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Genetics in Human Reproduction 1st Edition Elisabeth
Hildt (Editor) Digital Instant Download
Author(s): Elisabeth Hildt (editor), Sigrid Graumann (editor)
ISBN(s): 9781138314979, 1138314978
Edition: 1
File Details: PDF, 18.26 MB
Year: 2018
Language: english
GENETICS IN HUMAN REPRODUCTION
G enetics in Human
Reproduction

Edited by
ELISABETH HILDT and SIGRID GRAUMANN
First published 1999 by Ashgate Publishing

Reissued 2018 by Routledge

2 Park Square, Milton Park, Abingdon, Oxon, OXl 4 4RN
711 ThirdAvenue, New York, NY 10017

Routledge is an imprint of the Taylor & Francis Group, an infonna business

Copyright © Elisabeth Hildt and Sigrid Graumann 1999

All rights reserved. No part of this book may be reprinted or reproduced or utilised in
any form or by any electronic, mechanical, or other means, now known or hereafter
invented, including photocopying and recording, or in any information storage or
retrieval system, without permission in writing from the publishers.

Notice:
Product or corporate names may be trademarks or registered trademarks, and are
used only for identification and explanation without intent to infringe.

Publisher's Note
The publisher has gone to great lengths to ensure the quality of this reprint but points
out that some imperfections in the original copies may be apparent.

Disclaimer
The publisher has made every effort to trace copyright holders and welcomes
correspondence from those they have been unable to contact.

A Library of Congress record exists under LC control number: 98074633

ISBN 13: 978-1-138-31497-9 (hbk)

ISBN 13: 978-1-138-31498-6 (pbk)
ISBN 13: 978-0-429-45661-9 (ebk)
Contents

List of Contributors Vili

Preface XI
Acknowledgements XIII
List of Abbreviations XV

Part One: Medical and scientific view

1 Clinical experience with PID and ICSI

Ingeborg Liebaers, K. Sermon, C. Staessen, H. Joris, W. Lissens,
E. Van Assche, P. Nagy, M. Bonduelle, M. Vandervorst, P. Devroey,
A. Van Steirteghem 3
2 The various micromanipulative procedures:
State of the art, chances, and risks
Dieter Meschede and Jürgen Horst 17
3 The relation between ICSI and genetic diagnosis
from an ethical point of view
Barbara Maier 23
4 Modification of IVF application and access to IVF services by PID?
Brian A. Lieberman 33
5 Examples for possible PID indications -
scientific background and reflections on effects
Ulrike A. Mau 37

V
6 Should there be a uniform list of genetic diseases
allowing access to PID?
Hansjakob Muller 47
7 Nuclear transplantation - medical and ethical aspects
Gerd Richter and Matthew D. Bacchetta 55

Part Two: Personal interests and moral implications

1 Ethics of preimplantation genetic diagnosis

Guido de Wert 75
2 Preimplantation diagnosis. A reflection in light of a personalist ethics
Paul Schotsmans 97
3 Ethical aspects of germline gene therapy
Alexandre Mauron 107
4 ‘Quality control’ in reproduction -
what can it mean, what should it mean?
Dieter Birnbacher 119
5 Does gene therapy have ethically problematic effects on identity?
Ingmar Persson 127

Part Three: Moral rights and duties

1 Does PID solve the moral problems of prenatal diagnosis?

A rights analysis
Deryck Beyleveld 13 5
2 Ethics of Research on Human Embryos
Reiner Wimmer 141
3 Categorical arguments - Pro life versus pro choice?
Maureen Junker-Kenny 147
4 Selection through prenatal diagnosis and preimplantation diagnosis
Hille Haker 157

Part Four: Social concepts and moral implications

1 Eugenics comes back with medically assisted procreation

Bernard Sèle and Jacques Testart 169
2 Germline gene ‘therapy’: Public opinions with regard to eugenics
Sigrid Graumann 175
3 Predictive genetic medicine - a new concept of disease
Lene Koch 185

VI
4 Animal Models: an anthropologist considers Dolly
Sarah Franklin 197
5 Issues surrounding preimplantation diagnosis
and germline gene therapy
Alexandre Quintanilha 209
6 Beside the point - reflections on passivity
Paul JM van Tongeren 213

Part Five: Choices and decision making

1 What claims can be based on the desire for a healthy child?

Towards an ethics of 'informed desires'
Walter Lesch 223
2 The European Alliance of Genetic Support Groups
Ysbrand Poortman 235
3 Some reflections on the use of the term 'prevention'
in reproductive medicine
Elisabeth Hildt 243
4 Preimplantation diagnosis - implications for genetic counselling
Ruth Chadwick 251

Part Six: Health care, justice and regulation

1 Legal regulations concerning preimplantation diagnosis

Jennifer Gunning 261
2 Reproductive technology and the slippery slope argument:
A message in Blood
Tony McGleenan 273
3 Measuring the benefits of IVF
Emma McIntosh and Mandy Ryan 285
4 Justice and preimplantation diagnosis
Joke de Witte 293
5 The role of ethics codes in medicine -
how can they be helpful in making decisions?
Stella Reiter-Theil 299

Index 311

VII
List o f Contributors

Matthew D. Bacchetta is Managing Director at the Cornell Medical Center, New

York Hospital, Department of General Surgery.

Deryck Beyleveld is Professor of Jurisprudence at the University of Sheffield

and Founding Director of the Sheffield Institute of Biotechnological Law and
Ethics (SIBLE).

Dieter Birnbacher is Professor of Philosophy at the University of Düsseldorf.

Ruth Chadwick is Head of the Centre for Professional Ethics and Professor
for Moral Philosophy at the University of Lancashire.

Sarah Franklin is Senior Lecturer in Anthropology at Lancaster University.

Sigrid Graumann is Scientific Coordinator of the Network for Biomedical

Ethics together with Hille Haker.

Jennifer Gunning is Research Associate in Medical Law and Ethics at

Cardiff Law School.

Hille Haker is Research Assistant at the Department of Ethics and Social

Sciences (University of Tübingen) and Scientific Coordinator of the European
Network for Biomedical Ethics together with Sigrid Graumann.

Elisabeth Hildt was the Scientific Coordinator of the European Network for
Biomedical Ethics from 1996 until March 1998.
VIII
Jürgen Horst is Chairman of the Department of Human Genetics at the University of
Münster.

Maureen Junker-Kenny is Head of the School of Hebrew, Biblical and

Theological Studies at Trinity College, Dublin.

Lene Koch is Senior Research Fellow at the University of Copenhagen,

Institute of Social Medicine.

Walter Lesch is Research Assistant at the Interdisciplinary Institute for Ethics

and Human Rights at the University of Fribourg.

Ingeborg Liebaers is Professor of Clinical and Experimental Genetics and

Head of Clinic of the Academic Hospital at the Dutch-speaking Brussels Free
University.

Brian A. Lieberman is Professor of Gynaecology at the Manchester Fertility

Services.

Barbara Maier is gynaecologist at the Salzburg Frauenklinik and teaches

medical ethics at Vienna University.

Ulrike A. Mau is Clinical Geneticist at the Department of Anthropology and

Human Genetics at the University of Tübingen.

Alexandre Mauron is Associate Professor of Bioethics at the University of

Geneva Medical School.

Tony McGleenan is Lecturer in Jurisprudence at the School of Law at the

Queen’s University of Belfast and legal adviser to the European Commission
Euroscreen Project.

Emma McIntosh is working on her PhD in the area of benefit assessment in

health economics with particular attention to the technique of conjoint
analysis.

Dieter Meschede is Research Assistant at the Institute of Human Genetics at

the University of Münster.

Hansjakob Müller is Professor of Human Genetics and Head of the

Department of Medical Genetics of the University Children's Hospital Basel
and of the Laboratory of Human Genetics of the Basel University Clinics.

Ingmar Persson is Professor at the Department of Philosophy at Lund

University.
IX
Ysbrand Poortman is Vice President of the European Alliance of Genetic
Support (European Alliance of Muscular Dystrophy Associations).

Alexandre Quintanilha is Director of the Centre for Experimental Cytology,

University of Porto, Portugal, and President of the Scientific Board of the
Institute of Biomedical Sciences Abel Salazar.

Stella Reiter-Theil is Research Coordinator at the Centre for Ethics and Law
in Medicine, University Hospital Freiburg i. Br.

Gerd Richter is Internist and Gastroenterologist at the Department of Internal

Medicine of the Philipps-University of Marburg.

