Novel Approaches in Addressing Ovarian Insufficiency in 2019: Are We There Yet?

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Review

Cell Transplantation
Volume 29: 1–21
Novel Approaches in Addressing Ovarian ª The Author(s) 2020
Article reuse guidelines:
sagepub.com/journals-permissions
Insufficiency in 2019: Are We There Yet? DOI: 10.1177/0963689720926154
journals.sagepub.com/home/cll

Konstantinos Sfakianoudis1, Anna Rapani2,3,


Sokratis Grigoriadis2,3, Dimitra Retsina2,4, Evangelos Maziotis2,3,
Petroula Tsioulou2,3, Polina Giannelou1,2, Konstantinos Pantos1,
Michael Koutsilieris2, Nikolaos Vlahos3,
George Mastorakos4, and Mara Simopoulou2,3

Abstract
Ovarian insufficiency is described as a multifaceted issue typically encountered in the field of assisted reproduction. The three
main identified diagnoses of ovarian insufficiency include premature ovarian failure (POF), poor ovarian response (POR), and
advanced maternal age (AMA). Patient heterogeneity in the era of individualized medicine drives research forward leading to
the emergence of novel approaches. This plethora of innovative treatments in the service of adequately managing ovarian
insufficiency is called to undertake the challenge of addressing infertile patients exploring their reproductive options. This
review provides an all-inclusive presentation and critical analysis on novel treatments that have not achieved routine clinical
practice status yet, but have recently emerged as promising. In light of the lack of randomized controlled trials conveying safety
and efficiency, clinicians are left puzzled in addressing the “how” and “for whom” these approaches may be beneficial. From
ovarian injection employing platelet-rich plasma (PRP) or stem cells to artificial gametes and ovaries, ovarian transplantation,
and mitochondrial replacement therapy, this descriptive review provides insight toward assisting the practitioner in decision
making regarding these cutting-edge treatments. Biological mechanisms, invasiveness levels, efficiency, as well as possible
complications, the current status along with bioethical concerns are discussed in the context of identifying future optimal
treatment.

Keywords
ovarian insufficiency, premature ovarian failure, poor ovarian response, stem cells, platelet rich plasma, mitochondria
replacement therapy

From the time of birth of the first in vitro fertilization (IVF)


1
child in 1978 till today, the common denominator in research Centre for Human Reproduction, Genesis Athens Clinic, Athens, Greece
2
Department of Physiology, Medical School, National and Kapodistrian
is the focus of clinicians’ efforts to override pathophysiolo- University of Athens, Athens, Greece
gical barriers leading to infertility and provide solutions. 3
Assisted Reproduction Unit, 2nd Department of Obstetrics and
From controlled ovarian hyperstimulation (COH) protocols Gynecology, Aretaieion Hospital, Medical School, National and
to the emergence of intracytoplasmic sperm injection (ICSI), Kapodistrian University of Athens, Athens, Greece
4
Unit of Endocrinology, Diabetes Mellitus and Metabolism, 2nd
embryo and gamete cryopreservation, and preimplantation
Department of Obstetrics and Gynecology, Aretaieion Hospital,
genetic screening (PGS), these—once introduced as—novel Medical School, National and Kapodistrian University of Athens,
approaches have been successfully established as clinical Athens, Greece
routine practice1.
Submitted: January 4, 2020. Revised: March 9, 2020. Accepted: April 2, 2020.
Despite the fact that several infertile couples can be suc-
cessfully treated, one category of infertile patients still Corresponding Author:
remains highly challenging for clinicians. This category Mara Simopoulou, Associate Professor of Experimental Physiology and
Embryology, Sr. Clinical Embryologist and Geneticist, Laboratory of
refers to patients diagnosed with ovarian insufficiency. Experimental Physiology, Medical School, National and Kapodistrian
Ovarian insufficiency is a term describing a wide range of University of Athens 75, Mikras Asias 11527, Athens, Greece.
pathophysiological conditions where ovarian function is Email: marasimopoulou@hotmail.com

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License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further
permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
2 Cell Transplantation

compromised. Several iatrogenic, pathophysiological, or efficiency, along with the possible complications, the current
physiological factors and processes can compromise ovarian status, and the bioethical implications of these novel
function namely maternal age, autoimmune diseases, muta- approaches.
tions in genes implicated in the regulation of ovarian func-
tion and development, chromosomal and developmental
abnormalities, infectious diseases, and gonadotoxic treat-
Stem Cell Transplantation
ments (e.g., chemotherapy or radiotherapy)2–5. In the context Stem cell therapy has noted considerable success in the field
of assisted reproductive technology (ART), women present- of regenerative medicine during the last two decades. The
ing with ovarian insufficiency tend to produce oocytes of term “stem cells” is used in order to describe a wide spec-
compromised quality, may be unable to ovulate normally, trum of undifferentiated cells of the human body having the
may not be responding to COH protocols or they may pres- ability to self-renewal, to proliferate and differentiate to sev-
ent with primary failure of ovarian function2–5. Considering eral organ and tissue-specific cell types. Due to their devel-
that ART success requires good quality oocytes that will in opmental ability, stem cells may represent the “pin of the
turn provide good quality embryos of high implantation arrow” in the field of regenerative medicine today.
potential, patients with ovarian insufficiency present with a As anticipated, following successful implementation of
significantly compromised reproductive potential. stem cell therapy on various systems (as mentioned above),
The three main patient categories with ovarian insuffi- investigating the potential use of stem cell therapy in the
ciency are defined as premature ovarian failure (POF), poor context of treating the female reproductive system became
ovarian response (POR), and advanced maternal age (AMA). the focal point of cutting edge research. This trend has fur-
Due to the wide spectrum of pathophysiological conditions ther focused on the context of infertility related to ovarian
leading to ovarian failure, managing patients with ovarian insufficiency. The explosion of interest witnessed by the
insufficiency is highly perplexing. These challenges have scientific community regarding the potential use of stem cell
served as a driver for the emergence of a new era in the field therapy in treating infertility is depicted in a recent systema-
of reproductive medicine which has risen dynamically in the tic review published by Fazelli et al. in 2018. The results
last decade, aiming to effectively address ovarian insuffi- provided, indicate that more than of 11,400 published studies
ciency in the context of treating infertility. Recent studies have investigated the potential use of stem cell as an effec-
investigating the regenerative dynamic potential of the ovar- tive therapy for several conditions leading to both male and
ies demonstrate that follicular growth could be stimulated female infertility 11 . From endometrial rejuvenation in
when an adequate ovarian environment is restored6. This patients presenting with Asherman syndrome to restoration
restoration can be achieved employing factors of known of ovarian function in poor responder patients, stem cells
regenerative potential such as stem cells, isolated growth presented as an appealing therapeutic tool in the field of
factors, or platelet rich plasma (PRP)7. Additionally, recent reproductive regenerative medicine12,13.
advances in the field of stem cell biology are enabling the Studies have hitherto focused on the use of the multi-
reprogramming of differentiated cells to stem cells and sub- potent stem cells and particularly of the mesenchymal
sequently to gamete-like cells also known as artificial stem cells (MSCs) as a potential therapeutic tool for ovar-
gametes8. Last but not least, recent studies suggest that mito- ian insufficiency. Their exceptional properties including
chondrial function is strongly related to oocyte aging and undifferentiation, proliferation, and renewal capacity are
fertilization failure9,10. These data, in combination with the of high significance rendering stem cells a remarkable
recent technological development allowing nearly complete “tool” in the service of transplantation and drug targeted
replacement of the cytoplasm of an oocyte, introduce the therapy. Additionally, the unique profile of the multipo-
novel approach of mitochondrial replacement therapy for tent stem cells reduces the rejection and tumorigenic risk
oocyte rejuvenation claiming to lead to an advanced and increases stem cell survival rate making therapy more
cellular dynamic. efficient and safer14,15.
Our team of experts provides herein a timely and essential Stem cell therapy could perhaps represent the missing
critical review analysis aiming to report on the novel piece of the puzzle in overcoming the challenges encoun-
approaches in addressing the challenging case of ovarian tered in the management and treatment of these infertile
insufficiency, expressing considerations and concerns couples. Multipotent stem cells can effectively induce tissue
toward clinical application in humans. This review presents regeneration, mediated by the two exceptional and unique
the prospective use of ovarian rejuvenation employing ovar- abilities characterizing them. On the one hand, these cells are
ian injection of stem cells, growth factors, and PRP, along able to migrate to damaged tissues and organs and differ-
with mitochondrial replacement therapy, and continuing to entiate to tissue-specific cells, promoting healing processes.
employment of artificial gametes, artificial ovaries, and On the other hand, several studies demonstrate that various
ovarian transplantation. This review provides a comprehen- multipotent stem cells have the ability to act as paracrine
sive and critical analysis with regards to the novel treatments modulators. These cells are able to secrete a wide spectrum
addressing ovarian insufficiency. The manuscript discusses of growth factors, chemokines, and mitogenic proteins
the biological mechanisms entailed, the invasiveness and and via these are able to promote proliferation and
Sfakianoudis et al 3

