Current Women`s Health Reviews

Transcription

Current Women’s Health Reviews
Volume 6, Number 3, August 2010
Current Concepts in Assisted Reproduction and
Fertility Preservation (Part II)
Guest Editors: Sajal Gupta and Ashok Agarwal
Contents
Biography of Contributors
197
Assisted Reproduction:
Evidence-Based Management of Infertile Couples with Repeated
Implantation Failure Following IVF
Kim D. Ly, Nabil Aziz, Joelle Safi and Ashok Agarwal
200
Single Blastocyst Transfer: Contemporary Experience
Chin-Kun Baw, Kim Dao Ly, Amr Kader, Ali Ahmady and Ashok Agarwal
219
The Role of Oxidative Stress and Antioxidants in Assisted Reproduction
Sajal Gupta, Lucky Sekhon, Yesul Kim and Ashok Agarwal
227
Coasting: What is the Cost?
Mohamed Aboulghar
239
PGD and Prenatal Diagnosis: Comparison and Review in Different
Genetic Disorders
Ling Sun, Lian Liu, Man Li, Nikoo Afifiyan and Ali Ahmady
245
Options to Prevent Multiple Pregnancies with ART
James M. Goldfarb and Nina Desai
250
Metabolomic Profiling for Selection of the Most Viable Embryos in Clinical IVF
George A. Thouas and David K. Gardner
254
Contd….
Fertility Preservation:
Creating A Standard of Care for Fertility Preservation
Kathryn D. Coyne, Amr Kader and Ashok Agarwal
261
Prognostic Role of Ovarian Reserve Testing
Jashoman Banerjee, Mona H. Mallikarjunaiah and John M. Murphy
267
Whole Ovarian Vitrification: A Viable Option for Fertility Preservation?
Bruno Salle and Jacqueline Lornage
273
Emerging Technologies for Fertility Preservation in Female Patients
Ahmed Nasr and Mohamed Ali Bedaiwy
276
Preface
Current Women’s Health Reviews, 2010, Vol. 6, No. 3
i
PREFACE
The Special Issue on "Recent Advances in Reproductive Endocrinology and Women’s Health" published by the Current
Women’s Health Reviews is a series of two volumes containing articles on cutting edge technology and contemporary topics
of importance to reproductive endocrinologists, general gynecologists and specialists alike. The second volume has excellent
articles on many recent advances and state-of-the-art technologies related to the field of assisted production and fertility
preservation in women.
Dr Kader et al. present recent updates on “Single Blastocyst Transfer: Contemporary Experience”. The authors state that the
cryopreserved single blastocyst transfer has been developed as a practical method that can be used to optimize pregnancy
outcomes following an unsuccessful initial transfer or in patients who do not meet initial transfer criteria.
In their article on “The role of oxidative stress in assisted reproduction”, Drs. Gupta et al. explain that sperm and oocytes used
in assisted reproductive techniques are subjected to oxidative stress because gametes lack the natural antioxidant defences that
are present during in vivo reproduction in the male and female reproductive tract. In the past, studies quantified OS using
inconvenient and labour-intensive biochemical methods to measure reactive oxygen species (ROS) and assess the antioxidant
status within follicular fluid and culture fluid. Metabolomic profiling is a faster, more accurate method of quantifying OS
during ART and may be used to identify gametes and embryos that are more likely to contribute to successful implantation and
pregnancy. Various studies on the benefits of oral antioxidant supplementation in male and female patients undergoing ART
procedures have yielded inconsistent and conflicting reports, and further research is required.
Dr Ahmady et al. from the Macdonald IVF and Fertility program discuss pre-implantation and prenatal testing techniques that
provide genetic information and detect birth defects or abnormalities in an embryo/fetus before implantation/birth. They
emphasize that the limitations of FISH and PCR will lead to the use of whole-genome analysis/CGH in the setting of in vitro
fertilization (IVF), replacing current PGD testing. Its preliminary application has been encouraging as more chromosomes can
be analyzed with higher sensitivity and accuracy.
Dr Aboulghar et al. from the The Egyptian IVF Center have contributed an excellent article on “coasting,” which is a popular
method of preventing ovarian hyperstimulation syndrome (OHSS). The authors point out that while coasting does not totally
avoid the risk of OHSS, it decreases its incidence in high-risk patients. They highlight the effectiveness of coasting in patients
undergoing intracytoplasmic sperm injection (ICSI) and the fact that it does not jeopardize outcomes. Coasting for >3 days is
associated with a moderate decrease in pregnancy rates. The authors state that coasting is a feasible option in patients
undergoing IVF cycles with GnRH antagonists.
Dr Gardner et al. have contributed an excellent article that discusses state-of-the-art technologies such as metabolomics and
their application in the ART field. The authors have stated that metabolomics technology is perceived as an important
diagnostic tool in clinical IVF that has the potential to assess embryo viability prior to transfer or cryopreservation. The authors
conclude that as an embryo selection technique, metabolomics screening will form an integral part of ART laboratories. This
will lead to an enhanced ability to determine the functional metabolic phenotype of an embryo as a key indicator of viability.
They also highlight that the affordability of the new techniques is likely to improve as they become more readily available and
tailored to clinical IVF and as they integrate with other cost effective and efficient technologies such as chip-based analysis.
This special issue also contains 4 articles on fertility preservation by researchers from Cleveland Clinic, the University of
Toledo and University Hospital-Case Western Reserve University. The article on the economics of female fertility preservation
discusses the increasing demand of fertility preservation and obstacles that hinder the creation of a standard of care. The authors
analyze the challenges to establishing higher quality care standards and provide suggestions for continued research and
multidisciplinary collaboration for a larger patient population.
Dr Nasr and Dr Bedaiwy have provided an overview of new technologies for fertility preservation in an article titled “Emerging
Technologies for Fertility Preservation in Female Patients.” The authors explain that most of the currently available strategies
to preserve fertility in women are still experimental and do not guarantee subsequent fertility. The only established method in
women is IVF with embryo cryopreservation prior to cancer therapy. Other proposed strategies to preserve fertility in women
with cancer include storage of frozen embryos, frozen ovarian tissue or isolated follicles for in vitro growth and maturation.
They emphasize that future research should focus on critically defining patient suitability, methods of tissue collection, optimal
tissue size, choice of cryoprotectants and cryopreservation protocols, and the possible use of in vitro maturation (IVM) of
oocytes for human ovarian tissue.
Dr Salle et al. from the Université Claude Bernard in Lyon France, write about the emerging technology of whole ovarian
vitrification. They state that it is an experimental procedure and the advantages expected from this procedure have yet to be
confirmed.
Dr Banerjee at al has delineated the importance of ovarian reserve testing in their article titled “Prognostic role of ovarian
reserve testing.” The authors state that in clinical practice, each patient must be evaluated individually to utilize the best
prognostic indicator of their reproductive outcome. They also express their view that with the development of predictive
markers of ovarian reserve testing, ART will also require further improvement to improve their outcome.
ii Current Women’s Health Reviews, 2010, Vol. 6, No. 3
Preface
We hope that the readers will enjoy reading the latest, informative and authoritative articles by some of the most recognized
and prolific leaders in reproductive endocrinology from across the globe. We would like to extend our appreciation to all the
authors for their hard work and valuable contributions. We are indebted to our colleagues and associates at Cleveland Clinic for
their valuable contributions. We gratefully acknowledge the generous support of Ms. Amy Slugg Moore (Manager, Medical
Writing Education Program) for her help. We are also grateful to Prof. Jose Belizan, Editor in Chief of Current Women’s
Health Review, for his constant encouragement and support. We are most thankful to the editorial team of CWHR for their
support and hard work. Finally, we extend our sincere thanks for the opportunity to serve as a Guest Editor on the special issue
of CWHR. We are confident that readers will benefit from the latest knowledge in these valuable articles from the experts in
their respective fields.
Sajal Gupta, MD, TS (ABB)
Ashok Agarwal, PhD, HCLD
(Co-Guest Editor)
Assistant Coordinator & Project Staff
Center for Reproductive Medicine
Glickman Urological and Kidney Institute &
OB/ GYN and Women’s Health Institute
Cleveland Clinic
Cleveland, OH 44195
USA
Tel: 216-444-9485
Fax: 216-445-6049
E-mail: [email protected]
(Guest Editor)
Professor, Lerner College of Medicine
and Case Western Reserve University
Director, Andrology Laboratory and
Reproductive Tissue Bank
Director, Center for Reproductive
Medicine Staff, Glickman Urological
& Kidney Institute and Ob-Gyn
and Women's Health Institute
Cleveland Clinic
Cleveland, OH 44195
USA
Tel: 216-444-9485
Fax: 216-445-6049
Current Women’s Health Reviews, 2010, 6, 197-199
BIOGRAPHY OF CONTRIBUTORS
Amr Kader, MD
Amr Kader, MD graduated from Alexandria University, Egypt in 1998 and joined the Cleveland
Clinic’s Center of Reproductive Medicine and the Minimally Invasive Surgery as a Research
Fellow in Gynecology from 2006 to 2009. During this period his research was focused on gametes and tissue vitrification and fertility preservation. He has many publications on vitrification
and he holds a patent for the invention of the Ohio-Cryo, a closed vitrification device for tissue
fragments. He earned many grants and awards such as the NIH trainee travel award (2007) and
the ASRM best video presentation award (2009). He is currently an Ob-Gyn resident at the
West Virginia University.
Ali Ahmady, PhD
Ali Ahmady is Assistant Professor of Reproductive Biology at Case Western Reserve Medical
School, and Director of IVF & Andrology at University Hospital Case Medical Center,
Cleveland, OH. He is a member of National Center for Regenerative Medicine. Dr. Ahmady
produced the first live birth in the mouse using ICSI and investigated the fertilizing ability
of dead sperm at the cell and molecular level. Dr. Ahmady is an author of a manual for IVF, coauthor of a chapter in the atlas of embryology, and has published more than 20 peer-reviewed
manuscripts.
Jashoman Banerjee, MD
Jashoman Banerjee, MD is a trained Ob-Gyn specialist from India. He is currently graduating
as a chief resident in Ob-Gyn from the University of Toledo Medical Center in Toledo, Ohio.
Dr. Banerjee will start his fellowship in reproductive endocrinology and infertility at Wayne
state University. He has actively participated in extensive research involving endometriosis and
infertility at the Cleveland Clinic. His other research interests are to explore effects of oxidative
stress on oocyte quality and ovarian cryopreservation as means of fertility preservation. He has
published his research work in peer reviewed journals.
Mohamed Bedaiwy, MD
Dr. Bedaiwy received his medical degree from Assiut University School of Medicine, Egypt,
where he graduated Valedictorian, summa cum laude. He is still on staff as an associate
professor. He did a REI/MIS research fellowship at the Cleveland Clinic, during which he
prepared a Doctorate thesis involving his research work in ovarian tissue cryopreservation and
transplantation. He also completed a clinical fellowship in laparoscopic surgery in the same
institution. Dr Bedaiwy has 67 peer-reviewed publications, 8 book chapters and 9 videos. He
has received 15 national and international awards.
Kathryn Coyne, BS
Kathryn Coyne is a senior at the University of Notre Dame and is majoring in pre-professional
studies. She is the President of the Irish Kids Fighting for St. Jude Kids club and volunteers at a
clinic that serves uninsured residents of St. Joseph County. Kathryn interned at the Cleveland
Clinic Center for Reproductive Medicine and past internships include working at the Spanish
Red Cross in Toledo, Spain and the Ireland Cancer of University Hospitals of Cleveland. She
has travelled on multiple medical brigades in Honduras and hails from Shaker Heights, Ohio.
She plans on attending medical school in the future.
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© 2010 Bentham Science Publishers Ltd.
197
198 Current Women’s Health Reviews, 2010, Vol. 6, No. 3
James Goldfarb, MD, MBA
Dr. Goldfarb is Director of Infertility Services and In Vitro Fertilization (IVF) for the Cleveland
Clinic. He is also the current President of the Society for Assisted Reproductive Technologies.
Dr. Goldfarb has been a pioneer in IVF. His program was responsible for the first IVF birth
in Ohio (1983) and the world’s first IVF surrogate birth (1986). Dr. Goldfarb is also the co
founder of the Partnership for Families Program, a charitable organization that provides funding
for second IVF cycles for qualifying couples and for fertility sparing procedures for qualifying
cancer patients.
Sajal Gupta, MD
Dr. Gupta holds the position of Project Staff in the Glickman Urological Institute and serves
as the Assistant Coordinator of Research at the Center for Reproductive Medicine. She has
published over 40 review articles and scientific papers in peer-reviewed scientific journals. She
is a member of several professional societies, including: American Society of Reproductive
Medicine, American Society of Andrology, and the Society for the Study of Male Reproduction. Dr. Gupta is an ad hoc reviewer for Human Reproduction, Fertility & Sterility, and
European Journal of Obstetrics and Gynecology. Dr. Gupta is a co-investigator or principal
investigator on 8 research grants. Her current research interests include the role of oxidative
stress in female infertility, endometriosis, assisted reproductive techniques and gamete
cryobiology.
Kim D. Ly, BS, MBA
Kim D. Ly obtained her Bachelor of Science in Biology with a minor in Chemistry from Baylor
University, Waco, TX, USA in 1998. She later obtained her Masters in Business Administration from the University of North Texas, Denton, TX, USA in 2000. She joins the Center
for Reproductive Medicine Cleveland Clinic, Cleveland, OH, USA as a researcher with an
interest in embryology, implantation failure, preimplantation genetic screening for aneuploidy,
and blastocyst transfer.
Mohamed Aboulghar, MD
Dr. Mohammed Aboulghar is Professor of Obstetrics and Gynecology at Cairo University.
He is the clinical director and founder of the Egyptian IVF-ET center at Maadi in Cairo, Egypt
and the founder and first president of the Middle East Fertility Society (MEFS). He is also the
editor in chief and the founder of the Middle East Fertility Society Journal (MEFS) since 1996.
He has over 100 published papers in top international medical journals and over 100 papers
in local and regional journals. He was awarded the Egyptian National Award for Excellency
in Medical Sciences in 2000 and Honorary Membership of the European Society of Human
Reproduction and Embryology at Berlin in 2004.
Bruno Salle, MD, PhD
Dr. Salle is a Professor of Reproductive Medicine at the Claude Bernard University, Lyon,
France and the Head of the Fertility Clinic at the Hospices Civils de Lyon, University Hospital.
He is in charge of a research program on fertility preservation at the INSERM. He has written
many papers on ovary transplantation and ovarian cryopreservation or vitrification. He is
a member of the European Society of Human Reproduction and Embryology, member of
the task force of fertility preservation and a member of the International Society of Fertility
Preservation.
Biography
Biography
George Thouas, PhD
George Thouas is a post-doctoral research fellow in reproductive biology, with specialization in
oocyte and preimplantation embryo mitochondrial biology, and development of new ARTs. He
has worked previously as a clinical embryologist, involved in specific research programs
at Monash IVF, and embryo-toxicity testing for quality control. More recently, Dr Thouas
has worked in biomedical engineering and biotechnology research, toward optimization of
in vitro technologies for the life sciences. His latest appointment with Professor David Gardner
at the University of Melbourne sees a return to his original specialization, to furthering the
understanding of embryo metabolism and its application to embryo diagnostics.
Ashok Agarwal, PhD, HCLD
Ashok Agarwal is a Professor in the Lerner College of Medicine at Case Western Reserve University and the Director of Center for Reproductive Medicine, and the Clinical Andrology Laboratory
at The Cleveland Clinic, Cleveland Ohio, United States. He has published over 500 scientific
articles and reviews and is currently editing 10 text books in different areas of andrology/
embryology, male and female infertility and fertility preservation. His research program is known
internationally for its focus on disease oriented cutting edge research in the field of human reproduction. His team has presented over 700 papers at national and international meetings. More than
200 scientists, clinicians and biologists have received their training in Ashok’s Lab. His long term
research interests include unraveling the role of oxidants-antioxidants, genomic integrity, and
apoptosis in the pathophysiology of male and female reproduction.
199
200
Evidence-Based Management of Infertile
Implantation Failure Following IVF
Couples
with
Repeated
Kim D. Ly1, Nabil Aziz2, Joelle Safi1 and Ashok Agarwal1,*
1
Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH 44195, USA; 2Liverpool Women’s Hospital and
The University of Liverpool, Liverpool, UK
Abstract: Embryo implantation depends on both embryo quality and the endometrial environment. Implantation failure
has a complex, variable pathophysiology and is detrimental to the outcome of in vitro fertilization (IVF). Thus, patients
with multiple implantation failure require an individualized approach to diagnosing and managing treatment options for
future IVF cycles. These options should be based on concrete, unambiguous, consistent scientific evidence with randomized, controlled trials.
We review and discuss 14 treatment options: (i) blastocyst transfer, (ii) assisted hatching, (iii) co-culture, (iv) preimplantation genetic screening, (v) hysteroscopy, (vi) sildenafil, (vii) salpingectomy for tubal disease, (viii) oocyte donation, (ix)
transfer of six or more embryos, (x), intratubal embryo transfer, (xi) natural cycle IVF, (xii) antiphospholipid antibodies
(APA) testing and treatment, (xiii) allogenic lymphocyte therapy, and (xiv) IV immunoglobin therapy. The approaches
were evaluated based on available information from studies, expert opinions, consensus, etc.
We conclude that blastocyst transfer, assisted hatching, salpingectomy for tubal disease, and hysteroscopy in IVF
procedures are clinically effective. This review serves as a summary of current treatment options for clinicians to counsel
patients and manage their expectations based on strong and reliable evidence.
Keywords: Repeat implantation failure, in vitro fertilization, endometrial receptivity, blastocyst transfer, assisted hatching,
hysteroscopy, salpingectomy.
INTRODUCTION
The world's first baby conceived by in vitro fertilization
(IVF) was Louise Joy Brown in 1978. Today, more than 3
million children worldwide have been born as a result of
IVF. The numbers continue to increase as science discovers
ways to overcome barriers for various subgroups of infertile
patients. However, despite the improvements in IVF technology and methods, many couples experience multiple IVF
failures. After each failed IVF attempt, pregnancy rates in
subsequent attempts decrease by as much as 57% with the
most remarkable decrease after the third attempt [1, 2]. The
main causes of multiple IVF failures include: (i) poor
response to ovarian stimulation, (ii) repeated fertilization
failure, (iii) repeated difficult transfers, and (iv) repeated
implantation failures (RIF).
Implantation failure is a major limiting step for IVF [3].
The process of embryo implantation is described as having
three phases: 1. Apposition: “unstable adhesion” of the transferred embryo to the surface of the uterine lining. 2. Attachment (adhesion): “stable adhesion,” believed to involve signaling back and forth between the embryo and the lining. 3.
Penetration (invasion): invasion of the trophectoderm cells
from the embryo through the surface of the lining deeper into
*Address correspondence to this author at the Center for Reproductive
Medicine Cleveland Clinic 9500 Euclid Avenue, Desk A19.1 Cleveland,
OH 44195 USA; Tel: 216-444-9485; Fax: 216-445-6049;
1573-4048/10 $55.00+.00
the stroma of the uterine lining, forming a vascular connection to the mother.
The etiological causes of implantation failure include
embryo quality; endometrial receptivity; immunological factors; uterine, tubal and peritoneal factors; and culture media
[3]. Poor response to superovulation and chromosomal aneuploidy due to advanced maternal age negatively affect embryo quality; suboptimal embryos are less likely to implant.
The disruption in prostaglandin synthesis is one of many
factors that decrease endometrial receptivity in some patients
prone to RIF [4]. Other cellular and adhesion pathways affected by abnormal gene expression in the endometrium
have been observed to be linked to RIF [5]. The cultured
endometrial cells of RIF patients were found to have different gene expressions than those found in the cultured endometrial cells of women who miscarried or had an ongoing
pregnancy [6]. From this observation, the differential gene
expression of RIF patients is assumed to negatively affect
critical signaling pathways important for the development of
adhesion molecules in the embryo-endometrium bond and
may be linked to implantation failures. Immunological factors such as antiphospholipid antibodies (APA), abnormal
expression of endometrial natural killer cells, cytokines [3],
local and systemic immune factors, anti-sperm antibodies,
and anti-thyroid antibodies [7] have been found in significant
amounts among RIF patients and have been reported to affect implantation. APA interferes with the normal function
of blood vessels by either causing narrowing/irregularity of
the blood vessels (vasculopathy) or by causing the develop© 2010 Bentham Science Publishers Ltd.
Evidence-Based Management of Infertile Couples
ment of blood clots in the blood vessels (thrombosis). In the
majority of cases, failed implantation appears to be related to
the quality of embryos transferred rather than to the endometrial receptivity. Part of the evidence stems from the significantly higher implantation rates found in egg donation
programs, even in couples that have failed IVF repeatedly
using their own eggs.
In this paper, we focus on the options supported by clinical evidence to improve implantation and pregnancy rates for
couples with multiple IVF failures. Evidence-based medicine
has three levels of recommendation. Level A recommendations are based on good and consistent scientific evidence
with randomized, controlled trials. At level B, recommendations are based on limited or inconsistent scientific evidence
with clinical controlled trials, cohort, etc. Level C includes
recommendations based primarily on consensus and expert
opinion. Clinicians would benefit from knowing to which
category a treatment option belongs to be able to counsel
patients and manage their expectations for future IVF cycles.
We will examine 14 approaches to repeated implantation
failure in IVF: (i) blastocyst transfer, (ii) assisted hatching,
(iii) co-culture, (iv) preimplantation genetic screening, (v)
hysteroscopy, (vi) sildenafil, (vii) salpingectomy for tubal
disease, (viii) oocyte donation, (ix) transfer of six or more
embryos, (x), intratubal embryo transfer, (xi) natural cycle
IVF, (xii) APA testing and treatment, (xiii) allogenic lymphocyte therapy, and (xiv) IV immunoglobin therapy. (See
also Table 1).
Table 1.
Fourteen Possible Approaches to the Management of
Infertile Couples with Repeat Implantation Failure
Blastocyst transfer
Assisted hatching (AH)
Co-culture
Preimplantation genetic screening (PGS)
Hysteroscopy
Sildenafil
Salpingectomy for tubal disease
Oocyte donation
Transfer of six or more embryos
Intra-tubal embryo transfer
Natural cycle IVF
Antiphospholipid antibodies (APA) testing and treatment
Allogenic lymphocyte therapy
I.V. immunoglobin therapy
201
and these advances have enabled researchers to culture zygotes to the blastocyst stage prior to implantation in the
uterus. The original intent of blastocyst transfer was to select
the healthiest embryos for transfer. Thus, a single or the fewest possible embryos may be transferred per cycle to reduce
high order pregnancies, which are associated with very real,
serious risks to mother and baby. Several advantages of culture and transfer of blastocysts make this option appealing
for RIF patients. Self-selection of blastocysts increases endometrial receptivity. The trophectoderm cells of the blastocyst cross-talk with the endometrium and develop the ability
to attach to the lining of the endometrial lining of the uterus,
both of which are likely to improve implantation success.
However, blastocyst culture and transfer usually is recommended for patients with a good prognosis--those who are
younger in age with > 6 oocytes available from an uneventful ovarian stimulation. Sequential media, a two-part culture
medium, commences with the embryo initially grown in media rich with pyruvate to Day 3. The embryo is then transferred to the second media, rich with glucose, on Day 3 to
support embryo growth from the eight-cell stage to the blastocyst stage.
A study by Weissman et al. observed a significant improvement in pregnancy rates from blastocyst culture and
transfer to patients who were young, did not have multiple
IVF failures, who produced multiple oocytes, and whose
zygotes developed into good quality cleavage-stage embryos
[1]. However, for patients in the general population with a
single failed IVF attempt, pregnancy and implantation rates
were observed to significantly decrease with subsequent IVF
attempts employing blastocyst culture and transfer strategy
[12]. In patients with multiple implantation failures, Cruz
et al. reported a positive correlation between blastocysts and
improved implantation and pregnancy rates for RIF patients
in a non-randomized population [13]. However, a prospective, randomized control study by Levitas et al. that specifically examined the effects of blastocyst transfer for RIF patients compared with Day 2 embryo transfer found that blastocyst transfer was beneficial only in patients with an acceptable response to ovarian stimulation [14]. However,
there was no difference in the incidence of multiple pregnancies between the groups. Blastocyst transfer on Day 5 for
RIF patients permits the selectivity of a higher quality embryo after embryonic genomic activation has occurred [15].
It may also decrease the rate of ectopic pregnancy because of
the larger diameter size of the Day 5 embryo; because the
blastocyst normally resides in the uterus, an improvement to
receptivity is also expected.
ASSISTED HATCHING (AH)
Embryo transfer occurs either during the cleavage stage
on Day 2/Day 3 or during the blastocyst stage (the embryo
has inner cell mass, trophectoderm layer, and blastocoele) on
Day 5/Day 6. Several studies have demonstrated higher rates
of implantation and pregnancy with blastocyst culture and
transfer compared with Day 3 cleavage stages [8-11].
Differences in embryo development during IVF treatment compared with in vivo are well-documented. One observed difference is the hardening of the zona pellucida (ZP),
which prevents the blastocyst from escaping the ZP during
hatching. The culture medium, which differs in its available
nutritive source from the in vivo environment, and the cryopreservation method have been implicated as possible causes
of implantation failure [16].
Recent advances in culture technology have been made
due to a better understanding of early embryo metabolism,
Assisted hatching was developed as a workaround to the
hardened ZP of in vitro embryos. It involves thinning or cre-
BLASTOCYST TRANSFER
ating a hole in the ZP, either of which can be performed by a
mechanical (glass pipette), chemical (acidic Tyrodes’s or
enzymes), or laser method (contact and non-contact) with
equal efficacy. The industry standard today includes the use
of laser-assisted hatching for rapid and precise drilling. AH
is not a benign procedure since the time between the zona
drilling and embryo transfer is a vulnerable period for the
blastocyst as it is exposed to foreign elements, undefined
antibodies, or surveillance cells in the endometrial environment. However, blastocysts are believed to be more prepared
for exposure to uterine lymphocytes and immune cells at this
stage [17]. In addition, an increased incidence of monozygotic twinning may occur during the ablation procedure,
which in some reports has caused herniation and division of
the inner cell mass into two [18]. This may result from (i) a
too-narrow opening of the ZP, trapping the blastomere in a
figure-eight formation, thus causing a subdivision of the
blastocyst to form twins or (ii) a premature hatching of the
blastocyst from a too-large opening combined with loosening of the tight junctions between blastomeres to cause division of the inner cell mass to form twins [18].
A Cochrane review of AH that included 28 randomized
control trials from 24 publications or abstracts shows an increase in clinical pregnancy rates for women with RIF but
suggests additional research is needed [19]. There is no evidence supporting AH after one failed IVF attempt, but
women with multiple implantation failures benefited most
from AH [19]. The review’s validity, coupled with multiple
good quality, randomized, controlled studies and strong recommendations, suggest that AH does improve implantation
rates for women with multiple implantation failures (3+) due
to hardening of the ZP, occurring mainly from cultured IVF
treatment or methods of embryo cryopreservation.
CO-CULTURE
Co-culture refers to the placement of human and nonhuman live cell types (feeder cells) alongside the embryo
during in vitro culture. It has been suggested that co-culture
improves embryo growth and development by improving in
vitro parameters--it removes toxic substances such as heavy
metals and ammonium and free radicals [20].
Endometrial (Simon, Mercader et al. 1999, Weichselbaum,
Paltieli et al. 2002) and granulose [21-23] cells are commonly used in co-culture. Studies have demonstrated conclusively the existence of cross-talk between endometrial cells
and embryonic cells, resulting in a paracrine release of molecules believed to improve implantation [20]. The presence
of uterine epithelial cells, the first cells in contact between
maternal and fetal cells, may initiate a signaling cascade
for cells from the blastocyst to become depolarized in preparation for endometrial attachment, thus improving embryo
competence for implantation [24].
The heterogeneous co-culture cell lines that have been
utilized include human and bovine oviductal cells [25-30],
bovine uterine epithelial cells [31, 32], African Green Monkey kidney cells (Vero cells) [28, 33-35], ovarian cancer
cells [36], buffalo rat liver cells [37], and human skin fibroblasts [38]. One randomized study using conventional media
compared the various types of co-culture cell lines and found
that granulosa cells and bovine oviductal uterine epithelial
Ly et al.
cells were associated with a higher percentage of early
embryos developing to the eight-cell stage.
In some countries, including the United States, nonhuman cell types have been banned by the Food and Drug
Administration due to concerns regarding disease transmission from the cell types to the developing embryo or mother.
Consequently, the use of autologous endometrial cells has
gained in popularity [39]. Several different human cell types
are now available, including cumulus cells, luteinized granulosa cells, fallopian ampullary epithelial cells (FAEC), and
endometrial epithelial cells [40]. No randomized controlled
studies are available to compare and determine which of the
human cell types provides maximum benefit.
In a study by Weichselbaum et al., very poor quality embryos, in which more than 50% of embryo volume had fragmentation and the blastomeres were of unequal size with
grainy to dark cytoplasm, were rescued from cleavage arrest
and degeneration when co-cultured with fallopian ampullary
epithelial cells. In addition, blastocyst formation increased
by 56%, suggesting that co-culture may help embryos overcome developmental incompetence [40]. Eyheremendy et al.
used the best embryos from women with multiple implantation failure to co-culture with monolayers of autologous endometrial cells [41]. Of the 68 patients who failed to become
pregnant after multiple IVF treatments, 39 become pregnant,
19 attained a live birth, and 10 remained pregnant beyond 12
weeks. The study also suggested that an endometrial biopsy
should be performed about 7 days after ovulation and that
the co-culture medium contain both stromal and glandular
cells in the monolayer to improve implantation rates [41]. In
2008, Desai et al. reported for the first time the success of a
human endometrial culture system in a clinical environment,
thus supporting the two previously mentioned studies [42].
Interestingly, although the benefits of co-culture media
have been demonstrated over the years, its prevalence is not
widespread, perhaps because it remains a labor-intensive and
unproven technique [41]. Several limitations must be overcome to realize its benefit. The most serious limitation is that
supplementation of human or non-human live cells with the
culture media results in an uncontrolled and undefined alteration to the conditions of the media with unknown growth
factors in unknown concentrations [43]. The introduction of
sequential media in the past decade has slowly replaced not
only non-human but also human cell types because of its
safety and ease of use [44]. Additional research is needed in
the area of co-culture, with special attention to the type of
cells and type of culture media (conventional or sequential
media), to support its use prior to its widespread adoption in
clinical settings.
PREIMPLANTATION GENETIC SCREENING
Chromosomal abnormalities have been widely reported
to be a major cause of early spontaneous abortions in as
much as 60% of the general population [45]. In couples with
multiple implantation failures undergoing fertility treatment,
the frequency of chromosomal abnormalities appears to be
higher regardless of maternal age [46-49]. Specifically, embryonic aneuploidy in patients with multiple implantation
failures was 54-57% compared with 35% in a control group
[50, 51]. Thus, preimplantation genetic screening (PGS)
could be utilized as an appropriate means for selecting
normal embryos to improve implantation and pregnancy
rates and, ultimately, live birth rates in couples with RIF.
However, recent studies present conflicting data supporting
the use of PGS for couples with RIF.
The European Society of Human Reproduction and
Embryology (ESHRE) PGD Consortium Steering Committee reports the use of PGS as having little impact on improving the pregnancy rate for women with RIF [52]. In addition,
Gianaroli et al. showed that PGS did not increase implantation or pregnancy rates per embryo transfer in RIF patients
[53]. Other data from ESHRE’s review, ESHRE PGD
Consortium IX, demonstrated that 57 IVF centers performed
748 IVF cycles with oocyte retrieval for RIF, which resulted
in a 27% clinical pregnancy rate, 24% implantation rate, and
an 11% delivery rate between January and December 2006
[54].
For comparison, the 27% clinical pregnancy rate for PGS
in RIF patients is better than the 18% clinical pregnancy rate
in the total patient population undergoing PGS for various
reasons. This latest data collection from 2006 highlighted
RIF as a main indication for PGS in fertility centers despite
the ongoing debate regarding its efficacy [54]. In a retrospective study of 121 first PGS for RIF, multivariate logistic regression analysis was utilized to generate a predictive model
[55]. The model demonstrated that to have a 90% probability
of having an embryo transfer after PGS, the patient should
have at least 10 mature oocytes, eight normally fertilized
oocytes, and six embryos for biopsy on Day 3.
The cause of aneuploidy among RIF patients differs from
other cases of random failed implantation. One main characteristic of chromosomal abnormality found in preimplantation embryos of couples with multiple implantation failures
is the low probability of meiotic errors resulting in chromosomal abnormalities [56, 57]. Two studies by Mantzouratou
et al. and Voullaire et al. have shown that meiotic errors are
an unlikely cause in this group of patients and that chromosomal abnormalities are reflective of an inefficiency in mitotic division due to abnormal cell cycle regulation by the
embryo. This may explain the lower rate of success in the
management of fertility for couples with RIF in studies using
polar body analysis.
In 2009, Fragouli et al. released a study comparing results of PGS between polar body I, polar body II and blastocyst stages in which the aneuploidy rates were 36.5%,
45.8%, and 45.2% after meiosis I, meiosis II, and mitosis,
respectively [48]. Though the numbers appear to indicate
that aneuploidy rates are higher after meiosis II, errors from
meiosis I carried into meiosis II should be considered. The
higher aneuploidy rate at the blastocyst stage may be explained by the fact that the embryo is more vulnerable to
developmental arrest between the four-cell and eight-cell
stage as it switches from maternal to embryo gene expression [58]. Maximum embryonic gene expression has been
shown not to occur until the blastocyst stage. Therefore, the
disturbed immature cell cycle regulation increases the likelihood of chromosomal abnormalities, which persists to the
blastocyst stage, thus reducing the likelihood of successful
implantation. These observations suggest that future studies
203
should focus on understanding the embryonic role in RIF by
sampling the blastocyst stage where maximum embryonic
gene expression occurs [59].
The occurrence and pathology underlying complex
chromosomal abnormality (three or more whole chromosome imbalance) was another characteristic abnormality
found in patients with RIF in a recent study using array
comparative genomic hybridization (CGH) analysis of
chromosomal material in Day 3 cleavage-stage embryos.