Mandy Ryan is MRC Senior Fellow, Health Economics Research Unit, at the
Department of Public Health, Aberdeen.

Paul Schotsmans is Professor of Medical Ethics and Director of the Centre

for Biomedical Ethics and Law at the School of Medicine, K.U. Leuven.

Bernard Sele is Geneticist and Professor of Reproductive and Developmental

Biology at the Grenoble University Medical School (France) and Director of
the IVF Laboratory.

Jacques Testart is Professor at the Institut de Physiologie et Psychologie de la

repoduction humaine, Centre de recherche de l’INSERM.

Paul J.M. van Tongeren is Professor of Philosophical Ethics at the Catholic

University of Nijmegen and Chairman of the Center for Ethics of the Catholic
University of Nijmegen.

Guido de Wert is a Senior Research Fellow at the Institute for Bioethics, Maastricht,
and Associate Professor in Medical Ethics at the Erasmus University Rotterdam.

Joke de Witte is biologist and philosopher and a staff member of the Center for
Ethics in Nijmegen.

Reiner Wimmer is Professor of Philosophy at the University of Tübingen.

X
Preface

Since the birth of the first child conceived by in vitro fertilisation almost two
decades ago the field of assisted reproduction is expanding continuously.
Though, in the beginning of this development there has been an intensive
discussion about the moral permittance o f artificial intervention in human
procreation and many aspects are still controversial in the public, today
assisted reproduction is widely established as infertility treatment in medical
practice.
In the 70s and 80s the ethical discussion was dominated by the problems
related with artificial procreation as such, poor success rates of IVF, surrogate
motherhood, split in social, biologic and genetic parenthood, cryoconservation
and spare embryos, male domination of women’s bodies, research with human
embryos to improve the methods and similar topics. In spite of the fact that
most of the stressed problems are still prevalent there is a change in the
concentration on points of emphasis perceptible during the last years. The
background for this alteration of the ethical discussion forms the experience of
the establishment of the clinical practice of assisted reproduction and in vitro
fertilisation as well as the presence of results of empirical follow-up studies on
the one hand and the technological innovations in this field on the other hand.
The new techniques pre-implantation diagnosis (PID), intracytoplasmic sperm
injection (ICSI), in vitro ovum nuclear transplantation (IVONT), and in the
fixture possibly germline gene therapy are bringing human genetics and
assisted reproduction together.
Though, the theoretical possibility to check up the embryo in vitro for
genetic “abnormalities” may have been from the beginning of in vitro

XI
fertilisation an idea of great influence on the part of the involved scientists, the
expected benefits and the feared dangerous consequences of pre-implantation
genetic diagnosis are rather new topics of public interest. Although not being
feasible in human beings at the moment, also germline gene therapy - for
which IVF is the presupposition - is a matter of intensive medical and ethical
discussion.
Medical, social and ethical issues relating to the latest developments in IVF
are discussed in the first book of the European Network fo r Biomedical Ethics
with the title “In Vitro Fertilisation in the 1990s - Towards a Medical, Social
and Ethical Evaluation” which has been issued 1998 at Ashgate with Elisabeth
Hildt and Dietmar Mieth being the editors.
The present volume concentrates on the issues related to the current as well
as to the possibly future technological progress in genetic technologies linked
to IVF, i.e. preimplantation diagnosis and germline gene therapy, from a
scientific and medical as well as from a social, juridical and ethical point of
view.
This book contents the contributions of the second symposium of the
European Network for Biomedical Ethics ‘Genetics in Human Reproduction’
which took place from February 26th to March 1st, 1998 in Maastricht,
Netherlands. It provides a multidimensional view on the moral questions
raised by PID and related technologies by collecting contributions from
researchers coming from various European countries, working in different
disciplines and arguing on various theoretical backgrounds.
The basic scientific data concerning preimplantation diagnosis and other
micromanipulative procedures, as well as considerations concerning the
chances and risks going along with these technologies from a scientific and
medical point of view are discussed in Part One of this volume. These
contributors are all physicians and scientists which does not mean that they
leave out the ethical questions. The individual interests playing a role in PID
and other micromanipulative procedures and their moral implications, e.g.
concerning the responsibilities of prospective parents, the scientists involved,
and society as a whole, are further examined in Part Two. Part Three
concentrates on moral rights and duties regarding the possibilities of the new
techniques on the one hand and the moral status of the embryo on the other.
Part Four collects contributions with controversial moral views on the social
implications of PID and related technologies. The contributors to Part Five are
stressing the moral significance of desires, moral implications of reproductive
choices and the role of counselling in the decision making process in the
context of PID and related technologies.
The book is completed by Part Six with questions of justice in health care
systems and legal regulation of PID and other micromanipulative technologies
in the European context.

XII
Acknowledgement s

This volume is a collection of the lectures given at the symposium “Genetics

in Human Reproduction” (Maastricht, February 1998) which was organised by
the European Network fo r Biomedical Ethics in co-operation with the Instituut
voor Gezondheitsethiek, Maastricht.
We want to heartily thank Ruud ter Meulen for making this Maastricht
conference possible. We are especially indebted to the local organisers Guido
de Wert and Angelique Heijnen.
Without the co-operation of a great number of persons, the symposium could
not have been held and this book could not have been prepared. In particular,
we would like to thank Katja Ruppel and Christof Mandry for invaluable help
with the organisation of the symposium. Katja Ruppel and Annika Thiem
carried out a great deal of the editorial work on this book, Glenn Patten and
Hille Haker gave us support with proof-reading. We also want to thank the
Center for Ethics in the Sciences and Humanities, University of Tübingen, for
technical support, and Michael von Doering for organisational help.
In particular, this volume owes its existence to the enormous co-operation of
all contributors and to the great support of the members of the European
Network fo r Biomedical Ethics. We want to take the opportunity to express our
thanks for their personal engagement.

XIII
 We are grateful to the European Commission, Dr. Christiane Bardoux, DG XII,
Science, Research and Development, for the generous funding of the European
Networkfor Biomedical Ethics, the symposium “Genetics in Human Reproduction”,
and the publication of its results.
Elisabeth Hildt
Sigrid Graumann

These lectures were also the contributions at the Second Symposium of ENBE in
Maastricht/NL in April 1998. The first symposium resulted in the publication of In
Vitro Fertilisation in the 1990s edited by Elisabeth Hildt and Dietmar Mieth, which
concentrated on interdisciplinary approach and dialogue about IVF in a general mean­
ing and in assisted procreation. This second volume focuses on PGD-techniques,
scientific, social, legal and ethical aspects. It will be followed by a third volume (from
the symposium in Sheffield in January 1999), the purpose of which is the social and
ethical debate on Human Procreation, promoting the controversy but also common
‘points to consider’.
As the Director of the Network I would like to thank the editors of this book but
also say thank you for the teamwork in the co-ordination of the whole project, includ­
ing management, newsletters and research activities.

Dietmar Mieth

XIV
List o f Abbreviations

AC Amniocentesis
ACGT Advisory Committee on Genetic Testing
AID Artificial insemination with donor sperm
ART Assisted reproductive technology
bp Base pairs
BRCA 1 (Breast cancer predisposition gene)
BRCA2 (Breast cancer predisposition gene)
CA Conjoint analysis
CBA Cost-benefit analysis
CBAVD Congenital bilateral absence of the vas deferens
CEA Cost-effectiveness analysis
CF Cystic fibrosis
CFTR (Cystic fibrosis gene)
CHA Catholic Health Assosiation of America
CUA Cost-utility analysis
CVS Chorionic villus sampling
DMD Duchenne’s muscular dystrophy
DNA Deoxyribonucleic acid
EAGS European Alliance of Genetic Support Groups
ECJ European Court of Justice
ESHG European Society of Human Genetics
ET Embryo transfer
FAP Familial adenomatose polyposis
FISH Fluorescence in situ hybridisation
GLGT Germline gene therapy

xv
HBOC Hereditary breast/ovarian cancer
HD Huntington’s disease
HEXAA Beta-N-acetylhexoaminidase A
HFE (Act) Human Fertilisation and Embryology (Act)
HFEA Human Fertilisation and Embryology Authority
HIV Human immunodeficiency virus
ICSI Intracytoplasmic sperm injection
IRB Institutional Review Board
IVF In vitro fertilisation
IVM In vitro maturation
IV ONT In vitro ovum nuclear transplantation
MELAS Mitochondrial encephalomyopathy, lactic acidosis, and
stroke-like episodes
MESA Microsurgical sperm aspiration
mtDNA Mitochondrial DNA
NABER National Advisory Board on Ethics in Reproduction
nDNA Nuclear DNA
NF 1 Neurofibromatosis type 1
NHS National Health Service
PCR Polymerase chain reaction
PEP Primer extension preamplification
PGC Principle of generic consistency
PGD Preimplantation genetic diagnosis
PGS Preimplantation genetic screening
PID Preimplantation diagnosis
PKU Phenylketonuria
PND Prenatal diagnosis
PPA Prospective purposive agents
TBHR Take baby home rate
TESE Testicular sperm extraction
TNT Therapeutic nuclear transfer
WHO World health organisation
WTP Willingness-to-pay

XVI
 Part One
MEDICAL AND SCIENTIFIC VIEW
1 Clinical experience with
PID and ICSI*
Ingeborg Liebaers ' , K. Sermon , C. Staessen ,
H. Joris2, W. Lissens1, E. Van Assche1, P. Nagy2,
M. Bonduelle1, M. Vandervorst2, P. Devroey2,
A. Van Steirteghem2

This article was reproduced with kind permission o f Human Reproduction.

