase preparations may contain components interfering with follicle quality.
The inconsistent results obtained with collagenase
thus prompted us to set up a new follicle isolation protocol using Liberase. Indeed, in order to enhance the
chances of follicular survival after isolation, enzymatic
digestion procedures for human ovarian tissue needed
to be optimized and standardized. Liberase is a blend
of highly purified enzymes that has been successfully
used to improve the quality of human pancreatic islet
isolation [30].
We were able to obtain good quality isolated
human follicles using the Liberase enzyme blend,
showing good morphology and integrity and high
viability (Figure 25.2). Morphology of the isolated
follicles was assessed using DAPI staining with the
Chapter 25: Cryopreservation of isolated follicles
from the stromal cells. After investigating for follicles in the different interfaces, we found 63% in the
phosphate-buffered saline (PBS)1.06 Ficoll interface
and 36.9% at the 1.061.09 Ficoll interface, which represents 99.9% of total recovered follicles. Analysis by
vital fluorescent staining showed that 95.8% of follicles treated with Ficoll were totally viable. The Ficoll
density gradient method thus allows us to maximize
recovery of isolated human ovarian follicles and minimize manipulation time, while maintaining high follicular viability [32].
Figure 25.2 Enzymatically isolated human follicles seen under a
stereomicroscope.
Concept of follicle transplantation
M1M4 classification [31], based on granulosa cell

layer integrity and oocyte extrusion. Viablity of the
isolated follicles was assessed after exposure of the
follicles to calcein and ethidium homodimer-I, while
ultrastructural investigation by transmission electron
microscopy provided us with additional qualitative
information on follicle morphology after enzymatic
isolation. Obtaining good quality isolated follicles is
an absolute prerequisite for their further processing, either for culture or transplantation. This purified endotoxin-free enzyme preparation is a promising
alternative for the reproducible isolation of intact primordial and primary follicles from human ovarian tissue. Moreover, with a view to clinical application and
in accordance with new European laws, good manufacturing practice (GMP) blends are now available.
After ovarian tissue digestion by enzymes, follicles need to be recovered one by one with a glass
micropipette under a stereomicroscope, which can be
very exacting step if the follicle suspension contains
a lot of isolated stromal cells and debris. This is why
we developed a method for the recovery of isolated
pre-antral follicles, based on the Ficoll discontinuous
density gradient separation technique [32]. In the literature, filtration of the digested cortical cell suspension is widely employed to remove stromal debris and
harvest follicles in different species. However, when
this method was used in our laboratory, extensive
follicular loss was noted on the mesh filter surface,
with follicles remaining stuck to the filter after rinsing. The Ficoll discontinuous density gradient separation technique successfully addresses this problem
and provides a suitable means of recovering large numbers of viable primordial follicles and separating them
Another approach to avoid transmission of malignant

cells could be to transplant a suspension of isolated follicles. Transplantation of frozenthawed isolated primordial follicles has been successfully carried out in
mice [33], yielding normal offspring. As the follicular
basal lamina encapsulating the membrana granulosa
excludes capillaries, white blood cells and nerve processes from the granulosa compartment [34], grafting
fully isolated follicles may be considered safe.
After isolation and recovery of human follicles, our
aim was to evaluate the developmental capacity and
viability of these isolated human follicles after transplantation. They were therefore embedded in plasma
clots, serving as vehicles to facilitate subsequent grafting, and xenografted for 1 week to nude mice. Our
study clearly showed that, after xenografting, isolated
human primordial follicles were able to survive and
grow [35]. Their survival and growth was evidenced by
their morphologically normal structure and follicular
stage at histology, as well as positive staining for Ki-67,
a nuclear antigen associated with cell proliferation.
Another finding of the study was follicular activation 1 week after transplantation, as demonstrated
by the decrease in the percentage of primordial follicles and increase in the percentage of primary follicles, accompanied by a much higher proportion of
Ki-67-labeled follicles in the grafts. This phenomenon
was observed after both cortical tissue and isolated
follicle grafting, and may lead to premature depletion of the graft. Further studies are clearly required
to better understand these events occurring during
the first post-transplantation days, in order to preserve the ovarian reserve and prolong graft survival.
A proper balance between inhibitory and stimulatory factors appears to be essential to control primordial follicle activation. Our hypothesis is that, after
307
grafting, this equilibrium might be disturbed due to a

lack of growing follicles and disruption of the vascular supply. Previous studies, including those from our
laboratory, have evidenced factors possibly involved in
the control of the primordial to primary follicle transition [36, 37].
We then extended the grafting period to assess the
developmental capacity of these enzymatically isolated
human follicles after 5 months xenografting to severe
combined immunodeficient (SCID) mice. The results
showed that isolated human primordial follicles are
able to survive after long-term xenografting, and can
develop into antral follicles after follicle stimulating
hormone (FSH) stimulation [38].
As this approach has successfully restored fertility
to mice [33], our optimization of follicle isolation and
recovery protocols now allows us to consider its development for humans.
Concept of artificial ovary

Isolation of human follicles could be done directly
after tissue harvesting on fresh ovarian tissue, as it has
been done in experimental studies so far. These isolated follicles could then be cryopreserved and banked
until use, as described previously with sheep follicles
[39]. Although the easiest and most logical way to
select is probably to cryopreserve the ovarian biopsy, in
strips or fragments, and thaw them the day of isolation
and grafting, it needs to be investigated whether there
could be an advantage in cryopreserving follicles over
tissue. The cryoprotectant permeation might be more
efficacious through a cell suspension than through tissue blocks.
Reseeding isolated follicles, free of cancer cells,
could be considered to restore fertility to patients
where the risk of re-introducing malignant cells by
ovarian tissue autografting cannot be excluded. This
technique requires new surgical approaches to increase
efficiency of graft recovery. Therefore, new artificial
matrices might play an important role. Indeed, tissue
engineering is an expanding field with more and more
applications. New matrixes have been developed based
on hydrogels, foams or natural polymers which could
be suitable to nestle the isolated follicles and form a
kind of artificial ovary before grafting to the patient.
This would allow patients at risk of ovarian metastasis to benefit also from ovarian tissue cryopreservation
and transplantation.
308
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Reproduction 2003; 126, 41524.
35. Dolmans MM, Martinez-Madrid B, Gadisseux E
et al. Short-term transplantation of isolated human
ovarian follicles and cortical tissue into nude mice.
36. Skinner MK. Regulation of primordial follicle
assembly and development. Hum Reprod Update 2005;
11: 46171.
37. Qu J, Godin PA, Nisolle M and Donnez J.
Distribution and epidermal growth factor receptor
expression of primordial follicles in human ovarian
tissue before and after cryopreservation. Hum Reprod
2000; 15: 30210.
38. Dolmans MM, Yuan Yuan W, Camboni A et al.
Development of antral follicles after xenografting of
isolated small human preantral follicles. Reprod
39. Amorim CA, Rondina D, Lucci CM et al.
Cryopreservation of sheep primordial follicles. Reprod
Domest Anim 2007; 42(1): 537.
309
Section 6
Chapter
26
ART and oocyte donation in cancer survivors

Ina N. Cholst, Glenn L. Schattman and Zev Rosenwaks
Introduction
This book is primarily about prevention; its emphasis is on interventions that can be done at the time
of cancer diagnosis modifications of treatment and
techniques for storing gametes, tissues or embryos for
future use. By contrast, this final chapter of Section 6
explores the options open to the cancer survivor some
time after the diagnosis has been made and treatment
completed. If preventive treatment was successful, in
some cases, normal fertility has been preserved. Other
survivors may conceive using the gametes, embryos
or tissue that were obtained and cryopreserved before
their gonadotoxic treatment.
However, in some cases, prevention may not have
been successful. This chapter is written to provide
insight into the fertility management of cancer survivors with compromised or absent ovarian function
and without cryopreserved material.
Before embarking on a pregnancy, no matter how
it is to be achieved, a cancer survivor and her physicians must look at her general health, at her uterine function, at her chance of recurrence and, finally,
at her prognosis for long-term survival. Since these
issues are common to a cancer survivors pregnancies
whether achieved in the natural cycle, through assisted
reproductive technology (ART) or through donor egg
(DE) they will be discussed together in the first part
of this chapter.
The second part of this chapter will focus on situations where the cancer survivor does not conceive
spontaneously but her fertility is amenable to treatment. In situations where ovarian function persists but
is attenuated, ART may be able to overcome treatmentrelated damage. For some cancer survivors, specific
modifications of ART may be indicated.
The third part of this chapter explores situations in

which prevention has failed, was not possible or was
not the right choice for a particular individual. We
emphasize here that DE is not always the last resort.
For a variety of different reasons that will be explored
later, some women may choose DE as the best reproductive choice in a difficult situation.
Evaluation of the cancer survivor

for pregnancy
We begin, then, with the evaluation of the cancer survivor for pregnancy. A flow-chart of cancer survivor
screening is shown in Figure 26.1.
Prognosis
Before other evaluation is done, the treating oncologist
should be consulted about the prospective mothers
prognosis. Has the survivor been disease free for a
sufficient period of time? Is her prognosis for cure
generally good? Is there a risk that pregnancy would
increase cancer recurrence? Kavic and Sauer have suggested that in some cases, but particularly in cases
where cancer is considered to be in remission but not
cured, a second opinion from an independent oncologist may be helpful [1]. Patients and their physicians
may elect to proceed with pregnancy in the presence
of a poor prognosis, or even in situations where pregnancy may increase the risk of recurrence. However,
this should not take place without full discussion and
fully informed consent from all parties involved.
Chronic health problems

Additionally, 75% of survivors of childhood cancer will have at least one chronic health problem.
310
Chapter 26: ART and oocyte donation in cancer survivors
No
Clearance from oncologist
Psychological evaluation
and ethical considerations
Yes
Figure 26.1 Screening of the cancer

survivor for pregnancy (spontaneous,
assisted reproductive technology [ART] or
oocyte donation). MFM, Maternal Fetal
Medicine.
Is the pregnancy high risk?

(e.g. Radiation to pelvis,
chemotherapy-induced
cardiotoxicity?)
Yes
No
No
Routine preconceptual
counseling
MFM consult
(Is pregnancy contraindicated?)
Yes
Test for ovarian function
Consider gestational
carrier
Yes
Attempt pregnancy
with ART
No
Donor egg
Cardiotoxicity from anthracyclines or mediastinal

irradiation is common. Nephrotoxicity may result
from ifosfamide and cis-platinum chemotherapy,
radiotherapy, surgery or immunotherapy. Finally, 1 in
25 survivors of childhood cancers will develop a second primary cancer [2]. Thus, a cancer survivor contemplating parenthood should be evaluated for organsystem damage, and counseled about risks to herself
and the fetus during pregnancy. When indicated, she
should consult with the obstetrician who will care for
her during pregnancy. She should also be made aware
of any significant health risks that might affect her ability to raise a child to adulthood.
Time interval between treatment

and pregnancy
Depending on the nature of the cancer and the type
of gonadotoxic treatment, oncologists may recommend that cancer survivors wait at least a year before
attempting pregnancy [3]. The interval gives the oncologist time to evaluate for recurrence. Furthermore,
it may minimize toxic effects of chemotherapy on
the developing fetus. Experimental studies in animals
have supported concerns about the risk to the fetus
from exposure to chemotherapeutic agents. For example, mice that were exposed to chemotherapy 3 weeks
prior to conception had high malformation rates and

malformation rates remained 10-fold higher than the
control group for conception up to 9 weeks after exposure [4]. In humans, pregnancy outcomes reported
in women previously exposed to potentially mutagenic therapies are reassuring; however, these pregnancies often occur many years after exposure [5, 6].
A wait of at least a year is generally recommended;
however, a safe time interval from chemotherapy to
pregnancy has not been determined. Younger women
with good ovarian reserve may wish to wait for
longer.
Breast cancer survivors, by contrast, are usually
advised to wait 2 years after completing chemotherapy before attempting pregnancy. Breast oncologists
make this recommendation primarily because of the
high rate of recurrence in the first years after diagnosis and not necessarily because of the effects of treatment on pregnancy outcome. The recommended time
interval may be different depending on the age of the
patient and stage of disease with longer intervals recommended for younger patients and those with more
advanced stage disease. Disease-free breast cancer survivors who conceive more than 2 years after finishing
treatment do not appear to be at any increased risk of
recurrence as compared to those who do not become
pregnant following treatment [3].
311
This imposed delay causes concern for many

women they are aware of the higher risk of premature ovarian failure and the reduced chance for pregnancy as time passes. We can help to ease the anxiety
by giving information and support and by not suggesting delay of treatment unless justified.
Uterine evaluation
If a cancer survivor does not achieve a pregnancy in the
first few months of attempting conception, she should
generally have a uterine cavity evaluation a hysterogram or sono-hysterogram to rule out adhesions,
intraluminal pathology or a significant septum. The
uterine evaluation is especially critical for a woman
who has had pelvic or total body irradiation.
Pelvic irradiation often results in damage to the
uterine musculature and vasculature. Childhood radiation, especially, may result in poor uterine growth
during puberty. The smaller uterine volume may
diminish implantation, cause second trimester loss,
preterm labor or placenta accretia [7]. A tripling of
spontaneous miscarriage and a 10-fold increase in lowbirth weight infants have been reported in patients
who had received pelvic radiation therapy (RT) compared to the general population [8].
These risks are affected by the dose of RT delivered to the uterus and by the temporal association
with puberty. There is, for example, a linear correlation between the size of the uterus, the response to hormone treatment and the age at which RT was administered [8]. Some investigators have emphasized that
many reported pregnancies post-pelvic radiation are
delivered prematurely and do not result in the delivery of a healthy child [9].
Although the adult uterus is less sensitive to
the effects of radiation, adult cancer survivors who
received RT to the pelvis should delay pregnancy for
at least a year following RT; it has been suggested that
pregnancies that occur 1 year after RT have a higher
rate of low birth weights and miscarriages [10]. (For a
more detailed discussion of the effects of pelvic radiation on uterine function, see Chapter 2.)
Use of a gestational carrier

In individuals in whom a pregnancy is contraindicated, i.e. where carrying a pregnancy poses a significant risk to the mother or child, a gestational carrier
may be considered. If ovarian function is sufficient and
there is no medical contraindication to ovarian stim-
312
ulation or retrieval, the patients own oocytes can be

retrieved and fertilized for transfer into a hormonally synchronized gestational carrier. In cases of low or
absent ovarian reserve or where there are contraindications to ovarian stimulation, oocytes from a donor
can be fertilized and transferred to the carrier. Fresh
embryos may be transferred when the cycles have been
synchronized in the same way as that used for oocyte
donation (see below) or the embryos may be cryopreserved for transfer into the carrier during a later cycle.
Screening and testing for infectious agents from all
gamete providers reduces the risk to the gestational
carrier. Finally, psychological and legal counsel for all
parties should be obtained before proceeding with a
gestational carrier pregnancy.
ART in cancer survivors

Testing ovarian function
Ovarian reserve, a measure of the quantity of oocytes
remaining in the ovary is difficult to assess accurately.
It can be useful to test ovarian reserve prior to initiating reproductive treatment in cancer survivors since
gonadotoxic treatments oftentimes reduce the primordial follicle pool and effectively age the ovary. Diminished ovarian reserve, even in young women, may
already have been present prior to gonadotoxic treatments [11] and it will almost certainly be exacerbated
after therapy. Early follicular phase follicle stimulating
hormone (FSH) and estradiol (E2), inhibin B, antral
follicle count, anti-Mullerian hormone (AMH) and
response to ovarian stimulation (in addition to other
measures) have all been utilized to estimate ovarian
reserve and indirectly assess the chance for pregnancy.
However, results of ovarian reserve tests in women
who have previously undergone gonadotoxic treatment or are undergoing treatment during the testing period must be interpreted with caution. Patients
who have received chemotherapy in the past have been
shown to have diminished ovarian reserve with abnormal elevations in FSH as well as lower AMH, inhibin
B and antral follicle count [12, 13].
None of the current tests, either alone or in combination, can predict with certainty whether a pregnancy
will be achieved. Therefore, it is not justified to withhold treatment from a properly informed patient.
A cancer survivor who maintains some follicles and
ovarian function should be given a chance to attempt
conception without utilizing her cryopreserved
tissue as long as she is advised of the presumed prognosis in doing so. By contrast, a patient who exhibits
extremely high FSH levels, very low inhibin or AMH
levels or has a negligible antral follicle count, may
wish to discuss oocyte donation or other reproductive
alternatives. If she has previously cryopreserved eggs
or embryos, she should be encouraged to use the
cryopreserved material.
Ovarian stimulation
The physician treating a cancer survivor who wishes
to have children will want to keep one central principle in mind: if the patient was treated with gonadotoxic
agents or pelvic RT, no matter what her present age or
ovarian function, she is likely to have reduced ovarian function earlier than her age would suggest. Therefore, as soon as she is ready, she should be encouraged to attempt conception. If she is not successful within 612 months, a thorough evaluation and
intervention should be undertaken promptly. Finally,
strategies to achieve pregnancy in the shortest time
frame possible are reasonable, if not imperative, in this
population.
For these reasons, we should recommend IVF for
the infertile cancer survivor who fails to conceive a
pregnancy after a limited trial of ovulation induction
and intrauterine insemination (IUI). In vitro fertilization (IVF) offers the greatest chance for pregnancy
in the shortest time interval. This is additionally
important in women with estrogen sensitive tumors in
order to minimize their exposure to multiple courses
of ovarian stimulation.
For women who have stored gametes or embryos
prior to gonadotoxic treatments, the following caveats
are equally important: when ovarian function still
exists and they are prepared to become pregnant, they
should be encouraged to conceive either on their own
or with ART. It should be emphasized that having
frozen oocytes and embryos does not guarantee pregnancy success; rather they should be considered as
the last option should the woman become sterile. One
should not allow women who can still conceive to delay
childbearing because of a false sense of security that
their future reproductive potential is guaranteed by
cryopreservation of either eggs or embryos. And, in
general, and especially if the patient is considering having more than one child, consideration should be given
to maintaining the cryopreserved eggs or embryos in
reserve. If the patient has reasonable ovarian function
and a normal fertility evaluation, she should attempt to

conceive a pregnancy first in a natural cycle with timed
intercourse. If she is unsuccessful after 36 months or
if there is tubal occlusion or male factor, ART would
be the best option. She gives herself the best chance
of fulfilling her reproductive desires by retaining her
frozen gametes or embryos until either her last attempt
at pregnancy or her ovaries fail.
Patients with a history of cancer (not known
to be hormonally sensitive) may be stimulated with
gonadotropins in the same way as patients who are not
cancer survivors. The main difference will be a generally lower response to gonadotropin stimulation and
an accompanying lower implantation and pregnancy
rate as compared to either age-matched controls with
infertility or cancer patients prior to chemotherapy
[14, 15].
Patients who received prior chemotherapy for a
variety of cancer types have significantly lower AMH
levels and fewer oocytes retrieved as compared to agematched patients undergoing fertility preservation
prior to chemotherapy. Additionally, the implantation
rate for patients who had undergone prior chemotherapy was only 7.9%, significantly lower than expected
for the infertile population in the same age group [16].
In a separate study, when a group of patients who had
received local treatments for their cancer was compared to a group who had received systemic therapies, response to stimulation was better in patients
who had received local treatment only. Additionally,
the study suggested that the pregnancy rate in patients
who had received systemic chemotherapy was lower
than that of those who had received only local treatments [14].
Finally, these results suggest that a liberal embryo
transfer policy should be applied. Patients should be
counseled that their chances for pregnancy as well as
the rate of multiple gestation, even with an increased
number of embryos transferred, will be lower as compared to other infertile patients of the same age. Documentation of this counseling should be placed in
the patients record and the reason for exceeding the
clinics transfer guidelines for patients in that age
group should be documented as well.
As in any other IVF situation, additional embryos
can be cryopreserved. There is no reason to believe
that cryopreserved good-quality embryos will not have
reasonable survival rates; however, there is no specific
data regarding outcomes with cryopreserved embryos
in cancer survivors.
313
Ovarian stimulation for hormonally

sensitive cancers
Standard ovarian stimulation for ART results in high
levels of circulating estrogen potentially 10 times the
peak levels of spontaneous cycles. Breast or endometrial cancer survivors and their doctors are appropriately concerned about the impact of these high
estradiol levels on the risk of recurrence. Because of
this theoretical risk, stimulation regimens that utilize
estrogen-receptor modulators such as tamoxifen or
agents which suppress estradiol synthesis (aromatase
inhibitors) have been explored and may be preferable
to conventional stimulation protocols.
Several alternative methods of ovarian stimulation
have been described. These use estrogen receptor modulators or aromatase inhibitors, either alone or in conjunction with gonadotropins, in an effort to stimulate
multifollicular development while reducing or avoiding high serum estradiol levels. Most protocols have
been developed and studied in the context of stimulation of newly diagnosed breast cancer patients for fertility preservation. However, the protocols may have
application for cancer survivors as well.
In a randomized controlled trial, our group compared tamoxifen, tamoxifen in combination with FSH
or letrozole in combination with FSH, to stimulate
follicle development (Figure 26.2). The highest number of total and mature oocytes was obtained in
the letrozole/FSH group (11 1.2 and 8.5 1.6,
respectively) compared to either the tamoxifen/FSH
group (6.9 1.1 and 5.1 1.1 respectively) or the
tamoxifen-alone group (1.7 0.3 and 1.5 0.3,
respectively). Despite high peak estrogen levels in the
tamoxifen, tamoxifen/FSH and letrozole/FSH groups
(419 39 pg/ml, 1182 271 pg/ml, 405 45 pg/ml,
respectively), cancer recurrence rates were not different between groups after a mean follow-up of 554 31
days [17]. The timing of final oocyte maturation with
human chorionic gonadotropin (hCG) in tamoxifen
or letrozole stimulation cycles is similar to protocols using clomiphene citrate: oocyte maturity is typically reached when the lead follicles are approximately 20 mm [18]. Additionally, when patients with
breast cancer underwent ovarian stimulation for fertility preservation using letrozole and gonadotropins at
our institution, there was no difference in disease-free
survival compared to patients who elected to proceed
directly to adjuvant treatments without ovarian stimulation [15]. Randomized studies with modifications
314
antag
hCG
FSH 150 IU
Tamoxifen
with FSH
Tamoxifen 60 mg/days
antag
Letrozole
with FSH
hCG
FSH 150 IU
10
Letrozole 5 mg/days
Letrozole 5 mg/day
12
Cycle days
Figure 26.2 Protocols for ovarian stimulation of the breast cancer

survivor. antag, antagonist; FSH, follicle stimulating hormone; hCG,
human chorionic gonadotropin.
to the above protocols are currently underway at our

institution and others in an effort to further improve
ovarian response and confirm safety. At this time,
we can reassure patients with a history of estrogensensitive tumors that ovarian stimulation, using protocols that modulate the estrogen receptor or the ovarian
hormonal response, do not appear to increase shortterm recurrence. Further studies need to be performed
to confirm and extend these findings.
Patients who are concerned about ovarian stimulation, either with or without modification, may express
interest in natural cycle IVF. The success rates for these
cycles are relatively low. Patients should be counseled
about the possibility of cycle cancellation due to premature ovulation, failure to retrieve an oocyte or lack
of an embryo to transfer. Additionally, the reduced viability of oocytes in patients who have undergone prior
chemotherapy renders natural cycle IVF in this population questionable.
Another option, which has demonstrated some
success in polycystic ovary (PCO) -like patients, is
retrieval of immature oocytes after hCG with or without a truncated course of gonadotropin stimulation
[19]. This field is reviewed in detail in Chapter 36.
The significantly reduced developmental potential of
embryos derived from in vitro matured oocytes (manifested in lower implantation rates and higher miscarriage rates), the need to transfer excess embryos in an
attempt to maintain acceptable pregnancy rates and
the requirement for prolonged hormone replacement
in these women limits the present applicability of this
approach for the cancer survivor [20].
As treatments for cancer improve and survival
extends, more women of reproductive age who have
been exposed to potentially gonadotoxic therapy will
present to their physicians desiring pregnancy. Many
of these women will achieve a pregnancy spontaneously. However, for others, fertility potential will be
reduced. In general, women who have been treated
with gonadotoxic therapy will experience diminished
ovarian reserve and menopause at earlier ages than
their peers.
Women with ovarian function, but diminished
ovarian reserve, can probably safely undergo ART and
can be reassured that, so far, recurrence rates are no
different when compared to women who elected not
to attempt pregnancy. Stimulation with standard protocols for IVF or novel protocols in patients with hormonally sensitive tumors can be undertaken. However,
the success rates in cancer survivors who received systemic chemotherapy or pelvic RT are not equivalent
to their age-matched peers without a history of cancer treatment. Despite advances, some will not achieve
genetic pregnancies. Some will choose to pursue adoption or child free living. Others may choose to pursue
pregnancy through oocyte donation.
Oocyte donation in cancer survivors

Egg donation is not suitable for everyone, and for some
it will be morally, religiously or emotionally out of
the question. However, for those for whom DE seems
a reasonable, less stressful alternative, for those for
whom methods of preserving genetic reproduction are
not feasible or carry risks to survival and for those who
carry a cancer predisposition gene that they prefer not
to transmit, DE has much to offer. It has been with
us for a quarter of a century and has a track record
of success [2123]. In the USA, for example, in 2006,
16 976 donor oocyte cycles were performed and 5393
or 32% of recipients achieved a delivered baby after
a single fresh donor oocyte transfer [24]. The success
rates are much higher approaching 100% if recipients undergo multiple attempts [25].
Depending on the cancer, its stage and grade and,
for some cancers, the presence or absence of estrogenreceptor positive cells, there may be cancer survival
risks associated with ART for fertility preservation.
These hypothetical risks may be either as a result of the
fertility treatments themselves or as a result of delay in
cancer treatment. In most cases, the actual (evidencebased) risk to survival is unknown, further complicating informed consent and decision making for the
young cancer patient.
In addition, some young women recently diagnosed with cancer will be carriers of cancer predis-
position genes. Hereditary breast cancer syndromes

(BrCa 1 and 2), familial adenomatous polyposis, multiple endocrine neoplasia syndromes, retinoblastoma
and hereditary non-polyposis colon cancer syndrome
are only a few examples [26, 27]. Without doubt, as
we begin to understand the biology of cancer, there
will be more in the future. We have already seen that
some of these young people will wish to have children
who are not carriers [2831]. Technology PGD with
transfer of unaffected embryos only, or prenatal diagnosis with termination of affected fetuses offers one
set of solutions. However, these are added interventions into what is already very high tech, high stress
reproduction. Both procedures carry risks and, when
the gene is dominant, half of the embryos or fetuses
so conceived will be carriers. Thus, for example, everything else being equal, a young woman, recently diagnosed with cancer, carrying a BrCa gene and wishing
to have a non-affected child will find that the success
rate for reproductive preventive care will be half that
of a recently diagnosed cancer patient who is not a
carrier. Some recently diagnosed cancer patients who
carry predisposition genes will see DE as a simpler
solution: a proven, highly successful way to build a
family and, at the same time, eliminate a deleterious
gene.
A young woman who has been diagnosed with cancer, along with other losses, may have lost confidence
in the normal functioning of her body. A pregnancy
genetic or donor egg offers her one of lifes most
normal and yet most miraculous bodily experiences. The chance to feel her body swell and fill is a
life-affirming occurrence. Likewise, for the adoptive
mother who delights in her babys first smile. We do
not mean here to minimize the cultural and emotional
meaning of genetic reproduction. We simply mean to
emphasize that, for some people, DE or adoption or
child-free living is the best possible solution to a difficult situation.
All of this means that, even with a good prognosis for fertility preservation, some cancer patients
may choose to demur. The young cancer patient faces
physical and emotional ordeals, as well as a daunting array of difficult choices to make in an impossibly
short time. Sometimes a plan for oocyte donation (or
adoption or child-free living) is a better option than
attempted fertility preservation. It is our hope that this
chapter will help the advisors oncologists, reproductive endocrinologists, psychologists and others to
support not only the patient who chooses to pursue
315
Table 26.1 Mechanics of oocyte donation
Donor
Recipient
Recruitment and screening
Screening
Medical/infectious disease
Medical
Psychological
Psychological/psycho-education
Genetic
Genetic screening of partner
Informed consent
Informed consent
Matching
Matching
Ovarian stimulation
Preparation/synchronization of endometrium
Retrieval of oocytes
Fertilization
Transfer
fertility preservation but also the one who chooses to

decline it.
To this end, we believe that it is important that
we discuss oocyte donation right from the beginning,
and sometimes even in some detail, along with other
options for fertility preservation. Some patients may
want to hear a lot about it, others may not. But, it is all
part of an ultimately hopeful message that, while life
may be different, it will go on.
Clinical practice of oocyte donation

In the future, the majority of oocyte donation cycles
may be done using cryopreserved and stored oocytes,
similar to what is done in present sperm banks [32].
At the time of this writing, though, almost all oocyte
donation is done using fresh oocytes, with the cycles
of donors and recipients synchronized. The mechanics of oocyte donation thus involve a number of steps:
the recruitment of suitable donors, informed consent
of donors and recipients, matching of donors to recipients, ovarian stimulation of the donor and the retrieval
of her oocytes. At the same time, we prepare the recipients endometrium hormonally for transfer and synchronize it to the donors stimulation cycle. Finally,
we fertilize the retrieved oocytes with the appropriate sperm and transfer the resulting embryos into the
recipients prepared uterus (Table 26.1).
Recruitment of donors
Third-party reproduction is one of the most ethically complex aspects of reproductive health care. Even
societies with generally similar values, legislate donor
recruitment very differently [33]. Thus, some countries
have mandated anonymity (Spain, France, Belgium,
316
Denmark) while others have mandated the opposite

that donors be identifiable to their genetic offspring
(UK, Sweden, Austria, Switzerland, the Netherlands,
Canada, New Zealand and the Australian state of Victoria) [34].
Some countries do not allow monetary compensation (UK) or strictly regulate it (in France donors can
be compensated for documented expenses) while others regulate more loosely (Spain allows a small monetary compensation and expenses) [34]. In others, compensation is unregulated (USA, India), though it may
be loosely limited by professional guidelines [35].
Some countries (Italy, Germany) do not allow
oocyte donation at all. In others, oocyte donation is
legal, but so strictly regulated as to make it impractical
(Chinas double blind regulations, for example, have
essentially eliminated oocyte donation in that country).
In general, these national regulations meet the
needs of their respective societies. However, differences in policy have fostered trans-border reproductive care [36, 37]. Not only do recipients travel for
oocyte donation services, but, also, centers actively
recruit donors across national borders [38, 39]. Not
surprisingly, generally, more prosperous countries
recruit from less prosperous countries. For some
donors, the small stipend, the travel, the meals and
the hotel stay are enticing indeed, if not undue inducement.
In 2005, European IVF centers performed 11 475
oocyte donation cycles, or 3% of the 418 111 IVF cycles
done in Europe that year [40]. By contrast, in the same
year, US IVF centers performed 134 260 IVF cycles
of which 16 161 or 12% were oocyte donation cycles
[41]. This difference is striking. The explanations are
various, but include cultural differences, the lack of

universal insurance coverage for infertility in the USA,
the significant compensation paid to US donors, the
predominance of anonymity in US gamete donation
and, most significantly, much less US government regulation.
Specifically, US law allows both anonymous and
known donation and the American Society for Reproductive Medicine (ASRM) guidelines suggest that both
are acceptable [35]. Compensation is not legislated. A
2007 ASRM Ethics Committee Report set the following guidelines for compensation:
Sums of $5000 or more require justification and sums above $10 000
are not appropriate . . .
To avoid putting a price on human gametes or selectively valuing
particular human traits, compensation should not vary according to
the planned use of the oocytes (e.g. research or clinical care), the
number or quality of oocytes retrieved, the outcome of prior donation cycles or the donors ethnic or other personal characteristics
[35].
Medical professionals screen and care for American

oocyte donors. Many IVF programs recruit donors
themselves. However, commercial, for-profit agencies
(run mostly by business people, although sometimes
by lawyers and, occasionally, by medical professionals)
recruit a large, but difficult to quantify, proportion of
American donors.
There are about 150 independent egg donor agencies in the USA. Only about a third have signed agreements to abide by ASRM guidelines regarding donor
compensation. Furthermore, a recent review of 53
websites of the assenting agencies found that, despite
their signed agreements, 24.5% advertise compensations that do not adhere to ASRM guidelines [43].
Thus, for many reasons, including both the relatively recent cultural pressure for delay of childbearing
and the comparative ease of availability of egg donors,
oocyte donation is a large and growing part of American fertility treatments. There are concerns about these
developments on several fronts. Nonetheless, the availability and relative social acceptance of DE services is
agreeable news for American cancer survivors.
Anonymous versus known donation

At the present time, most oocyte donation worldwide
is anonymous. However, family and known donation is
the best choice for some recipients. This may be especially true for some cancer survivors. A sister, cousin
or friend may have good reason to decide to donate

to a survivor and may derive great satisfaction from
the action. For the recipient, the opportunity to have a
child who is genetically related to her family (a sister
or cousin donation) or the kindness of the gift can also
make it a good choice.
By contrast, family and known donation also carries higher risk of coercion and higher risks of complicated family dynamics. This may be particularly true
when the recipient is a cancer survivor. For these reasons, we recommend psychological screening of all
involved parties prospective donor, recipient and
their respective partners before proceeding with
known donation [35, 44].
Infectious disease screening of donors

By law, donors in the USA must be screened for risk
factors for infectious diseases as well as tested for
exposure to specific infectious diseases according to
detailed Food and Drug Administration (FDA) guidelines [35, 45]. While the risk of infectious disease transmission is remote, FDA regulations have provided a
standard of care for this aspect of the screening of US
gamete donors.
Genetic screening
By contrast, genetic testing of US donors is not legally
mandated. The appropriate professional society sets
the standard of care: ASRM recommends that all
donors be tested for cystic fibrosis (CF) mutations
and that additional testing be based on risk factors
identified by history. According to ASRM guidelines,
donors and their first-degree relatives should be free
of mendelian disorders, major malformations, significant familial diseases with a known genetic component and mental retardation of undocumented etiology [35].
Genetics is progressing rapidly and recommended
screening tests change very quickly. The American
College of Medical Genetics has published excellent
guidelines regarding ethnicity and population-based
genetic screening (http://www.acmg.net). Of course,
the absence of legal mandates does not decrease US
physicians responsibility to act in the best interests
of the donor, the recipient parents and the potential
child. Of note is that screening gamete donors is not
the same as preconceptual testing in women attempting pregnancy. The testing of young, often unmarried oocyte donors and the dissemination of information so acquired carry different ethical, medical and
317
psychological implications than those same tests in

prospective parents. If at all possible, a genetic counselor should counsel every prospective oocyte donor
before testing. Ideally, it should also be a genetic counselor who conveys the test results and their often complex implications [46].
In our practice, a genetic counselor meets with
the prospective donor at the time of her initial visit.
The counselor takes a detailed family health history,
counsels the prospective donor about the implications of testing and then advises about screening tests
and any increased risk detected. Over more than a
decade, we have seen that a battery of tests does
not substitute for a professional genetic counselor. A
good genetic counselor will identify issues that are
detectable only through careful, directed history or,
in some cases, observation of physical characteristics
[4648]. Genetic health issues may be mild in the
donor herself or her family but may be of variable penetrance and may have serious consequences for potential offspring.
At the time of the writing of this book, our practice is to screen all donors for CF, fragile X, spinal
muscular atrophy (SMA), and a chromosome analysis. We do other testing based on the donors ethnic background. Examples would be thalassemia testing for donors with Mediterranean background or a
panel of 15 tests for donors with Ashkenazi Jewish
background. Although ASRM guidelines suggest that
recipients who have been appropriately consented can
accept a donor who is a carrier for a recessive disease
[35], the idea is controversial. It is our practice not to
accept such young women as donors, even if the male
partner of the prospective recipient is negative for the
identified disease. We, and others, are concerned about
the difficulty of informed consent in these often complex situations where as-yet unidentified mutations (or
de novo mutations) could result in potentially severe
disease despite negative male partner screening [47].
In addition to screening the donor, we routinely
test the male partner of the recipient for CF, SMA and
all diseases indicated by his ethnic background. If the
male partner is found to be a carrier, additional testing of the donor may be indicated and the recipient
family should be counseled about implications of the
findings.
Finally, we need to advise recipients not only of
the genetic evaluation findings, but also of their limits. Only a relatively small number of genetic diseases
are amenable to detection through either history or
318
testing. Birth defects are common in the population at

large 34% of all births and most of these defects
are not screenable [46].
We see meticulous genetic screening and counseling as part of our ethical responsibility to the families who come to us, to the donors and to the donorconceived persons who may result from our care.
Psychological screening
Psychological evaluation of the donor and the recipient and, when applicable, their families can be helpful
to all involved. Third-party reproduction is a complex
action with potential long-term repercussions. Participants have both the right and the responsibility to
explore the consequences and emotions that accompany these decisions. We can listen, explore feelings,
support and educate donors and recipients in a way
that helps all parties to make choices that are right for
them.
Donor
The donor should be free of significant psychiatric
pathology. The psychologist should assess current life
stressors, traumatic past events (including any unresolved history of abuse or neglect) and coping skills.
Psychosocial history should include such items as family history, interpersonal relationships, educational
background, employment history, sexual history and
any history of past or present substance abuse. Current and past prescribed psychoactive medication use
should also be assessed.
Informed consent will probably be formally
obtained at another point in the evaluation. However, the psychological interview provides a valuable
moment to ascertain that the prospective donor is
aware of the procedures involved and of their potential
risks.
The donor should be participating without coercion. If her donation is anonymous, we can give her
information about program precautions that are taken
to protect her anonymity. In addition, we want to
apprise her of the limits of anonymity in a complex
technological society and in a world in which values
and laws change rapidly [35].
When the donor is known, it is, perhaps, even more
important that situations of coercion be identified and
that the prospective known donor be supported by the
medical team if she is uncomfortable with egg donation. In family situations, in particular, and even more
so when the prospective recipient is a cancer survivor,
coercion can have many faces. It can be intergenerational, it can threaten family membership and it can
stir up long dormant feelings of anger and resentment
[44].
Even when the prospective donor is happy that she
can be of help, there are details to be discussed. Both
families should have discussed and come to agreement about disclosure (and timing of disclosure) to the
donor-conceived person(s) and to other family members, including genetic half siblings. The cancer prognosis should be openly and truthfully discussed among
family members. In cases where the prognosis is poor,
painful as it is to do, a discussion should be held about
how relationships might change were the recipient to
die.
Recipient
Psychological consultation can be very helpful for all
recipients of donated oocytes and is specifically recommended for recipients considering known donation
[44, 49]. These sessions are, at least partly, psychoeducation.
The psychologist and recipient family can discuss
feelings about pregnancy, positive and negative aspects
of disclosure and the family can be provided with a bibliography of childrens and adult books related to ovum
donation. Hopefully, any differences between the partners in attitudes towards disclosure can be resolved
well in advance of the birth of a child.
Additionally, the session offers the recipient and
her partner the opportunity to discuss their feelings about the loss of the hypothetical genetic child
that they might have made together. Couples often
carry many fears and fantasies about gamete donation,
including concerns that the donor will try to reclaim
the offspring or that the child will wish to seek out
his/her real mother. Most recipient mothers wonder
how they will feel carrying another womens genetic
child and whether they will bond to that child in utero,
after birth and throughout his or her lifetime. In situations where either blame or guilt has colored the process, these feelings, too, can be discussed. To the extent
that resentment of the process might later surface as
resentment towards the child, these conversations are
helpful steps towards building a healthy family.
In the case of recipients who are cancer survivors,
other issues and feelings may be layered onto the decision for DE. Anger and resentment about the cancer
and its treatment, seemingly resolved, may resurface.
And, while a pregnancy may restore confidence in the
normal function of the body, a DE failure may renew

feelings of physical inadequacy.
In conclusion, we do psychological screening of
donors and recipients in order to address a number
of different issues and to protect all parties involved.
Our goal is to help the recipient and the donor navigate a complicated psychological terrain. By doing so,
we hope that we will build stronger families and fulfill
some of our responsibilities as advocates for the donorconceived person.
Matching of donors and recipients

Although there has been little scientific inquiry into
the criteria that make for success in families created
with donated gametes, current practice has widely
included phenotypic matching. Thus, most programs
worldwide include an attempt to match donors and
recipients for criteria such as coloration, height and
ethnic background. And, in fact, most recipients (and
donors) express interest in some degree of phenotypic
matching.
However, in general, European donor oocyte programs are less concerned about phenotypic matching than American ones. In addition, several societal
trends suggest that phenotypic matching may assume
a lesser role in the future. First, adoption practices
have increasingly relinquished proscriptions against
interfaith and interracial adoption [50, 51]. Some of
these historical proscriptions have come to seem oldfashioned. Secondly, there is an increased tendency
for, and increased professional advice in favor of, disclosure of means of origin to individuals conceived
through gamete donation [35]. Thirdly, there is a growing interest (some of it legally mandated) towards
identity-release donation. These trends decrease the
need for secrecy that may have been a part of phenotypic matching. Finally, families have become more
diverse and the concept of a family has become more
diverse. All of these tendencies together may lessen the
importance of phenotypic matching in the future.
Nonetheless, at the present time, most recipients
(and many donors) express interest in at least some
degree of phenotypic matching.
Care of the oocyte donor

The oocyte donor is a young healthy person who takes
on risks and receives no direct benefits from them.
From an ethical point of view, she is more like a
research subject than a patient [52, 53]. It is the guiding
principle of this section to emphasize that the aim of
319
the physician caring for the oocyte donor should be to

reduce the rate of complications to as near zero as possible, while still maintaining reasonable success rates
for the recipients.
Ultimately, if we commodify our donors, if we do
not treat them with the care and respect to which they
are entitled, we will lose the confidence of the public,
of the recipients and of the donors themselves. Caring
well for donors is both a public trust and a professional
duty.
Ovarian stimulation of the oocyte donor

Increased success in oocyte donation, as in IVF generally, is linked to an increased number of healthy
mature oocytes that can be retrieved and fertilized. To
achieve this goal, physicians override two basic physiological controls. First, in the early follicular phase of
the natural cycle, negative feedback of estrogen and
peptides on the pituitary results in a steadily decreasing secretion of FSH. It is the exposure to this decreasing concentration of FSH which allows a single dominant follicle to emerge from the cohort of recruited
follicles. By contrast, in stimulated cycles, exogenous
gonadotropins override the negative feedback of rising
estrogen and allow multiple follicles in the recruited
cohort to mature.
Secondly, the rising levels of estradiol in a natural cycle will trigger a positive feedback response at
the pituitary resulting in a surge of luteinizing hormone (LH), which will trigger ovulation. If not controlled, the rising levels of estradiol in the stimulated
cycle may trigger the LH surge prematurely, resulting in immature oocytes. Thus, stimulation cycles for
IVF include either a gonadotropin-releasing hormone
(GnRH) agonist or antagonist to prevent premature
LH surges. Because the endogenous trigger for ovulation has been suppressed, an exogenous trigger must
be given at the appropriate time. Usually, this exogenous trigger is hCG. However, for reasons that will be
discussed, we may find advantages for the donor in the
use of a GnRH agonist to trigger an endogenous pituitary LH surge.
Stimulation of donors is, in most ways, similar to
the stimulation of infertile IVF patients. However, the
stimulation of donors differs in two important ways.
First, by definition, donors are young, healthy and presumably fertile. Thus, they generally respond briskly,
have a higher risk of early onset ovarian hyperstimulation syndrome (OHSS) and need to be stimulated more
gently than most IVF patients. Secondly, and more
320
importantly, donors experience risk but achieve no

benefit from their stimulation cycles. The donors risks
include OHSS, ovarian torsion, bleeding, infection and
anesthesia complications, amongst others. When all
care is taken, the risks are low reported as 1%
but may be serious [5457]. In worst-case scenarios,
a complication may affect the ongoing well-being and
fertility of a healthy young woman.
Dual suppression
In order to minimize OHSS, stimulation protocols
should utilize relatively low doses of gonadotropins
and should include appropriate step down regimens
once the cohort of follicles has been recruited. One
such protocol, commonly used in patients at high risk
for OHSS, and highly appropriate for donors, is a dual
suppression with oral contraceptive pills (OCPs) and
GnRH agonist overlap [58]. Because the OCP cycle
length can be varied, this protocol in donors has the
additional advantage of simplifying synchronization
with the recipient cycle (Figure 26.3 [59]).
GnRH agonist trigger for ovulation
Over the last few years, increasing evidence suggests that the incidence of OHSS can be significantly
decreased by the use of antagonist suppression along
with a GnRH agonist trigger without compromising oocyte or embryo quality [60]. Human chorionic
gonadotropin has a dramatically longer half-life than
LH (2.3 days versus 21 min) [61, 62], and therefore
generates a prolonged luteotrophic effect. This is beneficial in the IVF patient (it is the corpus luteum that
prepares the endometrium and sustains the early pregnancy), but results in risk without benefit in the oocyte
donor. Specifically, early GnRH agonist trigger studies in IVF patients seemed to result in lower pregnancy rates, which, in retrospect, were almost certainly related to early luteolysis and lack of support
of the endometrium [63]. By contrast, when the early
pregnancy was appropriately supported with estrogen
and progesterone, comparable pregnancy rates were
achieved with GnRH trigger as compared to hCG [64].
Because the donor herself has no early pregnancy
to support, she may be the ideal candidate for agonist trigger protocols. Not surprisingly, two recent
large prospective studies of these protocols in donors
have shown significant decreases in OHSS without any
compromise in recipient pregnancy rates [65, 66]. As
physicians gain more experience with agonist trigger
hCG
Finding threshold
FSH
1 mg leuprolide acetate daily
OCP 25 days
Day 1
21
25
14
Retrieval
Figure 26.3 A dual suppression protocol used by us in 1986, still valid today. FSH, follicle stimulating hormone; hCG, human chorionic
gonadotropin; OCP, oral contraceptive pill. Reprinted with permission from Rosenwacks [59], courtesy of Elsevier,
Reproductive Medicine.
GnRH analogue
trigger
GnRH antag
FSH
Figure 26.4 A GnRH agonist trigger

protocol from our program today. antag,
antagonist; FSH, follicle stimulating
hormone; GnRH, gonadotropin-releasing
hormone; LH, luteinizing hormone.
Confirm LH
Surge
FSH + LH
Menses
protocols, these protocols may become standard of

care for oocyte donor stimulation (Figure 26.4).
Oocyte retrieval
Donor oocyte retrieval does not differ from oocyte
retrieval for IVF. Under conscious sedation, and using
a transvaginal ultrasound-guided needle, the follicles
are aspirated and the oocytes identified in the follicular fluid. Complications of the procedure include
infection and intraperitoneal bleeding, as well as anesthetic complications. All complications are rare and yet
potentially serious.
The incidence of pelvic infection following
transvaginal ultrasound-guided oocyte retrieval
ranges from 0 to 1.3% [67, 68]. In a review of 2670
oocyte retrieval procedures, Bennett et al. reported 18
cases resulting in infection (0.6%), 9 of which were
severe with pelvic abscess formation (0.3%) [56]. The
severity of infection can range from a minor infection
with pyrexia, leukocytosis and abdominal pain, to a
12 13 14
Retrieval
major medical event such as pelvic abscess formation

or sepsis. Signs and symptoms can appear within
hours to days after the retrieval in an acute infection,
while pelvic abscess may not present clinically for
several weeks. Pelvic infection is a particularly concerning complication for a young egg donor because
even a relatively minor event, especially if not treated
promptly, may result in infertility later in life.
Minimizing infectious complication involves careful initial screening and testing for any history of pelvic
inflammatory disease or sexually transmitted disease,
meticulous preparation of the vagina with povidineiodine, attention to making the fewest vaginal punctures possible, and, possibly, prophylactic antibiotics at
the time of retrieval.
Intraperitoneal bleeding is another rare but potentially serious complication. Bleeding can arise from
inadvertent puncture of an arterial vessel. More commonly, it is probably secondary to generalized oozing from the highly vascularized ovaries, similar, but
321
in exaggerated form, to the intraperitoneal bleeding that can occur with natural ovulation. Clinical
observations suggest that intraperitoneal bleeding, like
OHSS, occurs more commonly when more oocytes are
retrieved. This is physiologically plausible: the larger,
more highly vascularized ovaries and the increased
number of follicular punctures would likely predispose
to bleeding.
Screening of the recipient

A cancer specific evaluation, as outlined in the first
part of this chapter, should be done before any other
screening of the recipient. Only with this cancerspecific evaluation complete, do we move on to routine preconception screening, including infectious disease screening, blood type, thyroid stimulating hormone (TSH), rubella and varicella titers. In addition,
we screen the male partner for infectious disease and
ask him to provide a sample for semen analysis. He
should be counseled and screened for recessive genetic
disease. As discussed previously, genetic evaluation of
the male partner has benefits even when the donor has
been screened.
Although some controversy exists about the
importance of a mock or preparatory cycle for most
recipients [69], recipients who have been previously
treated with pelvic irradiation, as discussed in the
first section of this chapter, should have particular
attention paid to the function of the uterus and should
have a preparatory cycle done. In these cases, a mock
replacement cycle with evaluation of the endometrial
response to hormonal treatment by ultrasound measurement of endometrial thickness and endometrial
biopsy can be very helpful. It will alert us to situations
that require individualized estradiol dosing to prepare
the endometrium and will highlight cases that should
be considered for single embryo transfer. In some
cases, uterine evaluation will show that oocyte donation is too risky to be entertained. The patient will
need to choose between a gestational carrier for the
donated oocytes and other alternative ways to form a
family.
Preparation and synchronization of the

recipients endometrium
In the natural cycle, events in the endometrium are
physiologically synchronized to events in the ovary.
The steadily increasing serum levels of estradiol pro-
322
duced by the ovary as the oocyte matures result in

proliferation of endometrial glands and stroma and the
development of endometrial LH receptors. Following
ovulation, granulosa cells in the dominant follicle, now
the corpus luteum, continue to produce estrogen, but
now also produce increasing amounts of progesterone.
The endometrium responds with tightening and coiling of glands and blood vessels and with the accumulation and secretion of glycogen into the endometrial
cavity. This is, not coincidentally, the ideal environment for the support of the early embryo. The embryo
first migrates freely in the cavity, making use of the
rich glycogen secretions, and then begins implantation
into the endometrial wall, seeking and finding vascular
support from the abundant, coiled vasculature.
Many cancer survivors seeking DE treatment have
little or no ovarian function. For those with ovarian
failure we can proceed directly with estrogen and progesterone, while for those patients who exhibit ovarian function, we use GnRH analogues to shut down
pituitary stimulation of the ovary before beginning
steroid replacement. In either case, there will be no
follicles to prepare the endometrium and no corpus
luteum to support the early pregnancy. Instead, we
must supply estrogen and progesterone exogenously.
In doing so, we seek to mimic both the preparation of
the endometrium and its synchrony with the developing embryo.
In general, preparation of the donor oocyte recipients endometrium has remained unchanged since the
inception of egg donation. Figure 26.5 shows a replacement protocol used by the author in 1986 [59].
At the time, three ongoing pregnancies from IVF
oocyte donation had been reported in the USA and
only a few others worldwide. Amazingly, in a field
in which so much has changed so quickly, this protocol from 25 years ago could be used, essentially
unchanged, to teach a patient or train a new nurse
today.
Basically, estradiol, delivered either as oral
micronized estradiol or through a transdermal patch,
is used to initiate proliferation of the glandular
and stromal cells of the endometrium. Our current
practice is to use slowly increasing doses of estrogen
delivered via transdermal estrogen patches. The
patches have the advantage of being relatively physiological; estradiol is delivered slowly and continuously
and the hepatic first pass is avoided [70]. We usually start with a dose of 0.1 mg/day, increase this to
Estrace oral
Progesterone vaginal
Intramuscular progesterone
150
125
100
75
50
25
1
9 10 11 12 13 14
Progesterone (mg)
Estrace (mg)
15 16 17 18 19 20 21 22 23 24 25 26 27 28
Figure 26.5 A replacement protocol used by us in 1986, still valid today. Reproduced with permission from the American Society for
0.2 mg/day as the donor begins her gonadotropin stimulation, and increase to 0.4 mg/day for approximately
4 days prior to the donors ovulation. During the luteal
phase, patches are decreased back to 0.2 mg/day [71).
Most patients tolerate the patches well although
redness or rash at the patch site may be seen in up to
1% of patients. As in the natural cycle (where follicular
phase length may vary considerably without affecting
fertility), there is considerable latitude in the length
of the estrogen-only phase of the replacement cycle
[72, 73]. This is fortunate; without this flexibility,
synchronization would be difficult.
Progesterone replacement, however, is a different matter. Its administration must be synchronized
closely to the donors cycle. The presence of adequate
endometrial levels of progesterone at the correct time
in the cycle is critical both for the preparation of the
endometrium for implantation and for the support of
the early embryo [73].
Progesterone can be administered either by the
vaginal route or as intramuscular injections of progesterone in oil [74]. Intramuscular progesterone has
the disadvantage of being irritating to muscle; patients
often experience pain at the injection site and occasionally develop fever and leukocytosis. On the other
hand, serum levels can be reliably used to monitor and
titrate the dose, which is not true of the better-tolerated
vaginal suppositories. (Oral micronized progesterone
is variably absorbed and is rarely used for recipient
replacement cycles.)
In our practice, we begin progesterone on the day
after the donor receives her ovulatory trigger at a dose
of 2 mg/day (im) or, more rarely, 300 mg/day (suppository). On the following day, we double the dose and
continue it either until the pregnancy test is negative
or, if positive, until the placenta takes over steroidogenesis, usually by 10 or 11 weeks of pregnancy [75].
If intramuscular progesterone is used, we monitor
serum levels and adjust the dose to keep the serum
concentration above 20 ng/ml. The estradiol dose is
titrated to yield a serum concentration between 100
and 200 pg/ml (Figure 26.6).
Conclusion
A young cancer patient may find significant psychological benefit in preserving oocytes or embryos prior
to embarking on a grueling course of chemotherapy.
This is true even if she ultimately does not need to
use the cryopreserved tissue. Many times, especially
if she is young, ovarian function will return following
treatment. However, even those survivors with regular
menses, may find that ovarian reserve and egg quality have been compromised. The cancer survivor with
regular menses needs to understand this and, after a
safe interval, be encouraged to attempt conception as
soon as she is ready. Physicians should discuss the
advantages of an abbreviated evaluation for a couple
unable to conceive within a few months. Often, effective intervention will shorten the time to pregnancy.
Sometimes, ART is the best initial approach.
When conception does not occur and when prevention has not been done, or has not been successful, a cancer survivor may consider pregnancy through
323
0.6
50
50 mg/day P4
0.4
0.4 mg/day E2
Estradiol
Progesterone
25
25 mg/day P4
0.2
0.2 mg/day E2
Progesterone im (mg)
Transdermal micronized E2
0.8
Figure 26.6 A similar replacement

protocol from our program today. BhCG,
beta human chorionic gonadotropin; E2,
estradiol; hCG, human chorionic
gonadotropin; im, intramuscular; P4,
progesterone.
0.2 mg/day E2
0.1 mg/day E2
.1
0 2
28
10
12
14 15 16 18 22 23 24 26
hCG
Embryo transfer
BhCG
Aspiration
Progesterone day 15
oocyte donation. As long as physicians screen both

donors and recipients meticulously, use techniques
to minimize risk to donors and are careful about
informed consent to both parties, pregnancy through
oocyte donation can be a positive experience for all
involved.
Finally, we have an understandable desire to be
optimistic and encouraging to our young cancer
patients. However, these patients also need support as
they adjust to the very real losses that accompany a
cancer diagnosis. The losses range from a loss of innocence and invulnerability, to the physical losses that
accompany surgery, chemotherapy and radiation, to
changes in life expectations, including potential loss of
reproductive options, and, finally, to the potential loss
of life itself. A focus on technological solutions alone
for quality of life issues may deny the newly diagnosed
cancer patient the opportunity to reconsider values
and to put life events in perspective.
We know that genetic reproduction is important to
living things, humans included, but we also know that
the genetic imprint that an individual makes is erased
in a few generations. This chapter addresses, among
other things, issues that occur when preservation of
fertility has not been possible or has not been the best
choice for a given individual. We can help our patients
by expanding their options and choices, but we can
help them also by affirming that there are many paths
to a fulfilled and satisfying life.
324
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327
Section 7
Chapter
27
Ovarian cryopreservation and transplantation
General overview of ovarian cryobanking

S. Samuel Kim
Introduction
Fertility preservation is now recognized as the most
essential quality of life issue in young cancer survivors. Although several strategies to preserve fertility in women have been developed, most of them are
still experimental. Ovarian cryobanking, either freezing ovarian tissue or the whole ovary, is currently perceived as a promising technology for fertility preservation which draws enormous attention not only from
scientific communities but also from the general public.
Although still in its developmental stage, ovarian tissue cryopreservation followed by transplantation has proven to be successful in many animals.
Furthermore, we have been witnessing the successful
restoration of fertility after ovarian transplantation in
humans since 2004. To date, 14 healthy babies have
been born worldwide after transplantation of frozen
thawed ovarian tissue [17].
It is exhilarating to see the steady progress and
increasing enthusiasm for clinical applications of this
technology. However, ovarian cryopreservation and
transplantation should remain experimental until the
efficacy of this technology is proven. Indeed, there are
numerous technical and ethical issues that should be
resolved with this technology. In this chapter, three
urgent and critical problems involved with ovarian
tissue cryopreservation and transplantation (cryoinjury, ischemic tissue damage, cancer cell transmission) are discussed. In addition, the current status of
human ovarian tissue transplantation and whole ovary
transplantation by vascular anastomosis are briefly
addressed.
Synoptic history of ovarian

transplantation
The history of ovarian transplantation dates to the
eighteenth century. Although many animal experiments were performed in nineteenth-century Europe,
the first human ovarian tissue transplantation was
reported by Robert Morris in New York in 1895 [8].
By 1901, Morris had performed 12 ovarian transplantations (autograft as well as allograft). In 1906, he
claimed a live birth after autografting ovarian tissue to
the broad ligament of a 33-year-old woman with polycystic ovary syndrome.
The discovery of cryoprotectants (CPAs) in London in 1948 was a scientific breakthrough which
made it possible to cryopreserve living cells and tissue. Just after discovery of CPAs, there was a flurry
of experiments on freezing gonadal tissue followed
by transplantation. In 1960, restoration of fertility
was reported after orthotopic isografting of frozen
thawed ovarian tissue in oophorectomized mice [9].
Over the next 30 years, however, there was no further
progress in this field. In 1994, Gosden et al. succeeded
in restoring fertility in sheep after autotransplantaion
of frozenthawed ovarian tissue, which rekindled the
interest in this technology with new perspectives, especially as a potential strategy to preserve fertility in cancer patients [10]. Ten years later, in 2004, the first baby
was born after orthotopic autotransplantation of cryopreserved human ovarian tissue in a woman with
Hodgkins lymphoma [1].
Transplantation of the whole ovary with vascular
anastomosis is not a new procedure either. In 1906,
Alexis Carrel in New York, who later won a Nobel
328
Chapter 27: General overview of ovarian cryobanking
Prize, reported the first ovarian transplantation by vascular anastomosis in cats. Since then, successful transplantation of the whole ovary with microanastomosis
of vascular pedicles has been reported in many animals including dogs, cats, rodents, rabbits, sheep and
primates [1113]. In 1987, Michel Leporrier in France
reported the successful heterotopic transplantation of
the whole ovary with vascular anastomosis before
pelvic irradiation to treat Hodgkins disease [14], the
first successful whole ovary transplantation (heterotopic) in humans. In 2009, the first baby was born
as a result of orthotopic transplantation of the intact
ovary by vascular anastomosis between monozygotic
twins [15]. Indeed, the surgical complexity of vascular anastomosis is no longer a barrier of human ovary
transplantation. The real challenge of vascular transplantation of the whole ovary is perfecting cryotechnology for organ cryopreservation. The first success in
restoring fertility after vascular transplantation of the
cryopreserved whole ovary was achieved in 2002 in
rats [16].
Clinical guidelines for

ovarian cryobanking
It is important to provide full counseling before any
procedures to protect patients and to prevent misuse
of technology. As ovarian tissue banking is not yet an
established technology, the current status and experimental nature of the technology should be fully and
accurately explained. At the same time, some details of
ovarian tissue banking should be discussed, including
the surgical procedure and its risks, efficacy of freezing and storage, and options of future use of cryopreserved tissues. In addition, it is imperative to communicate with the patients oncologist before and after the
procedure.
The physical and psychological conditions of the
patient should be evaluated and considered before the
procedure. The age of the patient is another crucial factor to consider as the chance of restoration of ovarian function and fertility is closely correlated to the
number of follicles in the ovarian graft. Current experiences with human ovarian transplantation suggest that
women over 40 years of age may not be good candidates for ovarian tissue banking as the chance of fertility restoration after transplantation is extremely low
[17].
Nevertheless, advanced reproductive age cannot be
an absolute indicator for low ovarian reserve in view
of individual variations. It is therefore recommended

that ovarian reserve is assessed with endocrine tests
as well as pelvic ultrasound (antral follicle count)
to guide clinical decision making. The serum follicle stimulating hormone (FSH) level has been used
widely to assess ovarian reserve, but its accuracy to
predict ovarian reserve is limited. In my opinion, the
single best test to assess the ovarian reserve before
ovarian tissue banking (especially in cancer patients)
is the serum anti-Mullerian hormone (AMH) level as
it is a direct assay for ovarian reserve (since it is produced from granulosa cells of the ovarian follicles) and
can be tested any time of the menstrual cycle (unlike
FSH).
The safety of transplanting stored ovarian tissue
is crucial as the risk of re-introduction of cancer
cells exists in certain cancers. At present, the type of
malignancy, the type of treatment and the prognosis after treatment should all be considered to determine if the candidate is suitable for ovarian cryobanking. To date, autotransplantation of ovarian tissue in Hodgkins lymphoma patients appears to be
safe [17]. While patients with Hodgkins lymphoma
are indeed good candidates for ovarian banking, the
types and doses of chemotherapeutic regimen should
be considered before offering ovarian cryobanking.
The chance of losing fertility with an ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine)
regimen in young patients with Hodgkins lymphoma is 15%, which cannot justify routine use
of ovarian tissue banking in this population. Furthermore, ovarian tissue banking should be discouraged in patients with systemic or disseminated malignancies.
Ovarian tissue banking will be most useful for
patients who need to undergo hematopoietic cell
transplantation, since the risk of premature ovarian
failure is extremely high due to highly gonadotoxic
preparatory regimens. The use of hematopoietic cell
transplantation is no longer limited to leukemia and
lymphoma but has been extended to solid malignant
tumors, such as breast cancer, and non-malignant conditions, such as lupus, rheumatoid arthritis, aplastic
anemia and sickle cell disease.
As the efficacy and safety of ovarian tissue banking has not been established and there is no consensus
for indications for ovarian tissue cryobanking, for the
time being it is prudent to offer this technology selectively to women who are at a high risk of losing their
fertility and with a good long-term prognosis.
329
Section 7: Ovarian cryopreservation and transplantation
Table 27.1 Challenging issues with human ovarian

tissue cryopreservation and transplantation
Patient selection criteria
Cryoinjury/optimization of freezing technique
Safety issues/prevention of cancer cell re-introduction
Ischemic-reperfusion injury
Effective graft sites
Effective in-vitro follicle culture technique
Quality of oocytes matured in a graft
Efficacy for restoration of fertility
Ethical issues, especially in children
Challenging issues with ovarian tissue

cryopreservation and transplantation
The landscape of ovarian tissue transplantation has
been changed since 1994. It is no longer staying in
the research arena but entering into the clinical realm.
We have solid evidence that the strategy of ovarian
tissue cryopreservation followed by autotransplantation works to restore fertility in cancer patients. Nevertheless, this strategy contains numerous technical
and scientific problems as well as ethical issues (Table
27.1). Of these, three challenging issues (cryoinjury,
ischemic tissue damage and cancer cell transmission)
are discussed in this chapter.
Cryoinjury
Cryopreservation of ovarian tissue by slow freezing
and rapid thawing is successful (5080% follicle survival rates), but current methods are still not perfect
and require further optimization to minimize the loss
of follicles and ovarian function. Two main mechanisms of cryoinjury are intracellular ice formation and
salt deposits. The most damaging phase (increased ice
formation and growth) of slow freezing is during cooling between 10 and 40 C, especially when the liquid phase is supercooled. However, significant cryoinjury can occur during the thawing (re-expansion)
phase because of changes in the composition of the
surrounding milieu, possibly mediated by temporary
leakage of the plasma membrane [18]. Indeed, the
thawing rate is important in maintaining cell viability.
Newton and Illingworth noticed the higher follicle survival and in-vitro maturation rates when samples were
thawed at 27 C rather than at 37 C in a mouse model
[19].
330
5C
Rapid cooling
Optimal
cooling
Slow cooling
<10C
Lethal
intracellular
ice formation
External ice
formation, no
intracellular
freezing
Excessive
dehydration,
high internal
and external
salt concentrations
Figure 27.1 Effects of cooling rates during cryopreservation of

living cells.
To minimize cryoinjury, cooling rates need to be

fast enough to reduce the exposure of cells to high
intracellular concentrations of electrolytes, but they
should be slow enough to dehydrate cells and avoid
intracellular ice formation (Figure 27.1). The thawing rates should be fast enough to prevent formation
and growth of ice crystals. Cryopreservation of living cells requires CPAs, which can be cytotoxic. The
toxicity of CPAs depends on the inherent characteristics of the chemical itself, duration of exposure and
temperature.
It is much more difficult to optimize freezing and
thawing conditions for tissue compared with those
for isolated cells. Tissue is composed of various cell
types with different physical parameters that influence cryostability during cooling and CPA penetration. Naturally, longer exposure time to CPAs increases
the toxicity. Furthermore, extracellular ice formation
is as detrimental as intracellular ice in multicellular
systems. Nevertheless, almost two-thirds of immature
follicles survive in human ovarian tissue after slow
freezing and rapid thawing [20, 21]. The majority of
these follicles are morphologically normal by light
microscopy, but distinctive ultrastructural changes
can be detected in frozenthawed tissue by electron
microscopy (e.g. mitochondrial and membrane damage, vacuoles in the cytoplasm) (Figure 27.2a,b).
Vitrification with a high concentration of CPAs and
ultra-rapid cooling, in which the aqueous phase turns
directly into a solid amorphous phase, can be advantageous for tissue freezing as it can eliminate both intraand extra-ice formation, which directly affects the
Figure 27.2 Ultrastructural changes in

human primordial follicles before (a) and
after (b) cryopreservation of ovarian tissue
(slow freezing) detected by transmission
electron microscopy (TEM). (a) A
primordial follicle from fresh ovarian
tissue showing intact nuclear and cell
membranes. Normal-shaped
mitochondria are clustered around the
nucleus. (b) A primordial follicle from
frozenthawed ovarian tissue showing
extensive vacuolation (V) throughout the
cytoplasm. Nuclear and cell membranes
are still intact, but mitochondrial damage
is evidenced by dilated cristae in the
mitochondria (arrows). CM, cell
membrane (oolemma); M, mitochondria;
N, nucleus; NM, nuclear membrane; PG,
pre-granulosa cell; V, vacuoles.
(a)
(b)
survival of cells in ovarian tissue. However, there are

limitations to tissue cryopreservation by vitrification.
A key limiting factor of vitrification is the toxic effects
of CPAs (chemical and osmotic). The high concentrations of cryoprotectant required for vitrification neces-
sitate a short equilibration time to minimize the toxicity. Unlike individual cells, tissue requires a longer
exposure to high concentration of CPAs to reach
optimal CPA penetration. This is a dilemma for
vitrification of ovarian tissue. The antifreeze proteins
331
(a)
(b)
(c)
Figure 27.3 Protein expression patterns assessed by two-dimensional gel electrophoresis in: (a) fresh ovarian tissue; (b) slow frozenrapidly
thawed ovarian tissue; (c) vitrifiedwarmed ovarian tissue.
(non-colligative CPA) that inhibit ice-nucleating

events may reduce the toxicity of CPAs by allowing
lower concentrations of CPAs to be used for vitrification. The second factor that can compromise tissue
survival is re-crystallization at warming (devitrification). Complete elimination of devitrification is not
easy unless very high concentrations of CPAs are used.
The probability of devitrification can be significantly
reduced by the warming rate equal to those imposed
during the cooling.
To date, there is no standard vitrification protocol
and it is rather confusing to see the many variations,
which include types and concentrations of CPAs, durations and steps of equilibration, methods of cooling
(straws, grids, aluminum foils, Cryovials, solid surface
vitrification, direct plunging), warming temperatures
and solutions. Detailed discussions are in Chapter 28.
Overall, we see more favorable results with the use of
both permeating and non-permeating CPAs for vitrification (e.g. 2040% of dimethyl sulfoxide and ethylene
glycol as permeating CPAs and sucrose or trehalose as
non-permeating CPAs), a two-step or multi-step equilibration, direct contact to liquid nitrogen, high warming temperature (3740 C) and serial dilution in solutions with sucrose. Our study showed that vitrification
of bovine ovarian tissue after equilibrating in 5.5 M
ethylene glycol for 20 min at room temperature was
as effective as slow freezing [22]. Currently, we have
adopted a two-step equilibration method using 20%
dimethyl sulfoxide and 20% ethylene glycol as colligative cryoprotective agents for vitrification of ovarian
tissue.
Although we have accumulated some knowledge of
cryoinjury after freezing and thawing, cryotechnology
332
for ovarian tissue cannot be perfected without further

basic research on cryobiology, especially at the molecular level. As a part of studies investigating molecular and biochemical changes with cryopreservation, we
have analyzed the protein expression in ovarian tissue between the fresh, slow cryopreserved and vitrified group before and after transplantation using twodimensional gel and mass spectrometry technologies
(Figure 27.3). By comparing the protein spots with
significant intensity differences between samples, we
could identify the proteins of significance including
RAB4B, actin, Chain A and B (lectin), serpinb 1a protein, 33 laminin receptor homolog and glutathioneS-transferase. Of note, these proteins are related to tissue survival and metabolism [23].
Ischemic injury
It has been proven that autotransplantation of frozen
thawed human ovarian tissue can restore endocrine
function as well as fertility. However, the follicular
loss in the grafted ovarian tissue is unacceptably high,
and it is mainly caused by tissue hypoxia after grafting while waiting for angiogenesis. In rodents, ovarian tissue slices become revascularized within 23 days
after grafting [24]. If the ischemic period is longer than
24 h, irreversible hypoxic tissue damage is unavoidable in the ovarian graft [25]. Primordial follicles are
more resistant to ischemia than growing follicle or cortical stromal cells. Nevertheless, most primordial follicles die of ischemia rather than of cryoinjury, and
only between 5 and 50% of follicles survive after grafting [26, 27]. The future of ovarian tissue transplantation depends on the development of new strategies to
facilitate angiogenesis or to protect the graft from

ischemia (especially within 24 h after transplantation).
Indeed, many researchers have begun to investigate
different strategies to minimize ischemic injury in the
ovarian graft, such as applying antioxidants and angiogenic factors [23].
Nugent et al. demonstrated that antioxidant treatment using vitamin E improved the survival of follicles
(45% in the control group versus 72% in the treated
group) 7 days after transplantation [28]. In addition,
the vitamin E supplemented group showed a significant reduction in lipid peroxidation in ovarian grafts
on day 3 after grafting. The results of this study indicate
that antioxidants can reduce damage from lipid peroxidation during ischemia in ovarian grafts.
Our study also demonstrated that ascorbic acid,
an antioxidant, can effectively protect bovine ovarian
grafts from hypoxic damage [25]. In this study, we
measured the rates of oxygen consumption and apoptosis as parameters of tissue damage after incubating
ovarian tissue at 37 C for different time periods (1,
3, 24 and 48 h of ischemia) with or without ascorbic acid. The significant tissue damage was evidenced
by the decrease in the oxygen consumption rate and
the increase in apoptosis after 24 h of ischemia, and
antioxidant treatment significantly reduced apoptosis
in ovarian cortical stroma.
Ovarian tissue is endowed with abundant genes
for angiogenic factors including vascular endothelial
growth factors (VEGF), transforming growth factors
(TGF), fibroblast growth factors (FGF), and angiopoietins. Expression of these genes is stimulated by
hypoxia through hypoxia inducible factors (HIF) that
regulate transcription of key angiogenic growth factors. There are many factors that can stimulate angiogenesis. One of the factors that can be clinically useful is gonadotropin, as gonadotropins stimulate angiogenesis by upregulating the angiogenic growth factors
including VEGF and angiopoietin.
Imthurn et al. showed that exogenous
gonadotropin could increase the number of developing follicles by facilitating angiogensis, but the
magnitude of the effect was influenced by the timing
of the gonadotropin administration relative to the
time of grafting [29]. They found that gonadotropin
injection started at or after surgery was not effective.
To maximize the number of follicles after grafting, gonadotropin stimulation should be started
2 days before surgery and continued 2 days after
transplantation.
To investigate the effect of exogenous administration of gonadotropin on angiogeneisis after ovarian grafting, we evaluated gene expression of angiogenic growth factors including HIF-1, VEGF, TGF
and angiopoietin-1, 2 and 3 in rat ovarian grafts 2, 7
and 30 days after transplantation. The mRNA expression of HIF-1 and angiopoietin-1 were low on day 2
but increased on days 7 and 30. Angiopoietin-2 and
3, VEGF and TGF were increased from day 2 and
peaked on day 30 after transplantation. When we compared the VEGF expression and apoptosis in the ovarian graft between the gonadotropin-treated and control groups, we did not find any added benefits of
exogenous gonadotropin for angiogensis or follicular
development in ovariectomized animals [30]. Nevertheless, a significant increase in the VEGF188 isoform
in the gonadotropin-treated group suggested the positive role of gonadotropin in the early stage of angiogensis in ovarian grafts. Of note, the mRNA expression
of VEGF188 increased on day 2 and returned to normal on days 7 and 30. On the other hand, VEGF164
and VEGF120 showed no changes on days 2 and 7 but
increased on day 30.
The optimal graft sites should be further determined to minimize ischemic damage and to improve
follicular survival after avascular ovarian grafting. It is
reasonable to expect better graft survival when ovarian cortical tissue is grafted to vascular sites, such as
muscle tissue or kidney capsule, rather than subcutaneous tissue. Furthermore, the importance of vascular smooth muscle cells and pericytes in sustaining vascular and tissue integrity after transplantation
has been demonstrated [31]. Perhaps, the secure strategy to prevent ischemic damage is using whole ovary
transplantation with vascular anastomosis. However,
cryopreservation of the whole human ovary along with
its vascular pedicles is a huge technical challenge.
Cancer cell transmission

The risk of cancer cell transmission is a serious safety
issue related to ovarian autotransplantation in cancer patients. Shaw et al. reported that healthy AKR
mice that received ovarian grafts from donor mice with
lymphoma died of the same disease within 23 weeks
after transplantation [32]. However, Kim et al. demonstrated the safety of transplanting human ovarian tissue from lymphoma patients using a xenotransplantation model; human ovarian tissue harvested from
18 lymphoma patients with high-grade disease was
333
xenografted to non-obese diabetes/severe combined

immunodeficient (NOD/LtSz-SCID) mice [33]. None
of the animals grafted with ovarian tissue from lymphoma patients developed disease, whereas all positive control animals that received lymph-node sections
containing non-Hodgkins lymphoma cells developed
human B-cell lymphoma. To date, there is no sign
of relapse in more than 10 women with Hodgkins
lymphoma who underwent autotransplantation of
cryobanked ovarian tissue worldwide [17].
Ovarian metastasis is clinically rare in most cancers of young people, and its risk depends on the disease type, activity, stage and grade. The chance of ovarian metastasis of Wilms tumor or Hodgkins disease
is negligible, whereas the risk of minimal residual disease (MDR) in ovarian tissue from leukemia patients
is a real concern. Indeed, MDR in the ovarian tissue from a chronic myelogenous lymphoma (CML)
patient has been detected by highly sensitive real-time
polymerase chain reaction (RT-PCR) for BCR-ABL
transcript [34]. It is thus imperative to screen ovarian tissue thoroughly for MDR before transplantation
using sensitive markers to prevent re-introduction of
cancer cells. Currently, the available methods to detect
MDR include histology/cytology, immunohistochemistry, flow cytometry and PCR. Preoperative imaging
can detect disease in the ovaries and prevent unnecessary surgery and storage. To date, there is no reported
case of cancer recurrence due to autotransplantation
of frozenthawed ovarian tissue, which should not
be interpreted as the proof of the safety. Indeed, it is
too premature to assess the risks of cancer recurrence
involved with this procedure.
Current status of human ovarian

tissue transplantation
There are three strategies, at least in theory, to mature
follicles in frozen-stored ovarian tissue: autotransplantation; xenotransplantation; and in-vitro culture (Figure 27.4). Recently, significant progress has been made
in immature follicle culture techniques. In particular, three-dimensional culture techniques and two or
three-stage culture strategies are promising (see Chapter 32). There are, nevertheless, many variables and
obstacles to overcome before perfecting these culture
methods for clinical applications.
Although full development of human oocytes can
be achieved after grafting ovarian tissue in the animal host (xenotransplantation), its clinical application
334
is problematic because of safety and ethical issues.