Chromosomal breakage and failure of mitotic cell cycle
checkpoints to detect abnormalities has been suggested as
the cause of mosaic complex chromosomal abnormalities in
nearly 30% of RIF patients [57, 60]. This study was further
supported by the use of CGH analysis of blastocysts [48].
Abnormal replication and segregation of chromosomes during early embryo development of RIF patients is likely
caused by maternal cytoplasmic factors or mutation in the
cell cycle control genes [61]. Thus, the use of PGS in identifying abnormal embryos in advance to improve implantation
is restricted.
Fluorescent in situ hybridization (FISH) is the most
commonly used method today to detect chromosomal aneuploidy. However, FISH techniques can only analyze a small
subset of the chromosomes, usually the most commonly involved in aneuploidy. Embryonic mosaicism (multiple germ
cell lines in one embryo) further complicates analysis of test
results in terms of reliability since only one blastomere, representing the entire embryo, is removed for testing. According to one study, 38% of embryos were incorrectly graded as
normal using a five-probe set on blastomeres [57]. In another
study using a nine-probe set, 25% of embryos were misdiagnosed [62]. In the most recent study, which used a 12-probe
set, 19% of embryos were incorrectly graded as normal [48].
The acceptable error rates from the studies suggest FISH is
able to reliably detect aneuploidy in mosaic embryos and
further implies that mosaic embryos have a sufficiently high
ratio of abnormal-to-normal blastomeres for cleavage-stage
biopsy to serve as clinically useful. Despite several recent
advances in diagnostic methods, including whole genome
amplification with comparative genomic hybridization and
the use of microarrays to overcome the limitations in FISH,
identifying complex chromosomal abnormalities has limited
success, is labor-intensive, and costly [65].
Currently, convincing evidence for the wide use of PGS
in RIF is insufficient. The technique did not improve implantation rates for RIF patients [53, 63, 64]. Moreover, some
normal embryos might be lost due to the error rate. Furthermore, with the advent of less invasive methods for predicting
better quality embryos such as metabolomics and proteomics, PGS may become a less popular option. It is not
likely to happen anytime soon because no studies to date
have identified the significant metabolites or proteins involved in early development that are unique to embryos with
a low implantation success rate. The role of PGS in the management of RIF patients remains unresolved.
HYSTEROSCOPY
Hysteroscopy is an invasive diagnostic procedure that is
used to visualize uterine pathologies, including submucous
fibroids, polyps, intrauterine adhesions, and uterine malfor-
Ly et al.
mations (i.e. septate uterus) that may be associated with infertility. Studies have shown the procedure to be more sensitive, specific and accurate than pelvic ultrasound in evaluating uterine pathology for patients with RIF [65]. Currently,
hysteroscopy has not been adopted as part of the routine
workup for infertility but rather as a secondary investigative
measure to evaluate patients after three or more implantation
failures [66, 67].
plasminogen activator inhibitor 1(PAI-1), and (iii) vascular
endothelial growth factor (VEGF) – 1154. The study found a
link between one or more of the genes and patients with repeat implantation failure. A significantly higher correlation
existed for RIF patients than the control group. More studies
are needed to confirm the findings, which could facilitate the
clinician's ability to identify and counsel patients prone to
implantation failure [72].
Women with RIF have been shown to have a higher incidence of uterine pathologies. In the most recent observational study of 1475 women with RIF, an abnormality was
found in the uterus of 36.6% of these women (16.7% endometrial polyps, 12.5% endometrial adhesions, 1.5% endocervical adhesions, 4.3% endometritis, 0.9% uterine septa, and
0.8% submucous fibroids) [68]. The study reported that
22.2% of the population had a prior ultrasound screening
with a false negative result and subsequent hysteroscopy
intrauterine pathology (endometrial adhesions in 12.5%, endometritis in 4.3%, endometrial polyps in 3.3%, endocervical adhesions in 1.5%, uterine septa in 0.5%, and submucuos
myomas in 0.1%). The same study also compared the use
and non-use of hysteroscopy for women with RIF in a new
IVF attempt and showed a significant increase in the
implantation rate and pregnancy rate in the former group
[68]. It strongly suggests the use of hysteroscopy for women
after two failed IVF attempts for two reasons: (i) a
significant number of uterine pathologies are undetected by
ultrasound and (ii) the significant success rate of ongoing
pregnancy after hysteroscopy [68]. For women with
endometrial polyps, a hysteroscopic polypectomy (surgical
removal of polyps) may be more efficacious than nonintervening hysteroscopy [69].
Several studies have focused on increasing endometrium
thickness with sildenafil therapy for women with RIF. In
2000, Sher and Fisch applied sildenafil vaginally in women
with RIF and a thin endometrium to increase blood flow for
endometrial growth. They hypothesized that a thicker endometrium (> 8 mm) may improve implantation and pregnancy
rates [73]. The preliminary study showed that three of the
four women had a successful pregnancy outcome after sildenafil treatment during the proliferative phase. Two years
later, Sher and Fisch released a follow-up study on 105 patients who were given sildenafil; 70% developed endometrial
thickness 9 mm with the remaining patients (30%) being 9 mm. Of the patients who developed endometrial thickness
9 mm, 45% had a significant implantation rate [74]. The
study does not explain why the majority (55%) of women
treated with sildenafil experienced optimal endometrial
growth but subsequent IVF failure. However, it highlighted
the importance of endometrium quality [75].
A novel approach to the use hysteroscopy for evaluating
endometrial cell integrity has been described [70]. The technique involves the application of chemical agents to highlight cellular-level or mucosal anomalies of the endometrium
(chromohysteroscopy). Preliminary studies on a small patient population have shown promising results for women
with RIF [70].
SILDENAFIL (VIAGRA)
Sildenafil (Viagra®, Pfizer, New York, N.Y.), most notable for the treatment of male erectile dysfunction, is a vasodilator used to improve vascular supply [71]. Endometrial
receptivity is believed to improve with increased blood flow
in the uterine arteries of women with thin endometrium to
increase the thickness of the endometrium, the portion of the
uterus associated with successful implantation. Both the
quality and quantity of endometrial tissue are important to
consider in a strategy aimed at improving the endometrial
environment for a successful pregnancy.
A recent case control study by Goodman et al. investigated three gene polymorphisms that have been linked to
patients with repeat implantation failures in prior individual
studies [72]. Successful implantation depends on the blastocyst’s ability to infiltrate the endometrium and develop a
sustaining blood supply, which requires the following genes
to produce the necessary proteins for digesting the endometrial cellular matrix, regulate cell growth, and induce angiogenesis: (i) p53 codon 7 tumor suppressor factor, (ii)
In a case report on two patients with Asherman’s
syndrome, sildenafil improved endometrial thickness but did
so by a much smaller margin. The endometrium of one
woman increased from 6.5 mm to 8.9 mm, and another
woman’s increased from 5.0 mm to 6.6 mm. Despite suboptimal endometrial measurements of 9 mm, both women
had healthy offspring [76]. In a study by Paulus et al., the
data did not support Sher and Fisch’s findings or those
of Zinger et al. In 10 women with at least one IVF failure,
sildenafil increased endometrial thickness, but only three
of 10 patients had a successful pregnancy. However, the
heterogeneous and small patient population may have
affected the results [77].
Natural killer cell activity levels, which have been reported as a predictor for recurrent miscarriages, may also be
involved in RIF [78-80]. An increase in both peripheral
blood natural killer cells and endometrial natural killer cells
appears to be associated with lower pregnancy rates in patients with recurrent miscarriages. Upon activation with nitric oxide, the natural killer cells release cytokines such as
tumor necrosis factor-, which has been implicated as a
cause of implantation failure [81]. One study extended the
hypothesis to suggest that repeated implantation failures may
be caused by high levels of peripheral natural killer cell activities. It showed that sildenafil lowered peripheral blood
natural killer cell activity, thereby improving the local endometrial immunological environment in women with multiple IVF failure. However, more randomized control studies
must be done to confirm these findings. It also will be important to determine the appropriate period to prescribe sildenafil therapy [75].
In a pilot study, the amount of NO released by the embryo in vitro correlated with implantation success. It was
reported that higher quality embryos producing an elevated
amount of NO in culture media had a higher success rate
than the controls [82]. Thus, the use of sildenafil to block the
breakdown of cyclic guanosine monophosphate (cGMP)
causes an accumulation of NO. As a result of increased NO,
radial uterine blood vessels dilate to increase the vasculature
and perpetuate endometrial growth. However, as NO induces
natural killer cells to produce cytokines that can cause implantation failure, it has been recommended that sildenafil
not be used five or more days prior to embryo transfer [74].
On the other hand, one study using mouse embryos reported
that higher concentrations of NO in vitro and in vivo resulted
in lower implantation rates in a dose-dependent manner [81].
The same higher concentration of NO (1 mM) inhibited implantation in vitro as it did for mouse embryos. Cytostatic
and cytotoxic effects resulting from an extended production
period of NO in reproductive tissues to protect against infection, immunological reactions, and pathological conditions
(e.g. endometriosis, reproductive tract infections) is a suggested cause for the lower implantation rates found in the
mouse embryo study [81].
The effects of sildenafil therapy on the endometrium and
nearby environments should be better understood before it is
widely adopted in IVF clinics for RIF patients. Natural killer
cells and NO play important roles in the female reproductive
tract. Alterations in NO-synthase (an enzyme that converts
the nitrogen in L-arginine to NO in the presence of NADPH
and dioxygen) and/or NO production of these tissues could
directly affect the development of human embryos, especially during the early stages of pregnancy.
SALPINGECTOMY FOR TUBAL DISEASE
Of all the different tubal pathologies, hydrosalpinx (distension of the fingered portion of the fallopian tube due to an
abnormal accumulation of fluid or water most likely resulting from an inflammatory response) has the most detrimental
effect on implantation [83]. Generally, hydrosalpinx is
caused by abortion, pelvic inflammatory disease, endometriosis, previous operations, a history of tuberculosis and peritonitis, or an unknown reason [84]. It usually occurs on both
sides but can occur exclusively on one side. Interestingly, a
one-sided hydrosalpinx will usually correspond with an abnormal fallopian tube on the opposite side. For women with
infertility problems, removing the fallopian tubes is a major
decision. Nonetheless, hydrosalpingectomy has been reported as a promising option for a subgroup of RIF women
with severe tubal factor infertility [85, 86]. The main theoretical reasons for its use as a treatment option are (i) the
embryotoxic effect of the fluid in the region by leakage of
Table 2.
205
hydrosalpinx fluid, resulting in either endometrial alterations
that are hostile to embryo implantation and development
[87], (ii) the mechanical wash out of the embryo [88] before
it has a chance to implant, (iii) altered endometrial receptivity due to variations in the levels of certain biomarkers such
as LIF [89] and v3 integrin [90, 91], (iv) release of intrauterine cytokines, prostaglandins, leukotrienes, and other inflammatory compounds directly to the endometrium or via
the circulatory or lymphatic system [92, 93], or (v) chronic
endometriosis caused mainly by asymptomatic Chlamydia
trachomatis [94]. Thus, some studies support preventative
salpingectomy for infertile patients meeting two criteria: (i)
large hydrosalpinges visible by ultrasound and (ii) bilateral
hydrosalpinges [85, 86]. The emphasis on the severity of the
tubal disease (see Table 2 for hydrosalpinx scoring system)
for salpingectomy as a treatment option is important since it
limits or removes chances for a natural conception and is
likely to result in disruptions to ovarian blood flow and nerve
supply and reduce ovarian reserves [95] -- all of which are
important for follicle production, hormone production, and
the number and quality of the ova [84]. An additional criterion for irreversibility of the tubal condition should also be
included so that patients have the option to maintain their
fallopian tubes.
A study by Dechaud et al. reported significantly higher
implantation rates per transfer among women <41 years of
age who had experienced RIF after laparoscopic bilateral
hydrosalpingectomy compared with those who did not [96].
The pregnancy rate was also higher for the group with
salpingectomy than the group without (23.5% and 9.9 %,
respectively, P value = 0.01). Bilateral salpingectomy also
was reported to not only increase implantation rates but also
decrease the time to pregnancy. A group of women who underwent salpingectomy became pregnant within three IVF
attempts as opposed to a group who did not undergo
salpingectomy in which some patients required as many as
11 IVF attempts to become pregnant [96]. Furthermore, a
recent Cochrane study for the surgical treatment of tubal
diseases performed a meta-analysis on five randomized trials
and found a significant increase in clinical pregnancy rates
and ongoing pregnancy rates for patients with hydrosalpinges who underwent salpingectomy and IVF (for
the first time) as compared with those who did not undergo
surgical intervention [97]; thus, salpingectomy should
be considered for all patients with ultrasound-visible
hydrosalpinges.
In contrast, there is an argument in preference of tubal
microsurgery, which is a less invasive procedure that pre-
Hydrosalpinx Scoring System (Puttemans and Brosens 1996)
I.
Simple hydrosalpinx represented by a thin-walled translucent hydrosalpinx with flattened and separated mucosal folds in a single lumen but
without mucosal adhesions.
II.
Hydrosalpinx foliculans represented by a thin-walled hydrosalpinx with mucosal adhesions which can be focal or extensive. As a result the tubal
lumen may become divided into locules by agglutinated folds forming compartments or pseudoglandular spaces.
III.
Thick-walled hydrosalpinx represented by an ampullary wall >2 mm thick and absent mucosal folds or some fibrotic fold remnants at most; tubal distension is less marked and the amount of invisible intralumina fluid is less abundant.
Source: Puttemans PJ, Brosens IA. Salpingectomy improves in-vitro fertilization outcome in patients with a hydrosalpinx: blind victimization of the fallopian tube?. Hum Reprod
1996; 11(10): 2079-81.
serves the fallopian tubes, for women with less severe tubal
pathology [84]. The higher cost and greater time commitment make it a less popular option among clinicians. Also,
no studies have been done to compare efficacy, recovery,
psychological factors, risks, invasiveness of tubal microsurgery (a surgical procedure to repair and open the fallopian
tubes that is sometimes referred to as tuboplasty) versus hydrosalpingectomy [98]. Perhaps a randomized controlled
study to compare salpingectomy and tubal microsurgery for
RIF patients with various severity of hydrosalpinx could
determine the optimal procedure in the appropriate patient
population. Additional randomized controlled studies are
needed to clarify whether unilateral or bilateral salpingectomy is more appropriate and necessary than tubal surgery to
minimize unnecessary removal of healthy fallopian tubes
[97].
OOCYTE DONATION
In the past two decades, oocyte donation has become a
valuable and effective therapeutic option for infertile patients
who choose assisted reproductive technology (ART) and has
become an acceptable indication for patients with multiple
implantation failures [99]. Egg donors are prescreened for
medical and physical history of infectious disease, genetic
disorders, polycystic ovarian syndrome, serious malformations (resulting in severe functional or cosmetic handicap
such as spina bifida or heart malformations), mental health,
and uterine pathologies (for accessibility to oocyte retrieval)
and then selected based on age and phenotypic match to the
recipient [99, 100]. The screening process also extends to the
oocyte recipients (medical, physical, psychological, and social) and their partners (for infectious disease and paternal
factors) to identify other possible sources that may adversely
affect the outcome.
Three groups of patients, those with (i) repeat implantation failure and no other indications, (ii) RIF in combination
with advanced age, or (iii) RIF in patients with balanced
translocations in homologous chromosomes, may benefit
Table 3.
I.
II.
Ly et al.
from oocyte donation [100-102] to overcome factors that
have been implicated in implantation failure such as genotype and age of the oocyte. [100]. Two committees, the
American Society for Reproductive Medicine (ASRM) and
Society for Assisted Reproductive Technology (SART) published the 2008 Guideline for Gamete and Embryo Donation,
which provides the most recent recommendations and information from the U.S. Center for Disease Control (CDC),
U.S. Food and Drug Administration (FDA), and American
Association of Tissue Banks (AATB) for optimal screening
and testing of oocyte donors and recipients (see Table 3).
The guideline specifically identifies women with “multiple
previous failed attempts to conceive via ART” as an indication for oocyte donation [99].
More recently, the advancement of cryopreservation
methods and technologies for freezing and storing of oocytes
has increased the number of available unused oocytes [103].
Patients undergoing controlled ovarian stimulation to cryopreserve their oocytes prior to treatment for illnesses that
pose a serious threat to their future fertility (e.g., cancer)
have the option to donate their extra oocytes for research or a
donor bank once they no longer need the oocytes. Accordingly, RIF patients have a choice between using fresh and
cryopreserved oocytes. Although the latter option is available and supported by the ASRM as a fertility preservation
strategy, there is not enough evidence to support its safety
and efficacy at this time [104]. For the first time, a recent
prospective, randomized study comparing fertilization and
embryo development rates and ongoing pregnancy rates
found no difference between fresh oocytes and vitrified oocytes fertilized via intracytoplasmic sperm injection and then
developed in vitro [105]. Further clinical studies are needed
to clarify the long-term safety concerns to support the use of
cryopreserved oocytes.
TRANSFER OF SIX OR MORE EMBRYOS
A definitive answer to the question of what constitutes
the proper number of embryos for transfer in IVF has eluded
ASRM and SART Guidelines for Evaluating the Oocyte Recipient
Provide psychological counseling by a mental health professional and further psychological consultation if necessary prior to consent.
Obtain medical physical examination and reproductive history to detect reproductive abnormalities. Provide treatment as appropriate prior to use
of donor oocytes.
III.
Complete a general physical exam and pelvic exam.
IV.
Assess the uterine cavity with hysterosalpingography or similar device to detect any significant uterine abnormality.
V.
Other recommended tests including:
a.
Blood type, RH factor, and antibody screen
b.
Rubella and varicella titers
c.
HIV-1 and HIV-2
d.
Serologic test for syphilis
e.
Hepatitis B surface antigen
f.
Hepatitis B core antibody (IgG and IgM)
g.
Hepatitis C antibody
h.
Cervical cultures or similar tests for Neisseria gonorrhoeae and Chlamydia trachomatis
Source: Guidelines for gamete and embryo donation: a Practice Committee report. Fertil Steril (2008); 90(5 Suppl): S30-44.
clinicians and scientists in the field for the last decade.
In 1995, Azem et al. reported a significant increase in
pregnancy rates with the transfer of six or more embryos in
comparison with five in women with repeated implantation
failure (at least four prior failed IVF-ET attempts) [106]. No
other published evidence has demonstrated improved pregnancy or live birth rates after the transfer of more embryos in
subsequent IVF-ET cycles than the number transferred in
previous failed cycles. Despite this lack of evidence, the
transfer of greater numbers of embryos than the recommended guidelines in women with multiple fresh IVF-ET
failures is commonly performed in practice. This has also
been extended to women predicted to have a poor conception
prognosis based on indicators such as embryo quality, also
with little supportive evidence regarding efficacy [107].
strictions by considering the transfer of more embryos than
recommended only “in exceptional cases when women
with poor prognoses have had multiple failed fresh IVF-ET
cycles” with a level of evidence/recommendation III-C
[111]. The most recent Cochrane systematic review on the
number of embryos to transfer in IVF compares pregnancy
rates and chances of multiple pregnancies following single
versus double, three and four embryo transfer in fresh IVF
treatments with various results, reflecting the experience of
selected young women in a single fresh cycle of IVF/ICSI.
As such, data concerning older women and women with
previous multiple failed IVF attempts have yet to be assessed
[112].
IVF practices will most likely be modified as scientific
advances allow a more accurate assessment of the implantation potential of a given embryo, which will most likely
include the determination of the embryonic genome and
the metabolic and proteomic fingerprints of viable versus
nonviable embryos using microarray technology.
On the other hand, women undergoing IVF with multiple
embryo transfer face an increased risk of higher-order multiple pregnancies (HOMP) with their known medical, social,
and economic consequences.
The first guidelines on the number of embryos to transfer
were issued by the ASRM in 1996 [108]. These guidelines
were revised four times since then, with a subsequent reduction in HOMP. As recently as 2003, the triplet rate for IVF in
women younger than 40 was approximately 6%. In 2007, it
was less than 2%, and this decrease is directly related to the
decrease in the number of embryos transferred per cycle
[109].
INTRATUBAL EMBRYO TRANSFER
Embryo transfer is a vital step of IVF treatments. Therefore, in patients with repeated implantation failure, clinicians
frequently focus on the transfer procedures to enhance embryonic implantation following IVF [113, 114]. Tubal transfer of embryos or zygotes has been widely utilized as part of
the arsenal in the treatment of difficult cases of repeated IVF
failure [115, 116] although it has never been proven to be
beneficial beyond any doubt.
In the latest ASRM guidelines, issued in November 2009
[110] (see Table 4) the maximum recommended number of
embryos to transfer is five, and this number only applies to
women aged 41-42. For all patients with one or two previous
failed fresh IVF cycles, the guidelines also recommend
transfer of one supplementary embryo (in comparison with
the standard recommended number according to age group,
see Table 4), after proper counseling regarding the risk of
HOMP and justification in the patient’s medical records
[110]. This only brings the number of embryos to transfer to
six in the 41-42 age group if previous failed IVF attempts are
documented. For all other age groups, the numbers are even
more limited (as low as 1-2 transferred embryos for women
younger than 35). The Society of Obstetricians and Gynecologists of Canada (SOGC) goes even further in their reTable 4.
Zygote intrafallopian transfer (ZIFT) has been hypothesized to have many advantages over transcervical embryo
transfer (ET), mainly that it provides a “natural” growth milieu for zygotes under physiological regulation with numerous growth factors and cytokines from the tubal fluid, which
helps these zygotes attach to the uterus with greater synchronization, thus enhancing implantation potential [117]. After
all, natural Day 1/Day 2 embryos belong in the fallopian
tubes and not the uterus. Environment or in vitro culture systems play a crucial role in the early development stages of
the embryo, and a suboptimal environment may have adverse effects. The ZIFT technique also prevents spillage of
embryos after transcervical ET and solves the problem of
Recommended Limits on the Numbers of Embryos to Transfer
Age
Prognosis
207
< 37 yrs
35-37 yrs
38-40 yrs
42-42 yrs
Favorable*
1-2
2
3
5
All others
2
3
4
5
Favorable*
1
2
2
3
All others
2
2
3
3
Cleavage-stage embryos
Blastocysts
*Favorable = first cycle of IVF, good embryo quality, excess embryos available for cryopreservation, or previous successful IVF cycle.
technically difficult ET in patients with cervical stenosis
[61]. Nowadays, the environmental advantage of tubal transfer seems limited since laboratory conditions have improved
along with the composition of culture media over the last
two decades.
Although initial retrospective reports of ZIFT showed
higher pregnancy rates than with intrauterine ET [118-120],
other investigators have found that the main value of ZIFT is
limited to RIF cases [115]. ZIFT was reported to be a valid
alternative to standard ET in most subgroups of patients with
either first or multiple failed attempts at IVF [114, 115]. It
was demonstrated that in patients with tubal factor and a
confirmed patency of one fallopian tube, ZIFT can be applied successfully as a treatment for RIF; the pregnancy rates
and implantation rates for all ZIFT cycles in RIF patients
were 35.1% and 11.1% - significantly higher PRs and IRs as
compared to standard transcervical ET [121]. This study
concluded that ZIFT should be recommended to IVF patients
with a mild form of tubal factor and proved patency of one
tube, whereas in severe forms, salpingectomy remains the
recommended treatment.
The interest in intratubal transfer was greatly diminished
after the results of a meta-analysis and a randomized, controlled trial that failed to demonstrate any advantage for
ZIFT over standard IVF/ET [122]. Furthermore, a study by
Aslan et al. including 229 patients with RIF showed comparable outcomes following ZIFT and transcervical ET [123] .
However, these studies are not readily comparable due to
different methods and selection criteria. It is likely that a
patient’s age, number of previous IVF attempts and etiology
of infertility may influence the results [123]. A number of
drawbacks to the use of ZIFT exist, such as the need for general anesthesia, laparoscopy, and heavy medical equipment,
which increases the medical risks to the patients, as well as
the expenses related to the need for operative conditions
[123]. An association between tubal embryo transfer and the
increased risk of ectopic gestation has been inconsistently
reported [122, 123].
ZIFT is normally performed in the pronuclear stage, one
day after egg retrieval. For practical reasons, ZIFT is sometimes deferred by one day, and cleavage-stage embryos are
transferred two days after egg retrieval--a procedure termed
“embryo intra-Fallopian transfer,” or EIFT [114]. In a recent
retrospective study by Weissman et al., ZIFT and EIFT
transfers had comparable outcomes in regards to implantation and pregnancy rates as well as in all other study parameters such as miscarriage rates, ectopic pregnancy rates, and
multiple pregnancy rates, which were found to be unacceptably high [124].
In conclusion, RIF patients are a heterogeneous group
with unclear and various pathophysiological diagnoses. The
potentially high efficiency of ZIFT in RIF patients is only
partially understood, with a commonly accepted interpretation being that ZIFT embryos possibly are protected from
expulsion from the uterine cavity by junctional zone contractions as well as from the introduction of cervical microorganisms into the uterine cavity by the transfer catheter
[116, 125]. Meanwhile, and despite the scant available evi-
Ly et al.
dence, ZIFT continues to be proposed in clinical practice
exclusively to RIF patients [124].
NATURAL CYCLE IVF
Despite the fact that the first IVF/ET baby was born in
1978 after a natural unstimulated cycle, this technique soon
was practically abandoned, mainly because of the very high
cancellation rates. Controlled pharmacological ovarian hyperstimulation became the standard treatment in IVF cycles
of high- and normo-responder patients [126]. However, natural cycles have regained attention in poor-responder patients,
where only very few follicles can be recruited and very few
oocytes can be retrieved after controlled ovarian hyperstimulation. In these patients, natural IVF cycles may offer comparable outcomes (comparable number of follicles) [127128], reduced side effects such as multiple pregnancy and
ovarian hyperstimulation syndrome, and may represent a
more cost-effective and patient-friendly alternative [129].
A recent retrospective study of 500 consecutive natural
cycles demonstrated that IVF in natural cycles is an affordable and valid alternative in poor-responder patients [129], in
accordance with the data reported in an earlier meta-analysis
[130]. Controversial data exist as to the efficacy of cycles
with minimal stimulation(i.e. GnRH antagonist plus mild
gonadotropin stimulation), and there are currently no studies
in the literature comparing natural versus minimal stimulation cycles [129].
Moreover, to address the specific issue of implantation
failure, a better understanding of the key determinants of
successful implantation is warranted, one of which is endometrial receptivity. The endometrium is receptive to blastocyst implantation during a limited spatio-temporal window,
called “the implantation window” [131]. In humans, this
period begins 6–10 days after the LH surge and lasts for 48 h
[132, 133]. Successful implantation depends on synchronization between the developmental stages of the embryo itself
and the complex endocrine environment [134, 135]. There is
evidence in the literature suggesting that controlled ovarian
hyperstimulation (COH) can alter endometrial receptivity
[136, 137]. Supraphysiologic doses of hormones can cause
asynchronism between the embryo and endometrium and
altered concentrations of growth and adhesion factors, causing implantation failure [138, 139].
Ledee-Bataille et al. conducted a study in which endometrial CD56 bright cells (uterine natural killers or uNK)
were immunostained. They found elevated numbers of endometrial NK cells in RIF patients after COH cycles and
significantly lowered numbers after natural cycles [140].
Data suggest that uNK may be directly or indirectly involved
in controlling the early steps of the implantation process, in
part because of their role in vascular remodeling, specifically
spiral uterine arteries [141]. Furthermore, on the molecular
biology level, alterations in endometrial gene expression
have been reported with the use of gonadotropins in stimulated cycles [142]. More recently, Haouzi et al., using DNA
microarrays of endometrial biopsies, identified for the first
time five genes that are up-regulated during the implantation
window and proposed them as new biomarkers for exploration of endometrial receptiveness, including during a natural
cycle. This novel strategy could prove useful in patients with
poor implantation after IVF or ICSI [143].
In conclusion, in light of the many potential advantages
of natural cycle IVF, and with the many improvements in
laboratory conditions and fertilization techniques such as
ICSI, it seems worthwhile to re-evaluate the place of natural
cycle IVF in the arsenal of fertility treatments, especially in
RIF patients. A randomized, controlled trial, comparing
natural cycle IVF with current standard practice, is justified.
ANTIPHOSPHOLIPID ANTIBODY (APA) TESTING
AND TREATMENT
Antiphospholipid antibodies (APAs) [144] are acquired
immunoglobulins or monoclonal antibodies (IgG, IgM,
and/or IgA) directed against negatively charged membrane
phospholipids, which were characterized as thrombophilic
factors because of their association with slow progressive
thrombosis and infarction in the placenta, leading to uteroplacental insufficiency [145]. With regard to implantation
and pregnancy, it is more appropriate to classify APAs as
autoimmune factors because of the complex nature of their
interactions [7]. APAs have been shown experimentally to
block in vitro trophoblast migration, invasion, and syncytialization, to reduce trophoblast production of the vital
hormone human chorionic gonadotrophin [144] and activate
complement on the trophoblast surface inducing an inflammatory response [146]. Increased concentration of APA in
the follicular fluid, which was once thought to be an explanation for the direct effect of APA on the implanting embryo
[147], is still debatable. A recent study demonstrated that
this increased concentration had no adverse effect on the
reproductive outcome of women undergoing IVF [148]; another study found a significant relationship between follicular fluid APA concentrations and fertilization rates in IVF
failure patients [149].
APAs associated with reproductive failure are lupus anticoagulant (LCA), anti-cardiolipin (ACL), anti-phosphatidyl
serine (APS), anti-phosphatidyl inositol (API), antiphosphatidyl glycerol (APG), anti-phosphatidyl ethanolamine
(APE), and phosphatidic acid (PA) [150]. Not only do APAs
bind to their direct antigens, they also bind to plasma-bound
proteins and co-factors such as 2 glycoprotein I (2GPI),
the most studied anti-phospholipid protein, and prothrombin,
which is also important in this role [151]. There is evidence
that the presence of 2GPI on trophoblast and decidual cell
membranes might explain the clinical association between
recurrent fetal loss and 2GPI-dependent APA and the
pathogenic role for these antibodies at the same time [152].
Antinuclear antibodies (ANA) also may be associated with
reproductive failure, but they were shown to be positive in
9% of normal fertile women and appear to lack specificity in
low titre [153].
Even after 20 years of investigation, the role of APA remains elusive when it comes to assisted reproduction, mainly
because most of the groups reported an increased prevalence
of APA in infertile patients [154, 155], but the evidence is
much less definite with respect to IVF outcome [156, 157].
An explanation for the conflicting evidence might be related
to differences in antibodies tested. Some groups solely tested
for ACL, LCA, or ANA [154], while others evaluated a
209
more comprehensive range of APA [156]. Coulam et al.
found a 22% prevalence of seven APAs in women experiencing implantation failure after IVF/ET. Only 4% of
women with positive antibodies would have been detected if
only ACL were tested and only 14% if APS were added to
ACA [156]. Unfortunately, with the exception of ACL, no
universally accepted standard for the determination of APA
concentrations exists, which adds to the confusion. Actually,
the most broadly used clinical assays for these antibodies test
for ACL antibodies using enzyme linked immunosorbent
assay (ELISA) and lupus anticoagulant (LA) [156]. It is
noteworthy here to emphasize the fact that APA can be
found in low concentrations in as many as 16% of “normal”
controls, i.e. healthy fertile women [153].
The association of antiphospholipids with RIF has been
shown in some early studies [145, 153, 154, 156, 158], but
large prospective studies failed to reveal an association [155,
157, 159, 160]. A meta-analysis considered the effect of
APAs on the likelihood of IVF success and concluded that
testing for APAs was unjustified in patients undergoing IVF
[161]. However, these results did not close the debate because of the heterogeneity of the cohort studies, the populations included, because the RIF group of patients was not
addressed specifically [162]. A strong association was demonstrated between antibodies to the cofactor 2 glycoprotein
1 and IVF implantation failure [153]. Antibodies to annexinV, which acts as an inhibitor of phospholipid-dependent coagulation and may be necessary for trophoblast differentiation, were found to have a significantly greater incidence
(8.3%) in women with RIF than in controls (1.1%) [163].
Other findings by Geva et al. suggest that although APAs
may be important in recurrent fetal loss and spontaneous
abortions, neither the serum concentration nor the number of
positive APAs appear to have significance in recurrent implantation failure, cumulative pregnancy, or live birth rates
[164]. According to the ASRM (2008), no association is present between APA abnormalities and IVF success, there is
no indication for the assessment of APA in couples undergoing IVF, and therapy is not justified on the basis of existing
data [165].
Despite the uncertainty concerning the pathophysiology
of APAs in reproductive failure, their presumed thrombotic
effects have led to the widespread use of heparin and aspirin
for women with RIF [145]. Heparin is thought to protect the
trophoblast from injury by inhibiting the binding of phospholipids with antibodies, thus promoting implantation and
placentation [166]. Only a very few randomized, placebocontrolled studies evaluating the benefits of heparin and aspirin for APA-positive women with RIF have been undertaken. While some evidence exists that treatment with
unfractionated heparin and low-dose aspirin can improve
live birth rates [167], other studies have shown that neither
implantation nor pregnancy rates are improved with heparin
and aspirin [168, 169]. A recent randomized, placebocontrolled cross-over study in patients with strictly defined
RIF did not show any benefit of heparin and low-dose aspirin in patients seropositive for at least one antiphospholipid
(APA), antinuclear (ANA), or beta(2) glycoprotein I autoantibody, when the outcome measured was ongoing pregnancy
or implantation rates [169].
The most recent Cochrane review by Empson et al. examined the outcomes of all treatments to maintain pregnancy
in women with prior miscarriages and positive APA. The
results found that only unfractionated heparin combined with
aspirin appeared to reduce pregnancy losses (by 54%) when
compared with aspirin alone. However, these results were
only based on two small trials, one of which lacked satisfactory allocation concealment. Low molecular weight heparin
(LMWH) combined with aspirin had no statistically significant effect when compared with aspirin or intravenous immunoglobulin (IVIg) [170]. Aspirin alone had no significant
effect on any of the outcomes examined; corticosteroids did
not show any benefit but demonstrated increased adverse
outcomes [170]. IVIg did not significantly differ from prednisone or aspirin in outcomes [171] and was shown to have
lower live births rates than LMWH plus aspirin [172, 173].