‘Centre for Medical Genetics and 2 Centre for Reproductive Medicine, Dutch speaking
Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium.
3To whom correspondence should be addressed.

Abstract

Preimplantation genetic diagnosis (PID) is a novel procedure which may be

considered as a very early prenatal diagnosis for couples at risk for
transmitting genetic diseases. Using the polymerase chain reaction (PCR) or
fluorescence in situ hybridisation (FISH) the genotype or the sex of biopsied
cleavage stage embryos obtained after in vitro fertilisation can be determined
and selected embryos are then transferred. In vitro fertilisation with
intracytoplasmic sperminjection is the method of choice to obtain embryos
analysed through PCR to reduce contamination by residual sperm DNA. In our
series of 61 PID cycles for 29 couples at risk since a period of 4 years the
ongoing pregnancy rate per cycle was 15 %, per transfer 19 % and per patient
31 %. O f the 6 morphologically normal children bom, one who is still alive
and doing well, weighed 850 gr. because it was bom at 25 weeks after a
complicated triplet pregnancy. More experience is needed to correctly evaluate
the efficiency and safety of this novel technique as well as its place in the
prevention of genetic disease.

3
Introduction

Preimplantation genetic diagnosis (PID) is a novel technique which permits

determination of the genotype of an oocyte before fertilisation or of an embryo
before implantation. On the one hand, this procedure became possible because
of the almost simultaneous development of IVF, micromanipulation, PCR and
fluorescence in-situ hybridisation (FISH). On the other hand, patients or
couples with a recurrence risk for genetic diseases were asking such a
procedure so as to avoid the need for pregnancy termination after conventional
prenatal diagnosis. The first clinical PID was reported by Handyside and
Winston in 1990 (Handyside et al. 1990). Preclinical studies had convinced
them that their was apparently no harm in biopsying a cleavage-stage embryo
at the 6- to 8-cell stage (Hardy et al. 1990). Since then several groups have
performed PID successfully using either PCR or FISH to analyse blastomeres
from 3-day-old embryos (Harper 1996, Lissens et al.l 996) or to analyse polar
bodies from oocytes before fertilisation (Verlinsky et al. 1996).
Intracytoplasmic sperm injection (ICSI) certainly has its use in PID. In the
first place it has an advantage over conventional in vitro fertilisation (IVF) in
that it avoids contamination by sperm in PID of a cleavage-stage embryo,
using the polymerase chain reaction (PCR). Moreover, for some couples with a
concurrent genetic risk such as cystic fibrosis (CF) in cases of congenital
bilateral absence of the vas deferens (CBAVD), pregnancy will be obtained
more often after ICSI with spermatozoa retrieved from the epidydimis than
after regular IVF (Silber et al. 1994). Klinefelter patients who on rare
occasions may produce a very low number of spermatozoa may try to father a
child through IVF and ICSI with spermatozoa extracted from an ejaculate or
more often from a testicular biopsy and in this experimental setting the number
of sex chromosomes in the embryos will have to be evaluated before transfer.
The outcome of these treatment cycles in Klinefelter patients have partially
been published and will be updated separately (Staessen et al. 1996, Toumaye
et al. 1996). In this article our clinical PID experience in case of a recurrence
risk for monogenic diseases will be reported and the place of ICSI will briefly
be discussed.

Materials and methods

Twenty-nine couples were counselled at the Centre for Medical Genetics

between February 1993 and February 1997 prior to PID. Except for 3 of them
with a lower risk (see appendix) they had a 25% or 50% risk of having
children with CF (with or without CBAVD in the male) (n=8), with non-
fragile X mental retardation (n=3), with hemophilia A (n=2), with Duchenne’s
muscular dystrophy (DMD) (n=4), with retinitis pigmentosa (n=l) or with

4
myotonic dystrophy (n= ll). The reason why these couples chose PID rather
than regular prenatal diagnosis were in general (A) infertility or subfertility
necessitating IVF as well as the genetic risk (n=15), (B) one or several
pregnancy terminations after chorionic villus sampling (CVS) or
amniocentesis (AC) (n=8) and (C) moral, emotional or religious objections
against abortion in itself (n=6) or in combination with another indication
(n=4). Table 1 summarises the indications and the outcome of PID in Brussels
over a period of 4 years. Couples were prepared for IVF (4 cycles) or for IVF
with ICSI (57 cycles) according to standard protocols (Staessen et al. 1993,
Van Steirteghem et al. 1993, 1995). A brief history of each couple is given in
the appendix.

Blastomere biopsy

Embryos were biopsied in the morning of day 3 after insemination or

microinjection. From the 7-cell stage on, two blastomeres per embryo were
removed, while from 4-cell to 6-cell embryos only one blastomere was taken.
A micropipette was used with an inner diameter of 40 to 45 pm while the
embryos were immobilised by means of a holding pipette. These biopsies were
performed in HEPES-buffered Earle’s medium. First, a hole was made in the
zona pellucida. This was done by blowing a stream of Acidic Tyrode’s
solution until the zona pellucida ruptured. Two different procedures to obtain
blastomeres have been used. In the first cycles, the hole in the zona was turned
to the 12 o’clock position. One or 2 blastomeres were pushed through the hole
by pushing with a bevelled pipette with an inner diameter of 40 pm. Later, a
blunt pipette with an inner diameter of 40 pm to 45 pm and a smoothened
opening was passed through the hole. The hole was placed at the equatorial
plane of a blastomere containing a nucleus before aspiration (Ao et al. 1996).

Diagnosis by the PCR method

Under continuous microscopical supervision, blastomeres were washed three

times in Ca2+ and Mg2+ free M2 medium and placed in a 0.5 or 0.2 ml PCR
tube. Lysis conditions and reaction conditions were worked out to detect the
concerned mutations or DNA sequences in the most efficient and accurate way
at the single-cell level. The resulting DNA fragments were further analysed on
a polyacrylamide or Metaphor® agarose gel (Liu et al. 1994a, 1994b, 1995,
Sermon et al. 1997).

5
Diagnosis by the FISH method

The individual blastomeres were first rinsed in medium, then transferred to a

1-2 pi droplet o f 0.01N HC1/0.1 % Tween 20 solution on a slide and the FISH
procedure as described by Coonen et al. (1994) was used. Double target FISH
was performed using directly labelled DNA-probes specific for chromosomes
X and Y. The X (Vysis, ?-satellite DNA probe, Spectrum Green) and Y (Vysis,
satellite III DNA probe, Spectrum Orange) were used for gender
determination. To counterstain the nuclei 4’6-diamidino-2-phenyindole
(DAPI) was used. The nuclei were then examined using a Zeiss Axioskop
fluorescence microscope with the appropriate filter set (filter 10 for fluorescein
isothiocyanate (FITC), filter 02 (DAPI) and Omega filter (FITC/Texas Red)
(West et al. 1989, Griffin et al. 1991, Harper et al.1994). All nuclei were
observed and FISH results (two green spots in case of the presence of female
blastomere or one green and one orange spot in case of a male blastomere)
were interpreted by two independent observers.