Grafting stored ovarian tissue back to the patients
own body (autotransplantation) therefore appears to
be the most practical strategy in the clinical setting.
In spite of skepticism, the first baby was born in 2004
after orthotopic autotransplantation of frozenthawed
ovarian tissue in a woman with Hodgkins lymphoma.
This is another milestone in the history of human ovarian transplantation, and it validates the clinical feasibility of ovarian transplantation for fertility preservation.
Orthotopic autotransplantation
Although its efficacy should be further probed,
restoration of fertility by orthotopic autotransplantation has been demonstrated in humans as well as in
many animals. For orthotopic transplantation, ovarian tissue can be either transplanted onto the remaining ovary or into the peritoneal pocket of the fossa
ovarica. It appears that grafting ovarian tissue in or
onto the remaining ovary has advantages and more
likely results in natural conception, unless the size of
the ovary is too small as a result of atrophy [35]. In
the 5 years since the first report of a live birth in
2004, the total number of babies born after orthotopic transplantation of frozenthawed ovarian tissue
has increased to 11 (including unpublished data). It is
still too early to determine the efficacy of this technology, but the current data is encouraging: 6 live births
out of 12 pregnancies following 30 transplantations
and several ongoing pregnancies as of the end of 2008
[17]. Six pregnancies occurred spontaneously and five
by in vitro fertilization (IVF) and embryo transfer. In
the IVF group, 15 embryos (including 2 blastocysts)
were transferred, which resulted in 6 pregnancies
(2 miscarriages, 1 ectopic pregnancy and 3 live births).
The details of orthotopic autotransplantation of ovarian tissue is discussed in Chapter 29.
Heterotopic autotransplantation
Heterotopic autotransplantation is an attractive alternative to orthotopic autotransplantation as it can avoid
invasive procedures and make the recovery of oocytes
easy. In particular, it is a practical and cost-effective
technology when repeated transplantation is required,
because of the shortened life span of the ovarian grafts;
or a hostile pelvic environment due to previous radiation; or severe pelvic adhesions precluding orthotopic
transplantation. The duration of ovarian function
Ovarian transplantation
Oophorectomy
Tissue processing
Whole ovary
prepared
with vessels
FREEZE
(Slow freeze/vitrifit)
FREEZE
(Slow freeze/
vitrifit)
THAW
THAW
Autotransplantation
Or thotopic
Autotransplantation
Heterotopic
Xenotransplantation
In vitro culture
Heterotopic
with vascular
anastomosis
Orthotopic
with vascular
anastomosis
Figure 27.4 Theoretical strategies for oocyte maturation in cryopreserved ovarian tissue and intact ovary. To date, the only strategy that
produced live births is orthotopic autotransplantation of ovarian tissue. See plate section for color version.
after heterotopic transplantation of human ovarian tissue varies widely (between 3 and 60 months).
In theory, the optimal site should be rich in blood
supply, convenient to implant, easy for oocyte recovery
and mimic the surrounding environment of the ovary.
Various heterotopic sites have been tested, which
include subcutaneous tissue of the abdomen [36], forearm [37] or hip [23], rectus muscle [38], breast tissue [38], uterus [D. Nugent, unpublished data], as
well as subperitoneal tissue beneath the abdominal fascia [39]. Nevertheless, the optimal site for heterotopic
transplantation of human ovarian tissue is still elusive.
335
Table 27.2 The advantages and disadvantages of heterotopic transplantation
Advantages
Disadvantages
Convenient for repeated multiple transplantations
In vitro fertilization (IVF) procedure required
Non-invasive procedure
Efficacy not proven (no live birth yet)
Easy access for oocyte retrieval without anesthesia
Suboptimal (unknown) environmental effects on follicle growth

and maturation
Feasible for patients with severe pelvic adhesion
Possible poor quality oocytes
Albeit frozenthawed ovarian tissue can be easily

transplanted to the heterotopic site, obtaining healthy
oocytes for IVF is a tremendous challenge. In fact, we
noted compromised follicular growth and poor quality
of oocytes retrieved from heterotopic ovarian grafts,
which may be caused by the suboptimal environmental factors for follicle development such as temperature, local pressure, paracrine factors and blood
supplies.
The re-establishment of endocrine function as well
as oocyte retrieval after heterotopic transplantation
of human ovarian tissue has been demonstrated by
several investigators [3741]. Nevertheless, no baby
has been born after heterotopic transplantation. In
2004, Oktay et al. reported a case of embryo development from the oocytes retrieved from ovarian tissue
implanted beneath the skin of the lower abdomen in a
woman with breast cancer [40]. A total 20 oocytes were
recovered (after 8 egg retrievals), but only 8 oocytes
were suitable for IVF. Of these, only one fertilized normally and developed to a four-cell embryo, but no
pregnancy ensued. In 2006, a Danish group reported
a biochemical pregnancy after transferring a four-cell
stage embryo which was generated by intracytoplasmic sperm injection (ICSI) following the retrieval of
metaphase-II (MII) stage oocytes from ovarian tissue
grafted to the heterotopic site [39]. In this case, frozen
thawed ovarian tissue was grafted to a midline subperitoneal pocket on the lower abdominal wall as a heterotopic site.
Over the past 8 years, Kim et al. transplanted
frozenthawed ovarian tissue heterotopically in cancer patients to assess the long-term ovarian function
and restoration of fertility [41]. Four study patients
(3 with cervical cancer, 1 with breast cancer), with
an age range between 28 and 35 years, were identified and consented for heterotopic transplantation.
All ovarian tissue had been cryopreserved using a
slow-freezing method before cancer treatment. Het-
336
erotopic ovarian transplantation (to the space between

rectus muscle and fascia in the abdomen) was performed between 2002 and 2005 (Figure 27.5). The reestablishment and maintenance of ovarian function
was confirmed by serial blood test (FSH, luteinizing
hormone [LH], estradiol, progesterone, testosterone)
and ultrasound monitoring. Three patients were stimulated with gonadotropin followed by oocyte retrievals
to investigate the restoration of fertility. The retrieved
oocytes were matured in vitro and fertilized with partners sperm.
The hormonal profile of all four patients was
consistent with the postmenopausal level before
transplantation. The return of ovarian function
was evidenced by the elevation of serum estradiol
levels and by the decrease of FSH levels below
20 mIU/ml between 12 and 20 weeks after transplantation in all 4 patients. However, restored
ovarian function lasted only 35 months, and 3
patients (except 1 with relapsed disease) agreed to
undergo a second transplantation. The return of
ovarian function after the second transplantation
was faster in all 3 patients (between 13 months)
(Figure 27.6).
In contrast to first transplantation, we observed
the establishment of long-term ovarian function (lasting for 960 months) after the second transplantation. All 3 patients maintained the FSH levels below
15 mIU/ml during this period. We were able to retrieve
7 oocytes (2 germinal vesicle [GV], 4 MI, 1 MII) from
ovarian grafts in 2 patients between August 2003 and
November 2006. Three of four MI oocytes were developed to full maturity in vitro. Four oocytes at the MII
stage were fertilized and cultured in vitro for 2 or
3 days before cryopreservation. Currently, 4 embryos
(at 6-cell, 3-cell, 2-cell and pronuclear [PN] stage)
are stored in liquid nitrogen. Although these results
are encouraging, it is difficult to predict whether it
could be a clinically practicable technology since the
Figure 27.5 The process of heterotopic transplantation of frozenthawed human ovarian tissue to the space between the rectus muscle
and the rectus sheath. Reproduced with permission from Elsevier. See plate section for color version.
120
FSH (IU/l)
100
80
60
40
20
0
0
(a)
4
5
6
7
Months after transplant
10
11
12
Figure 27.6 Monthly follicle stimulating

hormone (FSH) (a) and estradiol levels (b)
after the second transplantation of
ovarian tissue in three cancer patients
(AC). Estradiol production from ovarian
grafts was noticed 2 months after
transplantation, and serum FSH levels
decreased below10 mIU/ml in 2 patients
3 months after transplantation.
Reproduced with permission from
Elsevier. See plate section for color
version.
Estradiol (pg/ml)
250
200
A
B
C
150
100
50
0
0
(b)
4
5
6
7
environment of heterotopic sites may not be as favorable for normal follicle development.
Xenotransplantation
Numerous animal and human studies support the
value of xenotransplantation of ovarian tissue as a
strategy to preserve fertility and to conserve rare and
endangered species. It has already been demonstrated
that xenotranplantation of ovarian tissue from cat,
wombat, sheep, African elephant, monkey and human
to immunodeficient mice can support follicular development up to the antral stage [23]. Furthermore, generation of live young from xenografted rodent ovaries
proved that it is a valid technology for animal conservation as well as fertility preservation [42].
10
11
12
Historically, xenotransplantation of human ovarian tissue was explored as a strategy to restore fertility in cancer patients with a high risk of cancer cell
re-introduction with autotransplantation. Successful
follicular development, ovulation and corpus luteum
formation in the human ovarian tissue xenografted
after cryopreservation have been demonstrated [43,
44]. Furthermore, Kim et al. succeeded in retrieving human oocytes that had developed in the frozen
thawed ovarian tissue xenografted to SCID mice [45].
All host animals were stimulated with gonadotropin
from 20 weeks after xenografting. Grafts were recovered 36 h after human chorionic gonadotropin (hCG)
administration. Fifty-seven grafts from 30 animals
(total 60 grafts) were recovered, and 12 oocytes were
collected from 26 antral follicles (size larger than
337
Table 27.3 The use of xenotransplantation of human ovarian

tissue for studies
Angiogenesis
Ischemic tissue damage
Follicular biology and physiology
Oocyte quality
Steroidogenesis
Minimal residual disease (MRD) of cancer
Gonadotoxicity of various agents
2 mm in diameter). After in vitro maturation for 36 h,

only 2 oocytes were matured to the MII stage. Most
oocytes retrieved from xenografts, however, showed
aberrant microtubule organization and chromatin
patterns.
This study indicated that immature oocytes in
ovarian grafts can grow to maturity, but obtaining
fully competent and healthy oocytes for fertilization
can be problematic. We can speculate that the compromised follicular development and oocyte quality
may be influenced by multiple factors such as cryoinjury, ischemic damage or in vitro maturation. However, most of all, the environment of the xenograft site,
which is very different from that of the human pelvic
cavity, may not provide optimal conditions for human
follicular growth and maturation.
Even if we can overcome these technical difficulties, clinical application of xenotransplantation of
human ovarian tissue will remain problematic until
the safety and ethical issues are resolved. Nevertheless,
xenotransplantation of ovarian tissue will continue to
provide a useful experimental model to study follicular development, angiogenesis, ischemic tissue damage, MRD of cancer, gonadotoxicities of various agents
and much more (Table 27.3).
Whole ovary transplantation by

vascular anastomosis
As an alternative to ovarian tissue transplantation,
whole ovary transplantation has been explored. In theory, whole intact ovary transplantation with vascular anastomosis can restore the full function of the
ovary. The main challenge of whole ovary transplantation for fertility preservation is the development
of effective cryotechnology for the whole organ. The
whole human ovary is vulnerable to cryoinjury, as it
is bulky and composed of various cells and tissues.
Unless we develop the optimal crytechnology for the
338
organ, extensive cryoinjury to the ovary while freezing

and thawing is unavoidable, and leads to the destruction of numerous cellular ultrastructures and, subsequently, their function. In particular, vascular injury
with freezing and thawing is of great concern.
When freezing multicellular systems, extracellular
ice can be as lethal as intracellular ice. In fact, there has
been no documented success in freezing vital organs
(such as livers and kidneys) because of the difficulty
of preventing extracellular ice formation, particularly
the formation of intravascular ice. Additional factors
also make cryopreservation of the organ difficult, such
as delivery of adequate concentrations of CPA evenly
into all cells in a timely manner.
Nevertheless, it may be beneficial to transplant
the whole ovary by vascular anastomosis as it should
theoretically minimize ischemic damage. Many studies, however, showed that the follicular survival rate
after whole ovary transplantation is no better than
that of ovarian tissue transplantation. Courbiere et al.
reported successful microsurgical transplantation of
the fresh intact ovary, but follicular survival rate was
only 6% [46]. This poor follicular survival is most likely
due to prolonged warm ischemia time and significant
thrombosis.
The results of transplantation of the frozenthawed
whole ovary are even more disappointing. Microvascular anastomosis of the whole cryopreserved ovary
in sheep revealed large fibrotic areas with an absence
of follicles (3050%) in the transplanted ovary. As a
consequence, the follicular survival rate was 8% [47].
This severe follicular loss may be caused not only by
direct cryoinjury to the follicles due to suboptimal
cryotechnology but also by ischemia induced by
thromboembolism in the vascular system of the ovary
after transplantation.
Viability data following whole bovine ovary cryopreservation have shown a significant detrimental
effect of cryopreservation on the extent of arterial endothelial cell layer detachment and arterial
smooth muscle damage [48]. In fact, Bedaiwy et al.
demonstrated poor long-term vascular patency after
autotransplantation of intact frozenthawed bovine
ovaries with microvascular anastomosis [49]. The
anastomosed vessels were completely occluded in 8 of
11 cases, leading to immense follicular loss.
In summary, the main advantage of transplantation by vascular anastomosis is that it can restore blood
supply immediately after transplantation. By minimizing ischemia time with whole ovary transplantation,
Table 27.4 Advantages and disadvantages of whole ovary

transplantation
Advantages
Disadvantages
Immediate blood
supply to the graft
Technical difficulties of organ

cryopreservation
Minimizing tissue
ischemia
Surgical complexity and longer

surgical time
Potential long-term
ovarian function
Ischemia-reperfusion injury
Thromboembolism
Increased morbidity(and
mortality)
Potential risks of cancer
recurrence (especially with
minimal residual disease in
ovarian medulla)
greater longevity of ovarian function can be expected.

On the contrary, many disadvantages of vascular transplantation of the frozenthawed whole ovary are evident (Table 27.4). First, no reliable and successful cryopreservation for whole organ freezing is available, and
it will take a while before perfecting this technology. Second, ischemia-reperfusion injury cannot be
avoided even with vascular transplantation. Indeed,
surgical time can be prolonged due to the technical complexities of vascular surgery, which can accelerate follicular loss through warm ischemia. Moreover, thromboembolism after vascular transplantation
can cause extensive cell death and be life threatening under certain circumstances. Finally, whole ovary
transplantation can increase cancer cell recurrence
to a greater degree if the risk of residual ovarian
medullary pathology is high, whereas it is unlikely
with transplantation of small pieces of ovarian cortical
sections.
Conclusions
Ovarian tissue cryobanking has rapidly become a
promising strategy for fertility preservation in cancer patients, and its use will extend beyond women
with cancer. Although healthy babies have been born
with orthotopic autotransplantation of frozenthawed
ovarian tissue worldwide, its clinical efficacy and practicability for fertility preservation is still unclear. There
are many unresolved issues with ovarian tissue cryopreservation and transplantation, such as cryoinjury,
ischemic injury, risks of cancer cell transmission and
its efficacy. Heterotopic autotransplantation may be
an alternative method to restore fertility with use of
cyrobanked ovarian tissue. To date, there is no report

of a live birth after heterotopic transplantation of ovarian tissue in humans, and its clinical practicability is
questionable. However, it is too premature to abandon this strategy without further investigations, as heterotopic transplantation has several advantages over
orthotopic transplantation.
Due to the limited life span of ovarian tissue transplants, many investigators have been exploring the
feasibilities of whole ovary transplantation by vascular anastomosis. To date, no significant advantage of
transplanting the whole intact ovary has been demonstrated, whereas the risks involved with the whole
ovary transplantation by vascular anastomosis (especially after freezing and thawing) appear to be higher
than expected. The safety and efficacy of these emerging technologies should be carefully examined and
tested before full clinical application. Detailed discussions on ovarian cryobanking and transplantation are
found in Chapters 2831.
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341
Section 7
Chapter
28

Debra A. Gook and David H. Edgar
Introduction
Early detection and aggressive chemotherapy/
radiotherapy treatments have improved the long-term
survival rates for many young women with various
types of cancer. As a consequence of these cytotoxic
treatments, their reproductive future can be either
short lived or eradicated. For young single women
with cancer, oocyte cryopreservation offers the best
potential option for achieving a future pregnancy
using their own gametes. Unfortunately, the urgent
need to commence cytotoxic treatment often does not
permit adequate time for cryopreservation of mature
oocytes. Conversely, cryopreservation of ovarian
tissue eliminates the delay necessary to obtain mature
oocytes, but the subsequent potential for establishing
pregnancy is currently unknown. Although ovarian
tissue cryopreservation is an attractive alternative and
frequently used for patients with these conditions,
little has been published on the efficacy of various
protocols. Cryopreservation of ovarian tissue is more
complex than that of gametes or embryos, requiring preservation of multiple cell types, which may
vary in volume and water permeability. Essentially,
ovarian tissue cryopreservation is more similar to
organ cryopreservation than to that of gametes or
embryos.
Early attempts to cryopreserve mouse ovarian tissue had shown the potential of this technology with
evidence of intact follicles in cryopreserved tissue
after grafting [1] and subsequent litters from grafts
of cryopreserved tissue [2]. A resurgence of interest in ovarian tissue cryopreservation stemmed from
the birth of a lamb following cryopreservation and
grafting of ovarian tissue reported by Gosden et al.
in 1994 [3]. This success has now been translated
into the human with the demonstration of resump-
tion of cycling and live births following grafting of

cryopreserved ovarian tissue. However, there is little
information on the relative efficiency of the methods
presently being used for human ovarian tissue cryopreservation. In fact, unequivocal clinical evidence
of preservation of function in autografts using current methods is limited to eight patients [411; C. J.
Stern, personal communication], in which embryos
were formed in vitro from oocytes aspirated from
heterotopic grafts although no pregnancies resulted
(Table 28.1 [1011]). Despite providing proof of principle, this highlights the paucity of systematic information regarding cryopreservation of human ovarian
tissue.
Human ovarian tissue

The structure of the human ovary is a crucial consideration in the potential success of cryopreservation. The
human ovarian cortex is predominantly (80%) populated with quiescent primordial stage follicles [12
14], each consisting of an oocyte (approximately one
third the diameter of a mature oocyte) surrounded by
a single layer of flattened pre-granulosa cells (Figure
28.1a) or with a mixture of flattened and cuboidal pregranulosa cells (Figure 28.1b). Follicle classification is
reported in Table 28.2 [1417] using both the BD system reported by Gougeon and Chainy in 1987 [15] or
by name [14, 17]. A much lower proportion of follicles will have initiated development (primary follicle;
Figure 28.1c) and in approximately 3% of follicles division of granulosa cells will have occurred (proliferating
follicle; Figure 28.1d). In contrast to the rodent ovary,
in which much of the initial ovarian tissue cryopreservation was performed, more advanced follicles characterized by at least two layers of granulosa cells (secondary follicle; Figure 28.1e) and follicles with small
342
1.5 M EG
+0.1 M sucrose
1.5 M DMSO
+0.1 M sucrose
1.5 M DMSO
+0.1 M sucrose
1.5 M DMSO
+0.2 M sucrose
1.5 M DMSO
+0.1 M sucrose
1.5 M PROH
+0.1 M sucrose
551
551
and
10 10 1
552
10 10 1
NP
321
4 C
4 C
4 C
22 C
4 C
30 min
4 C
30 min
22 C
30 or 90 min
NP
NP
0 C
4 C
30 min
4 C
30 min
4 C
30 min
4 C
30 min
Start temp.
7 C
7 C
9 C
7 C
7 C
9 C
7 C
Seed temp.
First-thaw
solution
1.5 M DMSO
1.0 MDMSO
+ 0.1 M sucrose
0.75 M EG
+ 0.25 M sucrose
37 C
2 min
1.0 M PROH +0.2

M sucrose
Air 30 s then 37 C 1.4 M DMSO

2 min
+ 0.2 M sucrose
Air 30 s then 37 C
2 min
Air 2 min then

25 C 2 min
35 C
23 min
37 C
Air 30 s then 37 C 1.5 M DMSO

2 min
+ 0.1 Msucrose
Thaw temp.
14 oocytes
10 MII
2 2 cell ET
2 6 cell ET
10 oocytes
9 embryos
4 blastocysts
2 blastocysts ET
20 oocytes
6 MII
1 7 cell (no ET)
2 oocytes
1 3 cell ET
6 oocytes
4 2 PN
1 6 cell
1 3 cell
1 2 cell (no ET)
3 oocytes
2 MII
1 4 cell ET
1 5 cell ET
20 oocytes
8 MII
2 2 PN
1 3 cell
1 4 cell ET
Outcome
All cryopreserved using controlled rate slow freezing at 2 C/min to seed temperature followed by 0.3 C/min to 40 C or 50 C.
DMSO, dimethyl sulfoxide; EG, ethylene glycol; ET, embryo transfer; MII, metaphase-II oocyte; NP, not provided; PN, pronuclei; PROH, propanediol.
1.5 M DMSO
+0.1 M sucrose
Cryoprotectants Dehydration
551
and
15 5 2
Size of tissue
slices (mm)
Table 28.1 Cryopreservation methodology used in clinical cases where oocytes and embryos have been derived from heterotopic grafts
C. J. Stern,
personal
communication
Piver et al. [9]
Poirot et al. [5]

Poirot et al. [11]
Demeestere et al.
[7]
Kim et al. [8]
Rosendahl et al.
[6]
Schmidt et al. [10]
Oktay et al. [4]
Ref.
Table 28.2 Follicle classification
Typea
Nameb
Characteristic
Follicle diameter
(m)
Population
(%)
Primordial
A layer of flattened pre-granulosa cells
3540
4156
B/C
Transitory/ intermediary
primordial
Mixture of flattened and cuboidal pre-granulosa cells
3744
4122
Primary
A single layer of cuboidal granulosa cells
4654
1521
C/D
Proliferating
An incomplete second layer of granulosa cells
6077
Secondary
Two or more layers of granulosa cells
100
Antral
Multiple layers of granulosa cells with antral cavity
a
b
13
<1
Gougeon and Chainy [15]; Gougeon [16].

Westergaard et al. [14]; Gook et al. [17].
Figure 28.1 Types of follicles observed in

human ovarian tissue: (a) primordial; (b) two
intermediary primordial; (c) an intermediary
primordial (top) and primary (bottom); (d)
proliferating; (e) secondary and an
intermediary primordial; (f) an antral and a
primordial (top right corner). See plate section
for color version.
(a)
(b)
(c)
(d)
(e)
(f)
antral cavities (Figure 28.1f) are rarely observed in the

human ovary.
Primordial follicles in the human are situated
approximately 1 mm below the cortical epithelium
344
(Figure 28.2a) embedded in a dense cortex of stromal cells and collagen bundles. At birth, it is estimated
that between 250 000 and 500 000 primordial follicles
[12] are present within the human ovary, decreasing
Chapter 28: Ovarian tissue cryopreservation
Figure 28.2 Ovarian cortex from 2

patients: (a) an 18-year-old patient with
abundant primordial follicles throughout
the cortex; and (b) a 34-year-old patient
with a cluster of primordial follicles. See
plate section for color version.
(a)
(b)
to approximately 25 000 by the age of 37 [18]. Obviously these are the candidate follicles to preserve with
any cryopreservation regimen. The aim of cryopreservation is not only to preserve the structure and function of both the pre-granulosa cells and oocyte within
individual primordial follicles but also to preserve the
majority of the primordial follicles. This is complicated by the enormous variation in the density of
the ovarian cortex and the distribution of follicles
between patients. Histological examination of ovarian tissue destined for cryopreservation from over 150
patients, ranging in age from the early teens to over
40 years of age [D. A. Gook, unpublished observations] shows that the cortex of the adult human ovary
is extremely fibrous with highly variable distribution
of follicles [19]. In the young teenage ovary, abundant follicles appear to be evenly distributed around
the cortex (Figure 28.2a). However, in ovaries from
women of more advanced reproductive age, follicles
are depleted from specific regions, altering the appearance to that of follicle clusters with fewer individual
follicles which are, in turn, becoming more sparsely
distributed (Figure 28.2b). This lack of uniformity
across the cortex will impact not only on the evaluation
of methodology but also the potential clinical success
in older women requesting ovarian tissue cryopreservation.
Methodology
Preparation of ovarian tissue for
cryopreservation
The most significant loss of follicles results not from
cryopreservation but from ischemia; 65% of sheep follicles are lost following exposure to 0 C for 23 h in a
cell culture medium (Leibovitz L-15) and only a further 7% as a result of cryopreservation [20]. Under the
same conditions, a slightly higher loss due to ischemia

(78%) was observed in mouse ovaries with no further
reduction as a consequence of cryopreservation [21].
Despite its critical impact, the preparation of ovarian tissue has received little attention. This is probably a consequence of the apparent normality of tissue as assessed by routine histological assessment of
non-frozen tissue fixed at the end of preparation [D. A.
Gook, unpublished observations] and the high level of
viability staining after thawing [22]. This fails to detect
the consequences of ischemic exposure during preparation, which only becomes evident when the tissue is
grafted and begins to function.
It is routine practice for organs destined for transplantation to be perfused with and transported for a
number of hours in a basic salt solution at 4 C in
order to reduce ischemia. However, it is questionable
whether this rationale would apply to ovarian tissue in
which the follicles are located just under the surface
epithelium where there is minimal circulation.
Collection and preparation of ovarian tissue is
most commonly carried out in Leibovitz L-15 medium
at 4 C. Due to the limited number of centers performing ovarian tissue cryopreservation, tissue is frequently transported for 45 h [23] and has been transported in some cases for up to 28 h [24]. The consequences of these conditions are largely unknown. A
reduction in both primordial (60%) and developing
follicles (40%) has been observed following exposure
of intact rat ovaries to 4 C for 24 h prior to subsequent
transplantation [25].
It is possible that cold ischemia may be reduced
by transport in a more appropriate medium. A comparison of exposure of human ovarian tissue to 4 C
for 24 h in a Leibovitz-based medium or a histidine
tryptophanketoglutarade solution (HTK, an organ
transport medium) showed enhanced follicle survival
and a lower level of lipid peroxidation in the HTK
345
[26]. The HTK medium is currently used for extended

duration transport of ovarian tissue [26]. However, a
similar comparison of a culture medium designed for
gametes and embryos (Quinns Hepes modified HTF;
Figure 28.3a) and HTK (Figure 28.3b) for a shorter
duration (2 h) followed by xenografting resulted in a
dramatic loss of stromal tissue structure and follicles
regardless of the medium used [D. A. Gook, unpublished observations].
Whether ambient temperature would be more
appropriate for transport is difficult to ascertain. Similar levels of ischemia (measured by the rate of oxygen consumption) were observed in bovine ovaries following 24 and 48 h incubation at 4 C compared to
ambient temperature [27]. In animal studies, it is clear
that exposure of antral follicles in a whole ovary to
4 C results in impaired developmental potential in the
oocytes following fertilization [28], but information
relating to the impact on primordial follicles is scarce.
In our study, less ischemic damage was observed following exposure of human ovarian tissue to ambient temperature (22 C) for 2 h (Figure 28.3c) than
cold exposure. However, both warm and cold ischemia
result in loss of cell structure and a reduction in the
volume of ovarian tissue, with the damage reaching
a maximum at 4 days, generally when it appears that
neovascularization has occurred [29]. In contrast to
this study, bovine ovaries exposed for 3 h to cold
or warm conditions showed no increase in ischemia
(as measured by oxygen consumption) irrespective of
treatment [27]. A reduction in ischemic damage and
increased follicle survival has been reported following daily dietary supplementation of vitamin E in mice
after ovarian grafting [30].
(a)
(b)
Tissue dissection
Obviously, incubation at 37 C in an in vitro fertilization (IVF) medium is also beneficial for the collection of oocyte cumulus complexes (OCC) from
antral follicles during tissue preparation. Generally,
two types of OCC are recovered: those with very few
layers of corona cells, frequently with corona cells in
the perivitelline space (an indicator of atresia); and
those with a dense compact mass of corona cells in
which only a vague outline of the oocyte can be seen.
Our experience suggests that only a minor proportion of these oocytes will mature in culture and this
may take as long as 72 h, at which stage they can
be denuded and cryopreserved. In 2004, Isachenko
346
(c)
Figure 28.3 Fresh ovarian tissue exposed to different

temperatures for 2 h prior to xenografting under the kidney capsule
in immunodeficient mice and subsequently examined 2 days after
grafting. (a) Tissue in Quinns Hepes modified human tubal fluid
(HTF) at 4 C. (b) Histidinetryptophanketoglutarade solution (HTK)
medium at 4 C. (c) Quinns Hepes modified HTF at 22 C. (a) and (b)
magnification 10, (c) 5. See plate section for color version.
et al. reported two cases where oocytes were recovered

during ovarian tissue preparation with 50% maturing
after 36 h in culture in an in vitro maturation (IVM)
culture system [31]. In some patients with endometrial cancers there is an associated predisposition to
polycystic ovaries and priming with human chorionic gonadotropin (hCG) prior to ovarian harvest [32]
may be beneficial in these situations, resulting in a
higher proportion of mature oocytes [33]. Follicles can
also be collected from human ovarian tissue following
enzyme and mechanical isolation [3436]. Although
these follicles can potentially be cryopreserved, the
efficiency of this has yet to be accurately assessed in
the human. However, isolated murine follicles have
been successfully cryopreserved using both slow freezing [3740] and, more recently, using vitrification
[4143].
Attempts have been made to cryopreserve the
intact ovary (see Chapter 30). However, due to the
extremely fibrous structure of the human ovarian cortex and the location of the primordial follicles, the
general consensus approach has been to minimize the
size of the tissue pieces. Human ovarian tissue is prepared by first removing all medulla and reducing the
cortical thickness to approximately 1 mm. This facilitates cryoprotectant and water movement with the
ultimate aim of balancing effective dehydration with
minimal injury due to osmotic toxicity. To increase
surface area exposure to cryoprotectants, and facilitate
storage, the 1 mm thick cortical tissue is further sliced
into pieces ranging in surface area from 115 mm2
[44]. There is, however, no clear evidence that can be
used to establish a relationship between the dimensions of a tissue slice and the successful preservation of
follicles.
Cryopreservation
In contrast to cryopreservation of gametes and
embryos, ovarian tissue cryopreservation is more
closely related to organ cryopreservation [45]. The
underlying problem is that the optimal cryopreservation procedure for one cell type may not be optimal
for other cells within the tissue. Therefore, a compromise may be necessary to preserve a high proportion
of all cell types present in the tissue. There is obviously disparity of cell size between the stromal cells,
the oocyte and the pre-granulosa cells within the primordial follicle, which will limit the ability to optimize the cryopreservation procedure for human ovar-
ian tissue. The rate at which water traverses the cell

membrane, the membrane hydraulic permeability (Lp)
[46], is a fundamental consideration in cryopreservation and will be specific for a cell type. In some cases,
such as the human oocyte, the Lp varies between cells
of the same type [47]. This cellular property, together
with the surface area and free water content of a cell,
will dictate the rate of dehydration necessary prior to
cryopreservation regardless of whether cryopreservation is achieved via controlled rate cooling or vitrification. In addition, cell size, which will contribute to
determining the optimal rate of cooling [45], is not
uniform within ovarian tissue.
Cryoprotectants
Cellular density and tissue geometry will affect infiltration of cryoprotectants. Diffusion of cryoprotectants
is relatively rapid in murine ovaries, which consist
almost completely of abutting developing follicles with
almost no fibrotic material and are therefore relatively
porous. However, the densely fibrotic human ovarian
cortex is extremely different. Murine ovarian tissue is
therefore an unsuitable model for the human in this
respect.
In the case of permeating cryoprotectants, the aim
is to gradually displace cellular water without inducing
excessive shrinkage. To overcome the difficulties associated with the density of human ovarian tissue, the use
of higher concentrations of cryoprotectants has been
suggested. However, under these circumstances, the
reduced aqueous phase and hyperosmotic conditions
result in excessive shrinkage of cells and loss of cell
cell communication [48]. This, in turn may result in
subsequent impairment of tissue function even though
morphology may appear normal. The reduction in filamentous actin traversing the zona of oocyte cumulus complexes isolated from cryopreserved murine
ovaries indicates that this may occur in ovarian
tissue [49].
Four permeating cryoprotectants: glycerol (GLY),
dimethyl sulfoxide (DMSO), ethylene glycol (EG) and
propanediol (propylene glycol; PROH) have been used
in human and animal ovarian tissue cryopreservation. For all permeating cryoprotectants, a concentration of 1.4 or 1.5 mol/l has generally been used for
slices of human and animal ovarian tissue [44]. However, higher concentrations of PROH (2 and 4 mol/l at
ambient temperature) do not appear to cause follicular toxicity when compared to control tissue exposed
347
to no cryoprotectant [50]. In contrast, concentrations

of DMSO above 2 mol/l were toxic. Follicular toxicity
has also been shown for concentrations of EG above
2.0 mol/l [51].
Although GLY was used in initial rodent studies [1, 2, 52], it has not been used clinically for
human tissue. This is probably due to the reported
low proportion of follicles (10%) surviving after cryopreservation of human tissue with glycerol, the subsequent lack of follicles observed in the majority of
grafts using this tissue [53] and the failure of follicles to survive in vitro culture [54]. The poor outcome observed with glycerol is likely to be a consequence of the slow rate at which glycerol permeates tissue relative to other cryoprotectants [55], although this
can be compensated for by increasing the dehydration
time [56].
The rate of cryoprotectant penetration through
tissue is also a function of temperature. Newton
et al. showed that both DMSO and EG had penetrated
through 76% of human ovarian tissue in 20 min at
4 C but that this occurred twice as rapidly at 37 C
[55]. Of interest is the additional observation that neither of these cryoprotectants had permeated all of the
tissue even after 90 min at 37 C. In contrast, PROH
penetration was slower at 4 C than DMSO and EG,
requiring 30 min to achieve 76% penetration but at
37 C PROH had a significantly higher rate of diffusion,
achieving 100% penetration by 15 min. It is of no surprise then that reduced follicle survival was observed
following dehydration of larger pieces of human ovarian tissue at 4 C for 30 min in PROH (44%) relative to DMSO (84%) and EG (74%) [53]. High follicle survival following cryopreservation under the
same conditions using EG has also been observed by
others [57]. Similar levels of survival (80%) have
been achieved using appropriate conditions for individual cryoprotectants i.e DMSO at 4 C and PROH at
room temperature [58]. However, to achieve a similar survival rate using a short (10 min) exposure time
with larger (2 10 10 mm) pieces of sheep ovarian cortex, which is also relatively fibrous, a higher
concentration of either PROH or DMSO (2 mol/l)
was necessary [50]. Effective dehydration of tissue is
therefore dependent on the tissue geometry together
with the rate of penetration of the cryoprotectant
which is in turn a function of temperature. Therefore,
to conclude from some of the above studies that a
cryoprotectant is unsuitable for ovarian tissue cryopreservation is not justified. It would appear that
348
insufficient dehydration may have resulted from suboptimal conditions for a specific cryoprotectant and
that, with optimization, a variety of protocols may be
appropriate.
Non-permeating
cryoprotectants,
generally
sucrose at a concentration of 0.1 mol/l, have also
been used to facilitate dehydration of ovarian tissue
[11, 13, 5861]. Recent studies have shown that
increasing the sucrose concentration significantly
improves outcomes for embryo cryopreservation [62]
but addition of various concentrations of sucrose in
combination with DMSO did not appear to result in
improved cryopreservation [55] and no other studies
have specifically compared protocols with and without
sucrose.
The use of sucrose with the permeating cryoprotectant PROH [13] has been investigated in an attempt
to optimize dehydration of both pre-granulosa cells
and oocytes within primordial follicles [63]. Equivalent proportions of morphologically intact oocytes
could be achieved with shorter time exposure when
the sucrose concentration was increased from 0.1
to 0.2 mol/l. However, extended exposure to higher
sucrose resulted in decreased oocyte survival [63]
and the observation, within the surviving oocytes,
that the majority of cytoplasm consisted of vacuolation and lysed mitochondria. This phenomena was
not observed with the lower sucrose concentration
(0.1 mol/l) which, in contrast, resulted in an improvement in the proportion of morphologically intact
oocytes and pre-granulosa cells with time. The proportion of surviving oocytes with normal cytoplasm also
increased with time in 0.1 mol/l sucrose [13]. Extended
exposure to the elevated sucrose concentration may
result in inappropriate osmotic gradients which is also
the likely explanation for the low survival of pregranulosa cells (Figure 28.4b) and poor cytoplasmic
morphology in oocytes when applying two-step compared to single-step dehydration with equivalent final
cryoprotectant concentrations [13]. By manipulation
of dehydration using PROH and sucrose at ambient
temperature, a high proportion of intact pre-granulosa
cells (74%), intact oocytes (91%) and oocytes with normal cytoplasmic appearance (95%) could be achieved
(Figure 28.4c). However, morphology of the stromal
tissue was consistently poor irrespective of the regimen (Figure 28.4b,c) compared to non-cryopreserved
(Figure 28.4a), highlighting the relative complexity
associated with dehydration of tissue containing multiple cell types.
Rate of cooling
(a)
(b)
(c)
Figure 28.4 Primordial follicles present in ovarian tissue following

cryopreservation: (a) non-cryopreserved; (b) dehydrated using 1.5 M
propanediol (PROH) and 0.2 M sucrose; (c) dehydrated using 1.5 M
PROH and 0.1 M sucrose. See plate section for color version.
In the majority of the above studies the rate of cooling used has been similar to the rates used for controlled rate embryo cryopreservation (2 C/min to ice
seeding temperature followed by 0.3 C/min). As with
embryo and oocyte cryopreservation, the rate of cooling for ovarian tissue has never been systematically
optimized. Mazur estimated that the theoretical rate
of cooling for organs or tissues should be 1 C/min
[45], but a comparison of the standard slow rate
(0.3 C/min) and a faster cooling rate (2 C/min) with
large pieces (200 mm3 ) of ovine ovarian tissue demonstrated a significant improvement in follicular survival
at the faster rate [50]. In an attempt to investigate this
for human ovarian tissue, the slow rate (0.3 C/min)
was compared to two faster cooling rates following
the same prefreeze dehydration and the morphology
of each cell type was determined [13]. Poor morphology of the whole tissue was observed with a rapid rate
(Figure 28.5a; to be discussed in more detail later in
the context of vitrification). At an intermediate rate
(36 C/min), a high proportion of the stromal cells
and collagen bundles appeared normal but only half
the oocytes were intact and almost all pre-granulosa
cells were abnormal (swollen and with swollen nuclei;
Figure 28.5b), again emphasizing the importance of
variation in cell size. With the slow rate, approximately
half of both the stromal and pre-granulosa cells and
over 80% of the oocytes were normal. Normal appearance in the pre-granulosa cells and oocytes could be
further enhanced by applying the slow rate after more
extensive dehydration prior to cryopreservation but
this was at the expense of stromal cell survival (Figure
28.4c) [63, 64].
During controlled rate cryopreservation, cryoprotectant crystallization will occur, and the temperature
at which this occurs is specific for the cryoprotectant.
For PROH or DMSO this occurs at 6 to 8 C. Without manual seeding, crystallization will be initiated at
any solid surface i.e. throughout the tissue. Demirci
et al. [50] reported a dramatic deviation from the normal cooling curve resulting in reduced follicular survival in the absence of manual seeding. Manual seeding at a slightly higher temperature (5 compared to
7 C) appeared to improve follicle survival [54].
Similarly, damage can occur during thawing as
a result of crystallization of water or cryoprotectant. Irrespective of the cryopreservation method used
(controlled rate or vitrification), thawing should be as
349
Figure 28.5 Ovarian tissue

cryopreserved using: (a) a rapid cooling
rate showing remnants of a primordial
follicle; and (b) an intermediate rate of
cooling showing a primordial follicle
containing lysed pre-granulosa cells and
oocyte. See plate section for color version.
(a)
(b)
rapid as possible. Although critical, the thawing temperature is rarely reported and, again, almost no studies have systematically investigated this aspect of cryopreservation with animal or human ovarian tissue.
Exposure to some cryoprotectants, such as DMSO,
at higher temperatures may increase their toxicity. A
comparison of thawing of murine ovarian tissue, cryopreserved in DMSO, at 27, 37 or 47 C showed a significant reduction in follicle survival at the higher temperatures [54]. In our experience, thawing tissue cryopreserved in PROH in a 1 ml volume in a Cryovial
requires 3 min at 37 C to achieve liquefaction prior to
immediate removal of the tissue. At this temperature,
at least in the case of PROH, there appears to be no
affect on developmental potential (see later).
Vitrification offers the potential benefit of overcoming many of the issues associated with crystallization discussed above. The problem associated with vitrification of ovarian tissue, however, is how to achieve
the high cooling and warming rates required. Vitrification of murine ovaries has been successful with
subsequent births of pups reported [65, 66] following vitrification in cryostraws and plunging in liquid nitrogen. However, when a faster cooling rate was
achieved by direct contact with liquid nitrogen, it
resulted in better preservation as evidenced by significantly more morphologically normal, viable follicles
and pups [65]. This improvement, however, may also
be partly due to more appropriate dehydration prior
to vitrification. Vitrification in cryostraws resulted in
poorer outcomes relative to controlled rate cryopreservation for all parameters measured. Wang et al. [67]
have also shown the importance of the cooling rate
for vitrification with both mouse and human ovarian
tissue.
Ovine hemi-ovaries have also been successfully vitrified resulting in live births although one of the four
350
births was a large lamb and died short after delivery as

a result of malformations [68]. The cocktail of cryoprotectants (2.6 M DMSO, 2.6 M acetamide, 1.3 M PROH
and 7.5 mM polyethylene glycol) used in this study has
also been used for human ovarian tissue with evidence
of preservation of normal follicular morphology [69].
In contrast, poor developmental capacity was observed
following vitrification of murine embryos with this
protocol [70].
It is of some concern that, in order to achieve
dehydration of ovarian tissue and facilitate vitrification with rapid instead of ultra-rapid rates of cooling, exposure to very high concentrations of a cocktail of cryoprotectants may be necessary. In contrast
to embryo and oocyte vitrification, in which exposure
to these very high concentrations of cryoprotectants is
limited to 30 s and many normal births have been
reported, ovarian tissue may be exposed for 15 min.
Dehydration prior to controlled rate cryopreservation
in DMSO (1.5 M) has routinely been performed at
4 C to limit toxicity [71]. Using this approach, successfully vitrification of human ovarian tissue has been
achieved with dehydration in 1.5 M DMSO at 4 C in
combination with PROH and EG [72]. However, in a
another recent vitrification procedure, human ovarian tissue was exposed to 1.0 M DMSO followed by
2.8 M DMSO at ambient temperature for 15 min at
each concentration [73]. The safety of the vitrification procedures required for ovarian tissue will require
careful evaluation in animal models before clinical
application.
Storage
Irrespective of the methodology used for cryopreservation, tissue has generally been stored in Cryovials,
which do not constitute a fully sealed system. A
theoretical risk of cross contamination is associated

with storage of biological material in Cryovials under
liquid nitrogen and the relatively extended duration of
storage which may be anticipated with ovarian tissue
would potentially increase this risk. As such, storage in
liquid nitrogen vapor may be the preferred option for
facilities offering clinical ovarian tissue cryopreservation.
Evaluating the efficiency of ovarian

tissue cryopreservation
In contrast to evaluation of outcomes from embryo
or gamete cryopreservation, assessing the survival and
viability of cryopreserved ovarian tissue poses specific
challenges. In many of the studies discussed previously, success has been measured in terms of follicle
survival after isolation from thawed tissue. The validity of this approach depends on three fundamental
assumptions: (a) that lysed or destroyed follicles will
be detected after isolation; (b) that loss of follicles as a
result of enzyme digestion will be equivalent for cryopreserved and fresh tissue; and (c), specifically in the
case of human ovarian tissue, that there is an even distribution of follicles throughout the tissue. However,
once an oocyte within a primordial follicle has lysed,
it is essentially impossible to identify the follicle (Figure 28.5a,b). Therefore, only follicles with an intact
oocyte will be included in these studies, resulting in
over estimation of survival. In addition, much of the
stromal tissue is damaged with controlled rate cryopreservation rendering follicles within it more vulnerable to enzymes and this is also likely to vary between
protocols (Figures 28.4 and 28.5). Finally, due to the
extent of variation in follicle distribution in human
ovarian tissue, it is invalid to express the number of follicles present after cryopreservation as a proportion of
the number in a non-cryopreserved sample. Although
these criticisms weaken some of the conclusions which
have been drawn above there are no other available
studies of this type on cryopreservation of ovarian
tissue.
In contrast, it is potentially possible to overcome
these problems by histological evaluation of the entire
tissue but there are very few studies which have
attempted this [21] and expressed normal morphology
as a proportion of the total number of follicles within a
piece of tissue [13, 63, 74]. Histological examination, at
both the light and electron microscope level, has generally assessed only a small sample to estimate overall
follicle integrity [75], although this has allowed detection of abnormalities such as oocyte shrinkage [76],
vacuolated areas within the oocyte cytoplasm [77], loss
of mitochondrial cristae [78] and lysis of pre-granulosa
cells [79].
There are only two morphometric studies of
human ovarian tissue which assess cryopreservation
[13, 74], one of which is an evaluation of the most
commonly used procedure [3] using DMSO as a cryoprotectant and controlled rate cooling on tissue from
six patients [74]. In this study, almost half of the follicles and the vast majority of oocytes (81%) were damaged. Parallel assessment of apoptosis confirmed these
observations. The other study assessed the proportion of intact pre-granulosa cells and oocytes together
with the relative normality of the oocyte cytoplasm
as estimated by vacoulation and normal mitochondria within every oocyte for a range of cryopreservation procedures using PROH and sucrose as cryoprotectants [13, 63]. Observations were verified by electron microscopic evaluation of a small number of follicles (Figure 28.6a,b). The highest proportion of both
oocytes (85%) and pre-granulosa cells (74%) with normal morphology was observed following dehydration
for 90 min in 1.5 M PROH + 0.1 M sucrose at ambient temperature followed by a slow controlled rate of
freezing. Although this type of morphometric assessment has provided evidence of morphological normality after cryopreservation, it gives no indication of viability or function.
Viability staining has also been used to assess follicles within a piece of tissue [80, 81] following cryopreservation. Generally, this staining will identify live
cells on the basis of an intact membrane and cytoplasm
which is functionally capable of cleaving a chromagen,
and dead cells on the basis of their inability to exclude
a nuclear stain [51, 57]. Again, this form of assessment has limitations [76]. Tissue pieces must be very
small to facilitate diffusion of dye and only follicles
with a live oocyte are detected. Although the nuclear
stain will detect the germinal vesicles (GV) of lysed
oocytes, these are indistinguishable from the nuclei of
stromal or pre-granulosa cells, resulting in the potential for overestimation of follicle viability. An example
of a lysed oocyte which would be unlikely to be distinguished from a granulosa cell is shown in Figure 28.5a.
Finally, it is important to remember that we cannot
infer, on the basis of viability staining, that the developmental potential of these primordial follicles has been
retained following cryopreservation.
351
Figure 28.6 Electron micrographs of

oocyte cytoplasm within primordial
follicles after cryopreservation using: (a)
dehydration in 1.5 M propanediol (PROH)
and 0.1 M sucrose for 90 min followed by
slow cooling showing normal
mitochondria and cytoplasm; and (b)
following same regimen with a shorter
time of 30 min showing loss of
mitochondria, vacuolation and abnormal
cytoplasm.
(a)
(b)
Developmental potential
Assessment of expression of developmental potential in vitro is an attractive possibility, but requires
an understanding of the requirements for initiation
of growth in primordial follicles. Additionally, many
months of culture may be required. More advanced follicles isolated at the secondary stage, in which initiation of granulosa cell proliferation has occurred, can
be successfully grown in culture [8284]. However, the
predominant follicle present in the human ovary is the
primordial. There is evidence, that murine primordial
follicles, grown in a two-phase culture system, can produce live pups [85, 86], but this has not been established using cryopreserved ovarian tissue. The culture
of isolated primordial follicles, whether from fresh or
cryopreserved tissue, has proved problematic in animal models [8789] and in human [59, 90, 91]. However, follicular development to the secondary stage
has been established by culturing primordial follicles
within stromal tissue [92, 93]. More recently, development from primordial to early antral stage in vitro
has been demonstrated using a two-stage culture system with fresh tissue [35], and there is a preliminary
report of this approach being used to demonstrate
developmental potential of human primordial follicles
following vitrification [E. E. Telfer, unpublished data].
Results to date on demonstration of developmental
potential using in-vitro systems, while promising, are
limited to relatively few follicles.
As a result of the difficulties associated with culture of primordial follicles in vitro, various in vivo
approaches involving grafting of cryopreserved ovarian tissue, either alone or in combination with final
maturation in vitro, have been applied. Clear evidence
of preservation of developmental potential has been
established by the birth of live offspring from primordial follicles following grafting of ovine and murine
352
cryopreserved ovarian tissue [3, 71, 94]. Heterotopic

grafting of murine cryopreserved tissue with subsequent IVM has also resulted in live births [95].
In the human situation, xenografting of cryopreserved tissue into immunodeficient mice has been
used to assess preservation of developmental potential
in a number of studies and has established that follicles
are viable and capable of development [21, 60, 9698].
Developmental capacity to the antral stage has been
shown to be preserved with the commonly used cryopreservation regimen using DMSO [97, 98] and also
the PROH/ sucrose procedure [96]. Although primary
and secondary follicles within human ovarian tissue
may have survived cryopreservation, the time required
for these antral follicles to develop post-grafting (5
6 months) suggests that they have developed from
primordial follicles. Reproducibility of preservation
has been established by development of numerous
antral follicles using tissue cryopreserved from multiple patients with both procedures [99101]. Full developmental competence has been shown to be preserved
with both procedures with evidence of ovulation (corpora lutea) [98, 102] and mature oocytes [99, 100,
102]. Unfortunately, although mature oocytes were
aspirated from follicles in our laboratory, experiments
to determine subsequent fertilization were prohibited
by law in Australia.
Clinical evidence has established that fertilization
and embryo development can occur in oocytes recovered from cryopreserved ovarian tissue transplanted
at a heterotopic site [411; C. J. Stern, personal communication], but no pregnancy has resulted following
transfer of these embryos to date. The cryopreservation procedures which have been shown to be associated with preservation of full developmental potential
are reported in Table 28.1. At present, consistent follicle development and recovery of mature oocytes has
proved difficult.
Refrences
1. Green SH, Smith AU and Zuckerman S. The numbers
of oocytes in ovarian autografts after freezing and
thawing. J Endocrinol 1956; 13: 3304.
2. Parrott DMV. The fertility of mice with orthotopic
ovarian grafts derived from frozen tissue. J Reprod
Fertil 1960; 1: 23041.
3. Gosden RG, Baird DT, Wade JC et al. Restoration of
fertility to oophorectomized sheep by ovarian
autografts stored at 196 C. Hum Reprod 1994; 9(4):
597603.
4. Oktay K, Buyuk E, Veeck L et al. Embryo development
after heterotopic transplantation of cryopreserved
5. Poirot C, Piver P, Fayet P et al. Human embryo
development after subcutaneous autograft of
cryopreserved ovarian tissue. Hum Reprod 2006; 21
(Suppl. 1): i155 [AbstrP404].
6. Rosendahl M, Loft A, Byskov AG et al. Biochemical
pregnancy after fertilization of an oocyte aspirated
from a heterotopic autotransplant of cryopreserved
ovarian tissue: case report. Hum Reprod 2006; 21(8):
20069.
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Section 7
Chapter
29
Ovarian tissue transplantation

Jacques Donnez, Jean Squifflet and Marie-Madeleine Dolmans
Introduction
According to previous reports, around 700 000 new
cancer cases are expected every year among the female
population of the USA [1] and 8% of these women are
likely to be under the age of 40. In fact, it is now estimated that 1 in every 250 people in the adult population is a childhood cancer survivor [2].
Advances in the diagnosis and treatment of
childhood, adolescent and adult cancer have greatly
increased the life expectancy of premenopausal
women with the disease. Indeed, aggressive chemotherapy and radiotherapy, as well as bone marrow
transplantation, can cure more than 90% of girls
affected by childhood malignancies [3], but have
resulted in a growing population of adolescent and
adult long-term survivors of childhood cancer [2] who
may experience infertility problems due to induced
premature ovarian failure (POF).
Indications for ovarian

tissue cryopreservation
Oncological indications for ovarian tissue cryopreservation are summarized in Table 29.1 [4, 5]. In case
of gynecological malignancy, a conservative fertility
approach is only valuable if the uterus can be spared
during surgery. This includes cases of early cervical
carcinoma, early vaginal carcinoma, early endometrial adenocarcinoma, ovarian tumors of low malignancy and some selected cases of unilateral ovarian
carcinoma (stage IA) [4, 5]. The choice of a possible
conservative surgical approach in these patients and
the question of implementing such treatment alone
remain controversial, and all published results were
obtained on the basis of retrospective studies and/or

case reports. The fertility outcome is conditioned by
the adjuvant therapy administered, i.e. local radiotherapy and/or chemotherapy. Respective indications
in the case of malignant disease are presented in
Table 29.1.
However, cryopreservation should not be reserved
solely for women with malignant disease [6]. Indeed,
hematopoietic stem cell transplantation (HSCT) has
been increasingly used in recent decades for noncancerous diseases, such as benign hematological disease (sickle cell anemia, thalassemia major and aplastic anemia) and autoimmune diseases previously unresponsive to immunosuppressive therapy (systemic
lupus erythematosus, autoimmune thrombocytopenia) [6, 710]. Other benign diseases, such as recurrent
ovarian endometriosis or recurrent ovarian mucinous
cysts, are also indications for ovarian cryopreservation. Patients undergoing oophorectomy for prophylaxis may potentially benefit from ovarian cryopreservation too.
Autotransplantation of cryopreserved
human ovarian tissue
There have been numerous reported cases of autotransplantation of cryopreserved ovarian tissue, either
to an orthotopic or heterotopic site [6, 924].
Orthotopic autotransplantation of
cryopreserved human ovarian tissue
In theory, natural pregnancy may be achieved via
orthotopic tissue transplantation if the fallopian tubes
remain intact.
357
Table 29.1 Indications for cryopreservation of ovarian tissue in

cases of malignant and non-malignant disease.
A. Malignant
(a) Extrapelvic diseases
Bone cancer (osteosarcoma, Ewings sarcoma)
Breast cancer
Melanoma
Neuroblastoma
Bowel malignancy
(b) Pelvic diseases

Non-gynecological malignancies
Pelvic sarcoma
Rhabdomyosarcoma
Sacral tumors
Rectosigmoid tumors
Gynecological malignancies
Early cervical carcinoma
Early vaginal carcinoma
Early vulvar carcinoma
Selected cases of ovarian carcinoma (stage IA)
Borderline ovarian tumors
Systemic diseases
Hodgkins disease
Leukemia
Medulloblastoma
B. Non-malignant
(a) Uni/bilateral oophorectomy
Benign ovarian tumors
Severe and recurrent endometriosis
BRCA-1 or BRCA-2 mutation carriers
(b) Risk of premature menopause

Turners syndrome
Family history
Benign diseases requiring chemotherapy: autoimmune
diseases (systemic lupus erythematosus, rheumatoid
arthritis, Behcets disease, Wegeners granulomatosis)
(c) Bone marrow transplantation

Benign hematological diseases: sickle cell anemia,
thalassemia major, aplastic anemia
Autoimmune diseases unresponsive to immunosuppressive
therapy
From Donnez et al. [5]
Technique
Ovarian biopsy and freezing
Ovarian tissue cryopreservation is undertaken before
chemotherapy. Written informed consent is obtained
358
from the patient or her parents if she is under 18 years

of age. It is always considered an emergency and ovarian biopsy is performed as soon as possible in order
not to delay the start of chemotherapy.
As follicles are located inside the ovarian cortex, tissue samples collected for cryopreservation have
to come from the surface of the organ. The biopsy
can be taken during any gynecological procedure, by
laparoscopy or laparotomy, and may be composed of
one or several cortical fragments, or even a whole
ovary, depending on the surgical indications and the
risk of POF after treatment. Biopsies may be obtained
by simple laparoscopy carried out under general anesthesia. Biopsy forceps [25] are inserted through one of
the 5-mm trocars placed in the iliac fossa and are used
to grasp the ovary and cut a fragment from its surface
(Figure 29.1). The cortical biopsy can also be easily performed with laparoscopic scissors.
It is important to remove only the cortical surface
to a depth of 12 mm to be sure to obtain tissue from
the area rich in primordial follicles.
The number of biopsies taken varies according to
the size of the patients ovaries and the estimated risk of
POF. Indeed, POF after chemotherapy is dependent on
age, drug used and dose given [26]. Biopsy samples are
immediately transferred to the laboratory in Leibovitz
L-15 medium supplemented with GlutamaxTM (Invitrogen, Paisley, UK) on ice. To minimize any tissue
damage due to ischemia, the samples are transferred
within the shortest possible interval (minutes) to the
laboratory for processing. Unilateral (left) oophorectomy is only performed where there is a 100% risk
of ovarian failure, like after pelvic radiotherapy, bone
marrow transplantation or chemotherapy with 2 alkylating agents.
Slow freezing procedure

The whole procedure is performed on a laminar airflux table using sterile disposable materials to ensure
optimal sterility of the tissue fragments. Samples are
transferred to a Petri dish containing a sterilized glass
slide and 12 ml of Leibovitz L-15 medium. Tissue
temperature is kept close to 4 C by placing the dish
on top of a glass box containing crushed ice. The ovarian medulla is then separated from the cortex using
forceps and surgical scissors, and disposed of. The
remaining cortex is cut on the glass slide into strips
of 2 68 mm. These strips are transferred into 2 ml
Cryovials (Simport, Quebec, Canada) containing
800 l of L-15 medium and stored at 0 C in a cooler
Chapter 29: Ovarian tissue transplantation
Cancer diagnosis
Pregnancy
Biopsy of ovarian tissue

through laparoscopy
Restoration
of menstrual cycle
Uterus
Cryopreservation
of tissue
samples
Cancer treatment
Fallopian
tubes
Ovary
Vagina
Complete remission
Grafting or placing
of fragments in the
perinatal cavity
Thawing of tissue
samples
LE SOIR - 10.12.09
Figure 29.1 Illustration describing the different steps from biopsy to re-implantation and pregnancy. See plate section for color version.
box (NalgeneTM Labtop cooler, Cat. No. 51160032;

Nalge Nunc International, Rochester, NY, USA), each
of the tubes containing 25 strips. One strip is randomly put aside and immersed in a 37% paraformaldehyde solution for histological examination and evaluation of the ovarian reserve. Leibovitz L-15 medium
is then twice replaced with 800 l of freshly prepared
cryoprotective solution containing 88% Leibovitz L-15
medium, 2% human albumin 20% (Red Cross, Brussels, Belgium) and 10% dimethyl sulfoxide (SigmaAldrich Co., Irvine, UK).
Freezing of ovarian tissue is carried out according
to the protocol described by Gosden et al. [27]. The
Cryovials are cooled in a programmable freezer (Kryo
10, Series III; Planer, Sunbury-on-Thames, UK) following the steps below:
r At 0 C, place Cryovials inside the freezer and
keep stable at 0 C for 15 min.
r From 0 to 8 C, cool at a rate of 2 C/min.
r Keep stable at 8 C for 8 min for soaking.
r Seed manually (induction of ice crystal

formation) by grasping the Cryovials (for 510 s
each) with forceps prechilled in liquid nitrogen.
r Keep stable at 8 C for 15 min.
r From 8 to 40 C, cool at a rate of 0.3 C/min.
r From 40 to 150 C, cool at a rate of 30 C/min.
The Cryovials are then transferred to liquid nitrogen
(196 C) for storage.
Thawing [16]
For thawing, the Cryovials are removed from the liquid nitrogen and left at room temperature for 2 min.
Thawing is subsequently completed by immersing the
Cryovials in a warm (37 C) water bath for 2 min. The
tissue samples are then grasped with small forceps and
placed in a Petri dish containing L-15 medium, which
is replaced 3 times (5 min each wash) to remove the
cryoprotectant completely.
359
Fallopian
tube
Creation of
peritoneal window
Atrophic
ovary
Fimbria
Reimplantation of ovarian
graft in peritoneal window
(a)
(b)
Figure 29.2 (a) Creation of a peritoneal window close to the atrophic ovary. (b) Transplantation of ovarian cortex pieces into the peritoneal
window. See plate section for color version.
Re-implantation (Figure 29.2)

In two cases, we performed a first laparoscopy 7 days
before re-implantation to create a peritoneal window
by means of a large incision just beneath the right ovarian hilus, followed by coagulation of the edges of the
window [6, 16]. The goal was to induce angiogenesis
and neovascularization in this area.
A second laparoscopy was performed 7 days after
creation of the peritoneal window. A biopsy of 45 mm
in size was taken from each of the atrophic ovaries to
check for the presence or absence of primordial follicles. The cryogenic vials were thawed at room temperature (between 21 and 23 C) for 2 min and immersed
in a water bath at 37 C for another 2 min. Ovarian
tissue was immediately transferred from the vials to
tissue culture dishes (Becton Dickinson, NY, USA)
in Leibovitz L-15 medium and subsequently washed
three times with fresh medium to remove cryoprotectant before further processing. Thawed ovarian cortical tissue was placed in sterile medium and immediately transferred to the operating theater.
In the first case mentioned above, we pushed the
large strip and 35 small cubes of frozenthawed ovarian tissue into the furrow created by the peritoneal
360
window, very close to the ovarian vessels and fimbria

on the right side [16]. No suture was used. An extensive
neovascular network was clearly visible in this space
(Figure 29.3).
After a long discussion with the oncologists and
patient, a third laparoscopy was proposed. At least
Figure 29.3 An important vascular network is observed 7 days

after the creation of the peritoneal window. See plate section for
color version.
three reasons were given to justify the procedure: (1)

to check the viability of the orthotopic grafts 4 months
after transplantation by laparoscopic visualization and
histological analysis; (2) to check for the absence of
any cellular growth anomalies (peritoneal fluid, histology), the cortical strip and cubes having been biopsied before chemotherapy; and (3) to re-implant the
remaining ovarian cortical cubes, by request of the
patient, who was now aged 32 years. Indeed, if pregnancy had not ensued from the re-implanted tissue,
she would have considered oocyte donation. A validated technique will probably not require so many surgical procedures in the future.
In the second case, a first laparoscopy was performed 7 days before re-implantation, not only to create a peritoneal window just beneath the left ovarian
hilus, as previously described, but also to perform an
ovarian incision along the longitudinal ovarian axis
[6]. The edges of the window and the ovarian incision were coagulated in order to induce neovascularization in this area. Knowing from experimental data
that the ovary itself, even if atrophic, may be an ideal
site for re-implantation, we decided to simultaneously
prepare two sites for re-implantation [5, 10, 28; M.-M.
Dolmans et al., personal communication]. Both sides
were found to be effective as follicular development
was demonstrated by vaginal ultrasound [6].
In the other cases, pieces of cryopreserved ovarian tissue were sutured to the medulla of the remaining ovary, either by laparotomy (Figure 29.4) or by
laparoscopy (Figure 29.5).
Figure 29.4 shows transplantation of large ovarian
cortical strips to the remaining ovary. Figure 29.5 illustrates the transplantation by laparoscopy.
By either laparotomy or by laparoscopy, the different steps are identical.
1. Decortication of the remaining ovary. The ovarian
medulla is then denuded (Figure 29.4a).
2. If the ovarian strips are large enough, they are
sutured on the ovarian medulla (Figure 29.4b)
3. If only small cubes or strips are available, they are
placed on the ovarian medulla (Figure 29.5a) and

covered by Interceed (Johnson and Johnson,
USA) (Figure 29.5b).
R
Results from the literature

In 2000, Oktay and Karlikaya reported a laparoscopic
transplantation of frozenthawed ovarian tissue in
a 29-year-old patient, who had undergone bilateral
(a)
(b)
Figure 29.4 (a) Decortication of the ovarian cortex from the

remaining ovary. (b) Suture of the cryopreserved thawed cortical
strips on the ovarian medulla. See plate section for color version.
oophorectomy for a non-malignant disease [11]. Follicular development was demonstrated only once by
ultrasonography 15 weeks after transplantation. The
reason why this patient had undergone oophorectomy
and re-implantation is not clear and no further data
were available.
Radford et al. reported a patient with a history
of Hodgkins disease treated by chemotherapy [13].
Ovarian tissue had been cryopreserved before receiving high doses of chemotherapy for a third recurrence
of disease. Histological section of the ovarian cortical tissue revealed only a few primordial follicles due
to the previous chemotherapy. Eight months after reimplantation, estradiol was detected and the follicle
stimulating hormone (FSH) and luteinizing hormone
(LH) levels decreased. One month later, LH and FSH
concentrations returned definitively to menopausal
levels.
361
(a)
(b)
Figure 29.5 (a) After decortication, the cryopreservedthawed

cortical cubes are placed on the ovarian
medulla. (b) The ovarian

cubes are covered with Interceed . See plate section for color
version.
R
We reported the first successful transplantation of

cryopreserved ovarian tissue resulting in a pregnancy
and live birth [16]. In 1997, a 25-year-old woman presented with clinical stage IV Hodgkins lymphoma.
According to Schilsky et al., the risk of POF after such
a regimen in a woman of 26 years of age is more
than 90% [29], while according to Wallace et al. and
Lobo et al., the risk of subfertility after Hodgkins treatment with alkylating agents is more than 80% [26,
30]. Ovarian tissue cryopreservation was undertaken
before chemotherapy. After laparoscopy, the patient
received hybrid chemotherapy from August 1997 to
February 1998, followed by supradiaphragmatic radiotherapy (38 Gy).
362
In 2003, once the patient had been declared completely disease-free, re-implantation was carried out
in orthotopic sites (see Donnez et al. for techniques
[6, 16]). From 5 to 9 months after re-implantation,
concentrations of FSH, 17 -estradiol and progesterone showed the occurrence of ovulatory cycles. At
11 months, the patient became pregnant and subsequently delivered a healthy baby. This birth was
announced in the Lancet in September 2004 [16].
In 2005, Meirow et al. also published details of
a live birth after orthotopic autotransplantation of
cryopreserved ovarian tissue in a patient with POF
after chemotherapy [17]. Eight months after orthotopic transplantation, the patient spontaneously menstruated. Nine months after transplantation, she experienced a second spontaneous menstrual period. After
a modified natural cycle, a single mature oocyte was
retrieved and fertilized. Two days later, a four-cell
embryo was transferred. The patient became pregnant from this embryo transfer and delivered a healthy
infant weighing 3000 g.
Demeestere et al. reported a pregnancy after natural conception in a woman who had undergone
orthotopic and heterotopic transplantation of cryopreserved ovarian tissue [19]. Unfortunately, this pregnancy, obtained by natural conception, ended in miscarriage at 7 weeks due to aneuploidy. The same team
performed a second re-implantation to an orthotopic
site in the same patient after cessation of graft secretion was evidenced [20]. The patient became pregnant
and delivered a healthy baby. She recently delivered a
second healthy child.
Silber et al. reported a pregnancy following reimplantation of cryopreserved ovarian tissue between
monozygotic twins [21]. It should be noted, however,
that the same woman had already delivered a first
healthy baby after re-implantation of fresh tissue [21].
Andersen et al. described a series of six orthotopic re-implantations of cryopreserved ovarian cortex. In this series, two women became pregnant and
delivered healthy infants [22]. Single mature oocytes
were retrieved during optimized cycles, fertilized and
transferred on day 3. One of the two women was
later naturally pregnant and delivered a second healthy
child.
In 2009, Piver et al. reported the birth of a
healthy baby after orthotopic re-implantation of cryopreserved tissue [31]. The patient became pregnant
naturally. Very recently, Sanchez-Serrano reported a
twin pregnancy after an in vitro fertilization (IVF)
Figure 29.6 Significant follicle

stimulating hormone (FSH) decrease and
estradiol increase are observed 4 months
after ovarian tissue re-implantation. With
permission from RMB Online.
180
160
140
UI/ml
120
100
Mean FSH
Mean estradiol
80
60
40
20
0
0
2
3
4
Months post-transplantation
procedure in a woman who have undergone orthotopic

re-implantation of cryopreserved ovarian tissue [32].
Restoration of ovarian function

In our series, orthotopic transplantation of ovarian
tissue induced restoration of ovarian function [10].
Analysis of these cases raises some important points
for discussion. First of all, in all cases, it took between
31/2 and 5 months after re-implantation before a follicle could be seen by ultrasonography and a decrease
in FSH level observed (Figure 29.6). The process of
folliculogenesis takes around 46 months, during
which time the oocyte and surrounding somatic cells
undergo a series of changes that eventually result in
the development of a large antral follicle, capable of
producing a mature oocyte [33]. Thus, the appearance
of the first follicle originating from grafted tissue 45
months after re-implantation, proved by laparoscopy
in one case, is totally consistent with the expected time
course. This time interval between implantation of cortical tissue and the first estradiol peak is also consistent with data obtained from sheep [34, 35] and human
beings, although some variations may be observed.
Indeed, the delay between transplantation and follicular development was found to vary from 6 weeks to 8
months. Such a variation could be explained by a difference in follicular reserve at the time of cryopreservation.
Another very interesting finding in our series was
the persistence of relatively high FSH levels during the
follicular phase. The FSH levels remained as high as
25 mIU/ml during the follicular phase until ovulation, and then decreased to 15 mIU/ml during the
luteal phase. This may constitute an argument against

the use of gonadotropin injections. The relatively high
FSH levels may be explained by the relatively low number of surviving primordial follicles in the graft. The
patient should be considered a poor responder, with
reduced inhibin B secretion. These results are in agreement with those obtained in sheep by Campbell et al.
[36].
A further significant observation was the return to
an FSH level of 25 mIU/ml immediately after each
menstrual bleed, which supports the theory suggested
by Baird et al. that some inhibitory mechanisms, such
as inhibin B or anti-Mullerian hormones (AMH) normally produced by developing follicles in intact human
ovaries, are probably almost non-existent in transplanted tissue [35]. After transplantation, the patient
would have been regarded a poor responder because,
of the 5001000 primordial follicles that would have
been transplanted, more than 50% would have been
lost owing to hypoxia [16].
The crucial issue of revascularization

Van Eyck et al. recently characterized the oxygen environment in human ovarian xenografts in the early
post-grafting period (up to day 21) using electron
paramagnetic resonance oximetry [37]. This technique
allows sensitive, non-invasive and repeated measurement of PO2 in vivo. Before day 5, grafts were exposed
to hypoxia. From day 5 to day 10, progressive reoxygenation was observed, suggesting an active process of
graft revascularization.
Using a combined method of perfusion study
and double immunohistochemical staining of human
363
and murine vessels, the same team evaluated the

revascularization process of human ovarian tissue in
this model [38].
On day 5, reperfusion of ovarian grafts was initiated by host angiogenesis, as evidenced by the
appearance of murine neovessels penetrating from the
periphery and colocalized with perfused areas. By day
10, the center of the fragments was perfused and ovarian graft angiogenesis contributed to the vascular pattern of the ovarian transplants.
Host and graft angiogenesis thus both appear to
contribute to post-transplantation vascular behavior
and could be potential targets to improve the mechanisms leading to perfusion of grafts with the aim of
reducing the avascular period.
Is there still a place for heterotopic

transplantation?
There are only a few existing reports on this subject. Callejo et al. evaluated the long-term function of
cryopreserved heterotopic grafts, but no conclusions
could be drawn since the patient was perimenopausal
at the time of ovarian biopsy for cryopreservation [23].
In 2004, Kim et al. reported a case of a 37-yearold woman who underwent heterotopic (rectus and
pectoralis muscle) transplantation of cryopreserved
ovarian tissue [14]. By 14 weeks of transplantation,
restoration of endocrine function was demonstrated
but, approximately 28 weeks after transplantation, cessation of ovarian function was evidenced by very high
FSH levels (6299 mIU/L) and very low estradiol
levels.
The same year, Oktay et al. reported transplantation of frozenthawed ovarian tissue beneath the skin
of the abdomen [12]. A 4-cell embryo was obtained
from 20 oocytes retrieved from an ovarian graft, but
no pregnancy occurred after transfer. Oocyte quality
might have been compromised by transplantation to a
heterotopic site.
Kim et al. reported in 2004 on the heterotopic
patient cured of squamous cell carcinoma of cervix
[14]. Tissue was transplanted to two heterotopic sites:
abdominal (rectus muscle) and breast site (pectoralis
muscle). Growing follicles were seen in the abdominal
site from 14 weeks after transplantation, but ovarian
function ceased around 28 weeks after transplantation.
Wolner-Hanssen et al. reported on the subcutaneous transplantation of frozenthawed tissue to the
364
forearm in 2005 [9]. Two follicles developed, but only

to a maximum diameter of 12.6 and 6.7 mm, respectively, and the tissue survived 7 months.
Recently, Kim et al. reported on the heterotopic
autotransplantation of cryopreserved ovarian tissue in
four patients (three with cervical cancer and one with
breast cancer) [15]. Thawed ovarian fragments were
transplanted into a space between the rectus muscle and the rectus sheath. Recovery of ovarian function was evidenced in 3 patients by hormone profiles
obtained between 12 and 20 weeks after transplantation, but this only lasted 35 months. These three
patients subsequently underwent a second transplantation. Long-term ovarian function (1536 months)
was then established. Ovarian grafts were stimulated
daily with FSH, until a dominant follicle size of 14
16 mm was reached. During a 27-month follow-up
period in 2 patients, 6 oocytes were retrieved (1 germinal vesicle [GV], 4 metaphase-I [MI], 1 metaphase-II
[MII]). The MI oocytes were subjected to in vitro maturation. All 4 MII oocytes then fertilized and developed to cleavage-stage embryos (up to 6 cells on day 3)
before being frozen for transfer to a surrogate.
Papers describing heterotopic transplantation have
all reported follicular development, but with follicles
always 16 mm in size [14, 19]. As stressed by WolnerHanssen et al. and Oktay et al., differences in temperature and pressure could interfere with follicular development in heterotopic sites [9, 12].
In our opinion, there is no place for heterotopic
transplantation if the goal is to restore fertility.
Conclusion
Approximately one third of young women exposed
to chemotherapy develop ovarian failure. In 2010, we
believe it is our ethical responsibility to propose cryopreservation of ovarian tissue to all adolescents and
young women under Institutional Review Board (IRB)
protocols having to undergo chemotherapy with alkylating agents.
In conclusion, the 11 live births obtained after
transplantation of frozenthawed ovarian tissue in
humans give hope to young cancer patients, but there
is still much work to be done. Research programs
need to determine whether active angiogenesis can be
induced to accelerate the process of neovascularization in grafted tissue, if isolated human follicles can be
grafted or, indeed, if microvascular re-anatomosis of
an entire cryopreserved ovary is a valuable option.
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cryopreserved ovarian tissue: a pilot experience.
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Steril 2004; 82: 9302.
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18. Schmidt KL, Yding Andersen C, Loft A et al.
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following ovarian cryopreservation and
transplantation. Hum Reprod 2005; 20: 353946.
19. Demeestere I, Simon P, Buxant F et al. Ovarian
function and spontaneous pregnancy after combined
heterotopic and orthotopic cryopreserved ovarian
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with bone marrow transplantation: case report. Hum
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12. Oktay K, Buyuk E, Veeck L et al. Embryo development

after heterotopic transplantation of cryopreserved

heterotopic ovarian transplantation: is there a
germline stem cell connection? Hum Reprod 2006;
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13. Radford JA, Lieberman BA, Brison D et al.