The beneficial effect of IVIg has only been proved only in
uncontrolled studies [174].
To date, the combined use of low-dose aspirin and heparin is considered standard therapy for women seropositive
for APAs, despite the lack of adequate, prospective, randomized, placebo-controlled studies addressing the RIF group of
patients specifically. Caution must be exercised in recommending any given treatment.
In conclusion, in women with RIF, no consensus exists
regarding testing for APA, assays to be used, auto-antibodies
to test, definitions of patient groups or therapy for seropositive patients [175]. Patients must, therefore, be counseled
prior to starting any treatment that no clear evidence of
benefit for anticoagulation exists [116].
ROLE OF IMMUNE MECHANISMS IN RECURRENT
PREGNANCY LOSS AND RIF
Background
The immune system of a patient with a successful pregnancy has been considered a paradox since Medawar in 1953
described the embryo as a semi-allogen, but one that is protected from allogenic recognition by antigenic immaturity,
possibly explained by non-classical class I HLA molecules.
Since then, numerous studies have supported the theory of
alloimmune causes as an explanation for miscarriages and
implantation failure. The hypothesis is that the absence of
such alloimmunoprotective mechanisms would result in alloimmune-mediated miscarriage [176]. Moreover, Wegmann
et al. suggested that successful pregnancy might result from
the predominance of T helper 2 (Th2) cytokines over T
helper (Th1) cytokines [177]. In spite of the central role attributed to immunology in reproductive failure and the intense debates on its scope, no appropriate diagnostic strategy
has been established to date [176]. Genetic and immunological factors interact with each other in a complex network of
antibodies, adhesion molecules, metalloproteinases, natural
killer cells, and cytokines [150]. Other factors influencing
reproduction and implantation include human leukocyte antigen expression, antisperm antibodies, integrins, leukemia
inhibitory factor, cytokines, antiphospholipid antibodies,
endometrial adhesion factors, mucin-l, and uterine natural
killers [116, 150].
Ly et al.
NATURAL KILLER CELLS
A difference is thought to exist between the uterine and
peripheral natural killer (NK) cells of women with recurrent
pregnancy loss (RPL) compared with controls. Higher numbers of uterine NK cells have been found in the preimplantation endometrium of women with RIF [178]. However, this
abnormality was only part of a composite range of immune
and vascular abnormalities found in the endometrium of RIF
patients [178, 179]. The uNK from nonpregnant RPL patients
who exhibit lower CD56 expression (classified as CD16+
CD56dim) are more frequent than CD16+CD56bright, as
opposed to fertile controls [180]. Non-pregnant RPL patients
also show evidence of increased numbers of activated NK
cells in peripheral blood mononuclear cells [181]. Kwak et
al. also observed the up-regulated expression of CD56＋,
CD56+-CD16＋, and CD19＋cells in peripheral blood lymphocytes in pregnant women with RPL [182]. Moreover,
Aoki et al. also reported that high preconceptional NK cell
activity was associated with higher abortion rates in the next
pregnancy [80]. Studies of immune factors investigated at
the time of miscarriage showed that deficiency of CD56
bright natural killer cells in the decidua and high natural killer cell cytotoxicity in peripheral blood monocyte cells
(PBMCs) increase the risk of euploid miscarriage [183-185].
CYTOKINES
Strong evidence exists that locally secreted cytokines
control the implantation process and can cause implantation
failure [186, 187]. The Th1 cytokines include IL-2, IFN and
TNF, and the Th2 cytokines include IL-4, IL-5 and IL-10.
Evidence suggests that the mean of the Th1:Th2 ratio in
patients with RPL and in patients with multiple implantation
failure after IVF-embryo transfer [188] is significantly
higher than in normal fertile women. This predominance of
Th1 cytokines was demonstrated to exist in endometrial cells
as well as peripheral blood mononuclear cells before pregnancy [187, 189, 190] and at the time of miscarriage in decidual cells [191]. However, there are significant discrepancies in the results of the different studies, as some suggest
that Th1 cytokines production was higher in normal women
than in RPL patients in early pregnancy [192], and others
even found that the production of Th1 and Th2 cytokines
was similar in RPL patients who subsequently had successful
or failed pregnancies [193]. Th1 dominance may well be a
result of the miscarriage rather than a cause, and much more
basic knowledge is needed about the complex cytokine networks in pregnancy and the correlation between cytokine
production in peripheral mononuclear cells and decidual
lymphocytes [194] before tests measuring cytokines can be
introduced in clinical practice. With further research and
newly discovered cytokines, it is now clear that acceptance
of the Thl:Th2 paradigm as a single explanation for implantation failure would be an overly simplistic approach to a
very complex mechanism [195]. Other cytokines, particularly leukemia inhibitory factor (LIF), recently have been
shown to play a role in women with RIF [196]. Mannosebinding lectin, a constituent of the innate immune system
that modulates cytokine production by monocytes [197], was
shown to have significantly lower levels in women with RPL
[198-200].
TREATMENT WITH
GLOBULINS (IVIg)
INTRAVENOUS
IMMUNO-
The mechanism of action of IVIg, a fractionated blood
product, is multifactorial [201]. It is involved in a number of
processes, including modulation of T cells, B cells, NK cells,
monocytes, and macrophages; down-regulation of antibody
production; inhibition of antibody function; and modulation
of complement activation [202]. Immunoglobulins develop
their suppressive activity in vitro through the CD200 tolerance-signaling molecule, which is released from the surface
of subsets of blood mononuclear leukocytes and may bind to
IVIg [203]. CD200 is known to promote generation of regulatory T cells in mice [204]. A more recent report suggests
IVIg suppresses NK activity, specifically the CD56 bright
subset of NK cells found at the feto-maternal surface [205].
Additionally, one underlying mechanism may be the restoration of Th1/Th2 balance with dominant Th2 [201]. Highdose IVIgs nearly always have been combined with corticosteroids or anti-thrombotics, so that their precise efficacy
cannot be readily estimated and is practically hard to assess
[201].
A review of the literature concerning IVIg treatments
yields conflicting results. A meta-analysis of six trials by
Daya et al. demonstrated a lack of clinical efficacy of IVIg
on live birth rates [206], and a prospective, randomized,
double-blinded, and placebo-controlled study by Stephenson
et al. observed no differences between the IVIg-treated and
the placebo groups [207]. A prospective, randomized, double-blinded, and placebo-controlled study by Coulam et al.
demonstrated the efficacy of IVIg treatment in increasing the
percentage of live births among women experiencing unexplained RIF [208]. In a randomized study by Triolo et al.,
IVIg was less efficacious than low-dose aspirin and low molecular weight heparin in increasing live births rate [172]. On
the other hand, a randomized controlled trial by Christiansen
et al. showed no improvement in live birth rates with IVIg
compared with placebo, but thye suggested a possible beneficial role of IVIg in women with secondary recurrent miscarriage [209]. This effect was confirmed by the review of
Hutton et al., although the data concerning primary recurrent
miscarriage was inconclusive [210]. A meta-analysis of randomized and cohort controlled trials of IVIg in RIF patients
showed a significant increase in the live birth rate per
woman (p=0.012) and number needed to treat for one additional live birth = 6) [211]. Relevant variables appeared to
be selection of patients with abnormal immune test results
and properties of IVIg preparations, as different biological
preparations vary significantly in their ability to suppress NK
activity in vitro. Another variable is the scheduling of IVIg
treatment, as it is argued that pre-conception treatment is
better in both primary recurrent miscarriage patients and in
IVF failure patients [211, 212]. An observational pilot study
found that elevated numbers of NK cytotoxic CD16+ CD56+
cells are independent predictors of treatment success, and
that IVIg ameliorated the numbers of these NK cells in RIF
[213]. Another observational study by Winger et al. studied
for the first time a subset of IVF patients with elevated
Th1/Th2 cytokines and showed an improvement of implantation and live birth rates for the groups treated with IVIg and
both IVIg and TNF-alpha inhibitors as compared with the
211
no-treatment group [214]. However, the most current
Cochrane review shows no significant increase in the overall
pregnancy success rate over placebo or no treatment [215].
In conclusion, IVIg, an expensive treatment with possible
side effects, is widely used off-label in the treatment of early
reproductive failure. Systematic reviews have generated inconclusive results so evidence concerning its efficacy is controversial. Rigorous randomized, controlled trials studying
the efficacy of the treatment on various subgroups of RIF
patients and additional measurements of CD200-dependent
IVIg effects should be undertaken to solve the current controversy [211].
ALLOGENIC LYMPHOCYTE THERAPY
During pregnancy, the paternal human leukocyte antigen
(HLA) is recognized by the maternal immune system, which
induces production of several alloantibodies. These alloantibodies include anti-paternal cytotoxic antibodies (APCA),
anti-idiotypic antibodies (Ab2), and mixed lymphocyte reaction blocking antibodies (MLR-Bf). Once expressed, they
may coat the trophoblast to render it undetectable by the maternal immune response system [216]. A reduction in or the
absence of these alloantibodies during pregnancy may cause
fetal loss [201, 217, 218]. As has been shown repeatedly,
increased sharing of HLA may prohibit the mother from
producing these alloantibodies, leading to an increased tendency toward repeated fetal loss [201]. Maternal immunomodulation via transfusion of paternal leukocytes (lymphocytes) prior to conception has been proposed as a solution to
RIF [219-221], while the use of third party donor white cells
or trophoblast membranes transfusions have been largely
abandoned because of doubts about efficacy [201]. Allogenic
lymphocyte isoimmunization (ALT) also has been proposed
to solve the Th1/Th2 paradigm by shifting the balance towards Th2 cytokines, thus enhancing the implantation process [181, 188].
The first randomized, controlled study using ALT with
paternal PBMCs showed a 24% increase in successful pregnancy rates [222]. However, subsequent trials provided conflicting results due to variations in cell numbers, number of
injections, routes of administration, and types of placebo.
These differences make comparisons and meta-analyses difficult to realize [209]. A subsequent intention-to-treat metaanalysis by the Recurrent Miscarriage Immunotherapy Trialists Group showed an increased live birth rate of approximately 9–10% [223]. The number needed to treat for an additional live birth was limited to three to four women with
primary recurrent pregnancy losses who were seronegative
for autoantibodies (ANA and ACL) [219]. Since then, the
beneficial effects of ALT in RIF patients has been demonstrated primarily by non-randomized studies. In a retrospective, non-randomized review of 686 couples referred for
ALT, Kling et al. found a temporary beneficial effect lasting
for six months after immunization; this effect seemed to be
most pronounced in couples who failed three or more cycles
of IVF-embryo transfer [224]. Despite the lack of randomized trials for RIF, a large randomized trial in RSA patients
failed to show any beneficial effect of ALT [225]. Pooling
the results of the randomized and non-randomized studies,
Pandey et al. showed that 67% of RSA patients who re-
ceived paternal lymphocyte immunotherapy had successful
pregnancy outcomes in comparison to 36% success in
women with RSA in the control group who received either
autologous lymphocytes or no therapy [220].
In a double-blind, placebo-controlled trial in women with
unexplained RSA, immunization with paternal lymphocytes
proved to be beneficial over autologous (maternal) lymphocyte therapy [221]. On the other hand, a Cochrane metaanalysis of relevant trials [215] concluded that paternal and
third-party ALT provide no significant beneficial effect over
placebo in preventing miscarriages. The results of this metaanalysis are extremely controversial because it included a
large negative trial using immunization with paternal lymphocytes stored overnight [225]. This factor impairs the protective anti-abortive effect of the procedure and causes loss
in surface CD200, at least in mice [226]. The results of the
Ober study, despite the debate over its design, decisively
influenced the issues of the most current Cochrane review
cited above [215], as well as the US FDA regulation, [227]
which states that administration of such cells as allogenic
lymphocytes or cellular products in humans should only be
performed as part of a clinical research project and then only
if an Investigational New Drug application is in effect.
Moreover, concerns over possible adverse effects of LIT
have been raised. These include transfusion-related problems, autoimmune disorders, graft-versus-host reaction, and
transmission of infection such as hepatitis B virus or HIV
[176], or even cancer and gestational pathology [228]. Adverse neonatal outcomes are rare, but a case of neonatal alloimmune thrombocytopenia and intracranial hemorrhage in an
infant whose mother received immunizations of paternal
mononuclear cells has been reported [229]. However, a prospective study by Kling et al., with follow-up after 2-3 years,
showed that the acute side effects of intradermal ALT were
comparable to those reported after intradermal vaccination
for infectious diseases and that specific risks for anaphylaxis,
autoimmune, or graft- versus- host disease were not significant [228].
In conclusion, proposing ALT to RIF patients in the absence of standard and broadly applicable diagnostic tests of
immune-mediated pregnancy losses, of reliable methods for
judging the immunization effects, and of unified protocols of
immunization should await further randomized, controlled
trials based on adequate patient selection and more complete
knowledge of the underlying pathophysiology of the assumed alloimmune causes of recurrent miscarriage.
CONCLUSIONS
Randomized, controlled trials have shown that blastocyst
transfer, assisted hatching, salpingectomy for tubal disease,
and hysteroscopy in IVF procedures are beneficial for improving treatment outcome in patients with repeated implantation failure. Studies also demonstrate that treatment with
aspirin and heparin with IVIg does not have a clear impact
on treatment outcome. Allogenic lymphocyte therapy,
ZIFT/EIFT, co-cultures, sildenafil, use of donor oocytes,
transfer of six embryos, natural IVF, and PGS await further
clinical assessment. The management of RIF should be individualized because the pathophysiology is so variable and
often complex.
Ly et al.
EXPERT COMMENTARY
Having to endure not one but two or more failed rounds
of IVF is painfully frustrating to the patient as well as to the
clinicians and technicians involved. We discuss 14 current
options that are supported by varying degrees of scientific
evidence. Clinicians would benefit from knowing which
treatment options are proven in order to counsel patients
effectively and manage their expectations for future IVF
cycles. Unfortunately, even the more promising ones
are successful only to a certain subgroup of patients with
specific conditions. Our understanding of the mechanism
behind embryo implantation remains poorly understood,
which hampers our ability to find a solution. To date, strong
evidence supports the use of blastocyst transfer, assisted
hatching, salpingectomy for tubal disease, and hysteroscopy
in the management of couples with previous implantation
failures and clearly dismisses the use of aspirin and heparin
with IVIg. Other treatment options such as allogenic
lymphocyte therapy, ZIFT/EIFT, co-cultures, sildenafil,
use of donor oocytes, transfer of six embryos, natural IVF
and PGS are controversial, and their efficacy remains to be
elucidated.
FIVE-YEAR VIEW
Endometrial receptivity is a vital component for embryo
implantation. A better understanding of the critical success
factors required for successful implantation is recognized.
What is the optimal condition of the embryo? What is the
optimal condition of the endometrium? Recent genetic research focused on cultured endometrial cells from RIF
women has demonstrated a difference in transcriptional activity during the implantation window. A possible disruption
in genetic expression of specific genes that regulate the cell
cycle has been implicated. Further research is needed to fully
understand the genes involved in the defunct pathway of
endometrial cells during the implantation window, thus resulting in implantation failure.
KEY POINTS
• Implantation failure and embryo quality are major limiting steps for IVF treatment.
• Etiological sources of implantation failure include embryo quality; endometrial receptivity; immunological factors; uterine, tubal, and peritoneal factors; and culture
media. The treatment options discussed in the article are
intended to address the particular causes and improve
implantation rates.
• Differences in the local environment of the endometrium
of RIF patients compared with other infertile patients
have been found. These differences (i.e. gene expression)
possibly affect cross-talks between the embryo and the
endometrium and thus implantation.
• Blastocyst transfer, assisted hatching, salpingectomy for
tubal disease, and hysteroscopy are highly recommended
treatment options based on good, consistent scientific
evidence with randomized, controlled studies.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Weissman A, Biran G, Nahum H, Glezerman M, Levran D.
Blastocyst culture and transfer: lessons from an unselected,
difficult IVF population. Reprod Biomed Online 2008; 17(2): 2208.
Martin-Johnston MK, Uhler ML, Grotjan HE, Lifchez AS, Nani
JM, Beltsos AN. Lower chance of pregnancy with repeated cycles
with in vitro fertilization. J Reprod Med 2009; 54(2): 67-72.
Ola, Bolarinde; Li, Tin-Chiu Implantation failure following in-vitro
fertilization. Curr Opin Obstet Gynecol 2006; 18(4): 440-5.
Achache H, Tsafrir A, Prus D, Reich R, Revel A. Defective endometrial prostaglandin synthesis identified in patients with repeated
implantation failure undergoing in vitro fertilization. Fertil Steril
2009. [Epub ahead of print]
Koler M, Achache H, Tsafrir A, Smith Y, Revel A, Reich R. Disrupted gene pattern in patients with repeated in vitro fertilization
(IVF) failure. Hum Reprod 2009; 24(10): 2541-8.
Bersinger NA, Wunder DM, Birkhäuser MH, Mueller MD.
Gene expression in cultured endometrium from women with different outcomes following IVF. Mol Hum Reprod 2008; 14(8): 47584.
Cline AM, Kutteh WH.Is there a role of autoimmunity in implantation failure after in-vitro fertilization? Curr Opin Obstet Gynecol
2009; 21(3): 291-5.
Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B. Comparison
of blastocyst transfer with day 3 embryo transfer in similar patient
populations. Fertil Steril 2000; 73(1): 126-9.
Wilson M, Hartke K, Kiehl M, Rodgers J, Brabec C, Lyles R. Integration of blastocyst transfer for all patients. Fertil Steril 2002;
77(4): 693-6.
Levron J, Shulman A, Bider D, Seidman D, Levin T, Dor J. A
prospective randomized study comparing day 3 with blastocyststage embryo transfer. Fertil Steril 2002; 77(6): 1300-1.
Papanikolaou EG, D'haeseleer E, Verheyen G, et al. Live birth rate
is significantly higher after blastocyst transfer than after cleavagestage embryo transfer when at least four embryos are available on
day 3 of embryo culture. A randomized prospective study. Hum
Reprod 2005; 20(11): 3198-203.
Shapiro BS, Richter KS, Harris DC, Daneshmand ST. Dramatic
declines in implantation and pregnancy rates in patients who undergo repeated cycles of in vitro fertilization with blastocyst transfer after one or more failed attempts. Fertil Steril 2001; 76(3): 53842.
Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR.Is
blastocyst transfer useful as an alternative treatment for patients
with multiple in vitro fertilization failures? Fertil Steril 1999;
72(2): 218-20.
Levitas E, Lunenfeld E, Har-Vardi I, et al. Blastocyst-stage embryo
transfer in patients who failed to conceive in three or more day 2-3
embryo transfer cycles: a prospective, randomized study. Fertil
Steril 2004; 81(3): 567-71.
Barrenetxea G, López de Larruzea A, Ganzabal T, Jiménez R,
Carbonero K, Mandiola M.Blastocyst culture after repeated failure
of cleavage-stage embryo transfers: a comparison of day 5 and day
6 transfers. Fertil Steril 2005; 83(1): 49-53.
Gabrielsen A, Agerholm I, Toft B, et al. Assisted hatching improves implantation rates on cryopreserved-thawed embryos. A
randomized prospective study. Hum Reprod 2004; 19(10): 225862.
Germond M, Primi MP, Senn A. Hatching: how to select the clinical indications. Ann N Y Acad Sci 2004; 1034: 145-51.
Sheen TC, Chen SR, Au HK, Chien YY, Wu KY, Tzeng CR.
Herniated blastomere following chemically assisted hatching may
result in monozygotic twins. Fertil Steril 2001; 75(2): 442-4.
Das S, Blake D, Farquhar C, Seif MM. Assisted hatching on
assisted conception (IVF and ICSI). Cochrane Database Syst Rev
2009; (2): CD001894.
Simón C, Mercader A, Garcia-Velasco J, et al. Coculture of human
embryos with autologous human endometrial epithelial cells in
patients with implantation failure. J Clin Endocrinol Metab 1999;
84(8): 2638-46.
Carrell DT, Peterson CM, Jones KP, et al. A simplified coculture
system using homologous, attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in
vitro fertilization. J Assist Reprod Genet 1999; 16(7): 344-9.
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
213
Fabbri R, Porcu E, Marsella T, et al. Human embryo development
and pregnancies in an homologous granulosa cell coculture system.
J Assist Reprod Genet 2000; 17(1): 1-12.
Dirnfeld M, Goldman S, Gonen Y, Koifman M, Calderon I,
Abramovici H. A simplified coculture system with luteinized
granulosa cells improves embryo quality and implantation rates: a
controlled study. Fertil Steril 1997; 67(1): 120-2.
Escribá MJ, Escobedo-Lucea C, Mercader A, de los Santos MJ,
Pellicer A, Remohí J.Ultrastructure of preimplantation genetic
diagnosis-derived human blastocysts grown in a coculture system
after vitrification. Fertil Steril 2006; 86(3): 664-71.
Bongso A, Ng SC, Ratnam S. Co-cultures: their relevance to assisted reproduction. Hum Reprod 1990; 5(8): 893-900.
Bongso A, Soon-Chye N, Sathananthan H, Lian NP, Rauff M,
Ratnam S. alImproved quality of human embryos when co-cultured
with human ampullary cells. Hum Reprod 1989; 4(6): 706-13.
Morgan K, Wiemer K, Steuerwald N, Hoffman D, Maxson W,
Godke R.Use of videocinematography to assess morphological
qualities of conventionally cultured and cocultured embryos. Hum
Reprod 1995; 10(9): 2371-6.
Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Fiorentino A,
D'Errico A. Human embryo co-culture: results of a randomized
prospective study. Int J Fertil Menopausal Stud 1995; 40(5): 254-9.
Tucker MJ, Morton PC, Wright G, et al. Enhancement of outcome
from intracytoplasmic sperm injection: does co-culture or assisted
hatching improve implantation rates? Hum Reprod 1996; 11(11):
2434-7.
Wiemer KE, Garrisi J, Steuerwald N, et al. Beneficial aspects of
co-culture with assisted hatching when applied to multiple-failure
in-vitro fertilization patients. Hum Reprod 1996; 11(11): 2429-33.
Wiemer KE, Cohen J, Amborski GF, et al. In-vitro development
and implantation of human embryos following culture on fetal bovine uterine fibroblast cells. Hum Reprod 1989; 4(5): 595-600.
Wiemer KE, Cohen J, Wiker SR, Malter HE, Wright G, Godke RA.
Coculture of human zygotes on fetal bovine uterine fibroblasts:
embryonic morphology and implantation. Fertil Steril 1989; 52(3):
503-8.
Van Blerkom J. Development of human embryos to the hatched
blastocyst stage in the presence or absence of a monolayer of Vero
cells. Hum Reprod 1993; 8(9): 1525-39.
Sakkas D, Jaquenoud N, Leppens G, Campana A. Comparison of
results after in vitro fertilized human embryos are cultured in routine medium and in coculture on Vero cells: a randomized study.
Fertil Steril 1994; 61(3): 521-5.
Turner K, Lenton EA. The influence of Vero cell culture on human
embryo development and chorionic gonadotrophin production in
vitro. Hum Reprod 1996; 11(9): 1966-74.
Ben-Chetrit A, Jurisicova A, Casper RF. Coculture with ovarian
cancer cell enhances human blastocyst formation in vitro. Fertil
Steril 1996; 65(3): 664-6.
Hu Y, Maxson W, Hoffman D, et al. Co-culture with assisted
hatching of human embryos using Buffalo rat liver cells. Hum Reprod 1998; 13(1): 165-8.
Wetzels AM, Bastiaans BA, Hendriks JC, et al. The effects of coculture with human fibroblasts on human embryo development in
vitro and implantation. Hum Reprod 1998; 13(5): 1325-30.
Kattal N, Cohen J, Barmat LI. Role of coculture in human in vitro
fertilization: a meta-analysis. Fertil Steril 2008; 90(4): 1069-76.
Weichselbaum A, Paltieli Y, Philosoph R, et al. Improved development of very-poor-quality human preembryos by coculture with
human fallopian ampullary cells. J Assist Reprod Genet 2002;
19(1): 7-13.
Eyheremendy V, Raffo FG, Papayannis M, Barnes J, Granados C,
Blaquier J. Beneficial effect of autologous endometrial cell coculture in patients with repeated implantation failure. Fertil Steril
2010; 93(3): 769-73
Desai N, Abdelhafez F, Bedaiwy MA, Goldfarb J. Live births in
poor prognosis IVF patients using a novel non-contact human endometrial co-culture system. Reprod Biomed Online 2008; 16(6):
869-74.
Richter KS. The importance of growth factors for preimplantation
embryo development and in-vitro culture. Curr Opin Obstet Gynecol 2008; 20(3): 292-304.
d'Estaing SG, Lornage J, Hadj S, Boulieu D, Salle B, Guérin JF.
Comparison of two blastocyst culture systems: coculture on Vero
cells and sequential media. Fertil Steril 2001; 76(5): 1032-5.
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
Griffin DK. The incidence, origin, and etiology of aneuploidy. Int
Rev Cytol 1996; 167: 263-96.
Maroulis GB, Koutlaki N. Preimplantation genetic diagnosis. Ann
N Y Acad Sci 2006; 1092: 279-84.
Farfalli VI, Magli MC, Ferraretti AP, Gianaroli L.Role of aneuploidy on embryo implantation. Gynecol Obstet Invest 2007;
64(3): 161-5.
Fragouli E, Katz-Jaffe M, Alfarawati S, et al. Comprehensive
chromosome screening of polar bodies and blastocysts from
couples experiencing repeated implantation failure. Fertil Steril
2009. [Epub ahead of print]
Raziel A, Friedler S, Schachter M, Kasterstein E, Strassburger D,
Ron-El R. Increased frequency of female partner chromosomal
abnormalities in patients with high-order implantation failure after
in vitro fertilization. Fertil Steril 2002; 78(3): 515-9.
Gianaroli L, Magli MC, Munné S, Fiorentino A, Montanaro N,
Ferraretti AP. Will preimplantation genetic diagnosis assist patients
with a poor prognosis to achieve pregnancy? Hum Reprod 1997;
12(8): 1762-7.
Pehlivan T, Rubio C, Rodrigo L, et al. Impact of preimplantation
genetic diagnosis on IVF outcome in implantation failure patients.
Reprod Biomed Online 2003; 6(2): 232-7.
ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May
2001). Hum Reprod 2002; 17(1): 233-46.
Gianaroli L, Magli MC, Ferraretti AP, Munné S. Preimplantation
diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for
which it should be proposed. Fertil Steril 1999; 72(5): 837-44.
Goossens V, Harton G, Moutou C, Traeger-Synodinos J, Van Rij
M, Harper JC. ESHRE PGD Consortium data collection IX: cycles
from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009; 24(8): 1786-810.
Platteau P, Staessen C, Michiels A, Van Steirteghem A, Liebaers I,
Devroey P. Which patients with recurrent implantation failure after
IVF benefit from PGD for aneuploidy screening? Reprod Biomed
Online 2006; 12(3): 334-9.
Mantzouratou A, Mania A, Fragouli E, et al. Variable aneuploidy
mechanisms in embryos from couples with poor reproductive histories undergoing preimplantation genetic screening. Hum Reprod
2007; 22(7): p. 1844-53.
Voullaire L, Wilton L, McBain J, Callaghan T, Williamson R.
Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation
failure. Mol Hum Reprod 2002; 8(11): 1035-41.
Braude P, Bolton V, Moore S. Human gene expression first occurs
between the four- and eight-cell stages of preimplantation development. Nature 1988; 332(6163): 459-61.
Wells D, Bermudez MG, Steuerwald N, et al. Expression of genes
regulating chromosome segregation, the cell cycle and apoptosis
during human preimplantation development. Hum Reprod 2005;
20(5): 1339-48.
Voullaire L, Collins V, Callaghan T, McBain J, Williamson R,
Wilton L. High incidence of complex chromosome abnormality in
cleavage embryos from patients with repeated implantation failure.
Fertil Steril 2007; 87(5): 1053-8.
Margalioth EJ, Ben-Chetrit A, Gal M, Eldar-Geva T. Investigation
and treatment of repeated implantation failure following IVF-ET.
Hum Reprod 2006; 21(12): 3036-43.
Munné S, Magli C, Bahçe M, et al. Preimplantation diagnosis of
the aneuploidies most commonly found in spontaneous abortions
and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat Diagn
1998;18(13): 1459-66.
Kahraman S, Bahçe M, Samli H, et al. Healthy births and ongoing
pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure. Hum Reprod 2000; 15(9): 2003-7.
Munné S, Sandalinas M, Escudero T, et al. Improved implantation
after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003; 7(1): 91-7.
Arefi S, Soltanghoraei H, Sadeghi MR, Tabaei AS, Zeraati H,
Zarnani H. Findings on hysteroscopy in patients with in vitro fertilization by intra cytoplasmic single sperm injection and embryo
transfer failures. Saudi Med J 2008; 29(8): 1201-2.
Lorusso F, Ceci O, Bettocchi S, et al. Office hysteroscopy in an in
vitro fertilization program. Gynecol Endocrinol 2008; 24(8): 465-9.
Ly et al.
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
Oliveira FG, Abdelmassih VG, Diamond MP, et al. Uterine cavity
findings and hysteroscopic interventions in patients undergoing in
vitro fertilization-embryo transfer who repeatedly cannot conceive.
Fertil Steril 2003; 80(6): 1371-5.
Makrakis E, Hassiakos D, Stathis D, Vaxevanoglou T, Orfanoudaki
E, Pantos K. Hysteroscopy in women with implantation failures after in vitro fertilization: findings and effect on subsequent pregnancy rates. J Minim Invasive Gynecol 2009; 16(2): 181-7.
Madani T, Ghaffari F, Kiani K, Hosseini F. Hysteroscopic polypectomy without cycle cancellation in IVF cycles. Reprod Biomed Online 2009; 18(3):412-5.
Küçük T, Safali M. "Chromohysteroscopy" for evaluation of endometrium in recurrent in vitro fertilization failure. J Assist Reprod
Genet 2008; 25(2-3): 79-82.
Boolell M, Allen MJ, Ballard SA, et al. Sildenafil: an orally active
type 5 cyclic GMP-specific phosphodiesterase inhibitor for the
treatment of penile erectile dysfunction. Int J Impot Res 1996; 8(2):
47-52.
Goodman C, Jeyendran RS, Coulam CB. Coulam, P53 tumor suppressor factor, plasminogen activator inhibitor, and vascular endothelial growth factor gene polymorphisms and recurrent implantation failure. Fertil Steril 2009; 92(2): 494-8.
Sher G, Fisch JD. Vaginal sildenafil (Viagra): a preliminary report
of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF. Hum Reprod
2000; 15(4): 806-9.
Sher G, Fisch JD. Effect of vaginal sildenafil on the outcome of in
vitro fertilization (IVF) after multiple IVF failures attributed to
poor endometrial development. Fertil Steril 2002; 78(5): 1073-6.
Check JH, Graziano V. Multiple confounders--measured with
error? Fertil Steril 2003; 79(5): 1256-7; author reply 1257-8.
Zinger M, Liu JH, Thomas MA. Successful use of vaginal sildenafil citrate in two infertility patients with Asherman's syndrome. J
Womens Health (Larchmt) 2006; 15(4): 442-4.
Paulus WE, Strehler E, Zhang M, Jelinkova L, El-Danasouri I,
Sterzik K. Benefit of vaginal sildenafil citrate in assisted reproduction therapy. Fertil Steril 2002; 77(4): 846-7.
Quenby S, Bates M, Doig T, et al. Pre-implantation endometrial
leukocytes in women with recurrent miscarriage. Hum Reprod
1999; 14(9): 2386-91.
Gilman-Sachs A, DuChateau BK, Aslakson CJ, et al. Natural killer
(NK) cell subsets and NK cell cytotoxicity in women with histories
of recurrent spontaneous abortions. Am J Reprod Immunol 1999;
41(1): 99-105.
Aoki K, Kajiura S, Matsumoto Y, et al. Preconceptional naturalkiller-cell activity as a predictor of miscarriage. Lancet 1995;
345(8961): 1340-2.
Strandell A, Lindhard A, Waldenström U, Thorburn J. Nitric oxide
inhibits development of embryos and implantation in mice. Mol
Hum Reprod 1998; 4(5): 503-7.
Battaglia C, Ciotti P, Notarangelo L, Fratto R, Facchinetti F, de
Aloysio D. Embryonic production of nitric oxide and its role in implantation: a pilot study. J Assist Reprod Genet 2003; 20(11): 44954.
Strandell A, Waldenström U, Nilsson L, Hamberger L. Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates.
Hum Reprod 1994; 9(5): 861-3.
Bontis JN, Dinas KD. Management of hydrosalpinx: reconstructive
surgery or IVF? Ann N Y Acad Sci 2000; 900: 260-71.
Strandell A, Lindhard A, Waldenström U, Thorburn J. Hydrosalpinx and IVF outcome: cumulative results after salpingectomy in a
randomized controlled trial. Hum Reprod 2001; 16(11): 2403-10.
Mansour R, Aboulghar M, Serour GI. Controversies in the surgical
management of hydrosalpinx. Curr Opin Obstet Gynecol 2000;
12(4): 297-301.
Granot I, Dekel N, Segal I, Fieldust S, Shoham Z, Barash A. Is
hydrosalpinx fluid cytotoxic? Hum Reprod 1998; 13(6): 1620-4.
Sharara FI. The role of hydrosalpinx in IVF: simply mechanical?
Hum Reprod 1999; 14(3): 577-8.
Seli E, Kayisli UA, Cakmak H, et al. Removal of hydrosalpinges
increases endometrial leukaemia inhibitory factor (LIF) expression
at the time of the implantation window. Hum Reprod 2005; 20(11):
3012-7.
Bildirici I, Bukulmez O, Ensari A, Yarali H, Gurgan T. A prospective evaluation of the effect of salpingectomy on endometrial re-
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
ceptivity in cases of women with communicating hydrosalpinges.
Hum Reprod 2001; 16(11): 2422-6.
Meyer WR, Lessey BA, Castelbaum AJ.Hydrosalpinx and altered
uterine receptivity. Fertil Steril 1997;68(5): 944-5.