vascularization. Among these factors are the transforming regarding ovarian function restoration in POF, POR, and
growth factor a (TGF-a), the TGF-b, the epidermal growth AMA patients29,30. Following the encouraging published
factor, and the insulin-like growth factor 1 (IGF-1)16–18. data indicating the prospective use of BMSCs in addressing
Furthermore, multipotent stem cells are able to modulate ovarian insufficiency, the therapeutic potential of other
immune response and present with anti-inflammatory prop- sources of stem cells indicating their ability to induce similar
erties. These cells are capable of secreting several growth regenerative phenomena. The results from the aforemen-
factors and anti-inflammatory cytokines such as members of tioned studies demonstrate that there are several easily
interleukin superfamily, tumor necrosis factor, and inter- accessible novel sources of stem cells that may be used
feron g (INF-g). Moreover, several studies demonstrate that toward ovarian rejuvenation.
multipotent stem cells exert anti-apoptotic properties limit- Hitherto, clinical studies investigating the potential ther-
ing tissue degeneration19,20. In regard to the ovarian func- apeutic value of these cells in humans are limited. This may
tion, several studies demonstrate that the ovarian niche is be attributed to the fact that there are several limitations
able to attract several types of undifferentiated cells from entailed in stem cell transplantation, albeit promising out-
other organs and tissues and especially from bone marrow. comes have been documented. In the study of Eddesy et al.,
These cells are able to migrate into the ovaries supporting 10 women presenting with idiopathic POF were subjected to
folliculogenesis in a procedure known as “homing”21. Fol- MSC autologous ovarian transplantation. Recovery of men-
lowing the migration to ovaries, these cells secrete several struation was achieved 3 months following autologous MSC
growth factors and hormones orchestrating ovarian function. transplantation for two patients and a subsequent live birth of
Considering the pathophysiological base of ovarian insuffi- a healthy offspring has been documented31. In another study
ciency involving degenerative phenomena leading to the published by Gabr et al., 30 women with idiopathic POF
collapse of the ovarian niche and the disruption of the mole- were also subjected to MSC autologous ovarian transplanta-
cular network controlling the ovarian vascularization, it is tion. Ovarian function restoration, including gonadotropin
easy to understand the rationale behind the use of the multi- level reduction, and estrogen and anti-Müllerian hormone
potent stem cells as a potential therapeutic tool. Stem cells (AMH) level increase, was observed 1 month post-
could restore ovarian function via the secretion of several treatment. The therapeutic effectiveness was impressively
hormones and growth factors inducing angiogenesis and tis- sustained during a 48 week follow up. Three patients
sue regeneration. achieved pregnancy via IVF treatment and one patient
The potential therapeutic value of stem cells in ovarian reported a pregnancy following natural conception32. In the
failure was reported for the first time approximately 25 years prospective observational pilot study published by Herraiz
ago. Sanders et al. published a retrospective observational et al., the effectiveness of an autologous stem cell ovarian
study indicating that POF patients were able to achieve nat- transplantation protocol (ASCOT protocol) was assessed in
ural conceptions following bone marrow transplantation22. POR patients. This was the first clinical study investigating
Fast forward to today and this approach albeit promising still stem cell therapy for ovarian rejuvenation in POR patients.
retains its experimental procedure status. Several other pub- Following bone marrow mobilization with granulocyte-
lished reports indicated that ovarian function restoration colony stimulating factor (G-CSF) administration and stem
could be achieved following bone marrow transplantation cell collection by apheresis from peripheral blood, the 17
in some POF patients previously treated with high-dose of patients participating in the study were submitted to
chemotherapy or/and total body irradiation23,24. It has been CD133þ BMSC ovarian infusion via intra-arterial catheter-
voiced that the mechanism of action for bone marrow stem ization under ultrasonography guidance. The time frame
cells (BMSCs) is to migrate to ovaries through the circula- between stem cell collection and stem cell infusion was less
tion, exhibiting regenerative properties via the secretion of than 24 h for all patients. Two weeks following ASCOT
several hormones and growth factors. Following “homing”, treatment, a significant increase in the antral follicle count
BMSCs could restore the function of pre-existing unda- (AFC) was observed. As anticipated, an increase in AMH
maged follicles, leading finally to restoration of ovarian levels was also observed. Following COH treatment, an
function25–28. increase in the number of AFC and oocytes obtained was
Following the publication of aforementioned observa- recorded in comparison to the patients’ previous medical
tions, numerous studies investigating the potential use of history. However, the embryo euploidy rate was reported
stem cells for ovarian insufficiency were conducted in ani- to be low. Five pregnancies were achieved, two following
mal models prior to initiating human studies. As reported, an IVF/embryo transfer (ET) cycle and three via natural
the compromised function of ovaries in POF or POR models conception13. The potential effectiveness of stem cell ther-
was reversed, estrogen secretion from the compromised tis- apy was also recorded in a 45 years old perimenopausal
sue was restored, and follicular growth was reactivated, woman. Eight weeks following stem cell therapy, a signifi-
while granulosa cell apoptosis was mitigated accompanied cant improvement regarding AFC number and AMH levels
with the occurrence of angiogenesis. What is more, fertility was observed. The patient achieved pregnancy via an IVF-
restoration and live births were documented suggesting that frozen cycle. The woman was subjected to noninvasive pre-
BMSCs transplantation could be an effective treatment natal testing indicating a normal karyotype. A live birth was
4 Cell Transplantation