Orthotopic reimplantation of cryopreserved ovarian
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Hodgkins lymphoma. Lancet 2001; 357: 11725.
25. Meirow D, Fasouliotis SJ, Nugent D et al.

Laparoscopic technique for obtaining ovarian cortical
biopsy specimens for fertility conservation in patients
with cancer. Fertil Steril 1999; 71: 94851.
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26. Wallace WH, Thomson AB, Saran F and Kelsey TW.

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Phys 2005; 62: 73844.
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Fertil Steril 2010; 93(5): 167685.
Figure 1.1 John Hunter (172893). The first reported person to

successfully perform artificial insemination in a human.
Figure 1.2 William Harvey (15781657). The first person to

describe the egg as responsible for the production of all creatures.
Figure 1.3 Anton van Leeuwenhoek (16321723). The first person

to study animal and human sperm under microscopes, which he
constructed himself.
Figure 1.6 Patrick Steptoe and Robert Edwards at the birth of the worlds first in-vitro
fertilization conceived baby Louise Brown on July 25, 1978. Courtesy of Bourn Hall Clinic.
10 000 000
NGF population (log10 scale)
1 000 000
n =325
r 2 =0.81
100 000
Observed values
WallaceKelsey model
Lower 95% Cl for model
Upper 95% Cl for model
Lower 95% prediction limit
Upper 95% prediction limit
10 000
1000
100
10
5
0
5
10
15
20
25
30
35
40
45
50
Age (in months from conception to birth; in years from birth to menopause)
Figure 2.1 The best model for the establishment of the non-growing follicle (NGF)
population after conception and the subsequent decline until age at menopause is described
by an ADC model. The model has a correlation coefficient r2 = 0.81, fit standard error = 0.46
and F-value = 364. The figure shows the dataset (n = 325), the model, the 95% prediction limits
of the model and the 95% confidence interval (CI) for the model. The horizontal axis denotes
age in months up to birth at age 0 and age in years from birth to 51 years. From Wallace and
Kelsey [8].
55
Figure 10.1 Primordial follicle to

secondary follicle formation
(gonadotrophin independent phase):
The oocyte, under the stimulatory
effects of retinoic acid, recruits
squamous type granulosa cells (GCs) to
form the primordial follicle. Steroid
hormones inhibit the formation of
primordial follicles. Under the activating
effects of stem cell factor (SCF)/kit
ligand, basic fibroblast growth factor
(bFGF), bone morphogenetic proteins 4
and 7 (BMP-4 and -7), platelet derived
growth factor (PDGF), leukemia
inhibitory factor (LIF) and keratinocyte
growth factor, the primordial follicle
further develops into the primary follicle
with accompanying morphological
changes of the supportive cells, i.e. from
a monolayer of squamous type GCs to
cuboidal type GCs. Thecal cells then
surround the now multiple layers of GCs
which encloses the developing oocyte
to form the secondary follicle.
Repressive signals like phosphatase and
tensin homolog (PTEN), Foxo3 and
anti-Mullerian
hormone (AMH),
tuberous sclerosis complexes (TSC) 1
and 2 and FOXL2 inhibits the
development of primordial follicles to
secondary follicles.
AMH
Secondary follicle
FSH, activins,
inhibins
androgens,
estrogens,
GDF9, BMP15
Primary
oocyte
with zona
pellucida
Basement
membrane
Pre-antral follicle
Thecal layer
Antrum formation
Cumulus cell
layer
enclosing the
oocyte
FSH, insulin-like growth

factors, inhibins,
androgens, estrogens
Antral follicle
Figure 10.2 Pre-antral phase to antral

phase transition. As the secondary follicle
develops, it becomes more
gonadotrophin sensitive with the
expression of gonadotrophin receptors.
Under the activation of follicle
stimulating hormone (FSH) and the
effects of activins and inhibins, androgen
synthesis with estrogen production take
place, resulting in the development of
the secondary follicle into the pre-antral
follicle. Anti-Mullerian
hormone (AMH)
has been shown to suppress early stages
of follicular growth and onset of
responsiveness to FSH in vitro, exerting a
controlling influence on the rate at which
follicles become available for
pre-ovulatory development. Enhancing
effects from growth differentiation factor
(GDF-9), bone morphogenetic protein 15
(BMP-15) and insulin-like growth factors
enables the pre-antral follicle to develop
further into the antral follicle with the
formation of the antrum. At the same
time, the GCs proliferate and differentiate
into the cumulus cell layer which
encloses the developing oocyte. With the
antral cavity filled with follicular fluid, the
pre-antral follicle now becomes the
antral follicle.
Figure 10.3 Antral phase to ovulation:

The antral follicle progresses to develop
into the pre-ovulatory follicle under the
stimulation of luteinizing hormone (LH).
This transition is augmented by
epidermal growth factors (EGF), EGF-like
growth factors and follicular fluid
meiosis-activating sterol (FF-MAS). A
critical step in this transition is the
completion of meiosis I in the maturing
oocyte with the extrusion of the first
polar body. This ensures that the oocyte
achieves meiotic competence prior to
ovulation. Under the LH surge and
increased expression of
metalloproteinases and proteolytic
enzymes, the pre-ovulatory follicle
subsequently ruptures and ovulation
takes place with the release of the
cumulus oocyte complex. The follicle
then collapses and proceeds to form
the corpus luteum.
Gonadotrophin-independent phase
Ovarian follicular
development
Gonadotrophin-dependent phase
Figure 10.4 In the ovary, follicular development

constitutes of a gonadotrophin-independent and a
gonadotrophin-dependent phase. Coordination of
multiple growth factors, hormones and
biochemical molecules signaling at timely intervals
with the activation of several pathways, e.g. PI3K,
JAK/STAT, WNT/-catenin and MAPK between the
growing oocyte, surrounding somatic cells and
wider endocrine system ensures optimum follicular
development with the release of a meiotically and
developmentally competent oocyte.
Required CPA for vitrification (M)
11
10
Pool boiling
Forced flow
Boiling
New
technology
Figure 11.1 The effect of sample size on

the cryoprotectant agent (CPA)
concentration required to achieve
vitrification at various cooling rates.
Plunging samples into liquid nitrogen
(LN2 ) is an example of a pool boiling
approach. Flowing LN2 over a sample is an
example of a forced flow boiling
approach. Oscillating heat pipe (OHP)
technology (data from Jiao et al. [41]) is an
example of new technologies that are
being developed to increase our ability to
apply vitrification approaches to biological
samples.
h = 103 W/m2 K
h = 104 W/m2 K
h = 105 W/m2 K
6
5
h = 106 W/m2 K
4
0.0
0.2
0.4
0.6
0.8
Sample dimension (cm)
1.0
Figure 14.1 Damaged and undamaged

DNA after warming. Fluorescent staining
was performed using SYBR green stain
(working concentration 1:200). In healthy
cells, the fluorescence was confined to the
nucleoid: undamaged DNA is supercoiled
and does not migrate very far from the
nucleoid. In cells that have incurred
damage to the DNA, the alkali treatment
unwinds the DNA, releasing fragments
that migrate from the nucleoid and form
the so-called comet-tail (circled).
Figure 14.3 Scheme of the spermatozoa

vitrification procedure. (1) Single channel
pipettes with adjustable volume (30 l).
(2) Spermatozoa suspension. (3) Strainer.
(4) Foam box. (5) Liquid nitrogen. (6)
Distance between bottom of strainer and
surface of liquid nitrogen (minimum
3 cm). With permission from Isachenko
et al. [115].
(b)
(a)
(c)
(d)
Figure 14.4 Example of non-capacitated

(a), capacitated (b), acrosome-reacted (c)
and non-viable (d) spermatozoa. At least
200 spermatozoa were observed in each
plate and 3 patterns were identified (see
Figure 14.2). (a) A uniform fluorescence on
the head of the spermatozoa
(non-capacitated spermatozoa). (b) A
band of fluorescence diminished in the
post-acrosomal region and a relatively
shining fluorescence in the acrosomal
region (capacitated spermatozoa). (c) A
fluorescence in the complete head of the
spermatozoa, except a tenuous band of
fluorescence in the equatorial segment
(acrosome-reacted spermatozoa). The
slides were viewed using a Zeiss Axiolab
Epifluorescence microscope that was
equipped with an excitation/emission
filter of 485 nm/520 nm under 400
magnification. The non-viable
spermatozoa were observed with the filter
set 09 (450490 nm). The dead
spermatozoa displayed a pattern of blue
fluorescence in the whole head (Figure
14.2d). With permission from Isachenko
et al. [115].
(a)
Figure 14.7 Example of undamaged (a) and

damaged mitochondria (b). In undamaged
mitochondria the mitochondrial membrane
potential (M ) is intact and the JC-1 reagent
aggregate inside of the non-damaged
mitochondria and fluoresces red. In our case,
the midpiece is yellow, as expected for the red
fluorescence from JC-1 aggregates merging
with the green fluorescence of JC-1 monomer
dispersed throughout the cell plasma
membrane. In damaged mitochondria, the M
is broken down and the JC-1 reagent
disperses though the entire cells and fluoresces
green. The changes in M is measured using
an unique fluorescent cationic dye,
5,5,6,6-tetachloro-11,3,3-tetraethylbenzamidazolocarbocyanin iodide, commonly
known as JC-1.
(b)
*
*
*
(a)
(d)
(b)
(e)
(g)
(h)
(c)
(f)
Figure 14.12 The vitrification procedure for big volume of spermatozoa suspension. (a) The 0.25 ml plastic straws are halved and
dark-marked from one side (dark asterisks) and a 0.5 ml packaging straw (white asterisks). (b) The non-marked side of the half-straw is filled
with spermatozoa suspension. (c) A 0.25 ml half-straw, hermetically closed from both sides, is filled with spermatozoa suspension. (d)
Holding it in a horizontal position, the 0.25 ml half-straw filled with spermatozoa suspension is marked from one side (arrowed) and
placed into a 0.5 ml packaging straw, closed from both sides. (e) Using tweezers and keeping it in a horizontal position, this closed
packaging system is directly immersed into liquid nitrogen (LN2 ) and submerged for over 5 s to prevent the flow of spermatozoa
suspension spreading into packaging straw. (f) The vitrified sample is stored in LN2 . Using tweezers, the dark-marked part of the Sealed
Pulled Straw (SPS), approximately 1.01.5 cm, is removed from the LN2 and the end of packaging straw is cut. (g) With the help of a 200 l
pipette tip (Eppendorf AG, Hamburg, Germany), the suspension-filled fixed straw is quickly removed from the packaged straw and (h)
immersed into a 15 ml plastic tube containing 6 ml of human tubal fluid (HTF) and human serum albumin (HSA) prewarmed to 37 C with
gentle agitation to accelerate the melting and removing of content.
Culture
Transplantation
Cryopreservation
Sperm
production
Testis
biopsy
Chemotherapy
or irradiation
Figure 15.1 Male germline stem cell preservation. Before treatment for cancer by chemotherapy or irradiation, a boy
could undergo a testicular biopsy to recover stem cells. The stem cells could be cryopreserved or, after development
of the necessary techniques, could be cultured. After treatment, the stem cells would be transplanted to the patients
testes for the production of spermatozoa. From Brinster [14] with permission.
Germ
cells
Enzyme
In situ
injection
digestion
Testis with
transgene
35 months
Donor genes
transmitted
X Mate
Progeny
Figure 15.2 Testis cell transplantation method. A single-cell suspension is produced from a fertile donor testis. The
cells can be cultured or microinjected into the lumen of seminiferous tubules of an infertile mouse. Only a
spermatogonial stem cell can generate a colony of spermatogenesis in the recipient testis. When testis cells carry a
reporter transgene that allows the cells to be stained blue, colonies of donor cell-derived spermatogenesis are
identified easily in the recipient testes as blue stretches of tubule. Mating the recipient male to a wild-typed female
produces progeny, which carry donor genes. From Brinster [11] with permission.
Tight
junction
Sertoli cell
Sertoli
cell
GFR
-1
Human
spermatogonial
stem cell
SFK
PI3 /AKT
PI3K
AKT
Etv5, Bcl6b
Self-renewal
Differentiation
Figure 15.3 A proposed model of human spermatogonial stem cell (SSC) self-renewal
regulation by glial cell line-derived neurotrophic factor (GDNF), which has been demonstrated
to have an essential role in regulating rodent SSC self-renewal. The model is similar to those
suggested for mouse SSC self-renewal. In this model, GDNF binds to RET and the GFR1
coreceptor with possible intracellular protein kinase signaling through SFK and PI3K/AKT
downstream pathways to regulate the expression of specific genes, such as Etv5 and Bcl6b,
which are involved in SSC self-renewal. However, other genes not regulated by GDNF (e.g.
Zbtb16, Taf4b and Lin28), are likely controlled by different signals and may block differentiation
but not be involved directly in self-renewal. Genes for these regulatory molecules have been
shown to be highly expressed in pre-pubertal human spermatogonia, mouse gonocytes and
mouse SSCs. The basement membrane (green), on which the SSC rests, is generated by the
peritubular myoid cells (dark brown) and Sertoli cells (tan). From Wu et al. [27] with permission.
(a)
(b)
Figure 15.4 Detection of human germ cells transplanted into seminiferous tubules of
recipient mouse testes using a baboon testis specific antibody that identifies human
spermatogonia. (a) Donor human spermatogonia in mouse tubule 4 months after
transplantation of cryopreserved cells. This panel shows that cryopreserved cells also colonize
mouse testes as observed with freshly transplanted cells. (b) Donor human spermatogonia in
mouse testis 5 months after transplantation. These donor cells were transplanted without
cryopreservation. Bar = 100 m (a) and 40 m (b). From Nagano et al. [17] with permission
C 2002 American Society for Reproductive Medicine.
from Elsevier Science, Inc.
Figure 16.1 Histological appearance (hematoxylin/eosin sections) of donor testicular tissue

from a 44-year-old man after 3 weeks orthotopic xenografting at 200 magnification. Most
tubules show degenerative changes, i.e. sclerosis, while the rest contain mainly Sertoli cells.
(a)
(b)
(c)
Figure 16.2 Histological appearance (hematoxylin/eosin sections) of donor testicular tissue

from a 12-year-old boy (a) after 6 months orthotopic xenografting at 200 magnification; (b)
showing pachytene spermatocytes (arrow) and spermatid-like cells (inset) at 400
magnification; and (c) spermatid-like cells at 1000 magnification.
Control
3
HSD
N
Nm
50 m
TEM
Fresh
L
M
Cm
Fresh
2.8 m
50 m
SER
F/T/G
L
Nm
Cm
SER
F/T/G
2.8 m
50 m
Figure 16.3 Steroidogenic activity in Leydig cells evaluated by transmission electron

microscopy (TEM) (left) and immunohistochemistry (IHC) (right). The TEM shows fresh and
frozen/thawed/grafted Leydig cells showing intact structures of nuclear and cytoplasmic
components and activity. Magnification 12 000. Bm, basement membrane; Cm, cell
membrane; F/T/G, frozen, thawed and grafted for 6 months; L, lipid droplets; M, mitochondria;
N, nucleus; Nm, nuclear membrane; SER, smooth endoplasmic reticulum: site of conversion of
pregnenolone to testosterone. The IHC shows fresh and frozen/thawed/grafted Leydig cells
that are stained for 3 -hydroxysteroid dehydrogenase (3 HSD), converting pregnenolone to
progesterone.
Figure 17.2 Intraoperative picture of tubules more likely to harbor spermatogenesis, as

indicated by forceps.
Figure 18.2 Percentage of women with

a return of ovarian function following
chemotherapy. Each study is a different
color. GnRHa, gonadotropin-releasing
hormone agonist.
100%
80%
60%
40%
20%
0%
With GnRHa
Figure 19.1 Radiation field.
No GnRHa
Figure 19.2 Surgical anatomy.
Figure 19.3 Intensity modulated radiation therapy (IMRT).

R
Figure 20.1 Visualization of right pelvic lymphatic channels through laparoscopy after Patent Blue injection in the
cervix.
Figure 20.2 Dissection of a right pelvic sentinel node.
Figure 20.3 Performance of the vaginal cuff.
Figure 20.4 Dissection of bladder pillars and identification of the left ureter.
Figure 20.6 Section of the operative specimen at the level of the uterine isthmus.
Figure 20.8 Operative specimen of a radical trachelectomy showing the vaginal cuff and the proximal parametrial
resection.
Figure 20.9 Set up of the isthmic cerclage.
Figure 20.10 Pre-treatment epidermoid cervical carcinoma stage IB2 (45 mm) in a 25-year-old young woman,
neoadjuvant chemotherapy is planned.
Figure 20.11 Same patient after four courses of neoadjuvant chemotherapy: complete regression of the lesion.
3.8
3.2 2.7 2
0.6
Leukemia
11.3
Hematological
malignancies
44.3
9.6
Hodgkin's
lymphoma
22
22.6
B re a s t c a n c e r
O v a ri a n c a n c e r
R h a b d o m y o s a rc o m a
C e rv i c a l c a n c e r
L i p o s a rc o m a
Le u k e m ia
Non-Hodgkin's
lymphoma
11
O t h e r c a n c e rs (p a n c re a s , s t o m a c h , )
O ste o sa rc o m a
C o l o re c ta l c a n c e r
E w i n g 's s a r c o m a
H o d g k i n 's l y m p h o m a
N o n - H o d g k i n 's l y m p h o m a
Figure 25.1 Indications for ovarian tissue cryopreservation in case of malignant disease at
Saint Lucs University Hospital, Brussels, Belguim in 2008.
Oophorectomy
Tissue processing
Whole ovary
prepared
with vessels
FREEZE
(Slow freeze/vitrifit)
FREEZE
(Slow freeze/
vitrifit)
THAW
THAW
Autotransplantation
Or thotopic
Autotransplantation
Heterotopic
Xenotransplantation
In vitro culture
Heterotopic
with vascular
anastomosis
Orthotopic
with vascular
anastomosis
Figure 27.4 Theoretical strategies for oocyte maturation in cryopreserved ovarian tissue and intact ovary. To date,
the only strategy that produced live births is orthotopic autotransplantation of ovarian tissue.
Figure 27.5 The process of heterotopic transplantation of frozenthawed human ovarian

tissue to the space between the rectus muscle and the rectus sheath. Reproduced with
permission from Elsevier.
120
FSH (IU/l)
100
80
60
40
20
0
0
(a)
4
5
6
7
10
11
12
Estradiol (pg/ml)
250
200
A
B
C
150
100
50
0
0
(b)
4
5
6
7
Figure 27.6 Monthly follicle stimulating hormone (FSH) (a) and estradiol levels (b) after the
second transplantation of ovarian tissue in three cancer patients (AC). Estradiol production
from ovarian grafts was noticed 2 months after transplantation, and serum FSH levels
decreased below10 mIU/ml in 2 patients 3 months after transplantation. Reproduced with
permission from Elsevier.
10
11
12
(a)
(b)
(c)
(d)
(e)
(f)
Figure 28.1 Types of follicles observed in human ovarian tissue: (a) primordial; (b) two
intermediary primordial; (c) an intermediary primordial (top) and primary (bottom); (d)
proliferating; (e) secondary and an intermediary primordial; (f) an antral and a primordial (top
right corner).
(a)
(b)
Figure 28.2 Ovarian cortex from 2 patients: (a) an 18-year-old patient with abundant primordial follicles throughout
the cortex; and (b) a 34-year-old patient with a cluster of primordial follicles.
(a)
(c)
Figure 28.3 Fresh ovarian tissue exposed to different

temperatures for 2 h prior to xenografting under the kidney
capsule in immunodeficient mice and subsequently examined 2
days after grafting. (a) Tissue in Quinns Hepes modified human
tubal fluid (HTF) at 4 C. (b) Histidinetryptophanketoglutarade
solution (HTK) medium at 4 C. (c) Quinns Hepes modified HTF at
22 C. (a) and (b) magnification 10, (c) 5.
(b)
Figure 28.4 Primordial follicles present in ovarian tissue

following cryopreservation: (a) non-cryopreserved; (b)
dehydrated using 1.5 M propanediol (PROH) and 0.2 M sucrose;
(c) dehydrated using 1.5 M PROH and 0.1 M sucrose.
(a)
(b)
(c)
(a)
(b)
Figure 28.5 Ovarian tissue cryopreserved using: (a) a rapid cooling rate showing remnants of
a primordial follicle; and (b) an intermediate rate of cooling showing a primordial follicle
containing lysed pre-granulosa cells and oocyte.
Cancer diagnosis
Pregnancy
Biopsy of ovarian tissue

through laparoscopy
Restoration
of menstrual cycle
Uterus
Cryopreservation
of tissue
samples
Cancer treatment
Fallopian
tubes
Ovary
Vagina
Complete remission
Grafting or placing
of fragments in the
perinatal cavity
Thawing of tissue
samples
LE SOIR - 10.12.09
Figure 29.1 Illustration describing the different steps from biopsy to re-implantation and pregnancy.
Fallopian
tube
Creation of
peritoneal window
Atrophic
ovary
Fimbria
Reimplantation of ovarian
graft in peritoneal window
(a)
(b)
Figure 29.2 (a) Creation of a peritoneal window close to the atrophic ovary.
(b) Transplantation of ovarian cortex pieces into the peritoneal window.
Figure 29.3 An important vascular network is observed 7 days after the creation of the peritoneal window.
(a)
Figure 29.4 (a) Decortication of the ovarian cortex from the remaining ovary.
(b)
(b) Suture of the cryopreserved thawed cortical strips on the ovarian medulla.
(a)
(b)
Figure 29.5 (a) After decortication,

the cryopreservedthawed cortical cubes are placed on the ovarian medulla. (b) The ovarian cubes are

covered with Interceed .
R
Sheep no. 1131
2.5
15
10
1.5
1
0.5
3
Progesterone
levels (ngr/ml)
2
1
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
71
Weeks after transplantation
30
20
10
38
1
Progesterone
level
(ngr/ml)
40
Estradiol
level
(pmol/ml)
Progesterone
level
(ngr/ml)
37
73
Sheep no. 2075
Sheep no. 2076
36
72
300
200
0.5
100
0
Estradiol
level
(pmol/ml)
Estradiol level
(ngr/ml)
Progesterone level
(pmol/ml)
Sheep no. 1199 *
20
34
35
36
(a)
(b)
Figure 30.1 Endocrine function and fertility after cryopreservation and re-transplant of intact sheep ovaries at 2 and 6
years post-transplantation. (a) Hormonal levels 2 years post-transplantation [50]. (b) Follicles by transillumination 6 years
post-transplantation [51].
Figure 30.2 Whole human ovary cryopreservation with the vascular pedicle utilizing the
same Multi-Thermal-Gradient device and slow cooling, rapid thawing protocol as described in
the text.
(a) Stages of follicle development from primordial to ovulatory. All growing follicles (primary onwards)
must be activated from the finite resting pool of primordial follicles.
Primordial
pool
Growth
activation
Primary
Pre-antral
Early antral
Mid antral
Pre-ovulatory
(b) P13K signaling

Growth factors
PTEN
PIP2
PIP3
PDK1
RPTK
Pk
p85
p110
PIP3
Akt
mTORC2
Thr308
Ser473
P13K
PTEN inhibits activation
of primordial follicles
TSC1/TSC2
mTORC1
mTORC1 promotes activation
PDK1
P
Thr309
P
S6K1
Thr225
rpS6
Figure 33.1 (a) Digrammatic representation of follicle growth from the non-proliferating pool
of primordial follicles. Primordial follicles are continuously activated into the growing
population where they become primary follicles consisting of an oocye arrested at the dictyate
stage of prophaseI of meiosis (yellow) surrounded by granulosa cells (green). Primary follicles
undergo oocyte growth and granulosa cell proliferation and differentiation (purple) when they
form an antral cavity. Antral follicles continue to grow and granulosa cells differentiate into two
subpopulations of cells: (1) cumulus surrounding the oocyte (blue); and (2) mural lining the wall
of the follicle (orange). Exact timings for this developmental sequence to occur in humans are
not known but estimations suggest several months. However, it is not known whether the
growth profile is continuous or whether there are resting phases through follicle
development. (b) Simplified version of the PI3K pathway. The factors initiating this process are
largely unknown but a body of evidence is emerging to show that the
phosphatidylinositol-3-kinase (PI3K-AKT) signaling pathway is a major regulator of early
follicle/oocyte development and that components of this pathway are involved in controlling
the rate of activation from the non-growing population of follicles. The phosphatase PTEN
converts PIP3 to PIP2, which negatively regulates PI3K activity. Signaling mediated by PI3Ks
converge at PDK1. PDK1 phosphorylates Akt and activates it. Akt can phosphorylate and
inactivate tuberous sclerosis complex 2 (TSC2 or tuberin), which leads to the activation of
mTOR complex (mTORC1). mTORC1 can phosphorylate (activate) S6K1. S6K1 subsequently
phosphorylates and activates rpS6, which enhances protein translation that is needed for cell
growth. mTORC1 can be inhibited pharmacologically with Rapamycin and stimulated by
leucine. The manipulation of this pathway could have important clinical applications in the field
of fertility preservation.
Micro-cortex
culture
Removal of pre-antral
follicles by
micro-dissection
Individually cultured
and monitored for
oocyte/follicle
health markers
(b)
(a)
(c)
Aliginate encapsulated culture

for further growth and
development then isolation of
oocytecumulus complexes
(d)
(e)
IVM of isolated
and subsequent fertilization
Step 1
1. Preparation of the tissue

(a) is crucial: The shape and
structure of the cortical tissue
is important and underlying
stroma should be removed
and the tissue flattened
Step 2
Step 3
2. Multilaminar follicles (b) can be

removed within 6 days of culture;
however, there may be many
primordial and primary follicles
remaining. The remaining pieces can
be replaced into culture for a further
period to maximize yield
Step 4
3. Once antral formation

has been achieved (c),
follicles can be cultured
within alginate (d) or on
membranes and cultured
for up to 21 days
Figure 33.2 Proposed multi-step culture system for activation of human primordial follicles and subsequent follicle/oocyte development.
The stages required for a multi-step culture system are as follows. Activation of primordial follicles within cortical strips (a). Removal of all
growing follicles and most of the underlying stromal cells increases the rate of activation [6]. Flattened strips are cultured free floating in
medium containing human serum albumin (HSA), ascorbic acid and basal levels of follicle stimulating hormone (FSH) [6]. Once follicles have
reached multilaminar stages they are isolated mechanically using needles and cultured individually Isolated follicle culture is to support
development from pre-antral to antral stages (b). The addition of activin at this stage results in improved follicle development and increased
antral formation (c) [6]. Follicles of similar stages that have been grown in vivo have been isolated and grown with alginate drops [7] (d), and
oocytes grow to almost full size within a total of 30 days [7]. The final stages of oocyte growth and development could be achieved by
culturing the oocyte and its surrounding somatic cells outwith the constraints of the large follicle (e).
(a)
(b)
(d)
(e)
(c)
(f)
Figure 33.3 (a) A cluster of quiescent follicles in freshly fixed human ovary. (b) After 6 days in vitro, growing follicles () appear on
the surface of a cultured fragment of human ovarian cortex. (c) A growing follicle protruding from the edge of a fragment of cultured
human cortex. (d) Intact secondary human mechanically dissected with presumptive theca layers attached. (e) Histological image of a
secondary human follicle fixed after 6 days in vitro growth within a cortical fragment. (f) Histological image of human antral follicle
fixed after a total of 10 days in vitro growth.
Antral
Secondary
Primordial
Antrum
Primary
Organ culture
(stromal cells)
Oocyte
Granulosa
Theca
(a)
(b)
Figure 34.1 Folliculogenesis. Primordial

follicles develop to antral follicles, which
are capable of producing fertilizable
oocytes. Ovarian stromal cells are
hypothesized to have significant roles in
the activation of primordial follicles and the
recruitment/differentiation of theca cells.
Most follicle culture systems focus on
secondary or multilayer follicles and
produce antral follicles with fertilizable
oocytes. Primordial and primary follicles do
not activate or mature in vitro. The culture
of these follicles is typically performed as
an organ culture.
Figure 34.2 In-vitro follicle culture

systems. (a) In two-dimensional systems,
follicles are cultured on flat surfaces such as
tissue culture plastic (polystyrene). The
unnatural geometry/mechanics of these
systems disrupts cellcell communication
and causes the granulosa cells to break
though the basement membrane, migrate
away from the oocyte, and attach to the
two-dimensional surface. Two-dimensional
systems lack the ability to support large
follicles for extended culture times. (b) In
three-dimensional systems, follicles are
cultured within biomaterial scaffolds, such
as alginate. These systems maintain the
natural spherical geometry and cellcell
interactions of the follicle.
Figure 34.3 Stromal cell co-culture

approaches. (a) Stromal cells can be
cultured separately on a flat surface below
the encapsulated follicle. This set up allows
for paracrine signaling between the follicle
and the stromal cells. (b) Stromal cells can
be encapsulated inside the biomaterial
scaffold with the follicle. This set up allows
for paracrine signaling as well as cellcell
attachment and interaction with secreted
extracellular matrix proteins.
(a)
(b)
Section 7
Chapter
30
Whole ovary freezing

J. Ryan Martin, Jason G. Bromer and Pasquale Patrizio
Introduction
Patients with cancer who desire to preserve their future
reproductive potential but require immediate gonadotoxic treatments (chemo and/or radiotherapy), are
left with few options for fertility preservation. These
options include: (a) cryopreservation of ovarian tissues
as cortical strips; (b) dual cryopreservation of both
ovarian cortical tissue and cryopreservation, after in
vitro maturation, of immature oocytes extracted from
the small antral follicles visible within the ovarian cortex at the time of the harvest; (c) cryopreservation of
one whole ovary [19]. Each of these options is still
considered experimental (thus requiring Institutional
Review Board approval and patients informed consent).
[1011], either as heterotopic or orthotopic allografts,
has shown some reproductive success [1214]. At
the time of writing, a total of seven live births from
re-transplantation of ovarian cortical tissue to an
orthotopic location have been reported [1519], while
four more have been announced at a meeting, but not
yet published. Typically, it takes about 45 months
for resumption of endocrine function as evidenced by
menses or serological hormonal evaluation. However,
the re-transplanted cortical pieces only retain ovarian
function for a short time and almost all ceased to
function by 3 years [6, 20, 21].
There are several reasons to explain this transient return of ovarian function followed by the rapid
decline. One reason is that the amount of cryopreserved/thawed cortical tissue re-transplanted during a
graft is limited. Another reason for the short longevity
is that the cortical tissue is grafted without a vascular anastomosis and is, therefore, completely dependent for its survival on the development of a new vas-
culature; a process which requires at least a week [22,

23]. By the time neovascularization occurs, the grafts
will have already sustained significant ischemic damage resulting in massive loss of primordial follicles [13,
2427], ultimately responsible for the limited functional life span of the graft [28, 29]. This process was
illustrated in a sheep model that found that 6070% of
follicles were lost at transplantation, but only 7% of the
loss was dependent upon the cyropreservation procedure itself [30].
Cryopreservation of the whole ovary with its intact
pedicle and vascular supply has recently been proposed as an experimental strategy that could potentially overcome the problem of ischemic damage; in
these instances a successful re-anastomosis will provide immediate reperfusion so, theoretically, the ovary
should maintain long-term endocrine and reproductive functions [2, 24, 3133].
This chapter summarizes the technical challenges
that had to be overcome for the freezing/thawing of
intact ovaries with animal experiments first and then
with humans.
Challenges of whole ovary

cryopreservation
Two main problems with whole organ cryopreservation and re-transplantation have caused technical difficulties: the first related to the feasibility of executing
a perfect vascular re-anastomosis of the whole organ
with re-establishment of a prompt vascular flow; the
second was related to the development of a successful
cryopreservation protocol.
Experiments with whole fresh ovary retransplants, have proved that the re-anastomosis
of the ovarian pedicle is technically possible in rabbits
367
[34], sheep [3537], dogs [38], monkeys [39]

and also in humans [24, 28, 4042]. Wang et al.
demonstrated that cryopreservation and subsequent
re-transplantation of the reproductive system en bloc
could be performed in rats [33]. To date, two cases of
human re-transplant of fresh whole ovaries and one
live birth have been reported [43]; however, no cases
of frozen/thawed whole ovary re-transplants have
been performed yet.
The second challenge, i.e. creating a successful
cryopreservation protocol for large-sized intact
ovaries, has proved more problematic due to: (a) heat
and mass transfer problems; (b) the physical constraints related to the heat transfer between the core
and periphery of a large organ; and (c) the establishment of adequate stromal and cortical diffusion of the
cryoprotectants [44, 45] to prevent the formation of
intravascular ice [24]. Large amounts of ice crystals are
a major destructive force for cells and are known to be
mechanically disruptive. Inadequate perfusion of the
cryoprotectants into the vascular compartment may
lead to intravascular ice formation with subsequent
vascular injury and irreversible endothelial disruption
[46]. Slow cooling methods were developed to allow
a slow enough process to dehydrate cells in order to
prevent intracellular crystallization but, at the same
time, fast enough to minimize osmotic stress to cells.
Whole ovary freezing in rat model

Wang et al. investigated cryopreservation after immersion in liquid nitrogen and re-transplantation with
microscopic re-anastomosis of whole rat ovaries [33].
In this landmark study, the authors used adult female
rats and removed the right ovary and the upper segment of the uterus en bloc, with the ovarian vessels dissected to create short cuffs of aorta and vena
cava. Seven dissections were perfused for 30 min at
0.35 ml/min with M2 medium containing 0.1 M fructose and increasing concentrations of dimethyl sulfoxide (DMSO). The treated organs were then cooled
slowly in Cryovials and, after overnight storage in liquid nitrogen, were rapidly thawed and the cryoprotectant was removed.
After re-transplantation, four of the seven rats had
subsequent follicular development, with corpora lutea
indicating recent ovulation, and one animal achieved
pregnancy. Importantly, tubal and uterine morphology and architecture were indistinguishable from nonoperated controls. However, these rats had higher
368
serum follicle stimulating hormone (FSH) levels, fewer

follicles and lower estradiol levels and uterine weights
than controls, indicating that the freezing had compromised the ovarian function.
Yin et al. continued this work using a similar model
to investigate the long-term longevity of ovarian grafts,
as well as to assess the effect of ischemia after cryopreservation for 24 h prior to transplant [47]. Graft
survival with endocrine function was seen at 2 months,
and ovulatory response to FSH was seen at 4 months,
suggesting good graft survival after vascular anastomosis. These grafts however had fewer surviving follicles than the controls, emphasizing that even minimal
ischemia time significantly reduces the follicular pool.
Whole ovary freezing in sheep model

To improve the technique of freezethaw and transplantation of whole ovaries, adult female sheep have
become the preferred animal model to study both slow
cooling and vitrification methods. Sheep have ovaries
that are similar in size to humans and are therefore
ideal animal models. Despite human ovaries having
different vascular pedicle anatomy, the sheep ovaries
have dense fibrous stroma and a relatively high primordial follicles density in the cortex similar to human
ovaries [48].
Slow cooling
In 2002, Jeremias et al. attempted orthotopic transplantation of a whole ovary in an adult sheep by anastomosis of the vascular pedicle [36]. After bilateral
laparoscopic oophorectomy, ovaries were autotransplanted into the abdominal wall, and microsurgical
vascular anastomosis of the ovarian to the inferior epigastric vessels was performed. After noting promising resumption of endocrine function post-transplant
and a high follicular count, the authors concluded that,
in conjunction with an improved protocol for cryopreservation, ovarian autotransplantation with vascular anastomosis may be superior to ovarian tissue
banking and grafting techniques [36].
The same team also performed also one of the
first transplants of an intact frozenthawed ovary via
microvascular anastomosis [44]. After laparoscopic
dissection, the whole ovaries were immediately perfused with heparin, followed by perfusion and immersion in a bath containing Leibovitz L-15 medium,
10% fetal bovine serum (FBS) and 1.5 M DMSO.
Ovaries were perfused via the ovarian artery with the
Chapter 30: Whole ovary freezing
cryoprotectant solution at a rate of 1.3 ml/min. After

perfusion, the ovaries were transferred into a Cryovial and cooling began at 4 C and at 2 C/min until
ice nucleation was induced at 7 C. The temperature
was then reduced by 2 C/min until 35 C and, subsequently, by 25 C/min until 140 C after which the
Cryovials were plunged into liquid nitrogen. Thawing
occurred 1 week later, and was achieved by first plunging and swirling Cryovials in water bath at 37 C. The
ovaries were then immediately perfused with Leibovitz
L-15 and 10% FBS for 20 min, thus gradually eliminating the cryoprotectant.
After microvascular transplantation immediate
patency was documented in 100% of the grafts. However, after 810 days, 77% of the ovaries showed complete occlusion of the anastomosis. While no significant differences were found in the mean values of
apoptosis and follicular viability compared to ovarian cortical strip cryopreservation and autotransplantation by TUNEL assay and histology, postoperative
FSH levels were much lower in the whole ovary graft
group (P = 0.03) and similar to preoperative values in
animals with patent vessels [44].
After establishing long-term patency of the anastomosis, the next objective was to assess whether the
post-transplanted ovaries could respond to in vivo
stimulation with FSH and produce viable oocytes.
Grazul-Bilska et al. treated ewes for approximately 5
months post-transplant with FSH and then the ovaries
were removed [49]. In all ovaries, primordial, primary, secondary, antral and pre-ovulatory follicles
were found along with fully functional vascularization, which was manifested by the expression of factor VIII, vascular endothelial growth factor (VEGF)
and smooth muscle cell actin (SMCA). Proliferating
cells were detected in follicles, and the rate of apoptosis was minimal. One ewe had four visible follicles
from which three oocytes were collected, but none fertilized. The morphology of autotransplanted and control ovaries was similar. The authors concluded that
autotransplantation of intact frozenthawed ovaries is
feasible because vascular and cellular function may be
restored.
Using both in vitro and in vivo methods, Arav et al.
showed no significant difference in follicular survival
between fresh ovaries and frozenthawed ovaries, as
well as similar histological morphology and normal
immunohistochemical expression of factor VIII, suggesting normally restored vascular pedicles [50]. In
addition, six oocytes were aspirated from two sheep,
and subsequent fertilization and embryo development

occurred (Figure 30.1 [50, 51]). Two of the sheep continued to show normal hormonal cyclicity by progesterone levels up to 36 months post-transplantation,
and follow-up MRI studies confirmed normal ovarian size and intact vasculature. Furthermore, in a very
recent study, Arav et al. demonstrated that the follicular and endocrine function lasted up to 6 years
post re-transplantation [51]. This is the longest documented functional survival of a frozen/thawed whole
ovary. The experiments performed by the Arav group
used 812-month-old sheep where, after dissection of
the right ovarian artery and vein via laparotomy, the
ovaries were removed and perfused through the ovarian artery with 4 C University of Wisconsin (UW)
solution containing 10% DMSO for 3 min. Freezing was performed using a novel freezing device, the
Multi-Thermal-Gradient (MTG; Coredynamics, Ness
Ziona, Israel), which utilizes a directional freezing gradient [48, 50]. By advancing the freezing test tube at
a constant velocity of 0.01 mm/s through predetermined temperature gradients, freezing was performed
at 0.6 C/min until a seeding temperature was reached,
and then at 0.3 C/min until 30 C, after which time,
the tubes were plunged into liquid nitrogen.
Thawing and grafting was performed 314 days
later by plunging the Cryovials into a 68 C water
bath for 20 s and then into a 37 C water bath for
2 min. The cryoprotectant was removed by reperfusion of the ovarian artery with UW supplemented
with 0.5 M sucrose and 10 IU/ml heparin. Ovarian retransplantation was performed via end-to-end anastomosis of the ovarian artery and vein to the contralateral (the left side) ovarian vascular pedicle of
the same sheep via repeat laparotomy. Successful reanastomosis was documented in five of nine sheep.
Progesterone cyclicity was seen 3471 weeks after
transplantation, thus documenting the functional survival of primordial follicles after the freeze/thaw transplant process.
Imhof et al. first explored cryopreservation by
cannulating the ovarian artery from freshly retrieved
porcine ovaries, and flushing with RPMI-1640 solution containing 1.5 M DMSO and 10% human albumin
for 30 min on ice [52]. The ovaries were transferred
to a programmable freezer at a starting temperature
of 4 C, and cooled at 2 C/min to 0 C. The temperature was then lowered by 1.5 C/min to 9 C and then
by 0.5 C/min to 40 C. Cooling was then continued at
10 C/min to 150 C. The vials were then plunged into
369
Sheep no. 1131
2.5
15
10
1.5
1
0.5
3
Progesterone
levels (ngr/ml)
2
1
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
71
30
20
10
1
Progesterone
level
(ngr/ml)
40
Estradiol
level
(pmol/ml)
Progesterone
level
(ngr/ml)
37
73
Sheep no. 2075
Sheep no. 2076
36
72
38
300
200
0.5
100
0
Estradiol
level
(pmol/ml)
Estradiol level
(ngr/ml)
Progesterone level
(pmol/ml)
Sheep no. 1199 *
20
34
35
36
(a)
(b)
Figure 30.1 Endocrine function and fertility after cryopreservation and re-transplant of intact sheep ovaries at 2 and 6 years
post-transplantation. (a) Hormonal levels 2 years post-transplantation [50]. (b) Follicles by transillumination 6 years post-transplantation [51].
liquid nitrogen. Thawing was performed 3 weeks later

by re-warming in air for 2 min before the ovaries were
immersed in a water bath at 25 C, and the cryoprotectant was removed from the tissue by washing in saline
and fresh medium [52]. Light and electron microscopy
were used to evaluate follicular and oocyte survival. Of
the primordial follicles, 84.4% in the frozenthawed
ovaries appeared histologically, with 73% of the follicles looking similar to those from the unfrozen contralateral ovary [52].
After success using the porcine model, Imhof et al.
then cryopreserved and re-transplanted whole ovaries
in sheep [53]. Using a similar method for cryopreservation, the authors performed a second laparotomy
35 weeks later, and the frozenthawed ovary was
autografted by microvascular end-to-end anastomo-
370
sis to the contralateral pedicle. Ischemia time before

complete re-anastomosis was 30 min. The FSH and
progesterone levels were used to evaluate ovarian function. Initially FSH levels kept rising for 3 months but
reached normal physiological levels about 6 months
after transplantation. Progesterone was first detected
1214 months after implantation and 2 of 9 sheep
resumed normal ovarian function. One sheep achieved
a spontaneous pregnancy, with delivery of a healthy
lamb [53].
Wallin et al. conducted a study to assess methods
for the evaluation of viability and function of frozen
thawed whole ovaries [37]. Histology and a viability assays were used to evaluate the ovaries. Fourteen ewes underwent oophorectomy via laparotomy
and their ovaries were frozen using the slow cooling
method. A solution containing 1.5 M propanediol,

0.1 M sucrose and 2% human serum albumin in Leibowitz L-15 medium was used as the cryoprotectant
in one group of ovaries. Ovaries were stored between 1
week and 9 months. No antral follicles were seen in the
cryoprotectant group and edema was noted within the
stroma. There was no histological difference between
the two groups.
Vitrification
While many attempts have been made to cryopreserve
whole ovaries by slow freezing techniques, there have
been few studies that have attempted utilizing vitrification. This method is becoming increasingly recognized as alternative to organ and tissue preservation by
slow freezing as it circumvents the mechanical damage
cause by ice crystal formation [54].
Fahy and colleagues have described two vitrification solutions that may be useful for whole ovary cryopreservation. The first, VS1, contains 20.5% wt/vol.
DMSO, 15.5% wt/vol. acetamide, 10% wt/vol. propylene glycol and 6% wt/vol. polyethylene glycol in a
modified Dulbeccos saline (HB1) [55]. The second,
VS4, contains 2.75 M DMSO, 2.76 M formamide and
1.97 M propylene glycol diluted in BM1 medium [56].
The two solutions, VS1 and VS4, were compared by
Courbiere et al. in a sheep model of whole ovary cryopreservation for toxicity to primordial follicles and
vessels by collecting ovaries with intact pedicles from
5 to 6-month-old lambs [57]. Each ovary was perfused via the ovarian artery with heparinized Ringers
solution, followed by perfusion and immersion in a
bath with VS1 or VS4 solution. Perfusion rate was performed at 0.35 ml/min with a stepwise increase in concentration of cryoprotectant. After perfusion, ovaries
were transferred into cryobags containing the cryoprotectant mix and then plunged into liquid nitrogen.
After storage, the vitrified samples were rapidly rewarmed in a 37 C water bath, and the cryoprotectant
was removed by a reversed concentration gradient perfusion, and then washed in BM1 medium for 5 min
[57].
Follicle viability fell from 75.6% 1.1% without
vitrification to 68.2% 1.9% after vitrification with
VS1, and from 68.0% 3.8% to 60.7% 2.4% after
vitrification with VS4. While these differences between
VS1 and VS4 were not statistically significant, follicle density remained significantly higher in vitrification with VS4 (P 0.05). Histologically, the percent-
age of normal primordial follicles fell after vitrification, with 25.2% 7.0% of follicles remaining normal
with VS1 and 53.5% 3.2% remaining normal with
VS4. There were also more post-vitrification cytoplasmic anomalies with VS4 (P 0.05) but more nuclear
and combined anomalies with VS1 (P 0.05). Fractures occurred in vessels during thawing in 3 of 5 cases
with VS1 and in 8 of 10 cases with VS4, although the
authors noted that the catheters used to re-perfuse the
ovarian artery were fitted without difficulty. This study
showed that whole sheep ovaries can survive vitrification with good immediate follicular viability via histological evaluation; however, the blood vessels were
cryodamaged.
Similar to previous to slow-freezing studies, Courbiere et al. performed a follow-up study in 2008,
attempting vascular anastomosis to the contralateral pedicle in sheep ovaries either fresh or after
vitrification [58]. Successful microsurgical transplantation was performed in both groups, but, not surprisingly, the median ischemia time was significantly longer in the cryopreservation group. Only
one out of five ewes undergoing ovarian vitrification recovered endocrine function six months after
transplantation compared to four out of five in the
fresh transplant group. However, histological evaluation showed total follicle loss in the vitrification
group, suggesting that attempts at cryopreservation
with vitrification were unsuccessful, despite technical
feasibility [58].
Whole human ovary cryopreservation

Despite the many technical challenges involved in
the choice of cryoprotectants and tissue viability after
freezing and thawing, there have been several attempts
at whole ovary cryopreservation in humans (Table 30.1
[59, 60]). In 2004, Martinez-Madrid et al. tested the
feasibility of freezing intact human ovaries using a
passive cooling device [24]. Ovaries from three premenopausal women undergoing oopohorectomy were
resected with their vascular pedicle intact. The ovarian
artery was cannulated and the ovary was perfused first
with isotonic heparinized solution, and then with a
solution of Leibovitz L-15, 10% DMSO and 2% human
serum albumin for 5 min at 2.5 ml/min. The ovary
was then placed in a Cryovial and cooled at a rate
of 1 C/min to 80 C, at which time it was transferred to liquid nitrogen. For thawing, the Cryovial
was directly transferred to a water bath at 60 C. To
371
Table 30.1 Summary of whole human ovary cryopreservation experiments listed according to type of freezing method, cryoprotectant
used and outcomes measured to indicate post-thaw viability
Study
N=
Surgical
method
Freezing method
Cryoprotectant
Outcomes measured
Martinez-Madrid et al. [24]
Laparoscopy
Slow cooling
DMSO
Follicle, stromal cell, vascular

viability, histological morphology
Bedaiwy et al. [25]
Laparoscopy
Slow cooling
(ovaries bisected)
DMSO
Follicle viability, apoptosis
Martinez-Madrid et al. [31]
Laparoscopy
Slow cooling
DMSO
Apoptosis, ultrastructural
assessment
Jadoul et al. [46]
Laparoscopy
Slow cooling
DMSO
Technical feasibility of
oophorectomy and freezing
Patrizio et al. [59, 60]
11
Laparoscopy,
laparotomy
Slow cooling
EG
Apoptosis, histological
morphology
DMSO, dimethyl sulfoxide; EG, ethylene glycol.
remove the cryoprotectant, the ovary was reperfused

at room temperature, with a reversed sucrose concentration gradient of 0.25, 0.1 and 0 M sucrose in L-15
medium.
Viability evaluation by vital fluorescent staining
revealed that the percentage of live follicles was 99.4%
in fresh tissue, 98.1% after cryoprotectant exposure
and 75.1% after thawing. Viability assessment also
showed live stromal cells and small vessels after thawing. On histological evaluation, the morphology of follicles and cortical and medullar tissue was similar in all
three groups. While this work utilized a much more
simplified cryopreservation algorithm than in previous studies, and it did not report on vascular endothelial viability [61], it did suggest that a slow cooling
method could be utilized preserving whole human
ovaries [24]. Martinez-Madrid et al. showed high survival rates of follicles, small blood vessels and stromal
cells in an intact human ovary using an accessible cryopreservation protocol [24].
Martinez-Madrid et al. have subsequently reported
on additional viability assessments in frozen/thawed
human ovaries [31]. They assessed apoptosis via
TUNEL-assay as well as immunohistochemistry for
active caspase-3. No primordial or primary follicles
were found to be positive for either TUNEL or active
caspase-3, and they found no significant differences
in mean TUNEL-positive surface area values between
fresh control and frozen/thawed ovaries. Electron
microscopy also showed well-preserved ultrastructure, healthy-appearing primordial and primary follicles and normal endothelial cells [31].
372
In 2007, the same group described the technique of laparoscopic oophorectomy with the intent to
cryopreserve the whole ovary and its vascular pedicle
[46]. The whole ovary was successfully removed and
cryopreserved by arterial catheterization in all nine
patients in the study. The authors had two main conclusions: first, that the ischemic interval before cryopreservation must be as short as possible, as this is the
time when significant damage is done to the organ; second, that surgeons must leave a long infundibulopelvic
ligament (5 cm) in order to allow easier access to the
ovarian vessels during canalization and perfusion of
the cryoprotectant.
More recently, Bedaiwy et al. described the successful cryopreservation of the human ovary [25].
They performed bilateral oophorectomy in two premenopausal women. In each case, one ovary was cryopreserved intact with its vascular pedicle in the same
method as described earlier. Ovaries were thawed 7
days later, and follicular viability and histology were
assessed, as well as apoptosis via TUNEL assay and
Bcl-2 and p53 protein expression profiles. They found
that overall viability of the primordial follicles in the
two ovaries was 75 and 78% in frozen/thawed ovaries,
and that there were similar primordial follicle counts,
absence of features of necrosis and mean values of
apoptosis when compared to control ovaries. After
demonstrating comparable survival rates and limited
molecular alterations, the authors concluded that this
represents further evidence that an intact human ovary
could be cryopreserved using a slow freezing protocol.
However, there has been confusion about whether the
Figure 30.2 Whole human ovary cryopreservation with the

vascular pedicle utilizing the same Multi-Thermal-Gradient device
and slow cooling, rapid thawing protocol as described in the text.
ovaries in this study were bisected after perfusion with

a cryoprotective agent [62].
More recently, we have reported a successful whole
human ovary cryopreservation with the vascular pedicle utilizing the same MTG device and slow cooling, rapid thawing protocol described earlier [48, 63]
for the sheep (Figure 30.2). With this method, 11
ovaries from premenopausal women undergoing hysterectomy and bilateral salpingo-oophorectomy have
been successfully cryopreserved for 4896 h following either laparotomy, laparoscopic or robotic-assisted
laparoscopic oophorectomy [59]. In all cases, the ovarian artery was successfully cannulated for perfusion
of cryoprotectant and the contralateral ovary was utilized as a fresh control. A pathologist was unable to distinguish between the frozen/thawed ovary and control
in a blinded histological analysis. Immunohistochemistry and Western blot assays showed modest increase
in anti-caspase 3 and p53 phospho-serine expression,
suggesting a non-significant increase of apoptosis in
the frozen thawed specimens [59]. In addition, in three
cases, the ipsilateral fallopian tube was also cryopreserved intact with the associated ovary and pedicle. In
these cases, the histological architecture was also preserved, suggesting that cryopreservation of the entire
adnexa en bloc may be technically feasible [60].
exploring their options for fertility preservation. As

cryopreservation protocols have improved and technical challenges related to whole organ cryopreservation have been partially resolved, the prospect of fertility preservation by whole ovary cryopreservation has
become more of a reality. While no human studies
of whole ovary transplant after cryopreservation have
been performed, studies in large animals have been
encouraging.
The strategy of whole human ovary cryopreservation has a major potential advantage over the cortical
strips: it allows for immediate perfusion of the transplanted organ thereby reducing the ischemic damage,
thus theoretically resulting in long-term resumption
of ovarian and endocrine function. However, whole
ovary cryopreservation may not be a realistic option
for many patients due to inherent technical difficulties.
The process requires a challenging surgery due to the
small diameter of the ovarian artery, further exacerbated by the inadequate length of the vascular pedicle.
If the microvascular anastomosis fails, then the whole
organ survival is irreversibly compromised, preventing
a second attempt at transplantation. This is in contrast
to failure of transplanted cortical strips, when another
attempt can be performed with the remaining frozen
strips.
An issue that remains unresolved is the handling
of ovarian tissue containing metastasis from systemic
cancers such as leukemia. In the future, patients with
malignancies at high risk of ovarian metastasis could
have a whole ovary removed and perfused in vitro,
to stimulate folliculogenesis in vitro. If successful,
oocytes could be harvested for cryopreservation or for
fertilization and subsequent embryo cryopreservation.
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Section 7
Chapter
31
Whole ovary transplantation
Mohamed A. Bedaiwy and Tommaso Falcone
Introduction
Over the past few decades, considerable attention
has been given to the long-term reproductive function of females undergoing cancer treatment. Many
treatments, such as non-specific chemotherapeutic
agents and ionizing radiation, damage the ovaries
and induce premature ovarian failure (POF), making
future pregnancy impossible. Indeed, the number of
cancer patients at risk of POF who are seeking help to
preserve their fertility has increased dramatically.
One method of preserving fertility in female cancer patients is cryopreservation of ovarian cortical
strips. In this procedure, strips of ovarian tissue are
harvested before cancer treatment, cryopreserved and
then thawed for subsequent use. The practice has
become well established within the last decade. However, the subsequent use of the frozenthawed ovarian
strips to restore fertility remains a challenge. Transplantation is currently the only available option to
restore fertility using cryopreserved ovarian tissue
because in-vitro follicular culture technology is far
from fully developed in humans, although it has been
successful in the rodent model [1].
An option for those undergoing pelvic radiation
therapy is fresh whole ovary autotransplantation. Typically, transposition of the ovaries can be performed.
However, in cases where the radiation beam involves
much of the pelvis and abdomen transposition may
not be possible. In this case, the ovary can be surgically
removed and immediately implanted into an alternate
site that is not exposed to pelvicabdominal radiation. Currently, this procedure can be done using fresh
whole ovaries only. Although whole ovaries have been
successfully frozen and later transplanted back into the
donors in animal models, the procedures needed to
do so safely and successfully in humans are still under

development.
In this chapter, we review the state of the art of
ovarian transplantation as it pertains to the whole
ovary.
Ovarian tissue transplantation: current

status and limitations
In a previous study, we systematically reviewed the
literature to assess the outcomes after ovarian tissue
transplantation (OTT) in women who were at high risk
for POF [2]. In that report, we identified 46 unique
cases from 25 studies in which cryopreserved or fresh
OTT was performed. In most cases, the transplantation was done using ovarian tissue strips. The indications for the procedure were: treatment for POF
(n = 27), prevention of POF (n = 15), treatment for
infertility (n = 2) and accidentally (n = 1).
The procedure restored spontaneous menstrual
cycles for several months in almost all cases. In 23
women with a follicle stimulation hormone level (FSH)
of 30 at the time of OTT, ovarian function (OVF)
was re-established; the median time to return of function (ROF) was 120 days (range, 60 to 244). Within 6
months of ROF, 4 of the 23 women experienced recurrent ovarian failure. There were insufficient data to
evaluate the long-term effects of the transplantations,
including OVF (12 months).
Of particular note, the use of fresh grafts increased
the chances for return of OVF and decreased the likelihood for recurrent ovarian failure more so than the
use of cryopreserved grafts. Of 25 women who sought
pregnancy, 8 had 9 pregnancies 12 months after OTT
giving a cumulative pregnancy rate of 37%.
377
Live births after autologous transplantation of

frozenthawed cortical tissue have been reported [3
6]. Pregnancies and live births have also been reported
after heterologous transplantation of fresh and frozen
thawed ovarian tissue between twins discordant for
POF [710].
The time needed to re-establish ovarian function
after transplantation can take as long as 45 months
as the follicles usually require at least 120 days to start
growing, and another 85 days or so are needed for
them to fully mature [2, 11]. The longevity of the grafts
after transplantation is variable; OVF may last only
for a few months or many years [2]. Although transplantation restores menstrual function, basal FSH levels generally remain elevated after surgery, reflecting
poor ovarian reserve [12]. In addition, the oocytes
harvested from these transplanted tissues tend to be
immature and of poor quality [13]. Of the pregnancies that have been reported after OTT thus far, all
occurred within the first year after transplantation,
once menstrual function was re-established and FSH
levels normalized [310]. These observations demonstrate the importance of achieving adequate ovarian
endocrine function before attempting pregnancy.
Post-transplantation ischemic
ovarian damage: morphological and
functional consequences
Avascular OTT is limited by one important fact
the graft completely depends on the establishment of
a new blood supply via the process of neovascularization. Avascular grafts experience an initial period
of ischemia after transplantation while waiting for
neovascularization to occur and consequently, a large
proportion of follicles are lost during this time [14
20]. The main feature in all of the OTT reports that
used cortical strips is the limited graft longevity and
high risk of recurrent ovarian failure (Box 31.1) [2].
The key reason behind this phenomenon is the fact
that most of these experimental transplants were performed without vascular re-anastomosis. As a result,
ischemic injury occurred before full revascularization
of the transplanted tissue from the surrounding vessels
could take place.
The exact mechanism by which the neovascularization process occurs is unknown and so is the time
needed for this process to be completed. Theoretically,
the shorter the time needed for neovascularization,
378
Box 31.1 Effects of ischemia on ovarian

transplants.
Morphological
r Decreased size
r Accelerated brosis
r Decreased number of primordial follicle
Functional
r Reduced longevity
r Increased basal follicle stimulating hormone
(FSH) levels
r Decreased inhibin B level
r Decreased anti-Mullerian
hormone (AMH)
the longer the graft survival. In animal experiments,

the time needed for revascularization was 3 days posttransplantation in mice [21] and up to 1 week in rats
[22, 23]. In an in vitro experiment, the neovascularization process was observed after only 3 days following
human OTT onto the chorioallantoic membrane of a
chick [24]. The ischemic exposure is critical for the survival of not only primordial follicles but also ovarian
stroma as well [25, 26].
It has been estimated that ischemic exposure is
associated with accelerated loss of the follicular pool.
In a series of xenografting experiments, approximately
25% of the primordial follicles were lost as a result
of transplanting cryopreserved xenografts of human
ovarian tissue into mice [14]. In autografting experiments, it was noted that ischemic injury was associated with up to a 95% loss of follicular reserve [17,
27]. In addition to follicular depletion, ischemic exposure was associated with abnormal hormonal function
of the graft in the form of up to a three- to fourfold
increase in FSH levels during the estrus cycle in the
sheep animal model. This increase in FSH levels could
have occurred if the growing follicles did not produce enough inhibin A [28]. These hormonal changes
could also be explained in part by the granulosa cell
dysfunction observed in the grafted tissue [29]. In
addition, low anti-Mullerian hormone levels reflecting
poor ovarian reserve have been reported [30].
Strategies to prevent
post-transplantation ischemic
ovarian damage
Many strategies have been devised to minimize the initial post-transplantation ovarian ischemia (Box 31.2).
Chapter 31: Whole ovary transplantation
Box 31.2 Strategies to nullify the effect of

ischemia on ovarian transplants.
Microvascular anastomosis
Neoangiogenic factors
r Fibroblast growth factor (FGF)
r Transforming growth factor (TGF)
r Vascular endothelial growth factor (VEGF)
Antioxidant
r Vitamin C
r Vitamin E
r Erythropoeitin
r Melatonin
Hormones
r Follicle stimulating hormone (FSH)
r Luteinizing hormone (LH)
r Human menopausal gonadotropin (HMG)
r Gonadotropin-releasing hormone (GnRH)
analogues
r Estrogenprogesterone
Implantation on granulation tissue
Steps to improve neoangiogenesis have been successfully applied by experiments that have shown early
perfusion of ovarian cortical strips upon their transplantation into granulation tissue [31]. Free oxygen
radicals are produced as a result of the ischemia perfusion process. They have the potential to damage cell
membranes, endothelial membranes and mitochondrial function [32]. The use of exogenous antioxidants
to augment ovarian transplant resistance to oxidative stress-associated damage has been evaluated by
many investigators. In one study, for example, ascorbic
acid and mannitol reduced surgically-induced ovarian ischemic injury in rats [33]. Moreover, injection
of vitamin E before ovarian transplantation improved
the follicular survival rate [21]. These preliminary data
have not been confirmed by others, however [34]. Sapmaz et al. locally administered melatonin and oxytetracycline during intraperitoneal rat ovarian grafting
and assessed the effects on graft function. They found
that the injection reduced ovarian tissue necrosis [35].
In an in vitro model, Kim et al. found that incubating
ovarian tissue with ascorbic acid for up to a maximum
of 24 h reduced apoptosis [26].
It is expected that neoangiogenic growth factors
such as fibroblast growth factor (FGF), transforming
growth factor (TGF) and vascular endothelial growth

factor (VEGF) somehow aid in the establishment
of graft function. Preliminary data from an animal
study (monkey model) showed that the most angiogenic growth factor, VEGF, was not associated with
improved graft function [36]. This was due in part to
the systemic administration of VEGF. Local administration at the transplant site may be more beneficial.
In lower animals, it was shown that the invasion of the
rat cortex by vessels was associated with a significant
increase in the expression of mRNA in the outer cortex for both TGF and VEGF [22].
A wide variety of hormonal treatments designed to
be given before or after ovarian grafting have been created in an effort to the consequences of the ischemic
exposure. These hormones include, but are not limited to, recombinant FSH, luteinizing hormone (LH),
human menopausal gonadotrophins, gonadotropinreleasing hormone (GnRH) agonists, estrogen and
progesterone [37]. Data from several animal experiments showed that pre-treating the graft recipient
and/or the donor with gonadotrophin stimulation
before and after transplantation may have a positive
effect on the viable growth follicle rate [21, 38, 39].
However, the impact of such treatments on long-term
ovarian function and fertility is still questionable and
needs further investigation.
Whole ovary transplantation

Parenchymatous organ transplantation has been practiced with variable success rates all over the world
for many decades, particularly with kidneys and livers. These transplantations are typically heterologous
between HLA-matched genetically different individuals. On the other hand, transplantation of reproductive organs can be done via autotransplantation or heterologous transplantation. Autotransplantation is the
process of transplanting tissue back into the body such
as with OTT or from one part of the body to another
in the same individual. With heterologous transplantation, the donor tissue is transplanted into another
person. The dynamics and the logistics of whole ovary
transplantation are not the same as those for nonreproductive organs.
It has been almost two decades since whole ovary
autotransplantation was reported in early human studies. In those reports, ovaries were removed from their
pelvic location and immediately transplanted into
other sites. The use of heterotopic sites for ovarian
379
autotransplantation dates back to 1988, when the first

case was reported [40]. These authors documented
normal follicular growth and regularity of menstrual
cycles after the end of pelvic radiotherapy.
Whole ovary transplantation with a vascular anastomosis was proposed as a mechanism to reduce
ischemic time and, in theory, prolong the longevity of
the graft [41]. In this technique, the whole ovary with
its vascular pedicle is removed, cryopreserved, thawed
and then transplanted with a microvascular anastomosis into a heterotopic or orthotopic site. Transplantation of an intact ovary with vascular anastomosis
reduces the ischemic interval between transplantation
and revascularization by allowing immediate revascularization of the transplanted tissue [42].
Bedaiwy et al. reported the restoration of ovarian
function after autotransplanting intact frozenthawed
sheep ovaries with microvascular anastomosis [43].
Imhof et al. autotransplanted whole cryopreserved
sheep ovaries with microanastomosis of the ovarian
vascular pedicle, which lead to pregnancy and delivery
[44]. An intact human ovary with its vascular pedicle
could be cryopreserved without affecting the follicular viability, vascular density or molecular integrity of
different ovarian components [45].
Whole fresh ovary transplantation