Embryo transfer, cryopreservation and follow-up

Whenever possible up to 3 unaffected embryos of grade A (no anucleated

fragments), B (1 to 20% anucleated fragments) or C (21-50 % anucleated
fragments), were transferred per cycle as indicated (female age, rank of trial,
embryo quality). Spare unaffected embryos were cryopreserved and transferred
in a subsequent cycle. The luteal phase was supplemented by micronised
progresterone (600 mg daily) administered intravaginally or HCG (5000 units
5 days after ovum pick-up) administrated intramuscularly and serum HCG was
determined from day 10 onwards. Where possible, a close pregnancy follow­
up was organised, including regular ultrasound examinations, chorionic villus
sampling or amniocentesis to confirm the result of the PID, registration o f the
pregnancy outcome and clinical evaluation o f the child at birth and thereafter
(Wisanto et al.1996, Bonduelle et al.1996). If no pregnancy ensued follow-up
information on the couples was collected.

Results

So far 61 cycles for preimplantation diagnosis have been performed for 29

patients as reported in table 1. O f these cycles, 36 were performed during the
last year. The mean age of the women at their first attempt was 30.4 years,
with a range between 24 and 37. The number of attempts per couple ranged
from 1 to 5 with an average of 2.1 cycles per couple (see appendix).

6
 The number of cumulus-oocyte complexes recovered per cycle was between
2 and 43, providing a mean of 13.2 (805/61). Fertilisation, i.e., the presence of
two pronuclei (2PN) was observed in 456 oocytes which corresponds to a
mean of 7.5 per cycle. In 4 cycles there was no further development of the
fertilised oocytes and therefore no further analysis. In 333 cleavage-stage
embryos between the 4- and the 10-cell stage a biopsy was performed. The
mean number of biopsied embryos per cycle was 5.8 (333/57). In 43 (12.9%)
of the 333 embryos no diagnosis was possible because of no amplification,
inconsistent results or contamination.
One hundred and twenty-nine unaffected embryos, a mean of 2.3 per cycle
were available for transfer; except for 1 embryo of grade A, they were all grade
B or C. In 12 cycles no embryos could be transferred; in 4 of these because no
embryos developed and in the remaining 8 because no unaffected embryos
were available. In 16 cycles only 1 embryo was transferred. Unaffected
embryos were cryopreserved in 5 cycles and most of these were transferred in
three additional cycles but without success.
So far 10 pregnancies have ensued from fresh transfers. One miscarriage has
occurred, 4 singleton pregnancies are ongoing and 6 children have been bom
from the remaining 5 pregnancies. The children are between 3 months and
more then 2 years of age. One of them is a boy, the others are girls.

Discussion

The success rate in terms of pregnancies is 10 out of 61 cycles or 16 %. Per

transfer the pregnancy rate is 10/48 (21 %) and per couple it is 10/29 (34 %).
Numbers are too small to calculate the take-home baby rate but if we subtract
the one miscarriage and consider the ongoing pregnancies (n=4) plus the
deliveries (n=5), the take-home baby rates are 15% per cycle, 19% per transfer
and 31 % per couple. In our regular IVF or IVF/ICSI cycles the pregnancy rate
per cycle is currently around 30 % and the take-home baby rate per cycle well
over 20 %, and in the world figures for PID the pregnancy rate per cycle was
25 % and per transfer 29 % (Harper, 1996). The lower success rate in this
small series cannot be explained by the age of our patients which is quite
similar. One of the reasons for a lower pregnancy rate is most probably the
higher number of cycles in which none or only 1 unaffected embryo of grade B
or C were available for transfer. From our availabe data we therefore decided
that cycles for PID with less then 9 cumulus-oocyte-complexes should be
cancelled. Another reason for a lower success rate may be a “subfertility” of
the myotonic-dystrophy patients due to their disease, since 25 out of the 61
cycles were performed in 11 couples at risk for this disease (Sermon et al.
1997).

7
 None of the 4 couples at risk of CF has become pregnant so far. Two of these
had a subfertility problem as well as the genetic risk but, nevertheless one of
them has since had two spontaneous pregnancies followed by the birth of non-
affected children after CVS. Prior to the pregnancies the couple was intending
to have another PID cycle. One couple had 4 cycles so far without success.
After CF had been diagnosed in 1 of their 2 children 8 years ago, they waited
for the development of PID so as to be able to have at least 1 other healthy
child, especially since the wife could not cope with the idea of prenatal
diagnosis followed by a possible pregnancy termination. Although this couple
was proven to be fertile, the oocytes and embryos produced during the 4
treatment cycles were always low in number and of extremely poor quality.
Pregnancies have ensued in 2 of the 4 couples at risk for CF because the
wives of CBAVD-men were carriers. In 1 case the pregnancy occurred during
the first treatment cycle after replacement of 3 embryos and a healthy boy now
over 2 years of age was bom (Liu et al. 1994a). Subsequent cycles were
unsuccessful.
For the second couple, 5 cycles were needed to obtain a singleton pregnancy
after transfer of 3 embryos.
In 6 out of 10 patients at risk for an X-linked disease, pregnancies have
ensued. The mean age of these patients was 28 years. Four of the 10 patients
were at risk of DMD. Two of them now have girls. In the first case the
pregnancy occurred during the second cycle and the diagnosis was based on a
PCR assay detecting the presence or absence of a dystrophin gene deletion.
Two embryos were transferred (Liu et al.1995). The girl is now over 2 years of
age and healthy. In the second case a triplet pregnancy occurred during the
second cycle after transfer of 3 embryos. The triplet was one singleton and one
twin (monochorionic, biammniotic) one of which was shown to be an
acardiacus between 13 and 14 weeks of pregnancy. Five weeks later selective
reduction of the malformed twin was performed extramuros and another 4
weeks later, the children were prematurely bom at almost 25 weeks of
pregnancy. The morphologically normal twin weighed 450 g and subsequently
died. The singleton baby girl weighed 850g and is doing well according to the
information we obtained so far. In 2 patients at risk for hemophilia A, 1
healthy girl was bom after transfer of 2 embryos and 2 healthy twin girls have
recently been bom after the replacement of 3 embryos in the second patient
respectively. Of the 3 patients at risk of non-fragile-X-linked mental
retardation, 1 patient became pregnant after replacement of 3 embryos in the
first cycle but a miscarriage occurred. Finally, 1 patient at risk of retinitis
pigmentosa is currently pregnant after 1 treatment cycle with transfer of 2
embryos.

8
 Two out of 11 couples at risk of myotonic dystrophy are currently pregnant
with singletons, both after a 3rd cycle in which respectively 2 and 4 embryos
were transferred (Sermon et al. 1997).
The mean age of all the preceding pregnant women was 29.8 years (range 24-
37); the mean number of embryos transferred per cycle was 2.5 (range 2 to 3
except in one case where 4 were transferred).
In our population of 29 couples who had requested PID, the indications,
apart from the genetic risk, were infertility in the 4 cases with CBAVD,
subfertility in 11 cases (most of which belong to the myotonic dystrophy
group), a previous history of affected pregnancies which had to be terminated
in 8 cases and moral problems with termination of pregnancy in 6 cases. The
high pregnancy rate of 60% in the group of patients at risk of sex-linked
diseases might be explained by the lack of subfertility problems (only 1 out of
10) and the younger mean age (28 years) of these patients. The one miscarriage
occurred in the subfertile couple with a previous history of G4P1A3.
Only the first 4 cycles in couples without CBAVD involved classical IVF.
Since then IVF with ICSI has been used for insemination. The aim was to
reduce the risk for contamination in PCR reactions from residual sperm-DNA.
We still consider this to be the insemination method of choice in PCR- based
PID. In FISH-based PID for couples with no known subfertility or infertility,
conventional IVF is probably equally valid as an option.
Before starting the treatment, PID patients were asked to agree to a prenatal
diagnosis through CVS or amniocentesis to confirm the result of the PID
should they become pregnant, since at least in PCR-based assays misdiagnoses
have been reported (Harper 1996) and since diagnostic errors may occur as a
result of contamination or allele-specific drop-out during the PCR reaction. O f
the 10 pregnancies, 1 miscarried before prenatal diagnosis. Two patients
pregnant after a FISH-based sex-determination and one patient pregnant after
CF-diagnosis declined to have prenatal diagnosis. In 6 cases (1 CVS and 5
amniocenteses) the PID was confirmed or refined (CF carrier boy, non-carrier
DMD girl).
The age of the 6 children bom so far ranges from 3 months to over 2 years of
age. Four of these are girls because female embryos were selected for transfer
as a result of a risk of a sex-linked disease. The fifth girl was bom to a carrier
of Duchenne’s muscular dystrophy but the PCR-based PID indicated affected
boys (absence of fragment) versus unaffected male embryos and non-carrier as
well as carrier female embryos (presence of fragment). This girl and the boy
bom at term in 1994 were morphologically normal (Liu et al. 1994a, 1995). At
birth and at 2 years of age their growth and developmental milestones were
within the normal range. One of the 4 girls bom in 1996 issued from the triplet
pregnancy mentioned earlier; she was bom at 25 weeks o f pregnancy and
weighed 850 g. At 4 months of age the girl weighed 3.2 kg and measured 49