finally achieved and the patient delivered via cesarean sec- severity— requires an individualized standardization by the
tion at 38 weeks. No complications were observed in the practitioner in the era of precision medicine. Nonetheless, it
pediatric follow up33. is imperative to thoroughly understand the underlying
Stem cell therapy seems to be a promising approach. mechanisms regulating stem cell treatment.
Nonetheless, in order to safely recruit it in clinical applica- Regarding the appropriate administration method for
tion toward treating ovarian insufficiency in the context of ovarian stem cell transplantation, data that have been
infertility, it should be highlighted that it is equally impera- obtained till date are inconclusive and lacking clarity. Two
tive to consider both strengths and weaknesses. potential administration methods have been proposed
Despite the fact that mesenchymal stem cells are charac- namely stem cells injection into the ovarian cortex via
terized by a low immunologic profile, allogeneic stem cell laparoscopy and intra-arterial ovarian catheterization under
transplantation could lead to severe graft rejection, posing a ultrasonography guidance. Both of these methods appear to
risk for the patients’ health. Studies in the field demonstrate be equally efficient. Considering the level of invasiveness,
that autologous stem cell transplantation is related with intra-arterial catheterization may be viewed as more patient-
enhanced therapeutic efficiency compared to allogeneic friendly. However, in certain cohorts of patients such as
transplantation in one out of the four women treated with AMA women, the practice of intra-arterial catheterization
allogeneic transplantation could face severe chronic gyneco- targeting atrophic ovaries of compromised volume due to
logical graft disease30,34. One of the antipodes, autologous aging renders the procedure challenging43. Future rando-
stem cell transplantation could serve as a potential solution mized controlled trials (RCTs) comparing the two methods
in an effort to eliminate the graft rejection risk. However, with respect to the patients’ pathophysiology will provide a
hitherto, the practice of autologous stem cell transplantation deeper impact in categorically delineating this issue and
entails two levels of invasiveness. The first one refers to the addressing the benefits entailed.
harvesting procedures and methods of stem cell retrieval via In conclusion, robust data provided from future RCTs and
bone marrow aspiration. This fact coupled with the second well-designed clinical trials are vital in order to ascertain
level of invasiveness encountered at the stage of treatment safe conclusions with respect to an array of crucial questions
application entailing stem cell injection into the ovaries, remaining unanswered and perhaps even undefined. This
performed via laparoscopy or intra-arterial catheterization, very fact justifies why beyond the promising nature of its
significantly increases concerns. Thus, it is of high signifi- application it still retains its experimental status. The appro-
cance to investigate the potential use of other sources of stem priate dose and specific administration time following stem
cells for autologous transplantation, except from BMSCs, cell harvesting, the appropriate administration method, the
namely endometrial stem cells (EnSCs) and adipose derived duration of the recovery period and the potential treatment
stem cells (ADSCs). These categories of stem cells have replication are all issues of value that merit further investi-
successfully been used as a potential therapeutic tool for gation. Furthermore, cost-effectiveness studies along with
addressing ovarian insufficiency in animal models. None- studies focusing on assessing patients’ behavioral patterns
theless, studies in humans have not been conducted till date. and response comparing stem cell therapy to the conven-
An overview of the presented studies on the novel approach tional treatment options should be submitted. Regarding the
of stem cells administration indicating study design, meth- cost of stem cell therapy, it may be safe to conclude that it
ods employed, outcome, and any adverse effects reported is typically corresponds to a particularly high charge per treat-
provided in Table 1. ment cycle one that could range from over two to fivefold
Reporting on the safety of stem cell therapy in ovarian higher than the cost for a conventional IVF cycle depending
insufficiency, serious considerations should be highlighted, on the country where treatment is provided44,45. This fact
especially in light of the lack of published evidence addres- alone may raise considerable bioethical issues. Considering
sing any side effects related to this novel approach. The most the multifaceted nature of ovarian insufficiency implicating
severe potential adverse effect is that unfortunately, intense several different pathophysiological conditions, meticulous
cell proliferation events that occur following stem cell trans- observations and documentations are needed in order to
plantation may induce malignant formation. It is well docu- determine the specific cohorts of patients for whom stem
mented that long-term cultured mesenchymal stem cells cell therapy may be efficient and safe. Till then, stem cell
could induce tumorigenesis and metastasis. Following stem transplantation for ovarian rejuvenation should maintain the
cells, isolation from their source of origin, in vitro expansion status and classification of an experimental treatment and
of cell population is typically required to achieve a clinically practitioners should abstain from embarking on clinical
suitable grade of stem cells. However, stem cells at higher application outside the scope of an approved research
passages could lead to malignant cell transformation29. Con- protocol.
sidering the aforementioned, it is of added value to deter-
mine both the appropriate dose and the specific
administration time following stem cell harvesting, in order
Platelet-Rich Plasma Intra-Ovarian Infusion
to achieve a balance between safety and efficiency. It seems PRP is comprised of crucial ingredients identified in repair
that the appropriate management of patient—defined by mechanisms instigating fundamental physiological
Table 1. An Overview of Published Studies on the Novel Approach of Stem Cells Administration Indicating Study Design, Methods Employed, Outcome, and Adverse Effects Reported.
Type of ovarian
Publication Study design insufficiency Intervention Method employed Outcome Adverse effects
23
Salooja et al. (1994) Retrospective observational Iatrogenic POF Treatment for AML with high-dose Autologous BM transplantation Cycle restoration in young NR
study chemotherapy with autologous BM patients (age 21–32);
transplantation spontaneous pregnancies
Sanders et al. Retrospective observational Iatrogenic POF High-dose chemotherapy with TBI and Autologous BM transplantation Cycle restoration; ovulation; Risk of spontaneous
(1996)22 study BM transplantation and spontaneous abortion; risk of
for aplastic anemia pregnancies preterm labor;
and risk of SGA
Salooja et al. (2001)24 Retrospective observational Iatrogenic POF Chemotherapy, total body irradiation Peripheral blood or BM transplantation Cycle restoration; ovulation; High risk for
study with peripheral blood or BM allogeneic or autologous and spontaneous maternal and
transplantation pregnancies fetal
complications in
allograft patients
Johnson et al. Animal study (mice) Iatrogenic POF model; Mice sterilized by chemotherapy, as BM transplantation Cycle restoration; ovulation; NR
(2005)25 ataxia telangiectasia- well as in ataxia telangiectasia- and
mutated gene- mutated gene-deficient mice BM could sustain oocyte
deficient model production in adulthood
Eggan et al. (2006)27 Animal study (mice) Iatrogenic POF model Parabiotic mice treated with Parabiotic model Cycle restoration; ovulation; NR
chemotherapy and
BMSCs “homing”
Veitia et al. (2007)28 Case report Amenorrheic patient Allogeneic BM transplantation due to Allogeneic BM transplantation Cycle restoration; ovulation; NR
with Fanconi aplastic anemia Hormone recovery; and
anemia spontaneous pregnancy
Lee et al. (2007)35 Animal study (mice) Iatrogenic POF model Chemotherapy and BM transplantation Allogeneic BM transplantation via tail vein Fertility rescue in mice treated NR
with nonlethal doses of
chemotherapy
Abd-Allah et al. Animal study (rats) Iatrogenic POF model Chemotherapy and BM transplantation Allogeneic BM transplantation via ear vein Hormone recovery; follicle NR
(2013)36 number increase; and VEGF
production
Guo et al. (2013)37 Animal study (rats) Iatrogenic POF model; Chemotherapy and GCs and BMSCs Isolated GSs co-cultured with BMSCs Increased GCs apoptosis in NR
perimenopausal model isolation perimenopausal rats;
apoptosis reduction
following BMSCs co-
culture
Lai et al. (2013)38 Animal study (mice) Iatrogenic POF model Chemotherapy and hAFCs hAFCs injection into ovaries Hormone recovery and GCs NR
transplantation proliferation
Sun et al. (2013)39 Animal study (mice) Iatrogenic POF model Chemotherapy and ADSCs ADSCs transplantation via injection into Follicle number increase and NR
transplantation ovaries or/and intravenous injection ovulation
Liu et al. (2014)21 Animal study (rats) Iatrogenic POF model Chemotherapy and BMSCs injection BMSCs injection via tail vain Hormone recovery and follicle NR
number increase
Lai et al. (2015)40 Animal study (mice) Iatrogenic POF model Chemotherapy and EnSCs EnSCs injection via tail vain Cycle restoration and GSCs NR
transplantation depletion reduction
31
Edessy et al. (2016) Prospective observational Idiopathic POF BM aspiration and autologous BMSCs BMSCs injected into the ovaries via Cycle restoration; hormone NR
study transplantation laparoscopy recovery; spontaneous
pregnancy; and live birth

(continued)

5
6
Table 1. (continued)
Type of ovarian
Publication Study design insufficiency Intervention Method employed Outcome Adverse effects

Gabr et al. (2016)32 Prospective observational Idiopathic POF G-CSF administration for BM BMSCs injected into the ovaries in two Cycle restoration; hormone NR
study mobilization, BM aspiration, and different sides: BMSCs were recovery;
autologous BMSCs transplantation laparoscopically injected into the follicular growth reactivation;
ovarian cortex and into the ovarian and pregnancy via IVF or
artery natural conception
Wang et al. (2019)41 Animal study (mice) Iatrogenic POF model Chemotherapy and MenSCs MenSCs injection via tail vein Hormone recovery; follicular NR
transplantation growth reactivation; and
GCs apoptosis reduction
Herraiz et al. (2018)13 Animal study (mice) POR model SCID ovariectomized mice Human C133þ BMSCs injection via tail Hormone recovery; follicular NR
xenotransplanted with ovarian vein growth reactivation; GCs
cortical fragments from POR apoptosis reduction; and
patients and treated with human spontaneous pregnancy
BMSCs
Mohamed et al. Animal study (mice) Iatrogenic POF model Chemotherapy and BMSCs BMSCs intraovarian injection Hormone recovery; follicular NR
(2018)42 transplantation growth reactivation; and
spontaneous pregnancy
Herraiz et al. (2018)13 Pilot study POR G-CSF administration for BM BMSCs injected into the ovaries via intra- Hormone recovery; follicular Low embryo
mobilization, BM aspiration, and arterial catheterization under growth reactivation; and euploidy rate
autologous stem cell ovarian ultrasonography guidance pregnancy via IVF or natural
transplantation protocol (ASCOT conception
protocol)
Mohamed et al. Animal study (mice) Iatrogenic POF model Chemotherapy and UCMSCs UCMSCs ovarian transplantation via Hormone recovery and NR
(2019)42 transplantation laparotomy spontaneous pregnancy
Gupta et al. (2018)33 Case report Perimenopausal BM aspiration, autologous BMSCs BMSCs injected into the ovaries via Hormone recovery; follicular NR
women (AMA) ovarian transplantation laparoscopy growth reactivation;
pregnancy via IVF
Euploid embryo; and live birth