Fresh whole ovary transplantation with vascular anastomosis has been successfully performed using a wide
variety of orthotopic and heterotopic recipient sites.
In addition, a number of vessels have been used in a
wide variety of animal models. These include pelvic
vessels such as the ovarian artery and iliac artery,
parietal vessels such as the inferior epigastric vessels and extrapelvic vessels such as the carotid vessels
[39, 46]. In our preliminary experience with Merino
sheep, the revascularization process was compromised
in approximately 50% of the cases when fresh ovaries
were transplanted [41].
All human whole ovary transplantation has been
performed using fresh ovaries. A limited number of
human studies [9, 4749] have attempted this exhausting and technically challenging approach (Table 31.1).
A team of surgeons is needed, and the tissues must
undergo intraoperative microscopic evaluation. This
procedure is usually attempted in cancer patients who
need to start chemo- or radiotherapy without delay,
but other patient groups as monozygotic twins discor-
380
dant for POF [9] or Turners syndrome [49] have benefited from it.
The upper arm was the first recipient transplantation site tested in humans. There are two reports of successful whole fresh ovary transplantation in the upper
arm that was done prior to sterilizing pelvic irradiation [47, 48]. Leporrier et al. created a cavity in the
forearm arm for the ovary using a testicular prosthesis that was inserted 3 months before the transplantation. In that report, the ovary remained function
for the next 16 years [47]. In the second report, the
transplantation process was performed in the context
of radical hysterectomy for early stage cervical carcinoma. The ovary remained functional for at least 1 year
after transplantation [48].The procedure was unfortunately followed by local vault malignant recurrence.
No long-term follow-up has been reported. Mhatre
et al. successfully implemented the technique in two
patients with Turners syndrome (ovarian function was
restored and the patients developed secondary sexual
characteristics) [49]. An abdominal pfannenstiel incision was used in both cases.
More recently, Silber et al. culminated their efforts
in ovarian transplantation in monozygotic twins discordant for POF by reporting the first full-term pregnancy obtained using orthotopic whole fresh ovary
transplantation with microvascular anastomosis [9]. A
fresh ovary from the fertile twin was implanted in her
monozygotic twin with POF.
Whole cryopreservedthawed ovary

transplantation
Whole frozen ovary transplantation with microvascular anastomosis was first described in rats by
Wang et al. [39]. They described successful vascular
transplantation of frozenthawed rat ovaries, which
were transplanted along with the reproductive tract,
in 4 of 7 (57%) transplants; these transplants survived for 60 days, were ovulatory and resulted
in 1 pregnancy. Ovarian function was restored in
100% of cases when fresh organs were transplanted
[50]. Following their success, Courbiere et al. [51]
described cryopreservation (vitrification) of whole
ovaries with vascular pedicles in 5 to 6-month-old
sheep. There was no statistically significant difference in follicle viability or normal primordial follicle rates between the ovaries exposed to two cryoprotectants (VS1 and VS4 containing dimethyl sulfoxide, formamide, and propylene glycol) and those
Table 31.1 Studies reporting whole fresh ovary transplantation in humans
Indication for
transplantation
of ovarian tissue
Reference
No. of
patients
Ovarian volume
and site
Leporrier, 1987 [47]
Whole ovary,
heterotopic
Hodgkins disease
Ovarian cycles remained regular

despite radiotherapy, and follicle
growth occurred normally
Hilders et al., 2004

[48]
Whole ovary
Cervical cancer
Mhatre et al., 2005

[49]
Whole ovary,
orthotopic: Case 1,
vascular pedicle;
Case 2, avascular
transplantation
Turners syndrome
Whole fresh ovary

with microvascular
anastomosis
Premature ovarian
failure
Silber et al., 2008

[9]
A pair of monozygotic
twins discordant for
premature ovarian failure
that were not. The same observations were maintained

before and after vitrification with the cryoprotectant
solutions.
Although reports describing autotransplantation
of frozenthawed sheep ovarian cortex resulting in a
pregnancy [52] and prolonged normal hormone production date back to the 1990s [18], it was not until
2003 when the first report of successful cryopreservation and transplantation of an intact ovary in sheep
(defined as return of hormonal functions) occurred
[43]. Successful pregnancy and delivery of a lamb in
sheep was reported by Imhof et al. in 2006 following
autotransplantation of whole cryopreserved ovaries
with microanastomosis of the ovarian vascular pedicle
[44].
We have used deep inferior epigastric vessels, while
others used the ovarian vascular pedicle, to vascularize frozenthawed ovaries with success in sheep [43,
53]. The same success was also reported in rabbits [54].
In sheep, the success was suboptimal due to venous
thrombosis or a torn artery [41, 44, 55]. We have
Outcomes
Normal blood flow in the

anastomosed artery and vein. Cyclic
swellings of the upper arm without
major discomfort
Documented follicular activity at
different stages
Adequate blood flow and follicular
activity of the transplanted
ovary
Case 1: serum estradiol showed a

significant rise from the pretransplant value of <20 pg/ml to
50 pg/ml
Case 2 showed after 2.5 years:
developed uterus with
endometrium, normally functioning
transplanted ovary and her native
streak gonads. At the conclusion
of 2.5 years, the patient is having
spontaneous menstruation,
ovulation and excellent growth
of secondary sexual characteristics
First successful spontaneous

pregnancy
found evidence of endothelial cell damage caused by

the freezethaw process or by the ischemic time until
successful re-anastomosis [43].
The challenge of whole ovary cryopreservation
and transplantation technology is not only the surgical technique but the cryopreservation protocol for
an entire organ. Such a protocol should ensure that
the cryoprotectant(s) evenly diffuses throughout the
entire ovary. In addition, the frozen ovary should survive the thawing process and maintain functionality
after transplantation. Imhof reported that 18 months
after transplantation the follicular survival rate was
8% [44]. Other authors reported an even lower follicular survival rate (6%) and the depletion of the entire
follicular population after whole ovary cryopreservation and transplantation [56]. Although ovarian vessel
thrombosis is a potential complication, its incidence is
higher when vitrified ovarian tissue is used.
Similarly, in a more recent study in ewes, it
was shown that immediate vascular patency was
achieved in all ewes and maintained in seven of eight
381
cryopreserved and three of four control grafts. Functional corpora lutea were identified in 3 ewes (1 control; 2 cryopreserved) 1825 weeks after grafting. In
addition, inhibin A levels indicated resumption of follicular development in four cryopreserved and one
control ewes; however, castrate gonadotrophin levels
persisted in five cryopreserved and two control ewes.
The main prominent feature of this whole ovary transplantation experiment is the fact that primordial follicle density was significantly reduced following grafting
in both cryopreserved and non-frozen ovaries [57].
Although transplantation of whole cryopreserved
thawed ovary was not performed in humans, cryopreservation of a whole ovary using a slow-freezing
protocol has been successfully attempted [45]. The
results showed both vascular and follicular integrity
upon thawing after freezing and thawing. More
recently, a multi-gradient-freezing device was used
with promising results [58]. In that study a high follicular viability, normal histological architecture and preserved vessel integrity were reported, supporting the
potential for vascular re-anastomosis.
Technical aspects of whole ovary

transplantation: harvesting approaches
The procedures used to harvest ovaries for subsequent cryopreservation or transplantation should be
modified to reflect the need for a healthy and adequate pedicle. Consequently, surgeons must ensure
that the pedicle is long enough so that the ovarian artery and veins can be skeletonized and sutured
to recipient vessels of a similar diameter. During
this process, ischemia time must be minimized [42].
Although open surgery can help diminish ischemia
time, a minimally invasive laparoscopic approach is
the preferred approach when the ovary will be frozen
and subsequently transplanted [59]. These approaches
can facilitate the dissection of the ovarian pedicle
up to the pelvic brim, a technique that has been
used for a long time in ovarian transposition [19].
Using laparoscopy in cancer patients ensures better
wound healing, which is consistent with the patients
need to start their chemotherapeutic cycles immediately [42].
We have summarized the technical and microsurgical principles of the laparoscopic approach for
ovariectomy [59]. These included severing the uteroovarian ligament before advancing cephalad through
the mesosalpinx and ending at the infundibulopelvic
382
ligament, which should be dealt with last to minimize

ischemia time. All through the procedure, sharp dissection and suturing are preferred to electrocoagulation, which may induce desiccation of ovarian tissues through thermal injury to the vascular walls. The
length and width of the vessels can be maximized by
ligating the ovarian vessels as proximal to the origin as
possible. An endobag is placed through the 10 mm trocar to deliver the ovary with its vascular pedicle outside
the peritoneal cavity.
An extended port incision, which helps avoid
crushing the ovary and the blood vessels against the
narrow port site, can be used. Another approach,
though technically challenging, is the transvaginal
route. We reported one case of human ovarian harvesting using this approach, where the researchers combined oophorectomy with vaginal hysterectomy [59].
In order to minimize the ischemic time, we suggest
handling one ovary at a time for cryopreservation and
saving the infundibulopelvic ligament and the ovarian
vessels until the very end of the procedure. We found
that the dissection of a long portion of the ovarian
vessels via the vaginal route may not be as easy with
laparoscopy. The ovary and the uterus on one side has
to be released in order to obtain a longer portion of
the pedicle. With the advent of laproendoscopic single site surgery (LESS), it is expected that the harvesting of ovaries for fertility preservation will be even less
invasive.
Whole ovary freezing protocols

Several challenges need to be overcome before an ideal
intact ovary cryopreservation approach can be developed. Currently, we lack cryoprotective agents that
can adequately perfuse into the relatively large tissue
masses and prevent vascular injury following intravascular ice formation [42]. The freezethaw protocols
used today still require further optimization. Some
cryoprotective agents lead to zona hardening and meiotic spindle depolymerisation, which may lead to aneuploidy [60].
Modifications of slow-freezing cryopreservation
protocols were implemented in ovarian tissue freezing
as an integrated strategy for fertility preservation.
These modifications mainly entailed increasing the
concentration of sucrose used as non-penetrating
cryoprotectant and replacing sodium with choline.
These modifications improved survival rates by
80% [61].
Cryoprotectants must adequately penetrate the

stroma and granulosa cells without causing cryoprotectant toxicity. Newton et al. demonstrated the importance of the diffusion rate and the diffusion temperature [14, 62]. Ice crystal formation must also be
minimized by choosing optimal freezing and thawing rates. The cryoprotectant that will maximize permeation capacity and minimize toxicity and ice crystal formation is specific to each cell and tissue type
[63]. An effective technique of cryoperfusion where
the cryoprotectant is perfused into the ovary via
the ovarian artery was proposed [6466]. This technique lead to acceptable follicle survival rates and
relative ovarian function restoration with one case
of restored fertility [43, 44, 51, 55, 65]. However,
the loss of tissue viability amongst these studies was
common.
Thus, in the ovary, it is a matter of balance between
the stroma, the follicular cells and the oocytes [67].
The standard method for human ovarian cryopreservation is slow-programmed freezing using human
serum albumin-containing medium and propanediol,
dimethyl sulfoxide or ethylene glycol as a cryoprotectant, with or without sucrose [67].
Martinez-Madrid et al. described a cryopreservation protocol for intact human ovary with its vascular pedicle [68]. Ovarian perfusion with cryoprotective solution and slow freezing with a cryofreezing container was performed. Rapid thawing of the
ovaries was carried out by perfusion and bathing with
a decreased sucrose gradient. High survival rates of the
follicles (75.1%), small vessels and stroma as well as a
normal histological structure were documented in all
the ovarian components after thawing [68]. No postthawing induction of apoptosis was observed in any of
the cell types, assessed by both the terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) and immunohistochemistry for active
caspase-3 [69, 70]. Transmission electron microscopy
confirmed that the majority (96.7%) of primordial follicles were intact and that their endothelial cells had a
completely normal ultrastructure after cryopreservation [71]. The percentage of active caspase-3-positive
endothelial cells was 1%. It appears that in large
mammals and humans, cryopreserving a large, intact
ovary may prove more problematic than cryopreserving a small ovary from a small animal due to the
difficulty of achieving adequate cryoprotective diffusion and vascular injury caused by intravascular ice
formation.
Transplantation techniques for whole

ovaries with microvascular anastomosis
Several transplantation techniques of intact ovaries
have been described. Jadoul et al. suggested that
by applying clips on the utero-ovarian ligament, the
ischemic interval could be reduced prior to ovarian artery perfusion with heparinized solution and
cryoprotectants [42]. They also recommended that a
microsurgeon and biologist should attend the procedure and have a cooled sterile table with a stereomicroscope and microsurgical instruments ready. They
would enable surgeons to proceed immediately to
ovarian pedicle dissection and ovarian artery catheterization, thereby reducing ischemic time. Also, severing
the infundibulopelvic ligament at the end of the procedure could also help minimize ischemic time [59].
This approach has several limitations, especially in
regards to its ability to cryopreserve an entire organ.
In order to restore fertility after cryopreservation of
ovaries and testes in small laboratory animals, vascular transplants require technically challenging microsurgery to deal with the small blood vessels [7274].
In ewes, using an aortic patch, the ovarian artery was
re-anastomosed to the carotid artery during autotransplantation of an intact ovary with its vascular pedicle [75]. A primate model was tested for an orthotopic transplantation technique using a microsurgical
re-anastomosis of the ovarian blood vessels [76]. Both
techniques have a rather limited chance for application
in clinical practice since an aortic patch is used for the
anastomosis [41].
An intact human ovarian autotransplantation was
reported in a pair of 38-year-old monozygotic twins
discordant for POF [9]. The donor ovary was removed
laparoscopically from the fertile sister by dividing the
infundibulopelvic ligament at its base to maximize
the length. Using minilaparotomy, the donors ovarian
veins (3.0 mm in diameter) were anastomosed to the
recipients ovarian veins with 90 nylon sutures, and
the donors ovarian arteries (0.5 mm in diameter) were
anastomosed to the recipients ovarian arteries with
100 nylon interrupted sutures. A normal-appearing
blood flow through the ovarian vessels of the transplanted ovary was observed after an ischemic period
of 100 min. Subsequently, the recipient twin had 11
regular menstrual cycles. At day 427 after transplantation, she became pregnant and gave birth to a normal healthy baby girl. This case demonstrated the feasibility of using whole ovary transplantation between
383
monozygotic twins who are discordant for POF to

restore fertility in the affected twin [9]. Should intact
human ovary cryopreservation be optimized, the same
approach could be adopted for autotransplantation of
intact cryopreservedthawed ovary with a vascular
pedicle (Table 31.1).
Despite this success, determining the suitable
recipient vessel for this technique awaits experimental proof. In 2007, guided by the results of animal
experiments, we provided a model for human intact
ovary autotransplantation based on the human vascular anatomy [59]. The neck, pectoral region, antecubital fossa, lower part of the anterior abdominal
wall and the inguinal region are possible transplantation recipient sites in humans. The carotid vessels, the
cutaneous branches of the internal mammary vessels,
the antecubital vessels, the inferior epigastric vessels
and the femoral vessels are anastomosed to the ovarian vessels in their respective regions. Based on safety,
amenability to monitoring, caliber, accessibility, liability to trauma, surgical anatomy and cosmetic factor,
the deep inferior epigastric vessels stand as the best
available heterotopic option.
A discrepancy in diameter between the ovarian
vessels and the recipient vessels is an important challenge to the subsequent patency of the anastomosis.
The sudden change of caliber between the cut end
diameters of the vessels, encountered in approximately
one third of anastomoses, may cause turbulence in
the blood flow, thus predisposing the vessel to platelet
aggregation [77]. The choice of the technique of reanastomosis is affected by the degree of discrepancy.
There is no ideal technique to deal with every size
discrepancy, and choices may have to be individualized according to the specific case and body area [78].
Options range from dilatation with the use of a jewellers forceps [77], in the case of a simple discrepancy
of 1.0 : 1.5, to using the oblique cut, fish-mouth cut
or end-to-side anastomosis when discrepancies exceed
1.0 : 1.5 [77]. Sleeve anastomosis is performed when
discrepancies are larger and when the upstream donor
vessel is smaller than the recipient vessel [79].
Given the straight course and wider caliber of the
ovarian vessels, anastomosis of the ovarian vessels in
humans is expected to be less technically challenging, provided that the recipient vessels are similar in
diameter [59]. Based on animal studies, we suggested
the use of three different microvascular anastomosis
techniques that base the anastomosis on the caliber
of the ovarian and the deep inferior epigastric vessels:
384
end-to-end, end-to-side and fish-mouth anastomosis

[59]. The anastomosis of the ovarian vessels to the
deep inferior epigastric vessels was performed using
810 interrupted sutures (90 or 100 prolene). Obviously, end-to-end anastomosis appears to be the ideal
approach to performing the anastomosis procedure,
with a patency rate 60% [59]. However, if vascular
discrepancy between the ovarian vessels and the inferior epigastric vessels is inevitable, end-to-side anastomosis should be used. Vascular clips [42], sutureless approaches, glues and adhesives and laser-assisted
anastomosis are potentially useful [78].
Microvascular thrombosis after

transplantation
In the only reported intact fresh human ovary transplantation, the vascular anastomosis was functional
for more than a year after transplantation [9]. In
the animal experiment of Bedaiwy et al. , long-term
patency was lost in 8 of 11 transplanted sheep ovaries
due to thrombotic events in the re-anastomosed vascular pedicle [43]. Imhof et al. reported that in 6
of 8 ovaries, the major ovarian vessels were free of
thrombosis, with the structural integrity of the ovarian stroma largely retained 1819 months after transplantation [44]. These complications motivated Jadoul
et al. to highlight the necessity of cryopreserving the
contralateral ovarian cortical tissue when whole ovary
cryopreservation is attempted, until the results of ovarian transplantation are validated [42].
Another alternative was discussed by Yin et al.,
who suggested that one of a pair of ovaries should
be left in situ so that an intact pedicle is available for
exchanging the sterilized organ with the frozen and
thawed ones, once the patient is ready for autotransplantation [50]. It also allows for the possibility that the
ovary left behind may not be totally damaged by the
chemotherapy. There are many reports of pregnancies
after chemotherapy.
Heterologous whole ovary transplantation

A case of an allograft between two non-identical twins
was recently reported by Donnez et al. [80]. They used
ovarian cortical tissue from the donor sister that had
already been the donor of bone marrow for a transplant. Although this was done with cortical tissue,
these concepts can apply to whole ovary transplantation. The recipient had received chemotherapy, total
body radiation and bone marrow transplantation. The

recipient developed spontaneous cycles after receiving the transplant. Furthermore, two oocytes and two
embryos were obtained. Since the recipient of the ovarian tissue had also been the recipient of bone marrow,
the patients were able to avoid long-term immunosuppressive medication. The possibility of acute graft
rejection and long-term immunosuppressive complications in the mother, such as infection and obstetrical complications, may limit its use in allogeneic transplantation in patients that have not had a bone marrow transplantation and potential donor. The introduction of an ovary into a patient with a different
genetic makeup may make this procedure unacceptable to societies that do not accept donor oocytes.
Conclusion
The current evidence suggests that whole frozen
thawed ovary transplantation may be successful in
humans in the future. However, the significant depletion of the primordial follicle reserve observed after
transplantation in animals is a major problem that
needs to be further addressed at the experimental level
prior to attempting that approach in humans. With
the recent success of whole ovary transplantation with
microvascular anastomosis, reproductive organ transplantation researchers may need to learn from other
transplantation settings. Safer cooling techniques and
new operative steps that minimize vascular thrombosis are needed. Multi-team approaches should be
expanded [42]. Future research should operate within
the framework of the patients needs, namely fertility
and sexuality.
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Section 8
Chapter
32
In vitro follicle growth and maturation
Molecular and cellular integrity of

cultured follicles
David F. Albertini, Gokhan Akkoyunlu and S. Samuel Kim
Introduction
With the advent of culture models for studying the
process of ovarian folliculogenesis some 40 years ago,
opportunities arose for the more systematic evaluation
of the factors that regulated ovarian function [1]. The
initial focus of studies using cultured follicles emphasized two of the then widely recognized roles of the
follicle in mammals: the production of ovarian steroid
hormones and of viable oocytes during the process
of ovulation. As our understanding of the molecular
and cellular complexity of this tissue compartment has
evolved and deepened, so too has the need to redefine the major functions of the follicle at both local
ovarian and systemic levels in the context of reproduction in mammalian species, especially as it relates to
the origins and treatments for human infertility [2].
Thus, a shift in the motivation to use cultured follicles in humans has taken place owing primarily to the
rapidly evolving field of fertility preservation. Through
an interesting turn of events dictated by the need to
maintain and propagate human oocytes that would be
capable of supporting term gestations, a dire need has
been recognized that would enable optimization of follicle functions under in vitro conditions in order to
realize ovarian capacity for young women who have
had their fecundity seriously compromised as a result
of genetic, environmental or iatrogenic life-sparing
treatments such as those involved with the management of cancer [35].
The introduction of technology that permits the
cryopreservation on ovarian tissues has opened the
prospect of sustaining and storing primordial follicles
from individuals that could at a later time be thawed
and subjected to prolonged culture. Exactly what con-
ditions will be required to sustain follicular function

to support the growth phase of oogenesis has yet to
be worked out, as are the factors that would normally
be involved in this protracted and oocentric phase of
folliculogenesis. Working out conditions that could
recapitulate the structural, molecular and cellular
properties of the follicle in vitro remains a major challenge in the area of fertility preservation, as does the
development and implementation of novel technologies that would permit a reproducible and reliable
assessment of oocyte integrity. It is the intent of this
chapter to review methods that have been in practice
for the evaluation of follicle culture integrity and to
point out the strengths and deficiencies of these methods. Finally, the techniques that loom on the horizon
which could meet the criteria necessary for monitoring
follicular integrity will be considered, as new sentinels
or biomarkers could predict the developmental capacity of oocytes for the field of fertility preservation.
While direct measures of oocyte developmental
competence remain a lofty goal, more recent efforts
to characterize this aspect of follicular integrity have
tended to rely upon indirect ways to avoid unnecessary damage to the oocyte that might already have
been compromised by ex vivo conditions and/or cryopreservation [3, 6, 7]. For this reason, we propose
a viewpoint of follicle integrity that emphasizes the
syncitial heterocellular character, which includes the
somatic granulosa and theca elements and offers multiple parameters for assessment that in the larger picture may better represent the overall quality of the
enclosed oocyte [8]. This viewpoint is further supported by the recent appreciation of the level of
signaling and metabolic integration that reflects the
389
Section 8: In vitro follicle growth and maturation
Oocyte: growth, meiotic arrest,

prevent growth and differentiation
of follicle (GDF 9 et al.)
Granulosa: support oocyte
metabolism, induce thecastroma,
establish architecture, produce E2
Theca: produce androgenic
precursors, reinforce basal lamina,
support neovascularization
continuity of both negative and positive feedback

mechanisms between the oocyte, granulosa and theca
cells that comprise the ovarian follicle [2]. While this
perspective excludes formal contributions from the
surrounding stromal compartment within which the
follicle develops, the role of the stroma cannot be overlooked with respect to the contributions that it makes
during the course of in vivo folliculogenesis in relation
to thecal lineages including the microvasculature of
antral stage follicles. Thus, what follows is a summary
of the current and future biomarkers that may be useful in evaluating the integrity of cultured follicles. We
begin with somatic components of the follicle and finish with those assayable properties of cultured oocytes
that must be analyzed before the use of oocytes in
assisted reproductive technology (ART) applications
such as in vitro fertilization (IVF), embryo culture and
transfer.
Evaluating somatic cell components of

the follicle
The traditional array of biomarkers for the ovarian
follicle has emphasized endocrine performance, especially as related to the biosynthesis and secretion of
estradiol [9, 10]. Built on the classical two-cell model,
assays that monitor the production of thecal androgens
in response to luteinizing hormone (LH) and granulosa cell-derived estradiol in response to follicle stimulating hormone (FSH) remain the mainstay for evaluating follicular integrity under in vivo and in vitro
contexts [8]. It can be argued, however, that these valid
390
Figure 32.1 Diagram illustrating the

gradient of influences from the oocyte to
granulosa and thecal compartments of the
ovarian follicle that serve as guideposts for
monitoring the cellular and molecular
integrity of follicles maintained in culture.
Note that the primary function of oocyte
secreted factors such as growth
differentiation factor-9 (GDF-9) is to
prevent the proliferation (hyperplasia) and
differentiation (hypertrophy) of the
steroidogenic functions of the follicle prior
to and following ovulation. Thus, signs of
steroidogenesis in culture are likely to
reflect a loss in this command function of
the oocyte resulting in impaired viability
and developmental competence.
and predictive biomarkers for the endocrine health of

the follicle overshadow the most relevant attribute that
directly bears on the overall developmental status and
health of the oocyte (Figure 32.1).
Thus, the major phases of oogenesis, during which
both oocyte growth and preparations for the completion of meiosis and fertilization occur, occupy the earliest stages of follicle development between the activation of the primordial stage and entry into the secondary or antral stage when estrogen production commences upon the induction of aromatase activity in
response to FSH [1114]. That the latter property
appears commensurate with the rapid expansion of the
theca and granulosa by a burst of cell proliferation is
often identified as a measure of follicular health, but
the essential question is how do these FSH-induced
attributes of the follicle, increased proliferation and
aromatase activity, influence the terminal stages of
oogenesis if the goal of culturing follicles is to obtain
high quality oocytes?
There can be no question that the measure of any
organ or tissue culture system is the health and wellbeing of its constituent cells. Moreover, as in many
other developmental systems that are highly regulated
in a stage-specific fashion, the ovarian follicle engages
the basic cellular properties of differentiation, proliferation, survival and death, and each of these apply to
both the theca and granulosa at select stages of follicular development (Table 32.1).
It is not surprising then that the most commonly
used measure of cultured follicle integrity is hypertrophy over time, whether follicles are cultured under
Chapter 32: Integrity of cultured follicles
Table 32.1 Summary of major functional properties for ovarian follicles that have been used in the assessment of tissue integritya
Property
Follicle stage
Biomarker
Vital
Quiescence
Primordial
Chromatin/PTEN
Yes (Hoechst 33342)
Proliferation
Primary, secondary
BrdU, PH3, MPM2
Yes (Click-IT)
Apoptosis
Secondary, antral
TUNEL, caspase 3
Yes (Hoechst 33342)
Autophagy
Primordial (? others)
Beclin/ATG
Yes (LysoTracker Red, acridine orange)
Differentiation
Antral
Aromatase, LHr
No
Note that many of the biomarkers employed to date draw on the use of immunocytochemical and histological assays requiring tissue
destruction. Also, not all markers are pertinent to all stages of folliculogenesis. Some examples of vital biomarkers that are under development for determining cultured follicle integrity are shown in the last column.
adherent two-dimensional conditions or within matrices of various kinds that retain a three-dimensional
architecture [1520]. While standard protocols
deploying assays for cell proliferation (3H-thymidine,
BrdU incorporation or cell cycle markers such as
phosphohistone-3, PCNA, etc.) or apoptosis (TUNEL,
caspase-3) offer postscripts for the relative fraction
of viable cells within a follicle (Table 32.1), in the
end these are crude and retrospective assays that
add little to the immediate needs of the clinician
requiring a more real-time assessment of follicle
integrity. Towards this end, several new probes have
gained usage in the evaluation of tissue culture models
that take advantage of the speed, sensitivity and
spectral properties of microplate readers. This new
generation of reagents permits resolution of metabolic
activity, including reactive oxygen generation, cell
proliferation and even identification of rapid-versusslowly dividing cells within an organ or tissue culture
using multi-well formats, which should avail the
optimization of conditions that support oogenesis.
Moreover, as discussed below, the link between DNA
damage sensing and repair is fast becoming a major
determinant in the assessment of follicle integrity as
it relates to both somatic and germ cell components,
and these assays have introduced a range of sensitivity
and precision that will materially advance the field of
follicle culture.
As with most in vitro systems, culture environments create adverse conditions that are known to
affect DNA integrity, often due to the generation of
free radicals in response to high oxygen tension [21].
Given these deleterious side effects of culture environments, genomic integrity is one area of follicle evaluation that has received little attention and requires
closer inspection. Many new reagents are available for
evaluating the cascade of events associated with the
(a)
(b)
(c)
(d)
(e)
(a')
(b')
(c')
(d')
(e')
Figure 32.2 Range of DNA double-strand breaks detected in

bovine granulosa cell cultures observed before or after exposure to
cyclophosphamide. Top row (ae) illustrates staining profiles after
labeling with gamma-H2AX antibody that reveals solitary foci (a),
multiple foci (b) and various patterns (ce) that presumably reflect
earlier stages in DNA lesioning prior to foci formation as indicative
of active sites of DNA repair. Bottom row (a
e
) demonstrates total
chromatin. Scale bar represents 10 m.
detection of DNA damage, as well as the activation and

completion of the DNA-repair pathway that should be
active in both somatic and germ cells of the follicle.
We have recently been exploring the components of
this pathway in the mammalian ovary and find that a
variety of insults ranging from advancing maternal
age, reactive oxygen species (ROS) generation during
culture and exposure to chemotherapy agents such as
cyclophosphamide bring about rapid and reversible
changes in the degree of DNA damage and repair
in ovarian cells. As shown in Figure 32.2, granulosa
cells isolated from bovine ovarian follicles provide a
useful culture system for evaluating DNA damage by
immunofluorescence microscopy.
A wide variety of reagents are now available that
detect epitopes that appear in response to spontaneous
or induced double-strand breaks. Amongst these, antibodies that recognize histone modifications that occur
at the site of strand breaks reveal both the extent and
magnitude of lesions in the form of foci of varying
number and size within single cell nuclei. By fixing
391
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 32.3 Bovine granulosa cells cultured in the absence (ac)

or presence (di) of cyclophosphamide for 4 h (df) or 24 h (gi)
that have been fixed and stained for the demonstration of total
chromatin (a, d, g), gamma-H2AX (b, e, h) and RAD51 (c, f, i). With
progressive repair, as evidenced by the formation of discrete
gamma-H2AX positive nuclear foci, cytoplasmic RAD 51 assumes an
intranuclear location coincident with the sites of DNA
double-strand breaks. Scale bar represents 10 m.
cells in response at various times following exposure to

the chemotherapy agent cyclophosphamide, these foci
appear in increasing numbers that can be quantified
as the mean density/nucleus. This gives us an understanding of the relative time course of the induction of
DNA damage and the rate of repair upon drug removal
or after altering culture conditions (Figure 32.3).
When used in combination with reagents that
detect components of the repair complex, such as
RAD51 or BRCA1, further insight into the DNA repair
capacity can be obtained. This approach is applicable
to sections of ovarian tissue or cultured follicles, and
thus adds an important dimension to the assessment
of DNA integrity in both somatic and germ line components of the follicle under in vivo or in vitro conditions.
Center stage: supporting and

maintaining oogenesis
In the context of fertility preservation, the singular
objective of follicle culture is to provide an ex vivo con-
392
text within which the growth and maturative stages of

oogenesis can proceed unabated and without inflicting
damage to the oocyte as it acquires the capacity to support embryonic development [18, 22, 23]. As alluded
to above, to a certain extent a paradox emerges, since
the most valid predictors of the success of follicle culture will reside in the ability to document that indeed
oocyte quality has been established and maintained
for whatever duration of follicle culture is needed to
achieve this objective [5, 2426]. Thus, while a substantial body of evidence supports the idea that folliculogenesis consumes upwards of 100 days in humans
[1, 2, 8], we remain ignorant as to the exact chronology and duration of events that are required to support
the entire process of oogenesis as it occurs within the
confines of the follicle. Moreover, it remains unclear
as to whether the process of folliculogenesis reflects a
developmental continuum or one that is punctuated by
episodes that attend to key steps in oogenesis dictated
intrinsically or from cyclic variations in gonadotropin
availability or expression of receptors for LH or
FSH [2730]. Answers to these questions will inform
future approaches in this field in conjunction with
the emergence of assays that more directly target and
report upon the quality of the oocyte within cultured
follicles.
As noted above, short of assessing the ability of
oocytes to complete meiosis, engage successfully in fertilization in vitro and support pre-implantation development [1, 7, 31], there are few biomarkers that could
safely be considered as reliable or efficient in determining oocyte quality.
Those that have been proposed such as metabolic
substrates, visual indicators that rely on the use of fluorescent reporters or other vital indicating dyes ( Table
32.1) pose limitations pertaining to assay sensitivity,
free radical generation and metabolic perturbations
respectively, thus precluding their general utility. For
these reasons, non-invasive and non-perturbing optical assays remain a cornerstone for the assessment of
oocyte quality in materials that will be subjected to
IVF and embryo culture as a measure of developmental competence.
The more promising indirect biomarkers are those
that monitor the integrity of the interaction between
oocytes and granulosa cells, whereby both the differentiative state of the oocyte and its surrounding granulosa cells can be assayed [31, 32].
Based on the tenet that establishing contact
between the oocyte and granulosa is realized through
the anchoring of transzonal projections at early stages

of follicluogenesis, the effectiveness of these connections is likely to be a direct reflection of the metabolic
synergy required to acquire meiotic competence and
preserve the oocyte in a developmentally competent
state for the duration of culture [27, 33]. Earlier studies
using mouse ovarian follicles emphasized the importance of minimizing FSH exposure as this resulted
in the precocious resumption of meiosis, a consequence that would materially exacerbate the problem of oocyte aging [17]. Thus, more recent efforts to
achieve maintenance of oocyte health during oocyte
culture have viewed the inclusion of FSH at inappropriate times to be hazardous to the oocyte while
advancing follicle differentiation to the next level [1,
14, 18, 30]. Interestingly, these adverse effects of FSH
appear to be due to a direct action on the transzonal
projections (TZPs) as they are rapidly and irreversibly
retracted and may delimit the availability of nutrients
and/or signals that operate during both the acquisition of meiotic competence and maintenance of meiotic arrest [34]. It may well be that, besides identifying
more suitable media conditions favoring oocyte viability, other factors will be discovered that effect a more
stable interaction between oocytes and granulosa cells
based upon the composition of the extracellular matrix
[6, 10] and whether or not cultures are maintained in
a two- or three-dimensional context [1820, 35]. One
final factor that is likely to contribute to the stability of TZPs is growth differentiation factor-9 (GDF9). Originally discovered as a critical oocyte produced
factor required for the development of secondary follicles [36], GDF-9 was shown to be essential for the
establishment of TZPs in mouse knockout models
[37]. Whether exogenous recombinant forms of GDF9 or related oocyte signaling molecules will facilitate
metabolic synergy remains to be demonstrated, but
this variation in culture conditions would be worth
exploring in seeking alternative strategies that would
preserve the native architecture of the follicle in a fashion consonant with support of oocyte development
and metabolism [38].
Maintaining cellular interactions within the follicle is relevant to the effects of ovarian tissue cryopreservation or primordial follicle organization. The
hyperosmotic stress induced by cryoprotectants in
both slow freeze and vitrification protocols may disrupt cell interactions in distinct ways. As shown in
Figure 32.4, slow freeze protocols tend to perturb the
stromalbasement membrane interface whereas vitri-
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 32.4 Micrographs illustrating chromatin integrity in routine

histological preparations of primordial follicles from fresh (ac), slow
frozen (df) and vitrified (gi) bovine ovarian cortical strips. The top
row (ac) demonstrates the appearance of diffuse diplotene
germinal vesicle chromatins within primordial follicles viewed
under conventional bright field (a) or Nomarski differential optics (b,
c); note the distended configuration of chromatin towards the
nuclear margin and cytoplasmic homogeneity. Samples thawed for
30 min following slow freeze retain a fibrillar character that fills the
nucleus (df), whereas those recovered following vitrification
consistently demonstrate condensed chromatin retracted from the
nuclear periphery (g, h) and retraction of the oocyte cortex from the
interface with granulosa cells (i).
fication impacts the interface between the oocyte and

surrounding granulosa cells.
Importantly, these preliminary observations on
bovine cortical strips also suggest that the linkage of
chromatin to the nuclear envelope may be at greater
risk after vitrification when compared to tissues recovering from slow freeze. Together, measures of intercellular and intracellular protein interactions will represent an important direction for further studies on follicle integrity.
Future prospects for assessing the

integrity of cultured follicles
The evaluation of follicle integrity for future applications in fertility preservation will require changes
in our conceptual and technological approaches to
this problem. Throughout the course of this chapter,
the emphasis dictated by the need to obtain healthy
oocytes of high developmental competence warrants
393
conceptualizing folliculogenesis as an oocentric process rather than one that fulfills the endocrine functions so vital to reproductive physiology [8, 9]. In
this sense, future research into the phases of follicular development that are focused on supporting the
growth stage of oogenesis, as well as in anticipation of
the resumption and completion of meiosis, are likely
to uncover complex feedback pathways that are subject to regulation by the patterns of gene and protein
expression resident within the oocyte [39]. Defining
what these factors are, and how their presence signals
transformations in the behaviors of granulosa and thecal cells, should reveal arrays of biomarkers that in the
end reflect the ongoing role of the oocyte in determining its ultimate fate [2, 4044]. This may require application of more sensitive methods for the detection
of metabolites and substrates whose concentration is
maintained in a microenvironment more reminiscent
of what is present within the intact ovary. This goal is
achievable if the potential of microfluidic technologies
is realized for cultured follicles as it is being realized
for other model developmental systems requiring protracted periods of time in vitro.
A second dimension of future considerations for
the ex vivo maintenance of ovarian follicles concerns
that of time [3, 4, 18]. While the exact course of events
and their chronology that govern the development
of oocytes is not known, it is likely to require some
weeks for the entire process to be brought to fruition.
Besides being of an invasive nature, rote biochemical
and molecular studies while providing large quantities of data that might be expected to serve some value
as biomarkers are unlikely to contribute in a major
way to our understanding of the epigenetics of oogenesis. This rapidly evolving area of contemporary biology
has already entered the front-lines of human ARTs [18,
20, 24] given the well-documented effects that various
in vitro procedures have on the software that drives
the cytoplasmic and nuclear genomes of the early
embryo, all of it inherited from the process of oogenesis. Our biological clocks are changing the way in
which epigenetic modifications of the oocyte genome
play out during the natural course of folliculogenesis
[2, 27], warranting use of caution in future applications of follicle culture technology that would be relied
upon to preserve oocytes for individuals whose germ
line may have been compromised as a result of natural aging or the adverse effects of cancer treatment
[4, 18].
394
Acknowledgements
The support of the ESHE fund, the University of
Kansas Cancer Center and The Scientific and Technological Research Council of Turkey (TUBITAK) is graciously acknowledged.
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Section 8
Chapter
33
In vitro growth systems for human oocytes

From primordial to maturation
Evelyn E. Telfer and Marie McLaughlin
Introduction
Follicular development
Tissue banking of ovarian material is being increasingly offered to a variety of patients as a means of fertility preservation [1]. This tissue comprises of thin
cortical surface biopsies that contain predominantly
immature primordial follicles and currently is the only
option to restore fertility using this tissue is by transplantation [2]; however, this may not be a viable option
for all patients [3]. Increased options to maximize the
potential of this tissue to restore fertility could be realized by the development of in vitro systems to support complete growth from the early primordial stages
through to maturity. This technology would have many
therapeutic applications, including the production of
competent oocytes for assisted reproductive technology (ART); a model system to determine toxicological effects on germ cell development; a method for
the assessment of cryopreserved ovarian tissue prior
to transplantation; as well as providing an experimental model to address basic scientific questions concerning human oocyte development [4, 5]. Complete
oocyte development in vitro from the primordial stage
has been achieved in mice, but the larger size and
longer growth period of human follicles has made the
inter-species translation of these techniques difficult.
Recently progress has been made in defining conditions that support different stages of human follicle
development in vitro [6, 7], and these advances bring
the prospect of achieving a complete in vitro system
that supports oocytes from primordial to maturation
closer [8]. This chapter deals with our current understanding of in vitro development of human oocytes
and highlights the gaps that need to be bridged to
achieve a complete in vitro growth (IVG) system.
Female reproductive function requires cyclical development and maturation of ovarian follicles on a background of continuous activation from the pool of primordial follicles (Figure 33.1a). Primordial follicles are
formed prenatally and represent a finite population of
germ cells from which recruitment for growth will take
place throughout the womans reproductive life. Follicular growth and development involves a series of complex and precisely regulated events. It is characterized
by transition stages that begin with: (1) initiation of
primordial follicle growth and development to the preantral follicle stage; (2) the formation of antral follicles
where expansion to the pre-ovulatory or Graafian follicle is associated with granulosa cell proliferation and
antral fluid accumulation within the basement membrane; and (3) rupture of the Graafian follicle releasing
a cumulusoocyte complex at ovulation in response to
the mid-cycle luteinizing hormone (LH) surge (Figure
33.1a).
During its growth within the follicle the oocyte is
held in meiotic arrest, but as it grows it must acquire
the ability to resume meiosis (meiotic competence)
and the ability to support fertilization and embryonic development (developmental competence). Thus,
the oocyte is dependent upon the local environment
within the follicle for subsequent function as a gamete.
The development of follicles is regulated by a complex mixture of inhibitory and stimulatory endocrine,
paracrine and autocrine signaling by the somatic cells
(granulosa and surrounding theca cells) enhanced by
a range of oocyte specific regulatory factors mediated through bi-directional communication within the
follicle [9].
397
(a) Stages of follicle development from primordial to ovulatory. All growing follicles (primary onwards)
must be activated from the finite resting pool of primordial follicles.
Primordial
pool
Growth
activation
Primary
Pre-antral
Early antral
Mid antral
Pre-ovulatory
(b) P13K signaling

Growth factors
PTEN
PIP2
PIP3
PDK1
RPTK
Pk
p85
p110
PIP3
Akt
mTORC2
Thr308
Ser473
P13K
TSC1/TSC2
PTEN inhibits activation

mTORC1
mTORC1 promotes activation
PDK1
P
Thr309
P
S6K1
Thr225
rpS6
Figure 33.1 (a) Digrammatic representation of follicle growth from the non-proliferating pool of primordial follicles. Primordial follicles are
continuously activated into the growing population where they become primary follicles consisting of an oocye arrested at the dictyate stage
of prophaseI of meiosis (yellow) surrounded by granulosa cells (green). Primary follicles undergo oocyte growth and granulosa cell
proliferation and differentiation (purple) when they form an antral cavity. Antral follicles continue to grow and granulosa cells differentiate
into two subpopulations of cells: (1) cumulus surrounding the oocyte (blue); and (2) mural lining the wall of the follicle (orange). Exact timings
for this developmental sequence to occur in humans are not known but estimations suggest several months. However, it is not known
whether the growth profile is continuous or whether there are resting phases through follicle development. (b) Simplified version of the
PI3K pathway. The factors initiating this process are largely unknown but a body of evidence is emerging to show that the
phosphatidylinositol-3-kinase (PI3K-AKT) signaling pathway is a major regulator of early follicle/oocyte development and that components of
this pathway are involved in controlling the rate of activation from the non-growing population of follicles. The phosphatase PTEN converts
PIP3 to PIP2, which negatively regulates PI3K activity. Signaling mediated by PI3Ks converge at PDK1. PDK1 phosphorylates Akt and activates
it. Akt can phosphorylate and inactivate tuberous sclerosis complex 2 (TSC2 or tuberin), which leads to the activation of mTOR complex
(mTORC1). mTORC1 can phosphorylate (activate) S6K1. S6K1 subsequently phosphorylates and activates rpS6, which enhances protein
translation that is needed for cell growth. mTORC1 can be inhibited pharmacologically with Rapamycin and stimulated by leucine. The
manipulation of this pathway could have important clinical applications in the field of fertility preservation. See plate section for color version.
In humans the female germ cell population is

formed before birth and it is accepted that it is not
substantially replenished during adult life [10]. At any
age the majority of follicles within the ovary will be
at the primordial stage of development, and this represents a pool which is continually depleted during
reproductive life [11, 12]. The relative abundance and
lack of differentiation of primordial follicles makes this
population an ideal choice for IVG to obtain fertilizable oocytes for potential use in ART and fertility
preservation programs [5, 13]. The ability to develop
these immature follicles fully in vitro would have sev-
398
eral basic and clinical applications integral to fertility

preservation.
The capacity of immature mammalian oocytes
to develop fully in vitro has been demonstrated in
rodents with the birth of pups from in-vitro matured
oocytes derived from murine cumulus-oocyte complexes [14], primordial follicles [15, 16] and cultured
primary follicles [17]. However, this has yet to be
successfully repeated in humans or domestic species
where follicles undergo a protracted developmental
period in vivo. A great deal of basic scientific progress
has been made in developing systems designed to
Chapter 33: In vitro growth systems for human oocytes
support the partial growth of human follicles and several developmental milestones have been achieved,
namely, follicle activation [6, 1822] pre-antral follicle growth [6, 7, 19, 21, 2325], follicle differentiation
[6, 7] and oocyte maturation [26, 27]. These advances
have been made using both fresh and cryopreserved
human tissue. In the clinical setting progress has been
made with the ability to apply in vitro maturation
(IVM) techniques to immature human oocytes with
subsequent in vitro fertilization (IVF) of these oocytes
leading to pregnancy and live births [2830]. Despite
the successes, stumbling blocks still exist in putting
all of these systems together to achieve complete IVG.
A major difficulty associated with prolonged culture
is oocyte degeneration associated with the disruption
of the contact between the oocyte and its companion
somatic cells. Therefore it is important to understand
how culture conditions impact on the maintenance
of appropriate cell interactions during follicle/oocyte
development.
Activation and growth of

immature follicles
Central to our understanding of female fertility, reproductive aging and developing IVG systems is knowing how the resting population of primordial follicles is regulated. While some local factors regulating the initiation of primordial follicle development
have been identified, such as anti-Mullerian hormone
[31], the underlying mechanisms involved are largely
unknown [32]. A growing body of evidence is now
emerging to show that the phosphatidylinositol-3kinase (PI3K-AKT) signaling pathway is a major regulator of early follicle/oocyte development and that
components of this pathway are involved in controlling the rate of activation from the non-growing
population of follicles (Figure 33.1b) [33]. A recent
study showed that the phosphatase PTEN acts within
the oocyte as a negative regulator of PI3K-AKT and
suppresses initiation of follicle development in mice,
with global activation of primordial follicles demonstrated in mice with an oocyte-specific Pten knockout
[34]. This work also highlighted that PI3K-PDK1 (3phosphoinositide-dependent protein kinase-1)-AktS6K1 (p70 S6 kinase 1)-rpS6 (ribosomal protein S6)
cascade in oocytes controls ovarian aging by regulating survival of primordial follicles [34, 35]. Activation of S6K1-rpS6 is largely dependent on the mammalian target of rapamycin complex 1 (mTORC1), a
serine/threonine kinase that regulates cell growth and

proliferation in response to growth factors and nutrients [36]. Recent studies have implicated mTORC1 signaling in regulating dormancy and activation of the
primordial follicle population, which is equally important as PTEN/PI3K signaling [35, 37]. Thus there is
now an accumulation of evidence to show that PTEN
and mTORC1 play important roles both in initiating and regulating progression of follicle development, with PTEN actively suppressing and mTORC1
promoting activation (Figure 33.1b). Manipulation of
these pathways could lead to improvements in IVG
systems by supporting increased activation in a coordinated manner. Current work in our laboratory is focusing on these processes [38, 39].
In humans quiescent primordial follicles are continuously activated to grow and this is independent of
physiological status [40, 41]. Activation of growth is
marked by the gradual transformation of the flattened
epithelial cells surrounding the oocyte into cuboidal
cells which proliferate forming a multilaminar structure in which the germ cell will mature (Figure 33.1a)
[4, 11, 42]. In the human ovary, initiation of growth
and early follicle development is believed to be an
extremely protracted process taking many weeks to
complete [2, 11, 28]. However, whether this lengthy
period of time accurately represents the definitive rate
of somatic and germ cell development or whether in
vivo growth is modulated by ovarian molecular constraints is unclear. What is certain is that sustained normal follicle development critically depends upon intercellular communication between the growing oocyte
and the developing granulosa [43]. This communication takes the form of developmentally regulated cytoplasmic projections which extend from the membrane
of the granulosa and traverse the zona pellucida terminating close to the oolemma frequently forming
gap junctions on the oocyte surface [44, 45]. In vivo
follicle development comprises of a series of events
which occur sequentially and take varying lengths
of time to complete. During the protracted transformation of a primordial follicle into a primary follicle the oocyte does not increase in size [11, 46].
However, at the point of antral cavity formation, significant oocyte growth has been achieved [4, 9]. To
achieve competency, the fully grown oocyte must
undergo nuclear maturation and cytoplasmic differentiation, processes which occur chiefly in concert with
granulosa cell proliferation during pre-antral follicle
growth [4, 9, 47]. The ability of an oocyte to complete
399
Micro-cortex
culture
Removal of pre-antral
follicles by
micro-dissection
Individually cultured
and monitored for
oocyte/follicle
health markers
(b)
(a)
(c)
Aliginate encapsulated culture

for further growth and
development then isolation of
(d)
(e)
IVM of isolated
and subsequent fertilization
Step 1
1. Preparation of the tissue

(a) is crucial: The shape and
structure of the cortical tissue
is important and underlying
stroma should be removed
and the tissue flattened
Step 2
Step 3
2. Multilaminar follicles (b) can be

removed within 6 days of culture;
however, there may be many
primordial and primary follicles
remaining. The remaining pieces can
be replaced into culture for a further
period to maximize yield
Step 4
3. Once antral formation

has been achieved (c),
follicles can be cultured
within alginate (d) or on
membranes and cultured
for up to 21 days
Figure 33.2 Proposed multi-step culture system for activation of human primordial follicles and subsequent follicle/oocyte development.
The stages required for a multi-step culture system are as follows. Activation of primordial follicles within cortical strips (a). Removal of all
growing follicles and most of the underlying stromal cells increases the rate of activation [6]. Flattened strips are cultured free floating in
medium containing human serum albumin (HSA), ascorbic acid and basal levels of follicle stimulating hormone (FSH) [6]. Once follicles have
reached multilaminar stages they are isolated mechanically using needles and cultured individually. Isolated follicle culture is to support
development from pre-antral to antral stages (b). The addition of activin at this stage results in improved follicle development and increased
antral formation (c) [6]. Follicles of similar stages that have been grown in vivo have been isolated and grown with alginate drops [7] (d), and
oocytes grow to almost full size within a total of 30 days [7]. The final stages of oocyte growth and development could be achieved by
culturing the oocyte and its surrounding somatic cells outwith the constraints of the large follicle (e). See plate section for color version.
meiosis is acquired after its ability to resume the process; moreover, oocyte size is a determinant in reaching metaphase II [48, 49]. Growing oocytes accumulate cytoplasmic organelles and these are dispersed
to the periphery of the cell in readiness for fertilization and pre-implantation embryo development; only
oocytes that have completed both cytoplasmic and
nuclear maturation are capable of fertilization and subsequent embryo development [50]. The aim of invitro follicle development or IVG is to achieve developmentally competent oocytes. Therefore culture systems must provide an environment that will sustain
oocyte growth and support cytoplasmic and nuclear
maturation but without the necessity to develop large
follicular structures [13]. Attempts to recapitulate in
vivo development (timings and size) in vitro have not
been successful and therefore strategies to improve
IVG systems should concentrate on optimizing oocyte
growth within the context of surrounding somatic cells
in the shortest time possible.
Strategies to support follicle

development in vitro
Various approaches have been taken to promote early
human follicle development in vitro using fresh [6, 18]
400
and thawedcryopreserved [18, 20, 51] human cortical

tissue. It is clear that to achieve complete in vitro development of human oocytes a multi-step culture system needs to be developed. The follicle functions both
as an endocrine structure and as a vehicle to support
oocyte growth and development. In optimizing such a
culture system, the focus should be on oocyte development which may not require the development of
large follicular structures but rather the maintenance
of appropriately differentiated somatic cells in contact
with the developing oocyte. The multi-step approach
has been designed to support the changing requirements of the developing oocyte and its surrounding
somatic (granulosa) cells and the steps involve: (1) culturing small pieces of ovarian cortex to support activation of primordial follicles [6, 18, 21]; (2) isolation and
culture of growing pre-antral follicles to achieve oocyte
growth and development [6, 7, 19, 23, 25]; (3) aspiration and maturation of oocyte cumulus complexes [26,
52, 53]. Our work has been the first to combine these
steps and advances have been made in the first two
steps [6]. Figure 33.2 illustrates our proposed multistep IVG system to produce competent human oocytes
from ovarian cortical tissue. The current status of each
of these stages will be discussed in the following sections.
(a)
(b)
(d)
(e)
(c)
(f)
Figure 33.3 (a) A cluster of quiescent follicles in freshly fixed human ovary. (b) After 6 days in vitro, growing follicles () appear on the
surface of a cultured fragment of human ovarian cortex. (c) A growing follicle protruding from the edge of a fragment of cultured human
cortex. (d) Intact secondary human mechanically dissected with presumptive theca layers attached. (e) Histological image of a secondary
human follicle fixed after 6 days in vitro growth within a cortical fragment. (f) Histological image of human antral follicle fixed after a total of 10
days in vitro growth. See plate section for color version.
Culture of ovarian cortical biopsies

In fertility preservation programs many young women
(particularly pre-pubertal girls) opt for cryopreservation of ovarian cortical biopsies. The majority of viable
follicles within this tissue are at the quiescent primordial stage (Figure 33.3a). Therefore the priority for
developing an IVG system must be to optimize activation of primordial follicles in vitro and support early
follicle development. Recent work from our laboratory
[6] has shown that human primordial follicles grow
well within mechanically loosened cortical pieces and
can develop to multilaminar pre-antral (secondary)
stages within 6 days. The culture conditions in this system differ from those described in earlier studies [18,
19], as the culture medium is serum free and no supporting matrix is present. Preparation of the tissue is
crucial to success and this involves removal of most
of the underlying stromal tissue so that the cultured
pieces consist of predominantly ovarian cortex containing primordial and primary follicles (Figures 33.2
and 33.3a). When these small pieces of human ovarian cortex are cultured there is a significant shift of
follicles from the quiescent to the growing pool over
short culture periods of 610 days [6, 38, 51], an obser-
vation repeated in cattle where extensive primordial

activation has been reported within 2 days in vitro
[5456] indicating that activation results from a
release of intraovarian factors that act to inhibit the initiation of follicle growth [37, 55]. Observations made
during IVG and confirmed by histological analysis in
both human and bovine models demonstrate that the
growth of follicles in tissue fragments is differentiated, with the rate of growth enhanced in follicles lying
adjacent to tissue edges (Figure 33.3b,c) compared to
follicles embedded within dense stroma [McLaughlin and Telfer, unpublished observations]. Moreover,
when excessive stroma is removed from tissue pieces
prior to culture, the rate of follicle growth increases
(Figure 33.3 b,c) [6, 38, 51].
Cortical strip culture removes follicles from the
in vivo endocrine and paracrine processes regulating
growth rate. However, follicles will still be subject to
the effect of follicle interactions and the influence of
stromal cell factors. It is clear that tissue shape and
stromal density are important factors that regulate follicle growth initiation in vitro, as solid cubes of cortical tissue show a lower rate of growth initiation [18].
In contrast, when stromal cells are removed and the
401
tissue is cultured as flattened sheets, the initiation

rate is greater and follicles grow more quickly [6]. The
physical environment of the follicles within the cortical tissue affects their response to stimulatory and
inhibitory factors and therefore influences their ability to grow [57].
Culture medium to support activation

and early growth
The optimum culture medium that supports activation and early growth has yet to be developed. The first
culture systems used fetal calf serum (FCS) or human
serum-containing medium but this has now been
replaced by defined media substituted with human
serum albumin (HSA) and a combination of insulin,
selenium and transferrin (ITS) with the addition of
ascorbic acid to minimize cell death [5860]. The
choice of basal culture medium includes MEM alpha,
Waymouths medium and McCoys 5A, with our preference being for McCoys 5A [6]. The most basic medium
appears to support activation and growth, and the
addition of growth factors such as activin at this stage
leads to less activation [61], whilst in bovine systems
increased insulin leads to greater activation [55]. Further information is needed on how specific factors
affect activation and stromal cell support but a promising approach, which may lead to improvements in
the number and quality of growing follicles available
for harvesting, is directly manipulating key signaling
pathways that control activation (Figure 33.1b). This is
now a major focus in our laboratory [38, 39].
Once follicle growth has been initiated within the
strip, follicles can develop to multilaminar stages.
Growing follicles do not survive well within the cortical environment and it appears to be inhibitory to further growth resulting in a loss of follicle integrity and
oocyte survival [6, 19]. Therefore, in order to develop
further, follicles require to be released from the cortical stromal environment and cultured individually
[6, 38, 51].
Isolation of growing human follicles

Isolation of pre-antral follicles from cultured cortical tissue can be achieved by mechanical dissection,
enzymatic isolation or a combination of both. Enzymatic isolation commonly uses collagenase and DNase
to liberate primordial and pre-antral follicles from
stromal tissue and yields many more follicles than
402
mechanical dissection [6265]. However, collagenase

can exert a species-specific effect which has been
associated with follicle damage and poor survival in
large mammals [67, 68], but this may be overcome
by modified techniques and new purified enzyme
preparations including Liberase [6971]. Mechanical
isolation using fine needles has the advantage of preserving follicular integrity by maintaining the basal
lamina and thecal layers of the follicle (Figure 33.3d).
However, the yield is low and the procedure protracted
and laborious due to the dense fibrous cortical tissue in the ovaries of large mammals, particularly in
the human where follicles are embedded in the tough
fibrous cortex and, as such, are relatively inaccessible.
Enzymatic [19, 24, 25], partial enzymatic [7, 71] and
mechanical [6, 23] isolation methods have been used
to dissociate human pre-antral follicles from the cortical stroma with the resulting follicles being cultured
for up to 4 weeks.
The progression of human follicles following isolation from the cortex is remarkable. In the presence
of FSH, enzymatically isolated secondary human follicles can differentiate, become steroidogenically active
and complete oocyte growth in 30 days [7]. Furthermore, quiescent follicles activated to grow within cultured fragments of cortex and mechanically isolated
as secondary follicles (Figure 33.3e) become steroidogenic and undergo differentiation after a 10 day in
vitro period with and without activin (Figure 33.3f)
[6]. These observations would appear to confirm that
local ovarian factors indeed inhibit development of follicles: the question remains as to whether the growth
rate in vitro is accelerated or if it is growth without the
brakes on. Further studies on growth rates are required
and it is essential to determine how growth rate affects
oocyte quality.
Culture of isolated human follicles

Culture systems designed to support the development
of isolated human follicles can be divided into several
categories with some degree of overlap. The defining
aspects of current culture systems are: (1) the developmental stage of follicles on harvesting; (2) the type
of material support employed; and (3) the length of the
in vitro period. Although it is possible to enzymatically
retrieve viable human primordial follicles from cortical tissue, prolonged culture and survival of individual follicles has been unsuccessful with gross degeneration observed [24]. Whether this is due to enzymatic
damage of the basal lamina or the necessity of these

follicles to be in groups with stromal support or a combination of factors is unclear; indeed, it has been noted
that the presence of stromal cells can improve the
growth of cultured human primordial follicles indicating that support of extrafollicular cells is vital during initial growth [5, 6, 72]. A recent study of ovine
primordial follicle culture reports progress after using
lectin and kit ligand (KL) to promote oocyte granulosa cell aggregation and oocyte development [73],
an encouraging step towards defining the conditions
required to support the unilaminar to multilaminar
transition in cultured follicles of large mammals.
Growing follicles isolated for in vitro development can be derived from several sources; thawed
cryopreserved tissue, cultured cortical fragments or
uncultured ovarian tissue. In vitro growth of thawed
cryopreserved tissue will be discussed later. Although
the technique of harvesting in-vitro activated follicles at the secondary stage is still evolving and much
remains to be optimized, there are several advantages
to selecting these follicles for further development in
vitro, including homogeneity and abundance of population, relative lack of apoptotic sequelae [74, 75] and
reduced likelihood of disease infiltration [3]. Using our
two-step culture technique unilaminar human follicles are capable of growth, differentiation and steroidogenesis after a total in vitro period of 10 days and
normal oocyte morphology is maintained [6, 38, 51].
There are very few studies reporting the development
of secondary follicles isolated from fresh non-cultured
human cortical tissue. However, progress is encouraging with growth, differentiation, steroidogenesis and
complete oocyte growth being achieved within a total
of 30 days [7]. Optimal results require the inclusion of
a basal concentration of follicle stimulating hormone
(FSH) (1.01.5 IU/ml), and successful culture is promoted by material support of the follicular unit while
in vitro to prevent migration of the somatic cells away
from the oocyte.
Measures to support the three-dimensional follicular architecture and thereby maintain intrafollicular
cell association in vitro have been used in the culture of mammalian follicles for almost two decades
and it has been demonstrated, largely using rodent systems, that the use of suspension cultures, mineral oil
sheaths, hydrophobic membranes and follicle encapsulation result in the promotion of follicle growth
and the attaining of developmental milestones [5].
When culturing large mammalian follicles the use of
v-shaped micro-well plates has allowed the maintenance of three-dimensional follicular architecture in
vitro while promoting growth and differentiation in
bovine [7678] and human follicles [6, 38, 52] with
antral formation occurring within 10 days. Follicle differentiation has also been reported in bovine follicles embedded in collagen gels and cultured for 13
days [79] and, using a combination of media thickened with polyvinylpyrrolidone, a macromolecular
supplement and microporous membranes, one live calf
was produced from immature bovine follicles cultured
for 14 days [80]. In addition to v-shaped microwell culture plates, follicle encapsulation in alginate
hydrogels has been used to support secondary human
follicle growth in vitro [7]. The novelty of alginate
encapsulation is that it is believed to mimic the extracellular matrix in vivo in terms of its ability to facilitate molecular exchange between the follicle and the
culture medium, while its flexibility can accommodate
cell proliferation but its rigidity prevents dissociation
of the follicular unit. It would appear that the rigidity
of the alginate capsule is of vital importance to follicle development as inhibition of growth and reduced
steroidogenesis have been reported in murine follicles embedded in 1.0 and 1.5% alginate gels, respectively [81, 82], whereas fully grown human oocytes
have been produced using 0.5% gels [7]. The ability of
the reported systems to promote human follicle growth
in vitro from the earliest stages at rates that are accelerated in comparison to the in vivo environment is
indeed promising. The next step is to ascertain whether
the oocytes produced in these systems are capable of
IVM and to determine whether the altered growth is
deleterious to oocyte epigenetic health and normality.
Culture of thawed cryopreserved

human follicles
If IVG systems are to have a clinical application
within fertility preservation they must be capable of
supporting development from cryopreserved tissue.
The viability of human follicles isolated from cryopreserved tissue was established over 10 years ago
[83], and in the past decade human ovarian cryopreservation and tissue re-implantation has resulted in
live births [84, 85]. Although human follicles isolated
from thawedfrozen tissue can develop to the antral
stage following xenografting [70, 86, 87], there is a
paucity of information regarding the ability of human
follicles isolated from thawed tissue to survive in
403
culture. In 1997, Oktay et al. demonstrated that the

viability on isolation of primordial follicles enzymatically isolated from thawed human cortical tissue is
the same as that of primordial follicles isolated from
fresh tissue [83], yet when cultured individually survival rates are extremely poor [18, 24]. Work in our
laboratory in collaboration with Outi Hovattas group
in Stockholm is focusing on optimizing culture conditions to support cryopreserved tissue. Our preliminary results show that, following mechanical isolation, follicles harvested from thawed-vitrified or slow
frozen fragments of human cortex cultured for 6 days
are capable of further individual growth at a rate similar to those isolated from fresh tissue [51; Telfer and
McLaughlin, unpublished observations]. Poor results
observed in earlier studies [18, 24] may reflect the
method of follicle isolation and that the effect of enzymatic isolation and premature loss of somatic cell contact impacts negatively on the ability of follicles isolated from thawed tissue to survive in vitro.
Culture of human oocyte

cumulus complexes
The ultimate aim of a system supporting follicle
growth is to produce competent epigenetically normal oocytes. In order to achieve this, in-vitro grown
human oocytes need to be matured in vitro. Maturation of oocytes already exists as a separate strategy
and this is utilized routinely in human ART processes
with varying degrees of success [27, 88]. As discussed
earlier, achieving and sustaining oocyte growth is the
major objective of any complete in-vitro development
system as this is a size-specific indicator of the oocytes
ability to resume meiosis [11, 48, 49, 53]. Another consideration must be that the system should be capable
of supporting the nuclear maturation and cytoplasmic
differentiation of oocytes in vitro [11, 89], processes
that in vivo require precise timing and hormonal regulation. It appears that, despite numerous studies and
a plethora of media tested, no maturation protocol
has emerged as demonstrably superior [90]. While
4080% of immature human oocytes can successfully
complete IVM and IVF giving rise to live births, the
rate of maturation of immature oocytes lies well below
that of oocytes from stimulated ovaries indicating that
either the protocols are suboptimal or many of the harvested oocytes are intrinsically unable to undergo maturation. In-vitro grown oocytes may require a further
period of growth within the cumulus complex before
404
maturation (Figure 33.2), and oocyte growth within

human complexes has been demonstrated in vitro [53].
While there are no culture systems established to support the in vitro development of human oocytes from
the mid-growth stage, i.e. 9095 m, a live birth has
been reported using bovine oocytes of this size [80].
The immature bovine oocytes required a period of up
to 14 days in vitro to allow further oocye growth and
development to support maturation [80]. This suggests
that a similar system could be applied to human oocyte
cumulus complexes in order to achieve oocyte diameters suitable to undergo current IVM protocols.
Proposed multi-step culture system

for human oocytes
The successes reported at each of the stages of human
follicle development outlined above supports the theory that complete development of quiescent human
primordial follicles could be achieved in vitro. Currently there are culture systems for humans and
domestic species that have been designed to support
specific stages of development, but there are significant
gaps in oocyte development that have not yet been supported in vitro. Development of a complete multi-step
culture system requires us to fit each of these culture
systems together (Figure 33.2), and future research
needs to focus on the processes necessary to bridge
the current gaps that would allow continuous development from primordial to developmentally competent
stages. Significant advances have been made towards
achieving this, particularly with the development of a
system that supports activation and early growth [6]
and subsequent growth to antral stages [6, 7]. The challenge now is achieving the final steps of maturation
(IVM) and fertilization (IVF) of these in vitro grown
(IVG) oocytes without the need to culture whole antral
follicles.
The stages required for a multi-step culture system
are: (a) cortical strip culture to support initiation and
growth of primordial follicles to pre-antral stages, followed by (b) isolated follicle culture to support development from pre-antral to antral stages (c) and then,
subsequently, (d) a system to support later stages of
oocyte growth and development without the very large
antral follicles of humans and domestic species (Figure
33.2). This would involve: (1) improving growth of primordial follicles in vitro so that follicles develop to
a stage at which they can then be isolated, allowing
progression to the next culture step; (2) optimizing
conditions that enable the transition of these isolated follicles from pre-antral to antral stage of follicle development in vitro, since at present oocyte
somatic cell interactions are not fully supported at the
antral transition; and (3) developing in vitro systems
to support oocyte growth and subsequent maturation
of oocytegranulosa cell complexes taken from the
in-vitro grown early antral follicles, hence removing
the requirement to support large pre-ovulatory follicle
growth in vitro.
Summary
The achievement of complete mouse oocyte development in vitro has led to investigation into the potential of large mammal and human oocyte development
within culture systems. The individual success of several keys stages of this process, i.e. initiation of primordial follicle growth, follicle differentiation, completion of oocyte growth and IVM have been encouraging, although a unified system incorporating all of
these developmental milestones has yet to be defined
for human follicles. Although significant progress has
been made, much optimization is still required to routinely complete the in vitro development of the stages
detailed above. Translation of any in-vitro human follicle growth system into a clinical setting will require
rigorous testing to determine the normality and health
of in-vitro grown oocytes prior to the application of
IVF procedures. Basic research using rodent models suggest that oocyte development in vitro does not
result in adverse developmental outcomes or longterm effects [91]. Whilst this is encouraging, it is also
essential that good models, using large animal models,
are rigorously tested before proceeding with translating human culture systems to a clinical setting.
Whilst the therapeutic potential of in-vitro grown
human oocytes may not be imminently realized, the
methodology itself is central to fertility preservation
programs. The culture systems provide a reproducible
and effective technique to assess the viability of cryopreserved cortical strips prior to transplantation.
Furthermore, these systems allow many basic scientific questions regarding human oocyte development
to be addressed and, as a consequence, factors and
mechanisms involved in its regulation identified. The
knowledge gained from these basic studies will facilitate the development of optimized culture systems,
which could have the prospect of clinical application
to restore fertility in young women currently storing

tissue.
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Section 8
Chapter
34
Contributions of ovarian stromal

cells to follicle culture
David J. Tagler, Lonnie D. Shea and Teresa K. Woodruff
Introduction
In-vitro culture systems for ovarian follicles are
enabling tools for advancing the study of folliculogenesis and the development of fertility preservation techniques. Folliculogenesis is a complex process regulated
by endocrine, paracrine and autocrine factors, and can
be difficult to study in vivo. In-vitro culture systems
provide a controlled environment in which to investigate the mechanisms driving follicle development. The
usage of these systems has produced significant discoveries about the influence of hormones, mechanics and
extracellular matrix (ECM) proteins in folliculogenesis [16]. In addition to these fundamental observations, these culture systems are providing a foundation
for the development of systems for fertility preservation for cancer survivors [7, 8]. The increase in survival rates for young women with cancer has prompted
the need for fertility preservation techniques [9]. Lifesaving cancer treatments, such as chemotherapy and
radiation, threaten fertility by diminishing the immature follicle pool and triggering early menopause. Current treatments include cryopreservation and transplantation of ovarian tissue, which incurs the risk of
re-introducing cancer cells into the patient [10, 11].
The successful development of follicle culture systems
could circumvent this risk, by allowing in vitro follicle
maturation/fertilization to obtain fertilizable oocytes
from immature follicles. Follicle culture systems have
had some success; however, further developments are
necessary to achieve the consistent growth of human
follicles to produce fertilizable oocytes [12, 13].
Stromal tissue is essential for the development of
the earliest stage follicles. The ovarian stroma con-
tains interstitialtheca cells, neurons, blood vessels

and macrophages. Secondary follicles can be grown
individually using in-vitro culture systems; however,
primordial and primary follicles are typically activated
in organ culture-containing stromal components [13
18]. Hence, the stroma has a clear influence on follicle development. Stromal cells provide structural support and have complex bi-directional paracrine signaling with the follicle [1922]. Moreover, it is widely
hypothesized that stromal cells are recruited by the follicle and differentiate into theca cells [2327]. Nevertheless, the specific mechanisms of action remain
unclear. In-vitro culture systems that contain stromal cells may be an enabling tool for investigating
the mechanisms by which stromal cells activate the
early stage follicles and may ultimately be translated
toward strategies for fertility preservation for cancer
patients.
In this chapter, we discuss ovarian stromal cells
and in-vitro follicle culture systems. Integrating stromal cells into current follicle culture systems will better
simulate the natural ovarian microenvironment and
could lead to the elucidation of the mechanisms by
which these follicles are activated. In addition, stromal
cell co-culture could improve early follicle growth and
survival, which are essential for the successful translation of in-vitro culture systems to primate and human
follicles. The current knowledge base for the culture of
somatic and stromal cells is discussed, with an emphasis on three-dimensional culture systems using biomaterials. In addition, the potential and challenges of coculture is discussed by drawing from recent work with
co-culture systems in tissue engineering.
409
Antral
Secondary
Primordial
Antrum
Primary
Organ culture
(stromal cells)
Oocyte
Granulosa
Theca
Roles of ovarian stromal cells in

follicle development
Folliculogenesis is the development process during
which primordial follicles develop to Graafian follicles that ovulate fertilizable oocytes (Figure 34.1).
For comprehensive reviews of folliculogenesis, please
refer to references [2830]. The follicle is the functional unit of the ovary and is composed of a germ
cell (the oocyte) and layers of somatic cells (granulosa and theca cells). Primordial follicles are the
most immature class and are found during embryonic
(human) or immediately post-embryonic (murine)
life. The oocyte within primordial follicles is arrested
in the first meiotic prophase. Follicle activation from
the non-replenishable ovarian reserve is a process
that is not well understood. Selected primordial follicles are activated and form primary follicles, which
have larger oocytes and a layer of cuboidal granulosa
cells enclosed by a basement membrane. Subsequently,
the granulosa cells proliferate and form several layers around the oocyte. At the same time, theca cells
begin to surround the basement membrane of the follicle. These follicles are called secondary and multilayer follicles. Under the influence of follicle stimulating hormone (FSH), the granulosa cells proliferate and
differentiate into cumulus (surround the oocyte) and
mural (inside of basement membrane) granulosa cells.
Likewise, the theca cells differentiate into theca interna
(androgen secreting cells) and externa (connective and
supportive tissue). The follicle increases in size and
develops a fluid-filled cavity called an antrum. Under
the influence of luteinizing hormone (LH), the oocytes
in antral follicles complete the first meiosis cell division and then pause in the second meiotic metaphase.
These oocytes are capable of fertilization. The over-
410
Figure 34.1 Folliculogenesis. Primordial

follicles develop to antral follicles, which
are capable of producing fertilizable
oocytes. Ovarian stromal cells are
hypothesized to have significant roles in
the activation of primordial follicles and
the recruitment/differentiation of theca
cells. Most follicle culture systems focus on
secondary or multilayer follicles and
produce antral follicles with fertilizable
oocytes. Primordial and primary follicles do
not activate or mature in vitro. The culture
of these follicles is typically performed as
an organ culture. See plate section for
color version.
all goal of follicle culture systems is to reproduce this

entire process in vitro.
Ovarian stromal cells may have significant roles
in folliculogenesis, particularly in the activation of
primordial follicles and the differentiation of theca
cells. Stromal cells, which are similar in morphology to
fibroblasts, make up the connective tissue throughout
the ovary and surround follicles. Stromal cells are
assumed to arise from a population of unspecialized
mesenchymal stem cells [31]. The morphology of
the stromal tissue varies between the cortex and
medulla of the ovary [32]. In the cortex, the stromal
cells are organized parallel to the surface and have a
rounded structure. In the medulla, the cells exhibit
random organization and have an elongated structure, and they are often referred to as interstitial or
luteinized cells. The stromal cells in the medulla are
believed to be further differentiated towards theca
cells and have greater steroidogenic capacity than
the stromal cells in the cortex [33]. Specific stromal
cell markers have not yet been identified. Hence,
stromal cells are often identified as mesenchymal cells
that lack theca cell markers, such as the LH receptor
(LHR), steroidogenic acute regulatory protein (StAR),
3-hydroxysteroid dehydrogenase (3-HSD) and
17-hydroxylase (CYP17A1) [3335]. Stromal cells
signal via paracrine factors and influence early follicle
development. For example, bone morphogenetic
proteins 4 and 7 (BMP-4 and -7), secreted by stromal
and/or theca cells have been identified as positive
regulators of the primordial-to-primary follicle transition [3638]. Moreover, it is widely hypothesized
that stromal cells are recruited by the follicle via
paracrine factors and differentiate into theca cells.
This hypothesis was first proposed by Dubreuil in
1946 [23] and later reiterated by others [2427]. To
Chapter 34: Contributions of ovarian stromal cells
date, a number of paracrine factors that regulate

theca cell recruitment and differentiation have been
identified, such as insulin-like growth factor (IGF-1),
kit ligand (KL) and basic fibroblastic growth factor
(bFGF) [22, 39, 40]. Nevertheless, the conclusive
evidence for this hypothesis remains elusive. In-vitro
culture systems of follicles and stromal cells may be an
enabling tool to elucidate the functions of the ovarian
stroma, signaling mechanisms and roles in follicle
development.
Studies investigating the roles of the

ovarian stroma
Follicle-to-stroma paracrine signaling
Early stromal cell experiments established paracrine
signaling from the follicle to the stroma. In 1995,
Magoffin and Magarelli demonstrated that granulosa
cells of developing follicles secrete a signal that stimulates theca cell differentiation [19]. In this experiment,
isolated thecainterstitial cells from rat ovaries were
cultured in media conditioned by follicles and assayed
for androgen secretion. The conditioned media was
found to stimulate androgen secretion, which suggested the presence of a theca differentiation factor
secreted by the follicle. Later, Magarelli et al. identified an increase in the mRNA expression of the
LHR and various theca steroidogenic enzymes, including cholesterol side-chain cleavage (P450scc), 3-HSD
and CYP17A1 in response to this media [21]. Also,
Magarelli et al. showed that this differentiation signal was both gonadotropin (FSH) and developmentally regulated, as only pre-antral follicles with 25 layers of granulosa cells produced the signal [21]. To date,
the identity of this signal has not yet been purified;
however, proteins in the range of 1924 kD that interact synergistically may be responsible for this action
[20]. In a series of candidate signaling studies, the combination of two granulosa produced peptides, IGF-I
and KL, were found to increase androgen production
and gene expression of androgenic factors in rat theca
interstitial cells [22]. Other potential regulating factors include growth differentiation factor-9 (GDF-9),
activin, inhibin and follistatin [20, 4146]. In sum,
these experiments support the notion that follicle-tostroma signaling exists.
Additional studies have demonstrated the effects
of follicle-to-stroma signaling on theca cell recruitment, stromal cell proliferation and the primordial-to-
primary follicle transition. Parott and Skinner treated

ovary fragments and isolated stromalinterstitial cells
with KL, which was hypothesized to be a theca cell
organizer secreted by granulosa cells [39]. Kit ligand
was found to significantly increase the percentage of
theca cell layer thickness of primary follicles in organ
culture. This result suggests KL helps to recruit theca
cells from the stroma. Furthermore, KL was found to
stimulate ovarian stromal cell proliferation in a dosedependent manner. Treatment with KL did not affect
stromal cell androstenedione or progesterone production. Hence, KL did not promote theca cell differentiation, which is consistent with research that has determined that theca cell differentiation is controlled by
a synergy of multiple factors [2022]. Later, Nilsson
and Skinner identified the roles of KL and bFGF in
the primordial-to-primary follicle transition [40]. The
combination of these factors decreased the percentage of primordial follicles and increased the percentage of primary, pre-antral and antral follicles. Overall, these studies build upon the earlier work by identifying the possible roles of stromal cells in follicle
development.
To further explore the effects of paracrine signaling, stromal cells have been co-cultured with granulosa and theca cells. Orisaka et al. developed a coculture system that separates two cell populations via
a collagen membrane, which permits the diffusion of
factors smaller than 12.5 kD [33]. In these studies,
bovine stromal cells from the cortex and medulla of the
ovary were cultured with and without granulosa cells.
Co-culture with granulosa cells increased the number of secreted lipid droplets, filopodia and mitochondria. Co-culture also stimulated androgen secretion in
both cortex and medulla stromal cells. However, an
increase in LH receptor mRNA was only observed in
cortex stromal cells. Surprisingly, no increase in other
theca markers was detected. While these data suggests
theca cell differentiation, the evidence is not conclusive. No changes in the co-cultured granulosa cells
were reported. Nevertheless, these experiments successfully demonstrated paracrine signaling from granulosa cells to the stromal cells in a co-culture system.
Stroma-to-follicle paracrine signaling