Grifo JA, Jeremias J, Ledger WJ, Witkin SS.Interferon-gamma in
the diagnosis and pathogenesis of pelvic inflammatory disease. Am
J Obstet Gynecol 1989; 160(1): 26-31.
Ben-Rafael Z, Orvieto R. Cytokines--involvement in reproduction.
Fertil Steril 1992; 58(6): 1093-9.
Jones RB, Mammel JB, Shepard MK, Fisher RR. Recovery of
Chlamydia trachomatis from the endometrium of women at risk for
chlamydial infection. Am J Obstet Gynecol 1986; 155(1): 35-9.
Lass A. What effect does hydrosalpinx have on assisted reproduction? What is the preferred treatment for hydrosalpines? The
ovary's perspective. Hum Reprod 1999; 14(7): 1674-7.
Dechaud H, Anahory T, Aligier N, Arnal F, Humeau H, Hedon B.
Salpingectomy for repeated embryo nonimplantation after in vitro
fertilization in patients with severe tubal factor infertility. J Assist
Reprod Genet 2000; 17(4): 200-6.
Johnson NP, Mak W, Sowter MC. Surgical treatment for tubal
disease in women due to undergo in vitro fertilisation. Cochrane
Database Syst Rev 2004; (3): CD002125.
Johnson NP, Mak W, Sowter MC. Laparoscopic salpingectomy for
women with hydrosalpinges enhances the success of IVF: a Cochrane review. Hum Reprod 2002; 17(3): 543-8.
Practice Committee of American Society for Reproductive Medicine; Practice Committee of Society for Assisted Reproductive
Technology. Fertil Steril 2008; 90(5 Suppl): S30-44.
Klein J, Sauer MV.Oocyte donation. Best Pract Res Clin Obstet
Gynaecol 2002; 16(3): 277-91.
Sauer MV, Kavic SM.Oocyte and embryo donation 2006: reviewing two decades of innovation and controversy. Reprod Biomed
Online 2006; 12(2): 153-62.
Lee RM, Silver RM. Recurrent pregnancy loss: summary and clinical recommendations. Semin Reprod Med 2000; 18(4): 433-40.
Lee RM, Silver RM. The efficacy and safety of human oocyte
vitrification. Semin Reprod Med 2009; 27(6): 450-5.
ASRM Practice Committee response to Rybak and Lieman: elective self-donation of oocytes. Fertil Steril 2009; 92(5): 1513-4.
Rienzi L, Romano S, Albricci L, et al. Embryo development of
fresh 'versus' vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum Reprod 2010; 25(1):
66-73.
Azem F, Yaron Y, Amit A, et al. Transfer of six or more embryos
improves success rates in patients with repeated in vitro fertilization failures. Fertil Steril 1995; 63(5): 1043-6.
Hu Y, Maxson WS, Hoffman DI, et al. Maximizing pregnancy
rates and limiting higher-order multiple conceptions by determining the optimal number of embryos to transfer based on quality.
Fertil Steril 1998; 69(4): 650-7.
Stern JE, Cedars MI, Jain T, et al. Assisted reproductive technology practice patterns and the impact of embryo transfer guidelines
in the United States. Fertil Steril 2007; 88(2): 275-82.
Campbell B, Erickson L. The number of embryos to transfer: current practices in Minnesota. Minn Med 2009; 92(4): 40-1.
Guidelines on number of embryos transferred. Fertil Steril 2009;
92(5): 1518-9.
Guidelines for the number of embryos to transfer following in vitro
fertilization No. 182, September 2006. Int J Gynaecol Obstet 2008;
102(2): 203-16.
Pandian Z, Bhattacharya S, Ozturk O, Serour G, Templeton A.
Number of embryos for transfer following in-vitro fertilisation or
intra-cytoplasmic sperm injection. Cochrane Database Syst Rev
2009(2): CD003416.
Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001; 76(5):
863-70.
Levran D, Farhi J, Nahum H, Royburt M, Glezerman M, Weissman
A. Prospective evaluation of blastocyst stage transfer vs. zygote intrafallopian tube transfer in patients with repeated implantation
failure. Fertil Steril 2002; 77(5): 971-7.
Levran D, Mashiach S, Dor J, Levron J, Farhi J. Zygote intrafallopian transfer may improve pregnancy rate in patients with repeated
failure of implantation. Fertil Steril 1998; 69(1): 26-30.
Urman B, Yakin K, Balaban B. Recurrent implantation failure in
assisted reproduction: how to counsel and manage. B. Treatment
[117]
[118]
[119]
[120]
[121]
[122]
[123]
[124]
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
[138]
[139]
[140]
215
options that have not been proven to benefit the couple. Reprod
Biomed Online 2005; 11(3): 382-91.
Jansen RP. Endocrine response in the fallopian tube. Endocr Rev
1984; 5(4): 525-51.
Devroey P, Staessen C, Camus M, De Grauwe E, Wisanto A, Van
Steirteghem AC.Zygote intrafallopian transfer as a successful
treatment for unexplained infertility. Fertil Steril 1989; 52(2): 2469.
Hammitt DG, Syrop CH, Hahn SJ, Walker DL, Butkowski CR,
Donovan JF.Comparison of concurrent pregnancy rates for in-vitro
fertilization--embryo transfer, pronuclear stage embryo transfer and
gamete intra-fallopian transfer. Hum Reprod 1990; 5(8): 947-54.
Asch RH.Uterine versus tubal embryo transfer in the human. Comparative analysis of implantation, pregnancy, and live-birth rates.
Ann N Y Acad Sci 1991; 626: 461-6.
Farhi J, Weissman A, Nahum H, Levran D.Zygote intrafallopian
transfer in patients with tubal factor infertility after repeated failure
of implantation with in vitro fertilization-embryo transfer. Fertil
Steril 2000; 74(2): 390-3.
Habana AE, Palter SF. Is tubal embryo transfer of any value? A
meta-analysis and comparison with the Society for Assisted Reproductive Technology database. Fertil Steril 2001; 76(2): 286-93.
Aslan D, Elizur SE, Levron J, et al. Comparison of zygote intrafallopian tube transfer and transcervical uterine embryo transfer in patients with repeated implantation failure. Eur J Obstet Gynecol Reprod Biol 2005; 122(2): 191-4.
Weissman A, Eldar I, Ravhon A, et al. Timing intra-Fallopian
transfer procedures. Reprod Biomed Online 2007; 15(4): 445-50.
Weissman A, F.J., Levran D. Zygote intrafallopian transfer.
Reproductive Techniques: Laboratory and Clinical Perspectives,
2nd ed. W.A. Gardner DK, Howies CM, Shoham Z, Eds. London
Taylor and Francis 2004; pp. 735-48.
Ubaldi F, Rienzi L, Ferrero S, et al. Natural in vitro fertilization
cycles. Ann N Y Acad Sci 2004; 1034: 245-51.
Morgia F, Sbracia M, Schimberni M, et al. A controlled trial of
natural cycle versus microdose gonadotropin-releasing hormone
analog flare cycles in poor responders undergoing in vitro fertilization. Fertil Steril 2004; 81(6): 1542-7.
Bassil S, Godin PA, Donnez J. Outcome of in-vitro fertilization
through natural cycles in poor responders. Hum Reprod 1999;
14(5): 1262-5.
Schimberni M, Morgia F, Colabianchi J, et al. Natural-cycle in
vitro fertilization in poor responder patients: a survey of 500 consecutive cycles. Fertil Steril 2009; 92(4): 1297-301.
Pelinck MJ, Hoek A, Simons AH, Heineman MJ. Efficacy of natural cycle IVF: a review of the literature. Hum Reprod Update 2002;
8(2): 129-39.
Paria BC, Lim H, Das SK, Reese J, Dey SK. Molecular signaling in
uterine receptivity for implantation. Semin Cell Dev Biol 2000;
11(2): 67-76.
Paria BC, Lim H, Das SK, Reese J, Dey SK. Time of implantation
of the conceptus and loss of pregnancy. N Engl J Med 1999;
340(23): 1796-9.
Martín J, Domínguez F, Avila S, et al. Human endometrial receptivity: gene regulation. J Reprod Immunol 2002; 55(1-2): 131-9.
Davis GE, Senger DR. Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and
neovessel stabilization. Circ Res 2005; 97(11): 1093-107.
Bischof P, Campana A. Molecular mediators of implantation.
Baillieres Best Pract Res Clin Obstet Gynaecol 2000; 14(5): 80114.
Basir GS, O WS, Ng EH, Ho PC. Morphometric analysis of periimplantation endometrium in patients having excessively high
oestradiol concentrations after ovarian stimulation. Hum Reprod
2001; 16(3): 435-40.
Devroey P, Bourgain C, Macklon NS, Fauser BC. Reproductive
biology and IVF: ovarian stimulation and endometrial receptivity.
Trends Endocrinol Metab 2004; 15(2): 84-90.
Tavaniotou A, Albano C, Smitz J, Devroey P. Impact of ovarian
stimulation on corpus luteum function and embryonic implantation.
J Reprod Immunol 2002; 55(1-2): 123-30.
Basille C, Fay S, Hesters L, Frydman N, Frydman R. In vitro fertilization (IVF): why doing it in unstimulated cycles?. Gynecol Obstet Fertil 2007; 35(9): 877-80.
Lédée-Bataille N, Dubanchet S, Kadoch J, Castelo-Branco A,
Frydman R, Chaouat G. Controlled natural in vitro fertilization
[141]
[142]
[143]
[144]
[145]
[146]
[147]
[148]
[149]
[150]
[151]
[152]
[153]
[154]
[155]
[156]
[157]
[158]
[159]
[160]
[161]
[162]
may be an alternative for patients with repeated unexplained implantation failure and a high uterine natural killer cell count. Fertil
Steril 2004; 82(1): 234-6.
Croy BA, Ashkar AA, Minhas K, Greenwood JD.Can murine uterine natural killer cells give insights into the pathogenesis of
preeclampsia? J Soc Gynecol Investig 2000; 7(1): 12-20.
Horcajadas JA, Riesewijk A, Polman J, et al. Effect of controlled
ovarian hyperstimulation in IVF on endometrial gene expression
profiles. Mol Hum Reprod 2005; 11(3): 195-205.
Haouzi D, Mahmoud K, Fourar M, et al. Identification of new
biomarkers of human endometrial receptivity in the natural cycle.
Hum Reprod 2009; 24(1): 198-205.
Di Simone N, Meroni PL, de Papa N, et al. Antiphospholipid antibodies affect trophoblast gonadotropin secretion and invasiveness
by binding directly and through adhered beta2-glycoprotein I. Arthritis Rheum 2000; 43(1): 140-50.
Kutteh WH.Antiphospholipid antibodies and reproduction. J Reprod Immunol 1997; 35(2): 151-71.
Girardi G, Yarilin D, Thurman JM, Holers VM, Salmon
JE.Complement activation induces dysregulation of angiogenic factors and causes fetal rejection and growth restriction. J Exp Med
2006; 203(9): 2165-75.
el-Roeiy A, Gleicher N, Friberg J, Confino E, Dudkiewicz A. Correlation between peripheral blood and follicular fluid autoantibodies and impact on in vitro fertilization. Obstet Gynecol 1987; 70(2):
163-70.
Buckingham KL, Stone PR, Smith JF, Chamley LW. Antiphospholipid antibodies in serum and follicular fluid--is there a correlation with IVF implantation failure? Hum Reprod 2006; 21(3): 72834.
Matsubayashi H, Sugi T, Arai T, et al. IgG-antiphospholipid antibodies in follicular fluid of IVF-ET patients are related to low fertilization rate of their oocytes. Am J Reprod Immunol 2006; 55(5):
341-8.
Choudhury SR, Knapp LA.Human reproductive failure I: immunological factors. Hum Reprod Update 2001; 7(2): 113-34.
Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies
(ACA) directed not to cardiolipin but to a plasma protein cofactor.
Lancet 1990; 335(8705): 1544-7.
Meroni PL, Gerosa M, Raschi E, Scurati S, Grossi C, Borghi
MO.Updating on the pathogenic mechanisms 5 of the antiphospholipid antibodies-associated pregnancy loss. Clin Rev Allergy
Immunol 2008; 34(3): 332-7.
Stern C, Chamley L, Hale L, Kloss M, Speirs A, Baker HW. Antibodies to beta2 glycoprotein I are associated with in vitro fertilization implantation failure as well as recurrent miscarriage: results of
a prevalence study. Fertil Steril 1998; 70(5): 938-44.
Birkenfeld A, Mukaida T, Minichiello L, Jackson M, Kase NG,
Yemini M. Incidence of autoimmune antibodies in failed embryo
transfer cycles. Am J Reprod Immunol 1994; 31(2-3): 65-8.
Gleicher N, Liu HC, Dudkiewicz A, et al. Autoantibody profiles
and immunoglobulin levels as predictors of in vitro fertilization
success. Am J Obstet Gynecol 1994; 170(4): 1145-9.
Coulam CB, Kaider BD, Kaider AS, Janowicz P, Roussev
RG. Antiphospholipid antibodies associated with implantation
failure after IVF/ET. J Assist Reprod Genet 1997; 14(10): 6038.
Denis AL, Guido M, Adler RD, Bergh PA, Brenner C, Scott RT Jr.
Antiphospholipid antibodies and pregnancy rates and outcome in in
vitro fertilization patients. Fertil Steril 1997; 67(6): 1084-90.
Geva E, Yaron Y, Lessing JB, et al. Circulating autoimmune
antibodies may be responsible for implantation failure in in vitro
fertilization. Fertil Steril 1994; 62(4): 802-6.
Kowalik A, Vichnin M, Liu HC, Branch W, Berkeley AS. Midfollicular anticardiolipin and antiphosphatidylserine antibody titers do
not correlate with in vitro fertilization outcome. Fertil Steril 1997;
68(2): 298-304.
Hill JA, Choi BC. Maternal immunological aspects of pregnancy
success and failure. J Reprod Fertil Suppl 2000; 55: 91-7.
Hornstein MD, Davis OK, Massey JB, Paulson RJ, Collins JA.
Antiphospholipid antibodies and in vitro fertilization success: a
meta-analysis. Fertil Steril 2000; 73(2): 330-3.
Gleicher N, Vidali A, Karande V. The immunological "Wars of the
Roses": disagreements amongst reproductive immunologists. Hum
Reprod 2002; 17(3): 539-42.
Ly et al.
[163]
[164]
[165]
[166]
[167]
[168]
[169]
[170]
[171]
[172]
[173]
[174]
[175]
[176]
[177]
[178]
[179]
[180]
[181]
[182]
Matsubayashi H, Arai T, Izumi S, Sugi T, McIntyre JA, Makino T.
Anti-annexin V antibodies in patients with early pregnancy loss or
implantation failures. Fertil Steril 2001; 76(4): 694-9.
Eldar-Geva T, Wood C, Lolatgis N, et al. Cumulative pregnancy
and live birth rates in women with antiphospholipid antibodies undergoing assisted reproduction. Hum Reprod 1999; 14(6): 1461-6.
Practice Committee of American Society for Reproductive Medicine. Anti-phospholipid antibodies do not affect IVF success. Fertil
Steril 2008; 90(5 Suppl): S172-3.
Ermel LD, Marshburn PB, Kutteh WH. Interaction of heparin with
antiphospholipid antibodies (APA) from the sera of women with
recurrent pregnancy loss (RPL). Am J Reprod Immunol 1995;
33(1): 14-20.
Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior
to low-dose aspirin alone. Am J Obstet Gynecol 1996; 174(5):
1584-9.
Kutteh WH, Yetman DL, Chantilis SJ, Crain J. Effect of antiphospholipid antibodies in women undergoing in-vitro fertilization: role
of heparin and aspirin. Hum Reprod 1997; 12(6): 1171-5.
Stern C, Chamley L, Norris H, Hale L, Baker HW. A randomized,
double-blind, placebo-controlled trial of heparin and aspirin for
women with in vitro fertilization implantation failure and antiphospholipid or antinuclear antibodies. Fertil Steril 2003; 80(2): 376-83.
Empson M, Lassere M, Craig J, Scott J. Prevention of recurrent
miscarriage for women with antiphospholipid antibody or lupus anticoagulant. Cochrane Database Syst Rev 2005; (2): CD002859.
Vaquero E, Lazzarin N, Valensise Het, et al. Pregnancy outcome in
recurrent spontaneous abortion associated with antiphospholipid
antibodies: a comparative study of intravenous immunoglobulin
versus prednisone plus low-dose aspirin. Am J Reprod Immunol
2001; 45(3): 174-9.
Triolo G, Ferrante A, Ciccia F, et al. Randomized study of subcutaneous low molecular weight heparin plus aspirin versus intravenous immunoglobulin in the treatment of recurrent fetal loss associated with antiphospholipid antibodies. Arthritis Rheum 2003;
48(3): 728-31.
Dendrinos S, Sakkas E, Makrakis E. Low-molecular-weight heparin versus intravenous immunoglobulin for recurrent abortion associated with antiphospholipid antibody syndrome. Int J Gynaecol
Obstet 2009; 104(3): 223-5.
Clark AL, Branch DW, Silver RM, Harris EN, Pierangeli S, Spinnato JA.Pregnancy complicated by the antiphospholipid syndrome:
outcomes with intravenous immunoglobulin therapy. Obstet Gynecol 1999; 93(3): 437-41.
Wong RC, Favaloro EJ. Clinical features, diagnosis, and management of the antiphospholipid syndrome. Semin Thromb Hemost
2008; 34(3): 295-304.
Takeshita T. Diagnosis and treatment of recurrent miscarriage
associated with immunologic disorders: Is paternal lymphocyte
immunization a relic of the past? J Nippon Med Sch 2004; 71(5):
308-13.
Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional
cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 1993; 14(7):
353-6.
Lédée-Bataille N, Bonnet-Chea K, Hosny G, Dubanchet S,
Frydman R, Chaouat G. Role of the endometrial tripod interleukin18, -15, and -12 in inadequate uterine receptivity in patients with a
history of repeated in vitro fertilization-embryo transfer failure.
Fertil Steril 2005; 83(3): 598-605.
Quenby S, Farquharson R. Uterine natural killer cells, implantation
failure and recurrent miscarriage. Reprod Biomed Online 2006;
13(1): 24-8.
Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T,
B, and NK cells in patients with recurrent spontaneous abortion.
Altered profile and pregnancy outcome. J Immunol 1996; 156(10):
4027-34.
Ntrivalas EI, Kwak-Kim JY, Gilman-Sachs A, et al. Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology. Hum Reprod
2001; 16(5): 855-61.
Kwak JY, Beaman KD, Gilman-Sachs A, Ruiz JE, Schewitz D,
Beer AE. Up-regulated expression of CD56+, CD56+/CD16+, and
CD19+ cells in peripheral blood lymphocytes in pregnant women
[183]
[184]
[185]
[186]
[187]
[188]
[189]
[190]
[191]
[192]
[193]
[194]
[195]
[196]
[197]
[198]
[199]
[200]
[201]
[202]
with recurrent pregnancy losses. Am J Reprod Immunol 1995;
34(2): 93-9.
Yamada H, Kato EH, Kobashi G, et al. High NK cell activity in
early pregnancy correlates with subsequent abortion with normal
chromosomes in women with recurrent abortion. Am J Reprod
Immunol 2001; 46(2): 132-6.
Yamamoto T, Takahashi Y, Kase N, Mori H. Role of decidual
natural killer (NK) cells in patients with missed abortion: differences between cases with normal and abnormal chromosome. Clin
Exp Immunol 1999; 116(3): 449-52.
Quack KC, Vassiliadou N, Pudney J, Anderson DJ, Hill JA. Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion. Hum Reprod
2001; 16(5): 949-55.
Lédée-Bataille N, Laprée-Delage G, Taupin JL, Dubanchet S,
Frydman R, Chaouat G. Concentration of leukaemia inhibitory factor (LIF) in uterine flushing fluid is highly predictive of embryo
implantation. Hum Reprod 2002; 17(1):213-8.
Kwak-Kim JY, Chung-Bang HS, Ng SC, et al. Increased T helper 1
cytokine responses by circulating T cells are present in women with
recurrent pregnancy losses and in infertile women with multiple
implantation failures after IVF. Hum Reprod 2003; 18(4): 767-73.
Ng SC, Gilman-Sachs A, Thaker P, Beaman KD, Beer AE, KwakKim J. Expression of intracellular Th1 and Th2 cytokines in
women with recurrent spontaneous abortion, implantation failures
after IVF/ET or normal pregnancy. Am J Reprod Immunol 2002;
48(2): 77-86.
Lim KJ, Odukoya OA, Ajjan RA, Li TC, Weetman AP, Cooke ID.
The role of T-helper cytokines in human reproduction. Fertil Steril
2000; 73(1): 136-42.
Hill JA, Polgar K, Anderson DJ. T-helper 1-type immunity to trophoblast in women with recurrent spontaneous abortion. JAMA
1995; 273(24): 1933-6.
Piccinni MP, Beloni L, Livi C, Maggi E, Scarselli G, Romagnani S.
Defective production of both leukemia inhibitory factor and type 2
T-helper cytokines by decidual T cells in unexplained recurrent
abortions. Nat Med 1998; 4(9): 1020-4.
Bates MD, Quenby S, Takakuwa K, Johnson PM, Vince GS. Aberrant cytokine production by peripheral blood mononuclear cells in
recurrent pregnancy loss? Hum Reprod 2002; 17(9): 2439-44.
Kruse C, Varming K, Christiansen OB. Prospective, serial investigations of in-vitro lymphocyte cytokine production, CD62L expression and proliferative response to microbial antigens in women
with recurrent miscarriage. Hum Reprod 2003; 18(11): 2465-72.
Borzychowski AM, Croy BA, Chan WL, Redman CW, Sargent IL.
Changes in systemic type 1 and type 2 immunity in normal pregnancy and pre-eclampsia may be mediated by natural killer cells.
Eur J Immunol 2005; 35(10): 3054-63.
Chaouat G, Ledée-Bataille N, Dubanchet S, Zourbas S, Sandra O,
Martal J. TH1/TH2 paradigm in pregnancy: paradigm lost? Cytokines in pregnancy/early abortion: reexamining the TH1/TH2 paradigm. Int Arch Allergy Immunol 2004; 134(2): 93-119.
Steck T, Giess R, Suetterlin MW, et al. Leukaemia inhibitory factor
(LIF) gene mutations in women with unexplained infertility and recurrent failure of implantation after IVF and embryo transfer. Eur J
Obstet Gynecol Reprod Biol 2004; 112(1): 69-73.
Jack DL, Read RC, Tenner AJ, Frosch M, Turner MW, Klein NJ.
Mannose-binding lectin regulates the inflammatory response of
human professional phagocytes to Neisseria meningitidis serogroup
B. J Infect Dis 2001; 184(9): 1152-62.
Kilpatrick DC, Bevan BH, Liston WA. Association between mannan binding protein deficiency and recurrent miscarriage. Hum Reprod 1995; 10(9): 2501-5.
Dahl M, Tybjaerg-Hansen A, Schnohr P, Nordestgaard BG. Morbidity and mortality in persons with mannose-binding lectin deficiency. The Osterbro study. Ugeskr Laeger 2004; 166(50): 4606-9.
Kruse C, Rosgaard A, Steffensen R, Varming K, Jensenius JC,
Christiansen OB. Low serum level of mannan-binding lectin is a
determinant for pregnancy outcome in women with recurrent spontaneous abortion. Am J Obstet Gynecol 2002; 187(5): 1313-20.
Shetty S, Ghosh K. Anti-phospholipid antibodies and other immunological causes of recurrent foetal loss--a review of literature of
various therapeutic protocols. Am J Reprod Immunol 2009; 62(1):
9-24.
Morikawa M, Yamada H, Kato EH, et al. Massive intravenous
immunoglobulin treatment in women with four or more recurrent
[203]
[204]
[205]
[206]
[207]
[208]
[209]
[210]
[211]
[212]
[213]
[214]
[215]
[216]
[217]
[218]
[219]
[220]
[221]
[222]
[223]
217
spontaneous abortions of unexplained etiology: down-regulation of
NK cell activity and subsets. Am J Reprod Immunol 2001; 46(6):
399-404.
Clark DA, Chaouat G. Loss of surface CD200 on stored allogeneic
leukocytes may impair anti-abortive effect in vivo. Am J Reprod
Immunol 2005; 53(1): 13-20.
Gorczynski RM. Thymocyte/splenocyte-derived CD4+CD25+Treg
stimulated by anti-CD200R2 derived dendritic cells suppress mixed
leukocyte cultures and skin graft rejection. Transplantation 2006;
81(7): 1027-34.
Wright GJ, Cherwinski H, Foster-Cuevas M, et al. Characterization
of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol 2003; 171(6): 3034-46.
Daya S, Gunby J, Porter F, Scott J, Clark DA. Critical analysis of
intravenous immunoglobulin therapy for recurrent miscarriage.
Hum Reprod Update 1999; 5(5): 475-82.
Stephenson MD, Dreher K, Houlihan E, Wu V. Prevention of unexplained recurrent spontaneous abortion using intravenous immunoglobulin: a prospective, randomized, double-blinded, placebocontrolled trial. Am J Reprod Immunol 1998; 39(2): 82-8.
Coulam CB, Krysa L, Stern JJ, Bustillo M.Intravenous immunoglobulin for treatment of recurrent pregnancy loss. Am J Reprod
Immunol 1995; 34(6): 333-7.
Christiansen OB, Pedersen B, Rosgaard A, Husth M.A randomized,
double-blind, placebo-controlled trial of intravenous immunoglobulin in the prevention of recurrent miscarriage: evidence for a therapeutic effect in women with secondary recurrent miscarriage. Hum
Reprod 2002; 17(3): 809-16.
Hutton B, Sharma R, Fergusson D, et al. Use of intravenous immunoglobulin for treatment of recurrent miscarriage: a systematic review. BJOG 2007; 114(2): 134-42.
Clark DA, Coulam CB, Stricker RB. Is intravenous immunoglobulins (IVIG) efficacious in early pregnancy failure? A critical review
and meta-analysis for patients who fail in vitro fertilization and
embryo transfer (IVF). J Assist Reprod Genet 2006, 23(1): 1-13.
Carp HJ. Intravenous immunoglobulin: effect on infertility and
recurrent pregnancy loss. Isr Med Assoc J 2007; 9(12): 877-80.
van den Heuvel MJ, Peralta CG, Hatta K, Han VK, Clark DA.
Decline in number of elevated blood CD3(+) CD56(+) NKT cells
in response to intravenous immunoglobulin treatment correlates
with successful pregnancy. Am J Reprod Immunol 2007; 58(5):
447-59.
Winger EE, Reed JL, Ashoush S, Ahuja S, El-Toukhy T, Taranissi
M. Treatment with adalimumab (Humira) and intravenous immunoglobulin improves pregnancy rates in women undergoing IVF.
Am J Reprod Immunol 2009; 61(2): 113-20.
Porter TF, LaCoursiere Y, Scott JR. Immunotherapy for recurrent
miscarriage. Cochrane Database Syst Rev 2006; (2): CD000112.
Pandey MK, Rani R, Agrawal S. An update in recurrent spontaneous abortion. Arch Gynecol Obstet 2005; 272(2): 95-108.
McIntyre JA, Coulam CB, Faulk WP. Recurrent spontaneous abortion. Am J Reprod Immunol 1989; 21(3-4): 100-4.
Tamura M, Takakuwa K, Arakawa M, Yasuda M, Kazama Y,
Tanaka K. Relationship between MLR blocking antibodies and the
outcome of the third pregnancy in patients with two consecutive
spontaneous abortions. J Perinat Med 1998; 26(1): 49-53.
Daya S, Gunby J. The effectiveness of allogeneic leukocyte immunization in unexplained primary recurrent spontaneous abortion.
Recurrent Miscarriage Immunotherapy Trialists Group. Am J Reprod Immunol 1994; 32(4): 294-302.
Pandey MK, Thakur S, Agrawal S. Lymphocyte immunotherapy
and its probable mechanism in the maintenance of pregnancy in
women with recurrent spontaneous abortion. Arch Gynecol Obstet
2004; 269(3): 161-72.
Gatenby PA, Cameron K, Simes RJ, et al. Treatment of recurrent
spontaneous abortion by immunization with paternal lymphocytes:
results of a controlled trial. Am J Reprod Immunol 1993; 29(2): 8894.
Mowbray JF, Gibbings C, Liddell H, et al. Controlled trial of
treatment of recurrent spontaneous abortion by immunisation with
paternal cells. Lancet 1985; 1(8435): 941-3.
Worldwide collaborative observational study and meta-analysis on
allogenic leukocyte immunotherapy for recurrent spontaneous
abortion. Recurrent Miscarriage Immunotherapy Trialists Group.
Am J Reprod Immunol 1994; 32(2): 55-72.
[224]
[225]
[226]
Ly et al.
Kling C, Steinmann J, Westphal E, Magez J,Kabelitz D. Experience with allogeneic leukocyte immunization for implantation
failure in the in-vitro fertilization program. Am J Reprod Immunol
2002; 48: 147.
Ober C, Karrison T, Odem RR, et al. Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial.
Lancet 1999; 354(9176): 365-9.
Clark DA, Chaouat G. Immunomodulation by CD200 as a basis
for prevention of cytokine-dependent fetal loss syndrome. Am J
Reprod Immunol 2003; 49: 350-1.
Received: January 17, 2010
[227]
[228]
[229]
Research, C.f.B.E.a., Lymphocyte Immune Therapy. 2001.
Kling C, Steinmann J, Westphal E, Magez J, Kabelitz D. Adverse
effects of intradermal allogeneic lymphocyte immunotherapy: acute
reactions and role of autoimmunity. Hum Reprod 2006; 21(2): 42935.
Pearlman SA, Meek RS, Cowchock FS, Smith JB, McFarland J,
Aster RH. Neonatal alloimmune thrombocytopenia after maternal
immunization with paternal mononuclear cells: successful treatment with intravenous gamma globulin. Am J Perinatol 992; 9(56): 448-51.
Revised: March 29, 2010
Accepted: June 15, 2010
219
Chin-Kun Baw1, Kim Dao Ly1, Amr Kader 1, Ali Ahmady 2 and Ashok Agarwal1,*
1
Center for Reproductive Medicine, Glickman Urological & Kidney Institute and Ob/Gyn & Women’s Health
Institute, Cleveland Clinic, Cleveland, OH 44195, USA; 2University Hospitals Case Medical Center, Cleveland, OH
44106, USA
Abstract: Recent studies demonstrated an overwhelming success in single blastocyst transfer (SBT): implantation
rates (IR) were 60.9%-70.5% and pregnancy rates (PR) were 60.9%-76% while the multiple pregnancy rates (MPR) were
0%-3.2%. Most of these studies involved good prognosis patients not more than 37 years of age. The results indicated
that SBT decreased the number of multiple pregnancies while maintaining desirable pregnancy outcomes. However,
SBT and cryopreserved single blastocyst transfer (cSBT) in the field of in vitro fertilization (IVF) are still in their
infancy. Guidelines for the number of blastocysts being transferred and the techniques have not yet been standardized.
The method to estimate the most viable blastocyst has not yet been proposed. The success of SBT also was found to
be highly associated with the technique and patients’ and clinicians’ perceptions toward it.
Keywords: Single blastocyst transfer (SBT), embryo transfer, blastocyst grading, cryopreservation, in vitro fertilization (IVF),
assisted reproductive technology (ART).
DEVELOPMENT OF EMBRYO TRANSFER
DEVELOPMENT OF BLASTOCYST TRANSFER
Since the first success of human birth after the implantation of human embryo in 1978 [1], IVF has become the most
effective treatment for infertile patients regardless of the
cause of infertility [2]. To achieve higher IR, the practice of
transferring multiple embryos per transfer cycle has been
widely employed [3]. The results of multiple embryo transfer
have been shown to have an IR from 3% to 69% and a PR
from 24% to 66% [4-10]. However, with the relatively satisfying success of pregnancy outcomes, multiple gestations
have become a serious complication resulting from the transfer of multiple embryos at a time [3].
Blastocyst Culture: Higher Performance as the Culture
Technique Improves
The MPR revealed by many studies ranged from 17% to
75% [6-10]. With this high MPR, studies have been advocating reducing the number of transferred embryos and defining
the success of embryo transfer as the single live birth rate.
Luke et al. analyzed 69,028 data entries regarding embryo
transfer during 2004-2006 in the Society for Assisted Reproductive Technology (SART) Clinic Outcomes Reporting
System (CORS) database. The single live birth rate was
found to be 42.9%, 32.5%, 26.5%, and 23.2% for transferring of one, two, three, and more than four embryos at a
time, respectively [11]. The results showed that the single
live birth rate after single embryo transfer is significantly
higher than those of multiple embryo transfer (p<0.0001).
However, despite the support and advocating efforts in the
literature, single embryo transfer still represented a minority
of practice in the United States—12% overall in 2001 [12],
and 4.4% during 2004-2006 [11].
Medicine Cleveland Clinic 9500 Euclid Avenue, Desk A19.1 Cleveland,
OH 44195, USA; Tel: 216-444-9485; Fax: 216-445-6049;
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Recent improvements in culture technology have enabled
more robust embryo development to the blastocyst stage.
Furthermore, a better understanding of the dynamic uterine
and endometrial environment as the embryo develops from a
zygote to blastocyst has aided specifically in improvement of
the required nutritive contents in culture systems to more
closely mimic the reproductive tract. The addition of amino
acids, sugars, and other compounds permits early embryos to
develop into blastocysts [13], the embryonic stage known to
be associated with a higher IR, without compromising PR
[14]. With regard to nutrition requirements, the preimplantation period should be considered in two phases for correct
medium formulation, pre and post-compaction [15]. Prior to
Day 3 of embryo development the first couple of cell cycles
utilize reserves containing maternal transcripts and stored
mRNA; in other words, its metabolism is regulated at the
post-transcriptional level [16]. During post-compaction, the
embryonic genome has taken over and the embryo becomes
more susceptible to transcriptional inhibition from nutrient
deficiencies [13].