9
cm. According to the parents, who plan to visit us, she was doing fine. The
premature birth was probably the result of the selective reduction performed on
1 of the malformed twins at 18 to 19 weeks of pregnancy. The other twin
weighed only 450 g at birth and did not survive. The cause of the acardiacus
malformation is most probably linked to the twinning process and not to the
biopsy procedure. The 3 other girls bom in 1996 are doing well according to
information obtained from the parents and their physicians. One girl was bom
at 36 weeks of pregnancy and had a birthweight of 2.4 kg, a length of 47 cm
and a head circumference of 32 cm. She is now about 1 year old. The other
twin girls were bom at 35 weeks of gestation and weighed 2.6 and 2.1 kg
respectively; they are now 3 months old. So far, the number of children bom is
too small to draw any firm conclusions concerning possible problems with
morphology, growth or development. As in regular IVF and ICSI, multiple
pregnancies should be avoided where possible so as to reduce the risk of
complications (Bonduelle et al. 1996, Wisanto et al. 1996, Simpson and
Liebaers 1996).
Our PID programme is now well structured and based on a close
collaboration between the Centre for Medical Genetics and the Centre for
Reproductive Medicine. Before starting, patients are counselled extensively by
specialised physicians in both Centres. A nurse-coordinator schedules the
cycles and informs the team members who will be involved and especially the
laboratories dealing with cycle monitoring, IVF and ICSI, embryo biopsy and
FISH or PCR analysis. Patients are asked to come to the clinic for pick-up and
on day 3 post-insemination for a possible transfer. The outcome of the embryo
diagnosis is discussed with the couple at the clinic. In any case a follow-up
visit is scheduled with the geneticist as well as with the fertility specialist so as
either to organise a pregnancy follow-up with prenatal diagnosis, ultrasound
and finally a baby follow-up or to plan a subsequent cycle. Organising the
follow-up of patients from abroad is more complex and the data obtained are
less complete.
Possible reasons for the slow development of PID in our centres and
elsewhere are probably linked to its experimental character and to the
complexity of the procedure at the clinical as well as at the laboratory level.
Moreover the take-home baby-rate is low as a result of this complexity and the
cost is rather high. Finally, the availability of the procedure in general and of
specific procedures for specific diseases is still limited. Nevertheless, the
procedure does not appear to be too stressful for many of the patients, since
several of them have had repeated PID (see appendix). Further development in
diagnostic procedures as well as the evaluation of patient’s experience are
therefore to be expected. Moreover continuous data collection at the national
and international levels will be of great value to correctly appreciate the value

10
of this new procedure (ESHRE Special Interest Group on Reproduction and
Genetics, International Working Group on PID).

11
Table 1. PID in Brussels between February 1993 and February 1997 for monogenic diseases

Disease Couples PID indication Cycles Transfers Pregnancies Miscarriages Ongoing Births Children
pregnancies
Cf. 4 Subfertility 2 9 7
Historyc 1
' '

TOPf 1
CFa / 4 Infertile male 4 12 8 2 1 1 1
-
CBAVDb needing MESAg

MDC 11 Subfertility 8 25 22 2 2

Historyc 1
‘ '

TOPf 6
X-linkedd 10 Subfertility 1 15 11 6 1 1 4 5

Historye 6
TOPf 3
29 61 48 10 1 4 5 6

aCystic Fibrosis; bCongenital bilateral absence o f vas deferens; cmyotonic dystrophy; dX-linked diseases such as Duchenne’s muscular
dystrophy, Hemophilia A, X-linked mental retardation and retinitis pigmentosa; cprevious history o f prenatal diagnosis followed by
termination o f pregnancy; fmoral, emotional or religious objection to termination o f pregnancy (TOP); gmicrosurgical epidydymal sperm
aspiration.
Addendum

Since the publication of the above article, in total 170 PID cycles have been
performed for 84 couples. Twenty-nine pregnancies were established. Five of
these were multiple pregnancies.
One pregnancy was terminated because of a misdiagnosis detected at
prenatal diagnosis. Seventeen healthy children were bom, one acardiacs-twin
died. Twelve pregnancies are ongoing.
Genetic indications for preimplantationdiagnosis were for monogenic
conditions: several X-linked disorders such as Duchennes Muscular
Dystrophy, hemophilia A, Wiskott-Aldrich disease, adrenoleucodystrophy,
Charcot Marie Tooth disease, mental retardation, retinitis pigmentosa.
PID was also performed for autosomal recessive and dominant diseases such
as myotonic dystrophy, cystic fibrosis with our without (CBAVD), Marians
disease, Charcot Marie Tooth disease, p-thalassemia, 21-P-hydroxylase
deficiency, osteogenesis imperfecta and sickle cell anemia.
For chromosomal aberrations, PID has been performed for the velo-cardio-
facial syndrome due to a 22q deletion, for a translocation (11 ;22), for a Yq
deletion as well as for Klinefelter patients producing a few spermatozoa in
their testes.
The demand for PID has increased over the years. New diagnostic tests are
being developed and more centers are offering this new procedure. Evaluation
of patients experience with this new procedure is necessary and ongoing.

References

Ao, A., Ray, P., Harper, J., Lesko, J. et al. (1996), ‘Clinical experience with
preimplantation genetic diagnosis o f cystic fibrosis (AF508)’, Pren. Diagn.
16,137-142.
Bonduelle, M., Wilikens, A., Buysse, A. et al. (1996) ‘Prospective follow-up
study of 877 children bom after intracytoplasmic sperm injection (ICSI)
with ejaculated, epididymal and testicular spermatozoa and after
replacement of cryopreserved embryos after ICSI’, Hum. Reprod., 11(4),
131-159.
Coonen, E., Dumoulin, J.M.C., Ramaekers, F.C.S. et al. (1994), ‘Optimal
preparation of preimplantation embryos interphases nuclei for analysis by
fluorescences in situ hybridisation’, Hum. Reprod., 9, 533-537.
Griffin, D.K., Handyside, A.H., Penketh, R.J.A. et al. (1991), ‘Fluorescent in-
situ hybridization to interphase nuclei of human preimplantation embryos
with X and Y chromosome specific probes’, Hum. Reprod., 6 (1), 101-105.

13
Handyside, A.H., Kontiogianni, E.H., Hardy, K. et al. (1990), ‘Pregnancies
from biopsied human preimplantation embryos sexed by Y specific DNA
amplification’, Nature, 344,768-770.
Hardy, K., Martin, K.L., Leese, H. et al. (1990), ‘Human preimplantation
development in vitro is not adversely affected by biopsy at the 8-cell stage’,
Hum. Reprod, 5, 708-714.
Harper, J.C. (1996) ‘Preimplantation diagnosis of inherited disease by embryo
biopsy: an update of the world figures’, J. Assist. Reprod. Genet., 13(2), 90-
95.
Harper, J.C., Coonen, E., Ramaekers, F.C.S. et al. (1994), ‘Identification of the
sex of human preimplantation embryos is two hours using an improved
spreading method and fluorescence in situ hybridisation (FISH) using
directly labelled probes’, Hum. Reprod., 4,721-724.
Lissens, W., Sermon, K., Staessen, C. et al. (1996), ‘Review: Preimplantation
diagnosis of inherited disease’, J. Inker. Metab. Dis., 19,709-723.
Liu, J., Lissens, W., Devroey, P. et al. (1994), ‘Amplification of X- and Y-
chromosome specific regions from single human blastomeres by polymerase
chain reaction for sexing of preimplantation embryos’, Hum. Reprod., 9,
716-720.
Liu, J., Lissens, W., Silber, S.J. et al. (1994), ‘Birth after preimplantation
diagnosis of the cystic fibrosis AF508 mutation by polymerase chain
reaction in human embryos resulting from intracytoplasmic sperm injection
with epididymal sperm’, JAMA, 272,1858-1860.
Liu, J., Lissens, W., Van Broeckhoven, C. et al. (1995), ‘Normal pregnancy
after preimplantation DNA diagnosis of a dystrophin gene deletion’, Prenat.
Diagn., 15,351-358.
Sermon, K., Lissens, W., Joris, H. et al (1997,) ‘Clinical application of
preimplantation diagnosis for Myotonic Dystrophy’, Prenat. Diagn., in
press.
Silber, S.J., Nagy, Z.P., Liu, J. et al (1994), ‘Conventional in vitro fertilization
versus intracytoplasmic sperm injection for patients requiring microsurgical
sperm aspiration’, Hum Reprod ,9,1705-1709.
Simpson, J.L., Liebaers, I. (1996), ‘Assessing congenital anomalies after
preimplantation genetic diagnosis’, J. Assist. Reprod. Genet., 13(2), 170-
176.
Staessen C., Coonen E., Van Assche, E. et al. (1996), ‘Preimplantation
diagnosis for X and Y normality in embryos from three Klinefelter patients’,
Hum. Reprod., 11, 1650-1653.
Staessen, C., Janssenswillen, C., Van den Abbeel, E. et al. (1993), ‘Evidence
of triplet pregnancies by selective transfer of two quality embryos’, Hum.
Reprod., 8,1650-1653.