ADSCs: adipose derived stem cells; AMA: advanced maternal age; AML: acute myeloid leukemia; BM: bone marrow; BMSC: bone marrow stem cells; GC: granulosa cells; G-CSF: granulocyte-colony stimulating factor:
MenSCs: mesenchymal stem cells; NR: not reported; POF: premature ovarian failure; POR: poor ovarian response; SCID: severe combined immune deficiency; TBI: total-body irradiation; UCMSCs: umbilical cord-derived
mesenchymal stem cells.
Sfakianoudis et al 7

procedures in a healthy individual. Numerous physiological been investigated with reports revealing impressive hormo-
mechanisms are involved in the multifaceted phenomenon of nal restoration, improved oocyte accumulation, clinical
tissue restoration and repair process, including proliferation, pregnancies, and subsequent live births in a short time inter-
angiogenesis, programmed cell death46, and cell migration, val post-treatment59,60,62. Based on the observations regard-
all of which are attributed to the valuable synergy of factors ing hormonal restoration following the PRP application,
evoked by platelets namely vascular endothelial growth fac- these results indicate that ovaries of compromised function
tor (VEGF), platelet-derived growth factor AB (PDGF-AB), following PRP application seem to mimic the physiology
and TGF-b147–49. Additionally, growth factors are coupled and adopt respective pathways along with the endocrinolo-
by the presence of chemokines and cytokines engaging in gical behavior of a functional ovary even for a defined
order for tissue healing to be orchestrated50. The rationale shorter time frame. However, the lack of conclusive data
behind implementing PRP in the context of managing allows no further conclusions drawn on the mapping of the
pathologies affecting tissue integrity was based on the afore- activated pathways regarding the interaction between PRP
mentioned key components originally verified in participat- and the reproductive organs. An overview of the presented
ing in the tissue repair process. Its application may extent to studies on ovarian PRP injection indicating study design,
restore these physiological procedures in a system identified methods employed, outcome, and any adverse effects
with pathologies compromising function. reported is provided in Table 2.
PRP has noted considerable success in various systems51–55. In an effort to elucidate all aspects regarding PRP appli-
Following successful implementation of PRP in treating cation as a therapeutic option for ovarian insufficiency, it
pathologies related to various systems, investigating the should be highlighted that both strengths and weaknesses are
potential use of PRP in the context of the female reproduc- equally imperative to consider. PRP constitutes an autolo-
tive system was anticipated. In light of the fact that monthly gous product minimizing the potential of side effects that
ovulation in the female reproductive system has been may be related with the administration of a heterologous
described as micro-trauma56, the rational of recruiting PRP sample. Despite PRP application being described in the lit-
along with its vital components being involved in tissue erature as a straightforward procedure, nonetheless, the pro-
repair, may be viewed as one way of justifying its applica-
cedure of PRP application entails predicted challenges with
tion regarding ovarian function restoration.
respect to its invasive nature and the fact that the clinical
Intraovarian injection of PRP in animal models and sub-
application of intraovarian injection requires meticulous
sequent resurgence in oocyte production was initially intro-
handling and respective patient individualized standardiza-
duced in literature describing the injection of growth factors
tion by the practitioner. For certain cohorts of patients such
in the ovary. These studies resulted in increased follicular
as menopausal women, the practice of intraovarian infusion
development and a diminution in apoptotic events in animal
targeting atrophic ovaries of compromised volume due to
models57,58. This radical approach claims to trigger and
aging adds another level of complexity rendering the proce-
revive an otherwise described as a “comatose” ovary through
dure rather demanding43. What is more, multiple injection
autologous administration of enriched plasma providing a
concentrated cohort of platelets capable of producing essen- sites seem to be required, thus the level of invasiveness may
tial growth factors. Application of PRP regarding restoration be viewed as considerable. Regarding the cost of this novel
of ovarian function was introduced by Sfakianoudis et al. approach, an affordable technique has been indicatively pro-
and Sills et al.7,43,59,60. Regarding the method and technique posed for the preparation of blood samples with an estimated
describing intraovarian PRP treatment, various protocols cost of $1064. However, in clinical practice, the cost of this
have been described for the preparation of PRP including service is typically reaching over 100-fold of this corre-
either commercial kits or a more affordable in-house sponding to approximately 20% of a conventional IVF cycle
preparation43,60. cost depending on the country where treatment is provided65.
Albeit reports have surfaced regarding PRP employment PRP injection remains an invasive procedure performed
at the moment PRP is considered to be an experimental under ultrasound guidance with the anticipated risks not
procedure in the context of ovarian insufficiency until robust adequately elucidated. Lack of published evidence addres-
data are provided from RCTs. The first report on the clinical sing any side effects is far from reassuring leading to the
application of PRP in human ovaries included perimenopau- acknowledgment of the need for well-designed clinical
sal and prematurely menopausal IVF patients with poor trials. Aside from the promising results already published
ovarian reserve exploring options to address infertility and for some cohorts of patients, there has been some concern
pursue a pregnancy. These patients were treated employing voiced on the aftereffect of PRP application. The intense cell
intraovarian PRP injection61. Restoration of ovarian function proliferation events that occur may induce malignant forma-
along with restoration of the menstrual cycle was noted tions possibly due to differentiation of stem cells present in
1–3 months post-treatment. Further to that, the patients ovaries66. Nevertheless, the presence of stem cells in ovaries
included were subjected to natural IVF cycles yielding a represents a heated debate 67 with opposing schools of
range of 1–5 oocytes per retrieval. Poor responder patients thought emerging, supporting the norm of the restricted
as well as patients of diminished ovarian reserve have also reserve of oocytes versus the revolutionary concept of
8
Table 2. An Overview of Published Studies on the Novel Approach of PRP Administration Indicating Study Design, Methods Employed, Outcome, and Adverse Effect Reported.

Adverse
Publication Study design Type of ovarian insufficiency Intervention Method employed Outcome effects

Quintana et al. Animal study (mice) NA Ovarian injection of Growth factor administration Direct injection of VEGF into the NR
(2004)58 vascular endothelial mouse ovary results in the
growth factor development of an enhanced
vascular network promoting
follicular development and
diminishing apoptosis
Danfort et al. Animal study (rat) Immature ovaries Ovaries were injected Growth factor administration VEGF stimulates preantral follicular NR
(2003)57 with vascular development; exogenous estrogen
endothelial growth up-regulates VEGF expression in
factor the ovary and enhances early follicle
growth
Sills et al. (2018)60 Pilot study Poor prognosis (patients over age 35 PRP activation and PRP activation with calcium Improved ovarian function NR
with at least one ovary, infertility intraovarian injection gluconate and disposition
duration of >1 year, at least one under the ovarian capsule
prior failed (or canceled) IVF cycle,
or amenorrhea for at least 3
months)
Sfakianoudis et al. Case report Premature menopause PRP intraovarian injection PRP intraovarian injection Hormonal restoration; natural cycle NR
(2018)43 IVF attempt resulting to
biochemical pregnancy
Sills et al. (2019)63 Questionnaire study Low ovarian reserve patients and\or Ovarian PRP Ovarian PRP Ovarian hormonal rejuvenation; NR
patients with at least 1 failed IVF reversing low or absent ovarian
attempt reserve
Wang et al. (2019)41 EX vivo Mesenchymal stem cells from healthy PRP as a proliferation and Mesenchymal stem cells PRPs with different platelet NR
individuals differentiation proliferation and concentrations exerts osteogenic,
promoter differentiation induced by adipogenic, and chondrogenic
PRP differentiation
Farimani et al. Case series Poor ovarian response PRP intraovarian injection PRP intraovarian injection Improved oocyte yield; spontaneous NR
(2019)62 conceptions and live birth
Sfakianoudis et al. Case series Poor ovarian response PRP intraovarian injection PRP intraovarian injection Hormonal improvement; natural NR
(2019)59 conception and live birth
Pantos et al. (2019)7 Case series Premature ovarian failure PRP intraovarian injection PRP intraovarian injection Restoration of menstruation; NR
improvement in hormonal profile;
natural conception
IVF: in vitro fertilization; NR: not reported; PRP: platelet-rich plasma; VEGF: vascular endothelial growth factor.
Sfakianoudis et al 9