Two types of culture systems are involved in blastocyst
culture, monoculture media and sequential media. In a
monoculture system, a single medium is supplemented with
all the required components to sustain the embryo for growth
to the blastocyst stage. A sequential system requires growing
the early embryo until Day 3 in culture with amino acids
and low levels of glucose and phosphate to allow the embryo
to use its reserve (maternal transcripts and stored mRNA)
and then transfer the embryo to a more complex medium
with numerous metabolites (citrate, malate, various lipids,
etc.) to support higher demands and avoid metabolic blocks
[17]. As noted before, the embryo depends on its reserves
prior to compaction, and so it is reasonable for embryos
to grow in a medium with lower glucose levels during this
period.
Sequential media systems were introduced in the late
1990s and became a controversial issue. How can a simple
change in culture media have such a significant improvement
in embryo development to ensure a quality embryo is cultured to the blastocyst stage? Opponents of blastocyst culture
and transfer contend that the self-selection criteria, the main
underpinning benefit for use of sequential media for blastocyst culture, is thwarted by the preimplantation embryo’s
high incidence of chromosomal abnormality, an increased
risk for monozygotic twinning, and an increased risk for epigenetic mutation. A prospectively randomized control study
by Staessen et al. evaluated the incidence of chromosomal
abnormality of cleavage-stage versus blastocyst-stage embryos in women of advanced maternal age and noted a
difference: a reduction of aneuploidy incidence from 59%
on Day 3 to 35% on Day 5 [18]. Proponents argue that
considerable evidence suggests a significant improvement in
IR without compromising PR in blastocyst transfer as compared with Day 2 or 3 transfer [19]. Nonetheless, sequential
media is widely accepted today for blastocyst culture and
transfer.
Early studies evaluating sequential media culture systems
show evidence of improved PR and significant IR (32.3%)
following blastocyst transfer in a heterogeneous group of
infertile patients [20]. However, both Sepulveda et al. and
Reed et al. found it was unnecessary to transfer the embryo
to a subsequent media after Day 3 [21, 22]. Reed et al. demonstrated a significantly higher mean number of optimal
blastocysts on day five with the single medium compared to
the sequential medium (P<0.05) [21]. A total of 893 embryos
from patients and donors were divided into three groups: (i)
232 patient embryos cultured for Day 3 transfer, (ii) 480
patient embryos for Day 5 transfer, and (iii) 181 donor embryos for Day 5 transfer. Each group of 232, 480, and 181
oocytes was split in half to be cultured in sequential and continuous media. Embryos in the sequential medium group
were moved to the second sequential medium, while embryos in the single medium treatment remained in the original dish, without renewal of the microdrops. The mean number of embryos transferred for embryos cultured in sequential (1.3 embryos) versus continuous (1.6 embryos) for Day 3
transfer did not differ significantly (P>0.05). Also, the mean
quality score of Day 3 transfer was 2.5 and 2.4 (P>0.05) for
sequential and continuous media, respectively; and did not
show a significant difference. On the other hand, the mean
number of Day 5 blastocysts was higher in the continuous
media than in the sequential media. The same results were
found for the mean number of Day 5 blastocysts transferred.
Surprisingly, the mean number of Day 5 blastocysts to be
transferred from continuous media was two times that of
sequential media (P<0.01). Also, mean embryo quality
scores did not differ between the media for Day 5 blastocysts
[21].
Further support was demonstrated in a prospective, randomized, controlled study using donor oocytes cultured in
single medium versus sequential media in which no significant difference was found in number or quality of blastocysts
Baw et al.
on Day 5 when comparing embryo culture in either system,
single or sequential [22]. A total of 287 and 322 embryos
were cultured in continuous and sequential media, respectively. By Day 5, 24.7% of the 287 embryos in the continuous medium and 13.7% of 322 embryos from the sequential
medium (P=0.001) were in full to hatching status; thus, a
greater portion of embryos developed to Day 5 blastocysts in
continuous medium than in sequential [22]. In this study,
embryos in both groups of single and sequential medium
were moved to fresh droplets of single medium and the second sequential medium, respectively on Day 3. It is important to note that the single medium used in this study contained all 20 amino acids as opposed to the sequential media
with significantly fewer amino acids (alanyl-glutamine only)
[22]. To put it briefly, the single, continuous, uninterrupted
culture system was just as good as or better than the sequential media system when it was properly formulated with all
the required nutrients. If true, the monoculture system eliminates one step in handling the embryo - a more desirable
option to reduce any unknown risks associated with moving
the embryo to another medium. The extra step translates
into more work, increased cost, and a theoretical chance for
negative events to occur. A comparison of the efficacy of the
variety of single and sequential medium for culture to the
blastocyst stage also would be important.
Advance culture technology such as sequential media is
not the only critical success factor for improving SBT and
increasing IVF success. Prerequisites to successful blastocyst
culture are dependent on (i) lab protocols, (ii) quality
management system, and (iii) superior oocytes from a good
ovarian response in good prognosis patients [13]. Other
important features of the culture system also have an impact
on IVF success per transfer cycle, including gas phase,
embryo incubation volume and group size, and macromolecule supplementation [13]. The autocrine effect of volume
and group size on embryo is only proven in mouse and
remains controversial in humans. Quality control of procedures has long been declared as critical to protect embryos
from potential toxins, especially during the move to sequential media [15]. For instance, lab conditions must be stable,
and so close monitoring is required to protect against atmospheric fluctuations [21]. Unfortunately, comparisons of lab
quality conditions and lab protocols among various IVF
laboratories are not often studied and so only very limited
reports are available; no standard laboratory requirements
for performing single blastocyst culture and transfer are
documented.
In conclusion, we noted that sequential media has had a
positive effect on the outcome of single blastocyst culture
and transfer for a select patient population. Yet, the majority
of the studies regarding blastocyst culture and transfer have
eliminated patients with advanced maternal age. This suggests that extending studies to determine whether SBT would
be just as beneficial for older patients with good prognosis,
good response to ovarian stimulation, an optimal number (10
or more) of high quality embryos, and no other known indications would be worthwhile. Comparing the efficacy of the
variety of single and sequential medium for culture to the
blastocyst stage in the future may also be beneficial, especially in media with undefined contents and/or concentrations to standardize the components in complex media.
Multiple Blastocyst Transfer: Widely Employed to
Yield High IR
In the not too distant past, an IR above 10% was an
acceptable milestone for an IVF clinic. Given this low IR,
transferring two or more embryos was considered necessary
to achieve acceptable IR or PR. Milki et al. found that the IR
was 47% and the PR was 58% with 24 cycles of threeblastocyst transfer [23]. This result demonstrated the success
of multiple blastocyst transfer. Furthermore, Jain et al.
reported the results of 75 cycles of blastocyst transfer with
a mean number of 2.0±2 blastocysts being transferred
per cycle. The IR was found to be 45.3%, and the clinical
PR was found to be 50.7% [24]. In addition, Yamamoto
et al. reported the results of 290 cycles of multiple blastocyst
transfer with a mean number of 1.44±0.50 blastocysts being
transferred per cycle. The IR was found to be 35.2%, and
the clinical PR 42.4% [25]. These studies all indicated
the success of utilizing multiple blastocysts in blastocyst
transfer.
SBT: a Way to Curb High MPR in ART
Although great success was achieved with multiple blastocyst transfer, a high MPR also occurred. The high MPR
has been a problem for the Assisted Reproductive Technology (ART) industry over the past decade. Ryan et al. reported that the MPR approached 40% after double blastocyst
transfers (DBT) in their center [26]. Recently, IR above 50%
for transfers of top-quality cleavage-stage embryos have
been reported, while IR above 60% for transfer of selected
blastocysts have been reported. Stillman et al. demonstrated
that IR for double blastocyst transfers were almost 50% for
all patients regardless of age or blastocyst quality [27]. IRs
as high as these put patients at risk of multiple pregnancies if
more than one embryo or blastocyst is transferred. Stillman
et al. demonstrated that MPR were 44% among 4083 cycles
of fresh double blastocyst transfers [27].
Multiple pregnancy is associated with well-documented
increases in maternal morbidity and mortality from gestational diabetes, hypertension, cesarean delivery, pulmonary
emboli, and postpartum hemorrhage. It also increases the
risk of premature birth, resulting in fetal, neonatal, and
childhood complications from neurologic insults, ocular and
pulmonary damage, learning disabilities and retardation, and
congenital malformations [28, 29].
Reducing the MPR has become the crucial public health
requirement for the further development of effective IVF
practices. The only truly effective means by which to avoid
multiple pregnancies (aside from monozygotic twinning) is
to transfer a single embryo [27]. Recent studies demonstrated an overwhelming success in SBT: IRs were 60.9%70.5%, and PRs were 60.9%-76%, while the MPRs were
0%-3.2% [27, 30-35]. Most of these studies required good
prognosis patients not more than 37 years of age. While
other studies did not specify the diagnosis of the patients,
Styer et al. indicated that patients with infertility diagnoses
of idiopathic, male factor, and other female factors are all
included in their study. The IR was 70.5% with an MPR of
3.1%. The results suggested that SBT has the promising potential to significantly reduce the risk of multiple births
while maintaining the success of pregnancy outcomes.
221
Monozygotic Twinning Caused by SBT
The only possible mechanism for SBT to result in twins
is through monozygotic twinning (MZT). MZT is a rare phenomenon in humans, occurring in 0.42% of spontaneous
pregnancies. Guerif et al. demonstrated that MZT in 218
cycles of SBT is higher, although not statistically significant,
compared with 243 cycles of single cleavage-stage embryo
transfer (3.8% vs. 1.6%) [36]. da Costa et al. suggested
that one probable reason for the higher incidence of MZT in
blastocyst transfer could be the hardening of the zona pellucida resulting from prolonged exposure to embryo culture
[37]. As the blastocyst herniates through the abnormally
hardened zona pellucida, it might facilitate the blastocyst’s
division.
Moreover, Papanikolaou et al. demonstrated that MZT is
not increased after SBT (n=271) compared with single
cleavage-stage embryo transfer (n=308) (1.8% vs. 2.6%,
p>0.50) [38]. It is important to note that assisted hatching
and blastocyst coculture have not been performed in this
study. Several studies reported monozygotic twins and triplets in association with SBT [39, 40]. Lee et al. reported
three healthy boys each weighing 1.78 kg were born to a 28year-old woman after a SBT [40].
To summarize, although current studies revealed that the
incidence of MZT is increased following IVF compared with
spontaneous conception, evidence is insufficient to support
this observation. Further studies are needed to clarify the
association between extended culture to the blastocyst stage
and MZT.
CRYOPRESERVED SINGLE BLASTOCYST TRANSFER (cSBT)
With recent improvements in culture media and cryopreservation techniques, frozen embryo transfer has evolved.
Blastocyst cryopreservation can be accomplished by slowfreezing or vitrification. Slow-freezing utilizes cryoprotectants to minimize intercellular ice crystal formation. Vitrification was first reported in 1985 utilizing high concentrations of cryoprotectants to achieve a ultra-rapid freeze to
eliminate ice crystal formation [41, 42]. These two methods
have been readily employed at the blastocyst stage [43,
44].
Although the results of transfer for more than one cryopreserved blastocysts are well-documented [25, 32, 36, 45],
few studies have been published regarding the pregnancy
outcomes of cryopreserved SBT. Desai et al. examined 56
slow-freezing cycles of cSBT [46]. A two-step glycerol
freeze protocol was used in this study. The result demonstrated a 27% clinical PR and an 18% live birth rate.
Mukaida et al. showed that PR increased as the number
of transferred blastocysts increased [47] when a Cryoloop
technique was used to vitrify the blastocysts produced from
223 cycles. PR were 26% in cryopreserved single blastocyst
transfer, 34% in cryopreserved double blastocyst transfer,
and approaching 60% when four to five cryopreserved blastocysts were transferred. This study included blastocysts that
were either re-expanded or not re-expanded before embryo
transfer. In addition, Yanaihara et al. reported the pregnancy
outcomes of 412 cycles of vitrified single blastocyst transfer
in their center [48]. The PR and MPR were 40.7% and 2.3%,
respectively. This study demonstrated that vitrification is
a practical method for cSBT.
Baw et al.
20.7% to 34.1 % and from 30% to 40%, respectively [52].
This is good news for the future of IVF children who have
no voice to deny a future of complications and the movement
toward a single, healthy live birth for all patients.
CRITERIA FOR SBT
Current Guidelines
Together, the American Society of Reproductive Medicine (ASRM) and the Society for Assisted Reproductive
Technology (SART) have published several versions of
guidelines on the number of embryos to be transferred to
maintain pace with changing technology. In the most current
version, the number of blastocysts to transfer for ages <35,
35-37, 38-40, and 41-42 were 1, 2, 2, and 3 blastocysts, respectively, for patients with a favorable prognosis [49].
These include patients in their first cycle of IVF and/or those
with high quality embryos, high number of embryos available, or have had previous successful IVF cycles. Alternatively, all other patients within the same age ranges should
transfer the following (number of blastocysts): ages <35 (2),
35-37 (2), 38-40 (3), and 41-42 (3) [49].
A second set of guidelines published jointly by the Society of Obstetricians and Gynecologists of Canada and the
Board of the Canadian Fertility and Andrology Society also
includes recommendations for the number of blastocysts for
transfer, but it is important to note that their guidelines are
based mainly on studies of cleavage-stage embryos and not
of blastocyst-stage embryos [50]. In accordance with these
guidelines, good prognosis patients should transfer the
following (number of blastocysts) or less: ages <35 (1),
35-37 (2), 38-39 (2), 39+ (3). In all other cases, patients with
a less than desirable prognosis should transfer the following
(number of blastocysts) or less: ages <35 (2), 35-37 (3),
38-39 (3), 39+ (4) [50].
A recent study was done to evaluate the prevalence of
U.S. IVF clinics willing to act in accordance with the guidelines and led to additional interesting findings [51]. First, a
majority of U.S. IVF clinics (94% of those responding to the
survey) would follow the guidelines. Second, certain situations were likely to result in deviation from the guidelines;
those identified from the study include patient request, IVF
procedures involving the transfer of frozen embryos, and
IVF cycles preceded by a failed previous IVF attempt. Third,
a higher than expected number of patients (who had optimal
conditions for single embryo transfer) with insurance coverage had deviated from the guidelines and had a double embryo transfer. Fourth, clinicians are not likely to communicate the single embryo transfer policy to their patients, only
the risks involved with multiple pregnancies. These points
indicate a potential for improvement of the guidelines to take
patients’ and clinicians’ concerns into consideration in response to social, economic, and financial pressures. After the
publication of the ASRM-SART guidelines, the number of
higher order multiple deliveries in the United States declined
during subsequent years indicating a voluntary adoption
of single (or double) blastocyst transfers by IVF clinicians,
embryologists, and patients [52]. The data reveals a decrease
in the mean number of embryos transferred in women <35
years of age decreased from 3.12 in 1996 to 2.18 in 2003.
During the same period, IR and delivery rate increased from
Embryo Grading and Blastocyst Grading
IVF technique has long included embryo grading to aid
in selection of the most excellent quality embryos for implantation for the best results. Good embryo quality is associated with a high IVF success rate [53]. Embryos can be
graded at various stages (e.g. pronuclear, zygote, cleavage,
and blastocyst) by taking into account various markers,
including blastomere morphology, developmental rate,
fragmentation, metabolic markers, genetic markers, and
epigenetic markers [54]. Here we will focus on blastocyst
grading.
The two most popular blastocyst embryo grading systems
are the Dokras and Gardner grading system, both based on
morphology [14, 55-57]. Dokras grading is based on the
blastocoele’s rate of development and characteristics of the
blastocoele cavity; whereas Gardner’s system focuses on
blastocoele size and developmental characteristics of the
inner cell mass and trophectoderm. In the Dokras system,
blastocysts are graded as BG1, BG2, or BG3. BG1 and BG2
have been shown to have higher IRs and have been recommended for SBT [53, 58].
According to the Gardner grading system, the blastocoele
size, degree of expansion, and hatching status is initially
examined and graded from 1-6. Next, the blastocysts graded
3 through 6 are identified and their inner cell mass and trophectoderm are further graded.
Studies have shown that transfer one blastocyst with
grades 3AA using the Gardner system has had a significantly higher IR (54.3%) and higher clinical PR (69.6%)
than blastocysts with 3AA [56]. A more recent study by
Urman et al. had similar results when two blastocysts graded
as 3AA were transferred [54]. Finally, a randomized study
comparing the two grading systems illustrates a superior
Gardner system for predicting blastocysts with a higher
chance of implantation (37.6% vs. 25%, P=0.01) and clinical
pregnancy (66.7 % vs. 53.0%, P=0.11) than the Dokras
system. According to this study, both are valuable predictors
for selecting blastocysts with high implantation potential
and to implement elective SBT strategy to avoid multiple
pregnancies [59].
In some countries where blastocyst selection is illegal
(Germany), scoring is most effective when completed as
close to latest stage permitted for selection. Zygote scoring
for obtaining good quality blastocysts has not been promising [60-64]. The scoring system by Zollner et al. included an
examination of the pronuclear zygote for the following: (i)
number and size of pronuclei, (ii) juxtaposition of pronuclei,
(iii) halo effect, (iv) alignment and number of pronuclei
(evaluated separately) and nucleoli, (v) appearance of
vacuoles, (vi) and appearance of ooplasm. Each criteria was
evaluated and assigned a score with 10 being the best and 31
the worst. A pronuclear score of >15 correlated with poor
blastocyst development and a decrease in PR [62]. Even
though the study showed a correlation between pronuclear
morphology and blastocyst development, it was not strong
enough to identify the highest quality embryos with high
IRs.
The pronuclear scoring system by Scott and Smith (1998)
and Tesarik and Greco (1999) continue to be the most widely
used methods. Scott and Smith’s is based on the combination
of two scores, one for position of the pronuclei and the other
score for cytoplasmic appearance when two pronuclei and
two polar bodies are present [61]. Tesarik and Greco’s is
based on the appearance and distribution of the nucleolar
precursor bodies within the pronuclei and scored between 0
and 5 [63]. A score of 0 is associated with normal development, and all other patterns are considered irregular. The use
of scoring systems with blastocyst culture and transfer is a
valuable tool for the selection of an embryo with a higher
probability to be normal and result in a live birth, but is not
always reliable and should be pursued with caution. Patients
should be informed of the circumstances prior to their commitment.
Patients’ Clinical Conditions
Most studies practiced SBT in patients not more than 37
years old [27, 30, 34]. However, a couple of studies practiced SBT in patients with ages up to 43 years [31, 35]. Kalu
et al. compared PR after SBT in patients age 25-37 years and
38-43 years: 73.8% and 47.1%, respectively [31]. This study
suggested that SBT could result in better pregnancy outcomes in young women. Furthermore, most studies are restricted to patients who had a good prognosis. In addition to
young age, the good prognosis criteria often included no
previous history of failed IVF cycles, no moderate or severe
endometriosis, at least three (some studies at least five [34]),
blastocysts available for transfer, and normal uterine cavity,
etc [27, 30, 32]. However, Trout et al. also reported results in
patients at risk of ovarian hyperstimulation syndrome
(OHSS). Seven cases of SBT in women at risk of OHSS had
an ongoing PR of 57% [65].
PATIENTS’ PERCEPTIONS TOWARDS SBT
Patient Perceptions Differ from Clinicians
The patient’s perception of blastocyst culture and transfer
of one, two, or other multiples of embryos is important to
consider. It aids the clinician’s communication with couples
and improves patient satisfaction. In one study, men and
women were interviewed separately to remove influence
from their partners, and each was asked about their decisionmaking process in deciding on single or double blastocyst
transfer [66]. Several interesting points resulted from the
study. First, patients who chose a double transfer perceived a
higher chance of pregnancy. Second, patients who chose a
single transfer say it was a much more difficult decision than
deciding on a double transfer. Those that tended to select the
single transfer had similar characteristics, including: (i) having experienced a prior childbirth, (ii) being young, (iii) having no previous failed IVF treatment, and (iv) having spare
embryos to freeze. Thirdly, patients who were more likely to
accept a double transfer and perceive it as more desirable
were older, had no spare embryos to freeze, or perceived a
single embryo transfer might lower chances of pregnancy per
223
transfer based on evidence either from a study or a previously failed IVF attempt [66].
In another study, de Lacey et al. identified three factors
influencing the decision for a SBT, including repeated treatment, advanced age, and urgency to become pregnant [67].
This may be in relation to patients’ perception that the risk of
maternal and neonatal complications is low to moderate in
association with single or double transfers, despite being
fully informed of the risks [67]. Several studies also support
findings that patients are willing to accept a double transfer
along with the maternal and neonatal risks involved to have
children [66-68]. Thus, awareness of complications is not a
deterring factor in the decision for SBT, and so identifying
the points that influence patient decisions is important. Perhaps patients are more willing to accept SBT if there are no
financial pressures in case of a failed cycle of SBT [27]. For
this reason, determining what factors will motivate patients
to accept SBT over DBT in particular circumstances will be
important.
Business interests do not always coincide with patients’
best interests, and so clinicians may hesitate to decrease the
number of blastocysts to transfer despite the risk for unhealthy babies being born [69]. However, a recent study by
Stern et al. assessed the impact of embryo transfer guidelines
designed to minimize multiple births by lowering the number
of embryos transferred in IVF clinics. It reported a decrease
in the number of embryo transfers and proved the effectiveness of voluntary reduction of blastocyst transfer [52]. Not
surprisingly, this also has led to a decrease in the number of
higher order multiple births.
Patient Education is Essential for SBT
Ryan et al. reported the results of an educational campaign with a two-fold process: First, a one-page description
of the comparative risks of twins versus singletons to maternal, fetal, and neonatal health was given; second, this page
was then discussed between the couples and physicians [26].
Pre- and post- educational campaign questionnaires (n=110)
were collected and analyzed. Results showed that significantly more subjects ranked singletons as their most-desired
treatment outcome post-educational campaign compared
with pre-educational campaign (86% vs. 69%, p<0.001).
This observation suggests that an improved understanding of
twin risks appeared to affect patients’ declared desire for
numbers of embryos to transfer and for twin pregnancies.
Stillman et al. demonstrated the effect of a policy recommending SBT to patients identified as high risks for twins
[27]. In this study, an educational program for physician,
staff, and patient also was implemented to increase the program-wide proportion of SBT. This education program for
patients constitutes laminated sheets for each physician office outlining the recommendation criteria, statistics, and
risks regarding SBT and DBT. In addition to online education and in-person consulting with patients, the sheets also
were hung in each physician office. The education program
for physicians and staff took place periodically at physician
and staff seminars. Results have shown that the programwide number of embryos transferred was decreased significantly post-policy (n=2923) compared to pre-policy
(n=1556) (2.16±0.84 vs. 2.45±0.84, p<0.003). This observa-
tion suggested that patient education is an important factor
that could pave the future for SBT among good-prognosis
patients.
EXPERT COMMENTARY
The purpose of this article was to discuss various aspects
regarding the current practice of single blastocyst transfer.
Human blastocyst transfer has been evolving in the recent
decade since the successful development of blastocyst culture and transfer techniques. Given the unwanted high multiple pregnancies resulting from multiple embryo transfer and
multiple blastocyst transfer, single blastocyst transfer was
proposed to be a prospective method to resolve this problem.
Single blastocyst transfer was found to significantly decrease
the number of multiple pregnancies while maintaining desirable pregnancy outcomes. A number of recent studies have
demonstrated the potential role of single blastocyst transfer
in young and good prognosis women.
Baw et al.
KEY ISSUES
• Multiple embryo transfer was successful in terms of
pregnancy rates; however, it resulted in unwanted high
multiple pregnancy rates.
• Increased number of studies revealed that single embryo
transfer would be beneficial for young patients with good
prognosis.
• Two types of culture systems have been developed and
are widely available for culturing blastocysts: single media and sequential media. The single culture system was
just as good as or better than the sequential media system
when it is properly formulated with all the required nutrients.
• Multiple blastocyst transfer was able to generate satisfying implantation rates and pregnancy rates; however, it
resulted in the same problem encountered in single embryo transfer—high multiple pregnancy rates.
Single blastocyst transfer and cryopreserved single blastocyst transfer in the IVF are still in their infancy. The guidelines for number of blastocysts being transferred and the
techniques have not yet been standardized, and a method to
estimate the most viable blastocyst has not yet been proposed. The success of single blastocyst transfer is highly
dependent on the technique and patients’ and clinicians’ perceptions toward it. Further research is required to provide
more data on single blastocyst transfer in different patient
groups to standardize the practice based on different patient
variables.
• The only truly effective means by which to avoid multiple pregnancies is believed to be transfer of a single embryo or blastocyst.
FIVE-YEAR VIEW
• Determining for certain which blastocyst will implant is
not possible; however, many grading methods, including
morphology assessment could provide possible information on the blastocyst’ potential to implant.
The practice of single embryo transfer is less common in
the United States than in Europe. One reason for this has
been the concern that transferring two or more embryos
would lead to higher birth rates [12]. However, with more
robust studies proving the effectiveness and the advantage of
single blastocyst transfer [3], the unresolved problem of the
optimal number of embryos to transfer is about to be debunked. Although the success of single blastocyst transfer
has been overwhelming in controlled studies, it will require
more mature blastocyst culture, grading, and cryopreservation techniques before gaining wide clinical acceptance.
Clinics need to assess their own available techniques, laboratory conditions, and patients’ willingness to participate in
single blastocyst transfer.
Traditionally, the success of blastocyst transfer was determined by comparing implantation rates and pregnancy
rates. However, there has been a shift in focus to a single live
birth rate as the variable to be compared. In addition to patient characteristics, including age, BMI, and clinical conditions that need to be reported, the single live birth rate also
should be discussed in future studies to aid comparisons
across studies. Given more and more studies discussing the
effects of single blastocyst transfer in a variety of patient
populations, generating a standardized guideline for practitioners may be possible in the near future. It also is hoped
that many proposed blastocyst assessment methods can be
improved to more accurately predict the most viable blastocyst to transfer.
• Frozen single blastocyst transfer has been developed as a
practical method to optimize pregnancy outcomes following the possible failed initial transfer or to be employed
for patients with inappropriate initial transfer conditions.
• An improvement of current guidelines for blastocyst
transfer is possible when taking patients’ and clinicians’
concerns into consideration in response to social, economic, and financial pressures.
• The patient’s perception of blastocyst culture and transfer
of one, two, or other multiples of blastocysts is important
to consider. Physicians should communicate with patients
to educate them on the pros and cons of various blastocyst transfer choices.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
Steptoe PC, Edwards RG. Birth after the reimplantation of a human
embryo. Lancet 1978; 2: 366.
Van Voorhis BJ. In vitro fertilization. N Engl J Med 2007; 356:
379-86.
Papanikolaou EG, Camus M, Kolibianakis EM, Van Landuyt L,
Van Steirteghem A, Devroey P. In vitro fertilization with single
blastocyst-stage versus single cleavage-stage embryos. N Engl J
Med 2006; 354: 1139-46.
Johnson N, Blake D, Farquhar C. Blastocyst or cleavage-stage
embryo transfer? Best Pract Res Clin Obstet Gynaecol 2007; 21:
21-40.
Hreinsson J, Rosenlund B, Fridström M, et al. Embryo transfer is
equally effective at cleavage stage and blastocyst stage: a
randomized prospective study. Eur J Obstet Gynecol 2004; 117:
194-200.
Wilson M, Hartke K, Kiehl M, Rodgers J, Brabec C, Lyles R.
Integration of blastocyst transfer for all patients. Fertil Steril 2002;
77: 693-6.
Frattarelli JL, Leondires MP, McKeeby JL, et al. Blastocyst
transfer decreases multiple pregnancy rates in in vitro fertilization
cycles: a randomized controlled trial. Fertil Steril 2003; 79: 22830.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J,
Hesla J. A prospective randomized trial of blastocyst culture and
transfer in in-vitro fertilization. Hum Reprod 1998; 13: 3434-40.
Fauque P, Jouannet P, Davy C, et al. Cumulative results including
obstetrical and neonatal outcome of fresh and frozen-thawed cycles
in elective single versus double fresh embryo transfers. Fertil Steril
2009 [Epub ahead of print].
Schwarzler P, Zech H, Auer M, et al. Pregnancy outcome after
blastocyst transfer as compared to early cleavage stage embryo
transfer. Hum Reprod 2004; 19: 2097-102.
Luke B, Brown MB, Grainger DA, Cedars M, Klein N, Stern JE.
Practice patterns and outcomes with the use of single embryo
transfer in the United States. Fertil Steril 2010;93(2): 490-8.
Schieve LA, Schieve LA. The promise of single-embryo transfer. N
Engl J Med 2006; 354: 1190-3.
Gardner DK, Lane M, Schoolcraft WB. Physiology and culture of
the human blastocyst. J Reprod Immunol 2002; 55: 85-100.
Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F,
Meldrum DR. Blastocyst culture and transfer: analysis of results
and parameters affecting outcome in two in vitro fertilization
programs. Fertil Steril 1999; 72: 604-9.
Gardner DK, Lane M. Culture and selection of viable blastocysts: a
feasible proposition for human IVF? Hum Reprod Update 1997; 3:
367-82.
Gardner DK, Lane M, Schoolcraft WB. Culture and transfer of
viable blastocysts: a feasible proposition for human IVF. Hum
Reprod 2000; 15(Suppl 6): 9-23.
Menezo YJ, Hamamah S, Hazout A, Dale B. Time to switch from
co-culture to sequential defined media for transfer at the blastocyst
stage. Hum Reprod 1998; 13: 2043-4.
Staessen C, Platteau P, Van Assche E, et al. Comparison of
blastocyst transfer with or without preimplantation genetic
diagnosis for aneuploidy screening in couples with advanced
maternal age: a prospective randomized controlled trial. Hum
Reprod 2004; 19: 2849-58.
Blake D, Farquhar C, Johnson N, Proctor M. Cleavage stage versus
blastocyst stage embryo transfer in assisted conception. In:
Cochrane Database of Systematic Reviews. Chichester, UK: John
Wiley & Sons, Ltd, 2007.
Marek D, Langley M, Gardner DK, Confer N, Doody KM, Doody
KJ. Introduction of blastocyst culture and transfer for all patients in
an in vitro fertilization program. Fertil Steril 1999; 72: 1035-40.
Reed ML, Hamic A, Thompson DJ, Caperton CL. Continuous
uninterrupted single medium culture without medium renewal
versus sequential media culture: a sibling embryo study. Fertil
Steril 2009; 92: 1783-6.
Sepulveda S, Garcia J, Arriaga E, Diaz J, Noriega-Portella L,
Noriega-Hoces L. In vitro development and pregnancy outcomes
for human embryos cultured in either a single medium or in a
sequential media system. Fertil Steril 2009; 91: 1765-70.
Milki AA, Fisch JD, Behr B. Two-blastocyst transfer has similar
pregnancy rates and a decreased multiple gestation rate compared
with three-blastocyst transfer.[see comment]. Fertil Steril 1999; 72:
225-8.
Jain JK, Boostanfar R, Slater CC, Francis MM, Paulson RJ.
Monozygotic twins and triplets in association with blastocyst
transfer. J Assist Reprod Genet 2004; 21: 103-7.
Yamamoto S, Umeki M, Hamano T, Matsusita F, Kuwahara K.
Elective cryopreservation of all day 5 blastocysts is more effective
than using day 6 blastocysts for improving pregnancy outcome in
stimulated cycles. Reprod Med Biol 2008; 7: 75-83.
Ryan GL, Sparks AET, Sipe CS, Syrop CH, Dokras A, Van
Voorhis BJ. A mandatory single blastocyst transfer policy with
educational campaign in a United States IVF program reduces
multiple gestation rates without sacrificing pregnancy rates. Fertil
Steril 2007; 88: 354-60.
Stillman RJ, Richter KS, Banks NK, Graham JR. Elective single
embryo transfer: A 6-year progressive implementation of 784
single blastocyst transfers and the influence of payment method on
patient choice. Fertil Steril 2009; 92(6): 1895-1906.
Kogan MD, Alexander GR, Kotelchuck M, et al. Trends in twin
birth outcomes and prenatal care utilization in the United States,
1981-1997. JAMA 2000; 284: 335-41.
Scher AI, Petterson B, Blair E, et al. The risk of mortality or
cerebral palsy in twins: a collaborative population-based study.
Pediatr Res 2002; 52: 671-81.
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
225
Criniti A, Thyer A, Chow G, Lin P, Klein N, Soules M. Elective
single blastocyst transfer reduces twin rates without compromising
pregnancy rates. Fertil Steril 2005; 84: 1613-9.
Kalu E, Thum MY, Abdalla H. Reducing multiple pregnancy in
assisted reproduction technology: towards a policy of single
blastocyst transfer in younger women. BJOG 2008; 115: 114350.
Henman M, Catt JW, Wood T, Bowman MC, de Boer KA, Jansen
RPS. Elective transfer of single fresh blastocysts and later transfer
of cryostored blastocysts reduces the twin pregnancy rate and can
improve the in vitro fertilization live birth rate in younger women.
Fertil Steril 2005; 84: 1620-7.
Mullin CM, Fino ME, Talebian S, Krey LC, Licciardi F, Grifo JA.
Comparison of pregnancy outcomes in elective single blastocyst
transfer versus double blastocyst transfer stratified by age. Fertil
Steril 2010; 93(6): 1837-43.
Styer AK, Wright DL, Wolkovich AM, Veiga C, Toth TL. Singleblastocyst transfer decreases twin gestation without affecting
pregnancy outcome. Fertil Steril 2008; 89: 1702-8.
Gardner DK, Surrey E, Minjarez D, et al. Single blastocyst
transfer: a prospective randomized trial. Fertil Steril 2004; 81: 5515.
Guerif F, Lemseffer M, Bidault R, et al. Single Day 2 embryo
versus blastocyst-stage transfer: a prospective study integrating
fresh and frozen embryo transfers. Hum Reprod 2009; 24: 10518.
da Costa AA, Abdelmassih S, de Oliveira FG, et al. Monozygotic
twins and transfer at the blastocyst stage after ICSI. Hum Reprod
2001; 16: 333-6.
Papanikolaou EG, Fatemi H, Venetis C, et al. Monozygotic
twinning is not increased after single blastocyst transfer compared
with single cleavage-stage embryo transfer. Fertil Steril 2010;
93(2): 592-7.