14
Toumaye, H., Staessen, C., Liebaers, I. et al. (1996), ‘Testicular sperm
recovery in nine 47, XXY Klinefelter patients’, Hum. Reprod., 11, 1644-
1649.
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al (1993), ‘High fertilization and
implantation rates after intracytoplasmic sperm injection’, Hum. Reprod., 8,
1061-1066.
Van Steirteghem, A.C., Joris, H., Liu, J. et al. (1995), ‘Protocol for
intracytoplasmic sperm injection’, Hum. Reprod., Update 1, n° 3, CD-ROM.
Verlinsky, Y., Cieslack, J., Ivakhnenko, V. et al. (1996), ‘Birth of healthy
children after preimplantation diagnosis of common aneuploidies by polar
body fluorescent in situ hybridization’, Fertil. Steril., 66, 126-129.
West, J.D., Gosden, C.M., Gosden, J.R. et al. (1989), ‘Sexing the human fetus
and identification of polyploid nuclei by DNA-DNA in situ hybridisation in
interphase nuclei’, M ol Reprod. Develop. 1,129-137.
Wisanto, A., Bonduelle, M., Camus, M. et al. (1996), ‘Obstetric outcome of
904 pregnancies after intracytoplasmic sperm injection’, Hum. Reprod.,
11(4), 121-131.

Acknowledgements

Research funds of the university and the F.W.O.-Vlaanderen have made the
development of these new procedures possible. Besides the authors and all the
other members of the Centres for Medical Genetics and Reproductive
Medicine were helpful in taking care of the patients and their embryos. Special
acknowledgements go to F. Winter for correcting the grammar and style and J.
Heulaerts for typing the manuscript.

15
2 The various
micromanipulative
procedures: State o f the
art, chances, and risks
Dieter Meschede and Jürgen Horst

Micromanipulation has added a new dimension to reproductive medicine. It

entails the use of microtools that allow for a precise handling and manipulation
of single cells or their subcompartments such as the cytoplasm or nucleus.
With various types of micropipettes germ cells or parts of them as well as early
embryos can be individually selected, held, drilled, cut, injected, or biopsied.
The possible applications of this technology in research, diagnosis and therapy
are manifold. Cloning of humans, considered to be on the horizon, would also
have to rely on these micromanipulative technologies. But even some currently
available techniques such as oocyte cytoplasm donation make conventional in
vitro fertilisation (IVF) look like an old-fashioned and almost natural way of
inducing pregnancies.
In quantitative terms, intracytoplasmic sperm injection (ICSI) is by far the
most important procedure involving micromanipulation (Felberbaum and
Dahnke 1997). Its main application is severe male factor infertility, an entity
that in the pre-ICSI era had a dismal prognosis for a successful treatment
outcome. Centers with ample experience now report clinical pregnancy rates
exceeding 30 % per treatment cycle. The fact that this exceeds the natural
conception rate in fertile couples illustrates what dramatic progress ICSI
represents.
ICSI is supplemented by new techniques for surgical sperm retrieval in
azoospermic or severely oligozoospermic men. Patients with obstructions of
the seminal ducts may benefit from MESA (microsurgical epididymal sperm
aspiration), individuals with non-obstructive azoospermia from TESE

17
 (testicular sperm extraction) (Devroey et al. 1994). Both these procedures yield
physiologically immature and poorly motile germ cells so that combining them
with an ICSI procedure is almost mandatory in order to have a reasonable
chance for attaining a pregnancy. Similarly, ICSI facilitates the use of
cryopreserved sperm from men with malignant disorders who as a result of
radio- or chemotherapy lost their ability to sire children. There is a general
trend to go back to increasingly immature developmental germ cell stages for
ICSI - in humans normal pregnancies have been induced with spermatids
(Fishel et al. 1995), in mice even with secondary spermatocytes which have
not undergone the second meiotic division (Kimura and Yanagimachi 1995).
Freezing female gametes or ovarian biopsies has proven difficult, but with
technical improvements and the support of micromanipulation such
cryopreserved samples may soon become clinically useful. To have a ‘cryo-
reserve’ of eggs or ovarian tissue may in the future enable women to delay
childbearing into the postmenopausal age, or have children after fertility-
ablating cancer treatment. Cytoplasm donation may be another option for
‘reproductively old’ women to enhance their fertility potential. Their germ
cells can be freshened up with oocyte cytoplasm from a younger donor. This
procedure has already resulted in the birth of healthy children (Cohen et al.
1997), but its practical importance remains to be established. The technique
does not have any immediate genetic implicatons apart from the remote
possibility that mitochondrial disorders could be transmitted. In contrast, ovum
nuclear transfer would entail the exchange of the recipient's genome for a
donor genome. Possible reasons for the use of this micromanipulative
procedure could be advanced reproductive age or an inheritable disorder of the
recipient. In genetic terms, ovum nuclear transfer does not differ from the
donation of whole oocytes - any offspring resulting from this procedure would
not be a genetic child of the recipient. Whether there is any sound clinical
rationale for ovum nuclear transfer in humans is unclear.
Finally, cloning humans would entail the use of micromanipulative
technology. Cloning is defined as the intentional creation of genetically
identical individuals. In that regard, embryo splitting, a procedure employed in
veterinary medicine, would qualify as cloning. In less dramatic terms such a
procedure could be designated as the artifical induction of a monozygotic twin
(or higher multiple) pregnancy. In contrast, cloning in the narrower sense
means the creation of a genetically identical copy of an adult individual. As
demonstrated in sheep and cattle, mammals can be cloned by inserting a
somatic cell nucleus into an enucleated germ cell (Nash 1997). Genetically,
cloning means to circumvent the recombination of genes that occurs in
meiosis. In natural reproduction this process guarantees that children inherit a
non-predictable random sample of their parents' genetic repertoires. The

18
meiotic jumbling of genes is one safeguard for genetic diversity and a driving
force of evolution.
Preimplantation diagnosis (PIO) can also be counted among the
micromanipulative procedures as it entails the sampling of one or a few cells
from an early embryonic developmental stage (Lissens and Sermon 1997).
With this cornucopia of micromanipulative procedures already in clinical use
or on the horizon, what risks are to be considered? Direct side effects of the
hormonal or surgical pretreatment that is required prior to the use of many
micromanipulative techniques cannot be discussed here. Concerning the
outcome of pregnancies conceived through ICSI, MESA, TESE, injection of
spermatids or the use of cryopreserved ova data are still far from sufficient to
come to definite conclusions (Bonduelle et al. 1996, Kurinczuk and Bauer
1997)./ Until now, no convincing evidence has been brought forward that any
of these procedures implies health risks for the offspring significantly higher
than in natural pregnancies. However, this debate is still ongoing, and patients
opting for these treatments need to be fully informed about the incomplete
knowledgebase on malformation rates, long-term psychosocial and mental
development, and fertility of children conceived with the support of
micromanipulative procedures.
Late or even postmenopausal childbearing carries increased obstetric and
medical risks. Moreover, having old or very old parents may result in untoward
psychosocial and developmental effects on children and adolescents.
Obviously, the risk of early loss of one or both parents is increased in such
families.
Micromanipulative procedures pave the way for and are one technical
cornerstone of PIO. So far, it appears unlikely that PID will ever be employed
on as broad a basis as conventional prenatal diagnosis. As PID is only
applicable in IVF pregnancies,·this should preclude its widespread use. It has
to be conceded, however, that with improved technology it will become
increasingly tempting to subject all IVF or ICSI pregnancies to a genetic
'checkup' through PIO. The strongest driving force in that direction is the
claim that excluding chromosomal aneuploidies in the in vitro stage enhances
the success rate of ICSI (Verlinsky and Kuliev 1996). Behind these more
technical considerations looms the basic question of whether it is desirable to
diagnose or exclude as many genetic 'flaws' as possible by preimplantation or
conventional prenatal testing. The opposing views on this topic are well
known, incompatible as ever, and will not be further commented on here.
Since the recent announcements indicating that cloning of mammals is
technically feasible, cloning applied to the human species has become the
ultimate horror scenario for some, for others the promise of new possibilites
which were previously undreamed of. Consensus against cloning humans is
still strong, but the flawless frontline already seems to be giving way. It is