ovarian germline stem cells capable to rejuvenate the ovar- highlighted that natural conception may be achieved in cases
ian tissue. of orthotopic transplantation. The method of transplanting
While investigating the possibilities of PRP application, ovarian tissue may be considered technically appealing since
precise primary and secondary outcomes should be decided the utilized ovarian tissue is characterized by a solid extra-
upon, in order for a conclusive report on PRP’s effectiveness cellular matrix structure coupled with complex cellularity.
to be delivered. Undoubtedly, the molecular interaction Furthermore, biomaterials are introduced in order to enhance
needs to be elucidated prior to designing large RCTs that the structure and integrity of ovarian tissue by improving
aim to convey safety in practice. It has been voiced that adhesion, proliferation, and differentiation of the cells
PRP’s concentration may play a key role in cell proliferation involved. Natural biomaterials present with significant het-
and differentiation of mesenchymal cells. Managing various erogeneity in their composition spectrum, while synthetic
pathologies may require different optimal concentrations. biomaterials’ ability to promote cellular and tissue remodel-
This may explain the heterogeneity in the results and out- ing is restricted72. Thus, both sources present equally with
comes documented and observed following PRP application weaknesses regarding the configuration of an optimal model
amongst patients41. A personalized approach taking into for ovarian tissue transplantation.
account the underlying pathology should be pursued. Several Since undergoing autologous ovarian tissue transplanta-
factors potentially affecting the end result namely time inter- tion requires collecting tissue prior to acknowledging and
vals between PRP applications, volume administered, as identifying signs of ovarian deficiency, this strategy is typi-
well as the threshold of the maximum number of allowed cally addressing strictly a particular cohort of patients. These
interventions should be addressed. Pondering on whether patients are women that are destined to experience an antici-
PRP has a place in clinical practice in addressing ovarian pated ovarian shortage following cancer therapy, without
insufficiency, a study submitted the thesis that aside from excluding the patient group that will experience anticipated
promising outcomes in ovarian physiology, an essential gamete exhaustion due to a prognosis or even a diagnosis of
improvement on the treated patients’ quality of life has been POF. In cases of a diagnosis of malignancy, ovarian tissue
noted. This could be plausibly attributed to the hormonal cryopreservation for subsequent transplantation is suggested,
rebalance recorded following intervention63, encouraging since it could be performed in a short time-frame without
the employment of PRP and extending its consequences delaying the initiation of therapy. From the first attempts on
beyond a strict physiological and molecular context. ovarian tissue transplantation almost 20 years ago73 to
The ovarian reserve issue may be a multifaceted matter, numerous successful live births following this procedure
thus managing these patients may require a plethora of avail- published in literature since then 74–78 , a new line of
able tools and techniques. Hitherto, the complex dialog approaching ovarian deficiency has been introduced and
between PRP components and the ovary remains to be dec- established.
iphered. In anticipation of solid molecular and clinical evi- Valuable observations have been documented in litera-
dence to support the validity of the “ovarian rejuvenation” ture reporting on restoration of fertility, ovarian activity rein-
concept, PRP may face the risk of remaining a costly service. statement, endocrine function reactivation, and follicular
Nonetheless, preliminary data showcase its potential of recruitment70,79. What is more, transplanting ovarian tissue
being perceived as a ground-breaking medical innovation. has been proposed strictly on the grounds of ovarian rejuve-
Could that be enough to give it a try? Maybe. Nonetheless, nation leading to what may be described as a hormonal ovar-
we need to question whether evidence is accumulating to a ian reboot irrespectively of pursuing fertility treatment.
satisfactory degree leaving the scientific community content Ovarian tissue transplantation enabled a hormonal restora-
in deciding to introduce routine clinical application of PRP tion lasting an impressive 7-year period79, which was docu-
for ovarian insufficiency. mented to be extended to a 12-year restoration period when
the procedure was repeated70. Nonetheless, one should not
fail to highlight that this invasive procedure minimizes and
Ovarian Tissue Transplantation potentially jeopardizes the current ovarian reserve for the
Ovarian tissue cryopreservation may not be considered as a patient, thus bioethical concerns are raised.
unique novel approach. Since the first implementation, over A major limitation to consider is that autologous trans-
130 babies have been born68. However, it is still considered plantation entails the risk of reintroducing cancerous cells to
to be an experimental approach69. Various reports attempt to the treated ovary80. Moreover, immune rejections constitute
establish a commonly accepted technique for ovarian trans- a major complication for this line of approach, albeit the
plantation. Orthotopic transplantation may present as an autologous source of tissue may contribute to overcoming
optimal practice since the microenvironment around the this side effect. Ischemia has also been described as a detri-
transplanted tissue is the most suitable for follicular devel- mental side effect following ovarian tissue transplantation81.
opment70. On the other hand, there is limited number of The main cause of this event is the lack of vessel anastomo-
tissue fragments allowed to be transferred due to space con- sis during the tissue transplantation procedure. As a result,
striction, while the level of invasiveness presents as a major the transplanted ovarian tissue is exposed for a certain time
limitation resulting in severe pelvic adhesions71. It should be period of 5 days82 in a hypoxic and ischemic environment
10 Cell Transplantation

which demands an urgent intervention to induce neovascu- previous attempts have failed due to the common denomi-
larization. In this context, various approaches have been nator of implementing a solid structure albeit lacking ade-
attempted to optimize the outcome including employment quate mimicking of vital endocrine and paracrine pathways.
of growth factors as a supplementary tool to assist toward Three ovarian cell types were included namely theca cells,
completion of neovascularization in a timely fashion83. granulosa, and oocytes86. By including theca cells as a fun-
However, their application in human models is nowhere near damental component of the 3D ovary, hormonal production
consideration even though current experimentation on ani- was achieved. Interestingly, implementing collagen or algi-
mals has contributed favorable results. Concerns over sub- nate scaffolds failed to ascertain a similar endocrinology
sequent cancer diagnosis have emerged, nonetheless, they response. What is more, enhanced interaction between cell
were considered as a low probability event when tissue is types was observed conspiring toward the development of a
sourced from young women aged 20–2584. The technique of physiological model. Under these circumstances, maturation
cryopreservation and its respective efficiency should be fur- and development of the primordial oocytes may be enabled.
ther taken in to account as a challenging part of the ovarian The novelty behind this approach is indisputable; however,
tissue transplantation approach. It is the actual cryopreserva- the fact that it constitutes what may be described as an
tion technique that may perhaps result in compromised out- “isolated” system lacking the complexity of numerous inter-
comes misleadingly attributed to the transplantation actions with other physiological systems should be
technique. Contemplating on the logistics of performing highlighted.
ovarian tissue transplantation for restoring endocrine func- Bioengineering strategies to restore fertility and address
tion in menopausal patients, the procedure is less invasive ovarian insufficiency include transplantation of fresh or
since the graft site may not involve the pelvic cavity. cryopreserved ovarian tissue and tissue engineering involv-
Furthermore, it may be performed under local anesthesia, ing implementation of growth factors, stem cells, pluripotent
while in case of an ovarian neoplasm emergence, the ovarian stem cells, mesenchymal stem cells, and biomaterials72. The
tissue implant could be immediately extracted84. An over- percentage of cancer patient survivors is increasing, translat-
view of the presented studies on the novel approach of ovar- ing to patients who despite succeeding in overcoming a dis-
ian tissue transplantation indicating study design, methods ease, they will be reproductively challenged experiencing
employed, outcome, and any adverse effects reported is pro- infertility strain and concerns attributed to gonadal injury
vided in Table 3. during therapy87. Numerous successful attempts at perform-
All of the above constitute vital indications in favor of ing ovarian tissue cryopreservation and subsequent trans-
this treatment option. Nonetheless, similarly to all hormone plantation reveal valuable data on the efficiency of the
replacement approaches aiming to postpone the permanent approach. However, little is known in regard to whether this
hibernation of the ovaries for women reaching the end of approach should be considered as a method to delay the
their reproductive life-span, ovarian tissue transplantation onset of menopause—a trend that appears to be rapidly
entails risks. Apart from the concerns raised when rejuvena- evolving. In regard to the concept of the artificial ovary,
tion is the primary patients’ interest, practitioners appear further considerations should be taken into account. An
hesitant to embark on this suggested approach even in cases overview of the presented studies regarding the novel
where patients are facing a reproductive dead-end. This may approach of artificial ovary indicating study design, meth-
be attributed to the realization that ovarian transplantation ods employed, outcome, and any adverse effects reported is
has been around for several decades, failing nonetheless to provided in Table 4. It should be highlighted that exposing
be established as a foolproof approach in clinical practice sensitive tissue in a challenging environment may entail
one that could provide specialists with confidence regarding certain epigenetic risks which in turn may raise bioethical
application. issues. How far is too far when the—once considered as
science fiction—concept of harvesting gametes becomes a
reality? Until strong evidence accumulates, the concept of
Artificial Ovary artificial ovary clearly remains at the experimental stage.
An additional step in ovarian tissue preparation prior to With RCTs being an extrapolation at best, clinical practice
implantation has been proposed in an effort to address the will have to wait.
risk of reestablishing the treated disease. It includes the for-
mation of an ovarian tissue scaffold where oocytes appear,
grow, and subsequently “harvested.” Hitherto, this approach
Artificial Gametes
has been applied in animal models in which granulosa cell- There are four basic cell types employed in the formation of
oocyte complexes were collected and subjected to a 3D in artificial gametes, including germ line stem cells, induced
vitro culture. Following standard proliferation, oocyte yield pluripotent stem cells (iPSCs), somatic cell nuclear transfer
was higher than a conventional IVF approach could to embryonic stem cells (SCNT), and SCNT to donor
achieve85. Likewise, constructing a 3D artificial ovary has oocytes. Successful implementation resulting to live birth
been attempted in order to successfully incorporate, accom- following artificially generating gametes has only been
modate, and enable maturation of human oocytes86. Most reported in animal models88, thus hitherto artificial gametes
Table 3. An Overview of Published Studies on the Novel Approach of Ovarian Transplantation Indicating Study Design, Methods Employed, Outcome, and Adverse Effects Reported.
Type of ovarian
Publication Study design insufficiency Intervention Method employed Outcome Adverse effects