Yanaihara A, Yorimitsu T, Motoyama H, Watanabe H, Kawamura
T. Monozygotic multiple gestation following in vitro fertilization:
analysis of seven cases from Japan. J Exp Clin Assist Reprod 2007;
4: 4.
Lee SF, Chapman M, Bowyer L. Monozygotic triplets after single
blastocyst transfer: Case report and literature review. Aust N Z J
Obstet Gynaecol 2008; 48(6): 583-6.
Stehlik E, Stehlik J, Katayama KP, et al. Vitrification demonstrates
significant improvement versus slow freezing of human
blastocysts. Reprod Biomed Online 2005; 11: 53-7.
Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at
-196 degrees C by vitrification. Nature 1985; 313: 573-5.
Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M,
Takahashi K. Successful birth after transfer of vitrified human
blastocysts with use of a cryoloop containerless technique. Fertil
Steril 2001; 76: 618-20.
Varghese AC, Nagy ZP, Agarwal A. Current trends, biological
foundations and future prospects of oocyte and embryo
cryopreservation. Reprod Biomed Online 2009; 19: 126-40.
Behr B, Gebhardt J, Lyon J, et al. Factors relating to a successful
cryopreserved blastocyst transfer program. Fertil Steril 2002; 77:
697-9.
Desai N, Goldfarb J. Examination of frozen cycles with
replacement of a single thawed blastocyst. Reprod Biomed Online
2005; 11: 349-54.
Mukaida T, Nakamura S, Tomiyama T, et al. Vitrification of
human blastocysts using cryoloops: clinical outcome of 223 cycles.
Hum Reprod 2003; 18: 384-91.
Yanaihara A, Yorimitsu T, Motoyama H, Ohara M, Kawamura T.
Clinical outcome of frozen blastocyst transfer; single vs. double
transfer. J Assist Reprod Genet 2008; 25: 531-4.
Guidelines on number of embryos transferred. Fertil Steril 2009;
92: 1518-9.
Min JK, Claman P, Hughes E. Guidelines for the number of
embryos to transfer following in vitro fertilization. J Obstet
Gynaecol Can 2006; 28: 799-813.
Jungheim ES, Ryan GL, Levens ED, et al. Embryo transfer
practices in the United States: a survey of clinics registered with
the Society for Assisted Reproductive Technology. Fertil Steril
2009 [Epub ahead of print].
Stern JE, Cedars MI, Jain T, et al. Assisted reproductive
technology practice patterns and the impact of embryo transfer
guidelines in the United States. Fertil Steril 2007; 88: 275-82.
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
Baw et al.
Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R.
Blastocyst quality affects the success of blastocyst-stage embryo
transfer. Fertil Steril 2000; 74: 282-7.
Urman B, Yakin K, Ata B, Balaban B. How can we improve
current blastocyst grading systems? Curr Opin Obstet Gynecol
2007; 19: 273.
Dokras A, Sargent IL, Barlow DH. Human blastocyst grading: an
indicator of developmental potential? Hum Reprod 1993; 8: 211927.
Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB.
Blastocyst score affects implantation and pregnancy outcome:
towards a single blastocyst transfer. Fertil Steril 2000; 73: 1155-8.
Gardner DK, Schoolcraft WB. Culture and transfer of human
blastocysts. Curr Opin Obstet Gynecol 1999; 11: 307-11.
Balaban B, Urman B, Isiklar A, et al. The effect of pronuclear
morphology on embryo quality parameters and blastocyst transfer
outcome. Hum Reprod 2001; 16: 2357-61.
Balaban B, Yakin K, Urman B. Randomized comparison of two
different blastocyst grading systems. Fertil Steril 2006; 85: 559-63.
Wittemer C, Bettahar-Lebugle K, Ohl J, Rongieres C, Nisand I,
Gerlinger P. Zygote evaluation: an efficient tool for embryo
selection. Hum Reprod 2000; 15: 2591-7.
Scott LA, Smith S. The successful use of pronuclear embryo
transfers the day following oocyte retrieval. Hum Reprod 1998; 13:
1003-13.
Zollner U, Zollner KP, Hartl G, Dietl J, Steck T. Embryology. The
use of a detailed zygote score after IVF/ICSI to obtain good quality
[63]
[64]
[65]
[66]
[67]
[68]
[69]
blastocysts: the German experience. Hum Reprod 2002; 17: 132733.
Tesarik J, Greco E. The probability of abnormal preimplantation
development can be predicted by a single static observation
on pronuclear stage morphology. Hum Reprod 1999; 14: 131823.
Payne D, Flaherty SP, Barry MF, Matthews CD. Preliminary
observations on polar body extrusion and pronuclear formation in
human oocytes using time-lapse video cinematography. Hum
Reprod 1997; 12: 532-41.
Trout SW, Bohrer MK, Seifer DB. Single blastocyst transfer in
women at risk of ovarian hyperstimulation syndrome. Fertil Steril
2001; 76: 1066-7.
Blennborn M, Nilsson S, Hillervik C, Hellberg D. The couple's
decision-making in IVF: one or two embryos at transfer? Hum
Reprod 2005; 20: 1292-7.
de Lacey S, Davies M, Homan G, Briggs N, Norman RJ.
Factors and perceptions that influence women's decisions to have a
single embryo transferred. Reprod Biomed Online 2007; 15: 52631.
Scotland GS, McNamee P, Peddie VL, Bhattacharya S. Safety
versus success in elective single embryo transfer: women's
preferences for outcomes of in vitro fertilisation. BJOG 2007; 114:
977-83.
Gurgan T, Demirol A. Why and how should multiple pregnancies
be prevented in assisted reproduction treatment programmes?
Reprod Biomed Online 2004; 9: 237-44.
227
Sajal Gupta, Lucky Sekhon, Yesul Kim and Ashok Agarwal*
Center for Reproductive Medicine, Glickman Urological & Kidney Institute and Department of ObstetricsGynecology, Cleveland Clinic, Cleveland, OH, 44195, USA
Abstract: Aim: Oxidative stress contributes to the high rate of failure seen in assisted reproductive techniques in
achieving fertilization and pregnancy. Many studies have been done to elucidate the sources of oxidative stress in the
setting of assisted reproductive technology (ART) and interventions to overcome its negative influence on the outcome
of IVF and ICSI. This article explores the utility of metabolomics as a novel, non-invasive method of accurately and
efficiently quantifying oxidative stress. The aim of this study was to review the current literature on the effects of various
interventions, including the use of antioxidants supplementation of IVF culture media and patients to improve fertilization
and pregnancy rates in subfertile patients undergoing ART.
Methods: Review of recent publications through Pubmed and the Cochrane data base.
Results: Oxidative stress is correlated with negatives ART outcomes. Both exogenous and endogenous sources of reactive
oxygen species during IVF/ICSI are well established in the literature. Compared to IVF, ICSI is known to minimize
the exposure of gametes to endogenous sources of oxidative stress. Strategies to control exogenous sources of oxidative
stress within the ART setting include reducing visible/near UV light exposure, the addition of metal chelators to culture
media, maintenance of low oxygen tension in the environment and the use of antioxidant therapy. Antioxidant supplementation of culture media with vitamin C, vitamin E, and melatonin has been investigated and yielded conflicting results.
Whereas oral antioxidant supplementation of male patients has been accepted and is currently practiced, there is a lack of
consensus regarding the effectiveness of supplementation of vitamin C, vitamin E and melatonin in females undergoing
ART.
Conclusion: There is a need for further investigation with randomized controlled studies to confirm the efficacy and
safety of antioxidant supplementation of culture media and patients as well as the need to determine the dosage required to
improve fertilization rates and pregnancy outcome with IVF/ICSI.
Keywords: In vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), infertility, oxidative stress, antioxidants,
metabolomics.
INTRODUCTION
Infertility is a universal issue, affecting millions of men
and women in both developed and developing areas of the
world. Recent advances in the field of reproductive medicine
may allow for a select group of infertile couples to conceive
and bear offspring by utilizing assisted reproductive techniques (ART). However, any hope offered by these procedures is limited by persistently poor fertility outcomes. In
2003, a report by the Society for Assisted Reproductive
Technology conceded that only 35% of ART transfer procedures result in a live birth delivery. A myriad of physiological and environmental factors are known to influence the
success rates of ART in achieving fertilization and pregnancy culminating in living offspring. Reactive oxygen species (ROS) have been widely implicated in the literature to
render a significant impact on these outcomes.
ART methods such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are designed with a
Medicine Glickman Urological & Kidney Institute and Department of
Obstetrics & Gynecology, Cleveland Clinic, 9500 Euclid Avenue, Desk
A19.1, Cleveland, OH 44195, USA; Tel: (216) 444-9485;
Fax: (216) 445-6049; E-mail: [email protected]
1573-4048/10 $55.00+.00
view to emulate the process of natural, in vivo reproduction.
However, the in vitro environment exposes gametes and embryos to an excess of reactive oxygen species not normally
experienced during in vivo fertilization and pregnancy. Although reactive oxygen species are physiologically
required for various biochemical pathways necessary for
reproduction, their in vivo levels are tightly controlled by
enzymatic antioxidants that scavenge and neutralize free
radicals to maintain an optimal, physiologic oxygen tension
in the male and female reproductive system.
In contrast, in vitro ART procedures are carried out
without the protection of enzymatic antioxidants normally
found in vivo, leading to unopposed, elevated levels of ROS,
which have been shown to adversely affect gametes, gamete
interaction, fertilization and pregnancy rates. Given the
impact of redox status on ART outcomes, it is crucial to elucidate the mechanism by which ROS are generated, methods
to accurately quantify ROS levels, and strategies to regulate
the amount of ROS experienced to enhance embryo quality,
promote single embryo transfer and reduce multiple
pregnancy rates with ART.
Poor fertility outcomes with ART are particularly problematic when the high risk of failure in achieving reproduction is placed in an economic context. The high cost of ART
coupled with relatively low success rates is especially unacceptable in the developing world, where resources are scarce
and a high prevalence of infertility endures. Thus, it is imperative to investigate new strategies to improve the rate of
successful outcomes in ART. The aim of this discussion is to
clarify what is currently known regarding the role oxidative
stress plays in ART and potential ways to alleviate the
effects of oxidative stress, with specific interest in the use of
antioxidants to optimize ART outcomes.
WHAT IS ART?
Assisted reproductive techniques can serve as an effective method to overcome a variety of causative factors of
infertility such as pathology of the fallopian tubes, endometriosis, and male factor and unexplained infertility [1]. The
primary focus of this review is centered on the effect of oxidative stress on the outcome of in vitro ART procedures and
the potential role of antioxidants to protect against oxidative
stress to increase the likelihood of achieving successful
pregnancy with ART.
Intrauterine Insemination
Intrauterine insemination (IUI) is performed by threading
a very thin, flexible catheter through the cervix and injecting
washed spermatozoa into the uterus. This technique requires
large numbers of forward-progressing motile spermatozoa
[2]. Moreover, higher sperm counts increase the success
rates of this procedure. Therefore, male infertility patients
with a low number of spermatozoa are not suitable candidates for IUI. In addition, this method is not suitable for semen samples high in free radicals due to the presence of
large number of leukocytes, cellular debris, and/or immature
germ cells [2].
In vitro Fertilization
(IVF) is another assisted reproductive technique in which
sperm-oocyte interactions take place within culture media,
leading to fertilization. Most IVF cycles utilize fertility drugs
such as GnRH agonists and human menopausal gonadotropins to stimulate the ovaries to produce several mature eggs,
in addition to the the single egg normally produced each
month. If ovarian stimulation is not done, the oocyte may
simply be retrieved from the natural menstrual cycle, avoiding any risk of overstimulation and multiple pregnancy. The
oocytes are collected into a specially prepared culture medium, which then is microscopically evaluated for maturity.
Between 20,000-30,000 sperm are mixed with each oocyte in
a drop of specially prepared culture medium and then incubated to ensure an optimal environment to facilitate fertilization. After fertilization is complete, the resulting embryos are
qualitatively graded on the basis of morphology, and those
chosen for transfer are loaded in a minute volume of medium
into a transfer catheter. A catheter tip is advanced into the
uterus and the embryos are expelled. The pregnancy rate
increases with the number of embryos transferred.
This treatment is primarily for female-related infertility
problems such as hydrosalpinges, damaged or inoperable
fallopian tubes, endometriosis, and cervical mucus pathology. Idiopathic infertility, male infertility and immunologic
infertility also respond well to IVF. A major disadvantage of
Gupta et al.
IVF is the need for women to undergo ovarian stimulation
with hCG or progesterone hormonal therapy to support the
luteal phase, which can lead to ovarian hyperstimulation
syndrome- the most common and potentially serious complication for conventional IVF treatment [3].
Intracytoplasmic Sperm Injection
Intracytoplasmic sperm injection (ICSI) is a laboratory
procedure in which a single sperm is injected directly into an
oocyte’s cytoplasm using a very fine needle. The process
allows for oocyte fertilization regardless of the morphology
and motility characteristics of the single spermatozoon injected [4]. This procedure is used in cases in which unsuccessful conventional IVF led to fertilization failure or in
instances where a male-factor such as low sperm count is
involved [4]. This procedure is also used for non-obstructive
azoospermic patients, in which sperm may be retrieved using
testicular sperm aspiration [5].
ICSI is particularly beneficial in infertile males with
disordered sperm-zona pellucida interactions, in that it
bypasses all of the preliminary penetration and fusion steps
of fertilization. Despite these advantages, the injected sperm
in ICSI are at an increased risk for having oxidatively
damaged DNA which could adversely affect the outcome.
ICSI circumvents the process of natural selection and may
allow damaged spermatozoon to be directly injected into the
oocyte, which can negatively impact embryo development.
IVF is superior to ICSI with respect to avoiding this risk,
as collateral peroxidative damage to the sperm plasma
membrane will prevent fertilization by spermatozoa with
DNA damage [2].
Similarities Between IVF and ICSI
Both IVF and ICSI are very costly procedures not covered by most health plans. They require special facilities and
are subject to many regulatory restrictions. Although these
procedures provide the possibility of a potential cure for infertile couples, there are many weaknesses and factors that
often detract from successful reproductive outcomes. As in
vitro procedures, the environment in which IVF and ICSI are
conducted fails to mimic the intricate physiological conditions of an in vivo system. These methods are greatly affected by environmental factors without beneficial protective
factors such as antioxidants provided by the in vivo environment. Measures aimed at minimizing differences between
in vitro and physiologic in vivo conditions should be investigated and employed to maximize the efficiency of ART procedures. This article will review the role of oxidative stress
in both conventional IVF and ICSI and approaches to optimize the chance of achieving successful pregnancy with
these procedures.
WHAT IS OXIDATIVE STRESS?
Aerobic metabolism generates reactive oxygen specieshydroxyl radicals, superoxide anion, hydrogen peroxide, and
nitric oxide. They are small and highly reactive due to unpaired valence shell electrons that are capable of initiating an
uncontrolled cascade of chain reactions. Normally, this is
regulated by antioxidants, which have the ability to scavenge
and neutralize free radicals [2]. The controlled production of
such compounds is known to play a role in physiological
reproductive processes such as hormone signaling, oocyte
maturation, folliculogenesis, tubal function, ovarian steroidogenesis, cyclical endometrial changes, and germ cell function. However, at higher levels ROS may overhwhelm antioxidant capacity, leading to oxidative stress. The high energy
electrons of ROS are capable of modulating gene expression
and transcription factors, with the ability to modify and
damage DNA [2].
Intracellular homeostasis is achieved through a balance
between pro-oxidant compounds and antioxidants. Antioxidants have the ability to oppose the effects of pro-oxidants
by hindering ROS production, scavenging ROS, and repairing cell damage caused by ROS. Non-enzymatic antioxidants
consist of vitamin C, taurine, hypotauring, cysteamine, and
glutathione. Enzymatic antioxidants include superoxide dismutase, catalase, and glutathione peroxidase. Within the
body, enzymatic antioxidants protect and support gametes
and embryos during fertilization and pregnancy from OSinduced pathological changes. IVF and ICSI procedures lack
these natural antioxidants and, therefore, expose gametes and
embryos to a level of OS higher than that experienced in vivo
during physiologic reproduction.
WHAT IS THE ROLE OF OXIDATIVE STRESS
IN ART?
OS exerts toxic effects by altering cellular molecules
such as lipids, proteins and nucleic acids. This can lead to an
increase in membrane permeability, loss of membrane integrity, enzyme inactivation, structural damage to DNA, mitochondrial alterations, adenosine triphosphate depletion, and
apoptosis. Free radicals are thought to act as a determinant in
reproductive outcome due to their effects on oocytes, sperm,
and embryos in their follicular fluid, tubal fluid, and peritoneal fluid microenvironments. Oocytes and embryos can be
protected against OS by free radical- scavenging antioxidants that exist within the follicular and oviductal fluid [6].
However, this defense against OS is lost as sperm, oocytes,
and embryos are removed from their natural microenvironments to be used for ART [6]. This loss of protection leads
to increased exposure of gametes and embryos to OS, which
can lead to impaired oocyte maturation and embryo development [7].
Oocyte quality is thought to act as the main determinant
of IVF outcomes [8]. Generally, higher quality oocytes develop into healthy, high quality embryos, ultimately resulting
in a higher birth rate [8]. The compound 8-OHdG acts as a
sensitive marker of OS-induced DNA damage in granulosa
cells during ovulation. A study by Seino et al. demonstrated
an association between increased levels of 8-OHdG and decreased fertilization rates and embryo quality. Thus, increased OS in the granulosa cell environment that surrounds
oocytes adversely affects the oocytes themselves, leading to
lower success rates for IVF [8]. Wang et al. conducted a
study exposing mouse embryos to PMA-activated leukocyte
supernatant which generated ROS. The findings of the study
relate increased OS levels with a decreased blastocyst development rate [6]. Thus, the literature implicates OS and its
deleterious effects in low fertilization rates with IVF and
ICSI. Therefore, a thorough analysis of the endogenous and
229
exogenous factors that contribute to the oxidative stress
faced by gametes and embryos during IVF and ICSI is
necessary to appropriately manipulate the in vitro culture
medium to minimize oxidative stress and improve ART
outcomes.
WHAT IS THE ORIGIN OF ROS IN ART?
Endogenous Factors Affecting Both IVF and ICSI:
Embryo Development
ROS play a significant physiological role in the modulation of gamete interaction and successful fertilization. Oocyte metabolism generates ROS. This, coupled with the lack
of protective antioxidant mechanisms that normally would
be found in vivo [9], results in an overall increase in OS in
the in vitro environment. Preimplantation embryonic development is accompanied by a change in preference in energy
metabolism pathways [10]. Oxidative phosphorylation is
utilized throughout the preimplantation embryo development
period. Increased energy demand is met by a significant shift
from dependence on oxidative phosphorylation to glycolysis
to generate ATP [7]. Oxygen is needed for oxidative phosphorylation to convert ADP to ATP, which is required for
the processes of folliculogenesis and oocyte maturation. This
use of oxygen results in ROS production, which in excess
has a toxic influence on embryo development [7]. ROS are
primarily produced at the inner mitochondrial membrane
where electrons leak from the electron transport chain and
are transferred to the oxygen molecule, resulting in oxygen
with an extra unpaired electron [10, 11]. In addition, cytoplasmic or membrane-bound NADPH-oxidase, cytochrome
p450 enzymes and the xanthine-xanthine oxidase systems
[10] are also capable of generating an excess of ROS.
Endogenous Factors that Differ Between IVF and ICSI
There are differences between the potential sources of
ROS derived from conventional IVF and ICSI [12] (Fig. 1).
For instance, oocytes used in ICSI are initially denuded of
their cumulus cells so that the only possible source of ROS is
within the culture media environment, the oocyte, and the
injected spermatozoa [9]. With IVF, however, ROS may
arise from the multiple oocytes per dish, the large cumulus
cell mass, and from the spermatozoa used for insemination.
Unlike IVF, ICSI avoids contact time between the sperm and
oocyte, thus minimizing the opportunity for ROS generation
by defective spermatozoa [9, 13]. Lower sperm concentrations in the culture media are correlated with an improvement in fertilization, implantation, and pregnancy rates and
with yielding higher quality embryos [14]. Thus the ICSI
procedure, which utilizes single spermatozoa at a time,
minimizes OS development from the gametes themselves.
Exogenous Factors Affecting Both IVF and ICSI
The external environment and culture media surrounding
embryos during both IVF and ICSI are believed to significantly influence the outcomes of these procedures. IVF and
ICSI do not significantly differ with respect to the level of
exposure of sperm, oocytes, and embryos to ROS [8]- aside
from the fact that the incubation time interval is shorter in
the ICSI procedure, decreasing the amount of exposure of
Gupta et al.
Fig. (1). Endogenous sources of oxidative stress in ART.
the culture media to external environmental factors [9, 13].
An incubation time limited to 1-2 hours has been associated
with better ART outcomes. With IVF and ICSI, ROS
may arise from the male or female gamete, the embryo, or
indirectly from the external environment, which includes
cumulus cells, leukocytes, and the culture media.
As fertilization and embryo development in vivo are
known to occur in an environment of low oxygen tension, it
is hypothesized that OS experienced in the in vitro setting is
a major contributor to the persistently high failure rate of
ART. Therefore, ART outcomes should improve if in vivo
conditions are emulated by avoiding the various factors that
promote ROS production.
OXYGEN CONCENTRATION
ROS may be derived from exogenous factors that impact
both IVF and ICSI. This effect may be more pronounced in
IVF due to longer incubation time intervals leading to increased exposure to environmental oxygen concentration. A
hyperoxic environment promotes enzyme activity, which
generates an increased level of the superoxide radical. The
growth of a first trimester embryo in vivo has been demonstrated to take place in a microenvironment with low oxygen
tension [15]. This leads to the assertion that a low oxygen
concentration is necessary during ART, which attempts to
mimic the conditions of in vivo fertilization and pregnancy
[15].
Studies have shown that embryos cultured in an
environment with atmospheric oxygen concentration and
exposed to increased levels of hydrogen peroxide radicals
resulted in DNA fragmentation of approximately 20% of
embryos, compared with DNA damage seen in 5% of embryos grown in an environment with low oxygen tension
[16]. Reports of successful blastocyst development with embryos cultured in a low oxygen tension environment [16]
imply that the oxygen concentration experienced in the in
vitro setting plays an influential role in the outcome of ART
procedures. A study by Noda et al. confirmed the advantage
of lower oxygen concentrations in the in vitro environment
by measuring OS levels encountered when using a new IVF
culture system with low oxygen tension and a low level of
illumination [17]. This manipulation of the culture media
resulted in high blastulation rates and enhanced human embryo development [17]. Another study found that, in addition
to improved blastocyst outcomes in low-oxygen environments (5%) versus high-oxygen concentrations (19%), human embryos cultured in low oxygen tension are associated
with an increased live birth rate compared with embryos
incubated under the influence of high oxygen concentrations
[18].
METALLIC CATIONS
In the setting of ART, metallic cations may promote ROS
generation and act as an exogenous source of OS. According
to the Haber-Weiss reaction, traces of metallic ions such as
iron or copper in the culture medium may lead to increased
ROS generation [19]. The binding of metallic ions by metal
chelators such as ethylenediamine tetra-acetic acid (EDTA)
or transferrin can inhibit the ability of metals to react and
produce ROS, suggesting their potential use within the embryo culture media to block the induction of ROS by metals
present in the in vitro setting. Use of the iron chelator transferrin within the culture media has been shown to decrease
lipid peroxidation and the formation of hydroxy radicals
[20]. Minimizing the production of highly toxic hydroxy
radicals is protective against free-radical-mediated damage
to oocytes and preimplantation embryos and can lead to improved fertilization and pregnancy rates [20].
ILLUMINATION
Visible light acts as an exogenous source of OS in that it
promotes ROS production, causing cellular damage through
the oxidation of DNA bases and DNA strand breaks [21].
The findings of a study by Beehler et al. implicated UV
radiation in ROS-induced DNA base modifications. Furthermore, Takenaka et al. showed that smaller amounts of
short-wavelength visible light are advantageous in ART as
they expose oocytes and embryos to lower levels of OS than
cool white light [22]. These results suggest that minimizing
the exposure of oocytes, zygotes, and embryos to visible
light and near-UV light will serve to better mimic in vivo
conditions, thereby yielding more successful ART outcomes
[22].
SPERMATOZOA
Sperm quality is a crucial determinant of the potential for
successful fertilization and pregnancy achieved by ART
[23]. At physiologic levels, ROS facilitates normal sperm
function such as the acrosome reaction, oocyte fusion, and
capacitation. Intracellularly, sperm generate ROS at the level
of their plasma membrane and mitochondria. Leukocytes act
as a source of OS by generating extracellular ROS in
prostatic and seminal vesicle secretions [23, 24]. Leukocytes
can cause ROS-induced damage when seminal plasma is
removed during sperm preparation for assisted reproduction.
Seminal leukocytes stimulate ROS production by spermatozoa.
Male germ cells are extremely vulnerable to OS as the
sperm membrane is rich in unsaturated fatty acids [23] and
lacks the capacity for DNA repair [25]. Human spermatozoa
generate superoxide radicals that can cause lipid peroxidation, which occurs in a self-propagating manner. This is associated with a decrease in membrane fluidity and reduced
activity of membrane and ion channels which impairs spermatozoan fertilization capacity. In addition to these effects,
OS arising from spermatozoa induce peroxidative damage to
the oocyte and its DNA, reducing the likelihood of successful fertilization [25]. A study conducted by Hammadeh et al.
demonstrated that metaphase oocytes incubated with DNAdamaged spermatozoa during IVF were associated with high
rates of failed fertilization, defective embryo development,
implantation failure and early abortion [26]. Therefore, utilizing sperm preparation techniques that minimize ROS production within seminal fluid and by spermatozoa themselves
is likely to improve the success of IVF and ICSI procedures.
Swim-up preparation is a sperm preparation technique in
which a semen sample is centrifuged to form pellets that are
coated with culture medium. The most motile spermatozoa
will swim up into the culture medium, and they are selected
for use in the IVF procedure. This technique is not appropriate for semen samples with excessive leukocytes or immature or damaged spermatozoa [9] as close contact with defective spermatozoa and leukocytes [11] will lead to ROSinduced damage of functional spermatozoa. It is hypothesized that the risk of OS-induced damage may be reduced by
incorporating the use of antioxidants in the swim-up technique [27]. ROS levels have been shown to be significantly
lower in sperm suspensions washed with antioxidants than in
the control group without antioxidant protection. Antioxidant
supplementation was seen to significantly decrease DNA
fragmentation rates in spermatozoa, whereas it had no effect
on lipid peroxidation. These results suggest that antioxidant
supplementation may serve a beneficial role in sperm preparation to improve ART outcomes by enhancing the overall
231
functional parameters of spermatozoa through reducing levels of oxidative stress [27].
Density-gradient centrifugation is a sperm preparation
technique used to isolate mature, leukocyte-free spermatozoa
[9]. This method employs centrifugation to separate fractions
of spermatozoa based on motility, size, and density [3]. The
process of centrifugation can activate seminal leukocytes and
disturb the redox balance within a semen sample, with the
potential to adversely affect sperm function [28]. A study by
Shekarriz et al. demonstrated that even a single-step centrifugation for a short period could significantly increase
ROS formation in human semen [28]. Increased levels of
ROS are also thought to arise during this type of sperm
preparation technique due to mechanical perturbation of the
sperm plasma membrane. Thus, minimizing centrifugation
time minimizes the formation of excess ROS and may ensure
the use of high quality sperm in ART [28].
In general, the literature confirms an inverse correlation
between ROS levels and sperm functional characteristics,
including density, motility, and morphology. Oxidative
stress is related to DNA fragmentation of spermatozoa that is
sub-optimal for use in IVF or ICSI [26]. Thus, strategies
used to prepare sperm and decrease ROS levels in a semen
sample may allow for an increased chance of successful fertilization and pregnancy with ART.
HOW ARE ROS LEVELS MEASURED?
Follicular Fluid
Oxidative stress is thought to occur within the follicular
fluid of women undergoing IVF and ICSI [25]. The follicular
fluid microenvironment plays an integral role in shaping the
quality of the oocyte, which is a major determinant of successful ART outcomes [1, 25]. The results of a study by Van
Blerkom et al. demonstrate that hypoxic conditions, as evidenced by low intrafollicular oxygenation, are related to an
increased likelihood of oocyte cytoplasmic defects such as
impaired cleavage and abnormal chromosomal segregation
[29]. The degree of OS can be quantified by assessing biomarkers of lipid peroxidation and the total antioxidant capacity (TAC) within follicular fluid [25]. The compound
malondialdehyde (MDA) is a by-product of lipid peroxide
decomposition and can be used to measure ROS levels.
Pasqualotto et al. discovered that the mean MDA levels of
pregnant women were significantly lower than the mean
MDA levels seen in non-pregnant control subjects [30]. In
addition to its role as a biomarker for OS, MDA levels may
be a predictive marker of ART outcome. Follicular fluid of
oocytes that were later successfully fertilized exhibited an
increase in TAC [1]. Thus, lower TAC, which would lead to
a redox inbalance with overwhelming levels of unopposed
ROS, can be related to decreased fertilization potential as an
adverse effect of OS.
A study by Wiener-Megnazi et al. used a novel thermochemiluminescence assay to assess the degree of OS in follicular fluid [31]. Increased levels of OS within follicular
fluid were significantly related to poor fertilization rates and
post-fertilization outcomes, manifested as decreased blastocyst cleavage and development, and a lower likelihood for
completion of a successful pregnancy [31].
Culture Fluid
Within the culture fluid, the TAC levels of Day 1 culture
media are postulated to serve as a biochemical marker of
protection against OS in the early stages of embryo development [25]. Day 1 TAC levels have been shown to significantly correlate with increased clinical pregnancy rates in
ICSI cycles [25]. Bedaiwy et al. analyzed the effect of ROS
levels on early human embryonic development in IVF and
ICSI culture media on Day 1 post-insemination [12]. The
findings implicate increased Day 1 ROS levels in decreased
development rates, higher degrees of fragmentation, and
reduced formation of morphologically normal blastocysts
[12]. The results also showed that decreased fertilization
outcomes in ICSI were significantly related to increased Day
1 ROS levels in the culture fluid. However, this apparent
relationship was not exhibited by the conventional IVF
cycles studied. Another study by Bedaiwy et al. used TAC
levels in Day 1 culture media as a biochemical marker of
OS to demonstrate an association between increased Day 1
ROS levels and slower development, higher fragmentation
rates, and reduced formation of morphologically normal
blastocysts [13]. Day 1 ROS and TAC levels within culture
media serve as useful biomarkers of OS and can be utilized
to quantify the degree of OS present in the setting of IVF and
ICSI and relate OS to embryonic development parameters
and thus ART outcomes.
Gupta et al.
IVF. Morphological assessment consists of observing the
developmental pattern of embryos during culture, fragmentation, inclusion bodies, cell number, morphology of
the inner cell mass and trophoectoderm, and blastocoele
expansion at the blastocyst stage [37]. However, these
parameters may not accurately reflect functional status, as
normal-looking gametes and embryos can still harbour
genetic or epigenetic defects such as ROS-induced damage
[38]. The inability to precisely determine the reproductive
potential of gametes and embryos contributes to the high
failure rate in IVF. To minimize the risk of implantation
failure, multiple embryos often are transferred simultaneously, leading to an increased incidence of multiple pregnancies [35]. Metabolomic profiling used in conjunction
with morphological assessment of gametes and embryos may
serve to maintain or increase overall pregnancy rates while
decreasing the unfavorable occurrence of multiple gestations
[35].
Metabolomic Profiling to Assess ROS
In the past, evaluation of ROS and OS was based entirely
on biochemical methods that are inconvenient and laborintensive. Efforts have continued to determine a more
efficient way of identifying and measuring biomarkers that
accurately quantify and correlate OS to clinical reproductive
outcomes. OS may be assessed on a molecular level using
metabolomics- the systematic study of metabolites as smallmolecule biomarkers that contribute to the functional
phenotype of a cell, tissue, or organism [32]. The metabolome refers to the complete inventory of small molecules,
metabolic intermediates such as amino acids, lipids and
nucleotides, ATP, hormones, other signalling molecules, and
secondary metabolites [33]. Low-molecular weight metabolites represent end products of cell regulatory processes,
revealing the response of biological systems to a variety of
genetic, nutrient, or environmental influences [34]. Therefore, the metabolome represents the end product of gene
expression, illustrating the interaction of environmental
conditions with physiology [35] (Fig. 2).
Metabolomic profiling is a rapid, non-invasive method of
measuring OS markers such as –CH, -NH, -OH and ROH.
OS is reflected by the CH:ROH ratio. Gas chromatography,
high performance liquid chromatography, or capillary electrophoresis is used to separate biomarkers, while methods of
spectrometry are used to identify and quantify them [36].
Metabolomic profiling has been investigated for its use in
quantifying the degree of OS during ART and identifying
those gametes and embryos most likely to contribute to
successful implantation and pregnancy.
Currently, the morphology and cleavage rate of embryos
are used as a predictive measure of implantation potential in
Fig. (2). The use of metabolomic profiling to quantify oxidative
stress.
A quantitative, objective assessment of gametes retrieved
for use in IVF may provide a prognostic indication of IVF
outcome and clarification of the reasons for success or
failure of the procedure. Metabolomic profiling can be used
to assess the impact of OS on sperm function and fertilization capacity. A study by Agarwal et al. demonstrates the
existence of unique spectral ‘signatures’ of semen samples
that indicate statistically significant differences in the levels
of oxidative stress among men with various conditions
known to be associated with altered redox status including
varicocele, idiopathic male factor infertility, and vasectomy
reversal. The various degrees of OS were evidenced by
unique changes in the ratios of –CH to ROH. Significant
differences in –CH, -NH, and –OH concentrations were
observed among the groups of subjects [39]. These findings
assert that metabolomic analysis is a rapid, non-invasive
diagnostic method that can be used to assess semen for
abnormalities related to reactive oxygen species.
The metabolome of an oocyte is thought to be
‘fingerprinted’ by its interaction with its IVF culture medium
and exogenous factors such as environmental oxidative
stress [40]. Metabolomic profiles of follicular fluid samples
have demonstrated a significant relationship between high
levels of OS in follicular fluid and decreased oocyte viability
and poor embryo quality [41]. A study by Nagy et al.
correlated the metabolome of oocytes with corresponding
embryo fertilization, development, and viability and demonstrated the ability of NIR spectroscopy to predict fertility
potential as early as the pre-fertilization stage of oocytes.