19
beyond the scope of this paper to elaborate on the ethical issues arising from
endeavors to clone human beings. It may suffice to remark that we do not
currently have the slightest idea what medical risks such a procedure would
imply for the cloned offspring. The very feasibility of creating a viable
mammal from the nucleus of a once fully differentiated somatic cell breaks a
basic dogma of developmental biology. It may be envisioned that such clones
could suffer prematurely from the ravages of old age, or have an increased rate
of cancer, infertility, or other maladies. On the population level, cloning
performed on a large scale would reduce genetic diversity by precluding the
recombination of genes that under conditions of natural reproduction takes
place in every new generation. To what degree this would imperil the long­
term genetic health of humankind is a currently unanswerable question.

Table 1. Micromanipulative assisted reproduction: summary of major

chances and risks.

chances__________ risks
better treatment of infertility • treatment risks incompletely
understood
parenthood for cancer patients
facilitation of delayed • facilitation of delayed
childbearing childbearing
postmenopausal parenting • postmenopausal parenting
starting point for PID • starting point for PID
better understanding of gamete • usage of human embryos for
and embryo biology research
more reproductive autonomy • new legal dilemmas;
assault on human dignity
avoidance of genetic disease in • selective embryo transfer;
offspring affront to handicapped
individuals;
loss of genetic diversity
avenue to germ line therapy • avenue to germ line therapy
• avenue to genetic enhancement
• avenue to genetic engineering at
the population level

20
References

Bonduelle, M., Wilikens, A., Buysse, A., Van Assche, E., Wisanto, A.,
Devroey, P., van Steirteghem, A. and Liebaers, I. (1996), ‘Prospective
Follow-up Study of 877 Children Bom After Intracytoplasmic Sperm
Injection (ICSI), with Ejaculated, Epididymal and Testicular Spermatozoa
and After Replacement of Cryopreserved Embryos Obtained After ICST,
Human Reproduction, Vol. 11, Suppl. 4, pp. 131-55.
Cohen, J., Scott, R., Schimmel, T., Levron, J. and Willadsen, S. (1997), ‘Birth
of Infant After Transfer of Anucleate Donor Oocyte Cytoplasm Into
Recipient Eggs’, Lancet, Vol. 350, pp. 186-7.
Devroey, P., Liu, J., Nagy, Z., Tounaye, H., Silber, S.J. and van Steirteghem,
A.C. (1994), ‘Normal Fertilization of Human Oocytes After Testicular
Sperm Extraction and Intracytoplasmic Sperm Injection’, Fertility and
Sterility, Vol. 62, pp. 639-41.
Felberbaum, R. and Dahnke, W. (1997), ‘DIR - Deutsches IVF-Register.
Ergebnisse der Datenerhebung fur das Jahr 1996’, Fertilität, Vol. 13, pp. 99-
112.
Fishel, S., Green, S., Bishop, M., Thornton, S., Hunter, A., Fleming, S. and Al-
Hassan, S. (1995), ‘Pregnancy After Intracytoplasmic Injection of
Spermatid’, Lancet, Vol. 345, pp. 1641-2.
Kimura, Y. and Yanagimachi, R. (1995), ‘Development of Normal Mice From
Oocytes Injected With Secondary Spermatocyte Nuclei’, Biology o f
Reproduction, Vol. 53, pp. 855-62.
Kurinczuk, J J . and Bower, C. (1997), ‘Birth Defects in Infants Conceived by
Intracytoplasmic Sperm Injection: An Alternative Interpretation’, British
Medical Journal, Vol. 315, pp. 1260-6.
Lissens, W. and Sermon, K. (1997), ‘Preimplantation Genetic Diagnosis:
Current Status and New Developments’, Human Reproduction, Vol. 12, pp.
1756-61.
Nash, J.M. (1997), ‘The Age of Cloning’, Time, March 10, 1997, pp. 46-9.
Verlinsky, Y. and Kuliev, A. (1996), ‘Preimplantation Diagnosis of Common
Aneuploidies in Infertile Couples of Advanced Maternal Age’, Human
Reproduction, Vol. 11, pp. 2076-7.

21
3 The relation between ICSI
and genetic diagnosis from
an ethical point o f view
Barbara Maier

Introduction

The development of ICSI (Intracytoplasmic sperm injection) has

revolutionised the treatment of male infertility by bypassing natural barriers to
fertilisation. ICSI and particularly the two sperm retrieval techniques of
microsurgical epididymal sperm aspiration (MESA) and testicular sperm
extraction (TESE) may cause the transmission of this or other genetic
disorders to the next generation. Serious genetic diseases like cystic fibrosis
and the heritability of male infertility have become important issues for
discussion concerning some subpopulations of ICSI-candidates.
While the mechanical piercing of the oocyte which was initially considered
to damage its contents or ultrastructure, does not result in any significant
increase in birth defects in ICSI offspring, pregnancies may be obtained with
spermatozoa that carry genetic defects (Wisanto et al. 1995, Bonduelle et al.
1996). Thanks to ICSI in the past completely infertile men are able to father
their own children. The subsidiary interventions of donor insemination or
donor IVF with their special psychosocial implications have been ruled out in
a lot of cases by the ICSI method.
But the introduction of ICSI has raised new concerns both for clinicians
dealing with male infertility and for ICSI candidates themselves, the couples
concerned and their future children. Before the introduction of ICSI, the major
question to be asked was whether a man was able to reproduce. Now the

23
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 DREAM.

I dreamt that, standing on a height,

I wished to plunge me in the sea,
When, lo! a spirit of peace and light,
This wondrous song sang unto me:
‘Await the spring! I’ll soon be here;’
I’ll say, ‘Again let manhood rise!’
The mist from clouded brows I’ll clear,
And dreary dreams from heavy eyes.
Back to your Muse her voice I’ll give,
And once again you’ll find the days
All blessed—as you bind the sheaf—
Reaping your unmown upland ways.
 A SICK MAN’S JEALOUSY.

A heavy cross, the lot Fate laid upon her—

“Suffer! be silent! weep not! feign the smile!”
And he, to whom her love, her youth, her will,
Her all, she’d given, her torturer proved the while.

For years no greeting with a friend knew she;

Subdued, in sadness, and in trembling fear,
Bitter, unreasoning, sarcastic jeers,
Without a murmur, ’twas her lot to hear.

“Hush! tell me not you’ve lost your youth for me—

That you’re distracted by my jealousy;
Nay, tell me not! My grave is close at hand,
While you are fresher than spring’s blossoms be.

“That day, the day when you at first loved me,

And heard from me, ‘I love,’ in whispered breath,
Curse not that day! The grave is near for me!
I will right all, redeem all, by my death.

“Cease! Tell me not the days for you are sad;

This invalid a jailor cease to name.
For me remains the cold gloom of the grave;
For thee the embraces of another flame.

“Full well I know thou dost another love.

To spare, to wait, this seemed a tedious plan.
Oh, wait awhile! my grave is very near!
Let Fate end that which Fate in me began!”
Such cruel, torturing, insulting words—
Lovely, yet pale as chiselled marble—she
In silence heard, and only wrung her hands.
What could she answer to such jealousy?
 THE LANDLORD OF OLD TIMES.
(Loquitur.)

Before the Emancipation of the Serfs.

To whom I like I mercy show,

And whom I like I kill;
My fist—my only constable,
My only law—my will.
A blow from which the sparkle flits,
A blow that knocks the teeth to bits,
A blow that breaks the jaw!

After the Emancipation of the Serfs.

The mighty chain is snapped in twain,

Is snapped and bounds asunder.
The landlords clutch one broken end;
At t’other peasants blunder.