Oktay and Karlikaya Case report Iatrogenic POF Salpingo-oophorectomy Tissue sutured laparoscopically Long term graft survival NR
(2000)73 and ovarian tissue beneath left pelvic Follicle emergence; ovulation and subsequent
transplantation peritoneum menstruation following ovarian
stimulation
Donnez et al. (2004)74 Case report Iatrogenic POF Chemotherapy and ovarian Orthotopic autotransplantation Recovery of regular ovulatory cycles Risk for recurrence of disease
tissue transplantation of ovarian cortical tissue by following implantation, oocyte
laparoscopy quality compromised by
heterotopic site transplantation
Meirow et al. (2005)75 Case report Iatrogenic POF Chemotherapy and ovarian Strips of thawed ovarian tissue Menstruation and live birth following IVF Risk of grafting malignant cells
tissue transplantation transplanted to the left ovary employing a natural cycle
and small fragments injected
into the right ovary
Silber et al. (2005)76 Case report Premature menopause Ovarian transplantation Ovarian cortex graft Live birth following natural conception Risk of follicular atresia
between healthy
monozygotic twins
Demeestere et al. Case report Iatrogenic POF Chemotherapy and ovarian Orthotopic and heterotopic Recovery of ovarian function and live birth Reintroducing malignant cells and
(2007)77 tissue transplantation transplantation of ischemic damage during
cryopreserved ovarian tissue revascularization; limited life
span of the graft
Andersen et al. Case series Iatrogenic POF Autotransplantation of Orthotopic and heterotopic Restoration of menstrual cyclicity, NR
(2008)78 frozen/thawed ovarian autotransplantation of conception, and live birth
tissue and assisted ovarian cortical tissue
reproduction
Schubert et al. (2008)81 Animal study (mice) Iatrogenic POF Xenotransplantation of Fresh or frozen human ovarian Increase of primary and secondary follicles; Massive oxidative stress
ovarian tissue cortex grafted into mice decrease of primordial follicles
Van Eyck et al. (2009)82 Animal study (mice) Tissue deriving from Laparoscopic surgery for Frozen–thawed human ovarian Graft revascularization Ischemic tissue damage
patients with benign nonovarian benign tissue fragments grafted to
gynecological gynecological disorders intraperitoneal cavity
disorders and tissue
transplantation
Kim et al. (2012)79 Case series Iatrogenic POF Laparoscopic Heterotopic site transplantation Ovarian endocrine function restoration Primordial follicles cryoinjury
oophorectomy and
laparotomy
Kong et al. (2017)82 Animal study (mice) Iatrogenic POF Bilateral ovariectomy Bovine ovarian tissue Administration of Ang-2 and VEGF prior to NR
performed on mice xenotransplanted into mice transplantation alleviates ischemic
damage, maintains follicle normality and
density, and reduces levels of apoptosis
and fibrosis in the grafts

NR: not reported; POF: poor ovarian response.

11
12 Cell Transplantation

Table 4. An Overview of Published Studies on the Novel Approach of Artificial Ovary Indicating Study Design, Methods Employed,
Outcome, and Adverse Effects Reported.
Type of ovarian Adverse
Publication Study design insufficiency Intervention Method employed Outcome effects

Pangas et al. Animal study Dissected Granulosa oocyte BMSCs injection via tail vein Hormone recovery NR
(2003)85 (mice) ovaries complexes Follicle number increase
isolated from
mice
Krotz et al. Ex vivo study Oophorectomy Oophorectomy Theca and granulosa cells seeded into An artificial human ovary can be NR
(2010)86 for benign and artificial micro-molded gels and self- created with self-assembled
indications ovary creation assembled into complex 3D human theca and granulosa cell
microtissues microtissues

BMSC: bone marrow stem cells; NR: not reported.

Table 5. An Overview of Published Studies on the Novel Approach of Artificial Gametes Indicating Study Design, Methods Employed,
Outcome, and Adverse Effects Reported.
Type of ovarian Adverse
Publication Study design insufficiency Intervention Method employed Outcome effects

Aflatoonian Ex vivo study Cell lines Human embryonic Differentiation was Human primordial stem cells as well as NR
et al. stem cells induced postmeiotic spermatids were
(2009)91 formed in vitro; steroid hormones
production was detected
Cheng et al. Ex vivo study Human amniotic fluid Differentiation into Differentiation into Oocyte-like cells and zona pellucida- NR
(2012)92 stem cells oocyte-like cells oocyte-like cells like morphology were observed
White et al. Animal study Ovaries isolated from Intraovarian oogonial Intraovarian oogonial Formation of follicles NR
(2012)90 (mice) healthy young stem cells injection stem cells injection
women for sex and xenografting and xenografting
reassignment
purposes
Ma et al. Ex vivo study Human hepatic germ- Germ-line cells Germ-line cells Follicle-like structures, generating Hepatic
(2013)93 line formation formation oocyte-like cells, and subsequently teratomas
developing into blastocyst-like
structures in vitro were observed

NR: not reported.

constitute an experimental procedure. Based on observations stem cells have also been successfully recruited in an
in animal models where artificial gametes may be transferred effort to form artificial gametes92. Moreover, engaging
to ovaries enabling natural conception, the scientific com- human hepatic cell lines in order for germline cells to
munity cannot help but ponder whether this strategy may originate resulted in the formation of follicle-like struc-
present as a useful tool in the hands of practitioners manag- tures, subsequent blastocyst-like structures, and finally
ing ovarian insufficiency in the distant or not too distant germ cell/embryonic tumors following prolonged cul-
future. ture93. An overview of the presented studies on the novel
The option of employing artificial gametes has been approach of stem cells administration indicating study
introduced as an alternative solution for patients lacking design, methods employed, outcome, and any adverse
the potential to produce functional gametes such as effects reported is provided in Table 5.
women with premature ovarian insufficiency89. In human It is vital for concerns on safety to be thoroughly
models, the development of artificial oocytes has been addressed prior to even considering the route to clinical
described, while a successful fertilization has been con- application. With RCTs being a mere hypothesis, it
firmed in one case study88. As reported by White et al., appears that the experimental stage regarding artificial
human oogonial stem cells (OSCs) were transplanted to gametes may take longer than anticipated. Despite the
human ovary tissue xenografted to mice following in vitro biological risks entailed, there are significant ethical con-
proliferation, resulting in the development of artificial cerns raised in a societal level that should be addressed
oocytes90. A challenging process of gamete formation prior to pursuing this option as a solid novel approach for
originating from human embryonic stem cells has been management of ovarian insufficiency94 A clear presenta-
described; however, this route was outlined as insufficient tion of the patients for whom this approach may be ben-
since differentiation of embryonic cells in culture systems eficial and for whom access should be granted is
entails certain difficulties to perform91. Human amniotic imperative95.
Sfakianoudis et al 13