Metabolomic analysis of the spent culture media of oocytes
was shown to predict embryo development at Day 3 and
Day 5, with the potential to indicate embryo viability. NIR
analysis was shown to assess the metabolomic status of
oocytes with high sensitivity and significant correlation with
healthy embryo morphology and high implantation potential
[40].
Embryo metabolomic profiles also have been shown to
vary according to embryo implantation potential. A study by
Scott et al. used metabolomic profiling to calculate a
viability index for 41 spent media samples from 19 patients
with known reproductive potential. Higher viability indices
were observed in both Day 3 and Day 5 embryos with
proven reproductive potential than in those that failed to
implant. An overall diagnostic accuracy of 80.5% in
predicting delivery or a failed implantation demonstrates the
significant relationship between the reproductive potential
of embryos and modifications of their culture media [42].
Seli et al. used a multivariate analysis approach to compare
the spectral profiles between embryos that resulted in
live births with those that failed to implant. Markers of OS
discriminated between the two study populations and were
most predictive of pregnancy outcome. CH:ROH content
was significantly lower in the culture media of embryos that
progressed to pregnancy than in the culture media of
embryos that failed to implant [43]. Therefore, in vitro
cultured embryos with high reproductive potential may alter
their environment differently compared with embryos that do
not result in pregnancy [44].
A similar investigation by Agarwal et al. used
metabolomic profiling to identify the OS biomarkers R-OH,
233
CH, OH, and NH in 228 embryo media, 72 follicular fluid,
and 133 seminal plasma samples. The discarded culture
media, follicular fluid, and seminal plasma were shown to
consistently produce unique metabolomic OS profiles, which
correlated well with pregnancy versus non-pregnancy outcomes [45].
In addition to indices of redox status, metabolomic
profiling of amino acids may be helpful determining the
functional status of embryos. During the IVF process,
embryo culture media is often supplemented with mixtures
of amino acids that influence blastocyst formation [46]. A
study by Houghton et al. found that competent preimplantation embryos that develop into blastocysts demonstrate a
lower rate of amino acid turnover than embryos that did not
progress to the blastocyst stage. These results suggest that
embryos with higher metabolic activity are more likely to
arrest, contributing to failed outcomes in ART [47]. Brison
et al. reported an association between decreased glycine and
leucine and increased asparagine levels in the culture media
with increased clinical pregnancy and live birth rates. Lower
levels of amino acid metabolism in embryos were correlated
with increased viability [48]. Cryopreserved embryos used in
successful IVF procedures also have been shown to exhibit
lower rates of amino acid metabolism compared with
those that failed to implant [49]. Therefore, the use of metabolomic profiling to measure parameters such as CH:ROH
and amino acid turnover may allow for better discernment
when evaluating and selecting embryos for transfer in IVF.
These methods of objectively quantifying embryo viability
and implantation potential may minimize the number of
embryos needed to be transferred, thereby decreasing the
incidence of multiple infant births and improving overall
pregnancy outcomes.
In addition to predicting the potential of gametes and
embryos to give rise to favorable reproductive outcomes,
metabolomic profiling of the uterine endometrial lining may
indicate the degree of endometrial receptivity to blastocyst
implantation, providing the opportunity to optimally time
embryo transfer in ART [32]. Although the results of
many studies suggest various ways in which metabolomic
profiling may help to improve ART outcomes, this method
of evaluating the functional status and potential of gametes
and embryos has yet to be standardized and requires
validation by further studies.
WHAT IS THE ROLE OF ANTIOXIDANTS IN IVF
AND ICSI?
The culture media used in IVF and ICSI can be a source
of ROS generation during ART procedures [50]. The in vitro
environment exposes gametes to ROS in excess of what they
would normally face in vivo [50]. An excess of ROS has the
ability to damage lipids, proteins, nucleic acids, DNA, and
RNA [25]. Thus, IVF protocols must be revised to incorporate strategies that decrease or prevent the generation of ROS
[50].
Javiet et al. established that culture media riddled with a
toxic level of OS compromises oocyte and embryo integrity.
Because the success rates of IVF are influenced by the quality of the embryos transferred, antioxidant supplementation
to neutralize the effects of ROS on oocyte quality may have
a beneficial effect on ART outcome [25]. Repeated changes
of the culture media and the use of sequential culture
systems may help to minimize the ROS generated from
within the culture media itself [11]. The use of supplemental
antioxidants to modulate ROS levels in the culture media
is hypothesized to promote an ideal environment for
pre-implanted embryos produced by ART [25]. Ongoing
trials continue to investigate the role for oral antioxidant
supplementation in both infertile men and women with
the aim of optimizing the success rates of IVF and ICSI
procedures.
Antioxidant Supplementation Within the Culture Media
The success of ART depends on how closely the in vitro
setting mimics in vivo conditions. Therefore, it is crucial to
have physiological concentrations of individual amino acids,
antioxidants, vitamins, and energy sources within the culture
media of embryos to maximize blastulation hatching rates
[51]. However, the literature presents conflicting reports
regarding the validity of using any one specific antioxidant
therapy to improve ART outcomes. However, given the
strong evidence that OS plays a pathogenic role in decreasing the success rates of IVF and ICSI, the potential for the
use of antioxidants to control OS in the in vitro setting is a
promising concept that warrants further investigation in the
field of reproductive medicine (Fig. 3).
Gupta et al.
Supplementing the culture media with melatonin, a
powerful hormonal agent known to possess the ability
to neutralize ROS, also has been shown to improve the
efficiency of in vitro embryo production in experiments
using buffalos [54]. This has led to studies investigating
the merits of oral melatonin supplementation to improve
fertility.
L-carnitine also has been evaluated for its potential use as
a supplement in embryo culture medium and the effects of
this supplementation on developing mouse embryos [55]. Lcarnitine is known to possess antioxidant properties, consisting of free radical scavenging and metal-chelating properties
[55]. Specifically, this compound has the ability to neutralize
the embryotoxic effects of exogenous production of oxidative stress by hydrogen peroxide [55]. Embryo culture
medium supplementation with L-carnitine has been shown
to bring about a significant improvement in the blastocyst
development rate compared with a control group without
antioxidant supplementation. At moderate concentrations,
L-carnitine is has been proven effective in blocking the effect of ROS, resulting in a decreased level of DNA damage
[55].
Wang et al. elucidated the effects of adding the antioxidant vitamins C and E to culture media in ART. The results
of this study demonstrated that these antioxidants led to increased rates of blastocyst development, by way of counteracting the embryotoxic effects of ROS [6]. Embryo culture
supplementation with vitamin C was seen to exert a protective effect on embryo development in culture media containing an ROS-generating PMA-activated leukocyte supernatant [52]. Furthermore, the findings of Wang et al. suggest
that the beneficial, ROS-neutralizing effect of vitamin C
supplementation in culture media may be superior to the
effects seen with vitamin E supplementation or simultaneous
supplementation with both vitamins C and E [6]. The idea
that there is a critical composition and dosage of antioxidant
supplementation, below or beyond which the full potential
protective benefit may not be realized, is confirmed by the
findings of Choi et al. [53]. This study showed that vitamin
C administered at a higher dosage than needed is capable of
inducing damage. Moderate dosages, however, were seen to
reduce oxidative damage in the culture media, with no detrimental effect on mouse oocyte quality [53].
The investigations of Olson et al. determined that vitamin
E supplementation in culture media increased the number of
bovine embryos that reached the fully expanded blastocyst
stage [52]. This effect is thought to be mediated by the role
vitamin E plays in protecting unsaturated membrane fatty
acids. The peroxidation of these membrane lipids can lead to
structural damage and affect its membrane function. Furthermore, the results showed that vitamin E supplementation
alone improved conditions for embryonic development more
than that seen when vitamin E and C were employed in
combination.
Fig. (3). Interventions to control OS during IVF/ICSI.
Oral Antioxidant Supplementation
Studies investigating the benefits of oral antioxidant therapy in male and female patients undergoing IVF or ICSI
procedures have yielded inconsistent data and conflicting
reports.
FEMALE
Oral antioxidant supplementation in female patients undergoing conventional IVF or ICSI has been evaluated in
terms of the effects on fertilization rates and pregnancy outcome.
Melatonin, a free radical scavenger that acts within the
mitochondria to decrease protein damage, improve electron
transport chain activity, and reduce mitochondrial DNA
damage [56], has been shown to protect against the toxic
effects of oxidative stress on oocyte maturation, thus improving oocyte quality and fertilization rates [56]. Tamura et
al. used the biomarker 8-hydroxy-2-deoxyguanosine to
evaluate the association between OS in follicular fluid and
poor oocyte quality. Orally administered melatonin was seen
to improve both the redox status within follicular fluid and
oocyte quality [56]. In addition to confirming the toxic effects of OS on oocyte maturation, the study supported the
idea that oral melatonin supplementation can be used to
bring about a significant reduction in the number of degenerate oocytes and increase the number of fertilized embryos
[56].
Despite the findings of Tamura et al., clinical trials have
failed to demonstrate a specific regimen and dosage of antioxidant supplementation that will provide a definitive increase in ART success rates. Antioxidant supplementation
with ascorbic acid has long been hypothesized to have a favorable influence on ART procedures for female factor infertility. A significant correlation is known to exist between the
level of ascorbic acid in a woman’s blood serum and follicular fluid, with the follicles having a higher concentration of
ascorbic acid [54]. Crha et al. conducted an investigation in
which a group of women supplemented with vitamin C during the period of hormonal treatment in IVF had a statistically insignificant increase in the ability to achieve pregnancy compared with the control group that did not receive
oral antioxidant supplementation [57].
Based on the premise that ascorbic acid confers an improvement in fertility and ART outcomes, Griesinger et al.
evaluated the impact of ascorbic acid at different doses on
women undergoing IVF procedures [58]. In contrast to the
findings of Crha et al., the results of this study failed to reveal clinical evidence of any beneficial effect of ascorbic
acid on IVF [58]. Furthermore, Tarin et al. found that the
oral administration of pharmacological doses of vitamins C
and E on mice had no effect on time to pregnancy, age of
cessation of female reproductive life or pregnancy rate [59].
MALE
In contrast to the accepted management of female infertility, oral antioxidant supplementation in the management
and treatment of infertile men is widely accepted and practiced. A study by Geva et al. demonstrated that oral antioxidant supplementation with vitamin E led to a significant in-
235
crease in fertilization rates in IVF for male factor infertility
[60]. The results of this study provide evidence for the potential of vitamin E therapy to improve fertilization rates of
fertile normospermic patients with low fertilization rates
after one month of treatment. Greco et al. demonstrated a
significantly reduced percentage of DNA-fragmented spermatozoa in a study group treated with oral vitamins C and E
compared with control group subjects who were not given
oral antioxidant supplements [61]. However, this study failed
to show any improvement in pregnancy rates with oral antioxidant therapy. Another study by Greco et al., which examined the beneficial effect of oral antioxidant treatment in
cases of DNA-damaged sperm utilized for ICSI, revealed no
differences in fertilization and cleavage rates or in embryo
morphology with vitamin C and E antioxidant treatment.
However, a marked improvement in the number of clinical
pregnancies and implantation rates was observed in the antioxidant treatment group [62]. Vitamin E itself has been hypothesized to protect against the loss of sperm motility by
lipid peroxidation and improve sperm motility and increase
the possibility of successful fertilization with sperm from
asthenospermic patients [63]. Furthermore, Suleiman et al.
showed a significant decrease in ROS-induced sperm and
improvements in spontaneous pregnancy rates during the
next six months with oral vitamin E supplementation [63].
Tremellen et al. investigated Menevit (Bayer Health
Care, Pymble, NSW, Australia), an oral antioxidant treatment used by male patients, and found no differences in
quality of resultant embryos between the antioxidant-treated
and control groups. However, supplementation was related
to significant improvements in implantation rates and pregnancy outcomes. Therefore, treating infertile males with the
oral antioxidant Menevit may serve to improve pregnancy
rates by optimizing factors related to the amount of OS experienced during the course of IVF/ICSI treatment [64].
Meneco et al. [65] correlated oral vitamin intake with
reduced levels of ROS-induced DNA fragmentation, leading
to improved fertility. However, in addition to better semen
quality, oral antioxidant supplementation also was associated
with increased sperm decondensation, which may prevent
IVF/ICSI success [25]. A study by Rolf et al. failed to demonstrate any improvement in semen parameters or survival
rates of sperm from men with asthenozoospermia or moderate oligoasthenozoospermia who were supplemented with
oral vitamin C and E [66]. Although considerable data suggest a possible benefit from vitamin C and E supplementation on ART outcomes, several investigations have yielded
conflicting findings. This lack of consensus in the literature
complicates the debate as to whether oral antioxidant
supplementation has a definitive role in optimizing ART
outcomes.
CONCLUSION
Despite the many advances in ART, the issue of using
antioxidant therapy to alleviate the burden of infertility by
improving IVF and ICSI procedures and their outcomes continues to be the subject of much debate. Although data from
the existing literature fail to provide any definite conclusions
regarding whether specific antioxidant supplementation
of infertility patients and culture media used in ART will
increase successful ART outcomes, significant evidence
suggests that it has the potential to combat oxidative stress,
a known contributor to ART failure. There is no doubt
that there is an underlying link between OS and difficulty
in achieving fertilization and eventual pregnancy with IVF
and ICSI. Further controlled evaluations using large sample
size populations are needed to arrive at a consensus regarding the use of antioxidant supplementation in the culture
media and oral administration of these compounds in both
male and female patients undergoing ART.
EXPERT COMMENTARY
The purpose of this article was to discuss the role played
by oxidative stress in assisted reproductive technologies such
as IVF and ICSI. Controlled levels of OS are known to serve
a physiological role in the various biochemical pathways that
comprise human reproduction. However ART procedures
lack the protection of the natural antioxidant defense of the
female reproductive tract, the microenvironment where
fertilization normally takes place. Therefore, gametes used in
ART procedures are highly susceptible to damage from
overwhelming and unopposed degrees of oxidative stress
that may arise endogenously from the gametes themselves
and from exogenous influences within the laboratory
environment such as oxygen tension, light, and metallic
cations. Increased OS has been shown to hinder spermoocyte interaction and adversely affect fertility and
successful pregnancy rates. Given the evidence that
implicates oxidative stress in producing poor ART outcomes,
elucidating the precise mechanisms by which ROS arise in
the ART environment, current methods to accurately
quantify OS, and potential strategies to modulate the amount
of OS experienced by gametes within both the human
reproductive tract and the in vitro culture media must be a
top priority.
The field of metabolomics has given rise to novel
techniques to more accurately measure oxidative stress.
Oxidative stress quantification may allow for the selection of
the most viable gametes with the least OS-induced damage
to be used in ART to optimize fertility outcomes and ensure
greater efficiency. ART outcomes also may be improved by
modulating the amount of OS experienced by gametes and
embryos in vitro by supplementing the culture media with
protective antioxidants. Furthermore, increased levels of
oxidative stress that may be seen in vivo in both the male and
female partner participating in ART may be treated by oral
antioxidant supplementation. The use of oral antioxidants to
treat male factor causes of infertility are well-established and
in clinical use. However, the safety and efficacy of oral
antioxidant supplementation to treat female infertility
requires further investigation.
FIVE YEAR VIEW
Success rates in ART are influenced by maternal age,
number of oocytes retrieved, and the quality of the embryos
transferred. Embryo quality is influenced by extrinsic factors
like culture media. A large number of extrinsic factors that
modulate OS can influence successful outcomes of IVF and
embryo transfer including oxygen concentration, ionizing
radiation, and levels of the antioxidants EDTA, SOD, and
Gupta et al.
catalase. Evidence suggests that media supplementation with
antioxidants, disulphide reducing agents, or divalent chelators of cations may be beneficial to gametes/embryos. Repeated change of media and use of sequential culture systems may help reduce exposure to ROS. Oocytes also can be
protected from oxidant-induced early apoptosis by supplementing media with vitamin E and C. In addition to strategies to modulate OS to reduce the chance of fertilization and
pregnancy failure, more accurate methods to assess the degree of oxidative stress may allow for selection of healthy
gametes and embryos for use in IVF or ICSI. Optimizing the
procedure in this way may serve to reduce patient time and
costs associated with the need for repeat use of assisted reproductive technologies. The significance of improved
IVF/ICSI outcomes would be enormous to the many infertile
couples who seek assisted reproduction due to infertility issues.
KEY ISSUES
• IVF and ICSI are assisted reproductive techniques that
achieve fertilization by circumventing various anatomic,
mechanical and functional obstacles to fertility in vivo.
• Oxidative stress arises when high levels of ROS overwhelm antioxidant capacity, resulting in modified gene
expression and transcription factors and damaged DNA.
ART procedures are susceptible to increased OS as
gametes lack the natural antioxidant defence seen during
in vivo reproduction in the male and female reproductive
tract.
• Endogenous sources of oxidative stress in ART include
the multiple oocytes per dish, cumulus cell mass, and
spermatozoa used for insemination in IUI or incubation
with oocytes in IVF. ICSI avoids excess ROS that may
arise from defective spermatozoa in IVF.
• Exogenous sources of oxidative stress in ART include
oxygen concentration, metallic cations, and illumination.
• In the past, studies quantified OS by inconvenient and
labor-intensive biochemical methods to measure ROS
and antioxidant status within follicular fluid and culture
fluid. Metabolomic profiling is a faster, more accurate
method of quantifying OS during ART and may be used
to identify gametes and embryos more likely to
contribute to successful implantation and pregnancy.
• The success of ART is increased when the in vitro
culture setting mimics in vivo conditions. Therefore, the
use of the following antioxidants in culture media may
improve outcome: vitamin C, vitamin E, melatonin, and
L-carnitine.
• Various studies on the benefits of oral antioxidant
supplementation in male and female patients undergoing
ART procedures have yielded inconsistent and conflicting reports, and further research is required.
REFERENCES
[1]
[2]
Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in
female reproduction. Reprod Biol Endocrinol 2005; 3: 28.
Sikka SC. Role of oxidative stress and antioxidants in andrology
and assisted reproductive technology. J Androl 2003; 25(1): 5-18.
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Falcone T, Hurd W. Clinical reproductive medicine and surgery.
Philadelphia, PA Mosby Elsevier 2007; pp. 233-50.
Palermo GD, Neri QV, Takeuchi T, Rosenwaks Z. ICSI: Where we
have been and where we are going. Semin Reprod Med 2009;
27(2): 191-201.
Greco E, Scarselli F, Iacobelli M, et al. Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa. Hum Reprod 2005; 20(1): 226-30.
Wang X, Falcone T, Attaran M, Goldberg JM, Agarwal A, Sharma
RK. Vitamin C and vitamin E supplementation reduce oxidative
stress-induced embryo toxicity and improve the blastocyst development rate. Fertil Steril 2002; 78(6): 1272-7.
Harvey AJ, Kind KL, Thompson JG. REDOX regulation of early
embryo development. Reproduction 2002; 123(4): 479-86.
Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi
H. Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization- embryo transfer program. Fertil Steril 2002; 77(6): 1184-90.
Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG. Oxidative stress in an assisted reproductive techniques setting. Fertil
Steril 2006; 86(3): 503-12.
Gonzalez-Flecha B, Demple B. Biochemistry of redox signaling in
the activation of oxidative stress genes. In: Gilbert DL, Colton CA
Ed, Reactive oxygen species in biological systems: a multidisciplinary approach. New York, NY Kluwer/Plenum 1999; pp.
133-153.
Agarwal A, Gupta S, Sharma RK. Oxidative stress and its implications in female infertility- a clinician’s perspective. Reprod Biomed
Online 2005; II(5): 641-50.
Bedaiwy MA, Falcone T, Mohamed MS, et al. Differential growth
of human embryos in vitro: role of reactive oxygen species. Fertil
Steril 2004; 82(3): 593-600.
Bedaiwy MA, Agarwal A, Said TM, et al. Role of total antioxidant
capacity in the differential growth of human embryos in vitro. Fertil Steril 2006; 86: 304-9.
Gianaroli L, Fiorentino A, Magli MC, Ferraretti AP, Montanaro N.
Prolonged sperm-oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum Reprod
1996; 11(11): 2507-11.
Bedaiwy MA, Falcone T, Sharma RK, et al. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod 2002; 17(2): 426-31.
Bavister B. Oxygen concentration and preimplantation development. Reprod Biomed Online 2004; 9(5): 484-6.
Noda Y, Goto Y, Umaoka Y, Shiotani M, Nakayama T, Mori T.
Culture of human embryos in alpha modification of Eagle’s medium under low oxygen tension and low illumination. Fertil Steril
1994; 62(5): 1022-7.
Waldenstrom U, Engstrom AB, Hellberg D, Nilsson S. Lowoxygen compared with high-oxygen atmosphere in blastocyst
culture, a prospective randomized study. Fertil Steril 2009; 91(6):
2461-5.
Guerin P, Mouatassim S, Menezo Y. Oxidative stress and
protection against reactive oxygen species in the pre-implantation
embryo and its surroundings. Hum Reprod Update 2001; 7(2): 17589.
Nasr-Esfahani MH, Johnson MH. How does transferring overcome
the in vitro block to development of the mouse preimplantation
embryo? J Reprod Fertil 1992; 96: 41-8.
Beehler BC, Przybyszewski J, Box HB. Formation of 8hydoxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2. Carcinogenesis 1992; 13(11):
2003-7.
Takenaka M, Horiuchi T, Yanagimachi R. Effects of light on development of mammalian zygotes. Proc Natl Acad Sci USA 2007;
104(36): 14289-93.
Tarozzi N, Bizzaro D, Flamigni C, Borini A. Clinical relevance of
sperm DNA damage in assisted reproduction. Reprod Biomed Online 2007; 14(6): 746-57.
Jozwik M, Jozwik M, Kuczynski W, Szamatowicz M. Nonenzymatic antioxidant activity of human seminal plasma. Fertil Steril
1997; 68(1): 154-7.
Agarwal A, Gupta S, Sekhon L, Shah R. Redox considerations in
female reproductive function and assisted reproduction: from molecular mechanisms to health implications. Antioxid Redox Signal
2008; 10(8): 1375-403.
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
237
Hammadeh ME, Radwan M, Al-Hasani S, et al. Comparison of
reactive oxygen species concentration in seminal plasma and semen
parameters in partners of pregnant and non-pregnant patients after
IVF/ICSI. Reprod Biomed Online 2006; 13(5): 696-706.
Chi HJ, Kim JH, Ryu CS, et al. Protective effect of antioxidant
supplementation in sperm-preparation medium against oxidative
stress in human spermatozoa. Hum Reprod 2008; 23(5): 1023-8.
Shekarriz M, DeWire DM, Thomas AJ Jr, Agarwal A. A method of
human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol 1995; 28(1): 31-5.
Van Blerkom J, Antczak M, Schrader R. The developmental potential of the human oocyte is related to the dissolved oxygen content
of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod
1997; 12(5): 1047-55.
Pasqualotto EB, Agarwal A, Sharma RK, et al. Effect of oxidative
stress in follicular fluid on the outcome of assisted reproductive
procedures. Fertil Steril 2004; 81(4): 973-6.
Wiener-Megnazi Z, Vardi L, Lissak A, et al. Oxidative stress indices in follicular fluid as measured by the thermochemiluminscence
assay correlate with outcome parameters in in vitro fertilization.
Fertil Steril 2004; 82(Suppl 3): 1171-6.
Fnu Deepinder, Hyndhavi Chowdary, Ashok Agarwal. Role of
metabolomic analysis of biomarkers in the management of male
infertility. Expert Rev Mol Diagn 2007; 7(4): 351-8.
Posillico JT and the Metabolomics Study Group for Reproductive
Technologies. Selection of viable embryos and gametes by rapid,
noninvasive metabolomic profiling of oxidative stress biomarkers.
In: Cohen J, Elder K (eds). Human Preimplantation Embryo Selection. London, UK, Taylor and Francis 2007; 325–336.
Singh R, Sinclair KD. Metabolomics: approaches to assessing
oocyte and embryo quality. Theriogenology 2007; 68(Suppl 1):
S56-62.
Botros L, Sakkas D, Seli E. Metabolomics and its application for
non-invasive embryo assessment in IVF. Mol Hum Reprod 2008;
14(12): 679-90.
Dunn WB, Bailey NJ, Johnson HE. Measuring the metabolome:
current analytical technologies. Analyst 2005; 130(5): 606-25.
Alikani M, Sadowy S, Cohen J. Human embryo morphology
and developmental capacity. In: Van Soom A, Boerjan M, Eds.
Assessment of mammalian embryo quality, invasive and noninvasive techniques. Kluwer Academic Publishers 2002; pp. 1–31.
Sinclair KD, Lea RG, Rees WD, Young LE. The developmental
origins of health and disease: current theories and epigenetic
mechanisms. Reproduction 2007; 64(Suppl): 425–43.
Agarwal A, Sharma RK, Prabhkaran SA, et al. Assessment of
oxidative stress levels in semen using spectroscopy-based
metabolomic profiling: implications in male infertility. Presented
at: Annual Meeting of the American Society For Reproductive
Medicine, New Orleans, LA, USA, 2006; p. 131.
Nagy ZP, Jones-Colon S, Roos P, et al. Metabolomic assessment of
oocyte viability. Reprod Biomed Online 2009; 18(2): 219-25.
Agarwal A, Bungum M, Humaidan P, Botros LL, Burns DH.
Metabolomic assessment of oxidative stress in follicular fluid as a
possible predictor of oocyte viability. Presented at: 50th
Anniversary Conference of American Association of Bioanalysts,
Las Vegas, NV, USA 2006.
Scott R, Seli E, Miller K, Sakkas D, Scott K, Burns DH. Noninvasive metabolomic profiling of human embryo culture media using
Raman spectroscopy predicts embryonic reproductive potential: a
prospective blinded pilot study. Fertil Steril 2008; 90(1): 77-83.
Seli E, Sakkas D, Beht B et al. Non-invasive metabolomic profiling
of human embryo culture media correlates with pregnancy
outcome. Initial results of the metabolomics study group for ART.
Presented at: Annual Meeting of the American Society For
Reproductive Medicine, New Orleans, LA, USA, O-270; 2006.
Bromer JG, Seli E. Assessment of embryo viability in assisted
reproductive technology: shortcomings of current approaches and
the emerging role of metabolomics. Curr Opin Obstet Gynecol
2008; 20(3): 234-41.
Agarwal A, Behr B, Burns DH, Nagy P, Sakkas D, Scott R. Metabolomic biomarkers of oxidative stress in ART: A rapid noninvasive methodology to assess the selective viable gametes and
embryos from their non-viable cohorts. In: Proceedings of the Second International Conference on Cryopreservation of Human Oocyte; 2006.
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
Gupta et al.
Devreker F, Hardy L, Van den Bergh M, Vannin AS, Emiliani S,
Englert Y. Amino acids promote human blastocyst development.
Hum Reprod 2001; 16(4): 749-56.
Houghton FD, Hawkhead JA, Humpherson PG, et al. Non-invasive
amino acid turnover predicts human embryo developmental
capacity: Hum Reprod 2002; 17(4): 999-1005.
Brison DR, Houghton FD, Falconer D, et al. Identification of
viable embryos in IVF by non-invasive measurement of amino acid
turnover. Hum Reprod 2004; 19(10): 2319–24.
Sturmey RG, Hawkhead JA, Barker EA, Leese HJ. DNA damage
and metabolic activity in the preimplantation embryo. Hum Reprod
2009; 24(1):81-91.
Martin-Romero FJ, Miguel-Lasobras EM, Dominguez-Arroyo JA,
Gonzalez-Carrera E, Alvarez IS. Contribution of culture media to
oxidative stress and its effect on human oocytes. Reprod Biomed
Online 2008; 17(5): 652-61.
Ali AA, Bilodeau JF, Sirard MA. Antioxidant requirements for
bovine oocytes varies during in vitro maturation, fertilization, and
development Theriogenology 2003; 59(3-4):939-49.
Olson SE, Seidel GE Jr. Culture of in vitro-produced bovine
embryos with vitamin E improves development in vitro and after
transfer to recipients. Biol Reprod 2000; 62(2): 248-52.
Choi WJ, Banerjee J, Falcone T, Bena J, Agarwal A, Sharma RK.
Oxidative stress and tumor necrosis factor-alpha-induced alterations in metaphase II mouse oocyte spindle structure. Fertil Steril
2007; 88(4 Suppl): 1220-31.
Manjunatha BM, Devaraj M, Gupta PS, Ravindra JP, Nandi S.
Effect of taurine and melatonin in the culture medium on buffalo in
vitro embryo development. Reprod Domest Anim 2009; 44(1): 126.
Abdelrazik H, Sharma R, Mahfouz R, Agarwal A. L-Carnitine
decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos. Fertil Steril 2007; 91(2): 589-96.
Tamura H, Takasaki A, Miwa I, et al. Oxidative stress impairs
oocyte quality and melatonin protects oocytes from free radical
damage and improves fertilization rate. J Pineal Res 2008; 44(3):
280-7.
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
Crha I, Hruba D, Ventruba P, Fiala J, Totusek J, Visnova H.
Ascorbic acid and infertility treatment. Cent Eur J Public Health
2003; 11(2): 63-7.
Griesinger G, Franke K, Kinast C, et al. Ascorbic acid supplement
during luteal phase in IVF. J Assist Reprod Genet 2002; 19(4):
164-8.
Tarin JJ, Perez-Albala S, Pertusa JF, Cano A. Oral administration
of pharmacological doses of vitamins C and E reduces reproductive
fitness and impairs the ovarian and uterine functions of female
mice. Theriogenology 2002; 57(5): 1539-50.
Geva E, Bartoov B, Zabludovsky N, Lessing JB, Lerner-Geva L,
Amit A. The effect of antioxidant treatment on human spermatozoa
and fertilization rate in an in vitro fertilization program. Fertil Steril
1996; 66(3): 430-4.
Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J.
Reduction of the incidence of sperm DNA fragmentation by oral
antioxidant treatment. J Androl 2005; 26(3): 349-53.
Greco E, Romano S, Iacobelli M, et al. ICSI in cases of sperm
DNA damage: beneficial effect of oral antioxidant treatment. Hum
Reprod 2005; 20(9): 2590-4.
Suleiman SA, Ali ME, Zaki ZM, el-Malik EM, Nasr MA. Lipid
peroxidation and human sperm motility: protective role of vitamin
E. J Androl 1996; 17(5): 530-7.
Tremellen K, Miari G, Froiland D, Thompson J. A randomised
control trial examining the effect of an antioxidant (Menevit) on
pregnancy outcome during IVF-ICSI treatment. Aust N Z J Obstet
Gynaecol 2007; 47(3): 216-21.
Menezo YJ, Hazout A, Panteix G, et al. Antioxidants to reduce
sperm DNA fragmentation: an unexpected adverse effect. Reprod
Biomed Online 2007; 14(4): 418-21.
Rolf C, Cooper TG, Yeung CH, Nieschlag E. Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study. Hum Reprod 1999;
14(4): 1028-33.
239
Mohamed Aboulghar*
Department of Obstetrics and Gynecology, Faculty of Medicine, Cairo University, Cairo, Egypt; The Egyptian IVF
Center, Maadi, Cairo, Egypt
Abstract: The objective of this article is to review the current literature on the value of coasting for the prevention of
ovarian hyperstimulation syndrome (OHSS). Coasting is a common procedure that is performed in ovarian stimulation
cycles at risk of OHSS. Coasting is done by stopping FSH injections and monitoring E2 daily until it drops below 3000
pg/mL then hCG is given. Depriving granulosa cells of the FSH stimulus results in their apoptosis, thus reducing levels
of E2 and vascular endothelial growth factor (VEGF linked to the pathogenesis of OHSS). Meanwhile, the small follicles
that are dependent on FSH will undergo atresia, while large follicles will not be affected. Coasting is effective in reducing
the OHSS rate, but complete prevention is not possible. Prolonged coasting is associated with a significantly lower
pregnancy rate.
Keywords: Coasting, E2, Stoppage of FSH, Antagonist.
INTRODUCTION
Ovarian hyperstimulation syndrome is the most serious
complication of ovulation induction for in vitro fertilization (IVF) [1] as well as non- IVF cycles [2]. Rarely OHSS
occur after stimulation with clomiphene citrate or in a
spontaneous pregnancy [3]. Several methods have been used
for prevention of OHSS. Identification of patients at risk of
OHSS, namely, women with polycystic ovarian syndrome or
a history of previous OHSS, is the first step in prevention.
One of the effective measures for prevention is using a
small starting dose of FSH for stimulation together with
close monitoring. Other measures used for prevention of
OHSS include intravenous albumin [4], the use of GnRH
antagonist as a protocol of stimulation [5] and in patients
treated by a long GnRHa protocol with very high E2 levels
toward the end of ovarian stimulation [6]. Cancelling of the
cycle is the only choice if the risk of OHSS is very high.
Coasting was introduced in the 90s as a novel method of
OHSS prevention, and it gained wide acceptance worldwide.
The objective of this chapter is to review the literature on
coasting and discuss its place in OHSS prevention.
DEFINING COASTING
Coasting is a technique that involves withdrawing exogenous gonadotrophins and withholding hCG until the patient’s
serum estradiol concentration decreases to a safer concentration. Many studies have been published on the role of coasting for OHSS prevention. The technique appeals to physicians and patients, and it also allows for the timely transfer
of fresh embryos [7]. Coasting is performed when a large
number of follicles and/or rising serum estradiol concentrations are observed, and it is the most widely favored and
*Address correspondence to this author at the Egyptian IVF Center, Maadi,
Cairo, Egypt; Tel: + 202 25254944; Fax: +202 25253532;
1573-4048/10 $55.00+.00
used preventive measure for OHSS, as well as the most costeffective [8-10].
COASTING IN NON-IVF CYCLES
Coasting was introduced for gonadotrophin-induced cycles that were liable to develop hyperstimulation [2]. Treatment with HMG was stopped for 2–10 days, and pregnancies
occurred in three patients whose serum oestradiol concentrations continued to rise until the day of hCG. Urman et al.