The fields remain unploughed and bare;

The seed is left unsown;
No trace of order anywhere,
O mother-land, our own!
Not for ourselves thus sorrow we;
We grieve, O native land, for thee!
Oh, true-believing peasantry!
Russia’s your mother small;
The Tsar’s your little father.
And that for you is all!
THE RUSSIAN SOLDIER.

Then up there comes a veteran,

With medals on his breast;
He scarcely lives, but yet he strives
To drink with all the rest.
“A lucky man, am I,” he cries,
And thus to prove the fact he tries.
“In what consists a soldier’s luck?
Pray, listen while I tell.
In twenty fights, or more, I’ve been,
And yet I never fell.
And, what is more, in peaceful times
Full meal I never knew;
Yet, all the same, I have contrived
Not to give Death his due.
Again, for sins both great and small,
Full many a time they’ve me
With sticks unmercifully flogged,
Yet I’m alive, you see!”
FROM MAIKOF.
 A MIDSUMMER NIGHT’S DREAM.

For a long time last night I for sleep vainly yearned.

I arose, my room window wide throwing;
The night with its silence oppressed me, and burned,
O’er me odours intoxicant blowing.

Of a sudden the hedge ’neath my window-sill shook;

My curtain blew back with a shimmer;
And in floated a youth with a beaming look,
Just as if from the moonlight a glimmer.

Gliding up to my couch, came my wonderful guest,

Whispered he, as a smile his lips parted,
“Why from me, with your cheek ’neath the pillow prest,
Like a startled wee fish, have you darted?

“Look up! I’m a god—god of visions and dreams,

Secret friend of the innocent maiden;
And for thee, my own queen, for the first time, I ween,
With a bliss from on high come I laden!”

He spoke—and his hands my face lovingly seek;

From its nook he it tenderly presses;
Then a burning kiss fell on the curve of my cheek,
And his lips sought my lips in caresses.

Neath the breath of his mouth my strength seemed to have flown,

From my breast unclaspt arms I extended,
And there breathed in my ears, “You’re my own! you’re my own!”
Distant notes, with harp’s melody blended!
Swiftly glided the hours; when I opened my eyes,
Rosy dawn through my chamber was streaming;
Alone, locks dishevelled, I trembling arise,
And I know not the drift of my dreaming.
 WHO WAS HE?
A STORY OF PETER THE GREAT.

Upon the mighty Neva’s bank,

Along the winding woodland way,
A Horseman rode, in forest wilds
Of elm, of pine, of mosses grey.

Before him rose a Fisher’s hut;

Beneath a pine, by the blue stream,
An aged, bearded Fisherman
Was mending his boat’s broken beam.

The Horseman said, “Grandsire! Good-day!

God help thee, friend! how liveth thou?
Doth thou catch much? and tell me, pray,
Where doth thou sell thy takings now?”

The old man answered sullenly,

“Are fishes in the river few?
And other market have I none,
Except the town, there, close to you.

“And how am I to fish to-day?

What kind of turmoil’s here, you see!
You fight; and, in the fight, a shell
Has smashed my fishing-boat for me!”

The Horseman bounded from his horse,

Without a word the tools he grasped;
And in a twinkling planked the boat,
The rudder in the stern set fast.
“See, now, old friend, thy boat’s all right!
Out on the water boldly set;
And, in the name of Peter’s luck,
Cast forth into the deep thy net.”

He vanished. Mused the stern old man:

“I wonder who the de’il was he!
In every inch he looked a king,
But plied the hatchet splendidly.”
 THE EASTER KISS.[8]

Soon “the Sun-bright Feast-day” cometh,

I will claim my Easter kiss.
Others, then, will stand around us;
Pray, my Dora, mark you this!

Just as if I never kissed you,

Blushing red before the rest,
You must kiss, with downcast eyelids;
I will kiss, with smile represt.

FOOTNOTES:
[8] It is the custom in Russia for all friends meeting on Easter
morning (known as “Sun-bright Feast-day”) to exchange kisses
three times in the name of the Trinity.
 ON LOMONOSSOEF.[9]

God chose him from his earliest years;

Revealed, ’mid glittering icebergs stood,
In northern light, in gleam of stars,
In roar of wave, in hum of wood,
And bade him leave his Fisher’s net,
And led him forth from town to town,
That “Rus”[10] to him from gloomy cot
To sparkling palace, might be known;
And led him to famed Western climes,
That there his genius might obtain
All knowledge, from the earliest times
Made known to mighty chosen men;
That, from their torch of knowledge, he
Might light his own, and, with right hand
Uplifted high that all might see,
Illume with it his native land.

FOOTNOTES:
[9] Lomonossoef—the first great Russian scholar—was the son
of an Archangel fisherman.
[10] Ancient name of Russia.
 PROPRIETY.

Ferdinand, the King, was courtly!

Pink of nice refinement he;
All the naked plasts of Venus,
Placed he under lock and key.

But the Herculean statues,

Left he in their places bare!
Men he did not mind offending;
Hurt the ladies? He’d not dare!
 THE SINGER.

Beautiful I’m not, I know;

Useless I in fight;
How to men and maids am I,
Such a dear delight?
Songs, like sounds that ’mid strings stray,
Fill this breast of mine,
Smiling round my lips they play,
In my eyes they shine!
 A LITTLE PICTURE.
AFTER THE PROCLAMATION OF THE 19TH FEB., 1861,
FREEING THE SERFS.

See, in peasant’s cottage, flickering

Shines a little fire,
Where, around a little maiden,
Draws a circle nigher.

And from word to word her finger

Slowly pointing leads,
As, with effort, to the peasants
She a paper reads.

Deep in thought, intently listening,

They a silence keep;
Save when some one bids the women
Hush the babes to sleep.

Mothers soothe their crying infants

With the teething toy,
While they, too, to catch the reading
All their ears employ.

Seated in the chimney corner

Now for many years,
With bent head the grandsire gazes,
Though he nothing hears.

Is the maiden clever, that they

Thus to her give heed?
Nay! but simply in that household
She alone could read:
And her lot it was to read out,
To the peasants old,
The glad news of longed-for freedom,
Which the paper told.

The full meaning of the message

Knew not she nor they;
But all, darkly, felt the dawning
Of a better day.

Brothers! see, the day-dawn flushes!

Darkness yields its place,
Sons of yours, ere long, will look on
Daylight face to face.

More and more let darkness lighten!

Day arise in might!
Even now, in vision, see I
Rays of rising light.

They are shining on the forehead,

Gleaming in the look,
Of that thoughtful little maiden
With her little book.

Freedom, Brothers! This is only

First step on the way
To the kingdom, where, in knowledge,
Shines eternal day!
 THE ALPINE GLACIER.

Dank the darkness on the cliff-side;

Faintly outlined from below,
In their modest maiden gladness,
Glaciers in the dawn’s blush glow.

What new life upon me blowing,

Breathes from yonder snowy height,
From that depth of limpid turquoise
Flashing in the morning light?

There, I know, dread Terror dwelleth,

Track of man there is not there;
Yet my heart in answer swelleth
To the challenge, “Come thou here!”
 THE MOTHER.

Little sufferer—all on fire!

All’s to him so trying!
On my shoulder lean thy head,
On my bosom lying!
I will walk about with thee,
Sleep, my own sweet dearie.
Shall I tell a little tale?
“Once there lived a fairy”—
No? Thee likes not silly tales?
P’r’aps a song will take thee!
“Pine-wood rustling dark and dank,
Big fox, wee fox, wakes he.
In the dark pine-wood will I——”
Is my own pet sleeping?
“Gather blackberries for thee
Brimful baskets heaping.
In the dark pine-wood will I——”
Hush! he fast is sleeping.
Open wide his feverish lips,
Like a wee bird, keeping.

“In the dark pine-wood will I,”

Walks the mother, singing—
Till the long dark night declines,
Back the day-dawn bringing.
Singing—while her weary arms
With dull pain are tingling—
Walks the mother; with her sighs
Frequent tears are mingling;
And scarce stirs the restless child,
Tossing in its fever,
Ere again that song resounds,
Soft and low as ever.
With thy scythe depart, O Death,
Spare the tender blossom!
Fierce the fight ere she will yield
Baby from her bosom.
With her whole soul will she shield,
E’en though sore affrighted,
That mysterious flame of life
Which from her was lighted,
For scarce rose that little flame,
Ere to her revealed was
What of love,—of wondrous power,—
In her breast concealed was.
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