Mitochondrial Replacement Therapy group, for nine patients, embryo-transfer was not performed,
and 14 patients underwent embryo-transfer with embryos
Mitochondria are identified as “the powerhouse of the cell.”
originating from the AUGMENT group. Eight pregnancies
The role of mitochondria in the development of the preim-
were observed all originating from the experimental group.
plantation embryo and its implantation potential has been
A second, albeit smaller study, conducted by Oktay et al.104
studied for at least two decades96. The processes of fertiliza-
included 15 women undergoing an autologous germline
tion and proper embryonic development require great
mitochondrial energy transfer, described as autologous mito-
amounts of adenosine triphosphate (ATP) which is produced
chondrial injection (AMI) by the authors. Of the 15 women
by mitochondria9. Oocyte mitochondrial DNA (mtDNA) recruited, only 10 underwent embryo-transfer, and 4 of them
content has been associated with poorer oocyte quality and achieved a clinical pregnancy status.
ovarian insufficiency97. The prognostic value of the copy These promising results were contradicted by the interim
number has been a topic of controversy in literature since analysis of a discontinued large-scale triple blind RCT10.
a number of studies observed a statistically significant higher Following the enrollment of about 31% of the study’s total
number of mtDNA copies in embryos that resulted in population (n ¼ 59/190), an interim analysis was performed.
implantation failure98, whereas other studies observed no According to the results of the study, AUGMENT did not
statistically significant difference99. increase neither the fertilization rate nor the euploid blasto-
Nonetheless, it is evident that maternal aging is the cause cyst formation rate. On the contrary, blastocyst formation
for mitochondrial dysfunction through swelling and cristae rate was decreased in the experimental group. Moreover,
disruption in oocytes100. The employment of mitochondria embryo grading according to Spanish Association for the
from a young healthy donor was hypothesized to provide the study of Reproductive Biology (ASEBIR) criteria was
preimplantation embryo with a better and more potent devel- poorer, and live birth rates were similarly lower in the
opmental environment during the early stages101. This may experimental group. These observations in the interim anal-
have served as the driver leading to early attempts of mito- ysis rendered the recruitment of more patients unnecessary
chondrial replacement therapy, which was initially and the study was terminated, although the clinical preg-
approached by performing ooplasmic transfer, as first nancy and live birth rates presented in the study were better
described by Cohen101. Although ooplasmic transfer pre- when compared to the other studies regarding the autologous
sented with success in enabling an improvement in embryo mitochondrial transfer. It should be highlighted that patients
quality resulting to reports of clinical pregnancy and live included in the RCT were not necessarily of poor ovarian
birth102, the Food and Drug Administration (FDA) decided function as they were younger than 42 years old, with med-
to ban its application in 2001 due to ethical and medical ian AMH of 1.69 ng/ml, with an interquartile range from
concerns mainly related to heteroplasmy and interrelated 0.97 to 3.24 ng/ml. This is a reason for caution in all studies
adverse effects102. employing autologous mitochondrial transfer. The popula-
More than a decade following the FDA ban, novel tion enrolled is mainly a mixed population with only a few
approaches regarding mitochondrial transfer in order to participants being diagnosed with diminished or poor ovar-
achieve improved ovarian function have been implemented. ian response. It may be possible that the autologous mito-
To avoid concerns introduced by including a heterologous chondrial transfer is suitable for a specific population which
donor, the initial approach employed the autologous germ- still remains to be identified.
line mitochondrial energy transfer (AUGMENT). The pro- Apart from the autologous mitochondria transfer, hetero-
tocol of AUGMENT includes isolation of mitochondria from logous mitochondrial transfer, known as mitochondrial
the patient’s OSCs, processed and injected into the patient’s replacement therapy (MRT), has been employed mainly on
own oocytes during ICSI103. the grounds of avoiding transmission of mitochondrial dis-
The first application of AUGMENT was performed by eases105–107. There are four protocols available for MRT, the
Fakih et al.103 targeting women presenting with low quality pronuclear transfer (PNT), the polar body transfer (PBT), the
oocytes and/or embryos, diagnosed with diminished ovarian maternal spindle transfer (MST), and the germinal vesicle
reserve, ovulatory dysfunction, polycystic ovarian syndrome transfer (GVT)105,108. The three latter techniques require the
(PCOS), tubal factor, and endometriosis. A total of employment of donor enucleated MII oocytes, whereas PNT
93 women from two IVF centers were recruited. The results requires the employment of a donor zygote following
of the study supported a beneficial role for the AUGMENT removal of the pronuclei and polar bodies. In PNT, both
as the clinical pregnancy rate achieved was 35% and 22% for parental pronuclei are transferred to the donor zygote. GVT
each center respectively, in contrast to the 11% and 4% requires transfer of the maternal germinal vesicle enclosing
corresponding to the clinical pregnancy rate of the same the nuclear DNA to the donor enucleated oocyte, and PB
patients prior to AUGMENT. In the same study, sibling requires the transfer of the polar body of the maternal MII
oocytes from 25 patients were randomly allocated to two oocyte in the enucleated donor oocyte. MST requires the
groups, one designed to undergo the AUGMENT therapy transfer of the spindle transfer enclosing the nuclear DNA
and a control group. Out of the 25 patients, two underwent to an enucleated donor oocyte. PNT and PBT have been
embryo-transfer with embryos originating from the control evaluated as possible methods for MRT with embryos
14 Cell Transplantation

Table 6. An Overview of Published Studies on the Novel Approach of MRT Indicating Study Design, Methods Employed, Outcome, and
Adverse Effects Reported.

Type of ovarian Method


Publication Study design insufficiency Intervention employed Outcome Adverse effects
103
Fakih et al. (2015) Prospective Diminished ovarian MRT AUGMENT Enhanced clinical pregnancy NR
cohort study reserve rate
Oktay et al. (2015)104 Case series Mixed population MRT AUGMENT Enhanced clinical pregnancy NR
rate
Labarta et al. (2019)10 RCT Impaired embryo MRT AUGMENT No statistically significant Impaired blastocyst
quality improvement formation rate
ISRCTN Prospective Impaired embryo MRT Maternal spindle Results have not been NR
11455145 cohort study quality transfer reported
AUGMENT: autologous germline mitochondrial energy transfer; MRT: mitochondrial replacement therapy; NR: not reported; RCT: randomized controlled
trial.

reaching the blastocyst stage without progressing to implan- appears to be the sole option in providing favorable results
tation or pregnancy109,110. Zhang et al.111 employed PNT to in humans, all protocols should be evaluated, prior to decid-
achieve a pregnancy for a 30-year-old woman with mito- ing on superiority in the application. Although heteroplasmy
chondrial disease. The same group achieved a pregnancy levels seem to be low by employing the novel MRT tech-
employing MST, for a woman with Leigh syndrome112,113. niques, mito-nuclear interactions are a topic of research.
It should be mentioned that although initially a permission Two meta-analyses performed in animal models have
for long-term follow-up was granted, the parents of the off- reported contradicting results117,118. The first chronologi-
spring decided to forego the permission, thus pediatric cally published meta-analysis has reported that mito-
follow-up data are not available114. In the context of employ- nuclear interactions do not pose a side-effect of MRT117.
ing MRT, MST is the sole technique applied in an effort to The second meta-analysis has reported that MRT may rou-
address poor ovarian response. The clinical trial115 has so far tinely affect offspring biological and biochemical character-
resulted in one live birth, from a 32-year-old poor responder istics with large scale effect due to mito-nuclear interaction,
with four previous failed IVF attempts. This technique has which seem to be even greater in humans118. It is of interest
raised considerable ethical concerns. The European Society that the second meta-analysis has been questioned regarding
of Human Reproduction and Embryology (ESHRE) issued a its design by the author of the first119. Until large scale
formal thesis on strong discouragement and disapproval human studies are performed, the scientific community
regarding the technique’s application in the context of ought to practice cautiousness and maintain a vigilant stance
addressing poor ovarian response. Practitioners should in order to limit possible and perhaps probable long-term
refrain from adopting the clinical practice of MST for any- side effects that currently may not be efficiently identified,
thing else other than to avoid transmission of a mitochon- understood, treated, or reversed. At the moment, the research
drial inherited disorder for which case no other alternative is developing and fueling this novel approach is clearly not
viable. It is strictly this approach that has claimed regulation validated to justify clinical application. Perhaps this may
status by the Human Fertilisation and Embryology Authority be a good time for the scientific community to re-examine
(HFEA) in the United Kingdom116. It is clear that in the the “certainties” that fueled the idea that replacing mitochon-
context of ART treatment, there is a lack of robust or even dria may be the answer to ovarian insufficiency prior to
adequate data indicating safety and effectiveness of the proceeding with clinical application.
method, which currently maintains its experimental status.
An overview of the presented studies on the novel approach
of stem cells administration indicating study design, meth- Discussion
ods employed, outcome, and any adverse effects reported is The concept of ovarian reboot has recently attracted heated
provided in Table 6. interest. This may be attributed to the increased prevalence
Long term follow-up of MRT is required in order to report of the diagnosis of ovarian insufficiency for cases that
on the safety and efficacy of the procedure or even to but- demand effective options on managing their subsequent
tress the original hypothesis trail of thought driving its devel- infertility status. This increase may be justified on the
opment. Conducting more RCTs is a requirement in the grounds of the remarkable results and elevated survival rates
topic. The RCTs should present with strict inclusion and of treating cancer patients in the era of precision medicine.
exclusion criteria, and should not employ a mixed popula- These patients can now enjoy a prolonged life span which
tion. It is of imperative importance to evaluate the safety and nonetheless may harbor infertility and reproductive dilem-
the efficiency of a technique that may present as one of the mas. On another note, the concept of delaying aging has been
turning points of ART practice. Moreover, although MST intensively promoted in media. This reality reinforces
Sfakianoudis et al 15