In addition to paracrine signaling from the follicle to
the stroma, signaling from the stroma to the follicle
has been studied. The bone morphogenetic proteins
BMP-4 and BMP-7, which are expressed by stromal
411
and/or theca cells, have been linked to the primordialto-primary follicle transition. Lee et al. demonstrated
that BMP-7 promotes the activation of primordial follicles in vivo [38, 47]. BMP-7 was injected into the
ovarian bursa of rats, which produced a decrease in
the number of primordial follicles and an increase in
the number of primary, pre-antral and antral follicles.
Nilsson et al. achieved similar results using in-vitro
organ culture and BMP-4 [37]. Thus, these studies support the notion that stroma-to-follicle signaling exists.
Organ culture and the activation of

primordial follicles
Some of the most significant stromal cell experiments
have been ovary organ culture experiments (in situ).
These experiments culture follicles within thin fragments of ovarian stromal tissue. In 1996, Eppig and
OBrien achieved complete oocyte development in
vitro using oocytes from primordial follicles of newborn mice [14]. This result was accomplished using
a two-step strategy in which the ovaries of newborn
mice were grown in organ culture for 8 days and
then oocytegranulosa cell complexes were isolated
from the ovaries and cultured individually for an additional 14 days. Using this strategy, primordial follicles
developed to produce mature oocytes. These oocytes
were then fertilized in vitro and the resulting embryos
were implanted to produce live offspring. When cultured individually outside of stromal tissue, primordial
follicles rapidly lose their three-dimensional structure, pre-granulosa cells migrate away from the oocyte
and oocyte extrusion/degeneration occurs [48]. Follicle culture in ovary organ fragments provides a complex support system that closely resembles the in-vivo
ovary environment. Follicles maintain contact with
the supporting stromal cells, which provide the local
biochemical control pathways that trigger the activation of follicle growth [49]. Eppig and OBriens
method was later improved and translated to follicles from larger animals, such as cow and sheep [15
18]. Recently, Telfer et al. used this method to activate primordial human follicles [13]. Primordial follicles were matured to secondary follicles in organ culture and then cultured individually to the early antral
stage with the treatment of activin. Hence, these experiments motivate the need to integrate stromal cells into
the current follicle culture system in order to achieve
the activation of individual primordial and primary
follicles in vitro.
412
Three-dimensional culture systems for

ovarian follicles
Most follicle culture systems have focused on isolated
secondary/multilayer follicles or oocytegranulosa
cell complexes. Primordial and primary follicles do
not mature when cultured individually in vitro. The
majority of work has been completed using rodents
due to the low cost and relatively short-growth period
compared to larger animals. Follicle culture systems
can be divided into two approaches: two-dimensional
(flat) (Figure 34.2a) and three-dimensional (spherical) (Figure 34.2b). For detailed reviews of follicle culture systems see the following references [4951]. In
two-dimensional follicle culture systems, follicles are
cultured on flat surfaces such as tissue culture plastic
(polystyrene), collagen or polylysine. The first successful culture system using isolated mouse follicles was
developed by Eppig in 1977 [52]. This system was later
improved and achieved the birth of live offspring [14,
53, 54]. While two-dimensional culture systems have
proved to be successful at producing mature oocytes
in mice, this approach has proven difficult to replicate with follicles from large animals and humans. The
unnatural geometry of two-dimensional culture disrupts cellcell communication and causes the granulosa cells to break though the basement membrane,
migrate away from the oocyte and attach to the twodimensional surface [3, 48, 55, 56]. Thus, in order to
culture larger follicles and achieve the goal of maturing human follicles in vitro, three-dimensional culture
systems are necessary to properly support the developing follicle.
Three-dimensional follicle culture systems maintain the natural spherical geometry and cellcell
interactions of the follicle. Three-dimensional culture systems have utilized polylysine or collagencoated substrates [55, 57], hydrophobic membranes
[58], rotating walls and orbiting test tubes [59], mineral oil with daily follicle transfer [60], inverted
culture [61, 62] and serum-free media [13, 63].
These systems have been successful at maintaining
follicle geometry, preventing granulosa cell migration and minimizing attachment to the flat surface.
Nevertheless, these systems still lack the ability to support large follicles for extended culture times. Fortunately, the application of biomaterials to follicle culture offers the potential to overcome these limitations and provide a true three-dimensional culture
environment.
(a)
(b)
Figure 34.2 In-vitro follicle culture systems. (a) In two-dimensional systems, follicles are cultured on flat surfaces such as tissue culture
plastic (polystyrene). The unnatural geometry/mechanics of these systems disrupts cellcell communication and causes the granulosa cells to
break though the basement membrane, migrate away from the oocyte, and attach to the two-dimensional surface. Two-dimensional systems
lack the ability to support large follicles for extended culture times. (b) In three-dimensional systems, follicles are cultured within biomaterial
scaffolds, such as alginate. These systems maintain the natural spherical geometry and cellcell interactions of the follicle. See plate section
for color version.
Three-dimensional biomaterial scaffolds mimic in

vivo cellular microenvironments better than flat twodimensional surfaces [64, 65]. The usage of biomaterial scaffolds has demonstrated the effect of geometry
and mechanics on cell survival, proliferation, migration, gene expression and differentiation. For example, Bissell and coworkers demonstrated that human
mammary epithelial cells display a spread phenotype
when cultured on a two-dimensional surface, yet form
normal acinar structures when cultured in a threedimensional environment [66, 67]. Moreover, Tanaka
et al. found that enhanced chondrogenesis resulted
from the three-dimensional culture of embryonic stem
cells compared to flat monolayer culture [68]. In addition to geometry, mechanics has a substantial effect on
cell behavior. For example, Engler et al. identified that
stem cell lineage was controlled, in part, by scaffold
elasticity [69]. Mesenchymal stem cells were directed
to neurogenic, myogenic and osteogenic lineages using
soft, stiff and rigid matrices. Hence, these studies have
established the need to provide the proper environment for cell culture. This task has been accomplished
via the use of biomaterials that permit the modification
of physical properties. Thus, biomaterials have provided an enabling tool to mimic in vivo environments.
Highly water-soluble polymer networks, called
hydrogels, have been used as biomaterial scaffolds
for follicle culture. Hydrogels support natural follicle
growth by mimicking the natural stromal microenvironment of the ovary. Early hydrogels for follicle cul-
ture employed collagen [7074], which is an extracellular matrix protein that is prominent throughout the ovary [75]. Torrance et al. demonstrated the
growth and survival of primary mouse follicles in collagen hydrogels, but did not achieve antrum formation [71]. Later, Hirao et al. produced mature oocytes
from pre-antral pig follicles [74], and Sharma et al.
achieved antral follicles from pre-antral buffalo follicles [72]. In addition to collagen, alginate, a common tissue engineering hydrogel, has shown great
promise as a scaffold for follicle culture [1, 36, 12,
7678]. It is a naturally derived polysaccharide isolated from brown algae. Alginate is a block copolymer
composed of blocks of (14)-linked -D-mannuronic
acid (M units) and its C-5 epimer -L-guluronic acid
(G units) [79]. These blocks can be either similar or
alternating. Alginate chains can be crosslinked with
divalent cations, such as Ca2+ . As a result, alginate
avoids the use of harmful chemicals, ultraviolet light
or temperatures to crosslink the polymer network
[50]. Crosslinking occurs via interaction of the carboxylic acid functional groups in the G-blocks. This
crosslinking leads to the formation of a gel network
while retaining cell viability and the cellular interactions within the follicle. Due to its hydrophilic
nature, alginate discourages protein adsorption and
cell attachment. Thus, cells are unable to specifically
interact or bind with alginate. In order to overcome
this limitation, alginate can be covalently linked to
cell-adhesion molecules, such as RGD (Arg-Gly-Asp)
413
Figure 34.3 Stromal cell co-culture

approaches. (a) Stromal cells can be
cultured separately on a flat surface below
the encapsulated follicle. This set up
allows for paracrine signaling between
the follicle and the stromal cells. (b)
Stromal cells can be encapsulated inside
the biomaterial scaffold with the follicle.
This set up allows for paracrine signaling
as well as cellcell attachment and
interaction with secreted extracellular
matrix proteins. See plate section for color
version.
(a)
(b)
peptide ligands, via its carboxylic acid functional

group and carbodiimide chemistry [80]. In addition,
alginate can be degraded with alginate lyase, an alginate specific enzyme, in order to safely remove the follicle from the hydrogel. Hence, due to its inert nature,
gentle gelatin and modularity, alginate was a top candidate for follicle culture.
Alginate is a successful encapsulating matrix for
follicle culture. Experimentally, isolated follicles are
suspended within drops of alginate, which are subsequently crosslinked in a Ca2+ solution. Each resulting alginate bead is then transferred to an individual well in a standard cell culture plate containing
growth media. Alginate hydrogels have been shown
to maintain the natural three-dimensional morphology of the developing follicle [3]. This culture system has demonstrated the successful maturation of
secondary to antral follicles with mice, primate and
human follicles [1, 46, 12, 7678]. Follicles grow,
develop antrums and produce meiotically competent
oocytes. These oocytes have been successfully fertilized in vitro and implanted into mice, which has
yielded healthy, fertile offspring [76]. Follicle growth
was found to be a strongest function of alginate concentration or matrix mechanical properties. Permissive environments (low concentrations of alginate)
supported the most rapid follicle growth and highest
rates of fertilization [4, 5, 50]. In addition, the incorporation of extracellular matrix (ECM) proteins, such
as collagen, fibronectin and laminin, into the alginate
matrix has been found to improve follicle growth and
oocyte quality [3]. This culture system has also been
used in combination with ovary organ culture to produce mature oocytes from primordial follicles [15].
Furthermore, alginate has been used to culture human
414
follicles after cryopreservation [81]. Hence, alginate

has proven to be a powerful tool for the advancement
of follicle culture.
Additional functionalities are being incorporated
into the alginate system to enhance follicle development. A fibrin-alginate interpenetrating network
(FA-IPN) was developed to provide dynamic cellresponsive mechanical properties to the culture system [77]. Fibrin, the natural polymer involved in blood
clotting, and alginate are formulated simultaneously
into a single hydrogel. As the follicle grows, proteases
are secreted to degrade the fibrin, leaving only the
non-degradable alginate matrix to support the follicle. Fibrin alone was not successful in promoting follicle development. This FA combination produces a
more permissive environment than can be achieved
with alginate alone. The rate of meiotically competent oocytes produced was 82%, which is significantly
higher than alginate alone or any other reported invitro culture system. By promoting interaction with
the encapsulating matrix, degradable hydrogels better mimic the natural ovary microenvironment. Future
follicle culture systems will build upon this example in
order to further improve oocyte quality and survival,
which is essential for the complete translation to primate and human follicles.
Utility of co-culture for ovarian

follicle development
The next logical step in the development of follicle culture systems is the integration or co-culture
of stromal cells (Figure 34.3). As demonstrated via
stromal cell and ovary organ culture experiments,
stromal cells have a significant role in the activation of primordial/primary follicles and theca cell
recruitment/differentiation. The paracrine signaling
involved in these processes is hypothesized to be a
complex time-dependent synergy of unidentified factors [20]. Cellcell contact could also have an important role in these processes. Accordingly, the simple
addition of candidate hormones and growth factors
to the culture media has not yet achieved success.
Until the mechanisms of these processes is elucidated,
follicle culture systems could attempt to incorporate
stromal cells in order to potentially culture smaller
follicles and improve growth, survival and oocyte
quality.
While only a few co-culture studies with stromal
cells have been conducted, these experiments demonstrate positive results and motivate further investigation. Osborn et al. showed that the presence of stromal cells around isolated primordial follicles improved
initial culture success [82]. Building upon this observation, Itoh and Hoshi co-cultured small pre-antral
(primary and secondary) bovine follicles with ovarian mesenchymal cells, granulosa cells and skin fibroblasts for 30 days [83]. Compared to the non-coculture controls, follicle viability was significantly
increased in all three co-cultures (18.6, 17.1 and 10.0%,
respectively) and follicle growth was significantly
increased in the mesenchymal (15.4%) and fibroblast
(30.0%) co-cultures. In a similar fashion, Wu et al. cocultured pre-antral pig follicles with different follicular cells [84]. In contrast to Itoh and Hoshis results,
Wu et al. found that small pre-antral follicle growth
and survival was inhibited by co-culture with multiple follicles (with or without oocytes). The growth
and survival of these follicles was only enhanced
when co-cultured with cumulus cells from antral follicles 3 mm in diameter. Moreover, Ramesh et al.
co-cultured buffalo pre-antral follicles with different
somatic cells (cumulus, granulosa, mesenchymal and
epithelial) [85]. Co-culture with cumulus, granulosa
and mesenchymal cells resulted in better development,
growth rate and survival than the control and epithelial cells. Maximum growth and survival was achieved
via co-culture with cumulus cells, which supports Wus
results. Therefore, these studies clearly demonstrate
the utility and effect of co-culture. Nevertheless, these
co-culture experiments have not yet been conducted in
the current state-of-art three-dimensional culture systems with optimized culture media. Hence, the impact
of co-culture could be significantly enhanced by inte-
grating stromal cells into current three-dimensional

culture systems.
The integration of stromal cells into threedimensional culture systems is possible, but will
require some adaptations of the culture system. The
challenge will be to develop a hydrogel matrix that
promotes stromal cell survival within the constraints
of follicle growth. Unfortunately, stromal cells cannot
be added directly to the alginate follicle culture
system without considerable modifications. Alginate
does not support cell attachment. However, this can
be accomplished by covalently linking alginate to
full extracellular matrix proteins, such as collagen,
fibronectin and laminin or small peptides sequences
from these proteins. Without these attachment sites
the cells will undergo apoptosis or cell death. Alternatively, other biomaterials such as collagen, Matrigel
or polyethylene glycol (PEG) could be employed for
co-culture. Unquestionably, the modifications needed
for stromal cell culture will also influence follicle
growth. Hence, it will be difficult to find overlapping
culture conditions for both stromal cells and follicles.
For example, the concentration of stromal cells must
be fine-tuned to allow adequate paracrine signaling
while not starving the follicle of oxygen or nutrients.
Thus, developing a three-dimensional co-culture
system will be challenging, but it is definitely an
obtainable goal.
Examples of co-culture success in

tissue engineering
In considering the utility of co-culture for ovarian follicle development, we considered the success that has
been achieved for other biological systems. Perhaps the
foremost example of co-culture success in tissue engineering has been the development of an artificial bladder by Atala et al. [86]. Urothelial and muscle cells were
grown in culture and then seeded on a biodegradable
bladder-shaped scaffold made of collagen. The exterior surface of the scaffold was seeded with the smooth
muscle cells and the interior was seeded with urothelial cells. These artificial bladders were implanted into
seven patients with myelomeningocele (spina bifida)
and either high blood pressure or poorly compliant bladders. After surgery, bladder biopsies showed
an adequate structural architecture and phenotype.
These implants have been shown to be functional and
durable over a period of years [86]. This example
demonstrates that co-culturing multiple cell types via
415
biomaterial scaffolds is an effective strategy to recapitulate natural cellular environments and restore cell
cell communication.
Co-culture has also been successfully applied to
numerous cell types including skin, cartilage, bone,
liver, blood, nerve and stem cells [8791]. These experiments have demonstrated that co-culture can promote or inhibit differentiation of one or both cell
types. No correlation has yet been found between coculture methods, cell types or differentiation. Nevertheless, co-culture has proven to be effective at
improving the survival, proliferation and function of
cells. For example, Houchin-Ray et al. developed a
co-culture model consisting of primary neurons and
accessory cells in order to promote and direct neurite outgrowth [92, 93]. Co-culture promoted neuron survival and neurite extension. With respect to
reproductive technology, co-culture has been applied
to in vitro maturation (IVM) and in vitro fertilization (IVF). Oocytes and developing embryos have
been co-cultured with various feeder cells including oviduct endothelial, endometrial epithelial, fallopian tube ampullary, cumulus, granulosa and nonhuman cells such as the Vero cell line. While controversial, these experiments suggest that co-culture
may improve IVM and IVF results [9497]. Moreover,
the undifferentiated state of embryonic stem cells has
been maintained via co-culture with feeder cells such
as fibroblasts [98, 99]. In sum, these examples have
demonstrated the utility of co-culture, which potentially could have similar impacts in follicle culture.
Conclusion
The co-culture of ovarian stromal cells and follicles is
another step in the evolution of three-dimensional follicle culture systems. Stromal cell and ovary organ culture experiments have established the significant role
of stromal cells in primordial/primary follicle activation and theca cell recruitment/differentiation. Hence,
co-culture has the potential to activate individual primordial/primary follicles, which do not mature in
any in-vitro culture system and improve growth, survival and oocyte quality. Current follicle culture systems that utilize biomaterials, such as alginate, provide
three-dimensional scaffolds that maintain the natural
geometry of the follicle and could enable the translation to follicles of larger animals and humans. These
culture systems allows for the straightforward integration of stromal cells, which will help recapitulate the
416
natural ovarian environment. Accomplishing this task

will ensure that follicle culture systems continue to
advance the study of folliculogenesis and the development of fertility preservation techniques.
Acknowledgements
This research was supported by grants from the
National Institutes of Health (NIH) to the Oncofertility Consortium (Grant Numbers: RL1HD058295,
PL1EB008542) and the Eunice Kennedy Shriver
National Institute of Child Health and Human Development (NICHD) through cooperative agreement as
part of the Specialized Cooperative Center Program in
Reproduction and Infertility Research (Grant Number
U54HD41857). The content of this research is solely
the responsibility of the authors and does not necessarily represent the official views of the NIH or NICHD.
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Section 8
Chapter
35
In vitro maturation of GV oocytes

M. De Vos and J. Smitz
Introduction
Success rates of assisted reproductive technology
(ART) have tremendously improved over the past
three decades. Nevertheless, controlled ovarian stimulation protocols are still associated with a substantial
risk of ovarian hyperstimulation syndrome (OHSS)
which, in its severe form, may result in significant
morbidity and even mortality [1]. The overall incidence of severe OHSS is 2% [2], but in women
with polycystic ovary syndrome (PCOS), who represent up to 30% of women eligible for ART [3], the
risk of OHSS is higher. These patients are therefore at
increased risk of having the embryo transfer cancelled.
Strategies to prevent OHSS in patients with a high
response to gonadotropins include the administration
of a gonadotropin-releasing hormone (GnRH) agonist
instead of human chorionic gonadotropin (hCG) to
induce the final oocyte maturation [4] and in vitro
maturation (IVM) of oocytes. In vitro maturation
avoids the risks and side effects of conventional ovarian hormonal stimulation because it involves retrieving immature oocytes from unstimulated or minimally
stimulated ovaries. After immature oocyte collection,
these oocytes are cultured, matured and fertilized in
vitro. Selected embryos are then transferred to an
adequately primed endometrium or, alternatively, are
cryopreserved and subsequently thawed or warmed in
a natural or artificial cycle.
The first successful pregnancy and birth from IVM
in human was described more than 15 years ago. In
spite of technical advances since then to the IVM protocol and improvements of the maturation method
and culture media, implantation rates do not exceed
15%, they rather stagger at 10%. This often necessitates the transfer of more than one embryo, which
in young women may increase the risk of multiple
pregnancies. It also interferes with law restrictions in

a number of countries where multiple embryo transfer precludes ART cost reimbursement by the government. Lower pregnancy rates in comparison with conventional ART, as well as concerns about the genetic
health of IVM oocytes [5] and the long-term health
of embryos, fetuses and children born after IVM,
still preclude a more general acceptance of IVM in
ART centers. The causes and mechanisms of the lower
implantation rates of IVM-derived embryos are still
largely unknown, although they are almost certainly
linked to a lack of complete cytoplasmic maturation
[6] and to a suboptimal endometrial receptivity associated with IVM cycles. There is a need to optimize
IVM culture media [7], to standardize the IVM treatment protocol with regard to the use of estradiol
and/or gonadotropins to prime the follicles and the
endometrium [8], the aspiration technique and the
IVM timings [9].
There is evidence that oocyte maturation, fertilization rates and blastocyst production are compromised when compared with in-vivo matured oocytes
[6], possibly because of a dissynchronous nuclear
and cytoplasmic maturation. Promising technologies
have emerged in recent years to improve maturation rates and developmental competence of resulting embryos, based on methods that can compensate for the reduced maturation time (2436 h)
that oocytes undergo spontaneously when aspirated
out of follicles, compared to the situation where
they would remain within the ovary [10]. Recent
IVM culture system modifications include substances
that increase the cyclic adenosine monophosphate
(cAMP) levels in the oocyte environment, which
allows for a more physiological cascade of IVM triggering [11].
421
This chapter will summarize the scope of IVM as a

stand-alone ART and aims to contribute to the discussion of how IVM can become a valuable alternative to
conventional ovarian stimulation.
The emergence of IVM

The primordial follicle pool of the ovaries harbors
oocytes that are arrested at prophase stage I of meiosis. The majority of these follicles become apoptotic;
only a few follicles grow beyond the antral stage, at
which time the oocyte continues to mature until ovulation. The molecular events that drive and regulate
the process of oocyte maturation are not fully understood, although follicle stimulating hormone (FSH) is
thought to play a major role in influencing the maturation process. After selection of the dominant follicle,
this follicle becomes FSH dependent. The increasing
estrogen levels from the pre-ovulatory follicle induce a
luteinizing hormone (LH) rise, which in turn induces a
cascade of secondary factors synthetized in the mural
follicle cells that lead to breakdown of the germinal
vesicle (GV) in the oocyte [12]. The oocyte proceeds
from meiotic metaphase I to telophase I, which is associated with extrusion of the first polar body. The full
meiotic progress does not only occur after the LH
surge, but also when the oocytes become detached
from their follicular environment. Historical observations by Pincus and Enzmann in 1935 showed that
immature oocytes have the ability to resume meiosis
spontaneously when they are removed from the follicle [13]. Edwards et al. confirmed this concept by
showing that that they reach metaphase I approximately 2835 h after being released from the follicle,
that they extrude the first polar body after being in culture between 36 and 43 h and that they can be fertilized
[14].
In vitro maturaton has a number of important
advantages over in vitro fertilization (IVF). Ovarian hormonal stimulation is either limited or even
absent, which eliminates the risk of OHSS. Furthermore, the immediate side effects of the ovarian
stimulation drugs (i.e. nausea, vomiting, breast tenderness, abdominal swelling and mood swings) are
avoided. With IVM there is no risk of premature
luteinization and therefore the IVM protocol requires
less meticulous follow-up and reduces the number
of visits during the treatment cycle. Finally, IVM is
more affordable for the patients because the incurred
medication costs are significantly reduced. All of
422
the above make IVM a patient friendly protocol

in ART.
Cha et al. obtained the first IVM pregnancy in
1991 [15]. They had used the immature human oocytes
retrieved during gynecological operations in an oocyte
donation program. The first IVM pregnancy with a
patients own oocytes was achieved in 1994 and was
obtained by Trounson et al. [16]. However, pregnancy
rates after IVM in those years were disappointingly
low, until Chian et al. introduced hCG priming for
IVM in PCOS patients, 36 h prior to the oocyte
retrieval [17]. They reported implantation rates of 32%
and clinical pregnancy rates of 40%, which marked
an important step forward [17]. During the following
years the same group achieved clinical pregnancy rates
of up to 54% after IVM in unstimulated cycles [18].
However, such high pregnancy rates were obtained
after the transfer of 34 embryos irrespective of the age
of the patient.
In-vitro maturation pregnancy rates have consistently been lower than IVF pregnancy rates after conventional ovarian stimulation [19]. The reduced success rates have been attributed to the asynchrony in
the cytoplasmic and nuclear maturation of the oocyte
as well as to an inferior endometrial thickness and
receptivity [20]. Also, the final number of matured
oocytes obtained in unstimulated cycles followed by
IVM is relatively low as compared to the number of
mature oocytes obtained in conventional stimulated
cycles. Therefore, increasing the pregnancy and the live
birth rates in IVM treatment has become a major focus
of clinical and scientific research. A number of studies have focused on improving in-vitro culture media,
whereas other studies have been designed to improve
the quality and quantity of oocytes and the quality of
the endometrium.
Whereas in the early years IVM was advocated
as a particularly suitable technique for young women
with a high antral follicle count, current and future
improvements to the technology should contribute
to its use in a wider scope of applications, including
women with poor ovarian reserve, oocyte donors and
fertility preservation medicine.
Technical aspects of IVM

In patients with PCOS, a course of oral or vaginal progesterone to induce a withdrawal bleed is often prescribed [21]. However, whether this practice is truly
necessary prior to the start of an IVM cycle remains
Chapter 35: In vitro maturation of GV oocytes
to be verified. After a baseline ultrasound scan to

rule out the presence of cysts or other pathology,
serial ultrasound scans are scheduled to assess the
growth of the antral follicles and the thickness of the
endometrium. Once the endometrial thickness has
reached a minimum of 6 mm and the largest follicle is
1012 mm, most centers administer hCG. The timing
of egg retrieval has been shown to be important, as egg
retrieval 38 h after hCG trigger instead of 35 h appears
to yield an improved maturation rate in unstimulated
cycles [22].
There have been multiple published reports with
regard to the selection of the optimal day for egg
retrieval based on the follicular diameter. When the
leading follicle is larger than 13 mm, it has been
demonstrated that fewer oocytes are retrieved, the fertilization rate is lower and fewer embryos are generated
[23, 24], at least in unstimulated ovaries. On a similar note, Cobo et al. achieved higher rates of development to the blastocyst stage when the dominant follicle
is smaller than 10 mm [25]. These reports may suggest
the existence of a so-called dominant negative effect, in
that large follicles may compromise the developmental
potential of the oocytes in the smaller antral follicles,
although this has not been confirmed by some authors
[26, 27].
There is published evidence that the embryological and clinical outcome is improved in hCG primed
IVM cycles with in vivo matured oocyte(s) as compared to cycles with only immature oocytes at pickup [28, 29]. Retrieval of at least one in-vivo matured
oocyte is more likely in those cycles where the leading follicle has reached a diameter of 1012 mm before
oocyte retrieval.
How to improve implantation

rates in IVM?
Improving in-vitro culture techniques
The nuclear maturation through meiosis I and II is a
prerequisite for successful oocyte maturation. Cytoplasmic maturation is equally important and includes
relocation of organelles, synthesis and modification
of proteins and mRNAs, and regulation of biochemical processes that support subsequent fertilization and
embryonic development [30]. Regulation of oocyte
maturation in vivo involves complex signaling pathways that occur in the microenvironment of the maturing oocyte. The oocyte and cumulus cells communi-
cate through gap junctions [18] that allow passage of

regulatory molecules and growth factors. The oocyte
is in meiotic arrest until meiotic progress is triggered.
In vivo, maturation is triggered by the endogenous LH
surge and mediated by growth factors, such as epidermal growth factor (EGF) family members amphiregulin, epiregulin and beta-cellulin [12]. In vitro, oocyte
maturation occurs spontaneously when the oocyte is
removed from the follicular environment that inhibits
meiotic progression [31]. When immature oocytes are
removed from small antral follicles, meiotic resumption will occur precociously, i.e. before completion
of cytoplasmic maturation. Therefore, the timing of
resumption of meiosis is important in oocyte maturation. To solve this problem for IVM systems, some
authors suggest delaying spontaneous nuclear maturation while promoting development of the cytoplasm
at the same time [30]. The intracellular messenger
molecule cAMP plays a significant role in the regulation of mammalian oocyte maturation [32]. High
levels of cAMP and cAMP analogues prevent meiotic resumption [33]. Spontaneous oocyte maturation
in vitro can be inhibited or delayed by increasing
the cAMP level within the cumulusoocyte complex
(COC) environment by adding any of the following substances to the media: (1) cAMP analogues
such as dibutyryl cAMP; (2) activators of adenylate
cyclase, such as FSH, forskolin or invasive adenylate
cyclise; and (3) phosphodieserase (PDE) inhibitors,
such as the non-specific inhibitor IBMX, the PDE
type 4-specific inhibitor rolipram or the PDE type 3specific inhibitors milrinone, cilostamide or Org9935
[33]. These agents delay GV breakdown and simultaneously increase the extent and prolong the duration of oocytecumulus cell (CC) gap-junction communication during the meiotic resumption phase
[3335], which in turn extends the exchange of regulatory factors and metabolites between the oocyte
and the cumulus cells [36]. Although treating oocytes
in vitro with the addition of these cAMP elevating
agents prevents meiotic resumption of mouse [32] and
human oocytes [37], a beneficial effect on subsequent
oocyte developmental potential in human and ensuing pregnancy rates with any of these agents has not
yet been demonstrated. Recent controlled prospective
studies in the bovine, porcine and mouse model have
shown a clear improvement on blastocyst development
rate (porcine, bovine) and implantation rates (mouse)
in comparison to the conventional methods for doing
IVM [11, 34, 38, 39].
423
Also, premature interruption of the gap-junction

communication between the cumulus cells and the
oocyte may compromise the developmental competence of the oocyte. Although IVM rate and progression to the embryonic cleavage stage may not be
affected by absence of cumulus cells during maturation, the development to the blastocyst stage is significantly reduced in cumulus-free oocytes as compared
to cumulus-intact oocytes [40].
Hormonal priming
The introduction of hCG administration prior to
immature oocyte retrieval significantly improved maturation, fertilization and pregnancy rates [17, 41, 42],
although some authors did not find a beneficial effect
of hCG priming with regard to the number of oocytes
retrieved, maturation, fertilization or cleavage rates
[33]. The exact mechanism by which hCG exerts an
effect on small follicles is still unclear. At least there
may be a technical advantage to its administration,
in that the CC expansion of the oocytes secondary to
hCG may facilitate detachment of the COC from the
follicle during the aspiration. This in turn may improve
the oocyte yield in hCG-primed IVM cycles compared
to non-hCG primed IVM cycles. Priming with hCG
may also increase the likelihood to obtain some in-vivo
matured oocytes at retrieval, which is associated with
improved blastocyst development rates, and explain
the better implantation rates and clinical pregnancy
rates.
FSH priming
Compared to the use of hCG, the value of FSH priming in IVM cycles is more controversial. Follicle stimulating hormone (FSH) priming would lead to a technically easier oocyte retrieval, higher E2 levels and
improved maturational competence of the oocytes, as
well as improved endometrial priming [43].
In normo-ovulatory patients, Wynn et al. showed
that administration of FSH during the early follicular
phase is associated with a higher oocyte maturation
rate [43], whereas others reported that FSH priming
had no apparent effect on oocyte developmental competence [44, 45]. Suikkari et al. investigated the influence of low dose FSH priming in normo-ovulatory
women starting from the late luteal phase and came to
similar conclusions [46].
Conflicting results were reached in women
with anovulatory cycles; Mikkelsen and Lindenberg
424
demonstrated improved implantation and clinical pregnancy rates in FSH-primed cycles without
additional hCG administration [47], whereas Lin
et al., who prospectively compared FSH-priming
with hCG administration versus hCG alone, found
no additional benefit of FSH-priming in those cases
where FSH and hCG were both administered [48]. In
a large randomized study encompassing 400 women,
Fadini et al. demonstrated that FSH-priming with
150 IU/day FSH for 3 days from day 3 of the cycle
plus hCG priming led to significantly improved
maturation rates, implantation rates and clinical
pregnancy rates, whereas FSH priming and hCG
priming alone showed no significant effects on clinical
outcome [49].
Preparation of the endometrium

One of the major reasons why implantation rates
have remained low compared with conventional IVF
treatment is the insufficient development of the
endometrium during IVM cycles before embryo transfer [23]. In conventional IVF cycles, the endogenous
production of estradiol by the ovaries secondary to
exogenous gonadotropin stimulation is much higher
and allows for adequate endometrial proliferation
on the condition that progesterone supplementation
is provided in the luteal phase [50, 51]. But in
endometrium preparation for IVM there is a need
for estrogen supplementation to improve endometrial thickness, because of the shortened follicular
phase of IVM cycles. As a result of the dissynchrony
between the endometrial development and the embryonic development in IVM cycles, some centers prefer to freeze the embryos and to perform a thawed
embryo transfer in an artificial or a natural cycle [52].
In IVM cycles with a thin endometrium, a recent retrospective study showed that both low dose human
menopausal gonadotropin (HMG) and micronized
17-estradiol supplementation significantly improve
endometrial thickness, and that low dose hMG results
in larger follicles and a greater number of in-vivo
matured oocytes [53]. Nevertheless, there is a need
for adequately powered prospective studies to investigate the optimal protocol for endometrial priming in
IVM cycles and to correlate endometrial thickness in
these cycles with endometrial receptivity at the time
of embryo transfer, in normo-ovulatory women and in
women with PCOS.
Metformin
Metformin improves insulin sensitivity, lowers serum
LH, total and free testosterone concentrations, and
causes an elevation in serum FSH and sex-hormone
binding globulin levels in obese women with PCOS
[54]. Significantly higher implantation and clinical
pregnancy rates were obtained in a series of 56
metformin pre-treated clomiphene-resistant PCOS
patients undergoing IVM compared to the controls
[55], suggesting that metformin may improve IVM
outcome through optimization of the oocyte microenvironment, although the mechanism by which this
occurs needs to be elucidated.
Patient selection for IVM

A high yield of oocytes is a strong predictor of pregnancy following IVM treatment [56]. Although there
is sufficient published evidence regarding the value
of ovarian reserve tests (ORTs), including antral follicle count, anti-Mullerian hormone (AMH) and earlyfollicular-phase blood levels, in predicting poor ovarian response [57], the potential of these tests to predict
a high response to ovarian stimulation is only modest.
Women with PCOS or PCOS-like ovaries and those
with a high antral follicle count (AFC) are undoubtedly
the most suitable candidates to undergo IVM treatment [21]. The number of immature oocytes retrieved
at egg collection is correlated with the AFC, the ovarian volume and the peak ovarian stromal velocity measured by Doppler ultrasound during the early follicular phase [56]. In view of the correlation between AFC
and age [58], a low AFC, such as expected in the older
age group, will be associated with lower implantation
and clinical pregnancy rates, although advancing age
itself also contributes to poorer oocyte quality. Therefore, the majority of IVM pregnancies in the literature
have been reported in women with PCOS or PCOSlike ovaries and in women below 35 years of age [59].
In selected oocyte donors with high AFC, IVM is a
promising alternative to a conventionally stimulated
cycle, particularly in those donors who have concerns
about short-term side effects of the ovarian stimulation
or long-term health implications of the stimulation
hormones. Furthermore, endometrial quality is not an
issue in oocyte donors. Holzer et al. reported a clinical
pregnancy rate of 50% (6/12) in an IVM oocyte donation program where the donors had PCOS or PCOSlike ovaries [60].
In normo-ovulatory women, natural-cycle IVF

combined with immature oocyte retrieval followed
by IVM can also lead to reasonable pregnancy and
implantation rates. In a recently published series of
151 treatment cycles of natural cycle IVF, combined
by IVM, a clinical pregnancy rate of 40.4% has been
described [61].
Finally, IVM and oocyte or embryo cryopreservation have been presented as an attractive method
of fertility preservation for cancer patients who need
imminent cytotoxic treatment and do not have sufficient time to undergo conventional ovarian stimulation. Retrieval of immature oocytes from excised ovarian tissue was first reported by Revel et al. [62]. In a
recently published series of four consecutive patients
who underwent retrieval of immature oocytes from
the antral follicles of the excised ovarian tissue, eight
mature oocytes were eventually vitrified [63]. In cases
of important time pressure before the start of gonadotoxic treatment and when the patient declines ovarian tissue cryopreservation, egg collection followed by
IVM can also be applied during the luteal phase of
a natural cycle [64]. Patients with hormone sensitive
tumors are also excellent candidates for IVM. In a
series of 20 women who underwent ICSI of vitrified
thawed in-vitro matured oocytes, a 20% pregnancy
rate was reported [65].
IVM as a rescue in case

of threatening OHSS
When a high risk of OHSS emerges during conventional ovarian stimulation, the treatment cycle can be
rescued by final oocyte maturation triggering occuring with a GnRH agonist, as opposed to triggering
with hCG. This rescue mechanism was first reported
in 2000 by Itskovitz-Eldor et al. [66], but had disappointing reproductive outcomes due to suboptimal
luteal phase support. It was also known that without an hCG injection the oocyte recovery rate from
follicles 12 mm diameter is suboptimal in comparison to retrievals from small and medium-size
follicles.
Using adapted regimens of luteal support, Engmann et al. and Humaidan et al. independently
reported the avoidance of OHSS while comparably good pregnancy rates were achieved [4, 67]. As
an alternative, cancellation of further ovarian stimulation can be proposed, followed by retrieval of
the immature oocytes, IVM, subsequent fertilization
425
by ICSI and then embryo transfer. However, immature oocytes obtained from conventionally stimulated
ovaries have a lower fertilization rate [68], and the
resulting embryos have high incidence of non-cleavage
and chromosomal anomalies [69]. These observations
appear to indicate that oocytes that remain at the
GV stage after conventional ovarian stimulation are
of inferior quality or have a rare intrinsic follicular or
oocyte-specific defect.
a long-term mouse follicle culture model the primary

imprinting pattern for Snrpn, Peg3, H19 and Mest was
completely normal [74, 75, 76]. More research needs
to be done in animal models and in donated human
oocytes to evaluate further, on a larger number of
oocytes, what are the real risks of superovulation and
IVM on the acquisition of imprints in growing oocytes
and on the maternal-effect gene products subsequently
required for imprinting maintainance.
IVM outcome
IVM and fertility preservation
As compared to conventional ART protocols, IVM

is patient-friendly and IVM success rates have been
increasing in the past decade. In general, implantation rates of 1015% and clinical pregnancy rates of
3035% have been reported in women with PCOS and
PCOS-like ovaries, although usually three times more
embryos are transferred than in the conventional ART
setting.
All ART pregnancies are associated with an
increased risk of multiple pregnancy, cesarean delivery
and congenital abnormality. Babies born to date (presumably 15002000 children) following IVM treatment showed no increased risk of congenital abnormality or adverse perinatal outcome over that already
accepted for IVF or intracytoplasmic sperm injection (ICSI) [70]. Nevertheless, a higher rate of clinical miscarriage has been observed following IVM
as compared to IVF and ICSI. This appears to be
related to the patient characteristics of the treatment
group, i.e. patients with PCOS, rather than to the IVM
procedure itself since the miscarriage rates following IVM and IVF/ICSI were comparable in patients
with PCOS [70]. There is currently no strong evidence that IVM technology might have a higher risk
to the development of epigenetic pathologies, as the
GV oocytes studied were left-overs from normal
superovulated patients [71, 72]. These oocytes were
arrested in meiosis despite a normal FSH and hCG
stimulation and might have been intrinsically compromised [69]. To the contrary, recent experiments in
mouse models have demonstrated that in vivo superovulation with a higher dose of pregnant mares serum
gonadotropin (PMSG) induced methylation errors in
some imprinted genes [73]. Hence, absent or minimal
ovasian stimulation in an IVM procedure might obviate the increased risk of epigenetic abnormalities, as
seen with high doses of gonadotropins. As for the risk
of in vitro culture per se, it was demonstrated that in
Advances in oncological treatments have significantly

improved the survival rates for different malignant
tumors, including hematological pathologies, rectum and colon cancer and early-onset breast cancer. Potentially gonadotoxic chemotherapeutic agents
have largely contributed to this progress, although
this evolution has also led to a significant increase
of the incidence of premature ovarian insufficiency.
The development of oocyte vitrification as a cryopreservation method [77], has offered new perspectives for the application of IVM in fertility preservation medicine and is now considered as a valuable alternative for ovarian tissue cryopreservation.
Since IVM obviates the need for ovarian stimulation, the technique may be especially suitable for
patients with estrogen receptor-positive tumors. Chian
et al. described the first successful live birth following immature oocyte retrieval in a natural menstrual
cycle, IVM and vitrification of the IVM oocytes [78],
and outcomes of pregnancies achieved with vitrified
warmed oocytes after IVM treatment appear to be
favorable [79]. However, larger patient numbers are
needed to investigate the risks associated with vitrification of in-vitro matured oocytes. Although in a recent
study no difference was found between the fertilization
and cleavage rates when oocytes were vitrified at the
immature GV stage as opposed the mature MII stage,
embryos derived from oocytes that had been vitrified
at the immature GV stage are of poorer quality and
development to the blastocyst stage appear to be compromised [80].
Finally, the rapid developments in the field of IVM
of oocytes should ultimately be adapted to multi-step
in vitro follicle culture after ovarian tissue cryopreservation, which, when applied as a fertility preservation
strategy, can provide a maximal source of oocytes in
female cancer patients and in women with early-onset
incipient ovarian failure [81].
426
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Section 8
Chapter
36
Clinical potential of in vitro maturation

Baris Ata, Einat Shalom-Paz, Srinivasan Krishnamurthy,
Ri-Cheng Chian and Seang Lin Tan
Introduction
Over the last three decades there has been a significant delay in starting a family in modern Western
societies [1]. The most likely reason of this deliberate
action is extended life expectancy combined with everincreasing involvement of women in education and
the workforce. However, while men are able to reproduce until much later in life, female fertility declines
with increasing age [2, 3]. The current reproductive
paradigm suggests women are born with a finite quantity of oocytes and their reproductive potential usually does not last much beyond the end of the fourth
decade of life. This is a worrisome fact for many modern women deferring child bearing until their late thirties or even forties. In the year 2007, decreased ovarian
reserve was the sole indication for 14% of all reported
assisted reproductive technology (ART) cycles in the
USA [4]. Moreover, the female partner was older than
35 years of age in 71% of all ART cycles performed in
the same year [4].
The natural decline in fertility is markedly accelerated following gonadotoxic therapy for cancer and
other non-oncological conditions. Cancer continues
to be a major health problem despite advances in its
diagnosis and treatment. It is estimated that in 2009
approximately 713 220 women in the USA will be
diagnosed with cancer [5]. Survival rates have enjoyed
a stable increase over the last decades leading to an
increase in the number of cancer survivors every year
[5]. Similar advancements occurred in the treatment
of childhood cancers, and it is estimated that one in
every 250 adults will be a childhood cancer survivor
by the year 2010 [6]. Eventually, a growing number of
female cancer survivors are faced with the risk of infer-
tility resulting from gonadotoxic oncological treatment. Patients who are exposed to gonadotoxic agents
for the treatment of non-oncological diseases, such as
systemic lupus erythematosus, those who are undergoing surgery for endometriosis as well as women
with genetic disorders such as Turners syndrome and
fragile-X premutation face similar risks, further contributing to the population of women who need fertility preservation procedures [710].
Relatively recent advances in the field of reproductive medicine, particularly in cryopreservation methods, have rendered fertility preservation a realistic
option for such unfortunate women. With an increased
awareness of the options available for fertility preservation, a greater number of women are being offered
and are utilizing these technologies. Currently available options for preservation of female fertility are
cryopreservation of oocytes or embryos following in
vitro fertilization (IVF) or in vitro maturation (IVM)
or cryopreservation of ovarian tissue. Although successful transplantation of fresh whole ovary has been
reported in a pair of monozygotic twins discordant
for ovarian failure and encouraging results have been
achieved with frozen ovaries in animal models, cryopreservation of entire ovary has not been successfully performed in humans [11, 12]. Administration of gonadotropin-releasing hormone analogues or
inhibitors of apoptosis have been proposed as alternative strategies for patients undergoing chemotherapy;
however, effectiveness of these techniques remains to
be proved [13]. The most appropriate method for any
woman is determined by several factors including the
indication for fertility preservation, availability of a
male partner and patient preference.
431
Section 8: In-vitro follicle growth and maturation
IVM and its contribution to

While IVF involves collection and in vitro fertilization of multiple in-vivo matured oocytes collected
at the metaphase-II stage (MII), the IVM technique
aims to make use of the multiple immature oocytes
that already exist in the ovaries of a reproductiveaged woman. Early in the follicular phase, these immature oocytes are harbored in smaller-sized antral follicles, and are arrested at the prophase stage of meiotic division. These oocytes are shown to resume meiosis upon removal from the follicle, have the capacity to complete meiotic division and can be fertilized
in vitro. More than 80% of oocytes were reported to
resume meiosis independent of the menstrual cycle
day and gonadotropin support in IVM medium [14].
In fact, the first pregnancy and live birth from in-vitro
matured oocytes in humans was reported in 1983 in
the context of a stimulated IVF cycle [15]. However,
recovery of mature oocytes following controlled ovarian stimulation (COS) had already become the most
popular method, and so IVM did not attract much
attention until 1991, when Cha et al. reported collecting immature oocytes from women undergoing gynecological surgery, fertilizing these donated oocytes following IVM and transferring the resulting embryos
to a recipient woman with premature ovarian failure [16]. The recipient delivered healthy triplet girls.
Three years later, Trounson et al. from the Monash
IVF group reported the collection of immature oocytes
from women with polycystic ovary syndrome (PCOS)
[17]. The immature oocytes collected were matured in
vitro with gonadotropin-enriched medium, then fertilized, and a healthy live birth following transfer of
resultant embryos was reported [17]. However, the
initial pregnancy rates were low, and it took another
5 years to reach more satisfactory figures exceeding
30% per cycle in appropriately selected patient groups
[18, 19].
Overview of an IVM cycle for

Monitoring starts with a baseline scan performed in
the early follicular phase of the menstrual cycle, preferably between days 2 and 5 of a natural menstrual
cycle. The number and size of the antral follicles are
recorded. The ovaries are examined for any abnormalities. A second scan is performed about a week
432
later when it is anticipated that the largest follicle has

reached 1012 mm. The presence of a dominant follicle does not require cancellation of the treatment
cycle because smaller follicles are found to contain
viable oocytes, even in the presence of a dominant
follicle [20, 21]. Based on our own experience and
a favorable trend observed in trials of human chorionic gonadotropin (hCG) priming, the current routine IVM protocol at the McGill Reproductive Centre (MRC) involves hCG administration 10 000 IU im
38 h before oocyte collection. Although the decision
regarding timing of the hCG injection, and therefore
oocyte retrieval, requires that both the follicle size and
endometrial thickness be taken into consideration in
a regular IVM cycle, endometrial thickness is ignored
in fertility preservation cycles as there will not be an
embryo transfer in the same cycle.
Most patients easily tolerate the immature oocyte
collection procedure under conscious sedation with
intravenous midazolam and fentanyl. Paracervical
block is achieved with 1% bupivacaine injection after
cleaning the vagina with sterile saline. As the follicles are smaller than the mature follicles aspirated in
IVF cycles, a smaller-gauge needle (1920 G) with a
shorter bevel is preferred. The aspiration pressure is set
at 7580 mmHg, approximately half the conventional
IVF aspiration pressure, in order to minimize the risk
of oocyte denudation during aspiration. The follicles
are often widespread throughout the ovarian stroma
and aspirating all of them with a single puncture is
generally impossible. Moreover, the fine-bore needle
may be blocked frequently with bloodstained aspirate
and ovarian stroma. Therefore, multiple punctures are
often needed and flushing the needle lumen with heparinized saline between punctures is required. Sometimes external abdominal pressure may be required to
fix the mobile ovaries during collection. Patients with
difficult-to-reach ovaries or poor pain control may do
better under limited general anesthesia with propofol.
Obstetric outcome of IVM

In vitro maturation has become an effective treatment option for many infertile women resulting in
the birth of over 2000 healthy infants [18, 19, 22,
23]. We have found the mean birth weights of infants
conceived with IVM, IVF or intracytoplasmic sperm
injection (ICSI) to be similar, but lower than those of
spontaneous conceptions [24]. The proportion of lowbirth-weight and very low-birth-weight infants was
Chapter 36: Clinical potential of in vitro maturation
similar across ART children. The proportion of infants

with an Apgar score 6 at 1 and 5 min and the incidence of acidosis were all similar among IVM, IVF,
ICSI or spontaneous-conception deliveries [24]. Compared with spontaneous conceptions, the observed
odds ratios (ORs) for any congenital abnormality were
1.42 (95% confidence interval [CI] 0.523.91) for IVM,
1.21 (95% CI 0.632.32) for IVF and 1.69 (95% CI
0.883.26) for ICSI, respectively. None of these were
statistically significant [24]. This provides indirect evidence that the reported high congenital abnormality
rate with ICSI is due to poor sperm per se, because ICSI
with normal sperm did not increase the odds of congenital abnormality to the same extent in IVM cycles.
In a retrospective study, the chromosomal constitution
and mental development of 21 children born after IVM
were compared with 21 spontaneously conceived children. All of the IVM children were found to have normal karyotype and mean developmental index score
similar to controls in this study [25]. Another study of
46 IVM babies born to 40 women in Finland reported
similar findings [26]. The physical growth of IVM children seems to be similar to that of spontaneously conceived children [25, 26].
Young women with high antral follicle counts
seem to have the highest pregnancy rates with IVM
[27]. In vitro maturation has become an established
treatment option for women with polycystic ovaries
(PCO) or PCOS who need ART. However, the clinical application of IVM technology is not limited to
these women alone, and can be extended to benefit
other patient populations. In vitro maturation, especially when combined with oocyte vitrification, provides unique opportunities for women who wish to
preserve their reproductive potential.
IVM for fertility preservation

At present, embryo cryopreservation following IVF is
the only method endorsed by the American Society of
Clinical Oncology (ASCO) and the American Society
for Reproductive Medicine (ASRM), while the other
methods are still considered experimental [28, 29]. In
fact, it is the tried and true method as the successful cryopreservation of surplus embryos after IVF and
resultant pregnancy following frozenthawed embryo
transfer (FET) was first reported in 1983, and the first
child after embryo freezing was born in 1984 [30, 31].
It is estimated that almost one quarter of the children
born after ART are born following cryopreservation
GnRH antagonist
Two to five weeks from first
consultation to oocyte
retrieval depending on time
of first consult
>14mm
Scan on
day 2
Cycle 1
day
Orgalutron/cetrotide (antagonist)
10 11 12 13
FSH/hMG stimulation
Collection
hCG
Figure 36.1 Overview of the gonadotropin-releasing hormone

(GnRH) antagonist protocol. FSH, follicle stimulating hormone; hMG,
human menopausal gonadotropin. From a presentation on fertility
preservation by Dr. S. L. Tan, with permission.
of mostly cleavage-stage embryos and, less commonly,

blastocysts and oocytes [32]. However, several points
raise concern about IVFembryo freezing in cancer
patients. These are: (1) a possible delay of 25 weeks in
treatment of the primary disease due to ovarian stimulation depending on the timing of the first consultation with the reproductive endocrinologist in relation
to onset of the next menstrual cycle; (2) exposure to
supraphysiological estrogen levels induced by ovarian
stimulation; (3) the requirement for a male partner or
willingness to use donor sperm for embryo production; and (4) legal, ethical, religious issues related to
cryopreservation of embryos in general. These issues
and alternatives provided by IVM are discussed below.
Problems with ovarian stimulation in

cancer patients
The time required for completion of the fertility preservation procedure, which starts with the initial reproductive medicine consultation and technically ends
with oocyte collection, depends on the conditions
of any particular clinic. Ovarian stimulation takes
between 2 and 5 weeks, depending on the stimulation protocol employed and the timing of the following menstrual cycle of the patient (Figure 36.1). This
is a matter of concern for both the patients and treating physicians. The effect of such a delay in treatment
obviously depends on the underlying disease and must
be evaluated on a case-by-case basis together with the
treating oncology team.
433
Serum estradiol (E2) levels are increased during

ovarian stimulation for IVF and can reach levels 20
times higher than those of a natural cycle [33]. Breast
cancer remains the most common cancer in females,
representing 27% of all female cancers. Between 2002
and 2006, 12.4% of all breast cancer cases were diagnosed in women younger than 45 years, who are in
their reproductive period [34]. The risk of breast cancer is consistently found to be associated with persistently elevated blood estrogen levels [35]. Although
the effect of a temporary increase in serum E2 levels on the risk of recurrence of breast cancer is controversial, this remains another fact causing concern
among both physicians and patients. Such concerns
are not limited to women with estrogen-receptor positive breast cancer, because recent findings also suggest the presence of an indirect mitogenic effect of
estrogen on hormone-receptor negative breast cancer
[36]. Increased E2 levels can be relevant for patients
undergoing fertility preservation treatment due to
other oncological or non-oncological diseases considered to be estrogen sensitive, such as desmoid tumors,
systemic lupus erythematosus or severe endometriosis. Special stimulation protocols involving aromatase
inhibitors in order to limit the rise in estradiol levels
have been developed for such patients [37]. Despite
encouraging results with regard to disease-free survival and recurrence rates being reported by the
authors, it is interesting to note that 63.3% of breast
cancer patients referred for reproductive endocrinology and infertility (REI) consultation at the same center declined ovarian stimulation and IVF due to concerns about delay of chemotherapy, effect of ovarian
stimulation on cancer or costs associated with treatment [37].
Advantages of IVM
In vitro maturation avoids these two important concerns which prevent a substantial amount of patients
at risk of infertility using them. Immature oocyte
retrieval in an unstimulated menstrual cycle or from
ovarian tissue biopsies followed by IVM and oocyte
or embryo cryopreservation provides a novel fertility
preservation strategy [38, 39]. Avoiding ovarian stimulation provides several important advantages for cancer patients. Compared to 25 weeks required for a
stimulated IVF cycle, immature oocyte retrieval can
be done within 210 days, depending on the patients
menstrual status [39]. Immature oocytes can be col-
434
lected even in the luteal phase. We reported three

women without male partners seeking fertility preservation prior to chemotherapy who presented for the
first time in the luteal phase of their menstrual cycle
and were to undergo gonadotoxic treatment immediately. Five to seven immature oocytes were recovered
by luteal-phase oocyte retrieval from these women.
Three to five MII oocytes were vitrified following IVM.
Two of the three women later underwent one and
two more collections, respectively, in the follicular
phase of the next cycle(s) and additional immature
oocytes were vitrified following IVM [21]. Moreover,
immature oocyte collection in the luteal phase provides a rescue option for a patient who experiences a
premature LH surge during ovarian stimulation [40].
Although cancelling the treatment cycle can be an
option for the regular patient, cancer patients undergoing fertility preserving treatment usually dont have
time for a new treatment cycle and require immediate solutions. We were able to collect four immature oocytes in a breast cancer patient who had a premature LH surge during an ovarian stimulation cycle
started for fertility preservation in New York. She had
experienced an abrupt increase in serum LH level on
the 7th day of stimulation. Progesterone level reached
8.38 ng/ml on the 10th day of stimulation. On the
same day, the leading follicle size was 20 mm, and
there was no sonographic finding suggesting ovulation: 10 000 IU hCG was administered and collection
scheduled 35 h later. At the time of collection there
were approximately 10 follicles sized 10 mm, and 4
of them had the appearance of corpus lutei accompanied by free fluid around the ovaries. Nevertheless
all follicles were aspirated, resulting in collection of 4
immature oocytes at the germinal vesicle (GV) stage
from follicles 10 mm in average diamater. Two GV
oocytes were matured in vitro and fertilized successfully, resulting in vitrification of two embryos [40].
In addition to avoiding potential delay in treatment
and rise in estradiol levels, IVM eliminates the risk
of ovarian hyperstimulation syndrome (OHSS). This
syndrome is a major complication of COS, characterized with increased vascular permeability [41]. Clinically, it manifests as ascites, pleural effusion, hypovolemia, hemoconcentration and hypercoagulability.
Severe cases can be complicated by thromboembolism
or even death. When superimposed on cancer, OHSS
can have serious consequences on the health of these
patients and can cause further delay in pending oncological treatment.
Collection of immature oocytes from ovarian

tissue specimens for cryopreservation
Immature oocytes can also be harvested from ovarian biopsy specimens and fertilized or vitrified following IVM [38]. This combination of ovarian tissue
cryobanking and IVM represents a new strategy for
fertility preservation [9]. We retrieved 11 immature
oocytes from a wedge resection specimen in a 16-yearold patient with mosaic Turners syndrome. Eight of
these oocytes were vitrified following IVM [9]. In 4
women with cancer (2 Hodgkins lymphoma, 1 breast
cancer and 1 rectal cancer), we harvested 11 immature oocytes from wedge biopsy specimens collected
for ovarian tissue cryopreservation. Patient age ranged
between 18 and 38 years. In two patients, surgery
was performed in early follicular phase and another
two women underwent surgery in the luteal phase of
the menstrual cycle. The median number of immature oocytes collected was three. Eight of the eleven
immature oocytes reached MII stage following IVM
and were vitrified [38].
IVM: oocyte vitrification

Requirement for a male partner and ethical/legal/religious issues associated with embryo
cryopreservation are other aspects that require
attention when IVF-embryo cryopreservation is considered as a fertility preservation measure. Although
infertility affects a couple in general, cancer affects
the individual. Fertility preservation should aim to
preserve not only the individuals germ line, but also
her autonomy for her own reproductive potential.
For single women, generating then freezing embryos
conceived with donor sperm is obviously not the same
as cryopreserving unfertilized oocytes for possible use
with a future partner. On the other hand, while donor
sperm is not required for a woman who has a partner,
freezing embryos means sharing the control over the
embryos with him. Unfortunately, some couples split
up in the face of cancer. In the case of separation, the
former male partner also has rights over the embryos,
with all possible legal and ethical implications. The
ex-male partner may disagree to using the embryos
and to conceive a child, as in the case of Evans versus
Johnson [42]. In this famous case, Ms. Evans had
an IVF cycle before undergoing surgery for ovarian
cancer, and her oocytes were fertilized using sperm
from her fiancee at that time. The couple split up
prior to completion of her treatment. The ex-fiancee

withdrew his consent and asked for the embryos
to be destroyed. Ms. Evans started a lawsuit and
following the British High Court ruling against her
wish to continue with embryo transfer, the European
Council of Human Rights also ruled against her.
The embryos were eventually destroyed. If there is
any doubt, it seems better to freeze oocytes since
oocytes belong to the woman, while embryos belong
to the couple. We believe that in contrast to embryo
cryopreservation, oocyte cryopreservation provides
the most effective means of ensuring the reproductive
autonomy of the patient. Another advantage of oocyte
cryopreservation is that it avoids the ethical and
religious quandaries associated with the storage and
disposal of embryos.
Oocyte cryopreservation is considered an investigational procedure in the ASCO and ASRM reports
as well as in a working party report of the Royal Colleges of Physicians, Radiologists and Obstetricians and
Gynaecologists on the management of cancer patients
undergoing gonadotoxic treatment [28, 29, 43, 44]. The
major motivation behind this opinion was that a relatively better clinical outcome was being reported with
embryo than with oocyte cryopreservation.
However, these opinions by ASRM, ASCO and the
Royal Colleges are based on data published prior to
2005, 2006 and 2007, respectively. A meta-analysis of
the efficiency of oocyte cryopreservation published
in 2006 reported live birth rates of 1.9 and 2.0%
per oocyte thawed after slow freezing and vitrification, respectively [45]. The vast majority of data
on oocyte cryopreservation were from experience
with slow freezing at that time, and a substantial
amount of relevant data favoring vitrification has
subsequently been published [4651]. In a clinical
trial at the McGill Reproductive Centre, Montreal,
Canada involving 38 infertile women who underwent oocyte collection in a gonadotropin-stimulated
cycle, oocyte vitrification using the McGill CryoleafTM
resulted in a mean survival rate of 81% post-thawing,
a 76% fertilization rate, a clinical pregnancy rate
per cycle of 45%, a live birth rate of 40% and 22
healthy babies [52]. In a review of 165 pregnancies
and 200 infants conceived following oocyte vitrification, the birth weight and the incidence of congenital anomalies (2.5%) were comparable to those
following spontaneous conception or IVF treatment
[53]. A more recent review corroborates our observations [54].
435
Table 36.1 The number of patients to date who underwent different fertility preservation procedures at the McGill
Reproductive Centre, Montreal, Canada
Malignancy
IVM/EV
IVF/EV
IVM/OV
IVF/OV
Hematological
10
15
15
Breast
31
36
Gynecological
Brain
Sarcoma
Gastrointestinal tract
Melanoma
Autoimmune diseases
Desmoid tumor
Total
45
20
70
44
EV, embryo vitrification; IVF, in vitro fertilization; IVM, in vitro maturation; OV, oocyte vitrification.
Similar to in-vivo matured oocytes, in-vitro

matured oocytes can be successfully cryopreserved
with vitrification, further expanding the choices for
women who wish to preserve their fertility potential.
Although smaller sized immature oocytes without the
meiotic spindle can be anticipated to better survive
cryopreservation, laboratory and clinical outcomes
of cryopreservation of oocytes at the GV stage by
slow freezing or vitrification have not been as good
as those achieved with mature oocytes [46]. Despite
similar survival rates for GV and MII ocytes after
vitrification (85.4 versus 86.1%, respectively), the
maturation rate of vitrifiedwarmed GV oocytes is
significantly lower than that of fresh GV oocytes
(50.8 versus 70.4%, respectively) [46]. However,
the fertilization rate and embryo development rate
are similar for vitrified or fresh GV oocytes once
they reach MII stage. Given these facts, it seems
cryopreservation of immature oocytes after they have
matured to MII stage seems a better strategy. In a
pilot study at the McGill Reproductive Centre on
IVM oocyte vitrification, a live birth rate of 20% per
cycle was achieved, including the worlds first four live
births from vitrified IVM oocytes [55]. Compared to
in-vivo matured oocytes collected following ovarian
stimulation, IVM oocytes had a significantly lower
survival (81.4 versus 67.5%) and fertilization (75.6
versus 64.2%) rates following vitrificationwarming.
Despite a trend towards lower implantation (19.1
versus 9.6%), clinical pregnancy (44.7 versus 20.0%)
and live birth (39.5 versus 20%) rates with IVM oocyte
vitrification, none of the differences were statistically
436
significant [55]. The clinical outcome of IVM oocyte

vitrification is regarded satisfactory for patients whose
conditions preclude ovarian stimulation for any
reason. To date, the McGill Reproductive Centre has
provided fertility preservation to 180 patients with
breast, hematological, brain, soft tissue, colorectal and
gynecological cancers: more than 100 of these women
have oocytes or embryos cryopreserved following
IVM (Table 36.1). A suggested algorithm for cancer
patients is presented in Figure 36.2 [56].
IVM for social fertility preservation

Both the ASRM and European Society of Human
Reproduction and Embryology (ESHRE) recommendations are against ovarian tissue cryopreservation in
healthy women who are not faced with an immediate threat to fertility [43, 57]. Similar to ovarian tissue freezing, oocyte cryopreservation is also regarded
an experimental procedure, and both societies find it
early to recommend or encourage oocyte freezing
without a medical indication [43, 57]. The risk to benefit ratio of the procedure is one of the major concerns. In vitro maturation provides an unique opportunity of oocyte freezing without ovarian stimulation
for women who wish to do delay child bearing for
social reasons. In-vitro maturation oocyte vitrification
avoids risk of OHSS and the inconvenience of daily
gonadotropin injections for these healthy women. The
direct and indirect costs of treatment, i.e. cost of
gonadotropins, loss of working days for monitoring
scans etc., are less with IVM. Based on this, IVM can be
Figure 36.2 Suggested algorithm for

fertility preservation. Reproduced from Chian
et al. [56]. with permission from Elsevier
Science, Inc.
strategies for women
undergoing
gonadotoxic treatment
Ovarian wedge resection

or oophorectomy
Chemotherapy cannot be
delayed and/or hormonal
stimulation contraindicated
Chemotherapy can be
delayed and hormonal stimulation
not contraindicated
Immature oocyte retrieval
Immature oocyte retrieval

from ovarian tissue
Ovarian tissue
cryopreservation
Ovarian stimulation
Mature oocyte retrieval
In vitro maturation of oocyte
Male
partner
available
No male
partner
available
Male
partner
available
No male
partner
available
Embryo
cryopreservation
Oocyte
vitrification
Embryo
cryopreservation
Oocyte
vitrification
considered a simpler and safer procedure and regarded

more acceptable.
In conclusion, IVM combined with embryo or
oocyte vitrification provides previously unavailable
options for some patients and improves the services
provided by a fertility preservation program. Primary
care physicians and oncologists need to be made aware
of the available fertility preservation options in order
to allow early discussion with their patients followed
by referral, if desired, to an ART center that offers a
full range of fertility preservation options.
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439
Section 9
Chapter
37
Future technologies
From pluripotent stem cells to germ cells

Rosita Bergstrom and Outi Hovatta
Introduction
Embryonic stem cells
Infertility problems represent the biggest health issue

among people aged 2045 years, and a major concern
is that the magnitude of this problem may be increasing, as many couples wait until later in life trying to
build a family. It is speculated that the latter may be
part of the reason why the 2005 National Survey of
Family Growth report found a 20% increase in American couples experiencing infertility problems between
1995 and 2002 [1].
The number and quality of oocytes is reduced with
age, but it may also decrease as a consequence of a
disease or treatment [2, 3]. In males, spermatogenesis normally continues during adult life, but the number and quality of fertile sperm cells is, as for females,
affected by various factors, often following disease
[4, 5]. Yet very little is known in this area of research,
principally due to lack of models for human germ
cell development. Selected differentiation of pluripotent stem cells to germ cells opens up a door to not
only learning more about germ cell development in
general, but also as a tool to study specific causes of
infertility. Differentiation of gametes from pluripotent
human stem cells may constitute a therapeutic option
for infertile couples in the future.
Human embryonic stem cells (hESCs) are pluripotent,

meaning that they have the ability to give rise to a
wide range of cells belonging to all three germ layers (ectoderm, endoderm and mesoderm) as well as
into germline lineage [11]. These cells can be derived
from the inner cell mass of blastocysts [12], cleavage
stage embryos or single blastomers [1315] Extraembryonic tissues, such as the amniotic fluid, umbilical
cord blood and the placenta can give rise to multipotent stem cells. Such multipotent cells have more limited differentiation capacity than pluripotent embryonic stem cells (ESCs) and induced pluripotent cells.
Regarding reproductive biology and medicine, the
most interesting cells are two types of pluripotent stem
cells (i.e. human embryonic and induced) and testicular and ovarian stem cells.
Stem cells
Stem cells are undifferentiated cells characterized by
their ability both to renew themselves and to give rise
to various types of specialized cells. Adult stem cells are
multipotent, i.e. they have the ability to differentiate
to a limited number of cell fates, and can be obtained
from various tissues, including blood [6], bone marrow [7], fat [8], skin [9] and testis [10].
Stem cells from testis and ovary

Pluripotent stem cells have been identified in the seminiferous tubules of the testis in rodents and also, lately,
in humans [16, 17]. In the future, these cells may be
important tools for studying human spermatogenesis and for obtaining mature sperm in vitro after fertility preservation in pre-pubertal boys. It would be
less risky than transplantation of cells, particularly
in hematological malignancies. So far, spermatogenesis in vitro has not been completed, but there are
promising results in animal experiments, particularly
in three-dimensional cultures [18].
Stem cells in the ovary have been discussed intensively during the recent years and, for the time being,
this remains a controversial issue. Presently there is
no convincing evidence of oocyte-forming stem cells
being found in the postnatal human ovary.
440
Chapter 37: From pluripotent stem cells to germ cells
Induced pluripotent stem cells

In 2006 there was a major breakthrough in the field
of stem cell research. Yamanakas group were, for the
first time, able to reprogram somatic, specialized cells
into pluripotent stem cells. The cells in questions were
mouse fibroblasts, that upon transfection with retroviruses carrying the four key pluripotency genes Oct3/4, Sox-2, c-Myc and Klf4, spontaneously dedifferentiated into stem cell-like cells, so called induced
pluripotent stem cells (iPSCs) [19]. By altering the
expression of these genes alone, the fibroblasts spontaneously dedifferentiated into iPSCs. In the sense
of morphology, proliferation and teratoma formation
these cells were similar to hESCs, but there were severe
epigenetic errors such as abnormal DNA methylation
patterns and faulty gene expressions. Furthermore, the
iPSCs failed to produce viable chimeras when injected
into developing embryos.
Successful reprogramming of mouse fibroblasts
into iPSC and the production of viable chimeras were
obtained in 2007, by replacing Fbx15+ with Nanog for
isolation of cells by antibiotic selection [20]. However,
20% of the chimeric mice developed cancer, likely due
to the overexpression of the oncogene c-Myc, used to
induce the dedifferentiation and/or epigenetic errors.
At the end of 2007, Yamanakas team managed to
repeat their results in adult human cells using the same
four genes as in previous studies performed in mice
[21]. The concerns using c-Myc remained, but nevertheless this was a big step forward in the field of reproductive medicine in humans.
Also at the end of 2007, Thomson and colleagues
managed to create human induced pluripotent stem
cells (hiPSCs) avoiding the use of oncogenes. Instead
they used a lentiviral system based on the expression
of OCT4, SOX2, NANOG and LIN28 [22]. LIN28 is a
marker of undifferentiated hESC and is associated with
several epigenetic features, such as micro-RNA and the
regulation of the imprinted gene Igf2, but the exact
mechanisms behind this remain to be investigated.
The technical limitations obtaining iPSCs has been
a concern for its applications in humans. Using a virus
with random insertions in the human DNA is a dangerous approach as it may cause mutations in the DNA
code and/or cause epigenetic errors (epimutations).
Following these initial, but for therapeutic applications
insufficient, breakthroughs, inducible lentiviral systems have been developed to generate iPSCs [2325].
Using this strategy, however, the drawback of perma-
nent integration in the host genome remains. Lately

non-integrating approaches, like adenoviral delivery
[26], transient transfection [27] and direct delivery
of reprogramming factor proteins [28] have been
successfully used in the reprogramming of mouse and
human cells. The efficiency using these methods has
to be improved before they can be applied for clinical
purposes, but this gives great hope for the future.
Epigenetic regulation
The DNA code is more or less the same in all the cells
of the human body. What differs is the way this code
is utilized or, in other words, which combinations of
genes that are active in a specific cell at a given time.
The secret behind this is the epigenetics, the heritable
changes in gene function that occur without a change
in the sequence of the DNA [29, 30]. This includes a
wide range of mechanisms, such as DNA methylation,
histone modifications, genomic imprinting and higher
order chromatin conformation [29]. The overall epigenetic state of a cell is referred to as the epigenome.
The creation of the diverse cell types from pluripotent
stem cells depends ultimately on the establishment and
maintenance of specific patterns of gene expression,
a process under careful control of epigenetic features.
This information can be reversed and changed, but also
passed on through cell divisions and generations, such
as an epigenetic memory.
There is an extensive cross talk between different
types of epigenetic regulation that carefully adjust the
level of gene expression at any given moment. Some
adjust rapidly upon a stimulus and others remain
steady through cell divisions, distinguishing different
cell types. This fine-tuned control of gene expression is
the key factor behind stemness and differentiation, and
it is an absolute necessity that we learn to understand
these features in order to continue making progress
in the field of stem cell research and differentiation.
What is the difference between various types of cells?
How many different cell types are there in the human
body? This is a key question if we are to create one kind
of cell from another. However, first we need to define
cell type. Depending on who answers, the answer will
probably range from a few hundred to a few million. The epigeneticist would most likely state that
there are as many types of cells in the human body
as there are number of cells in total, because no cell is
epigenetically the same as another. The approximately
2 m of DNA is modified and folded in an unique way
441
in each and every cell. However, there are similarities

between cells with distinct functions. Germ cells are,
for example, epigenetically similar but not identical
to one another.
The three-dimensional positioning of the chromosomes in relation to each other, or chromosome territories, is one of the features that differ between groups
of cells. That is, the locations of the chromosomes, their
neighbors and the interactions/co-regulations vary
between different cells. This is important in the field
of germ cell differentiation and development, since
the degree of difference in the epigenome between the
starting material (the cells to be reprogrammed) and
end product (germ cells) will vary depending on which
starting material is utilized and the epigenetic statues
of these cells.
In line with this, it has been reported that using the
same protocol, differences in time and success rate for
reprogramming somatic cells to iPSCs varies with the
choice of starting material [31]. This is not surprising,
it makes sense that it is less of an effort to make one cell
from another that is more alike the end product. Due
to this, it might be a good strategy using explicit protocols for deriving iPSCs from various starting populations, and to consider that lines that are epigenetically
more different from pluripotent cells may need longer
time to adjust their epigenome in to a pluripotent state.
This strategy may very well also apply for differentiating stem cells of various kinds to germ cells. One may
not experience that the differentiation process will take
the same amount of time, depending on the potential
differences in the epigenomes.
A major concern in iPSCs derivation (and all other
kinds of reprogramming, such as any type of differentiation) is incomplete reprogramming [19, 32, 33].
Cells that are stuck somewhere in between two states
can be difficult to interpret. The expression of a handful of pluripotency markers is not enough to reprogram the cell on their own, and it is expected that DNA
methylation and histone modifications play important
roles [34]. For instance, the promoter regions of many
pluripotency associated genes are heavily methylated
in somatic cells, repressing the expression of the corresponding transcripts. For completion of reprogramming these promoters need to be hypomethylated,
as they are in ESCs [35], a process that takes several cell cycles to complete. The use of demethyationpromoting agents, such as 5-azacytidine during iPSCs
derivation, has proven to effectively support this process [32].
442
The patterns of histone modifications are other

key features for complete reprogramming. In differentiated cells, histones H3 and H4 are hypoacetylated in promoter areas of pluripotency genes, and
their expressions are silenced. In order for proper
pluripotency-specific gene expression, i.e. complete
reprogramming, hyperacetylation needs to occur. Use
of the histone deacetylation inhibitor valproic acid has
proven to efficiently enhance the generation of iPSCs,
probably due to the enhancement of acetylation [36,
37].
The purpose of iPSCs is that they should mimic
the nature of hESCs. Therefore, iPSCs should be cultured in the same way as hESCs. If they were not to
be cultured in the same manner, we would have completely failed making the kinds of cells we attempted.
However, there are concerns with iPSCs that we must
take under consideration during cell culture. First of
all, there is a risk that these cells are not fully reprogrammed and will differentiate back to the kind of cells
they were before reprogramming; therefore, there is a
concern that these cells will be more difficult to differentiate towards other lineages, such as germ cells. This
is possible if the epigenetic memory is not sufficiently
reprogrammed. Overexpression of a limited number
of factors alone will not keep cells in a pluripotent state
if the epigenome is set at a differentiated state.
Cell culturing is also challenging due to the problem with culture adaptation [38]. The iPSCs may even
be more unstable than ESCs, as the epigenome can be
out of equilibrium and the cells may fumble in the
dark, awaiting signals or stimuli, in order to rapidly
differentiate in to a more stable state. Recently, however, it has become clear that alternative culturing conditions, such as hypoxia, may improve the efficiency of
iPSCs [39].
There is no doubt that many epigenetic phenomena take place during reprogramming, and by learning
more about how these function there is great room for
improvement for controlled reprogramming of cells.
Perhaps epigenetic features can be used as a tool to
investigate the status of reprogramming. And perhaps
epigenetics can elucidate some of the many cases of
unexplained of infertility.
Epigenetic regulation of germ

cell development
The development of germ cells is a highly ordered process that begins during fetal growth and is completed
in the adult. Epigenetic modifications are sequentially

established and erased in the germ cell lineage. This
epigenetic reprogramming is essential for the acquisition of totipotency and the epigenetic marks called
imprints that distinguish the parental origin of about
80 genes in humans [40]. The imprinted marks are
erased in primordial germ cells (PGCs) and then reset
in a parent-of-origin specific manner, such that they
are in place at the time of fertilization.
In the 1980s, it was demonstrated how crucial
imprinting is for normal development. Two female
(biparental gynogenones) or two male (biparental
androgenones) pronuclei were used with the attempting to construct diploid mouse embryos [41, 42].
The ability of these embryos to develop to term was
compared with control nuclear-transplant embryos
in which either the male or female pronucleus was
replaced with an isoparental pronucleus from another
embryo. The results revealed that, in contrast to the
controls, neither diploid biparental gynogenetic or
androgenetic embryos completed normal embryogenesis. Interestingly, the outcome of the experiment also
showed to be different for the two setups. Whereas
the gynogenetic embryos showed relatively normal
embryonic development but poor placenta development, the androgenetic embryos showed poor embryonic development but normal placental development.
This experiment points out how important parental
imprinting is for certain stages of development, and
also explains why parthenogenesis does not exist in
humans.
Twenty years later, experimental manipulation of
the imprinting locus H19/Igf2, forcing unequal expression of this loci from the two pronuclei (i.e. forcing
an artificial imprinted state), allowed the creation of
rare individual mice with two maternal sets of chromosomes [43]. Interestingly, several other imprinting centers were affected due to manipulation of this region
alone. This observation is perhaps not that surprising,
as the imprinted locus in question has been pointed
out as a potential key point for higher order chromatin
conformation [44, 45] and is likely to affect the regulation of several other loci. Whether this is a unique feature of the H19/Igf2 locus or is true for all imprinted
regions is still to be determined.
The global degree of methylation is about the same
in the genomes of mature eggs and sperm as it is
in somatic cells, while the genome in stem cells is
hypomethylated. The specific pattern of methylation
is, however, unique in various cell types. Following
the genome-wide demethylation that occurs in both

male and female germ cells in the early development
of the PGCs, the cells enter mitotic (male) and meiotic
(female) arrest, respectively [46, 47]. Interestingly, the
subsequent remethylatin occurs much earlier in the
male germ line, at the prospermatogonia stage, than in
the female line where remethylation takes place after
birth during the growth of the oocytes.
Germ cells from ESC

Due to their plasticity and potentially unlimited capacity for self-renewal, hESCs have been popular candidates for various cell therapies, including regenerative medicine and tissue replacement. Lately there has
been a lot of focus on the potential use for hESCs in
reproductive medicine [11, 4850]. They offer excellent models for studying the regulation of human
oogenesis and spermatogenesis, which are not easily
accessible for research.
Establishing functional gametes, from pluripotent cells, particularly from patient-specific induced
pluripotent cells, would offer totally new options for
individuals who lose their own gametogenesis in connection with cancer therapies.
Oocytes from mouse ESCs

Differentiation of mouse embryonic stem cells
(mESCs) to oocytes was first described by Hubner
et al. [51]. Oocytes were differentiated in adherent
cultures from a mouse stem cell line which expressed
green fluorescent protein (GFP) under influence of
the Oct4 promoter. Populations were enriched by
selecting VASA and c-kit expressing cells. Starting
from day 12, increasing clusters of cells expressing early and more mature oocyte markers (such
as GDF-9) were detected. With time, the meiotic
marker synaptonemal comples protein 3 (SYCP3)
was activated, which also had several morphological
features on oocytes. More mature meiosis markers were not demonstrated. In long-term cultures,
embryo-like structures and blastocyst-like formation
were seen. The oocytes were not fertilized and must
have undergone parthenogenetic activation.
Oocyte formation from mESCs was carefully studied by Novak et al. [52]. Embryoid bodies were formed,
and the development of oocytes was enhanced in coculture with a bone morphogenetic protein 4 (BMP4)-producing cell line. A panel of meiosis specific
markers was systematically studied. Out of these,
443
SYCP3 alone was detected in the germ cell-like cells,

and the nuclear distribution of SYPCP3 was highly
abnormal. The germ cell-like cells did not contain
synapsed homogous chromosomes, but instead displayed a chromosomal organization normally seen in
somatic cells. The germ cell-like cells differenriated
from mESCs failed to undergo normal meiosis.
The obstacle of getting mature oocytes from
mESCs in vitro was overcome by Nicholas et al.
[53], who first established fundamental parameters
of oocyte development during ESC differentiation.
They created a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels with endogenous oocyte development in vivo
by single-cell expression profiling and analysis of
functional milestones, including responsiveness to
defined maturation media, shared genetic requirement of Dazl and entry into meiosis. Because ESCderived oocyte maturation ultimately failed in vitro,
they transplanted ESC-derived oocytes into an ovarian niche to direct their functional maturation. By
including this step, mature oocytes, which could be
fully characterized, were obtained and they could identify the regulational mechanisms controlling oocyte
development.
Male germ cells from mESCs

In 2003, Toyooka et al. [54] established functional male
germ cells from mESCs. They used knock-in ESCs, in
which GFP or lacZ was expressed from the endogenous mouse vasa homolog, MVH, which is specifically
expressed in differentiating germ cells. Using these
cells they visualized germ cell production during in
vitro differentiation. The appearance of MVH-positive
germ cells depended on embryoid body formation
and was greatly enhanced by the inductive effects of
bone morphogenic protein (BMP)4-producing cells.
After transplantation to seminiferous tubules these
cells reconstituted testicular tubules, demonstrating
that ESCs can produce functional germ cells in
vitro.
Primordial germ cells were identified from embryoid bodies by Geijsen et al. [55] and continuously
growing lines of embryonic germ cells were derived.
Embryonic germ cells showed erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a
property characteristic of the germ lineage. Embryoid
bodies supported maturation of the PGCs into haploid male gametes, which, when injected into oocytes,
444
restored the somatic diploid chromosome complement and developed into blastocysts.
Germ cells from hESCs

Germ cells were first differentiated from hESCs by
Clark et al. [49]. The hESCs were differentiated as
embryoid bodies, and a shift in expression from RNA
and protein markers of immature germ cells to those
indicative of mature germ cells, including expression
of VASA, BOL, SCP1, SCP3, GDF-9 and TEKT1, was
detected, with all markers specific to gonocytes.
The next step taken by the same team [48] was
to use stimulation of differentiation with growth factors, BMP-4, BMP-7 and BMP-8b. The cells which
expressed the germ-cell specific marker VASA, a factor
first identified in 2000 as specific for PGCs [56], were
enhanced in culture by BMP stimulation. These cells
also expressed SYCP3 as a meiotic marker.
In elegant experiments, the same team then differentiated hESCs in adherent culture instead of
embryoid bodies [11]. BMP stimulation was used as
before. Silencing and over-expression of the germcell specific RNA-binding proteins DAZL, DAZ and
BOULE significantly enhanced postmeiotic germ
cells in culture. First PGC markers (DAZ1, PRMI1,
Stella, VASA) appeared, followed by meiotic markers
gamma-H2AX and SYCP3. Typical epigenetic reprogramming, hypomethylation of H19, was detected.
Haploid chromosomes as revealed by fluorescence in
situ hybridization (FISH) were identified in the postmeiotic cells.
Recently, PGCs and, later, postmeiotic spermatids
were obtained from hESCs [57]. Spontaneous differentiation in embryoid bodies was carried out, and quantitative real-time polymerase chain reaction (RT-PCR)
and immunolocalization was used to identify stepwise the formation of PGCs and then spermatogonial
lineage cells, ending in sperm-specifc proteins, such as
protamine I and protamine 1.07. Gene expression profiles characteristic of oocyte development and folliclelike structures was seen, but no committed oocytes
with zona pellucida. Steroid secretion could be measured in these cultures.
The present knowledge regarding gametogenesis in
vitro starting from ESCs already gives us new insight
about regulation of human gametogenesis. It may be
possible to use this knowledge in identifying causes
of infertility and mechanisms of action of toxic factors; for example, those used in chemotherapy. Getting
functional gametes for research and for treatment is a

more distant goal.
Germ cell differentiation from induced

pluripotent cells
Renee Reijo Pera and co-workers were first managing to differentiate germ cells from hESCs in vitro
[49]. Recently, this method has been improved and
also applied to iPSCs [58]. By co-culture of the differentiating cells with human fetal gonadal cells, Park
et al. significantly improved the efficiency of generating in-vitro derived PGCs, using BMP stimulation,
from hESCs, and managed to repeat the experiments
using hiPSCs, originating from human skin fibroblasts
[58]. Interestingly, the authors did not detect any major
differences in the efficiency of germ cell differentiation between using hESCs or hiPSCs. After 7 days of
differentiation the in-vitro derived PGCs were transcriptionally distinct from the somatic cells (showing
expression of genes associated with pluripotency and
germ cell development). In addition, by use of bisulfite sequencing, signs of initiation of imprinting erasure was detected in the population originating from
hESCs but not from hiPSCs after this time period. In
conclusion, this suggests that iPSC derived from fetal
cells may differentiate to PGCs, but there is so far no
evidence if this is also true for iPSCs derived from
adult somatic cells. Neither is there currently any available evidence that iPS-derived PGCs can enter or go
through meiosis, a diagnostic property of germ cells.
Potential applications and

future prospects
The underlying genetic and epigenetic mechanisms
behind germ cell differentiation are poorly understood in humans due to a lack of models. Extensive, and essential, epigenetic modifications have however been observed to occur in Caenorhabditis elegans
and Drosophila, among other model organisms, during germ cell development [29]. Technical progress
in the field of molecular biology and epigenetics has
lead to a rapid development of the knowledge in this
area during the last decade. Nevertheless, there are
still major obstacles that need to be solved, not the
least in the area of germ cell development and fertility. Hopefully the combination of iPSCs derivation and
in-vitro germ cell differentiation will shed some light
over this process. The nature of the epigenome is that
it is always changing (as responses to various stimulus, from within the cell or the surrounding), and this
dynamic feature is technically challenging to investigate. In particular, there are very few single cell analyses available, where the epigenome of one induvidual
cell could be measured.
The use of stem cells has provided a research tool to
study development and disease in details never before
possible. But even though treatment using stem cells is
giving great hope towards the cure of various human
disorders, such as leukemia, brain damage, cancer and
infertility, there are many fundamental questions that
still need to be answered, not least at an epigenetic
level. How do we ensure that cells differentiated in vitro
have the same phenotype as corresponding cells differentiated in vivo? Which are the fundamental genetic
and epigenetic steps during differentiation and development?
In the future there may be potential for using
gametes differentiating from hESCs or hiPSCs as a
therapy for infertile couples. The advantage of using
iPSCs, compared to hESCs, is the hope to generate
patient specific cells, where somatic cells such as skin
cells from a patient could be utilized. The main benefit
to this would be that men and women lacking egg and
sperm, for any reason, could potentially get the opportunity to conceive children that are biologically their
own. The major obstacle for the moment is the technical difficulties deriving a sufficient amount of iPSCs
from differentiated cells without using a virus and/or
risking epimutations in the host genome.
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Section 9
Chapter
38
Future technologies
Artificial ovary
Introduction
The reader may feel it is somewhat fanciful to see a
chapter on artificial ovary assembly for further transplantation, when no studies have been published on
this topic to date, and no one has yet been able to
endorse its feasibility. However, we believe it is the
right time to consider the artificial ovary as a promising technology in reproductive medicine, as it could
represent an alternative to ovarian tissue transplantation. In addition, positive results with different types
of artificial organs and tissues, such as bone, cartilage,
skin, heart, liver, kidney, brain, cornea and teeth, indicate that construction of an artificial ovary is wholly
achievable. Since there are no available studies on artificial ovary assembly for grafting purposes, this chapter may provide the starting point for this technology,
where indications, advantages, strategies, techniques
and possibilities will be discussed.
The artificial ovary as a strategy to

re-establish fertility
Indications for patients
For cancer patients of childbearing age, fertility
restoration often becomes an important concern
after disease remission. In women, treatments such
as chemo/radiotherapy can be very harmful to the
ovaries, often causing loss of endocrine and reproductive functions, which results in premature menopause
and emotional distress. An option for these patients
is utilization of primordial follicles enclosed in ovarian cortex, since they represent 90% of the ovarian
follicle reserve and there are no oncological, legal
or ethical restrictions to their use. Before a patient
begins cancer treatment, a sample of her ovarian tis-
sue containing primordial follicles can be removed and

cryopreserved. Then, after disease remission, the tissue
fragments can be grafted back to the patient. This technique has led to successful ovarian function restoration, as well as pregnancy (for a review, see Donnez
et al. [1]).
Transplantation has so far been the only option
to re-establish ovarian function from cryopreserved
ovarian tissue in cancer survivors. Despite the promising results mentioned above, there is a legitimate concern about the possible presence of malignant cells
in the frozenthawed fragments, which could provoke a recurrence of the primary disease after reimplantation [2]. Although many types of cancer never
metastasize to the ovaries, leukemia is systemic in
nature and poses a greater threat to the patient, while
breast cancer is classed as moderate risk. Therefore, for
patients diagnosed with these types of cancer, transplantation of ovarian tissue after disease remission is
not advisable. Unfortunately, for these women, it is not
yet possible to transplant their tissue after their health
is restored.
A safe alternative for leukemia and breast cancer patients would be utilization of isolated follicles. Since the basement membrane encapsulating the
ovarian follicle excludes capillaries, white blood cells
and nerve processes from the granulosa compartment [3], grafting fully isolated follicles could be considered safer for these patients. Another option to
avoid cancer re-introduction could be purging isolated follicle suspensions of malignant cells with specific antibodies [4]. Since it is likely that stromal cells
are required for follicular growth, fully isolated follicles could be grafted together with autologous stromal cells (from a new ovarian biopsy after cancer
treatment) [5].
448
Chapter 38: Artificial ovary
Advantages
Although the main concern is being able to provide an
alternative to restore fertility in patients who cannot
benefit from ovarian tissue transplantation, the assembly and grafting of an artificial ovary would offer additional advantages compared to transplantation of ovarian tissue.
Improvement of follicular survival by decreasing

the ischemic period
After removal, freezing, thawing and transplantation,
ovarian tissue is subjected to hypoxia in the first
days post-grafting, and this deprivation of oxygen and
nutrients, as well as accumulation of metabolic waste,
may lead to cellular damage. Indeed, it has been estimated that a significant percentage (5095%) of primordial follicles may be lost due to ischemia [68],
which would directly affect the life span of the graft.
Therefore, the success of primordial follicle transplantation depends on the growth of new blood vessels
in order to restore adequate perfusion. This may be
improved by use of a scaffold with a porous structure, loaded with factors known to promote angiogenesis. An interconnected pore network in a scaffold has
been shown to enhance vascularization in prostheses
implanted in the abdominal aorta of rats [9], while
appropriate pore size and distribution would facilitate the diffusion of metabolites, oxygen and growth
factors [10], which would have a positive effect on
follicle survival and development. Angiogenic factors
could also be added to the scaffold, either chemically immobilized or physically entrapped [10]. Shi
et al. conducted studies to develop an artificial dermis and showed that when angiogenin, a polypeptide involved in angiogenesis, was added to a porous
collagen-chitosan scaffold subcutaneously grafted to
rabbits, vascularization increased [11]. Basic fibroblast growth factor (b-FGF) was also shown to have
a positive effect on vascularization in different studies. Peters et al. observed almost fourfold faster vascularization when polylactic-co-glycolic acid (PLGA)
microspheres were loaded with b-FBF. These authors
reported that released b-FGF induced the formation of
large and mature blood vessels in scaffolds implanted
in the mesenteric membrane of rats [12]. Tanihara
et al. also described induction of angiogenesis by
b-FGF in heparin/alginate scaffolds grafted to the dorsal area of rats [13]. Vascular endothelial growth factor
(VEGF) and platelet-derived growth factor (PDGF)
were found to increase vessel density, size and maturity when added to scaffolds grafted to mice [14].
According to Bergmann and West, these factors could
well influence the differentiation of mesenchymal stem
cells from the bloodstream into endothelial cells and
encourage microvascularization [10].
Control of follicular development

Premature recruitment of primordial follicles has also
been suggested after grafting of ovarian tissue, possibly caused by a deficiency in inhibitory mechanisms
implicated in the quiescence of primordial follicles in
intact ovaries [15]. This is indeed very likely, since tissue collected for cryopreservation and transplantation
comes from the ovarian cortex, where the vast majority
of the follicular population is made up of primordial
and primary follicles and there is a lack of larger follicles responsible for the production of inhibitory factors like anti-Mullerian hormone (AMH) and activin
A. Using a scaffold, follicular activation and development may be modulated through supplementation of
inhibitory and growth factors implicated in the different stages of folliculogenesis. These factors could,
for example, be added encapsulated in materials with
different degradation rates, according to requirements
after transplantation.
Improvement of follicular growth using

fresh ovarian cells
Apart from the interaction between granulosa cells
(GCs) and oocytes, follicles require neighboring stromal cells to support their growth. These cells are
recruited to differentiate into theca cells, which play an
essential role in follicular development through secretion of androgens as well as improving perifollicular
vascularity. Although freezing of ovarian tissue does
not negatively affect the morphology or ultrastructural
characteristics of primordial follicles [16], it is harmful to surrounding tissue, causing damage to the extracellular matrix (ECM) and stromal cell necrosis [17],
resulting in large areas of fibrosis [18]. The poor cellularity of tissue after freezing may influence the development of follicles, and could be involved in the lack
of a structured thecal layer around secondary follicles and asynchrony between oocyte and follicular cell
growth [19]. Therefore, in order to improve follicular
development in the scaffold, a fragment of ovarian tissue could be removed before the scaffold is grafted,
with the aim of isolating fresh stromal cells. These cells
449
would then be combined with isolated follicles from

frozen tissue and seeded in the scaffold.
Control of the number and quality of

follicles to be grafted
Due to the random distribution of primordial follicles in ovarian cortex, it is not possible to determine
the number, or even the presence, of follicles in ovarian tissue to be transplanted to a patient [20], which
could affect the life span of the graft. Grafting isolated
follicles would allow not only the introduction of a
high and known number of follicles into the host [21],
but also assessment of follicular quality before grafting
[22].
How to assemble an artificial ovary

As previously mentioned, there are no studies on the
construction of an artificial ovary for further transplantation and it is therefore necessary to start from
scratch, studying the ovary and its characteristics to
identify the main requirements, and analyzing studies
on the assembly of other artificial organs, applying tissue engineering strategies to meet these requirements.
The natural ovary

Although an understanding of mechanisms involved
in folliculogenesis is essential to build an artificial
ovary for transplantation, this process will not be discussed in the present chapter, since it can be found
in several well presented review papers [2325]. Nevertheless, it is important to bear in mind some basic
information about the ovary.
The ovary has two main functions: production
of oocytes capable of being fertilized, and secretion
of hormones required by the reproductive tract during oocyte fertilization and further pregnancy [25].
Both functions depend on folliculogenesis, a complex developmental process that is regulated by various
endocrine, paracrine and autocrine factors [26, 27], as
well as intraovarian cellcell and cellmatrix connections [3, 28]. Depending on developmental stage, follicles can be found in the outer or inner area of the ovary.
While the cortex contains the vast majority of ovarian
follicles, mostly in their initial stages of development,
the medulla is highly vascularized and responsible for
the maintenance of larger follicles.
In addition to follicles, different types of cells can
be found in ovarian tissue, such as epithelial, stromal, endothelial and theca cells. As previously stated,
450
these cells play an important role in the survival and

development of follicles through the production or
exchange of numerous factors essential for follicular
quiescence, nutrition, communication, growth, hormone production, oocyte maturation, etc. Furthermore, ECM proteins synthesized by some of these cells
serve as a scaffold to hold the ovary together and maintain the three-dimensional (3D) morphology of follicles. This 3D structure is indispensable to preserve
intercellular interactions between granulosa cells and
oocytes, regulating of many aspects of oocyte growth
and development [29, 30].
Requirements to assemble an artificial ovary

As in case of a natural ovary, the main goal of an artificial ovary is to offer an environment that allows folliculogenesis to occur. Therefore, just like the natural
organ, the scaffold should: (1) ensure proper communication between follicles and ovarian cells; (2) preserve their interaction with the ECM; (3) supply factors involved in follicular survival and development;
and (4) maintain the original structure of follicles. In
other words, the scaffold should spatially and temporally mimic the ECM, the natural scaffold of the
ovary. In order to do so, it should include some design
parameters, such as interaction with cells, physical
support of follicles, porosity, bioactivity, vascularization and biodegradability, which are all interconnected
and influence each other. It must also be biocompatible and, from a practical point of view, capable of being
handled.
Interaction with cells/follicles

Citing von der Mark et al., Cells are surrounded by
a wealth of information provided by the ECM, which
presents adhesive and bioactive peptide epitopes
located in matrix macromolecules and smaller glycoproteins, plus growth factors and cytokines trapped
and sequestered by the matrix [31]. The ECM thus
plays an essential role in cell fate: it regulates cell morphology, proliferation, migration, differentiation, orientation, production and secretion of molecules and
even death. For this reason, the scaffold should modulate the interactions of cells and follicles, supporting
cell adhesion, proliferation, migration and production
of matrix proteins necessary to form a substrate for
new cells required for follicular development. For follicles, the scaffold should act as a supporting matrix,
preserving their original 3D structure and intercellular
interactions between GCs and oocytes, which is essential to regulate follicular growth and development. The
3D arrangement of follicles is also influenced by cell
migration and proliferation, induced by interaction of
the scaffold and cells.
In order to modulate cell adhesion, behavior and
function, polymer properties, such as crystallinity,
morphology and surface, can be modified. Some of
these important properties are discussed below.
Crystallinity
Crystallinity refers to the degree to which molecules of
a polymer are oriented toward a repeating model. It is
therefore favored by polymers with a chemically simple structure. Crystallinity may influence the response
of cells to the scaffold, since it affects several surface characteristics, such as polarity and irregularity
[32]. Degirmenbasi et al. reported that growth rates of
fibroblasts varied according to the crystallinity range
of poly(L-lactide) (PLLA); when the polymer was
highly crystalline, fibroblasts showed lower growth
rates [33].
Morphology
Scaffold topography and dimensionality play an
important role in cell behavior. Topography can influence the morphology of cells, which consequently
affects their orientation, proliferation, gene expression
and function. Studies have shown that cells behave in
a completely different manner when cultured on irregular surfaces (e.g. edges, grooves and ridges) rather
than smooth surfaces [32, 34]. For example, ridges can
influence cell orientation and migration of different
types of cells. This cellular locomotory response is
termed contact guidance. Regarding dimensionality,
preservation of 3D structure is not only important for
follicles, but also for cells. A 3D scaffold would provide
normal polarity and spatial regulation of cells, and it
may also influence the cellular response to existing
physical and biochemical signals [32].
Surface
In tissue engineering, adhesion of cells to the scaffold surface is crucial because their activities (proliferation, migration, differentiation, etc.) depend on this.
In order to improve cell attachment and thereby its survival and behavior in the material, modifications can
be made to the scaffold surface. The advantage of treating polymer surfaces is that only the external part of
the material is changed, preserving the polymer bulk.
This is extremely useful, since materials with satisfactory bulk properties usually do not have the surface
characteristics required for clinical application [35]. In
this way, different polymers can be combined for specific purposes (i.e. surfaces with superior biocompatibility and bulk, with suitable mechanical properties).
Methods such as addition of adsorbed proteins
and immobilized functional groups have been proposed to alter the surface chemistry of scaffolds. Polymer surfaces can be pre-treated with matrix proteins
(fibronectin, collagen, laminin, etc) to increase cellular adhesion, which affects scaffold biocompatibility.
Although ECM proteins show high adhesiveness and
adsorb to practically all polymer surfaces, they may
induce an immunoresponse in the host, since they are
usually prepared from animal tissues. An alternative
could be isolation and purification of these proteins,
but the procedure is time-consuming and very expensive [31].
Incorporation of small biologically active functional groups, for example oligopeptides, saccharides
and glycolipids, has also been attempted with the
aim of isolating some features of ECM molecules
associated with cell adhesion [34]. Some amino acid
sequences, such as Arg-Gly-Asp (RGD), which is the
cell-binding domain of fibronectin, play an essential
role in cell binding and mediate cell adhesion. RGD
is not exclusively linked to fibronectin; it can also be
found in several ECM proteins, such as collagen, vitronectin and laminin. In addition, Tyr-Ile-Gly-SerArg (YIGSR) and Ile-Lys-Val-Ala-Val (KVAV) laminin
sequences also show cell binding activity and appear to
mediate adhesion in some cell types [34].
Alternatively, chemical groups can be added
through plasma, which consists of highly excited
atomic, molecular, ionic and radical species [35].
Plasma treatment has been used to attach chemical
groups or atoms to material surfaces. Use of oxygen
plasma, for example, has been shown to increase
the hydrophilicity of materials [35], which in turn
increases material biocompatibility. Addition of
chemical groups usually changes surface wettability
and, consequently, improves cell adhesion [34]. Wettability can be defined as the ability of a liquid to
adhere to a solid and spread over the surface. The
surface wettability of a material is a measure of
its hydrophilicity, hydrophobicity and surface-free
energy [32], and it is associated with the crystallinity
and chemical composition of the material [36].
Studying different types of mammalian cells, in 1978,
451
Grinnell demonstrated that surface wettability affects

cellular attachment and proliferation, and adhesion
favors water-wettable substrates [37]. Biocompatibility is also a vital parameter associated with surface
hydrophilicity. One can therefore assume that, in order
to improve cell adhesion and scaffold biocompatibility,
hydrophilic materials should be used. However, this
type of material usually has poor mechanical integrity
and is often utilized as a coating for a more robust and
less hydrophilic polymer. For instance, poly(ethylene
oxide) (PEO) has been used to create hydrophilic
surfaces that become easily hydrated [32].
Physical support for the follicles

As previously stated, for folliculogenesis to occur, it
is necessary to maintain contact between GCs and
oocytes because many aspects of oocyte growth and
development are regulated by interactions with adjacent GCs [29, 30]. A rupture in the GCoocyte connection would lead to uncoordinated growth and differentiation of somatic and germ cells [38]. In order to avoid
breakdown of the metabolic link between GCs and
oocytes, follicles need to maintain their 3D structure.
Three-dimensional scaffolds would be able to effectively mimic physiological conditions, since many cellular processes in organogenesis occur exclusively in
3D [39]. Previous studies on in vitro culture of isolated human follicles have shown that preservation of
their 3D structure positively affects their survival and
growth [20, 40].
Although the scaffold needs to be able to support
the 3D structure of follicles, it should not be excessively
stiff to prevent their exponential growth. An extremely
rigid scaffold may decrease the proliferation rate of
GCs and oocyte growth, and affect actin organization
in growing follicles [41], which could lead to diminished follicular growth and even apoptosis.
Porosity
Scaffolds of appropriate size with good pore distribution and interconnectivity are essential for uniform cell seeding and distribution and tissue ingrowth.
Porosity also influences invasion and migration of cells
from the host, which positively affects biodegradation
and neovascularization of the scaffold. In addition, an
interconnected pore network is necessary for transportation of signaling molecules, growth factors, oxygen and nutrients, as well as metabolic waste removal.
Another parameter to bear in mind is follicular
diameter. Human primordial follicles have a diame-
452
ter of around 30 m and it is important to ensure

correct pore size in order to allow easy follicle penetration and distribution in the scaffold. However, one
should be cautious in this regard because very large
pores decrease the surface area available for cellular attachment, consequently slowing tissue ingrowth,
and reduce the mechanical properties of the scaffold
due to increased void spaces [10].
Bioactivity
In order to assemble an artificial ovary, the scaffold
should act as a vehicle to graft isolated follicles, ensuring preservation of their 3D structure. However, it
should also have a bioactive function, regulating the
growth of these follicles. It is known that many biologically functional molecules, ECM components and
cells interact on the nanoscale, creating a highly specialized microenvironment that is essential for proper
cell development and continued function. For this reason, in order to induce and coordinate folliculogenesis in a grafted scaffold, it is necessary to program it
to deliver bioactive molecules, such as factors that may
positively influence neovascularization, follicle growth
and development and oocyte maturation. These factors
could be added using polymeric systems, which would
allow regulation of the localization, duration, delivery and availability of different inhibitory and growth
factors [42]. To this end, several strategies have been
applied, such as multiple levels of encapsulation, noncovalently bonding bioactive factors to peptides with a
range of dissociation constants that mimic the immobilization of growth factors in the ECM [43]. Encapsulation, for example, can protect these factors from
denaturation that could occur if they were directly
adsorbed onto the scaffold, which would result in their
complete degradation in a very short space of time.
The released quantity of factors may be modulated
by the encapsulated quantity in the microspheres, by
the number of microspheres incorporated in the scaffold or by the composition of the microspheres. Thus,
microspheres containing different factors implicated
in folliculogenesis, factors mitigating ischemic damage, as well as factors involved in angiogenesis can be
tested.
Vascularization
Vascularization is a crucial element in the success of
a scaffold, required to supply oxygenation and nutrients to grafted cells and follicles and remove metabolic
waste. However, formation of new capillaries in the
material is very challenging, as it necessitates interaction of different design properties, such as choice of
material, porosity and pore interconnectivity, bioactivity and biodegradation. When follicles and/or cells
are grafted with a scaffold, they are only oxygenated
by simple diffusion, which may be limited by the construct [10]. This oxygen and nutrient deprivation may
cause cellular damage and apoptosis. An additional
concern is that neovascularization may be slower
than cell proliferation and follicle development in the
grafted scaffold, creating a higher demand for oxygen
and nutrients than the new capillaries can meet, which
can also lead to follicular death.
Several strategies can be applied to promote vascularization. As for transplantation of ovarian tissue
fragments, the scaffold can be grafted to a peritoneal
window close to the ovarian vessels and fimbria. This
window should be created a few days earlier in order
to induce angiogenesis and neovascularization in the
area destined for scaffold grafting. Alternatively, the
cortex of the ovary remaining after cancer treatment
could be removed to place the scaffold in direct contact with the medulla, an extensively vascularized area
(for a review, see Donnez et al. [1]). Grafting of autologous endothelial cells may also be an option to promote vascularization in the scaffold, as well as addition of growth factors involved in neovascularization
(bFGF, VEGF, PDGF), as previously discussed (for a
review, see Bergmann and West [10]).
Biodegradability
Ovarian follicles are exceptional in that they can grow
to around 600 times their size during folliculogenesis
(the human follicle grows from 30 m in its primordial stage to 1824 mm when it is ready to ovulate). In
addition, they recruit cells and vessels to support their
development. An ideal scaffold would need to degrade
in order to allow exponential growth of follicles, formation of vessels, and proliferation of stromal cells.
Ideally, the artificial ovary should offer an appropriate
initial environment for follicles that would be replaced
by a new ovarian-like structure after a few weeks of
grafting. The degradation rate of the scaffold is thus an
essential parameter in the success of grafting.
Although biodegradation should be conceived
with follicular development in mind, other factors
should also be taken into account. The biodegradation rate cannot be faster than cell migration and proliferation or ECM synthesis and stabilization in the
scaffold, because cells would lose physiochemical fac-
tors for tissue regeneration [10] and isolated follicles

would lose their 3D support. On the other hand, slower
degradation would inhibit cell penetration and consequently ECM formation in the scaffold [10] and
negatively affect follicular growth. Therefore, the material should be carefully selected in order to control the
desired degradation rate.
Degradation of the material is characterized by different parameters loss of molecular weight, loss of
mass or loss of mechanical strength [44] and is associated with its molecular weight, wettability and crystallinity [10]. Knowledge of these parameters is therefore rooted in the comprehension of the degradation
rate of different polymers, which is fundamental to
choosing a material that will match the desired degradation kinetics.
The scaffold should also degrade into products that
can be easily eliminated through metabolic pathways
[36]. This is very important when considering the biocompatibility of the material [36], since these products
cannot be toxic to cells around the scaffold or organs of
the lymphatic system [10]. For instance, polylactic acid
(PLA) and polyglycolic acid (PGA), which are among
the few degradable polymers approved for human clinical use by the US Food and Drug Administration
(FDA) and have a clinical application as sutures, are
known to decrease pH in the area surrounding the
scaffold during their degradation. Such acidity has
been implicated in adverse tissue reactions [44] due to
the inflammatory response of surrounding tissue.
Biocompatibility
Biocompatibility is one of the most important characteristics of a scaffold and it has been shown to be
related to some of the above-mentioned parameters,
such as the degradation rate and byproduct and material wettability. Material biocompatibility can be evaluated by measuring the duration of adverse variations in
the homeostatic mechanisms that determine the host
response [45].
Scaffold grafting inevitably causes damage to surrounding tissue, inducing an inflammatory response,
foreign body reaction and cascade of wound healing
[45]. Depending on the magnitude of the inflammatory response triggered by the scaffold, the migration
of cells such as macrophages and neutrophils to the
grafting site may have undesirable effects on the success of the scaffold. These cells, involved in the inflammatory reaction, can secrete enzymes that increase the
degradation rate of the scaffold, and molecules that
453
block activation of factors essential to the survival of

cells in the scaffold [10]. In addition, a strong inflammatory response may also trigger the repair mechanism, which would result in the formation of scar tissue and a fibrous capsule around the scaffold. This
would negatively affect its function and lead to death
of cells and follicles present within.
Biocompatibility is usually excellent with natural
polymers, such as polysaccharides and proteins, since
their structure is very similar to the native cellular
environment, and with hydrogels because of their high
water content, which also mimics the natural ECM.
Scaffold handling
Having a scaffold lacking the mechanical strength to
be handled during surgery would not be ideal. For
instance, although hydrogels have the advantage of
mimicking the natural ECM, they usually do not have
adequate mechanical strength and may be damaged or
even destroyed during handling.
Mechanical strength is affected by scaffold composition, pore size and porosity. Therefore, while
hydrophilic and very porous materials are probably the
best choice for grafting isolated follicles, the effect of
these parameters on the mechanical properties of the
scaffold should also be taken into consideration.
The tissue engineering approach

In recent decades, scientists from all over the world
have been developing strategies to assemble different
types of artificial organs and tissues. At first, the idea
was to physically replace the lost structure, but increasingly, the metabolic function of tissues has been taken
into account. This has involved various tissue engineering strategies and, in the last decade, the construction of artificial organs has emerged as a leading
domain in this revolutionary and exciting new field.
Tissue engineering is a multidisciplinary field,
incorporating different areas of study, such as biochemistry, surgery, engineering, physics and physiology, thereby combining principles of life science with
material science. It aims to create new, or restore damaged or malfunctioning, tissues or organs [46] through
the introduction of biological products (e.g. proteins
and cells) in to a synthetic or natural matrix able to
support and organize them [34]. In this context, tissue engineering has been responsible for the creation
of many different materials capable of mimicking tissues and organs.
454
Among the many applications of tissue engineering, the most important for artificial ovary technology is the possibility of having a ready-made substitute for the ovary by seeding the patients own cells
and follicles to a biodegradable scaffold, with a view
to tissue regeneration or construction of a new ovary.
The question of scaffold manufacture is therefore crucial. Fabrication approaches must not only replicate
the properties of the ovary at the macroscopic level, but
also recreate nanoscale details observed in native tissue at the cellular level. Different synthetic and natural
polymers can be used for this purpose. Although natural polymers, like hyaluronic acid, poly -glutamic
acid and collagen, exhibit high biocompatibility and
biodegradability, they may pose a risk of antigenicity and show variations according to batch production. On the other hand, synthetic polymers, such
as alyphatic polyesters, polyanhydrides, polypropylene fumarates and polyphosphazenes, offer outstanding reproducibility, the ability to control degradation
kinetics, and high mechanical strength [47]. However,
they may show low biocompatibility compared to natural substances.
To process the different materials into porous scaffolds, several techniques have been developed [46, 48
50]:
r Gas foaming A biodegradable polymer is
saturated with carbon dioxide (CO2 ) at high
pressures. The solubility of the gas in the polymer
is then decreased rapidly by bringing the CO2
pressure back to atmospheric levels. This results in
nucleation and growth of gas bubbles.
r Fiber bonding/fiber meshes This technique
increases the mechanical properties of a scaffold
by dissolving PLA and casting it over a PGA mesh,
for example. The solvent is allowed to evaporate
and the construct is then heated to exceed the
melting point of PGA. Once the PLAPGA
construct has cooled, the PLA is removed by
dissolving it again. This treatment results in a
mesh of PGA fibers joined at the crosspoint.
r Phase separation The polymer solution
separates into two phases, a polymer-rich phase
and a polymer-lean phase. After the solvent is
removed, the polymer-rich phase solidifies.
Biologically active molecules can be added to the
polymer solution.
r Melt molding One of the techniques involved in
this process involves filling a Teflon mold with
Prototype failure
Prototype failure
ANIMAL
MODEL
IN VITRO
STUDIES
Scaffold
conception:
choice of
material and
fabrication
method
Prototype
Biocompatibility
tests
Toxicity testing
for follicles and
ovarian cells
Prototype
adaptation
(if necessary)
Biodegradation
tests
Follicular survival
and development
Biodegradation
tests
Neovascularization
studies
CLINICAL
TESTS
Figure 38.1 Schematic approach to the development of an artificial ovary.
polymer powder and gelatine microspheres of

specific diameter, and then heating the mold to
above the glass-transition temperature of the
polymer, while applying pressure to the mixture.
This treatment causes the polymer particles to
bond together. Once the mold is removed, the
gelatine component is leached out by immersing
the scaffold in water, before drying it.
r Emulsion freeze-drying This process involves
adding ultrapure water to a solution of methylene
chloride with PGA. The two immiscible layers are
then homogenized to form a water-in-oil
emulsion, which is quenched in liquid nitrogen
and freeze-dried to produce the porous structure.
r Freeze drying The polymer is dissolved in
glacial acetic acid or benzene and the resultant
solution is frozen and freeze-dried to yield porous
matrices.
r Solution casting The polymer is dissolved in
chloroform and then precipitated by the addition
of methanol before the material is pressed into a
mold and heated to 4548 C for 24 h.
r Solid freeform fabrication techniques (also

known as rapid prototype) These are
computer-controlled fabrication techniques that
create complex scaffold designs, with localized
pore morphologies and porosities, as well as
incorporated bioactive molecules to suit cell
requirements. The general process involves
producing a computer-generated model using
computer-aided design (CAD) software. This
CAD model is then expressed as a series of
cross-sectional layers. The data are then fed to the
solid freeform fabrication machine, which
produces the physical model.
r Indirect solid freeform fabrication technique
In this procedure, a negative mold is generated by
one of the solid freeform fabrication techniques
and the scaffold is formed by adding the casting
solution to the negative mold using the desired
polymer. After solidification, the negative mold is
removed by dissolution.
r Particulate-leaching With this technique, salt is
first ground into small particles and those of the
455
desired size are transferred into a mold. A

polymer solution is then cast into the salt-filled
mold. After evaporation of the solvent, the salt
crystals are leached away using water to form the
pores of the scaffold.
r Electrospinning This is a process capable of
producing ultra-fine fibers by electrically charging
a suspended droplet of polymer melt or solution.
r Vibrating particle fabrication technique In this
process, the polymer is dissolved in solvent and
the solution is molded with salt particles. The
particles are dispersed using vortex and, at
predetermined time intervals, more particles are
added. The solution then evaporates under
continuous vibration and the scaffold is subjected
to heat and vacuum.
Conclusion
Having provided a comprehensive description of the
assembly of an artificial ovary and the involvement of
tissue engineering strategies, we find ourselves at the
starting point of a new technology that may be termed
ovarian tissue engineering. The aim of this strategy is to replace not only the structure of lost tissue,
but rather its function, to potentially allow endocrine
activity and fertility to be restored in cancer patients.
Ovarian tissue engineering research should explore
different alternatives, involving a variety of materials that can be tested alone or in combination, techniques that can turn these materials into functioning
scaffolds, and many other factors that may be combined to give these scaffolds optimal bioactive properties. This will require numerous in vitro and in vivo
experiments on animal models, with data quantifiable
by functional and structural endpoints (Figure 38.1).
It is therefore likely that this innovative approach will
prove very challenging, probably taking many years
or even decades to achieve successful results. However, since it may represent a viable option for reestablishing fertility in cancer patients, it should be
extensively investigated.
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458
Section 9
Chapter
39
Future technologies
Predicting ovarian futures

The contribution of genetics
Elizabeth A. McGee and Jerome F. Strauss, III
Introduction
One of the consequences of cancer therapies, including radiation and chemotherapy, is gonadotoxicity. As
effective treatments have rendered a number of malignancies curable, or have delivered long-term survival,
post-treatment fertility has emerged as an important consideration for patients and their healthcare
providers. Unfortunately, there are currently no definitive ways to limit the injurious effects of these treatments on gonadal function, other than shielding the
gonads from direct exposure to ionizing radiation.
Suppression of gonadotropin secretion may have a
protective effect in some populations, but the efficacy
of this intervention for preserving fertility remains to
be conclusively established.
The banking of germ cells or embryos prior
to treatment represent options for preservation of
fertility. Sperm cryopreservation is a longstanding
option for sexually mature males, and embryo cryopreservation is an option for some women and couples. Though recent successes with cryopreservation
of ovarian cortex or oocytes are encouraging, options
for gamete or embryo preservation are more complicated for women, and entail procedural risks and
expense. Moreover the technologies have not advanced
to the point that female options for fertility preservation are as successful in outcomes as pre-treatment
sperm cryopreservation. The decision to pursue ovarian cortex or oocyte banking is complicated by the fact
that the gonadal response to radiation and chemotherapy varies among the population, and there is, at
present, no precise way to determine who will suffer irreversible damage and who will emerge from
treatment with fertility intact, obviating the need for
pre-treatment interventions. Additionally, preserving
the ovarian germ cell complement per se, while an
important determinant of fertility, does not necessarily insure it. Furthermore, the inability to make predictions regarding the extent of post-treatment gonadal
function impacts the design of research on interventions to spare fertility. Evaluations of such interventions could be conducted more efficiently with
smaller sample sizes based on a more precise knowledge of subject risk for significant post-treatment
gonadal dysfunction. In this chapter, we provide a
framework for thinking about factors that can predict gonadal function post-cancer therapy, focusing
on genetic contributions and reviewing outstanding
issues that need to be addressed in future research
with the aim of developing patient-specific algorithms that are predictive of post-treatment fertility or
infertility.
Predicting the weather requires knowing what the
current weather conditions are in the specified location such as air temperature and humidity, what fronts
and air masses are approaching and what other conditions might affect them. Local geography and previous
weather patterns must also be taken into consideration. These are all elements that a meteorologist must
assess before predicting future weather conditions.
Using the weather prediction rubric, we can identify
four general requirements for accurately predicting a
future biological event such as preservation of ovarian
function after cancer therapy:
1. The stability or flux of the current condition must
be known (i.e. baseline fertility, germ cell
complement and rate of germ cell depletion).
2. Knowledge of what events or conditions could
change the stability or rate of flux (i.e. types of
treatment, dosing, duration).
3. The likelihood of those events or conditions
happening (i.e. epidemiological data on
459
post-therapy fertility by types of agents, dose and

treatment duration and age-related effects).
4. Potential for interactions of those events affecting
outcome (i.e. host variables including differences
in drug metabolism and action, intrinsic factors
[i.e. genes] affecting follicular complement,
co-morbidities).
The current state of prediction of

future ovarian function following
cancer therapy
Alterations of the rate of follicle loss have been
investigated in patients undergoing radiation and
chemotherapy. However, other than age of the patient
and dosing of agents, specific predictors related to
infertility have not been established. The current
American Society of Clinical Oncologys categorization of risk for gonadal dysfunction is broad and
descriptive: (1) low; (2) medium; and (3) high. The
medium-risk category encompasses risk of permanent
cessation of menses (used as a surrogate marker of
ovarian function) of between 30 and 80% [1]. The large
range of probability in the medium-risk category is
frustrating to physicians counseling patients; it also
strongly suggests that there is substantial individual
variation to ovarian susceptibility to damage by radiation and chemotherapy. Thus, while useful in understanding the effects of toxic agents on ovarian function,
the existing classification of risk is not at all useful in
predicting an individuals future ovarian function.
Genetic risk factors for early ovarian

senescence that could impact ovarian
function after cancer treatment
In the last decade there has been a body of accumulated work that has identified genes that have a role in
placing women at risk of earlier ovarian senescence. It
is plausible that these risk alleles place women at an
even greater risk of infertility after cancer treatment,
although this notion has not been experimentally evaluated. Nonetheless, the wide range of ovarian compromise seen in women who have been treated for cancer,
even with the same therapies, strongly suggests that the
individual genetic variation controlling ovarian function may play a role in determining the level of ovarian
damage.
Eggs reside in groups of cells called follicles within
the ovary. Except for late in the reproductive life span,
460
the majority of follicles are in a resting state at the primordial stage of development. The complement of primordial follicles has been called the resting pool [2]
and represents ovarian reserve or the population of
oocytes that have not yet become committed to the
path of growth and ovulation. Since follicles (and eggs)
are progressively lost from the resting pool over time
by entering the growing pool or undergoing apoptosis,
the ovarian reserve is constantly declining over time in
women. In general, women with a diminished ovarian
reserve are considered to have reduced fecundity [3].
Decay curves modeling the decline of the resting
pool of follicles have been presented in the literature
in mice and humans [4, 5]. In women a bimodal decay
was described, with a sharp increase in the rate of follicle loss at an average age of 38 years. However, a
more recent model suggests that the rate of follicle loss
or resting pool decay is one of slight constant acceleration throughout the perimenopause [6]. Recently,
investigators have explored the impact that radiation
and chemotherapy have on shifting this follicle population decay curve to the left [7]. This model describes
diminished ovarian reserve ensuing at an earlier age
after the toxic treatments.
The ability to predict the consequences of decay
in the germ cell population depends upon determining a womans current ovarian reserve, and there is
presently no reliable method to accomplish this task.
Antral follicle counts can vary in the hands of different
observers, and biomarkers such as anti-Mullerian hormone and inhibin B suffer from the lack of standardized assays and insufficient normative data. Moreover,
predicting how that reserve might change over time is
even more challenging.
There are several environmental and genetic
conditions with clearly defined deleterious effects
on ovarian reserve, including exposure to environmental/occupational toxins like 4-vinylcyclohexene
diepoxide, heavy tobacco use, specific X-chromosome
deletions and genetic variants (e.g. FMR1 permutations). When present, these might reasonably be
expected to increase risk of ovarian failure following
exposure to a gonadotoxic therapy. However, there
are large number of genes involved in human ovarian
development and follicular growth and their potential
contributions to variability in response to cancer
therapies are largely unknown. There are also other
factors that could affect ovarian function when compounded with a gonadotoxic cancer therapy, whose
impact is less well understood including exposure to
Chapter 39: Predicting ovarian futures
bisphenols, dioxins, aniline dyes, immune issues or

even body mass.
Table 39.1 Selected genes and proteins with a reported role

in follicle formation and growth in mammals
Germ cell differentiation
Control of ovarian development may

impact future fertility (Table 39.1
[3, 813])
Female fertility is dependant upon a series of critical ovarian developmental events that are controlled
by a large number of genes on autosomes and the X
chromosome. Variation in these critical genes which
impact negatively on female fertility or fecundity
might have even greater impact on women post-cancer
treatment.
During ovarian development, primordial germ
cells must establish themselves in the yolk sac endoderm and then migrate to the gonadal ridge. Once
there, the presumptive oocytes must survive and
become inhabitants of the cords of cells that will
become granulosa cells. After the primordial follicle
pool is established, it is at risk of diminution from both
external and internal factors. Follicles can undergo
destruction by toxins such as radiation or chemotherapy. Initiation of growth of larger groups (cohorts) of
follicles can occur which would deplete the primordial
pool more rapidly. However, to achieve fertility, follicles must grow in an orderly fashion in the proper
endocrinologic milieu to allow ovulation of a mature
fertilizable oocyte that can be fertilized and implant
and grow within the uterus. Normal progression of folliculogenesis is just as important to continued ovarian
function as maintaining ovarian reserve [8]. Anything
that disrupts the process of gametogenesis from the
initiation of embryonic germ cell formation to implantation of a fertilized embryo can reduce fertility.
Gallardo et al. investigated the mouse genome for
candidate genes involved in murine ovarian fertility
and identified 348 candidate genes involved in different stages of folliculogenesis [9]. No similar study has
been performed in humans, but a number of genes
have been clearly defined as being essential for human
ovarian function and normal reproductive life span.
However, a review of candidate genes associated with
premature ovarian failure as well as linkage studies was
recently presented by van Doren et al. [14].
The X chromosome role in ovarian function

Human ovarian development requires the activity of
autosomal genes and two functional X chromosomes.
r
r
r
r
r
r
r
BMP-4
Smad1
Fragilis
Stella
GATA4
PUM2
DAZLA
Germ cell migration and survival

r
r
r
r
r
r
r
r
r
r
r
c-kit
kit ligand (SCF)
SOX3
BMP/GDF-9
TGF
TNF
LIF
SDF-1
CXCR4
Laminin
Fibronectin
Germ cell proliferation

r
r
r
r
r
POG
TGF
TIAR
FMR1
PIN1
Gonadal formation and colonization

r
r
r
r
r
r
r
r
r
TIAR
LIM (Lhx9)
SF1
WT1
DAX
FIG
FOXL2
Cadherin
1 integrin
Oocyte-specific early maturation

r
r
r
r
r
r
r
r
Nobox
IGF2bp2
Maelstrom
PTEN
TSC1
Foxo3a
P27
ZP 13
Follicle growth
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
r
Slc18a2
Smad3
FSHR
HSD17b1
Cyp11a1
Hsd3b1
Ihna
Dax1
Greb1
LHx8
Nrf2
Gdf-9
Mater
AMH/MIS
Activin
BMP-15
Fog2
ESR2
References: [3, 813].
461
Although the requirement for two X chromosomes

for ovarian development has long been recognized,
the essential human X chromosome genes and their
functions are largely unknown [10]. Cytogenetic studies have yielded clues as to the location of key ovarian function genes. Terminal deletions from Xp11 to
Xp22.1 are associated with primary amenorrhea, and
deletions from Xq13 to Xq27 are usually associated
with primary amenorrhea or premature senescence
[11]. The region encompassing Xq13 to Xq26 is considered to be the critical region and this domain
has been subdivided into two subregions Xq1321 ans
Xq2327 [15].
The study of X chromosome gene variants and
mutations has yielded a number of candidates for
loci controlling germ cell complement. Among these
is the fragile X syndrome gene (FMR1) on Xq27.3,
which encodes an RNA-binding protein. Premutations
in FMR1, represented by increased numbers of CGG
trinucleotide repeats in the 5-untranslated region, are
well established to be associated with premature ovarian dysfunction.
The human orthologue of Drosophila diaphanous
2 (DIAPH2), a gene located on Xq22, encodes a protein involved in cytokinesis that, when mutated, causes
sterility in flies. Premature ovarian failure associated
with a translocation that disrupted the DIAPH2 gene
has been reported. XPNPEP2, a gene located at Xp25,
which encodes aminopeptidase P, was disrupted by
a translocation associated with secondary amenorrhea. The zinc finger gene, ZFX, located on Xp22.1,
is known to be important in murine ovarian development because heterozygous and homozygous mutations are associated with a reduced germ cell number.
A mutation in the progesterone receptor membrane
component-1 (PGRMC1) gene, located on Xq22-q24,
has been found associated with premature ovarian failure. BMP-15 located at Xp11.2 is a candidate gene
for control of germ cell complement based on known
ovine variants that result in follicular growth arrest in
the Inverdale and Hanna sheep. However, genetic evidence for a role for BMP-15 in human ovarian dysfunction is weak [16, 17].
Autosomal genes and regions of interest

in ovarian function research
Autosomal genes are also play important roles in controlling follicular dynamics. Mutations in the follicle
stimulating hormone (FSH) receptor gene (FSHR), the
462
ataxia telangiectasia gene (ATM), which is implicated

in DNA repair and cell cycle control, the homeobox
gene, NOBOX, NR5A1, also known as steroidogenic
factor-1, a transcription factor in nuclear receptor
family, and the forkhead transcription factor, FOXL2,
are all associated with ovarian dysfunction or premature ovarian failure in certain populations. Mutations
in FOXL2 cause blepharophimosis/ptosis/epicanthus
inversus syndrome (BPES), the type 1 form of which
is associated with premature ovarian failure [12].
Mutations resulting in intranuclear aggregation
and cytoplasmic mislocalization of FOXL2 are predictors of ovarian dysfunction. Polymorphisms in the
gene encoding Inhibin alpha subunit (INHA) have
been associated with premature ovarian dysfunction in
some studies, but others dispute this association [18,
19].
Mutations in the catalytic subunit of the mitochondrial DNA polymerase gene, POLG, have been
reported to segregate with premature ovarian dysfunction and ophthalmoplegia [20].
There has been one genome-wide linkage scan in a
Dutch family that identified a region on chromosome
5 as a possible locus for familial premature ovarian
dysfunction [21]. Comparative genomic hybridization
profiling in a group of 99 Caucasian women with premature ovarian failure [22] resulted in identification of
8 regions with copy number variations (1p21.1, 5p14.3,
5q13.2, 6p25.3, 14q32.33, 16p11.2, 17q12 and Xq28).
It is evident that larger genomic studies conducted in
multiple populations are needed to identify gene variants associated with premature ovarian dysfunction
and mutations across biogeographical ancestry.
Genes that have been associated with early ovarian senescence observed in isolated family groups or
individuals are listed in Table 39.2 [3, 1013, 15
20, 2329]. Although it may be assumed that women
inheriting such genes or even groups of polymorphisms might have an earlier cessation of ovarian function, no markers have yet been shown to be predictive for individual fertility. There have been several
promising recent studies correlating specific gene variants/mutations with markers of diminished ovarian
reserve [14, 23, 30]. However, prospective studies validating these correlations are lacking at this time.
Outstanding questions
In order to build a clinically useful patient specific
prediction algorithm for post-treatment infertility, we
Table 39.2 Genes associated with early ovarian senescence

in humans
Autosomal
X-linked
FOXL2
FMR1
NOBOX
GDF-9
FSHR
FMR2
AIRE
DIAPH2
ATM
XPNPEP2
POLG
BMP-15
NR5A1
MSH5
NOG
INHA
FOXEI
B-glycan
PTHB1
AR
DMC1
ESR1
PCMT1
MCKDHB
ASCL6
PGRMC1
FIGLA
DMCI
SALL4
PTEN
TGFMR3
References [3, 1013, 1520, 2329].

Bolded genes have strong evidence for a role in ovarian
senescence.
need as a foundation the answers to several questions

regarding individual determinants of fertility.
Is fertility a heritable condition that can

be predicted?
The inheritance of fertility has been studied in
discrete populations by Blum et al. who used mitochondrial DNA samples from different human population groups to determined that the degree of imbalance of gene genealogy increases with fertility inheritance [31]. That such differences could be associated
with fertility differences, suggests that genomic analysis might be useful in fertility prediction.
Although there are genes and markers that are
associated with early ovarian senescence, there is limited data about the predictive value of testing for such
information. But the discrete limit of ovarian function
does create the desire to predict its course over time.
This academic pursuit has had a renewed driving force
from cancer survivors that want to predict accurately
if they will be able to have children after their therapy.
Should they delay cancer treatment to undergo expensive and unproven therapies to hold on to the possibility of future reproduction? Or should they get on with
their therapy and know that in all likelihood they will
continue to have ovarian function after chemotherapy
or radiation?
The complexities of the events of fertility go beyond
ovarian function. External factors such as individual
choices in fertility management or choice of a partner
also affect ultimate fecundity and fertility rates [32], as
well as factors such as fertilization opportunities, uterine function and implantation events [33]. Since predicting fertility is fraught with confounding factors, it
may be more practical clinically to attempt to predict
the likelihood of infertility after agents destructive to
oocytes are administered.
Can age at normal menopause be predicted?

There are several studies underway as well as a few
recent reports identifying genetic factors underlying
normal menopausal age, considered to range from
40 to 60 years [24]. Polymorphisms of CYP1B1 and
MTHFR have both been associated with timing of
menopause [25, 26]. Linkage studies have identified
loci on chromosome X as well as chromosome 9 with
variations in age at natural menopause [24] (Table 39.3
[24, 3436]).
Two recent genome-wide association studies have
identified loci associated with influencing age at
menopause. Stolk et al. identified 6 loci in European women associated with differences in age at
menopause on chromosomes 12, 19 and 20 [34]. They
predicted a 19% risk of menopause prior to age 50 with
the loci at 19q13.4 and 20p12.3 and similar magnitude
risk for increases in age at menopause for the other
loci. He et al. analyzed patients of European ancestry from the Nurses Health Study and the Womens
Genome Health study in the USA [35]. They found
4 regions of interest, 5q32.2, 6p24.2, 19q13.42 and
463
Table 39.3 Loci associated with natural age at menopause
What is the future of future fertility?
5q
35.2
5q
14.115
6p
24.2
9q
21.3
13q
34
We have identified a number of questions and very few

answers in predicting future ovarian function. But if
we revisit the original four key elements of the weather
prediction paradigm, a framework for predicting ovarian function begins to come into focus:
19q
13.42
20p
12.3
Compiled from references [24, 3436].