Table 7. A Critical Assessment Providing a Comparative Perspective between All Novel Approaches, Regarding Issues of Importance to the
Practitioner from Cost and Practical Aspects of Application to Novelty, Ethical Concerns Raised, and Potential for Acquiring Future Clinical
Routine Practice Status.

Invasiveness of Adverse effects of Potential for acquiring


application regarding application regarding Time frame Ethical status of clinical
Approach Cost the patient the patient of action* Novelty concerns routine practice

Stem cells þþ þþþþ þþþ þþþþ þþþ þþ þþþ


Platelet rich plasma þ þþþ þþ þþþþ þþ þ þþþþ
Ovarian transplantation þþþ þþþþþ þþþþ þþþ þ þ þþþ
Artificial ovary þþþþ þ þ þþ þþþþ þþþþ þ
Artificial gametes þþþþ þ þ þ þþþþ þþþþþ þ
Mitochondrial þþþ þþ þ þ þþþ þþþ þþ
replacement therapy
þ: very low; þþ: low; þþþ: moderate; þþþþ: high; þþþþþ: very high.

practitioners toward engaging and endeavoring novel the cause and effect relationship between novel therapeutic
approaches fueled by the patients’ request. On the antipode, intervention and the emerging improvement?” Despite the
understandably clinicians appear hesitant and practice skep- principles of physiological or pathological events and the
ticism in embarking on novel proposed options, since data respective unidentified molecular pathways leading to a
are often conflicting and lack robustness. Nonetheless, compromised ovary, “could a successful application in
reports in literature describing case series and pilot studies restoring function translate to rewinding time?” “Could the
communicate to the scientific community that clinical appli- resulting follicles and oocytes carry a distinctive element
cation of novel—albeit not yet having reached routine clin- compromising their potential and rendering them present,
ical application status—approaches may present a real but nonetheless of poor quality and potential?”
option. It may not be uncommon for certain practices to In order to properly assess these points of heated debate,
invest in optimizing a new line of treatment on premature certain guidelines should be proposed and established. The
ovarian insufficiency. The variety of approaches including common denominator of all novel approaches in the service
stem cells, platelet-rich plasma, ovarian tissue transplanta- of overcoming ovarian insufficiency as critically presented
tion, formation of artificial gametes, even the latest emer- herein lies in inconclusive but rather promising data. This
ging idea of mitochondrial replacement technique calls for very fact renders all of them to be at an experimental stage
specialization and expertise by clinics and practitioners. A rather than of established clinical routine practice. Reestab-
critical assessment providing a comparative perspective lishing fertility potential may be the main objective in pub-
between all novel approaches, regarding issues of impor- lished literature. Nonetheless, restoring fertile status
tance to the practitioner from cost and practical aspects of following the diagnosis of ovarian insufficient may be
application, to novelty, ethical concerns raised, and potential defined by numerous criteria, enabled by various
for acquiring future clinical routine practice status is pro- approaches, and confirmed employing different outcome
vided in Table 7. Despite the variation considering the measures depending on study design and population identity.
respective legal framework regarding application of novel Interestingly, and as shown herein, the perception of a suc-
approaches in ART, patients may have access to such treat- cessful outcome varies considerably amongst researchers.
ments enabled by cross-border reproductive care120. This may only be delineated by concrete studies examining
Staying true to their hippocratic oath “first do no harm” outcomes on a clinical as well as on a basic research level. It
practitioners are invited to see beyond the hype and hope is of high significance to highlight that all the approaches
entailed in cutting-edge treatments. In lack of robust RCT included herein have been proposed as promising techniques
data that could provide a safety net, and with publications on aiming to address the infertility related to ovarian insuffi-
novel approaches overwhelming the field of ART, it is our ciency. Societies of Reproductive Medicine have issued in
obligation to question the effectiveness and thoroughly the past statements and guidelines, clearly shifting the status
research these options. It is forming the apposite questions if a certain technique from being an experimental procedure
that will ascertain design of studies providing a consensus to one classified as ascertaining the status of clinical routine
on these options. “Could the hormonal and physiological practice. Such was the case for oocyte vitrification and
changes observed following application of novel warming121. Nonetheless, hitherto strong evidence is not in
approaches be truly attributed to the success of follicular place for any of the presented techniques in order for the
re-recruitment?” To extrapolate on that concern, since ovar- label of experimental procedure to be lifted. In the future,
ian function is programmed for termination, “how can we well-designed studies presenting clearly indicated outcome
definitively follow and confirm the exact trail along with measures, limitations, and complications, while ascertaining
16 Cell Transplantation

reproducibility may accelerate drawing a safe conclusion on Authors’ Contributions


whether introducing these methods to the clinical routine K.S., G.M., and M.S. conceived and designed the project. A.R.,
practice set up would be beneficial or detrimental. However, S.G., D.R., E.M., P.T., and P.G. performed the literature review.
understanding the outcome of such approaches presents one K.S., A.R., S.G., D.R., E.M., P.T., and P.G. contributed to drafting
side of the coin. Distinguishing the profiles of patients who and editing the manuscript. K.P., N.V., G.M., M.K., and M.S.
respond to certain approaches and to what extent these supervised the study and revised the manuscript. All authors
approved the final draft.
approaches restore their ovarian function remains to be deli-
neated in this era of individualized medicine. The molecular Ethical Approval
mechanism involved during the implementation of the afore-
Our institution does not require ethical approval for reporting
mentioned novel treatments may only be unraveled by basic review articles.
research studies that could hold the potential to conclusively
address the question “for whom these novel approaches may Statement of Human and Animal Rights
perform efficiently.” While estimating the cost and effec- This article does not contain any studies with human or animal
tiveness of the aforementioned approaches, one should not subjects.
fail to consider the financial and psychological cost
entailed—on an individualized basis—in addressing ovarian Statement of Informed Consent
insufficiency when opting to employ what may be described There are no human subjects in this article and informed consent is
as the conventional and traditional approach of oocyte dona- not applicable.
tion. The field of medical practice has experienced the phe-
nomenon of embarking on novel ideas solely on the grounds Declaration of Conflicting Interests
of limited-yet-promising data accompanying a novel tech- The author(s) declared no potential conflicts of interest with respect
nique covering the unchartered territory. It is important to be to the research, authorship, and/or publication of this article.
provided with published data conveying transparency to
Funding
enable a fair and impartial assessment, as not all of them
The author(s) received no financial support for the research, author-
may be perceived as equally promising. Taking into account
ship, and/or publication of this article.
the fact that there is an increase in the cost of health care
set up122 extending to infertility treatment, evidence-based ORCID iD
medicine in the field of ART is in the spotlight of research Mara Simopoulou https://orcid.org/0000-0002-1000-9100
aiming to a more collected and bullet proof approach to
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