Bolded loci were found in both studies [34] and [35].
20p12.3. The authors suggest that all 4 loci together

account for 2.69% of the variation of age at menopause.
Of note, two loci were identified by both studies
(19q13.42 and 20p12.3).
Clearly, factors other than just the markers
reported so far influence the age at menopause,
including environmental exposures, medications
and pregnancies. However genome-wide screening
has been able to reveal novel loci and its further
use may accelerate acquisition of knowledge of the
genetic factors involved in the reproductive life span.
Understanding which genetic factors are relevant will
likely require a combination of hypothesis-driven
evaluations based on surmised role of suspect genes
in ovarian function and non-hypothesis driven whole
genome surveys [3].
Can menopausal timing determinants

predict fertility?
Menopause on average occurs around age 52 years;
however, natural fecundity ends upon average around
age 41 years [33]. Thus, factors that control the cessation of menses may not directly correlate to an increase
or decrease in fertility. It makes sense that factors that
would result in earlier ovarian cessation would likely
also result in earlier loss of fertility [28]. If such factors result in a constant but increased rate of loss of
the resting pool of follicles, the left shift in the decay
curve proposed by Wallace might be appropriate for
predicting fertility as well as menopause. However, it
cannot be assumed that all conditions that might affect
age at menopause would necessarily also affect age at
diminution of fertility. However the predictive ability
of such proposed profiles will need to be clearly established before they can be used to advise patients clinically about risk profiles of their future fertility.
464
1. The stability or flux of the current condition

must be known. Data is rapidly accumulating
regarding serum and sonographic markers of
current ovarian reserve. These markers have not
yet been directly linked to predicting fertility, and
validation and standardization of these assays are
ongoing. Reliable assessment of an individuals
current ovarian reserve will be invaluable in
predicting future ovarian fertility.
2. Knowledge of what events or conditions could
change the stability or rate of flux. Data is slowly
accumulating, defining the diminution of ovarian
reserve from various doses of chemotherapeutic
agents though changes in chemotherapy
combinations and dosing regimens make
determinations of specific toxicities difficult.
Radiation parameters may be more rapidly
available [7].
3. The likelihood of those events or conditions
happening. Individual sensitivity to
chemotherapy toxicities is a topic under
investigation in pharmacogenomics studies.
Individual variations in chemotherapy
metabolism are just as likely to vary in ovarian
toxicity as lung, liver or bone toxicities. Specific
risks for diminished ovarian function need to be
better defined before their incidence can be
assessed.
4. Potential for interactions of those events
affecting outcome. There are no studies as yet that
report the baseline ovarian function, behavioral or
environmental risks, genetic markers and specific
chemotherapy and/or radiation treatments
correlated to fertility rates after treatment.
All of these will be necessary in establishing a useful
paradigm for risk assessment can be applied to individual patients to predict ovarian function following
cancer therapies.
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466
Section 10
Chapter
40
Ethical, legal and religious issues with fertility preservation
Psychological issues of cancer survivors

Allison B. Rosen, Kenny A. Rodriguez-Wallberg and Kutluk Oktay
Introduction
Patients whose fertility would be impaired due to
treatment for their medical condition face a daunting
task and need timely, accurate information about their
options. They need to know the short and long-term
side effects of medical treatment on their reproductive
system prior to initiating adjuvant chemotherapy. They
may require help finding fertility specialists. Many of
the currently available strategies to sustain fertility
are far from certain to succeed and fertility-sparing
options may carry their own risks. Cancer patients
may need psychological support for emotional distress
occurring because of the cancer diagnosis and/or for
psychological distress which exacerbates prior emotional difficulties. This chapter will focus on what we
know about the emotional needs of cancer patients and
others for whom fertility preservation is an option, as
well as the implications for cancer care providers. We
will discuss how to recognize psychosocial distress and
the type of communication skills that are necessary
when counseling patients. Finally, we will describe the
ethical implications of fertility preservation techniques
and provide resources for the healthcare provider.
Psychological needs of patients

undergoing fertility preservation
The rapidly growing field of fertility preservation has
focused primarily on medical aspects of treatment for
cancer survivors. Currently, less is understood about
the psychological needs of men, women and children who would benefit from fertility preservation.
Recently, Tschudin and Bitzer reviewed the literature
from 1998 to 2008 [1]. They identified only 24 studies that met their inclusion criteria on the psychological aspects of fertility preservation. Most the current
studies on patients experiences with fertility preservation have explored the issue in the context of sperm
banking [12]. At present, no studies exist to explore
systematically the emotional impact and effects of procedures such as oocyte freezing and ovarian or testicular cryopreservation [1, 36]. We do know that
fertility preservation is an important issue for cancer
patients [12, 710]. We also know that health professionals, patients and parents consider fertility preservation an important option for young cancer patients
[12, 1012]. Nevertheless, all parties involved in cancer care have information deficits and, despite the need
for counseling patients about fertility preservation, it is
not routinely offered to all patients needing it [1, 2, 8,
1318].
Individuals diagnosed with cancer during their
reproductive life span experience a life crisis in two
respects. Firstly, the diagnosis in itself may evoke emotional distress [19, 20]. Secondly, cancer treatments
may impair fertility and have profound implications
for the patients quality of life after treatment. We
know from investigations of infertility in non-cancer
patients that infertility in itself can be associated
with grief and depression, increased anxiety [21] and
reduced life satisfaction [2224]. Some patients may
experience infertility with the same emotional distress
as is typical of an AIDS diagnosis [25]. Although cancer survivors may be able to become parents though
egg and embryo donation, gestational surrogacy or
adoption, these paths to parenthood may not be available or may be prohibitively expensive. In the case of
adoption, survivors may face discrimination in their
efforts to adopt, particularly with international adoption, because of their history of cancer [26]. Many
countries, for instance, China, will not place a child
with an individual or family who has had a history of
467
Section 10: Ethical, legal and religious issues
any type of cancer, regardless of stage or years since

treatment [26]. Being deprived of the tasks of parenting can evoke profound feelings of loss [27, 28]. Thus,
the cancer care team may face an individual who is
highly distressed and the team must convey complex,
life-impacting information to an individual who has
many treatment decisions to make.
Women
A young woman with breast cancer must time her
appointments with a fertility specialist to fit with her
menstrual cycle and is often seen by her reproductive physician between surgery and chemotherapy. She
may have feelings about surgery in itself, fear losing
her life and dread losing her hair or suffer other worries about her body. She may experience the pain of
reduced self-esteem, decreased sexual feelings and she
may fear rejection from her current or future partner
[2931]. She may have had difficulty locating a reproductive endocrinologist. She must find the funds to pay
for fertility treatment along with the money needed for
cancer care, and she has no guarantee that the fertilitysparing treatments will work. A young woman without a partner may have to decide between freezing eggs
or embryos. If she chooses embryos, there is the additional issue of choosing a sperm donor and, if she has
a boyfriend, the complexity of anticipating the impact
of her choices on her current and future life. She may
not want to pick the father of her future children during this stressful time. She may worry about how pregnancy will affect her life (e.g. if she has hormonally
sensitive cancer) or the health of her children. Should
she see a genetic counselor? She may feel isolated and
alone, not part of the reproductive community and
too young for such a serious illness. If single, she may
worry about discussing her fertility status with a desirable mate, fearing rejection from her current or future
boyfriend and his family [29]. Cancer diagnosed during a young womans reproductive life may occur just
when she has worries about ever finding Mr. Right
accompanied by questions such as Whats wrong with
me? Potential loss of fertility can add to her already
shaky self-esteem and worries about her desirability
as a mate. Friends and relatives may try to reassure
with platitudes, Just be glad youre alive or You can
always adopt, not understanding the emotional complexity she is suffering. If she has children, she may
have work or have child-care pressures and may find
the many medical appointments to be a burden. She
468
may worry that she is not able to take care of her family as she would wish. For some women, the possibility
of not being able to complete her desired family size
hurts, and comments to the effect that You should
be glad you already have a child are very painful.
Although cancer usually brings couples closer, cancer
can occur during a stressful time in a couples life. If a
couple is in the midst of a stressful period, they must
decide whether or not to freeze oocytes or embryos or
both, and will this decision exacerbate their stress?
Men
In addition to anxiety and distress about cancer itself
[19], male cancer patients may worry about how
treatment will affect their masculinity, role, identity
and sexuality [29, 32]. If the patient anticipates role
changes because of cancer treatment (e.g. his partner must take care of him) these changes in domestic arrangements may affect how the patient feels
about himself and may exacerbate pre-existing couple conflict as well as negatively impacting on sexuality. Younger couples may find it particularly difficult to adapt to new domestic roles and to experiencing the many life and financial issues associated with
medical treatment [29]. Even under the best of times,
some couples find it difficult to discuss their intimate
lives with each other, and may find it especially challenging to discuss these concerns with their physicians
and medical staff. If treatment will cause infertility,
many men worry about the impact of infertility on
their present and future lives and fear for their childrens health [29].
Adolescents
Adolescence is challenging time to help young men
and women make appropriate decisions regarding fertility preservation and sperm or oocyte freezing. The
caregiver must be sensitive to the impact of the cancer on the patient. An adolescent may be concerned
about his or her body, appearance and sexuality [29].
In addition, this is a period where relationships with
peers and parents are changing and identity formation is occurring. Cancer may affect the patients sense
of value of him- or herself [28, 29]. Adolescents may
be pessimistic about their future, disillusioned with
parenthood and afraid of transmitting cancer to their
offspring [28, 29]. Balancing parental desires and the
authentic needs of the patient is a challenge. Is the
patient feeling embarrassed, afraid of failure, rejecting
Chapter 40: Psychological issues of cancer survivors
authority, etc., if he or she declines to freeze? Are

the patients parents unduly influencing decision making? Will the patients desires change in the future?
In addition, information appropriate for the adolescent patient may be lacking. Chapple et al. describe the
embarrassment and difficulty of discussions of sperm
storage with young men and their criticism of sperm
banking facilities [33].
Children
The challenges associated with fertility preservation
are even more pronounced with children. We know
parents usually want to preserve fertility for their children. Van Den Berg et al. interviewed the parents of
318 boys at the time of diagnosis and 2 years later [34].
They found that 61% of the parents would approve of
spermatogonial stem cell cryopreservation collected at
time of diagnosis. However, children may be too young
to comprehend the fertility implications of their cancer
treatment and parents may want to shelter their children from such discussions [35]. In addition, parents
may not know how to find specialized centers working with ethics board-approved research protocols for
fertility preservation involving tissue cryopreservation
for children. Presently, it is difficult for the caregiver to
counsel parents about the future efficacy of such experimental procedures.
Lesbian, gay, bisexual and transgender

Evidence based information addressing the emotional
and medical needs of gay and lesbian patients is lacking [36]. There is even less research on fertility preservation in the lesbian, gay, bisexual and transgender
(LGBT) community. The American Cancer Society
reports that approximately 7.5% of the US population
is gay or lesbian. Assuming similar rates of cancer in
LGBT patients, thousands of patients need counseling
about their family-building options. There are many
barriers to culturally competent quality health care.
Some of the barriers include lack of insurance policies covering unmarried partners, fear of or actual past
experience of discrimination by healthcare providers.
Anticipated or actual experiences with discrimination
may cause some men and women to ignore recommended screenings for breast, colon and prostate cancer, thus preventing early detection of cancers [37]. Not
every fertility program will work with a gay person or
couple. Many have policies that they will only work
with heterosexual, married couples.
Patients need timely, accurate

information
We know that the desire for biological parenthood and
genetically related children is important to cancer survivors [1, 2, 79, 11, 35, 3841]. Green et al., using
a series of semi-structured interviews with 15 young
male cancer patients, reported that all of his young
subjects found the possibility of infertility disturbing
[39]. Zebrack et al. found that 59% of a convenience
sample of 32 childhood cancer survivors between
1937 years old, who were interviewed 5 years beyond
diagnosis, reported being uncertain about their fertility status despite the fact that all of the cancer survivors
expressed a desire to have children in the future and
judged parenting and family as very important [35].
The percentage of patients with concerns about their
future fertility differ somewhat in the various surveys.
Schover et al. found 32% were concerned in a sample
of male patients from a tumor registry [10]. Zanagnolo et al. found that 57% of patients with ovarian cancer were concerned [40]; Partridge et al. found 57%
of breast cancer patients were concerned [8] and Saito
et al. found 60% of male cancer patients were concerned even though they had banked sperm [41].
Many surveys of cancer survivors have found that
patients are at risk for emotional distress if they
become infertile due to cancer treatment [e.g. 13, 5
7]. Patients (both men and women) who have not had
children prior to their medical diagnosis are more distressed about infertility than those who have already
had their children [7, 8, 11]. The desire to have children
may influence the type of treatment a patient chooses
[8] and may help the patient emotionally cope with
their cancer [41].
Yet despite the fact that patients may be very upset
about loss of fertility, there are many problems with
timely and effective communication between cancer
care specialists and their patients. The most significant barrier to fertility preservation is the fact that
many care providers simply do not convey information about fertility-sparing options to their patients [1,
2, 8, 1417, 42, 43]. Duffy et al. found that only 34% of
166 premenopausal women recalled discussions about
fertility preservation before beginning chemotherapy
with any of their healthcare providers [15]. Thewes
et al. reported that 71% recalled discussing fertilityrelated issues with their physicians, but that 45% said
that they initiated the discussion themselves [16]. Similarly, Goodwin et al. found that parents and patients
469
initiated discussions about fertility preservation themselves [44]. Partridge et al. found that 26% of women in
a web-based survey of 657 young breast cancer patients
felt that their concerns about fertility and reproduction
had not been adequately addressed at the time of diagnosis [8]. Oosterhuis et al. surveyed 97 parents of pediatric patients and 37 adolescent patients and found
that only 29% of the parents and adolescent patients
were satisfied with the information they received about
fertility-sparing options [42].
There is a debate about the meaning of lack of
recall about having been provided information (or
not) about fertility preservation, with evidence on each
side of the debate. Most simply, a lack of recall of any
discussion may indicate that information was not provided. In Duffy et al.s investigation, 34% recalled discussion about fertility-sparing options and 100% of
the patients recalled discussions about the impact of
chemotherapy on their everyday lives [15]. If the difference in recall is due to information given (but not
recalled), why do 100% of the patients recall discussing
the effects of chemotherapy on their everyday lives?
On the other side of the debate, Van Den Berg et al.
investigated 202 parents of 117 male childhood cancer
patients who had been informed about fertility during
consent [43]. Only 50% recalled statements about fertility and 36% denied receiving any information [43].
We know that anxiety and emotional distress can
impair the ability to register information [45]. We also
know that anxiety can inhibit information processing
[46, 47]. In addition, lack of information may lead to
emotional distress [4850]. Thus, a patients lack of
recall may be due to many factors, not simply due to
provider omission. Nevertheless, we know that there
are challenges to effective communication between
cancer care specialists and their patients. Quinn et al.
found that the physicians in their investigation usually
discuss fertility loss as a side effect of treatment for cancer [14, 51]. However, few actually provided specific
information for their patients to preserve their fertility.
Reasons cited by Quinn et al. [14, 51] and others [e.g.
8, 17, 52] include a lack of knowledge of options for
fertility preservation, a challenging medical setting, a
lack of time for the discussions and a lack of training in
how to discuss fertility issues [17]. Providers also indicated that gender and cancer site as well as the costs
of fertility preservation procedures and access to the
necessary resources were factors that influenced their
decisions regarding discussion about the full range of
options available [14, 51, 52].
470
At this point, there is general consensus that fertility preservation is very important to patients and
that patients affected by cancer during or prior to their
reproductive life span should be informed about possible fertility impairment due to cancer treatment [53
57]. Adult patients, parents of minors and (if possible)
minors should be provided up-to-date realistic expectations about the success rates of fertility preservation,
the cost of these procedures and referral to appropriate facilities. Parents of minors should be referred to
appropriate specialized research centers. The discussion of fertility-sparing options should address common patient concerns about whether or not fertility
preservation will decrease successful cancer treatment
or be harmful to the patient or to the patients offspring, short and long-term side effects of treatment on
the reproductive system, pregnancy risks and concerns
about the possibility of genetic risks to the offspring. In
addition, physicians and the cancer team should discuss the option of deciding not to preserve fertility.
We know that physician recommendations about
fertility preservation are very influential. Schover
et al. found that physicians encouragement to bank
sperm was almost as strong a predictor of whether
or not a patient banks sperm as the patients own
desire for future children [10]. Schover et al. noted
that the influence of provider recommendations is
consistent with what is known about the effectiveness of physician recommendations about smoking
and early cancer screening [10]. Saito et al. found
results consistent with Schover et al.s findings [41].
They interviewed 25 cancer survivors who had banked
sperm at their own initiative and 26 patients who
had banked sperm with their physicians recommendation. Patients who banked sperm with their physicians
instructions felt better about having cyropreserved
sperm. These results underscore the importance of
the cancer care teams communication and encouragement in exploring options to preserve the capacity for
biological parenthood.
Provider communication skills and

communication pitfalls
Information empowers patients to ask pertinent questions and may avoid emotional distress by providing
options and realistic expectations. Lack of information
can lead to emotional distress [48, 58, 59]. In addition,
poor communication skills can profoundly influence
the quality of life of patients. Kerr et al. prospectively
investigated the impact of patient-rated provider communication skills with 990 cancer patients [60]. They
found that 45% of these patients reported that some
aspect of communication with their physicians was
unclear and 59% of the patients wanted to speak to
their medical team more frequently. Age was a factor in patient satisfaction. Patients under 50 years of
age rated social and psychological help as more important than medical information. Patients who reported
that information was incomprehensible or incomplete
described significantly worse quality of life on 17 variables up to 4 years after diagnosis.
A number of studies have found that effective
communication significantly impacts patients satisfaction and quality of life [6170]. For instance,
Roberts et al. investigated the types of communication cancer patients find helpful [71]. They found
that the best communication and psychological adjustment occurred when physicians had a caring attitude, demonstrated empathy and spent sufficient time
with their patients. Patients found it very important that their physician engaged them in collaborative decision-making [71]. Common communication pitfalls are lecturing, stopping the patients from
addressing their own concerns, depending on routine
procedures and discussions and premature reassurance. Back et al. and others recommend assessing a
patients pre-existing knowledge and questions, providing information in small units, followed by understanding the patients comprehension and evaluation
of the information provided [72].
Thus, while discussions of response to treatment
and the types and severity of side effects is important, patients also need attention to their coping and
psychological well-being. They should be encouraged
to speak about the complex psychosocial issues they
are facing because of the cancer diagnosis and fertility treatments. Addressing these psychosocial issues is
important for achieving treatment goals and engaging
patients in their own care. Patients should be encouraged to bring trusted friends and family to provide
emotional support during and after the visit. Given the
anxiety and potential inability to register information,
friends and family can also help to process information and, if necessary, ask relevant questions. Patients
should also be provided a list of clearly written instructions and resources to help reinforce the information
given during an office visit and enhance treatment
adherence. Because of the time-sensitive nature of fertility treatments, patients should be helped in finding
fertility specialists who are familiar with the need to

fast-track cancer patients.
Emotional distress associated

with cancer
The prevalence rate of clinically significant emotional
distress (depression and anxiety) in cancer outpatients
varies considerably from study to study. Strong et al.
reviewed studies of clinically significant emotional distress of cancer patients and reports prevalence rates
from 15 to 42% [73]. The majority of studies have been
small, used different measures of depression and anxiety and had different sample characteristics. Strong
et al. investigated 3071 cancer outpatients with a variety of cancer types and found that nearly one quarter
had significant emotional distress [19]. Being young,
female and having active disease were independent
predictors of emotional distress.
Costanzo et al. wished to clarify the difference in
reported prevalence of psychological distress in cancer patients by conducting a longitudinal study of 206
patients with age, education, gender and demographically matched controls [74]. They found that survivors,
as compared with the matched controls with no cancer
history, had worse psychological functioning in a variety of areas including mental health, mood, environmental mastery and self-acceptance. Their investigation also showed that these differences existed prior to
diagnosis in the individuals who later developed cancer. They suggest that the cancer diagnosis exacerbated
prior psychological difficulties and that these difficulties may actually put individuals at risk for developing cancer. They are not suggesting that depression
and anxiety cause cancer. Rather, they hypothesize that
psychological distress may be related to poor health
behaviors such as smoking, poor diet and lack of physical exercise and that these behaviors put the individual
at risk for developing cancer.
Costanzo et al.s study is important in that it
was a prospective investigation with matched controls. However, this evidence must be weighed against
another prospective study of the prevalence of emotional distress in a normal population. In a longitudinal prospective study of depression, anxiety disorders, alcohol dependence and cannabis dependence,
Moffitt et al. report that nearly 60% of the population
experiences at least one of these mental disorders by
age 32 [75]. In their investigation Moffit et al. assessed
more than 1000 New Zealanders for mental disorders
471
11 times between ages 3 and 32. Their study focused

most intensively on the period from age 18 to 32. Most
investigations of the prevalence of emotional distress
in the USA and New Zealand rely on retrospective
self-reporting. Self-reporting may underestimate emotional distress because it relies on individuals ability
to remember and their willingness to disclose their
past experiences. On the other hand, it may also inflate
prevalence rates because researchers may assign mental ailments to people with mild symptoms of no real
concern.
Thus, psychological distress may precede, exacerbate or follow a cancer diagnosis. More research needs
to be done to clarify these complex interactions. However, we may safely say that many cancer patients suffer
significant emotional distress. The breast cancer literature suggests that younger women suffer more anxiety and depression than older women. Young women
typically report more emotional distress, more unmet
needs and worry about finances. They also report
more loss of time at work, more child-care problems
and decreased self-image when compared with older
women [7688]. A young womans stage of life may
create multiple stressors that interact with her cancer diagnosis leading to increased psychological distress [87, 8991]. Young women often experience psychological distress with the loss of menses during
chemotherapy. The loss may cause young women to
feel older and experience themselves as different from
other women their age. They may feel vulnerable and
have concerns about the potential loss of fertility [38,
92]. Even women who have completed their families
suffer from the loss of choice to have more children.
However, psychological distress is more significantly
related to infertility in women who have not yet started
their families and would like to do so [89, 92, 93].
Sexuality is often affected by chemotherapyinduced menopause. Younger women receiving
chemotherapy are at risk for lower levels of sexual
functioning, assessed by decreased libido, difficulty
reaching orgasm, vaginal dryness and reduced sexual
satisfaction. Younger women are more likely to view
themselves as less sexually attractive than they did
before therapy [94100].
Less research has been conducted on the psychosocial needs of men as compared to women [101]. Kiss
and Meryn reviewed studies of prostate and breast cancers to compare the psychosocial effects of cancer on
men and women and to compare their reactions [102].
They found that the diagnosis of cancer is distressing
472
for both men and women. Cancer site is less important

than disease stage, pain and absence of social support
[101, 102]. Both men and women benefit from social
support, though men are more comfortable turning to
their partner. Women often seek support from a larger
network of people.
Ream et al. investigated 1848 men who had been
diagnosed with prostate cancer during the previous
324 months [103]. In this postal survey, almost one
third of the men reported extreme anxiety or depression. The men also reported unmet needs for social
support. The men wanted help for their emotional distress, sexuality-related problems and treatment side
effects. The young men reported more unmet needs
about their sexuality, especially if their treatment
involved a radical prostatectomy.
More research needs to be done on the impact of
cancer on mens gender identity and sense of masculinity, particularly the interaction of such factors as stage
of life, sociocultural background and fertility status.
Green et al. investigated the psychological reactions of
young men who were infertile due to cancer treatment
[104]. The young men described intense reactions such
as depression and anger about their cancer diagnosis and treatment. Younger men who had not known
that their ability to parent a biological child would
be affected and who wished to have a child reported
the most powerful reactions. They were angry that
they had been denied a highly significant life choice;
expressed rage at the injustice of human suffering; and
were irritated they had not been informed about the
side effect of infertility.
Predictors of psychosocial distress

Green et al. studied 160 stage I and stage II nodenegative breast cancer patients to assess psychological and demographical predictors of psychological
distress [105]. Women with past histories of trauma,
previous psychological distress or those experiencing
additional stressful events, are most at risk. Younger
women with children at home are most vulnerable to
psychological suffering. The investigators recommend
that the cancer team assess psychological risk by collecting information about the womens histories. Since
cancer can trigger painful unresolved emotions, the
cancer team needs to address prior trauma such as sexual abuse. For some women, the invasive nature of the
procedures and the loss of control of their bodies can
re-traumatize them [106113].
There has been extensive investigation of coping

styles in cancer patients [114, 115]. Some coping styles
are associated with less emotional distress and better psychological adaptation to cancer. Adopting an
internal or personal locus-of-control, being optimistic
and taking a minimizing perspective are associated
with reduced emotional distress. In addition, problemsolving, having a fighting spirit, positively reinterpreting problems and seeking social support, are other
helpful strategies for dealing with cancer. In contrast,
avoidance or escapism is associated with more psychological distress. Behaviors such as wishful thinking, blaming oneself and adopting a resigned attitude
are associated with more emotional distress. Stanton
et al. studied 92 breast cancer patients and found that
actively processing and expressing emotions enhanced
physical and emotional adjustment over a 3 month
period [116].
The cancer team can provide better care by understanding the emotional needs, psychosocial predictors
of distress and methods of coping that benefit cancer
patients. It is important to note that trying to change a
patients style of coping by encouraging them to adopt
a fighting spirit or stop being fatalistic is not as
helpful as an empathic stance tailored to the emotional
needs of the individual patient [117].
Cross-border reproductive care

Although the first recorded instance of cross-border
reproductive care (CBRC) occurred in ancient Greece
over 2500 years ago, the phenomenon is rapidly growing [118]. Patients travel for a variety of reasons,
including technology advances; affordability; rapid
access to care; procedures not widely available; privacy concerns; and improved standards of care. The
cancer team needs to know common patient concerns,
whether or not they are a travel or destination country.
Patients need trusted sources of information; information on treatment options, risks and cost; international
accredited clinics; and counseling to understand the
psychosocial dimensions of their choices. Providers
also need trusted information, as well as data, on costs
and outcomes, standards of care and protocols for
advising patients on CBRC. The cancer team may be
asked such questions as, Where can I go to preserve
my fertility? Is it safe? Is the facility accredited
and by whom? Are the caregivers adequately trained
and certified?, etc. Patients need adequate counseling before departure, especially if gamete donation
or surrogacy is involved. They need information and

counseling in the destination country that is adequate
and provided in a language that is understandable to
the patient. Legal issues may be complex when traveling between countries (or even within countries). For
instance, within the USA gestational surrogacy is not
legal in some states. Referral to a lawyer experienced
in the issues in both sending and receiving countries is
very advisable.
Conclusion
Fertility preservation is a complex field involving many
specialists from different disciplines. Psychologists or
other mental health providers should be included in a
team approach to fertility preservation. Many cancer
patients would benefit from referral to a mental health
provider for additional attention and support. Mental
health providers can counsel patients about the complex decisions they are facing when considering fertility preservation. Patients may feel more at ease discussing mood changes, sexual difficulties, self-esteem
problems and other life stressors with mental health
providers than with their oncology physician or surgeon. Psychologists and social workers may have training, or know how to get information, about CBRC
and be aware of the issues involved in cultural competency. Thus, a team approach allows each care provider
the ability to stay within their professional scope of
practice. Most importantly, when the emotional needs
of patients are addressed, normalized and considered
standard of care, cancer patients may feel understood
and most fully cared for.
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Section 10
Chapter
41
Ethical considerations
Pasquale Patrizio and A. L. Caplan
Fertility preservation and ethical

considerations for adults and children
Fertility preservation is a new subfield of reproductive
medicine aimed at preserving the potential for genetic
parenthood in adults or children at risk of sterility before undergoing anti-cancer treatments. Modern and powerful chemo- and radiotherapy protocols are either curing or significantly extending the
survival for many young patients with cancer. Fiveyear survival rates for Caucasian and Hispanic American women have increased for Hodgkins lymphoma
from 86 to 98% in the quarter century before the year
2000 and for breast cancer from 78 to 91% [1]. At the
same time, diagnoses of some malignant diseases have
become more prevalent (e.g. breast and testicular cancer) [2]. The net effect has been an increase in numbers of patients in the reproductive age window (and
younger) at risk of sterilization or early menopause
by the effects of ionizing radiation or alkylating
agents such as cyclophosphamide and platinum-based
drugs [1].
As a result of this progress, quality of life issues
after cancer are emerging. Included in this quality of
life rubric is the possibility of protecting fertility from
the toxicity of these efficacious but noxious treatments.
Many strategies have been devised to pursue fertility preservation (Table 41.1) [3]. Embryo freezing
is well established but it is not always an applicable
option; for example, for women who are not married, for women requiring immediate treatment or
for young pre-pubertal girls. Others techniques like
oocyte freezing, ovarian tissue or whole ovary freezing are still considered experimental. Likewise for
men, the option of semen cryopreservation before
chemo/radiotherapy is well established. Spermatogonial harvesting and testicular tissue freezing for later
Table 41.1 Established and experimental options for

preserving fertility
Established
Ovarian translocation (oophoropexy) to avoid a radiation field
Radical trachelectomy for cervical cancer
Cryopreservation of spermatozoa
Cryopreservation of embryos
Experimental
Cryopreservation of oocytes
Cryopreservation of ovarian tissue
Cryopreservation of whole ovary
Folliculogenesis in vitro
In-vitro oocyte maturation
Cryopreservation testicular tissue
Cryopreservation spermatogonial cells
transplantation or even xenografting are highly experimental.

In using both therapeutic and experimental techniques informed consent is essential. In presenting the
option of therapies, women and men have the right to
know their options concerning fertility preservation
and the risks and costs involved. Consent to therapy
may require involving a surrogate decision maker in
the case of young children or mentally impaired persons. Providing thorough informed consent in recruiting persons to participate in research is the foundation of the ethical conduct of research. It is based
on three components: adequate, comprehensible information; a competent decision-maker; and a voluntary
decision process. Research should also be reviewed
and approved by Institutional Review Boards. Patients
have the right to know what will happen if they or
479
any children that are created are injured or disabled in

terms of health insurance and compensation. Unfortunately, much of the existing literature on informed
consent using reproductive technology has focused on
information disclosure with an eye toward minimizing
professional liability. Those involved in using experimental techniques must focus not simply on disclosure
but on comprehension. The use of quizzes and documenting responses to questions after information is
presented, are effective tools to assist in documenting
that patients understand the experimental or innovative nature of some modes of fertility preservation.
Adults
From an ethical standpoint, the key reason for pursuing fertility protection is to restore personal autonomy to those who are unable to conceive [4]. However, since many of the technologies are innovative but
yet highly experimental, it is difficult to design clinical trials: how to provide a proper informed consent
and insure respect for autonomy. Who to include or
exclude in trials of innovative techniques and how best
to recruit them? The presentation of risk information
is complicated by the fact that both the adult and their
offspring may be involved. A core principle of medical
ethics is to do no harm. If ovarian tissue or testicular
tissue cryopreservation is to be tested, then the level of
risk that can be tolerated should require essential careful animal studies and close oversight of research by
review committees. It is reasonable in the absence of
grant funds to seek reimbursement from patients to
cover the expenses of the research, but there should
be no charge for clinical fees until the experimental
options have been proved safe and effective.
Ideally the decision about who is candidate for
fertility preservation should be rendered by a team
including a medical oncologist, reproductive endocrinologist, a pathologist and a psychologist, all guided
by written protocols which can be shared with patients
[4]. Patients should not be provided with false hopes,
and alternative plans including no intervention with
the prospect of adoption or childlessness should also
be part of the discussion. Equity or ownership interests in novel technologies utilized in research must be
disclosed to potential subjects.
Children
Impaired future fertility is another possible consequence of exposure to cancer therapies even for
480
children. This risk may be difficult for children to

conceptualize, but potentially traumatic to them as
adults. Unfortunately, the modalities that are available to children to preserve their fertility are limited
by their sexual immaturity and are essentially experimental.
For boys who cannot produce mature sperm, harvesting and cryopreservation of testicular stem cells
with the hope of future autologous transplantation
or in vitro maturation represents potential methods
of fertility preservation. For girls, isolation and cryopreservation of ovarian cortical strips/primordial follicles followed by in vitro maturation of gametes
when fertility is desired is a possible option. Extensive research is still required to refine these modalities
in order to safely offer them to patients as therapies
[4]. Assisted reproductive technologies (ARTs) must
be scrutinized on the basis of efficacy and safety and
they must be subjected to rigorous ethical deliberation
by independent review committees before they can
be offered. The modalities involved in fertility preservation of young children are no exception to these
rules. In addition to ensuring that the basis for offering
the intervention is scientifically sound, the execution
of the intervention must be deemed ethically sound.
This determination requires that the intervention in
question be evaluated within an ethical framework
that considers it in terms of beneficence, respect for
persons (autonomy) and justice [5]. It can be argued
that fertility preservation aimed at children is ethical
because it prevents morbidity (reproductive and psychosocial) and it safeguards their reproductive autonomy [6]. Therefore, the main ethical question concerns
the process involved in achieving fertility preservation
and the techniques required. The answer is found in an
exploration of the potential risks of the intervention to
the patient and his or her progeny, the special situation
of children as research subjects and patients, and the
potential abuse of the technologies in the future [6, 7].
Programs must make every effort to minimize financial barriers to access for children and to work with
patient advocacy groups to seek coverage for children
and families who cannot afford to participate in fertility treatment or research.
Children represent a unique and vulnerable population with respect to medical research. They have
diminished autonomy, diminished capacity to understand risks and benefits of research objectives and lack
the ability to provide consent for research studies. As a
result, they require special protection against potential
Chapter 41: Fertility preservation ethical considerations
violation of their rights that may occur during research

investigations [5, 8]. Until very recently, institutional
attitudes impeded significant participation by children
in medical research for fear of exploitation [5]. This
attitude was attributed to several historical episodes
of the unethical targeting of children as medical
research subjects. Ethical guidelines to protect children as research subjects were outlined with the publication of the Belmont report in 1979, generated by
the National Commission for the Protection of Human
Subjects of Biomedical and Behavioral Research. Children should not be exploited to participate in pediatric research, nor should they be deprived of the benefits research has to offer because of their vulnerable
status. Research involved in childhood fertility preservation should be conducted on patients who could
experience personal benefit from the research, eliminating the prospect of exploitation for the gain of
others [4].
With respect to childhood fertility preservation,
proper attainment of informed consent from a legally
authorized representative (i.e. parent or guardian) and
of childhood assent must be ensured [5, 8]. Assent
the active affirmation by the research subject can
be obtained from incompetent minors and it should
be obtained from children whenever possible. While
the benefits of gamete cryopreservation are promising they are largely unquantifiable because human
data on the survival of gametes after the freezethaw
transplant process is limited. Until more data becomes
available we cannot tell patients what percentage of
gametes will survive and what the probability of conception is, and we must not provide them with false
hope. Alternatives to gamete cryopreservation should
be discussed and patients should be given the option
of no intervention [6]. Barriers to the consent process
for fertility preservation interventions may develop.
While parents may be competent to consent for their
children, the scenario is very complex clinically and
emotionally. It has been suggested that to overcome
some of the practical obstacles involved in the consent process, it should be performed in stages [9, 10].
If a two-stage process is adopted, the issues of gonadal
harvesting/storage and gamete manipulation can be
handled as two separate topics at distinct time points.
The decision to harvest gametes would be made at the
time of cancer diagnosis and consent for the procedure would be left to parents/guardians. The decision
of whether to use the gametes after they have been
isolated can then be made at a future point by the
child when they reach adulthood. At such a point in

time, the young patient would be better able to express
personal preferences about the handling of the tissue
based on an enhance capacity to understand the ramifications of the possible medical interventions available at that time.
Sterility after cancer and use of

donor gametes
Cancer survivors who did not preserve fertility and
became sterile after chemo- or radiotherapy may agree
to the use of donor gametes (oocytes or spermatozoa).
The pre-eminent ethical issues here are: (1) Should
couples resorting to the use of donor gametes be
obliged to disclose such use to their children once they
reach the age of understanding [11, 12]? (2) Should
the utilization of donor gametes follow policies analogous to those governing disclosure to children that are
adopted [13]? (3) What will be the welfare of a child if
he or she finds out that a gamete donor participated
in his or her creation and this was kept as a secret?
(4) Should a womans age and life expectancy factor
into a clinic policy concerning access to services? (5)
How hard should a clinic try to establish what parenting arrangements have been made in the event of the
death (or cancer recurrence) of the would-be mother
or father, since little is known about the capacity for
post-cancer women to parent infants and toddlers?
Without clear guidelines the ethical propriety of
the technologically driven options for parenting after
cancer (with donor gametes), becomes a matter of the
marketplace.
Mandating disclosure by a couple is problematic
on a number of fronts and the claim that the practice
of gamete donation is similar to adoption is not obvious [13]. With gamete donation, one of the parents
is the biological parent, while the other is the social
parent; with adoption, both parents are social parents.
With gamete donation the childs mother, whether biological or not, carries the pregnancy with all of the
accompanying psychological interplay. In the case of
a donor egg, to ask such a mother (and she is a mother
under any definition of motherhood) to tell the child
that technically she is not the mother because she is
not genetically related may strain the bond between
mother and child [13].
Certainly the pre-eminent concern is the welfare
of the child [14]. However, the benefits accruing to a
child from disclosure are difficult to assess and still
481
remain open to many interpretations. In debates about

child welfare with adoptions, researchers and policy
makers have not yet provided a clear consensus on
what is the best interest of the child [14]. In addition
to considerations of child welfare, one must address
the impact on the other participants in the process,
including the donor, the couple and the healthcare
providers. A donor has to be willing to donate his
sperm or her oocytes knowing that later he or she can
be identified; the couple has to agree in revealing the
origin of the gametes to their offspring; the providers
have to restrict reproductive services only to couples
who agree in writing to disclosure. Without uniform
requirements for the collection and maintenance of
records, the usefulness of revealing information is not
clear.
To force fertility specialists to participate in implementing disclosure is ethically problematic as well.
Including acquiescence to disclosure as part of screening criteria or recruitment to protocols may unjustly
interject social factors into criteria for program acceptance. It may also encourage desperate couples to lie
if they disagree. Since relationships vary greatly with
background, ethnic origin, and attitudes of both the
male and female partner, it would not be unreasonable
for a healthcare provider to raise the issue of informing
offspring of his or her genetic background at the time
the use of donor gametes is considered by a couple.
To do more than to raise the issue and present known
data on the potential psychological and health ramifications of disclosure and non-disclosure, could violate
the privacy of the couples relationship and interfere
with their ability to decide. Programs that use donor
gametes should, however, prepare policies on how to
handle in future requests of information from children of donor gametes. They may choose to disclose
only non-identifying information when the requesting
party has reached 18 years of age, or they may disclose
more based on the particular willingness of donors
to remove more of their information from anonymity
[15].
In countries where the identification of the donor
is required by law, such as the Netherlands, Sweden
and the UK, the pool of sperm donors has substantially decreased and many clinics have ceased using
this option. Despite this concern, legislation in the
UK enacted in April 2005 imposed mandatory disclosure for donor sperm. As a result of donors unwillingness to be identified, there has been a marked
reduction in the frequency of donor inseminations.
482
A BBC survey in September 2006 found that 90%

of UK sperm donors were recruited in just 10 of 87
licensed clinics for donor insemination and that after
the removal of donor anonymity, the cost of purchasing sperm rose very substantially; about an eightfold
increase. Between 2004 and 2006 there was a 30%
reduction in patients requiring donor insemination
but a much larger, about 45%, reduction in the number
of treatment cycles using donor sperm (http://www.
geneticsandsociety.org/search).
Pre-implantation genetic diagnosis

for designer babies
The ethical ramifications of pre-implantation genetic
diagnosis (PGD) are essentially related to the issue
of requesting embryo biopsy for non-medical indications such as family balancing (also known as gender
selection or preconception sex selection) and HLAmatching (also known as designer babies). Simply
stated, is it permissible to select embryos based on
HLA compatibility? And if not, what are the moral
justifications to deny it? Is it morally justified to create embryos and then transfer only the ones HLAmatched with an existing sibling, so to design a perfect tissue-donor? The medical reasons clearly offset the moral arguments of gender selection for the
prevention of gender-specific genetic disorders (e.g.
Hemophilia [XLR], muscular dystrophy or incontinentia pigmenti [XLD]). But in the absence of clear
medical indications like PGS for family balancing
the moral arguments to deny this service become
stronger. Using procreation and reproductive technologies as a means to save another sibling life could
be seen as exploitation. Creating a child (savior)
whose own value and identity could later be affected
by the act of being an organ donor for their sibling requires a full psychological evaluation of the
requesting families and the risks of instrumentation
and exploitation, albeit potential, need to be fully
addressed.
Currently, only a minority of couples seems to give
importance to the sex of their children and even less
seem to be willing to use the service of preconception sex selection for non-medical reasons [16]. It is
important however to establish ethical guidelines and
to address, for both HLA-matching and for gender
selection requests, the fates of the non-HLA matched
embryos and of the embryos of the undesired gender.
Ethical aspects of ovarian

cryopreservation and
re-transplantation
Ovarian tissue cryopreservation, thawing and transplantation, either as heterotopic or orthotopic allografts, has shown some reproductive success [1722].
In addition, many births have also been reported by
using fresh ovarian transplants between monozygotic
twins [23]. Taken together these reports of pregnancies and birth of healthy children (10 from the use
of cryopreservedthawed ovarian tissue at the time
of this writing), bode well for fertility preservation.
There is also the report of a birth from the transplant of a whole fresh ovary between two sisters
HLA-compatible [24]. However encouraging, these are
preliminary results and the technique requires more
follow-up.
The time is now for ethical considerations of ovarian cryopreservation and re-transplantation compared
to the other two options of oocyte and embryo cryopreservation.
Preserving ovarian tissue may result in less intervention both before and after re-transplantation (the
ovarian tissue could contain thousands of oocytes) and
can create more opportunities to conceive than oocyte
cryopreservation. Ovarian tissue harvesting does not
require cycles of hormonal stimulation as is needed
with oocyte extraction. Furthermore, once the ovary
is re-transplanted, pregnancies can follow within a
womans own natural endocrine cycle without the need
of in vitro fertilization (IVF). In contrast, with oocyte
cryopreservation each cycle of ovarian stimulation
usually yields 1015 oocytes, thus to bank an adequate number of oocytes (to ensure likelihood of a successful pregnancy in the future), multiple hormonal
interventions are required.
Lastly, faith traditions may favor ovarian tissue
transplantation over IVF because it is simply a reintroduction of the womens own ovarian tissue and
permits natural conception in vivo. This procedure
may therefore hold great promise for women whose
faith traditions prohibit other fertility treatment methods, though a thorough evaluation of reception of
ovarian cryopreservation by faith traditions has yet to
be performed.
Some disadvantages of ovarian cryopreservation
and re-transplantation, when compared to oocyte
cryopreservation and embryo cryopreservation, can
be anticipated. First, there is no opportunity to perform PGD without further intervention. Embryo and
oocyte cryopreservation can both provide an inherent option for PGD. As identifiable genes for diseases and phenotypic traits continue to be uncovered,
the option of PGD may become increasingly desirable. Though IVF could be performed and PGD could
be done post-transplantation of ovarian tissue, this
would require an extra step and an extra invasive
procedure.
The option of designing ones child may overshadow the positive aspects of ovarian cryopreservation and re-transplantation, especially if there is a family history of genetically transmitted disease. Ovarian
cryopreservation and re-transplantation could be considered unpredictable regarding which traits the child
inherits.
Second, ovarian tissue cryopreservation and retransplantation presents a multifaceted potential for
false hope. False hope in medicine can be defined as
based on a set of unrealistic expectations, encouraged through incomplete or faulty information or by
a patients unwillingness to acknowledge the limits
of medicine [25]. Currently, there is no guarantee
that ovarian cryopreservation and re-transplantation
will result in future offspring, just as in embryo and
oocyte cryopreservation. A reasonable success rate
should be established prior to the mainstream offering of this technique as non-experimental. The procedure should only be offered with a clear statement
of risks and benefits [26]. As this technology is further developed, a romanticized view of forever preserving fertility is imaginable, which may place unrealistic expectations on this technology. This reality must
be clearly indicated in order to avoid major disappointments. Though any new procedure has the potential for false hope, it is important to acknowledge the
limitations of the procedure to the best of ones ability, especially when the hope is for the creation of a
new life.
Future: stem cell research and the

manufacturing of gametes
Cancer survivors that have failed to preserve their
gametes prior to sterilizing treatment might benefit in
future of a type of stem cell research aimed at the creation in vitro of gametes derived from embryonic stem
cells [27].
483
The process would require the following steps:

(a) Use of a somatic cells nucleus to be inserted into
an ooplast (i.e. an oocyte from which its own
nucleus has been removed).
(b) Mechanical activation to induce cells division and
growth to blastocyst stage.
(c) Extraction of the inner cell mass, isolation and in
vitro growth of cells destined to form germ lines
(sperm or oocytes).
The subject of research on embryos created
through IVF presents a variety of ethical and legal
issues. The central part of the debate is the moral status of the embryo [27]. This debate is not unique to
the twenty-first century scientist or bioethic scholars; in fact it can be traced to Aristotle, who wrote
of the ensoulment of the human at a particular stage,
as did the pre-Socratic philosopher Heraclites before
him. Religious views of conception have been extensively debated in Judeo-Christian and Muslim scholarship dating to the earliest religious texts in those
traditions. Views on the moral status of the human
embryo normally take one of the following three
forms:
1. The human embryo has no intrinsic moral status;
it derives its value from others.
2. The human embryo has intrinsic moral status,
independent of how others value it.
3. Embryos begin with little or no moral status and
continue to achieve more and more status as they
develop.
The position that an embryo has no moral status can
be argued in several different manners. Because the
fetus fully depends on the pregnant woman for development, many ethicists believe that it cannot be viewed
as an unique entity. The moral concerns expressed by
those who hold this position about embryo research
are focused on the long-term social implications of
embryo research for the status of born persons, particularly those with disabilities. However, it is not held
that the destruction of an embryo is inherently morally
problematic.
The position that the fetus has intrinsic moral status is grounded in the view that a person is created
at a moment in time that can be linked both to the
consummation of an act by those who participate
in its creation, and to the physical and legal initiation of that persons participation in the human community. The metaphor most often used to describe
484
the status of the fetus for these purposes is that of

baby; the ever-increasing presence of the fetus in public and private life has contributed to the view that
from the moment of conception a person can be
identified, independent of the risks that face a person so defined, and regardless of the plain differences
between such a person (for example in the case of
a frozen embryo) and a person who participates as
a baby, child or adult in the institutional life of the
community. Given this view of conception and the
embryo, the use of an embryo for research purposes
is exactly tantamount to the use of any other vulnerable subject in research without consent, research
that poses not only a great risk but in many cases
has the clearly anticipatable outcome of death for the
subject.
The moral issues surrounding embryo research
leave the status of the embryo highly contested. The
lack of consensus about the status of the embryo and
the morality of research has resulted in what might be
somewhat contradictory and unclear legal definitions
in the USA at the state and federal level. Since it is
extremely difficult to define the status of the embryo
and the question still remains hotly contested, most of
the legislation tries to steer away from making a definitive statement.
The legality of embryo research also varies from
country to country. Experimentation on the embryo
for the purposes of developing stem cell and other
technologies, and for general knowledge, is legal in
the UK, Singapore and three Australian states under
certain circumstances. In Germany and Italy, embryo
research is banned completely. In Switzerland it is
highly restricted. In the USA, debates over the legality
of embryo research vary from state to state, with California having the most tolerant policy and Louisiana
among the most restrictive.
Whatever its religious or scientific underpinnings,
the ethical debate surrounding human embryonic
stem (hES) cells has recently centered on how the hES
cells are derived and on whether or not they should
be protected from destruction, much like an adult is
[27]. Using leftover IVF embryos for the purposes
of hES cell research raises complex questions about
the status of the embryo, the value of human life,
and whether there should be set limits regarding the
interventions into human cells and tissues. Furthermore, questions about adequate informed consent,
oversight and regulation also come prominently into
play.
Those who support hES cell research argue that an

embryonic stem cell, even though it is derived from
an embryo, is not itself an embryo and thereby would
never continue to develop into a fetus, child and adult.
Each stem cell is only a cell that can be triggered
to become a specific kind of tissue yet could not be
triggered to become an individual. Furthermore, the
embryo at the blastocyst stage has not developed any
kind of nervous tissue and thus extracting individual
stem cells would not be painful for the embryo. Since
the embryos used for stem cell research come mostly
from the leftover IVF embryos, which would otherwise be discarded, the proponents of stem cell research
argue that it is better to use such embryos to find cures
for debilitating diseases rather than to discard them,
benefiting no one.
One attempt to resolve the debate over stem
cell research involved the suggestion that researchers
might obtain stem cells from embryos without actually engaging in the destruction of those embryos
[28]. (It also was suggested that totipotent cells might
be removed from four- or eight-cell pre-implantation
embryos destined for PGD [28]).
Another central problem is the permissibility of
making embryos specifically for research purposes.
There are two different types of embryos used: those
classified as spare embryos which are left over from
unsuccessful in vitro fertilization and those produced
specifically for purposes of being tested. Some people
have ethical concerns about both of these methods;
however, those who support research are more likely
to question the ethical nature of the second of these
two alternatives.
The argument that it is acceptable to use spare
embryos but not to create embryos specifically for
that purpose centers on Kants categorical imperative,
specifically the formulation of that imperative that
centers on the claim that the ultimate moral wrong
is to treat someone as a means to some other end,
rather than as an end in him- or herself. Those who do
not support the use of embryos for the sole purpose
of enhancing research argue that it is morally unacceptable to use embryos for scientific purposes on the
grounds that this is a clear use of a person as a means.
Some of these same arguments can apply to the use of
embryos under any circumstances. In the case of spare
embryos, by contrast, many are too old or morphologically inappropriate to be implanted, and thus have
no other use; it is thus argued that the use of these for
research is not nearly as questionable [28].
Implications for ART clinics

The processes whereby embryos are created (whether
from donor eggs and/or sperm intended for research
purposes or as a byproduct of reproductive health
care), analyzed, stored, thawed or destroyed, are all
processes that require, the technologies, clinical expertise, patient population and institutions of ART. It is
thus no surprise that the largest research programs
in the field have employed reproductive endocrinologists, biologists, ART psychologists and social workers. Ethical issues related to participation in stem cell
research include three key problems.
1. First is the question of whether and under what
circumstances patients or research subjects should
be allowed to participate in the donation of
reproductive materials for stem cell research,
particularly where that research involves the
creation of embryos for research purposes.
2. Second is the question of whether reproductive
clinicians and technologists should be involved in
the non-reproductive use of cloning technologies
for the creation of nuclear transfer-derived stem
cells.
3. Third is whether and when clinicians involved in
the derivation of embryonic stem cells should be
held responsible for the failure of those cells in
clinical trials or therapies using those cells.
At this time, there is no real consensus about any
of these issues, although all three issues continue
to receive the attention of the ethics boards of professional societies, such as the American Society
for Reproductive Medicine (ASRM), and of bioethicists.
Conclusions
If they have lost their reproductive function, cancer survivors may wish to become parents by using
previously stored gametes or gonadal tissue. Fertility preservation serves such a wide range of medicosocial circumstances, some quite unique, that patient
care requires an individualized and multidisciplinary
approach. In particular, fertility specialists offering fertility preservation options to cancer patients should
be properly trained and knowledgeable to discuss
patients treatment plan, prognosis, as well as unusual
health risks for future offspring and the potential
harmful effects of pregnancy.
485
Overall there should be no ethical objections to

offer these services since they are offered with the aim
of preserving future fertility.
In practice, however, there are objections:
1. The options available, except sperm storage and
embryo cryopreservation, are all experimental.
There is lack of extended follow-up about their
safety.
2. Posthumous use of stored tissue or gametes.
When gametes or tissue is stored for later use,
written directives for posthumous use may be
given effect, and subsequently born children may
be recognized as legal offspring of the deceased.
Post-mortem reproduction with stored gametes or
tissue should be honored when the deceased has
given specific consent; programs storing gametes,
embryos or gonadic tissue from cancer patients
should be informed of the options for making
advance directives for future use. Whether
posthumously conceived or implanted offspring
will inherit property from the deceased or will
qualify for government benefits will depend on the
law of the jurisdiction in which death occurs [29].
3. Concerns about the welfare of offspring resulting
from an expected shortened life span of the
parent. This concern, however, should not be a
sufficient reason to deny cancer survivors
assistance in reproducing. Although the effect of
the early loss of a parent on a child is regrettable,
many children experience stress and sorrow from
other circumstances of their birth. The risk that a
cancer survivor will die sooner than other parents
does not impose an appreciably different burden
than the other causes of suffering and
unhappiness that persons face in their lives.
Protecting such children by preventing their birth
altogether is not a reasonable ground for denying
cancer survivors the chance to reproduce [29, 30].
4. Concerns about the welfare of children born using
gametes frozen after chemotherapy already
started.
5. Reseeding of cancer after transplanting
cryopreserved tissue.
Future successful production of germ cells de novo
could have applications in fertility preservation. Sterile
gonads would no longer limit reproduction as it will be
possible to produce artificial gametes by dedifferentiation of somatic cells.
486
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Hope. New York: Oxford University Press, 2007.
26. Lee S, Schover L, Partridge A et al. American Society
of Clinical Oncology recommendations on fertility
preservation in cancer patients. J Clin Oncol 2006;
28(18): 291731.
27. Mc Gee G, Patrizio P, Kuhn V and Kraft-Robertson
C. The ethics of stem cell therapy. In: Patrizio P,
Guelman V and Tucker M (eds.), A Color Atlas for
Human Assisted Reproduction, Laboratory and Clinical
Insights. Philadelphia PA: Lippincott, Williams and
Wilkins, 2003: pp. 297309.
28. Caplan AL and Patrizio P. The beginning of the end of
the embryo wars. Lancet 2009; 373: 10745.
29. Robertson JA. Procreative liberty, harm to offspring,
and assisted reproduction. Am J Law Med 2004; 30:
740.
30. Robertson JA. Cancer and fertility: ethical and legal
challenges. J Natl Cancer Inst Monogr 2005; 34: 1046.
487
Section 10
Chapter
42
Legal aspects of fertility preservation

Nanette R. Elster
Introduction
Give me children or else I die.
(Genesis 30:1)
This quote is even more poignant when discussing the
reproductive planning and options of a cancer patient
or cancer pre-vivor. For purposes of this chapter the
term pre-vivor refers to one who does not yet have
cancer but has an increased risk or predisposition to
having cancer, such as an individual who is identified
as having a BRCA1 or BRCA2 mutation. The imperative to have children is something that can be both psychological and biological, and it may be further exacerbated by ones own confrontation of mortality. Recent
scientific advances have made the once remote possibility of conception following cancer treatment more
tangible; however, these advances are not without risks
including legal and ethical risks.
Learning that one or ones child has cancer or that
one is at a greatly increased risk of developing cancer is life altering. Parents, patients and/or caregivers
are bombarded with innumerable treatment decisions,
many of which need to be made urgently with little time for deliberation. As such, meaningful discussion about fertility preservation often does not occur.
In fact a study published in the Clinical Journal of
Oncology Nursing of nurses attitudes regarding fertility preservation revealed that patients may be strongly
focused on survival and their cure and may not want
to think about other issues . . . [1]. According to a survey by Carlson, the vast majority of surveyed physicians discuss the potential for infertility with patients
receiving cancer treatment; however, fewer than 25%
responded that they made a referral to a reproductive specialist or provided educational information [2].
Even more striking in the survey was that only 38% of
respondents knew that the American Society of Clinical Oncology (ASCO) has developed guidelines on fertility preservation [2]. Another study, published in the
Journal of Clinical Oncology found that fewer than 25%
of men bank sperm and the most common reason for
not doing so was that the option was not made known
to them [3]. Nearly one half of the men in the study
had no recollection of any discussion about infertility before beginning their cancer treatment [3]. What
is not clear is whether their lack of recollection is the
result of not being provided with the information or
the result of the stress and strain of having to cope
with a cancer diagnosis and make immediate decisions directly impacting their cancer treatment. This
research does suggest, however, that more education
of and communication with patients about potential
infertility resulting from treatment as well as fertility
preservation options is necessary.
Advances in reproductive technology and oncology have made what was once impossible, possible,
by allowing young cancer patients extended periods
of survival and thus enabling them to contemplate
a future with children. Cryopreservation of sperm
has been an available option for men since the mid
twentieth century [4], but cryopreservation of oocytes
has only become an option for women in the last
decade or so and is still considered largely experimental despite the proliferation of publicity regarding
it. According to the American Society for Reproductive Medicine (ASRM), [a]lthough currently investigational, . . . oocyte cryopreservation holds promise for
future female fertility preservation, . . . [5]. Unlike
collection of sperm for cryopreservation, collection
of oocytes carries more risk and requires more time
potentially delaying cancer treatment.
Numerous other treatments or experimental procedures are also available. According to the ASCO,
488
Chapter 42: Legal aspects of fertility preservation
current options for fertility preservation in males

include sperm cryopreservation, gonadal shielding
during radiation, testicular tissue cryopreservation
and testicular suppression; and for females, embryo
cryopreservation, oocyte cryopreservation, ovarian
cryopreservation and ovarian transposition are some
of the available means of fertility preservation [6]. The
availability of any given option, however, may be limited by a number of factors including age of the patient,
risk of the procedure, prognosis of the patient as well as
accessibility to and cost of the procedure. Each of these
potential limitations has legal implications as well.
The majority of legal issues related to fertility
preservation revolve around decision making of the
patient or the patients guardian: informed consent
for both adult and minor patients; participation in a
research protocol or undergoing a more standard medical procedure; and future use and disposition of preserved gametes and/or reproductive tissue, including
the potential necessity for female patients to utilize
the services of a gestational surrogate. These issues
are all related to the standard of care for the healthcare professional and thus raise questions of potential
liability. Another significant legal issue that impacts
patients, regardless of age or gender, is the question
of insurance coverage. In this chapter each of these
issues will be discussed for both adult and minor
patients.
Informed consent
Informed consent is the embodiment of respect for
persons and reflects both a bioethical and a legal construct, applying to both treatment and research. The
essential elements of informed consent are the same in
both the treatment and research context, and include a
discussion with ones physician or investigator regarding the risks, benefits and alternatives to a particular
treatment or protocol. Informed consent is not merely
a signed document but rather an ongoing dialogue, the
purpose of which recognizes that adults are entitled to
accept or reject healthcare interventions on the basis of
their own personal values and in furtherance of their
own personal goals [7].
For those unable or incapable of this level of selfdetermination, such as minors, parents, acting in the
childs best interest, will be called upon to make such
determinations. The sections that follow will discuss
the issues of informed consent in both research and
treatment for both adults and minors undergoing can-
cer treatment in which their future fertility might be

compromised.
ASCO guidelines
In 2006, ASCO recommended that:
As part of education and informed consent before cancer therapy,
oncologists should address the possibility of infertility with patients
treated during their reproductive years and be prepared to discuss
possible fertility preservation options or refer appropriate and interested patients to reproductive specialists [6].
Unless this is part of the discussion between the

physician and the patient or patient representative,
it cannot and should not be assumed that the patient
is aware of any potential impairment of his or her
fertility, let alone any measures that might be taken
to preserve his or her fertility if warranted by the
patients prognosis. Once made aware of this possibility, however, the patient should then be provided
with information about what, if any, means might be
available for preserving fertility and how any such
interventions may or may not impact his or her cancer
treatment. The ASCO suggests that it is the role of the
oncologist to [a]nswer basic questions about whether
fertility preservation options decrease the chance
of successful cancer treatment, increase the risk of
maternal or perinatal complications, or compromise
the health of offspring [6]. Referrals to reproductive
medicine specialists and/or a mental health provider
may also be necessary or advisable [6, 8]. Despite
this recommendation, however, in one recent study
most oncologists in academic centers discuss the risk
of infertility with female patients, but rarely make a
referral to a reproductive endocrinologist [9].
Treatment adults
The legal principle of informed consent was solidified
nearly 40 years ago by the US Court of Appeals for
the District of Columbia in the seminal informed consent case Canterbury v. Spence, which delineated what
ought to be disclosed to patients. In that case the court
held that:
True consent to what happens to ones self is the informed exercise of
a choice, and that entails an opportunity to evaluate knowledgeably
the options available and the risks attendant upon each. The average patient has little or no understanding of the medical arts, and
ordinarily has only his physician to whom he can look for enlightenment with which to reach an intelligent decision. From these
almost axiomatic considerations springs the need, and in turn the
489
requirement, of a reasonable divulgence by physician to patient to

make such a decision possible [citations omitted] [10].
The ASCO and ASRM recommendations regarding

elements to include in the informed consent process
are in keeping with the principles set forth in Canterbury as that set forth the type of information that one
would likely need in order to make an informed choice.
Informing patients about potential risks to fertility of
cancer treatment, followed by a discussion of fertility preservation options is the starting point. Patients
need to also have information about how those options
impact their treatment, the potential success of these
options when the patient is seeking to reproduce, the
cost of the procedures, whether the procedure is experimental and the risks such options may have to their
own health as well as that of future offspring [6], [8].
While little has currently been written about the
information that should be provided to cancer previvors regarding fertility preservation, many of these
same issues merit consideration. For some pre-vivors,
the option of fertility preservation is what will enable
them to undertake prophylactic measures to reduce
their heightened risk of cancer. For example, a young
woman with a BRCA1 or BRCA2 mutation who has
not yet had children, may be reluctant to remove her
ovaries, a prophylactic surgery which would significantly reduce her risk of ovarian cancer [11]. However, if she becomes aware of fertility preservation
options, both in clinical practice and research, she may
be more comfortable considering this option. As such,
not only is it important for oncologists and reproductive endocrinologists to consider when, what and
with whom to discuss fertility preservation options,
it may also be important for geneticists and genetic
counselors to be aware of fertility preservation options
as well and to consider disclosing information about
these options to those patients who might benefit.
Given the depth and breadth of information that
needs to be communicated, reliance on a multidisciplinary team may be the most efficient and expeditious
means of communicating the information. Jeruss and
Woodruff have recommended that institutions create
a multidisciplinary board to aid in indentifying and
communicating options for the preservation of fertility
in patients with cancer [12]. Others also recommend
involving clergy and theologians in any fertility preservation program [13]. These are sound recommendations for patients and practitioners given the rapidly
changing technology, the paucity of specific legal guid-
490
ance and the likely increase in patient demand as more

individuals become aware of these options.
Treatment children
As mentioned above, parents are responsible for making medical decisions for their children. The general
rule seems to be that, unless there exists an emergency,
which prevents any delay, or other exceptional circumstances, a surgeon who performs an operation upon a
minor without the consent of his parents or guardian
is guilty of a trespass and battery [14]. This decisionmaking authority is part and parcel of the constitutionally recognized right of parents to the care, control
and custody of their children [15]. An historic rationale for giving parents this authority was based on the
notion that if anything went wrong with the medical
procedure the parents would be responsible for raising
the child and thus should make the decision with this
awareness [14, 16]. Still another rationale for bestowing the obligation upon parents is that parents are in
the best position to protect their childs welfare and to
consider what would be in the best interests of the child
[17].
Under some circumstances, however, minors can
make their own healthcare decisions, particularly
around issues of reproduction. States have exceptions
for emancipated minors, mature minors and for specific healthcare services [16]. For example, the US
Supreme Court in a series of cases found that minors
have a right to determine whether to bear a child and
the decision to do so should not be vetoed by another;
however, some limitation is considered permissible
[18, 19]. In addition, legislation exists which allows
minors to make treatment decisions regarding sexually transmitted infections without parental consent
or notification [20]; obtain information about contraception [21]; and to obtain medical treatment for
alleged sexual assault without parental consent [22].
These statutes are in keeping with the general understanding that the right to reproduce is a fundamental
one. Further supporting this is the recognition by most
states that parents cannot make the decision to sterilize
a minor without independent court review [23]. Sterilization touches upon the individuals right of privacy
and the fundamental right to procreate [24]. While
parental failure to consent to the pursuit of fertility
preservation likely is not the same deliberative process as consenting to the sterilization of ones child, the
consequence for the child might be the same and thus
indicates the need for thoughtful consideration of the

childs future reproductive needs and wishes.
Even outside the context of reproductive health,
the American Academy of Pediatrics asserts that
healthcare decisions involving adolescents and older
children should, to the greatest extent possible, involve
the assent of the patient [17]. Assent is, in essence, a
childs willingness to accept the treatment after having
had the treatment/procedures described to him or her
in understandable terms appropriate for his or her
level of development [17]. A childs unwillingness to
assent should be given great weight [17] and in the
context of research may, under some circumstances,
be binding [25].
In the context of cancer treatment, parental decision making has become increasingly complex when
parents are confronted with the option of fertility
preservation. In a Technical Report issued by the
American Academy of Pediatrics in 2008, the authors
acknowledged that for parents the act of preserving a
childs life must take precedence over the preservation
of the possibility of that childs ability to have children,
although the goals of each are intertwined [26]. Legal
and bioethics expert John Robertson supports parents
decision to preserve their childs fertility as long as the
minor assents and the intervention does not pose an
undue risk [27]. Dolin et al. reach a similar conclusion, but consider the question from the perspective of
the child [23]. They assert that [t]o the extent that the
minor in question can rationally consider her options
and express her preferences accordingly, that should be
the end of the matter [23]. In the event of a conflict
between the wishes of parent and child, if the minor is
above the age of 14 years and deemed mature enough
to give or refuse consent, there seems to be support to
allow the minor to do so [3].
While many of the fertility preservation procedures available to adults are also available for children,
not all are. This is, in part, due to psychosocial concerns. For example, Adolescent girls have not been
considered to be candidates for assisted reproductive
technology, largely because of the psychosocial issues
surrounding a delay in treatment and acquisition of
donor sperm [12]. Additionally, parents may, because
of religious or cultural beliefs, be uncomfortable with
some procedures such as cryopreservation of sperm
collected through masturbation [26].
Because parents cannot always remove their own
emotions and their own needs to contemplate what the
child might desire when he or she becomes an adult, it
may become necessary to consider the appointment of

a special advocate for the child or have an ethics committee or consultant review the decision. The law tends
to be reactive, not proactive, and thus little if any legal
guidance exists to dictate what should or should not
be done to preserve the fertility of minors. Given the
uncertainty, however, thoroughly discussing the issue
with the child, if mature enough to understand, and
with the childs decision makers is imperative, especially in light of the fact that while [i]mpaired future
fertility is difficult for children to understand [it is]
potentially traumatic to them as adults [6].
How, what, why and when the discussion about fertility preservation should occur with minors and their
parents will be somewhat case specific with consideration of a number of factors including but not limited to the age of the child; maturity of the child; family dynamic, particularly the nature of the relationship
between the parent and the child; cultural and religious
beliefs of the family; childs prognosis; cost; and availability and accessibility of any given fertility preservation option in the patients geographical area.
Research adults
Several fertility preservation techniques are still in the
experimental phase, and thus the elements of informed
consent for research should be adhered to regardless
of the funding source for such research. The goals of
research and practice differ and this must be reflected
in the consent process. The goal of treatment is to benefit an individual patient whereas the goal of research
is to contribute to generalizable knowledge [28]. Based
on this distinction, more protections are required of
human participants in research. One such protection
includes the Code of Federal Regulations requirement
that research involving human subjects be reviewed by
an Institutional Review Board (IRB) [29].
The role of the IRB is to determine that the risks
to patients are minimized; that the benefits of those
participating in the research will outweigh the risks;
that selection of participants is equitable; and that
informed consent is sought from the participant or
his or her representative [30]. The IRB review will
include a review of consent documents as well as the
consent process to determine whether all of the consent elements required by the Regulations are satisfied. Section 46.116 sets forth the general requirements
for informed consent. These requirements include,
among other things, an explanation of the purpose of
491
the research and a description of the procedures; a

description of the risks, benefits and alternatives; and a
statement that participation is voluntary and that the
participant is free to withdraw at any time without fear
of compromising his or her medical care [31].
Recognizing the experimental nature of oocyte
cryopreservation, for example, the ASRM has issued a
Practice Committee Opinion on the essential elements
of the informed consent process [32]. Among the
elements to be discussed is a description of the procedure or procedures to be followed; the costs, including annual storage fees for the oocytes; the likelihood
of success; and the need to determine the disposition
of any cryopreserved oocytes in the event of death
[32]. These requirements adhere to the requirements
set forth in the Code of Federal Regulations. Another
important factor is that patients recognize that the goal
of research differs from that of treatment and in fact,
the goal of research is really not treatment [28].
The less risky and more beneficial the research seems

to be, the fewer the requirements [16]. And this will
seemingly be the case with most of the experimental
fertility preservation procedures offered to children.
Robertson posits that since the use of some fertility preservation procedures are intended to benefit the minor subject, they might be done with the
minors assent and the consent of his or her parents
if an IRB finds that the potential benefit of preserving
fertility outweighs the burdens of retrieving gametes
or gonadal tissue [27]. Currently, ASRM, for example considers oocyte cryopreservation, ovarian tissue
cryopreservation and testicular tissue cryopreservation to be experimental [8]. Additionally, Fallat et al.
found that [c]urrent fertility-preservation options for
female children and adolescents should be considered
experimental and are offered only in selected institutions in the setting of a research protocol [26]. In this
way, the heightened protections of the Federal Regulations will be followed.
Research children
Standard of care?
When it comes to research, children are considered to

be a vulnerable population, in no small part due to the
long and disturbing history of research abuses involving children [16]. Because of the vulnerability of this
population, heightened protection of children participating in research is required by the Code of Federal Regulations. The Federal Regulations distinguish
between beneficial and non-beneficial research when
the participants are children, in that if the research
holds no direct benefit to the minor, then even a
mature minor as defined by a state law could not consent to the research [16]. With regard to fertility preservation, however, this would not seem to be the case
since arguably, the purpose of undertaking any experimental fertility preservation procedure would be to
directly benefit the child at some point in the future.
This does not, however, mean that such procedures
should not be considered experimental.
As with treatment of children, research involving child participants should seek the assent of the
minor and if the child dissents from participating in
research, even if his or her parents or guardian have
granted permission, the childs decision prevails [33].
With regard to research presenting minimal risk to the
child [34], or research in which there may be more than
minimal risk but with a direct benefit to the child, the
Federal Regulations require the consent of one parent
and the assent of the child [35]. According to Glantz,
Is this or should this be the standard of care for

oncologists and/or reproductive endocrinologists? If
so, could failure to adhere to comply with these recommendations result in potential future liability? The
standard of care regarding the information to be disclosed for informed consent for medical treatment can
be defined as that information which a reasonable person similarly situated would need to know in order
to make an informed decision [28]. Failure to provide
the necessary information for informed consent might
result in a negligence claim. The elements of a negligence cause of action include: duty, breach of that duty,
injury and proximate cause [28]. As the information
about fertility preservation continues to grow, its success continues to increase, and the leading professional
societies continue to recommend disclosure about fertility preservation there may, in fact, become a duty
to disclose such information in the treatment context.
If this becomes the case, then failure to do so may result
in liability if damages result and the failure to disclose
the information is the proximate cause of that injury.
492
Future use/disposition of gametes

and/or reproductive tissue
One cannot overlook the circumstances under which
gametes and/or reproductive tissue are being collected
and stored when working with cancer patients or
pre-vivors; and, as in any circumstance under which

reproductive material is collected and stored for future
use, an advanced directive regarding disposition of
gametes, embryos and/or reproductive tissue in the
event of death, divorce or incapacity must be discussed
and documented. Outside the fertility preservation
context, this has been at issue in several legal cases
involving posthumous reproduction and the use and
distribution of previously collected gametic material.
One of the first times this issue was tackled was
in the mid 1990s when Judith Hart was conceived
from her deceased fathers cryopreserved sperm 3
months after his death from cancer. No question
existed regarding the childs genetic make up, but when
Judiths mother sought Social Security Survivor benefits for her daughter, the child was denied those benefits when the Social Security Administration determined that under the law of the state of Louisiana,
where Judith was born, she was not considered a child
of the deceased [36]. Several years later an apposite result was reached by the Supreme Judicial Court
of Massachusetts. In Woodward v. Commissioner, the
court found that the children conceived from their
deceased fathers sperm were entitled to Social Security
benefits because their genetic relationship to the decedent was clear and the decedents affirmative consent
to the posthumous use of his sperm was also clearly
articulated [37].
Surviving spouses or partners of the deceased as
well as parents wanting to utilize a deceased childs
gametes to create a grandchild a legacy of their
beloved child are not far-fetched scenarios and have,
in fact, been a reality. In 1997, for example, the parents
of Julie Garber, a deceased 28-year-old woman who
had cryopreserved embryos comprised of her eggs
and donor sperm prior to undergoing treatment for
leukemia, sought to enlist the services of a gestational
surrogate to gestate and deliver Julies child [38]. Julies
parents, aged 62 and 68, inherited the embryos upon
their daughters death and then gifted them to Julies
brother and his wife who planned to raise the child
[38]. The surrogate ultimately miscarried [39], ending
the immediate need to address the legal and ethical
dilemmas raised, but this highly publicized news story
nevertheless brought to the fore many of the potential
legal and ethical quandaries that may arise in the event
that disposition determinations are not made at the
outset of any fertility preservation treatments. These
issues are especially pertinent with the expansion of
fertility preservation options.
Because of the panoply of legal and ethical issues

that may arise, the ASCO recommends that, Potential
legal issues, such as ownership of embryos and reproductive tissue in the event of a patients death, divorce
or incapacity, should . . . be discussed by the reproductive specialist [6]. Additionally, the ASRM, in its Practice Committee Guidelines on the Essential Elements
of Informed Consent for Elective Oocyte Cryopreservation recommend that the informed consent process should include information about [t]he disposition of any cryopreserved embryos not used . . . in
the event of death [32]. The dispositional options
would include, having the material discarded, utilized
for research purposes or used for posthumous reproductive purposes [8]. According to the recommendations of the Ethics Committee of the ASRM, Spouses
or family members with legal rights to dispose of a
deceased patients stored gametes or other material
should use them for posthumous reproduction only if
the deceased had previously consented to such posthumous use [8]. From a legal standpoint, having that
consent in writing would be the most reliable expression of the patients wishes and would avoid the need to
have friends and family members attempt to recall and
re-state what it is that the patient would have wanted.
The guidelines set forth by professional societies
provide useful guidance and may, as discussed above,
serve as evidence of the standard of care; however, they
do not have the force of law. Case law and statutory
law, though, are beginning to evolve. As posthumous
reproduction has continued to occur, some states have
developed specific legislation to assist in determining
the status of children conceived after a parents death
in order to facilitate such determinations as inheritance and parentage. Virginia, for example has enacted
a statute which reads:
Any person who dies before in utero implantation of an embryo
resulting from the union of his sperm or her ovum with another
gamete, whether or not the other gamete is that of the persons
spouse, is not the parent of any resulting child unless (i) implantation occurs before notice of the death can reasonably be communicated to the physician performing the procedure or (ii) the person
consents to be a parent in writing executed before the implantation
[40].
This language makes it clear that the wishes of the

deceased to become a parent posthumously must be
expressed in writing, thus stressing the importance
of advanced directives when preserving gametes or
reproductive tissue.
493
The complexity of gamete or tissue disposition is

further compounded when the patient is a minor and
parents or guardians have medical decision-making
authority for the child and, presumably, over gametic
material or reproductive tissue if the patient dies while
still a minor. Parents are likely to inherit sperm, eggs,
embryos or reproductive tissue of the deceased minor
and thus would have control over the disposition of
the material. Given the range of complex scenarios
that might arise in this instance, Rosoff and Kastur
have recommended that children and their parents
should sign a consent form declaring that they will
destroy the materials if the child dies before reaching his or her majority . . . [13], as well as having
parents and children sign directives regarding gamete
disposition in various scenarios. This is very much
the same as advanced directives that any patient is
required to sign prior to undergoing assisted reproduction in which egg, sperm or embryos might be
cryopreserved. Additionally, Fallat et al. have recommended that instructions concerning disposition of
stored gametes, embryos, or gonadal tissue in the event
of the patients death, unavailability, or other contingency should be legally outlined and understood by
all parties, including the patient if possible [26]. The
enforceability of such a written document by a child
is unclear, but, as discussed previously, mature minors
do have decision-making capacity with regard to their
own reproduction and, thus, should be able to control
the disposition of their own gametes.
In any posthumous reproduction scenario, legal,
ethical and psychosocial questions abound regarding the dispositional control over gametes or tissue remaining after the death of the gamete or tissue source. Such questions include: legal parentage of
a resulting child; inheritance rights of the resultant
child, including the ability to receive Social Security
Survivor benefits; the psychological ramifications of
being conceived after the death of a parent, especially
if that parent was a child him or herself at the time
of death; and the question of honoring the wishes of
the deceased, especially if such wishes have not been
clearly expressed in writing.
Insurance coverage for

Another important issue to discuss with patients and
their family members regarding fertility preservation
is the cost related to such treatments and the fact that
494
insurance is unlikely to cover not only the treatment

but the cryopreservation of gametes as well [27]. Discussion of cost should be included in the informed
consent process as this information may be determinative for some patients contemplating fertility preservation.
Currently, at least 14 US states have some type
of insurance mandate related to coverage of infertility services [41]. These mandates, however, also
impose many restrictions on use, which essentially
render them inapplicable to those individuals utilizing assisted reproductive services in order to preserve
fertility rather than to currently treat existing infertility. In fact, none of the existing statutes specifically
address coverage of fertility preservation and the terms
of coverage typically are only applicable to an individual with diagnosed infertility and do not cover cryopreservation. Infertility is most commonly defined as
the inability to conceive after 1 year of unprotected
sexual intercourse or the inability to sustain a successful pregnancy [42]. This definition would exclude
many undergoing reproductive procedures to preserve
fertility, particularly minors as they have not yet even
attempted to conceive.
Yet another potential exclusion presented by the
current language of state insurance mandates is a
requirement that the insured be legally married, which
again would exclude coverage of fertility preservation
services for minors and those who are single at the time
of undergoing the procedure. Utilization of experimental procedures would pose still another barrier
to insurance coverage as no mandate exists to cover
experimental procedures.
The issue of cost and insurance coverage for fertility preservation, while seemingly outside the traditional realm of informed consent is highly relevant to
patients and parents determination of whether fertility
preservation is appropriate for them. With all of these
barriers to obtaining insurance coverage, the cost of
this prophylactic treatment may be well out of reach
for many patients and their families. For this reason
it is important to discuss with patients other options
for family building following successful cancer treatment, including adoption, egg donation, sperm donation, embryo donation and gestational surrogacy.
Conclusion
As cancer patients and cancer pre-vivors continue
to have better prognosis for long-term survival and
fertility preservation techniques continue to develop

the law defining the rights and obligations of patients
and providers will evolve. With the goal of improved
cancer treatment and identification of genetic predisposition to certain cancers being increased, taking
a holistic approach to treating the patient is essential. Fallat et al. suggest that offering the technique
might provide some degree of comfort in light of a
life-threatening diagnosis, because if offer an optimistic perspective for the future that may conform
to a patient-centered philosophy of care [26]. Such
an approach would take into consideration not only
the patients immediate need for treatment and counseling, but also how such treatment and the resultant increased survival will impact his or her life plans
which may very well include family building.
References
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Oncology nurses perceptions of barriers to discussion
of fertility preservation with patients with cancer. Clin
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2. Carlson RH. Survey: little awareness of the ASCO
fertility-preservation guidelines by cancer specialists.
Oncol Times 2009; 31: 2630.
3. Schover LR, Brey K, Lichtin A, Lipshultz LI and
Jeha S. Oncologists attitudes and practices regarding
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4. Walters EM, Benson JD, Woods EJ and Crister JK.
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2009: pp. 117.
5. Practice Committee of the American Society for
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9. Forman EJ, Anders CK and Behera MA. A

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10. Canterbury v. Spence. 464 F.2d 772, 780 (D.C. Cir.
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11. Friedman LC and Kramer RM. Reproductive issues
for women with BRCA mutations. J Natl Cancer Instit
Monographs 2005; 34: 836.
patients with cancer. N Engl J Med 2009; 360:
90211.
13. Rosoff PM and Kastur ML. Preserving fertility in
young cancer patients: a medical, ethical and legal
challenge. J Philos Sci Law 2003; 3. http://www.
psljournal.com/archives/papers/preservingFert.cfm.
14. Lacey v. Laird, 166 Ohio St. 12; 139 N.E.2d 25; 1956
Ohio LEXIS 610; 1 Ohio Op. 2d 158, 1956.
15. Pierce v. Society of Sisters, 268 US 510 (1925).
16. Glantz LH. Research with children. Am J Law Med
1998; 24: 21344.
17. American Academy of Pediatrics, Committee on
Bioethics. Informed consent, parental permission, and
assent in pediatric practice. Pediatrics 1995; 95:
31417.
18. Ohio v. Akron Ctr. For Reprod. Health, 497 US 502,
510 (1990).
19. Bellotti v. Baird, 443 U.S. 622 (1979).
20. Capacity of minor to obtain treatment for venereal
disease without consent of parent. A.R.S. 44132.01
(2009).
21. Treatment for health-related problems. Md.
Health-general Code Ann. 20102 (2009).
22. Consent by assault victim to treatment. Cal. Fam.
Code 6928 (2009).
23. Dolin G, Roberts DE, Rodriguez LM and Woodruff
TK. Medical hope, legal pitfalls: potential legal issues
in the emerging field of oncofertility. Santa Clara L
Rev 2009; 49: 673716.

2006: 24; 116.
25. Additional protections for children involved as

subjects in research. 45 C.F.R. 46 401, et seq. 2009.
7. Presidents Commission for the Study of Ethical

Problems in Medicine and Biomedical and
Behavioral Research. Making Health Care Decisions.
Washington DC: Government Printing Office, 1982:
pp. 23.
26. Fallat ME, Hutter J, AAP Committee on Bioethics,

AAP Section on Hematology/Oncology and AAP
Section on Surgery. Preservation of fertility in
Pediatrics 2008; 121: e14619.

16228.
27. Robertson JA. Cancer and fertility: ethical and legal

challenges. J Natl Cancer Instit Monograph 2005; 34:
1046.
24. In re Guardianship of Hayes, 608 P.2d 635 (1980).
495
28. Morreim EH. Medical research litigation and

malpractice tort doctrines: courts on a learning curve.
Houst J Health Law Policy 2003; 4: 186.
35. Research involving greater than minimal risk but

presenting the prospect of direct benefit to the
individual subjects. 45 CFR 46.405.
29. Protection of human subjects. 45 C.F.R. 46, et. seq.

(2009).
36. Wharton J. Law and biology: miracle baby denied

benefits. Am Bar Assoc J 1996; 82: 38.
30. Criteria for IRB approval of research. 45 C.F.R. 46.

111.
37. Woodward v. Commissioner, 760 N.E. 2d 257

(2002).
31. General requirements for informed consent. 45

C.F.R. 46. 116.
38. Peres J. Children from beyond the grave: surrogate

carrying dead womans baby. Chicago Tribune.
December 15, 1997, p. 1.
32. The Practice Committee of the Society for Assisted

Reproductive Technology and the Practice
Committee of the American Society for
Reproductive Medicine. Essential Elements of
Informed Consent for Elective Oocyte
Cryopreservation: a Practice Committee opinion.
Fertil Steril 2008; 90: S1345.
33. Office for Human Research Protections. OHRP
research involving children frequently asked
questions. http://www.hhs.gov/ohrp/researchfaq.
html#q12. (Accessed Dec. 27, 2009)
34. Research not involving greater than minimal risk. 45
CFR 46.404.
496
39. Tribune Staff Writers. Surrogate. Chicago Tribune.

December 18, 1997, p. 13.
40. Parentage of child resulting from assisted
conception. Va. Code Ann. 20158 (2009).
41. National Conference of State Legislatures. State
Laws Related to Insurance Coverage for Infertility
Treatment, 2009.http://www.ncsl.org/IssuesResearch/
Health/InsuranceCoverageforInfertilityLaws/
tabid/14391/Default.aspx. (Accessed Dec. 22,
2009)
42. Infertility coverage. 215 ILCS 5/356m. (2009).
Section 10
Chapter
43
Christian ethics in fertility preservation

Brent Waters
The purpose of this chapter is to provide an interpretive framework for examining how Christian theological tenets and convictions might inform an ethical assessment of fertility preservation. In undertaking
this task I make no attempt to survey various religions,
other than to note that their respective beliefs inspire
disparate moral assessments of the medical procedures
deployed in preserving fertility. Subsequent inquiry in
comparative religious ethics and interfaith dialogue
are certainly required given the diverse character of
contemporary society, but will need to be pursued by
scholars with greater expertise. I also do not attempt
to make a systematic or normative assessment of fertility preservation in general, nor do I offer any detailed
evaluation of the discrete ethical issues accompanying
the particular medical procedure described in the previous chapters. Again, these are important tasks, but
beyond the limited scope of this chapter.
My intent, to invoke a crude analogy, is to visit
some important theological landmarks along the
moral turf in which fertility preservation is embedded.
My description of these landmarks is both informed
and limited by my perspective as a Christian theologian, but I use terminology which I hope will prove
informative to broader religious, secular and professional audiences. In most respects we will be revisiting
familiar landmarks, for many of the ethical issues associated with fertility preservation are similar or identical to earlier and ongoing disputes over the ethics
of assisted reproductive technology (ART) generally
(e.g. [13]). The principal contextual difference in this
instance is that fertility is being preserved, and thereby
assisted, in response to largely non-controversial therapies rather than treating infertility. Given the wide
variety of Christian churches and their respective
teachings and theological convictions, no universal
ethical position on the ethics of fertility preservation in
general or the associated medical treatments in particular can be formulated. Consequently, in conducting
this inquiry I examine four pertinent theological and
biblical precepts; describe four general moral stances
along a spectrum of options that may be derived from
these precepts; and identify a principal strength and
weakness, respectively, of each stance.
Theological and biblical precepts

The list of precepts examined below is not exhaustive,
but they have been selected as examples for how ethical
arguments based upon religious beliefs and convictions might be formulated. The first general precept
may be characterized as the procreative mandate. As
reported in Genesis 1.28, God commands humans to
be fruitful and multiply. Theologians have drawn
upon this passage in arguing that humans are in general commanded by God to procreate, and that offspring is the premier good of marriage (e.g. [4, 5]).
Fecundity is thereby held in high regard as a necessary means of fulfilling this religious obligation. It is in
light of this procreative mandate that infertility is often
portrayed in the Bible, especially in the Hebrew Scriptures or Old Testament, as a particularly severe curse
or tragic circumstance [6].
Although there is a long history of valuing fertility within the Christian tradition, there is no contemporary consensus regarding the ethics of ART in
general and fertility preservation in particular. On
the one hand, it can be argued that, despite the high
esteem afforded to fertility, preserving the biological
nature (sexual intercourse) of transmitting life and
inviolable structure of monogamous marriage precludes any artificial or technological interventions to
either treat infertility or preserve fertility. Specifically, artificial insemination by donor (AID) or by
497
husband (AIH), in vitro fertilization (IVF), embryo

or gamete storage, pre-implantation genetic diagnosis (PGD) and surrogacy would all be prohibited as
violating the biological or marital integrity of procreation. Consequently, this predominantly Catholic
teaching forbids any recourse to ART, which in turn
also prohibits virtually all of the medical treatments
associated with fertility preservation [7]. On the other
hand, it can be countered that the so-called biological and marital integrity of procreation is not sacrosanct. The natural and legitimate desire for offspring
should not be frustrated by a biological essentialism
and highly restricted view of marriage which effectively constrains the freedom to reproduce. This largely
liberal Protestant stance contends that using ART to
treat infertility is thereby morally permissible in treating infertility and, by extension, so too fertility preservation.
Closely related to the procreative mandate is
the second general precept of stewardship. Following Gods command to be fruitful and multiply,
humans are enjoined to exercise a stewardship of the
earths resources (see Genesis 1.2830). Asserting such
dominion or rule, however, is limited for ultimately the
earth belongs to God; humans are the caretakers and
not the owners of creation. Consequently, such human
governance should accord with divinely inspired concepts of what constitutes the larger or general good of
creation.
Subsequent theological reflection on the precept of
stewardship has developed, among other things, the
idea of the common good. Since material goods are
finite, the pursuit of the common good may require
that the desires of some individuals remain unfulfilled in order to promote a just distribution of scarce
goods and services. For example, the desire to be rich
is not necessarily wrong, but the desire should remain
unfulfilled if its fulfillment results in impoverishing
other individuals which in turn diminishes the common good.1 Likewise, assisting reproduction and preserving fertility might very well be good desires, but
whether or not they should be fulfilled needs to be
determined within a larger set of social, economic and
political considerations. Consequently, the ethics of
fertility preservation should not be evaluated solely in
terms of personal preferences and therapeutic safety
and efficacy, but also in respect to healthcare priori1
I am not convinced that this zero-sum argument regarding
the creation of wealth is necessarily correct.
498
ties, costs and accessibility. Given the general healthcare needs of civil community, does preserving the fertility of relatively few individuals justify the allocation
of scarce medical funding and personnel in achieving
this goal?
The third precept is derived from the doctrine
of the incarnation. The Gospel according to St John
asserts that the Word became flesh, a reference to Jesus
Christ (see John 1.118). The central tenet of this doctrinal teaching is that in Christ, as the second person
of the Trinity, God became a human being. This act
in turn affirms the embodied nature of human beings
and consequently the finitude of the human condition. Bodily health is therefore not a matter of indifference. Subsequent theological reflection has affirmed
the goodness of the body despite frequent heretical
attempts to disregard or malign the body as a mere
vessel, or worse, a prison of the soul. In Beth Felker
Jones evocative words, In the Christian tradition, the
temptation to denigrate the body has been continually
reasserted and consistently rejected[8].
The doctrine of the incarnation inspires a Christian
affirmation of the human body, but no obvious ethical
stance is forthcoming regarding modern health care in
general, or assisted reproduction and fertility preservation in particular. Although the life of embodied
human beings is highly valued and respected, efforts
to preserve or reproduce life are relative rather than
absolute [9]. For example, there is no corresponding moral duty to use every means possible to extend
the life of dying individuals for as long as possible
should such efforts prove unduly onerous or futile,
though euthanasia and assisted suicide are generally
proscribed [10, 11]. Similarly, the biological means of
reproducing human life is held in high esteem, but
it is again a relative rather than absolute good to be
pursued. An infertile couple, for instance, incurs no
moral obligation to employ every available medical
treatment. As noted above, a Catholic couple would
be prohibited from utilizing ART and could fulfill
their natural parental desires through such alternatives as foster care or adoption [12, 13]. Many Protestant couples, however, believe they are free to utilize
or refrain from utilizing ART in treating their infertility, which would presumably extend to attempting to
preserve or not preserve fertility as well. This expansive range of options is derived from the theological teaching of the incarnation which while affirming embodiment, and derivatively reproduction, also
indicates that with the birth of Jesus the urgency
Chapter 43: Christian ethics in fertility preservation
of the procreative mandate has been effectively

diminished [14].
The fourth precept entails healing and love of neighbor. The Gospels report that Jesus performed many
miraculous healings. Given the doctrine of the incarnation these acts are not surprising, for in affirming
the embodied nature of human life ameliorating the
pain and suffering that is inherent to the life of finite
and mortal creatures is also an act of love and compassion. Moreover, Jesus commands his followers to
love and care for their neighbors in need (see Matthew
22.3440), especially those suffering from illness, disability or injury (see, e.g., Luke 10.2537). In addition,
Jesus is portrayed as keeping company with the sick
and infirmed, especially those whose conditions have
made them outcasts from the larger community.
The complementary images of Jesus as both healer
and suffering servant have informed the subsequent
theological and moral tradition, which affirms the
importance of medicine and health care [15]. Christians have been instrumental in establishing hospitals
and were early proponents of medical research. This
religious commitment to relieving the human condition is exhibited not only in treating those suffering
illness and injury [16], but is extended, by many theologians and official church teachings, to include treating infertility and prenatal screening and monitoring
in order to prevent suffering [17, 18]. Presumably this
expansive support of ART could easily embrace fertility preservation. Moreover, modern medicine exhibits
in a highly visible manner the love for neighbor in
the contemporary world, embodying Jesus roles as
healer and suffering servant. In treating or preventing disease and injury the suffering of the neighbor
is alleviated, and, more importantly, medicine represents a moral commitment that the ill and infirmed
will not be abandoned by the civil community [19].
Arguably a case can be made that preserving fertility
is consonant with the precept of healing and love of
neighbor.
The preceding brief summaries of the theological and biblical precepts of the procreative mandate,
stewardship, incarnation and healing and love neighbor provide some useful starting points for examining how Christians might assess the ethics of fertility preservation. As these summaries suggest, however,
these precepts do not lead to a common moral position or stance. Although most Christians would affirm,
in varying ways, these precepts, subsequent ethical
reflection and discernment can lead to highly disparate
assessments of fertility preservation. The next section

examines the range of these ethical assessments and
how they might be formulated.
Moral stances
The following four ethical stances are derived from the
theological and biblical precepts summarized in the
preceding section. Each stance demonstrates how religious beliefs and convictions might inform particular ethical assessments of fertility preservation. These
stances do not reflect arguments promulgated by a
specific church or theologian. Rather, they serve as
heuristic devices that demonstrate varying and often
conflicting assessments. Furthermore, these stances
disclose how theological and biblical precepts may be
interpreted and applied in a variety of imaginative
ways in constructing a moral argument. As will be
seen, there is no given correspondence between a particular precept and a particular stance regarding the
ethics of fertility preservation. It should also be noted
that these stances do not exhaust the possible options
that could be formulated, but rather serve as examples
along a spectrum ranging from prohibition to encouragement.
In each instance I make the following assumptions: An individual is facing the prospect of a therapeutic application that may result in the loss of fertility. The various treatments that could be applied
in attempting to preserve fertility are accessible, relatively safe and potentially efficacious and adequate
funding is readily available. All legal safeguards and
recognized ethical practices and procedures are followed such as informed consent, protection of minors
and proper authorization and oversight of experimental protocols. In addition, I make no attempt to
assess the adequacy of the various moral stances, especially in regard to their respective interpretations and
applications of theological and biblical precepts and
coherency of argument, nor do I offer any counter
interpretations or arguments. Rather, I allow each
stance to stand in its own right in order to identify
various points along a spectrum of options concerning the ethics of fertility preservation that are based
upon selected religious beliefs and convictions. Furthermore, it should be noted that in some instances
these arguments do not accept medical designations
of embryonic development, for example, the difference between pre-embryos and embryos, as implying any inherent normative content. It is important
499
for medical practitioners to keep these potential discrepancies in mind when dealing with some patients
who may exhibit some reluctance in pursuing fertility
preservation. In short, when doctors and patients refer
to an embryo, they may not, in some instances, be
referring to a common perception. Although the religious reservations of these patients need to be honored, they do not necessarily call into the question
either the morality of the procedures or humane intentions motivating their development and deployment.
The first stance may be characterized as preserving the natural reproductive process. This stance draws
heavily upon the precepts of the incarnation and
the procreative mandate. The divine affirmation of
the embodied nature of human existence necessarily
entails the need of human beings to perpetuate themselves from one generation to the next; hence Gods
command to be fruitful and multiply. This affirmation and command, however, imposes constraints in
exercising an accompanying stewardship. In affirming
their embodiment and obeying the command to procreate, individuals are not free to do whatever they
might want. The affirmation and mandate acknowledges and consents to the frailty and limitations of the
human body, otherwise stewardship is distorted into a
form of mastery that rightfully belongs only to God.
Although this stance does not reject modern medicine
in general, indeed it is seen as an important means of
excising the stewardship of Gods creation and expressing the love of neighbor, it too is limited to restoring
the natural health of the body.2
Given these religious presuppositions, the morality
of fertility preservation is assessed largely, though not
exclusively, in respect to whether or not it preserves
or violates the natural reproductive process. Consequently, fertility preservation should not be used in
many, if not most, instances because the treatments
employed violate this natural integrity. Similar to the
objections against ART, gamete and embryo storage
are illicit because of the means required to procure the
gametes and embryos. Although such medical treatments are motivated by the legitimate desire to treat
infertility resulting from other therapeutic applications, this motive does not justify bypassing the nat2
The description of the basic religious principles underlying
this stance draws upon selected themes explicated in relevant
Catholic social teaching. See, e.g., Donum Vitae and Paul VI
[20]. For related Protestant themes see, e.g., Mitchell et al.
[21].
500
ural method of procreation. The creation and storage

of embryos is particularly objectionable since it might
entail the destruction of unneeded and, in the case
of PGD, unwanted embryos. The ensuing destruction
of embryos fails to exhibit a proper love of weak and
vulnerable beings.3 This stance, however, would presumably have no objections to repositioning ovaries
when undergoing radiation treatment in the lower
abdominal area. No attempt is being made to bypass
the methods of natural reproduction such as extracting oocytes, nor are any embryos artificially created
or willfully destroyed. Performing a radical trachelectomy in treating cervical cancer would presumably be
illicit since artificial means of initiating pregnancy following treatment would be required. Similar objections could also be raised against the experimental
procedure of ovarian tissue cypopreservation if IVFcreated embryos are implanted. In short, these treatments, with the exception of ovary repositioning, are
illicit not only because they violate the integrity of the
natural reproductive process but also because they represent an improper stewardship of medicine as they
seek to transcend rather than restore natural health,
thereby failing to honor the limitations of embodiment
which are affirmed in the incarnation and presumed in
fulfilling the procreative mandate.
The principal strength of this first stance is its seriousness concerning the embodied nature of human
beings. Since the incarnation affirms embodiment,
the inherent finitude and mortality set integral and
delimiting conditions that should be honored. To be
embodied, therefore, entails a natural reproductive
process which may be disabled by disease, dysfunction or therapies treating a condition unrelated to fertility. Although one may sympathize with individuals
who are infertile or may lose their fertility, this does
not justify recourse to technologies which bypass the
natural reproductive process. The principal weakness
3
Most often this argument is based on the belief that since

life begins at conception the resulting embryo is a person,
and therefore should be given full moral regard and protection. See, e.g., Grisez [22] and Meilaender [23]. It can also be
argued that even if embryos (or fetuses) are not persons their
status as human beings should nonetheless be sufficient for
protecting them against willful destruction. See, e.g., Grant
[24, 25]. In addition, even if agnosticism is invoked regarding the moral status of the human embryo a variety of ethical
arguments regarding the protection and ethical treatment of
prenatal life can still be made. See, e.g., pertinent chapters in
Waters and Cole-Turner [26] and Waters [27].
is its appeal to a biological essentialism that is not

applied consistently in respect to other medical practices. Given their status as finite and mortal creatures,
humans necessarily suffer the natural effects of disease,
dysfunction and degeneration, yet there are presumably few, if any, moral objections to treating these conditions. It is not clear why such rigid prohibitions are
set regarding the reproductive process in comparison
with other organic systems. The contention that ART,
and thereby fertility preservation, attempt to bypass
rather than restore natural fertility does not resolve
this inconsistency. Using suppressants in conjunction
with organ transplants, for instance, bypasses the natural immune system, yet few, if any, theologians would
now argue against these procedures in terms of violating the so-called natural integrity of the immune
system.4
A second stance entails assessing the ethics of fertility preservation in light of larger social priorities.
The precept of stewardship carries the heaviest weight
in formulating this moral perspective and evaluation.
Humans do not exercise their stewardship of creation as autonomous individuals, but through cooperative social and political relationships. Technology in
general and medicine in particular have undoubtedly
assisted humans in fulfilling this responsibility, but
their development and deployment should be determined in respect to larger social and political priorities rather than merely satisfying individual desires.
The common good and the needs of the many should
trump the interests of the few. This moral commitment
to the common good affirms the precept of the incarnation by recognizing the finite character of human
life within the finite constraints that are imposed by
creation. This scarcity must be taken into account in
ensuring that a just distribution of goods are services
is pursued in a manner which proves most beneficial to the greatest number of people. This same principle holds true in the allocation of scarce medical
resources. The relative urgency of the procreative mandate, therefore, should be determined in light of contemporary economic and political concerns related,
for instance, to population growth, and how these concerns are addressed in healthcare policies.5
4
Most of the ethical disputes now focus on such issues such

as fair access to scarce organs and public policies regulating
donors.
5
In describing the basic principles of this stance I have borrowed, rather loosely, from some prominent themes drawn
Given these religious assumptions it can be argued

that although fertility preservation need not be prohibited, the practice should nonetheless be discouraged.6
Although there is nothing necessarily objectionable
to the various treatments employed in preserving fertility, it marks a costly expenditure of scarce medical resources that serves a relatively small segment
of the population.7 These resources should instead be
deployed in addressing basic healthcare needs of the
broader population that is often deprived of such care
due to costs and limited accessibility, thereby promoting the common good rather than serving the interest
of a few individuals. Moreover, such a policy or strategy is more in line with Jesus role as healer and suffering servant, which were most often performed for
the sake of the poor and destitute. In addition, given
the array of social, economic and political problems
associated with overpopulation, it is hard to justify the
allocation of funds, the time of healthcare personnel
and technologies to meet the needs of relatively few
individuals. The ethical issue at stake is not so much
to preserve fertility (or treat infertility), but to ease the
longing of childless couples or individuals which can
be addressed through such options as foster care and
adoption. This approach would have the additional
benefit of providing parental care for orphans, abused
and neglected children, thereby once again promoting the common good, as well as recognizing the need
for tempering the urgency of the procreative mandate
given the pressing need to control population growth
and caring for needful children. In short, although
one may be sympathetic with the desire of individuals to preserve their fertility, a faithful stewardship of
creation requires that more pressing healthcare needs
are given priority. Consequently, fertility preservation
should be discouraged as an unwarranted consumption of scarce medical resources.
from what may be described as Christian environmentalism. See, e.g., Fern [28] and Scott [29]. In respect to setting
social priorities governing the development and distribution
of medical resources, see, e.g., Cahill [30].
6
This discouragement could be accomplished, e.g., through
a combination of informal social and religious disapproval,
refusal by public and private insurance carriers to cover costs
and other economic disincentives.
7
The same objection could also be raised against ART in
general. An individual holding this stance could also be dedicated to preserving the integrity of the natural reproductive
process as described in the previous moral stance.
501
The principal strength of this second stance is the

recognition that medicine and health care cannot be
separated from larger economic and political considerations. A medical decision is never made in isolation from broader social contexts, as demonstrated
by determining which treatments and procedures are
and are not funded by public and private insurance
carriers. Given limited economic resources, as well as
other ethical concerns such as overpopulation, good
stewardship requires limiting the provision of medical
treatments that affect a relatively small segment of the
population. Again, one may sympathize with individuals desiring to preserve their fertility, but it should not
be granted much priority, or even discouraged, given
more pressing and expansive healthcare needs. The
principal weakness is its implicit paternalism. Invoking a greater common good often entails an appeal to
abstract moral principles that are divorced from actual
practice. This disjuncture in turn effectively masks the
imposition of the emotive values and preferences of
some over those of others; those in a relative position of power know what is best for everyone, thereby
corrupting stewardship into an exercise of behavioral
control [31]. Yet it is not clear why fertility preservation should be discouraged on the basis of utilizing
scarce medical resources contributing to overpopulation when such a small segment of potential patients
are at stake. Rather, discouraging fertility preservation
by appealing to larger social priorities may effectively
serve as a more troubling wedge argument. If the common good is promoted by limiting births in general
and the births of individuals with potentially chronic
conditions in particular since both place strains upon
limited healthcare resources, then discouraging fertility must been seen within the context of social and
political agendas of utilizing medical technologies to
control both the quantitative and qualitative outcomes
of reproduction (e.g. [32, 33]).
A possible third moral stance can be characterized
as one of freedom of choice. The embodiment affirmed
in the precept of the incarnation does not diminish
the need for moral agency and the personal responsibility it entails. Constricting or denigrating the freedom to choose among various possible options effectively denies the human dignity which the incarnation affirms. Stewardship, therefore, should be directed
toward enabling the concrete and varying goods of
individuals rather than promoting an abstract common and collective good. In respect to medicine and
health care, it should be noted that Jesus was not
502
required to either heal or keep company with the ill

and infirmed, but freely embraced his roles as healer
and suffering servant. Likewise the Good Samaritan
was not compelled to stop and render aid but chose
to do so. Similarly, although the urgency of the procreative mandate may be muted in light of contemporary population concerns, it has not been rescinded
and individuals should be free to make responsible
reproductive choices. To portray individual freedom
and personal responsibility as antithetical to the common good, particularly in respect to health care and
procreation, is to effectively eviscerate the very meaning of ethics, especially Christian ethics, since the love
of neighbor expresses moral action based upon liberty
as opposed to compulsion.8
Given these basic religious convictions, this stance
would permit fertility preservation while neither
encouraging nor discouraging its use. Since the decision to either employ or forgo fertility preservation
is a matter of personal discernment and a wide variability of choices are to be expected. On the one
hand, for instance, if an individual does not believe
that the various medical treatments and social circumstances are morally objectionable then presumably there would be no compelling ethical reason to
refrain from attempting to preserve ones fertility.9 So
long as it is determined that, for ART and the potential destruction of unneeded embryos, these procedures are not jeopardizing basic healthcare provision
or substantially contributing to problems associated
with overpopulation, then an individual is free to pursue fertility preservation since it does not violate the
theological and biblical precepts outlined above. On
the other hand, if an individual believes that the medical treatments and social conditions are morally troubling then presumably there are strong ethical reasons
to refrain from attempting to preserve ones fertility.
If it is discerned that ART, the potential destruction of
unneeded embryos or that the provision of these treatments are unjust given the lack of basic healthcare provision or overpopulation concerns, then an individual
would choose not to pursue fertility preservation since
8
Although Victor Claar, Robin Klay and Michael Novak

do not address healthcare issues directly, their respective
accounts of economics help to explicate this broadly free
market account of stewardship. See Claar and Klay [34] and
Novak [35].
9
An individual would still be free not to pursue fertility
preservation as a matter of personal preference.
doing so would violate relevant theological and biblical

precepts. It should be noted that, although Christians
come to conflicting conclusions regarding the ethics
of fertility preservation, the source of moral authority resides in the conscience of the individual believer
rather than compliance with external sources. The act
of moral discernment and action stems from the will
of the individual rather than obedience to prohibitions
imposed by the larger community.
The primary strength of this third stance is that
it places the weight of moral responsibility upon the
individuals most directly affected by the treatments
in questions. Consequently, individuals should be free
to make whatever choices they might make regarding fertility preservation, so long as they do not violate their own religious and moral convictions or that
such decisions do not demonstrably harm others. In
the absence of freedom of choice, how else can individuals affirm their embodiment, pursue procreation and
exercise their stewardship as the responsible beings
they were created to be? The question of whether or not
to pursue fertility preservation is best left to patients
facing the prospect of undergoing therapies that may
compromise their fertility in consultation with appropriate medical expertise. The primary weakness is a
diminished understanding of freedom that is reduced
to license. Freedom is not merely the absence of external constraints against the will of individuals pursuing
their respective reproductive interests. Rather, freedom results from limitations necessarily imposed by
various relationships [36]. Responsible choices regarding fertility preservation cannot be made in isolation
from the interests of partners, spouses, family members, medical personnel and healthcare institutions,
religious communities and the civil community. In the
absence of these considerations, freedom is eviscerated
into a fictional autonomy that potentially distorts the
purported affirmation of embodiment, pursuit of procreation and stewardship into self-indulgence. Moreover, coupling freedom with such a diminished understanding of moral autonomy exacerbates the more
troubling aspects of the so-called procreative liberty
pervading contemporary society by adding fertility
preservation to a growing list of reproductive options
designed to bypass biological limitations and social
inequalities [37].10
10
For a religious account that both criticizes and utilizes,

albeit often obliquely, many of the arguments propounded
by Robertson, see Peters [38]. For a critique of Robertson,
A final possible stance may be characterized as

a technological affirmation of life. Although the procreative mandate has lost its urgency in the modern world, using medicine to preserve fertility may
nonetheless serve as a witness to the goodness of life
in a dominate culture of death [41].11 If medical
technologies are routinely used to prevent conception,
destroy embryos and abort fetuses, why cant they also
be used to assist the birth of children thereby promoting a culture of life? In this respect, fertility preservation serves as a powerful countervailing witness to the
dominant culture. Such a witness upholds the incarnations affirmation of embodiment, while also enabling
a stewardship of medical resources oriented toward
securing the good gift of life. Moreover, using medicine
to preserve fertility is consonant with Jesus ministry of
healing and ameliorating suffering.
Based on this admittedly highly speculative interpretation of the theological and biblical precepts discussed previously, this stance would not only permit
but would encourage fertility preservation; there is not
only permission but an implicit imperative to preserve
fertility.12 There are, however, some moral constraints
that should be honored in pursuing fertility preservation. Presumably the techniques of repositioning
ovaries, performing a radical trachelectomy, ovarian
tissue cryopreservation, artificially extracting and fertilizing gametes, storing and implanting embryos and
surrogacy are permissible. Yet if these treatments are
being employed, in part, as a witness to enabling a culture of life, then provisions need to be in place for
ensuring that unneeded embryos are not destroyed,
a goal that could be accomplished through embryo
donation or adoption (e.g. [44]). In addition, PGD
would also be prohibited since it implies the possible
destruction of embryos with genetic or chromosomal
abnormalities.
and more broadly the concept of procreative freedom, see
Meilaender [39]. For a critique of both Robertson and Peters,
see Waters [40].
11
It should not be construed that John Paul II or Catholic
social teaching and moral theology would endorse the hypothetical stance I am describing.
12
I am not aware of a Christian theologian or ethicist making this kind of imperative argument. Although Ronald
Cole-Turner does not address the question of fertility preservation specifically, he offers some broader themes, particularly in respect to human genetics, regarding the redemptive uses of medical technologies. See, e.g., Cole-Turner [42,
43].
503
The principal strength of this fourth stance is its

embrace of human ingenuity accompanying the affirmation of embodiment. Technology is what makes
Homo sapiens into human and humane beings; to a
large extent humans are rightfully becoming Homo
faber (e.g. [45]). Technology in general is a significant means for exercising stewardship, and medical
technologies in particular for pursuing healing and
ameliorating suffering. Moreover, fertility preservation in conjunction with ART provides a refreshing
and powerful witness to life in a culture of death in
which all too often medicine is used to prevent or
destroy life. Consequently, provided that the moral
constraints of avoiding embryo destruction and PGD
are honored, there are not only no compelling reason why fertility preservation should be either discouraged or greeted with indifference, but should instead
be encouraged. The principal weakness of this stance
is that it assumes that greater technological development is synonymous with moral, social and political progress. It fails to recognize sufficiently the often
unintended and unforeseen evil affects accompanying this so-called progress. This is not to simply parrot the simplistic slogan of technological neutrality in
which tools and instruments can be used for good
or evil purposes; a scalpel, for example, can be used
for surgery or to commit a murder. Rather, it fails to
acknowledge the extent to which modern technological development itself shapes or misshapes the moral
vision (e.g. [4650]). In respect to medicine, for example, the patient becomes subtly transformed into an
artifact of medical techniques, or in respect to ART,
and by extension fertility preservation, distorts procreation into reproductive projects. In short, encouraging fertility preservation may in the long run prove
to be one more little piece in forming a Homo faber
that is not necessarily comprised of more humane
individuals.
Concluding remarks
As the preceding section demonstrates, a singular or
universal Christian assessment of fertility preservation cannot be formulated. There are, rather, multiple assessments. This wide variety should not be surprising given the historical divisions among Christian churches in respect to doctrine and practice. Nor
should it be presumed that these divisions are uniquely
manifested in assessing the ethics of fertility preservation. Rather, ethical concerns over fertility preserva-
504
tion represent continuing disputes which were present

in the early development of contemporary bioethics,
particularly in respect to such issues as informed consent, experimentation, organ transplantation, genetic
manipulation and beginning and end of life issues (e.g.
[5155]).13 Given these early theological contributions
to the burgeoning field of bioethics, it is also not surprising that the moral stances described in this chapter
may share some similarities with prevalent secular, and
often conflicting, philosophical and ideological orientations. The preservation of the natural reproductive
process stance, for instance, draws heavily upon deontological moral theory, whereas the stance stressing
social priorities is utilitarian and communitarian; the
stance emphasizing freedom of choice is libertarian,
while the stance encouraging fertility preservation as
an affirmation of life expresses a technological progressivism.
It is important in treating patients and formulating policy guidelines to keep this wide ranging and
overlapping diversity in mind. Although Christians
may hold general biblical and theological precepts in
common, varying interpretations will lead to varying ethical assessments of fertility preservation. Moreover, the range of precepts and resulting moral stances
described in this chapter has been illustrative rather
than exhaustive. There are a number of other pertinent
doctrines and resulting stances that could have been
explored. Consequently, there is a need for further and
more detailed investigation not only within the various Christian traditions, but, more importantly, also
in respect to other religious traditions and communities if ethical assessments of fertility preservation are
to take into account the full and rich diversity of the
contemporary world in which medicine is now practiced.
Acknowledgements
I am indebted to my research assistant, Jason Gill, for
his highly efficient and invaluable assistance.
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506
Index
ABVD regimen (adriamycin,

bleomycin, vinblastine and
dacarbazine) 14, 18, 329
ACT regimen (adriamycin and
cyclophosphamide) 51
AC regimen (adriamycin and
cyclophosphamide) 51, 55, 66
achondroplasia 103
acrosomal membrane integrity
(AMI) 189
active specific immunotherapy 44
activins 116, 411
acute lymphoblastic leukemia
(ALL) 13, 75
acute promyelocytic leukemia
(APL) 39
adaptive immune response 43
adenomatous polyposis coli (APC) 36,
38
Adjuvant Online 52
adjuvant therapy
breast cancer
newer treatments and additional
benefits 512
risk of recurrence and
reduction 50
adolescent and young adult cancer
epidemiology 868
international classification 88
risk factors 95
cervical cancer 956
melanoma 95
testicular cancer 95
thyroid cancer 96
adoptive T-cell therapy 44
alcohol consumption, parental 94
alginate 413
alginate 414
all-trans-retinoic acid (ATRA) 39
allotransplantation 201
amenorrhea 12, 14, 49
breast cancer treatment 65
incidence 65
fertility loss 545
survival 501
risk 16
amino acid sequences 451
amphiregulin 121, 4234
anaplasia 39
anastomosis 251
end-to-side 384
microvascular 3834
sleeve 384
whole ovary transplantation 3389
advantages and
disadvantages 339
androgens 121
Angelmans syndrome 102
angiogenesis 449
angiogenic switch 41
angiogenin 449
angiopoeitin 333
anonymous donation
oocytes 317, 318
sperm 2313
anthracyclines 65
antibody dependent cellular
cytotoxicity (ADCC) 44
anti-emetics 64
anti-growth signaling 401
anti-Mullerian hormone (AMH) 13,
25, 115, 116, 329
antral follicle count (AFC) 13
apoptosis 38, 101, 110
evasion by cancer cells 41
germ line 103
ability 105
DNA damage 1035
prosurvival factors in
spermatozoa 10910
spermiogenesis 1056
chromatin remodeling 1067
importance 1079
Aristotle 1
aromatase inhibitors
teratogenicity 64
artificial bladder 415
artificial ovary 308, 44856
assembly 450
bioactivity 452
biocompatibility 4534
biodegradability 453
crystallinity 451
interaction with
cells/follicles 4501
morphology 451
natural ovary 450
physical support 452
porosity 452
requirements 450
scaffold handling 454
surface 4512
vascularization 4523
fertility re-establishment
strategy 448
advantages 449
follicle number and quality 450
follicular development 449
follicular growth 44950
follicular survival 449
tissue engineering 4546
ascorbic acid 379
Asperts syndrome 103
assisted conception 1
assisted reproductive technology
(ART) 1, 1012
cryocooling 132
cryopreserved embryos 138
ataxia telangiectasia 40
Austin, Colin Bunny 3
autoimmune diseases 289
autotransplantation 201, 213
5-azacytidine 442
azoospermia 18
chemotherapy 75, 76
BAD 109
base excision repair (BER) 40
basic fibroblast growth factor
(bFGF) 115, 202, 333, 411, 449
BEACOPP regimen (bleomycin,
etoposide, doxorubicin,
cyclophosphamide, vincristine,
procarbazine and
prednisolone) 14, 18
BeckwithWiedmann syndrome 102
benign tumors 35
benzo[a]pyrenes 103
BEP regimen (bleomycin, etoposide
and cisplatin) 274
betacellulin 121, 423
bilateral salpingo-oophorectomy 269,
270
biocompatibility 452
biparental androgenones 443
biparental gynogenones 443
birth defects 102
507
Index
blastoma 35
bone density and GnRH agonist
use 242
bone marrow transplantation
(BMT) 14
ovarian failure rates 15
bone morphogenic proteins
(BMPs) 115
BMP4 116, 410, 411
BMP7 116, 410, 411
BMP15 117, 118
borderline ovarian tumors
(BOTs) 2667
fertility results 26970
limits of surgery 2689
disease stage 268
histological subtype 268
serous tumor with MP
pattern 268
ovarian induction or IVF 270
recurrence rates 269
surgery and outcomes 267
survival following surgery 2678
Bourn Hall Clinic 5
brain tumors 91
BRCA mutations 62, 63
breast cancer 49, 57, 69
adjuvant therapy
newer treatments and additional
benefits 512
risk of recurrence and
reduction 50
amenorrhea 65
amenorrhea and fertility loss 545
amenorrhea and survival 501
ART
time between treatment and
pregnancy 311
contraception in aftermath 68
endocrine therapy and reproductive
function 66
fertility preservation 66
adjuvant therapy delay 56
barriers to 55
controlled ovarian stimulation
(COS) 56
cost 55
donor eggs and surrogacy 67
embryo cryopreservation 667
oocyte preservation 67
ovarian harvest 56
ovarian tissue
cryopreservation 67
suppression of ovarian
function 678
incidence 62
lactation in aftermath 69
occurrence 49
508
pregnancy following diagnosis 55

pregnancy in aftermath 689
pregnancy-associated 634
biopsy 63
chemotherapy 64
diagnosis 63
incidence 63
treatment 63
recurrence 68
risk of recurrence
controlled ovarian stimulation
(COS) 56
high risk patients 56
standard regimens and
modifications 51
ER+ tumors 523
ER tumors 534
therapy and reproduction 62
treatment
ovarian function 646
reproductive function 64
young women 623
breastfeeding 923
Brown, Louise 4
Burkitts lymphoma (BL) 92
buserelin 244
1-buthionine-[S,R]-sulphoximine
(BSO) 107
cancer pre-vivor 488
cancer treatments, effect on
reproductive system 11, 1819
chemoprotection 18
chemotherapy and testicular
function 1718
ovarian damage from chemotherapy
clinical detection 1213
nature of damage 1314
risk of ovarian failure 14, 15
radiotherapy and testis 18
radiotherapy and the hypothalamic
pituitaryovarian
axis 1417
radiotherapy and the uterus 17
cancer, biological basis 35, 45
hallmarks of cancer 40
angiogenesis, sustained 412
anti-growth signal
insensitivity 401
apoptosis evasion 41
genomic instability 423
growth signaling
self-sufficiency 40
invasion and metastasis 42
limitless replicative potential 41
molecular pathogenesis 37
cell cycle and checkpoint
abnormalities 38
cell death defects 389
differentiation aberrations 39
DNA damage repair system
failures 39
growth factor signaling 378
signal transduction molecules 38
origins
cancer stem cell theory
(CSC) 367
oncogenes and tumor suppression
genes 356
stepwise accumulation of
mutations 36
transformation of normal cells 35
carbohydrates for
cryopreservation 178
carboplatin 52, 55
carcinoembryonic antigen (CEA) 44
carcinoma 35
Cut Standard Straw (CSS) 186
-catenin 119
CEF regimen (cyclophosphamide,
epirubicin and
fluorouracil) 51, 65
cell cycle abnormalities 38
cell death defects 389
cell-mediated immune response 43
cervical cancer
surgery 2578, 263
assessment 2623
experience of surgical team 2612
experience worldwide 262
technique 25861
cervical intraepithelial neoplasia 262
Chang, M. C. 3
chemoprotection 18
chemotherapy
breast cancer 50, 64
ovarian damage
clinical detection 1213
nature of damage 1314
risk of ovarian failure 14, 15
ovaries 734
testes 756
childhood cancers 11
epidemiology 836
international classification 84
children from oocyte donation
fears of parents 319
children from sperm donation 231
disclosure 2335
age of disclosure 235
religious and cultural
influences 234
shame 233
chlorambucil 18, 25
azoospermia 75
chlortetracycline (CTC)Hoechst
33258 staining 181
Index
ChlVPP regimen (chlorambucil,

vinblastine, procarbazine and
prednisolone) 14, 18
Christian ethics in fertility
preservation 497, 504
moral stances 499504
theological and biblical
precepts 4979
chromatin protamination assay 104
chromosome location 442
chronic myelogeneous lymphoma
(CML) 334
cisplatin 27
CMF regimen (cyclophosphamide,
methotraxate and
fluorouracil) 50, 51, 55, 65
collagen 413, 414
collagenase 402
colonization 42
Comet assay 102
communication between oocyte and
granulosa 399
communication during therapy
lack of recall following fertility
information 470
provider communication 4701
confocal microscopy 286
congenital abnormalities 433
continuous-cooling transformation
(CCT) diagram 132
controlled ovarian stimulation (COS)
breast cancer 49, 56
high risk patients 56
risk of recurrence 56
cooling rate and additive
concentration relationship 132
corona cells 346
Cowdens syndrome 63
critical cooling rate (CCR) 132
cross-border reproductive care
(CBRC) 473
cryobiology 129, 140
classical techniques 12930
slow freezing 1301
toxicity and osmotic effects of
CPAs 1312
preservation of cells and tissues
embryos 1389
oocytes 1367
ovarian tissue 1378
spermatozoa 1356
vitrification 1323
equilibrium and non-equilibrium
methods 1335
cryoinjury
ovarian tissue 3302
Cryoloop 180, 297
cryopreservation
spermatozoa 176, 193

fertilization properties 187
cryoprotectant toxicity neutralization
(CTN) 156, 157
cryoprotectantwater mutual
diffusivity 138
cryoprotective agents (CPAs) 26,
130
cytotoxicity 330
diffusion rates 137
discovery 328
osmotic effects 284
ovarian tissue 3478
toxicity and osmotic effects 1312,
135
Cryotop method 26
cryptorchidism 95
crystal growth 149
CTNNB1 pathway 118
cumulus cells (CCs) 114
cumulusoocyte complex (COC) 115,
424
cyclic adenosine mono-phosphate
(cAMP) 119
cyclic guanosine mono-phosphate
(cGMP) 120
cyclin-dependent kinases (CDKs) 38
cyclins 38
cyclophosphamide 18, 25
azoospermia 75
ovarian damage 13, 14
premature ovarian failure
(POF) 14
SLE 14
cyproterone acetate 169
cystic fibrosis screening 317
cytokines 43
cytoplasmic membrane integrity
(CMI) 189
cytoxic therapies
testicular function 17
Dalenpatius 2
dangerous temperature region
(DTR) 132
delayed first pregnancy 431
deleted in azoospermia-like
(DAZL) 202
designer babies 482, 483
creating a donor child 482
sex selection 482
destruction complex 38
destruction of embryos 435
developmental competence 397
devitrification 134, 150, 152
dibromochloropropane (DBCP) 164
diet, parental 923
differentiation of cells 39
dimethyl sulfoxide (DMSO) 131, 134,

136, 146, 147, 157, 284
disease modifying antirheumatic drugs
(DMARDS) 25
DNA damage 110
germ line apoptosis 1035
spermatozoa 1012
tobacco smoking 103
spermiogenesis 1056
importance of apoptosis 1079
DNA damage repair systems 39
DNA fragmentation 189
donor insemination (DI) 233
donors of oocytes, recruitment 31617
doubly-unstable glass 150
drugs see recreational drugs
dysplasia 39
Edwards, Robert 3, 5
ejaculation, retrograde 78
elastic strain 153
elective limited insemination
(ELI) 295, 297
electrocoagulation 25
electroejaculation 78, 228
embryo cryopreservation 26, 667, 77,
1389
embryo freezing program 280
results 280
ethical concerns 31
fertility preservation 279, 281
outcomes 27980
ovarian stimulation
protocols 2801
results of replacements in
oncologic patients 281
successful births 279
vitrification 139, 280
embryo transfer (ET) 4
endocrine neoplasia 103
endometriosis 23, 25
indications for fertility
preservation 30
endormal sinus tumors (EST) 274
end-to-side anastomosis 384
epidermal growth factor (EGF) 121
epidermal growth factor receptor
(EGFR) 121, 122
epigenetic memory 441
epigenetic reprogramming 443
epigenic regulation 4412
epigenome 441
epiregulin 121, 423
epithelial ovarian cancer (EOC)
indications for surgery 2702
literature reviews 271
surgical procedure 272
509
Index
epithelialmesenchymal transition
(EMT) 42
epitopes 391
EpsteinBarr virus (EPV) 92
equilibrium cryopreservation 130
equilibrium vitrification 133
equilirium slow freezing 130
estradiol levels 29
estradiol patches 322
estrogen receptor-positive tumors 426
estrogen receptors (ERs) 50
1,N6-ethenoadenosine 104
1,N2-ethenoguanosine 104
ethical concerns 4856
ART clinics 485
embryo freezing 31
future use following death of
parent 280
adults 480
children 4801
financial compensation for
donors 316, 317
future trends 479, 4835
legality of donation 316
ovarian cryopreservation and
re-transplantation 483
ownership of frozen oocytes 297
pre-implantation genetic diagnosis
(PGD) 482
spermatogonia, transplantation of
cryopreserved 206
stem cell research 4835
embryo research 484
legality 484
moral status of embryo 484
spare embryos 485
testicular cell and tissue
transplantation 219
use of donor gametes 4812
disclosure to children 4812
international variations 482
ethylene glycol (EG) 151
Ewings sarcoma 75
extracellular matrix (ECM) 41, 409,
414, 449
extravasation 42
extremely low-frequency
electromagnetic fields
(ELF-MF) 901
FAC regimen (fluorouracil, adriamycin
and cyclophosphamide) 51, 66
FaddyGosden model for natural
oocyte decline 16
Fallopian tubes 115
false hope potential of therapies 483
familial cancers 40
family members as sperm donors 232
510
FEC regimen (fluorouracil, epirubicin

female fertility decline with age 431
female hormones, exogenous 29
Fertile Hope 57
fetal calf serum (FCS) 402
fibrinalginate interpenetrating
network (FA-IPN) 414
fibronectin 414
fluorescence activated cell sorting
(FACS) 204, 205, 213
follicle cryopreservation 3056
artificial ovary 308
isolation of follicles 3067
transplantation 3078
follicle population decay curve 460
follicle stimulating hormone (FSH) 12,
66, 114
abnormal levels 378
direct and indirect effects 245
flare response 2401
indirect effect of suppression 246
male hormone suppression 165
ovarian expression 246
ovarian tissue transplantation
(OTT) 377
follicles
co-culture systems 41415
three-dimensional culture
systems 41214
follicles, molecular and cellular
integrity in culture 38990
functional properties 391
future prospects 3934
somatic cell components 3902
genomic integrity 391
hypertrophy over time 390
supporting and maintaining
oogenesis 3923
follicle-to-stroma paracrine
signaling 411
follicular development 114, 123
classification 344
stages 11415
1. primordial to
pre-antral 11517, 342, 352
2. pre-antral to antral 11719
3. antral to meiosis
resumption 11921
4. ovulation and
luteinization 1213
follicular diameter 452
follicular fluid meiosis activating sterol
(FF-MAS) 121
folliculogenesis 409, 450
role of stromal cells 41011
stromal tissue 409
follistatin 411
FOXL2 116
Foxo3 115
Foxo3a 116
fracturing in vitrification solutions
154
freedom of choice 502
frozenthawed ovarian tissue 7, 26
frozenthawed embryo (FET) 433
gamete intrafallopian transfer
(GIFT) 1, 4, 6
GATA binding protein 4 (GATA4) 202
GDNF family receptor alpha-1
(GFR1) 202
genes and predictions 460
genomic integrity 391
germ stem cells in adults 11
germinal vesicle 119, 296
germinal vesicle breakdown 119
Gibbs free energy 148
glass transition temperature 132, 150
glial cell line-derived neurotrophic
factor (GDNF) 202, 203
glutathione transferase 246
glycerol 178, 284
GnRH agonists 6, 23, 27, 29, 239
breast cancer 51
clinical data about ovarian
reserve 242, 243, 245
control group studies 2424
clinical use
dose 241
flare response 2401
ovarian reserve 2412
side effects 241
direct and indirect effects 245
immunological effect 247
mechanism of action 23940
ovarian impact 244
ovarian protection, hypothetical
mechanisms 247
ovarian stromal blood flow 2445
suppression of ovarian function 67
GnRH analogues 18
gonadal medical protection 27
gonadotrophin deficiency 16
gonadotrophin-releasing hormone
(GnRH) 66
ovarian expression 2456
ovarian steroidogenesis 239
gonadotrophins 119
goserelin 51, 53, 244
Graff, Reiner de 2
Graffian follicles 410
granulocyte-colony stimulating
factors 64
granulosa cells (GCs) 12, 13, 24, 114
poliferation 352
green fluorescent protein (GFP) 200,
443
Index
growth differentiation factor 9

(GDF-9) 117, 118, 393, 411
growth factor signaling 378
growth signaling 40
Harvey, William 1
healing 499
Heap, Walter 3
hematopoietic stem cell
transplantation (HSCT) 209
Hemi-straw 297
hepatocyte growth factor (HGF) 115
Her-2 neu growth factor receptor 50
hereditary breast ovarian cancer
(HBOC) syndrome 63
heterotopic autotransplantation 251,
336, 3347
endocrine function
re-establishment 336
hormone profile 336
heterotropic grafting 352
Hippocrates 1
histidinetryptophanketoglutarade
(HTK) solution 345
historical perspective 18
HIV infection 92
Hodgkins lymphoma 14, 76
chemotherapy
testis 18
ovarian tissue
autotransplantation 329
risk of POF 14
homogeneous nucleation
temperature 149
human chorionic gonadotrophin 66
Human Fertilisation and Embryology
Authority (HFEA) 7
human follicular lymphoma 38
human papillomavirus (HPV) 95
human serum albumin 402
human tubal fluid (HTF) 186, 228
humoral immune response 43
Hunter, John 1
hyaluronan (HA) 122
hydrocarbons and cancer 91
hydrogels 413
hydrogen peroxide, exposure to
spermatozoa 107
hydrophilicity 451
hydrophobicity 451
8-hydroxy-2
-deoxyguanosine
(8OHdG) 103
4-hydroxy-2-nonenal 104
hyperstimulation syndrome 23
hypertrophy over time 390
hypothalamicpituitaryovarian axis
effect of radiotherapy 1417
hypoxia 332, 363
hypoxia inducible factors (HIF) 333
ice quantity during cooling 151

imatinib 18, 27
immortality of cancer cells 41
immune surveillance 43
immunoediting 44
immunology 43
tumors 434
immunotherapy 445
in vitro fertilization (IVF) 1, 4
in vitro maturation 399
ovarian stimulation protocols 6
in vitro growth systems for
oocytes 397, 405
activation and growth of immature
follicles 399400
culture medium 402
culture of isolated follicles 4023
culture of oocytecumulus
complexes 404
culture of thawedcryopreserved
follicles 4034
follicle development support
strategies 400
follicular development 3979
isolation of growing follicles 402
ovarian cortical biopsy
culture 4012
cortical pieces 401
cortical strip 401
proposed multi-step culture
system 4045
in vitro maturation (IVM) 7, 278,
296, 347, 399, 4212, 431
emergence 422
female fertility preservation 432,
433
advantages of IVM 434
immature oocyte collection 435
number of patients 436
obstetric outcome 4323
oocyte vitrification 4356
ovarian stimulation in cancer
patients 4334
typical cycle 432
improving implantation rates
culture techniques 423
FSH priming 424
hormonal priming 424
metformin 425
preparation of endometrium 424
outcomes 426
patient selection 425
pregnancy rates 422
rescue in OHSS 425
social fertility preservation 4367
technical aspects 4223
incarnation of the divine 498
incomplete reprogramming 442
infections and cancer 92

infertility counselors 233
infertility treatment 101
inflammatory bowel disease 25
inhibin 411
inhibin A 378, 382
inhibin B 12
innate immune response 43
Institutional Review Board (IRB) 206,
491
insulin, selenium and transferrin (ITS)
media 402
insulin-like growth factor (IGF) 411
International Federation of Fertility
Societies (IFFS) 8
International Society for Fertility
Preservation (ISFP) 57
interrupted slow freezing 131
intracellular ice formation (IIF) 130,
145
intracytoplasmic sperm injection
(ICSI) 1, 7, 135
intrauterine insemination (IUI) 6,
135
intravasation 42
ionizing radiation and cancer
intrauterine and postnatal
exposure 90
preconceptual exposure 90
thyroid cancer 96
ischemic injury
ovarian tissue 3323
IVF lite 6
Jones, Georgeanna 4
Jones, Howard 4
keratinocyte growth factor (KGF)
115
kit ligand (KL) 115, 411
Klinefelters syndrome 209
Kuwayama method for oocytes 156
laminin 414
laparoscopy 3, 27
laproendoscopic single site surgery
(LESS) 382
legal aspects 4889, 4945
future use/disposition of
gametes 4924
informed consent 489
ASCO guidelines 489
research upon adults 4912
research upon children 492
standard of care 492
treatment in adults 48990
treatment in children 4901
insurance coverage for fertility
preservation 494
511
Index
lesbian, gay, bisexual and transgender

(LGBT) fertility
preservation 469
letrozole 29, 56, 67
ovarian stimulation 280
leukemia 18
leukemia inhibitory factor (LIF) 116
leuprolide 241, 242, 247
Leydig cells 17, 18, 75, 165
dysfunction following
chemotherapy 18
effect of radiation on function 77
Liberase 306
LiFramenis syndrome 63
liquid helium 180
liquid nitrogen 129, 132, 133, 154
love of neighbor 499
luteal cells (LCs) 122
luteinizing hormone (LH) 16, 114
male hormone suppression 165
luteinizing hormone receptors
(LHRs) 118, 410, 411
luteinizing hormone releasing
hormone (LHRH) agonists 56
luteinizing hormone releasing
hormone (LHRH) receptors 55
magnetic-activated cell sorting
(MACS) 213
males, chemotherapeutic effects 225
anejaculatory men either
oligospermic or
normospermic 2278
chance recovery of
spermatogenesis 226
sperm banking 2256
sperm use following treatment 227
treatment options for azoospermic
men 226, 22830
azoospermic before
treatment 227
cryopreserved sperm 2267
third party donation 2301
underlying medical conditions 230
males, fertility preservation
strategies 209, 21920
beneficiaries 209
fertility restoration 211
testicular germ cell
transplantation 211
immature testicular tissue 210
cell suspensions 210
tissue pieces 21011
whole testis 211
indications 210
options before gonadotoxic
therapies 210
safety considerations
512
birth defect risks 219

cancer cell contamination 21718
infectious transmission 219
studies in cryopreservation 212
studies in xenografting 218
testicular cell grafting 21617
testicular stem cells, fresh
efficiency 213
outcome 21113
SSC enrichment and
expansion 213
testicular stem cells, frozen 21314
clinical applications 214
testicular tissue grafting 214
testicular tissue, fresh 21415
xenotransplantation 216
testicular tissue, frozen 215
xenotransplantation 216
males, hormonal suppression 164
animal models 1658
clinical trials 1689
hormone suppression
treatments 168
historical development 1645
interspecies differences 16972
summary of effects 170
testicular function, effect upon
165
malignant germ cell tumors
(MGCTs) 274
fertility 273
malignant tumors 35
mannitol 379
Maternal Antigen That Embryos
Require (MATER) 119
matrix metalloproteinases (MMPs) 41,
42, 122
maximum tensile stress 154
medroxyprogesterone acetate
(MPA) 246
meiosis in fetal ovary 114
meiotic competence 397
meiotic spindle 285
changes with age 285
recovery 286
melanoma
melatonin 379
menopause 54
predictability 4634
mental health professionals (MHPs),
counseling role in ART 230
addressing shame issues 233
infertility counselors 233
metastases 42
metformin 425
microdissection testicular sperm
extraction (mTESE) 226, 227,
228, 229
micropapillary serous carcinoma

(MP) 266
microtubule organizing center
(MTOC) 285
minimal residual disease (MRD) 334
Mirena progesterone-delivery
system 68
mismatch repair (MMR) enzymes 40
mitochondrial membrane potential
(MMP) 182, 188, 189
mitogen-activated protein kinase
(MAPK) 119, 121
molarity of water 156
molarity of water-bonding groups 156
MOPP regimen (mechlorethamine,
vincristine, procarbazine and
prednisolone) 14
Mudhopadhyay, Subash 5
multiple pregnancies 7
Multi-Thermal-Gradient freezing
device 369
mustine 18
mutations 36
nafarelin 244
nasopharyngeal carcinoma (NPC) 92
Nelson, Henry 3
Newtons law of cooling 133
Nijmegen breakage syndrome
(NBS) 40
nitric oxide (NO) 120
nitrogen mustard 76
non-anonymous donation
oocytes 317, 318
sperm donation 231
family members 232
non-cancer patients 234, 31
procedures 256
embryo cryopreservation 26
gonadal medical protection 27
in vitro maturation 278
oocyte vitrification 26, 29
ovarian tissue
cryopreservation 267, 28
transposition of ovaries 28
indications for fertility
preservation 28
bone marrow transplantation 30
endometriosis 30
ovarian borderline tumors 301
postponement of childbearing
(age) 2930
routine gynecological
situations 31
Turners syndrome 30
ovarian damage 245
age 24
Index
chemotherapy 24
disease modifying antirheumatic
drugs (DMARDS) 25
non-steroidal anti-inflammatory
drugs (NSAIDs) 25
radiotherapy 25
rheumatic disease treatments 25
surgery 25
non-epithelial ovarian cancer 274
(MGCTs) 274
sex cord stromal tumors
(SCSTs) 274
non-equilibrium slow freezing 131
non-growing follicles (NGFs) 11
non-homologous endjoining
(NHEJ) 40
non-permeating cryoprotective
agents 130, 178, 284, 348
non-specific immunotherapy 44
non-steroidal anti-inflammatory drugs
(NSAIDs) 25
nucleotide excision repair (NER) 40
obesity and GnRH agonist
effectiveness 241
odds ratios (ORs) 433
oligomenorrhea 14
oncogenes 356
oocyte cryopreservation 136, 283
chemical and physical aspects of
freezing 2835
cell volume change 284
equations explaining cell
damage 283
osmotic effects 284
immature 137
mature 1367
morphological variables 2857
slow-cooling protocols and
outcomes 2879
slow freezerapid thaw
program 288
oocyte cumulus complexes (OCC)
346
oocyte donation for cancer
survivors 310, 31516, 3234
ovarian function testing 31213
ART
ovarian stimulation, hormonally
sensitive cancers 31415
clinical practice of donation 31623
anonymous versus known
donation 317
care of donor 319
donor recruitment 31617
endometrium preparation and
synchronization 3223
genetic screening of
donors 31718
infectious screening of
donors 317
matching donors and
recipients 319
oocyte retrieval 3212
ovarian stimulation of donor 320
psychological screening 318
psychological screening of
donors 31819
psychological screening of
recipient 319
recipient screening 322
evaluation for pregnancy 310
chronic health problems 310
gestational carrier 312
prognosis 310
time between treatment and
pregnancy 31112
uterine evaluation 312
oocyte preservation 67
oocyte recovery with tubal
insemination (ORTI) 4
oocyte retrieval 56
complications
intraperitoneal bleeding 321
pelvic abscess 321
pelvic infection 321
oocyte secreted factors (OSFs) 117
oocyte vitrification 23, 26, 29,
2934
clinical use 295
continuing evolution 301
establishing and troubleshooting
oocyte cryostorage 298301
Kuwayama method 156
outcomes 297
post-warming survival 297
protocol improvement 2978
typical media 298
purpose 2945
relevance 2957
oocytes
development 114
see also follicular development
meiosis 115
meiosis 119
number at birth 73
number of mature eggs released 73
oophorectomy 270
Open Pulled Straw (OPS) method 132,
186
orthotopic autotransplantation 334
oscillating heat pipe (OHP) 133
ovarian borderline tumors 301
ovarian cryobanking 328, 339
clinical guidelines 329
current status 334
heterotopic
autotransplantation 336, 3347
orthotopic
autotransplantation 334
xenotransplantation 338, 3378
historical perspective 3289
issues and concerns 330, 330
cancer cell transmission 3334
cryoinjury 3302
ischemic injury 3323
whole ovary transplantation by
vascular anastomosis 3389
advantages and
disadvantages 339
ovarian cryopreservation, whole 367,
373
challenges 3678
human ovary 3713
summary of experiments 372
viability evaluation 372
rat model 368
sheep model 368
slow cooling 36871
vitrification 371
ovarian development 1112
ovarian function (OVF) 377, 378
ovarian hyperstimulation syndrome
(OHSS) 320, 421, 434
IVM rescue 4256
ovarian malignant tumors,
conservative management 266,
274
borderline ovarian tumors
(BOTs) 2667
limits of surgery 2689
surgery and outcomes 267
survival following surgery 2678
epithelial ovarian cancer (EOC)
indications for surgery 2702
surgical procedure 272
literature reviews
EOC 271
MGCTs 273
ovarian induction or IVF after
BOTs 270
recurrence rates of BOTs 269
non-epithelial ovarian cancer 274
(MGCTs) 274
sex cord stromal tumors
(SCSTs) 274
ovarian metastasis 334
ovarian reserve 11, 2412
clinical data from GnRH agonist
use 242, 243, 245
control group studies 2424
oocyte numbers 241
513
Index
ovarian reserve (cont.)

qualitative assessment 12
ultrasound assessment 13
ovarian reserve tests (ORTs) 425
ovarian senescence, early 4601, 463
ovarian stimulation 279, 313
hormonally sensitive
cancers 31415
oocyte donors 320
dual suppression 320
GnRH agonist trigger for
ovulation 3201
protocols 2801
IVF 6
letrozole 280
tamoxifen 280
ovarian tissue cryopreservation 23,
267, 28, 67, 77, 1378, 342,
364
cryopreservation technique 347
cooling rate 34950
cryoprotectants 3478
developmental potential 352
evaluating efficiency 351
storage 350
vitrification 350
diffusion of CPAs 137
heterotopic autotransplantation 364
human tissue characteristics 3425
follicle classification 344
primordial stage follicles 342, 352
indications 357
malignant and non-malignant
diseases 358
methodology 343
preparation 3456
tissue dissection 3467
orthotopic autotransplantation 357
literature results 3613
ovarian biopsy and freezing 358
re-implantation 3601
restoration of ovarian
function 363
revascularization 3634
slow freezing procedure 3589
thawing 359
ovarian transplantation 2504
eligibility criteria 250
failure incidence 253
ovarian transplantation, tissue 357
ovarian transplantation, whole 377,
37980, 385
cryopreservedthawed ovary 3802
current status and
transplantation 3778
indications 377
freezing protocols 3823
fresh ovary 380
harvesting approaches 382
514
heterologous transplantation 3845

microvascular anastomosis 3834
microvascular thrombosis 384
morphological and functional
consequences of ischemic
damage 378
preventing ischemic damage
3789
studies reporting 381
ovaries
chemotherapy 734
FSH expression 246
functions 450
GnRH expression 2456
normal physiology 73
radiotherapy 75, 745
steroidogenesis 239
stromal blood flow 2445
transposition of 28
ovary, artificial 308
ovulation 1213
lead up 122
oxytetracycline 379
paracervical block 432
paracrine signaling 118, 415
follicle-to-stroma 411
stroma-to-follicle 41112
parental factors as risks for pediatric
cancers
lifestyle factors 92
alcohol consumption 94
diet and breastfeeding 923
recreational drugs 945
tobacco smoking 934
occupational exposures 912
partially crystallized glass (PCG) 150
passive humoral immunotherapy 44
PautzJeghers syndrome 63
pediatric cancer 73, 83
epidemiology
adolescent and young adult 868
childhood 836
survival 8890
fertility following treatment 789
cost 79
females, preventative 778
females, treatment 78
males, preventative 78
males, treatment 78
gonadotoxic chemotherapeutic
agents 74
guidelines for parents and
patients 79
ovaries
chemotherapy 734
radiotherapy 75, 745
risk factors 96
adolescents and young
adults 956
extremely low-frequency
electromagnetic fields 901
infections 92
ionizing radiation 90
parental lifestyle factors 925
parental occupational
exposures 912
risk of infertility 767
role of physician 79
testes
chemotherapy 756
radiotherapy 76
pelvic abscess 321
pelvic infection 321
pelvic malignancies, incidence
250
peritubular myoid cells 17
permeating cryoprotective agents 130,
178, 284, 347
rate of penetration 348
pesticides and cancer 91
phagocytosis 109
phosphatase and tensin homolog
(PTEN) 115
phosphatidylinositol-3-kinase (PI3K)
pathway 115, 203
phosphatidylserine translocation
(PST) 189
phosphodiesterase 3A (PDE3A) 120
pituitary gonadotrophin 114
platelet derived growth factor
(PDGF) 115
platinum compounds
azoospermia 76
pluripotent stem cells 440
epigenic regulation 4412
germ cell development 4423
germ cells 443, 4445
induced 441
germ cell differentiation 445
male germ cells 444
oocytes 4434
potential applications and future
prospects 445
stem cells 440
embryonic (ESC) 440
testis and ovary 440
Poisson ratio 153
PolscopeR 286
poly (ADP-ribose) (PAR) 105
poly (ADP-ribose) polymerase
(PARP) 40
polycyclic aromatic
hydrocarbonDNA
adducts 104
Index
polycystic ovary syndrome

(PCOS) 240, 421, 422, 425
polyglycolic acid (PGA) 453
polylactic acid (PLA) 453
polylactic-co-glycolic acid (PLGA)
microspheres 449
population mixing 92
predicting ovarian futures 45960
control of ovarian development 461
autosomal genes 462
role of X chromosome 4612
current state of prediction 460
future prospects 464
genes associated with follicle
formation 461
genes associated with ovarian
senescence 463
genetic risk factors 4601
issues 462
menopausal timing and
fertility 464
predictability of fertility 463
predictability of
menopause 4634
prednisone 76
pregnancy
breast cancer 634
biopsy 63
chemotherapy 64
diagnosis 63
incidence 63
treatment 63
pre-implantation genetic diagnosis
(PGD) 7, 482
pre-implantation genetic screening
(PGS) 23
premature ovarian failure (POF) 357,
358, 377
cyclophosphamide 14
non-cancer patients 24
ovarian tissue transplantation
(OTT) 377
procarbazine 14
radiotherapy 14
SLE 27
pre-pubertal girls 401
preserving natural reproductive
process 500
pre-vivor 488
primordial germ cells (PGCs) 199, 443
procarbazine 76
azoospermia 75
premature ovarian failure (POF) 14
procreative mandate 497
progesterone 121, 123
polycystic ovary syndrome
(PCOS) 422
replacement for oocyte
recipients 323
progesterone receptors (PRs) 50

promyelocytic leukemia gene
(PML) 39
1,2-propanediol 139, 147, 284
propidium iodide 189
propylene glycol (PG) 131, 136, 139
prostaglandins (PGEs) 122
protein kinase A (PKA) 119
protein kinase C epsilon (PKC ) 119
proto-oncogenes 35
psycho-education 319
psychological issues 467, 473
cross-border reproductive care 473
emotional distress associated with
cancer 4712
men 472
sexuality 472
patients undergoing fertility
preservation 4678
adolescents 4689
children 469
lesbian, gay, bisexual and
transgender (LGBT) 469
men 468
women 468
predictors of psychosocial
distress 4723
provider communication 4701
timely information 46970
psychological screening 318
donors of oocytes 31819
quercetin 24
radiotherapy
hypothalamicpituitaryovarian
axis 1417
ovaries 75, 745
testes 18, 76
uterus 17
recreational drugs, parental use 945
re-crystallization 134
relationship breakdown following
cancer 435
religious and cultural attitudes to
ART 234
restriction point 38
Ret receptor tyrosine kinase (RET)
202
retinoic acid (RA) 115
retinoic acid receptor-alpha gene
(RARA) 39
retinoic acid response elements
(RAREs) 115
retrograde ejaculation 78
return of function (ROF) 377
rheumatic disease treatments 25
disease modifying antirheumatic
drugs (DMARDS) 25
non-steroidal anti-inflammatory
drugs (NSAIDs) 25
sarcoma 35
senescence 41
sentinel lymph node biopsy 63
sentinel node 258
serine proteases 122
serinethreonine kinase family
(AKT) 203
Sertoli cells 13, 17, 75, 165
severe combined immunodeficient
(SCID) mice 308, 337
sex cord stromal tumors (SCSTs) 274
signal transduction molecules 38
sleeve anastomosis 384
Smad2 and Smad3 117
smoking see tobacco smoking
social priorities 501
solution effects 296
Spallanzani, Lazzaro 2
spare embryos 485
sperm banking 2256
sperm chromatin structure assay
(SCSA) 102
sperm cryopreservation 78
Sperm DNA Degradation (SDD) 109
sperm donation and ART 225, 2301
anonymous versus known
donation 2313
disclosure to children 2335
religious and cultural
influences 234
sperm preparation medium (SPM) 180
spermatogenesis 101
spermatogonia (type A dark) 209
spermatogonia (type A pale) 209
spermatogonia, transplantation of
cryopreserved 199200, 206
culture of human cells 2034
development 2001
isolation, purification and culture of
murine cells 2023
morphological identification 2012
removal of malignant cells 205
spermatogonial stem cells (SSCs) 199
spermatozoa 101, 110
apoptosis and prosurvival
factors 10910
apoptosis in germ line 103
ability 105
DNA damage 1035
cryopreservation 1356, 176, 193
fertilization properties 187
DNA damage 1012
long-term storage 1923
male age and progeny disease 1023
515
Index
spermatozoa (cont.)
mammalian sperm head size 180
smoking and progeny disease 103
spermiogenesis 1056
importance of apoptosis 1079
vitrification 1767
IVF, ICSI and
insemination 18992
optimal cooling rates 180
suitability 1779
technique 17989
sphingosine-1-phosphate 210
squamous cell carcinoma of cervix 364
Src family kinase 203
stem cell factor (SCF) 115
stem cells 440
embryonic (ESC) 440
germ cells 443, 4445
male germ cells 444
oocytes 4434
testis and ovary 440
Steptoe, Patrick 3
Bourn Hall Clinic 5
sterilization of a minor 490
stewardship of the planet 498
StolesEinstein equation 154
strain 153
stress 153
stromal cells 409, 416
follicle co-culture systems 41415
organ culture and primordial follicle
activation 412
role in follicle development 41011
studies
follicle-to-stroma paracrine
signaling 411
stroma-to-follicle paracrine
signaling 41112
three-dimensional follicle culture
systems 41214
sulfasalazine 25
supercooled solutions 147, 149
supercooled water 134
surface free energy 451
surrogacy 67
systemic lupus erythematosus
(SLE) 14
premature ovarian failure (POF) 27
T lymphocytes 43
T regimen (taxotere) 51
T regulatory cells (Tregs) 44, 45
TAC regimen (taxotere, adriamycin
tamoxifen 50, 52, 53, 55, 244, 246
breast cancer during pregnancy 64
hormonal stimulation 67
516
irregular menses 66
teratogenicity 64
taxanes 52, 55, 65, 66
taxotere 55
technological affirmation of life 503
telomerase 41
telomeres 41
alternative lengthening 41
tensile stress, maximum 154
testes
chemotherapy 1718, 756
function 17
hormonal suppression, effect of 165
immature testicular tissue
cell suspensions 210
tissue pieces 21011
whole testis 211
radiotherapy 18, 76
undescended 102
testicular cancer
testicular sperm extraction (TESE) 7
testosterone, low levels 18
5, 5
, 6, 6
-tetrachloro-1, 1
, 3, 3
tetraethylbenzamidazolocarbocyanin iodide 183, 189
thalassemia screening 318
theca cell organizer 411
thecal cells (TCs) 114, 115
three-dimensional follicle culture
systems 412
thrombocytopenia 242
thyroid cancer
timetemperature transformation
(TTT) diagram 132
tissue engineering 41516
artificial ovary 4546
tobacco smoking, parental 934
DNA damage in spermatozoa 103
total body irradiation (TBI) 14
effect on ovaries 75
effect on testes 18
effect on uterus 17
transforming growth factor beta
(TGF) 40, 42, 333
superfamily 116, 117
trastuzamab 51, 64
tuberous sclerosis complexes
(TSCs) 116
tubulin 285
depolymerization of dimers 285
tumor-associated antigens (TAAs) 44
tumor-causing viruses 35
tumor-infiltrating lymphocytes
(TILs) 43, 44
tumor-specific antigens (TASs) 44

tumor suppression genes 356
tumors, immunology 434
TUNEL assay 104
Turners syndrome 30, 380
twins, monozygotic, discordant for
POF 380, 383
twins, non-identical 384
two-dimensional follicle culture
systems 412
ubiquitin carboxyl-terminal esterase
L1 (UCHL1) 202
ultrasound assessment of ovarian
reserve 13
unilateral (left) oophorectomy 358
upper arm as recipient ovary
transplantation site 380
uterus, evaluation for pregnancy 312
VAC regimen (vincristine,
actinomycin and
cyclophosphamide) 14
vaginal intraepithelial neoplasia 262
valproic acid 442
van Leeuwenhoek, Anton J 2
vascular endothelial growth factor
(VEGF) 41, 215, 333
viability staining 351
vincristine 76
viscous strain 153
vitrification 1323, 1456, 158
embryos 139, 280
historical origins 1468
oocytes
immature 137
mature 137
ovarian cryobanking 330
protocols 332
physical aspects
concentration dependence 150
glass fracture 1534
ice avoidance 1513
ice nucleation and growth 1489
necessity of vitreous state 149
storage below glass transition
temperature 1545
spermatozoa 1767
IVF, ICSI and
insemination 18992
optimal cooling rates 180
suitability 1779
technique 17989
vitrification solutions and
toxicity 1556
carrier solution selection 157
CPA toxicity 1567
defining needs 157
extracellular agents 157
Index
glass formation 156

osmotic effect elimination 158
special additives 157
VogelTammannFulcher (VTF)
equation 154
von Baer, Carl Ernst 2
WallaceKelsey model of ovarian
follicle decline 11
Warnock Report 7
wettability 451
wine, protection against cancer 94
xeroderma pigmentosum 40
xenografting 352
xenotransplantation 201, 213, 214,
338, 3378
fresh human testicular tissue 216
frozen human testicular tissue 216
zinc finger and BTB domain containg

16 (ZBTB16) 202
zona pellucida 287
hardening 287
zygote intrafallopian transfer (ZIFT) 1
Youngs modulus 153
517

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Principles and Practice of

Principles and Practice of

CAMBRID GE UNIVERSIT Y PRESS

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This book is dedicated to our courageous patients fighting cancer

Section 3: Reproductive biology

The evolution of ART

The effect of chemotherapy and

Fertility preservation in non-cancer

Basic cancer biology and immunology

Breast cancer and fertility

Breast cancer therapy and

Pediatric cancer therapy and fertility

Cancer epidemiology and

Life and death in the germ line:

10 Molecular aspects of follicular

Section 2: Cancer biology,

12 Fundamental aspects of vitrification

Section 4: Fertility preservation

13 Hormonal suppression for fertility

Section 5: Fertility preservation

Section 6: Fertility preservation

24 Cryopreservation of human oocytes

29 Ovarian tissue transplantation 357

Section 8: In vitro follicle growth

34 Contributions of ovarian stromal cells

36 Clinical potential of in vitro

Section 9: Future technologies

Section 10: Ethical, legal

43 Christian ethics in fertility

Karina Braga Ribeiro

and Gynaecology, University of Melbourne,

Anne Van Langendonckt

One of the first and most poignant cases that

the potential for biological parenthood after cancer.

Three decades have elapsed since the historic birth of

of their impending cancer therapy. Such individuals

Fertility preservation has become a very prominent

culture and ovarian cryopreservation and transplantation.

We, Jacques Donnez and S. Samuel Kim, would like to

The evolution of ART

To understand science, it is necessary to know its history.

Figure 1.1 John Hunter (172893). The first reported person to

hypospadias, and inseminated her with that specimen.

Figure 1.3 Anton van Leeuwenhoek (16321723). The first person

responsible for the production of all creatures. It was

Dalenpatius in 1699 stated that he could see a

Chapter 1: The evolution of ART

mammalian embryos. Von Baer is credited with being

Figure 1.5 Karl

Figure 1.6 Patrick Steptoe and Robert

from the British Army. There, under the supervision

Chapter 1: The evolution of ART

Figure 1.7 Worlds first in vitro

In the early years following the first IVF births in

late in their lives. In a few countries, treatment using

Chapter 1: The evolution of ART

only real option then was the use of donor sperm.