(1992), [11] in an attempt to avoid cancellation of 40 cycles
that had been overstimulated with exogenous gonadotrophins
in patients with PCOS. HCG was administered after a mean
2.8 days. The clinical pregnancy rate per cycle was 25%, and
only one patient (2.5%) developed severe OHSS.
It has been noted that prematurely reducing or withholding gonadotrophins prior to follicular maturation may lead to
arrest of follicular growth [12-16].
COASTING AND IVF
It has been suggested that coasting in GnRH agonist/hMG/FSH cycles might prevent life-threatening complications of OHSS [17]. These authors withheld menotrophins
in 17 patients whose serum E2 levels exceeded 6000 pg/ml,
and continued daily GnRH agonist treatment until serum E2
levels had fallen below 3000 pg/ml. hCG was then administered to trigger ovulation. The coasting period lasted between 4 and 9 days, after which six of the 17 cycles (35%)
produced viable pregnancies. All 17 patients developed signs
of grade 2 or 3 OHSS, but none developed severe OHSS. In
another trial, the same group [17] treated 51 women at great
risk of developing OHSS who underwent coasting until
plasma E2 level fell to <3000 pg/ml. The mean number of
embryos transferred per procedure was 5.4, and there were
21 clinical pregnancies (41% per oocyte retrieval), but none
of the women developed severe OHSS.
In a pilot study, 66 at-risk patients were coasted, receiving hCG when the serum E2 level reached 2500 pg/ml, but
four patients developed OHSS [12]. In a retrospective study,
120 women considered to be at risk of OHSS [15] were
coasted when serum E2 levels exceeded 2500 pg/ml, and
hCG was delayed until E2 levels fell below 2500 pg/ml.
Outcomes were compared with those from 120 matched
OHSS high-risk patients, but without coasting. Coasting signifcantly decreased the incidence of both moderate and severe OHSS.
In a multicenter trial [16], 65 IVF cycles were severely
hyperstimulated, and coasting was carried out until the serum E2 level fell below 10, 000 pmol/l (mean 4.3 days).
Four cycles were cancelled, and a pregnancy rate of 42% per
started cycle with an implantation rate of 31% was achieved.
Only one patient developed severe OHSS.
In another study [18], four out of 20 patients developed
severe OHSS despite coasting. The mean duration of coasting was 3 days and hCG was administered on the day that
serum E2 levels began to fall. The authors concluded was
that this was too early to administer hCG.
Three groups of IVF–ET (embryo transfer) patients, including a group of highly responsive coasted patients, a
group of equally responsive non-coasted patients, and an
age-matched normally responsive control group, were also
studied [19]. Two groups of coasted patients also were compared to assess the effect of estradiol concentrations at the
time that they met the follicular criteria for hCG administration. Lastly, the effect of varying coasting duration was examined by regression analysis. The authors concluded that
coasting had no detrimental effect on cycle outcome in the
subset studied. Regression analysis, however, suggested
an inverse relationship between coasting duration and the
number of mature oocytes retrieved, as well as the clinical
pregnancy rate.
In another study, 112 severely over-stimulated IVF/
intracytoplasmic sperm injection (ICSI) patients were treated
with coasting when the serum E2 level was >3000 pg/ml and
the leading follicles had attained a diameter of >18 mm [20].
Fertilization failure was noted in six couples, and in another
10 cases it was decided to freeze all of the embryos. A pregnancy rate per patient of 30.4%, with an implantation rate of
18.1%, was reported, while six patients developed moderate
and two severe OHSS.
Coasting was effective in preventing OHSS in five
patients with polycystic ovary syndrome (PCOS) who had
developed severe OHSS in a previous stimulation cycle [21].
Delvigne and Rozenberg, [22] assessed whether physicians
modify their preventive attitude in relation to clinical factors
and to the E2 response chart. Three case scenarios with
three levels of risk factors for OHSS were constructed.
At random, three out of the 12 artificially constructed case
scenarios were sent to 573 physicians who are members of
the European Society of Human Reproduction and Embryology (ESHRE). Coasting was by far the most popular choice
(60%) among the selected preventive measures. Most of
those physicians surveyed who would use coasting selected
an E2 level of 3000 pg/ml as a safe value for the hCG
administration.
A prospective, randomized study was conducted to
evaluate the incidence of OHSS and the cycle cancellation
rate in 49 high-risk patients using a reduced HMG dose in
Mohamed Aboulghar
one arm and continuation of the same dose in the other arm
before coasting [23]. The duration of coasting was significantly reduced when the HMG dose was reduced.
A systematic review was conducted to analyze whether
evidence is sufficient to justify the general acceptance of
coasting. The studies, which involved 493 patients in 12
studies, are very heterogeneous in the characteristics and
number of patients in the ovulation stimulation schemes.
Study designs, control groups, selection criteria for coasting,
and the OHSS classifications were variable. In most studies a
threshold value of E2 was used (often 3000 pg/ml) and/or the
number of follicles were considered. The fertilization rates
(36.7-71%) and the pregnancy rates (20-57%) were acceptable in terms of IVF results in comparison with those of
other large IVF databanks. In 16% of the cycles, ascites was
described and 2.5% of the patients required hospitalization.
In conclusion, while coasting does not avoid totally the risk
of OHSS, it decreases its incidence in high-risk patients.
Many questions remain unanswered about how coasting
should be managed, and we suggest that a randomized
prospective multicenter study is required [24].
DURATION OF COASTING AND IVF OUTCOME
The length of the coasting interval has been a matter for
research in recent years. If this period is too short, it may not
be effective in preventing OHSS, while a prolonged duration
may have deleterious effects on oocyte quality and/or endometrial receptivity [10]. Previous studies have suggested that
a duration of more than 4 days has a less favorable outcome
[19, 25, 26].
To investigate the effect of coasting on IVF outcome in
GnRH agonist cycles, in a retrospective analysis [27] the
average length of coasting was 2.2 days. Age and baseline
FSH were comparable to control cycles. The coasting group
had e were more follicles and oocytes, but the numbers of
fertilized oocytes and embryos transferred were similar. Implantation rate (22.4% vs. 13.9%) was higher in the control
group, but the pregnancy rates were comparable (45.1% vs.
38.5%). Within the coasting group, baseline, stimulation, and
embryology parameters were comparable between successful
and unsuccessful cycles. Pregnancy rates were comparable
after 1, 2, and 3 or more days of coasting (36.3% vs. 38.4%
vs. 40%). Pregnancy rates also were comparable (28.5%
vs. 35.7% vs. 44.4%) when groups were compared based
on change in E2 (<25%, 25%-50%, >50%). The authors
concluded that coasting for 3 days can be used successfully
in the management of hyper-responding patients during IVF
[27].
Another retrospective study compared 94 patients in
whom coasting was applied (group 1), a control group of 22
patients in whom coasting was not applied despite E > 3000
pg/mL (group 2), and a second control group of 111 normally responsive patients with peak E < 3000 pg/mL level
(group 3).When E levels were > than 3000 pg/mL in the
presence of at least 20 follicles, each measuring 10 mm in
diameter with 20% of them of diameter 15 mm, recombinant FSH administration was discontinued while GnRH agonist was maintained. There was no statistically significant
difference between number of total oocytes retrieved, metaphase II oocytes, and fertilization rates among group 1 vs.
group 2. However, implantation rates and pregnancy rates
were significantly higher in group 1 compared with group 2.
Group 1 had more total oocytes retrieved and metaphase II
oocytes compared with group 3. However, there was no significant difference in implantation and pregnancy rates between groups 1 and 3. The authors concluded that coasting
does not adversely affect assisted reproductive technology
outcome and can be applied safely to high-responder patients
in ICSI [28].
Aretrospective study evaluated the impact of the duration
of coasting on IVF cycle outcome in 132 patients who
showed a high response (estradiol >4500 pg/ml and/or more
than 20 follicles >17 mm) to ovarian stimulation and were
coasted due to their high risk of developing OHSS. Additionally, serum LH and progesterone concentrations were
studied to investigate whether premature luteinization was
present in these cycles and whether it might be related to
coasting duration. A significant decrease in implantation
rate was found when coasting was required for more than 4
days, together with a trend towards a higher cancellation
rate. Premature luteinization was significantly elevated in
women undergoing coasting compared with control women
(34% vs. 15.6%, P < 0.05). In the majority of patients who
showed premature luteinization, coasting lasted 3 days. The
conclusion was that prolonged coasting may affect the endometrium, affecting the implantation window [29].
Mansour et al. (2005) [30] reported a large series of patients (n = 1223) at risk of developing OHSS who underwent
coasting. Coasting started when the leading follicle reached
16 mm and continued until estradiol concentration fell to
3000 pg/ml. It fell to 2755 ± 650 pg/ml on the day of hCG
injection, after 2.89 ± 0.94 days. Results were analyzed according to the duration of coasting (Group I: n = 983, 3 days
or less; Group II: n = 240 more than 3 days). Number of oocytes retrieved was 16.45 ± 6.25 and 14.93 ± 6.01 in groups I
and II, respectively (P < 0.05). Fertilization rates were 63%
and 65% in groups I and II, respectively. Implantation and
clinical pregnancy rates were 26% and 52% in Group I compared with 18% and 36% in Group II, respectively (P <
0.05). Severe OHSS occurred in 16 patients, representing
0.13% of all stimulated cycles and 1.3% of patients who
were at risk of developing OHSS. Coasting was an effective
measure in OHSS prevention , without jeopardizing the ICSI
outcome. Coasting for >3 days is associated with a moderate
decrease in pregnancy rate.
Furthermore, whether there is a threshold for the percentage of estrogen decrement that would reduce oocyte quality
or that would have a negative impact on the implantation
capacity of the embryos transferred remains to be clarified.
Some investigators claim that there is no harm with extended
coasting, whereas others have demonstrated decreased fertilization, implantation, and pregnancy rates with coasting
lasting >4 days [19, 25, 27, 9, 29]. Similarly, the percentage
of E2 decrement during coasting did not jeopardize IVF cycle and did not alter pregnancy rates [24, 31].
On the other hand Atabekoglu et al. [32] reported two
patients with ongoing pregnancies despite a dramatically
sharp decrease in E levels after coasting. They were a 30year-old and a 25-year-old woman, both with unexplained
infertility, in whom E levels increased up to 6345 and
241
14,275 pg/mL during ovarian hyperstimulation and decreased by 79.5% and 75.5%, respectively, after coasting.
The result was two pregnancies ongoing at gestational weeks
20 and 14, respectively. The authors believe that a shar decline in E levels after coasting does not have deleterious
effects on implantation [32].
Using the oocyte donation model, prolonged coasting (>4
days) was found to affect both implantation and pregnancy
rates in oocyte recipients whose endometrial receptivity
was not altered by the coasting procedure. Thus, extended
coasting duration with prolonged follicular deprivation of
gonadotrophins had an impact on oocyte quality [25].
Endometrial receptivity also may be affected by the
coasting procedure. Despite numerous efforts to study endometrial receptivity by immunohistochemistry and electron
microscopy, and more recently by oligonucleotide microarrays and electrophoresis of secretory proteins, an unequivocal marker has not yet been found. Many authors have demonstrated that endometrium on the day of oocyte retrieval
shows a 2 day advancement as compared with natural
cycles [33]. This advancement was more pronounced in
those cycles with premature serum progesterone rise on the
day of hCG administration [34].
From the literature, it seems clear that coasting does not
compromise IVF cycle outcome as long as its duration is not
extended [9, 16, 25, 26, 35, 36].
WITHDRAWING BOTH FSH AND GNRHA FOR
COASTING
A retrospective study evaluated the effect of short coasting by withdrawing both gonadotropins and GnRH- agonist.
When >or=20 follicles >15 mm with E2 level of 4000 pg/mL
were detected, both gonadotropins and GnRH agonist
were withheld for 1 to 2 days. The mean serum E2 level fell
from 7915 pg/mL at the onset of coasting to 3908 pg/mL on
the day of hCG administration. Eighteen patients became
pregnant (43.9%), and the implantation rate was 12.7%.
Twenty-eight patients were coasted for 1 day, and 13 were
coasted for 2 days. After coasting, three mild and two severe
cases of OHSS occurred [37].
Coasting was tried with continuation versus stopping of
GnRHa during coasting. Fifty-nine IVF (ICSI)patients were
coasted for 3 or more days. In the GnRH-a withdrawn group,
E levels decreased by 63% (18,043–6,656 pmol/L) without
cycle cancellations or cases of severe OHSS. In the agonist
continuation group, the E decrease was 29% (14,205–
10,132 pmol/L) with cycle cancellation and severe OHSS
rates of 9.5% (4/42) and 4.8% (2/42), respectively. Oocyte
retrieval, fertilization, embryo transfer, and clinical pregnancy rates were not compromised by stopping the agonists.
Withdrawal of GnRH-a during coasting interrupted increasing E levels, prevented cycle cancellation, and mitigated the
risk of OHSS in this high-risk group without compromising
oocyte retrieval, fertilization, embryo transfer, or pregnancy
rates. The authors postulated that during prolonged coasting
[2] GnRH-a stimulated cycles [38] and withdrawal of the
agonists could interrupt the E increase and prevent cycle
cancellation in these patients.
HOW COASTING WORKS
The rationale behind coasting is similar to the step-down
protocols: mature follicles will survive for a few days without exogenous gonadotrophins while smaller follicles will
enter apoptosis/necrosis, reducing the granulosa cell population that will release VEGF among other vascular mediators
after hCG administration [36]. A total of 160 women (patients and oocyte donors) undergoing coasting and 116 controls were included in the study. Serum, follicular fluid and
granulosa cells were collected on the day of oocyte retrieval.
VEGF concentrations were determined using an enzymelinked immunosorbent assay (ELISA). Real-time PCR was
performed to evaluate VEGF gene expression in granulosa
cells. Cell death was studied by flow cytometry using annexin V-fluorescein isothiocyanate (FITC) and counterstaining by propidium iodide, and double staining with CD45
monoclonal antibody was performed to distinguish the contamination of apoptotic leukocytes. Follicular cells aspirated
from coasted patients showed a ratio in favor of apoptosis,
especially in smaller follicles (48 vs. 26%, P < 0.05). Follicular
fluid determinations confirmed that coasting reduces VEGF
protein secretion (1413 vs. 3538 pg/ml, P < 0.001) and gene
expression (2-fold decrease) in granulosa cells. Follicular
fluid VEGF protein levels positively correlated with follicular
size (r = 0.594, P = 0.001) and estradiol production (r = 0.558,
P = 0.038). Women who underwent coasting showed a
comparable IVF cycle outcome; however, a higher cancellation rate was found in cycles that were coasted. The conclusion was that coasting affects all follicles through apoptosis,
especially immature follicles, without affecting oocyte/
endometrial quality. The significant decrease found in VEGF
expression and secretion explains why coasting is clinically
effective in reducing the incidence and severity of OHSS
[36].
One of the mechanisms that explains the effectiveness of
coasting in preventing OHSS is by inducing apoptosis in
small/medium follicles (<14 mm) [36].
The reasons why coasting is effective in preventing
OHSS are speculative. Although high E2 levels are associated with OHSS, it is unlikely that they are causally involved
in its development. Hence, reduction of E2 levels in itself is
not the main goal of coasting [15]. It has been hypothesized
[19] that coasting may diminish the functional granulosa cell
cohort, resulting in the gradual decline in circulating levels
of E2 and, perhaps more importantly, in a reduction of the
chemical mediators or precursors that augment fluid extravasation. During coasting, E2 levels initially increased
before falling, illustrating that at least dominant follicles can
continue their growth in the face of a virtually absent stimulus, whereas intermediate follicles will undergo atresia.
Garcia-Velasco et al. [36] have recently shown that
coasting acts through down-regulation of VEGF gene expression and protein secretion [36]. One hundred and sixty
patients undergoing coasting and 116 controls were evaluated for granulosa and luteal cell cycle status. The authors
observed that follicular cells from coasted patients showed a
trend in favor of apoptosis, especially in smaller follicles
(48% vs. 26%, p<0.05). Follicular fluid determinations confirmed that coasting reduces VEGF protein secretion (1,413
Mohamed Aboulghar
vs. 3,538 pg/mL, P <0.001) and gene expression (twofold
decrease by real-time PCR) in granulosa cells.
GNRH ANTAGONIST FOR OHSS PREVENTION
In one case report, a GnRH antagonist was used to
stop the LH surge in a patient who had been stimulated
using the FSH/GnRH-antagonist protocol and who was
at risk of OHSS [39]. HCG was withheld and antagonist
continued, whereupon the serum estradiol concentrations
fell and OHSS was prevented. Coasting was also effective in
preventing OHSS in two high-risk patients receiving a
GnRH-antagonist/FSH protocol [40].
In another study Garcia-Velasco et al. [41] evaluated 151
coasted IVF cycles from January 2001 to January 2004, of
which 17 were patients who had been treated with GnRH
antagonist (cetrorelix 0.25 mg since stimulation day 6) and
144 were long standard protocols. The criteria for initiating
coasting were the same in both groups of patients (serum E2
levels >4,000 pg/mL and/or >20 follicles >17 mm). Patients
treated with GnRH antagonists received a significantly lower
dose of gonadotropins (1,780 IU vs. 2,682 IU, P=.012), were
coasted for a shorter period (1.7 vs. 3.9 days, P=.001), had a
lower serum E2 level at the onset of coasting (4,099 vs.
6,241 pg/mL, P=.001) but a similar E2 level on the day of
hCG administration (2,382 vs. 2,754 pg/mL, P=ns), similar
number of oocytes retrieved (19.7 vs. 20.6), comparable
numbers of mature oocytes (15.4 vs. 17.1), and analogous
implantation rates (25.2% vs. 26.1%).Thus, although serum
E2 levels are usually lower in IVF cycles with GnRH antagonists, coasting is a feasible option in these patients to
prevent OHSS.
It is believed that there is a need for a protocol that
avoids prolonged coasting with its drawbacks in women at
high risk of OHSS. The hypothesis of such a suggested protocol is that by using GnRH antagonist, estradiol concentration could be reduced rapidly to a safe level [42]. Meanwhile, the granulosa cells will not be totally deprived of FSH
by the administration of 75 IU of HMG daily, thus maintaining oocyte quality and providing better quality embryos. The
shorter intervention period in the antagonist arm and the administration of a small dose of HMG continuously may
maintain the granulosa cells and support the production of
good quality oocytes and embryos [6]. The mechanism of
action of the antagonist that resulted in the fall in estradiol
concentration is not clear. The mean drop in estradiol concentration in 94 women after 1 day of antagonist administration was 36% in the present study [6]. The reason for the
immediate effect of GnRH antagonist on estradiol concentration is not completely clear.
Aboulghar et al. [6] evaluated possible advantages of
GnRH antagonist administration as an alternative to coasting
in prevention of severe OHSS in women undergoing
IVF/ICSI. In a prospective, randomized study comparing a
coasting (group A, n = 96) and a GnRH antagonist administration (group B, n = 94) in patients at risk of OHSS, the
primary outcome measure was high quality embryos. The
secondary outcome measures were days of intervention,
number of oocytes, pregnancy rate, number of cryopreserved
embryos, and incidence of severe OHSS. The numbers of
high quality embryos (2.87 ± 1.2 vs. 2.21 ± 1.1; P < 0.0001)
and oocytes (16.5 ± 7.6 vs. 14.06 ± 5.2; P = 0.02) were significantly higher in group B as compared with group A.
Days of coasting were higher than days of antagonist administration (2.82 ± 0.97 versus 1.74 ± 0.91; P < 0.0001). In
conclusion, GnRH antagonist was superior to coasting in
producing significantly more high quality embryos and more
oocytes as well as reducing the time until hCG administration. There was no significant difference in pregnancy rate
between the two groups. No OHSS developed in either group
[6].
WHEN TO START AND WHEN TO STOP COASTING
Coasting is generally initiated when follicles are 15 to 16
mm in diameter and serum E2 levels are extremely high
(>3000 pg/ml). The large follicles have a low dependency on
FSH, and they can tolerate a few days without gonadotrophins administration. The immature follicles enter atresia as
they are very dependent on FSH hormone, and the mature
follicles will progress and be ready for oocyte retrieval [41].
The majority of publications suggest that E2 of 3000 pg/mL
is the limit below which coasting could come to an end and
hCG is administered.
3. While coasting does not avoid totally the risk of OHSS, it
decreases its incidence in high-risk patients.
4. Coasting is an effective measure in OHSS prevention,
without jeopardizing the ICSI outcome. Coasting for >3
days is associated with a moderate decrease in pregnancy
rate.
5. During coasting mature follicles will survive for a few
days without exogenous gonadotrophins while smaller
follicles will enter apoptosis/necrosis, reducing the granulosa cell population that will release VEGF.
6. Coasting is a feasible option in patients undergoing IVF
cycles with GnRH antagonists to prevent OHSS.
REFERENCES
[1]
[2]
[3]
[4]
EXPERT COMMENTARY
The purpose of this paper is to discuss the role of coasting in the prevention of OHSS. Unfortunately, there are no
available randomized studies that compare coasting versus
no coasting or any other procedure that could be an alternative to coasting in patients at risk of OHSS. However, multiple controlled studies and comparative studies show that
coasting is effective in OHSS prevention. The proposed of
action for coasting is apoptosis of granulosa cells which are
producing E2 and a possible significant decrease in VEGF,
which is thought to be the main etiological factor underlying
the development of OHSS. Coasting does not completely
prevent OHSS but markedly reduces its incidence. It could
be used effectively with GnRH agonist or GnRH antagonist
protocols.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
FIVE-YEAR VIEW
The procedure of coasting has been used extensively with
several modifications to improve its effectiveness. Major
changes to the procedure are unlikely in the near-term future.
On the other hand, it is likely that new drugs will be developed to prevent VEGF expression and secretion, and this
will be a real breakthrough in the prevention of OHSS.
However, this substance or drug to be developed should not
have an effect on the quality of the embryo or its implantation in the endometrium.
KEY ISSUES
1. Coasting is a technique that involves withdrawing exogenous gonadotrophins and withholding hCG until the serum estradiol concentration decreases to a safer concentration in patients at risk of OHSS.
2. Coasting was by far the most popular choice (60%)
among the selected preventive measures for preventing
OHSS.
243
[12]
[13]
[14]
[15]
[16]
[17]
Aboulghar MA, Mansour RT. Ovarian hysterstimulation sundrome:
classifications and critical analysis of preventive measures. Hum
Reprod Update 2003; 9: 275-89
Rabinovici J, Kushnir O, Shalev J, Goldenberg M, Blankstein J.
Rescue of menotrophin cycles prone to develop ovarian hyperstimulation. Br J Obstet Gynecol 1987; 94:1098-102.
Lussiana C, Guani B, Restagno G, et al. Ovarian hyperstimualtion
syndrome after spontaneous conception. Gynecol Endocrinol 2009;
25: 455-9
Aboulghar M, Evers JH, Al-Inany H. Intravenous albumin for
preventing severe ovarian hyperstimualtion syndrome: a Cochrane
review. Hum Reprod 2002; 17: 3027-32
Al-Inany HG, Abou-Setta AM, Aboulghar M. Gonadotrophinreleasing hormone antagonists for assisted conception. Cochrane
Database Syst Rev 2006; 3: CD001750
Aboulghar MA, Mansour RT, Amin YM, Al-Inany HG, Aboulghar
MM, Serour GI. A prospective randomized study comparing coasting with GnRH antagonist administration in patients at risk for severe OHSS. Reprod Biomed Online 2007; 15: 271-9
M Aboulghar. Symposium: Update on prediction and management
of OHSS Prevention of OHSS. Reprod Biomed Online 2009; 19:
33-42
Fatemi HM, Platteau P, Albano C, Van Steirteghem A, Devroey P.
Rescue IVF and coasting with the use of GnRH antagonist after
ovulation induction. Reprod Biomed Online 2002; 5: 273-5.
Chen D, Burmeister L, Goldschlag D, Rosenwaks Z. Ovarian
hyperstimulation syndrome: strategies for prevention. Reprod
Biomed Online 2003; 7: 43-9.
Levinsohn-Tavor O, Friedler S, Shachter M, et al. Coasting – what
is the best formula? Hum Reprod 2003; 18: 937-40.
Urman B, Pride SM, Ho Yuen B. Management of overstimulated
gonadotrophin cycles with a controlled drift period. Hum Reprod
1992; 7: 213-7.
Ben-Nun I, Shulman A, Ghetler Y, Shilon M, Maneti H, Beyth Y.
The significance of 17b-estradiol levels in highly responding
women during ovulation induction in IVF treatment: its impact and
prognostic value with respect to oocyte maturation and treatment
outcome. J Assist Reprod Genet 1993; 10: 213-5.
Sher G, Zouves C, Feinman M, Maassarani G. Prolonged coasting:
an effective method for preventing severe ovarian hyperstimulation
syndrome in patients undergoing in-vitro fertilization. Hum Reprod
1995; 10: 3107-9.
Benadiva CA, Davis O, Kilgman I, et al. Withholding gonadotropin administration is an effective alternative for the prevention
of ovarian hyperstimulation syndrome. Fertil Steril 1997; 67: 7247.
Dhont M, Van der Straeten F, De Sutter P. Prevention of severe
ovarian hyperstimulation by coasting. Fertil Steril 1998; 70: 84750.
Waldenstrom U, Kahn J, Marsk L, Nilsson S. High pregnancy rates
and successful prevention of severe ovarian hyperstimulation
syndrome by prolonged coasting of very hyperstimulated patients:
a multicentre study. Hum Reprod 1999; 14: 294-47.
Sher G, Salem R, Feinman M, Dodge S, Zouves C, Knotzen V.
Eliminating the risk of life-endangering complications following
overstimulation with menotropin fertility agents: a report on
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
Mohamed Aboulghar
women undergoing in vitro fertilization and embryo transfer.
Obstet Gynecol 1993; 81: 1009-11.
Lee C, Tummon I, Martin J, et al. Does withholding gonadotrophin
administration prevent severe ovarian hyperstimulation syndrome?
Hum Reprod 1998; 13: 1157-8.
Tortoriello DV, McGovern PG, Colon JM, et al. Coasting does
not adversely affect cycle outcome in a subset of highly responsive
in-vitro fertilization patients. Fertil Steril 1998; 69: 454-60.
Grochowski D, Wolczynski S, Kuczynski W, Domitrz J, Szamatowicz J, Szamatowicz M. Correctly timed coasting reduces the
risk of ovarian hyperstimulation syndrome and gives good cycle
outcome in an in vitro fertilization program. Gynecol Endocrinol
2001; 15: 234-8.
Ohata Y, Harada T, Ito M Yoshida S, Iwabe T, Terakawa N. Coasting may reduce the severity of the ovarian hyperstimulation
syndrome in patients with polycystic ovary syndrome. Gynecol
Obstet Invest 2000; 50: 186-8.
Delvigne A, Carlier C,Rozenberg S. Is coasting effective for
preventing ovarian hyperstimulation syndrome in patients receiving
a gonadotropin-releasing hormone antagonist during an in-vitro
fertilization cycle? Fertil Steril 2001; 76: 844-6.
Aboulghar MA, Mansour RT, Serour GI, et al. Reduction of human
menopausal gonadotrophin dose followed by coasting in prevention
of severe ovarian hyperstimulation syndrome. J Assist Reprod
Genet 2000; 17: 298-301.
Delvigne A, Rozenberg S. A qualitative systematic review of coasting, a procedure to avoid ovarian hyperstimulation syndrome in
IVF patients.Hum Reprod Update 2002; 8(3): 291-6.
Isaza V, Garcia-Velasco JA, Aragonies M, et al. Oocyte and
embryo quality after coasting: the experience from oocyte donation. Hum Reprod 2002; 17: 1777-82.
Ulug U, Bahceci M, Erden HF, et al. The significance of coasting
duration during ovarian stimulation for conception in assisted
fertilization cycles. Hum Reprod 2002; 17: 310-3.
Kovács P, Mátyás S, Kaali SG. Effect of coasting on cycle outcome
during in vitro fertilization/intracytoplasmic sperm injection cycles
in hyper-responders. Fertil Steril 2006; 85(4): 913-7.
Yilmaz N, Uygur D, Ozgu E, Batioglu S. Does coasting, a
procedure to avoid ovarian hyperstimulation syndrome, affect
assisted reproduction cycle outcome? Fertil Steril 2010; 94(1): 18993.
Moreno L, Diaz I, Pacheco A, Zúñiga A, Requena A, GarciaVelasco JA. Extended coasting duration exerts a negative impact
on IVF cycle outcome due to premature luteinization. Reprod
Biomed Online 2004; 9: 500-4.
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
Revised: April 07, 2010
Mansour R, Aboulghar M, Serour G, et al. Criteria of a successful
coasting protocol for the prevention of severe ovarian hyperstimuation syndrome. Hum Reprod 2005; 20: 3167-72.
Ulug U, Ben-Shlomo I, Bahceci M. Predictors of success during
the coasting period in high-responder patients undergoing controlled ovarian stimulation for assisted conception. Fertil Steril
2004; 82: 338-342.
Atabekoglu C, Sonmezer M, Özkavukcu S, Isbacar S. Unexpected
pregnancy despite extremely decreased estradiol levels during
ovarian stimulation. Fertil Steril 2008; 90(5): 2003
Fanchin R, Righini C, Olivennes F, et al. Consequences of premature progesterone elevation on the outcome of in vitro fertilization:
insights into a controversy. Fertil Steril 1997; 68: 799-805.
Ubaldi F, Bourgain C, Trounaye H, et al. Endometrial evaluation
by aspiration biopsy on the day of oocyte retrieval in the embryo
transfer cycles in patients with serum progesterone rise during
follicular phase. Fertil Steril 1997; 67: 521-6.
Al-Shawaf T, Zosmer A, Tozer A, et al. Prevention of severe ovarian hyperstimulation syndrome in IVF with or without ICSI and
embryo transfer: a modified ‘coasting’ strategy based on ultrasound
for identification of high-risk patients. Hum Reprod 2001; 16: 24-30.
Garcia-Velasco JA, Zúñiga A, Pacheco A, et al. Coasting acts
through downregulation of VEGF gene expression and protein
secretion. Hum Reprod 2004; 19: 1530-8.
Moon HS, Joo BS, Moon SE, Lee SK, Kim KS, Koo JS. Short
coasting of 1 or 2 days by withholding both gonadotropins and
gonadotropin-releasing hormone agonist prevents ovarian hyperstimulation syndrome without compromising the outcome. Fertil
Steril 2008; 90(6): 2172-8.
Urman B, Pride SM, Ho Yuen B. Management of overstimulated
gonadotrophin cycles with a controlled drift. Hum Reprod 1992; 7:
213-7.
de Jong D, Macklon NS, Mannaerts BMJL, et al. High dose gonadotrophin-releasing hormone antagonist (ganirelix) may prevent
ovarian hyperstimulation syndrome caused by ovarian stimulation
for in-vitro fertilization. Hum Reprod 1998; 13: 573-5.
Delvigne, A. and Rozenberg, S. Preventive attitude of physicians to
avoid OHSS in IVF patients. Hum. Reprod 2001: 16: 2491-5.
Garcia-Velasco JA, Isaza V, Quea G, Pellicer A. Coasting for the
prevention of ovarian hyperstimulaiton syndrome: much ado about
nothing? Fertil Steril 2006; 85: 547-54
Gustofson RL, Larsen FW, Bush MR, Segars JH. Treatment with
gonadotropin-releasing hormone (GnRH) antagonists in women
suppressed with GnRH agonist may avoid cycle cancellation in
patients at risk for ovarian hyperstimulation syndrome. Fertil Steril
2006; 85: 251-4.
245
PGD and Prenatal Diagnosis: Comparison and Review in Different
Genetic Disorders
Ling Sun1, Lian Liu2, Man Li2, Nikoo Afifiyan3 and Ali Ahmady4,5,*
1
Assisted Reproductive Technology Center, Guangzhou Women and Children’s Medical Center, Guangzhou, China;
PGD Science Inc., Westlake Village, California, USA; 3Isatis Scientific Inc., Palos Verdes Peninsula, California, USA;
4
MacDonald Fertility & IVF Program, University Hospitals Case Medical Center; 5Department of Reproductive
Biology, Case Western Reserve University, Cleveland, OH 44106, USA
2
Abstract: Pre-implantation and prenatal testing provide genetic information and detect birth defects or abnormalities in an
embryo/fetus before implantation/born. In this review, the process and details of the two testing are discussed.
Keywords: PGD, Prenatal Diagnosis, Blastomere Biopsy, FISH, CGH, Chorionic villus sampling, Amniocentesis.
INTRODUCTION
Prenatal diagnosis is a technique for couples to avoid
having a child affected by severe disease; it has been applied
routinely for more than 30 years in many countries [1]. Ideally, prenatal diagnosis aims to provide an accurate and rapid
result as early in pregnancy as possible. However, therapeutic abortion can not be avoided if genetic disease is diagnosed, which could hurt the woman physically as well as
emotionally. In the early 90s, following widespread use of in
vitro fertilization (IVF), a new diagnostic technique was developed in England termed pre-implantation genetic diagnosis (PGD), allowing genetic diagnosis to be made prior to
embryos being transferred into the uterus [2]. Because only
unaffected embryos are selected for transfer to the uterus for
implantation, it eliminates the dilemma of pregnancy termination following unfavorable prenatal diagnosis. The techniques used in PGD are much different than those in prenatal
diagnosis. This review discusses the details of these two
techniques.
PRE-IMPLANTATION GENETIC TESTING
Pre-implantation genetic diagnosis was first introduced
by Handyside in 1990 [3] to sex embryos for X-linked recessive disorders by PCR for Y-sequence amplification after
blastomere biopsy. The first diagnosis for Mendelian traits
involved -1-antitrypsin deficiency by Verlinsky et al. [4]
and F508 homozygosity cystic fibrosis by Handyside et al.
[5]. Fluorescence in situ hybridization (FISH) with chromosome specific probes was used [6, 7] to detect aneuploidy.
Two different terms are used in pre-implantation genetic
testing. The term “pre-implantation genetic diagnosis”
(PGD) applies when one or both genetic parents carry a gene
mutation or an unbalanced chromosome. The embryos are
*Address correspondence to this author at the IVF & Andrology Lab
Director, MacDonald IVF & Fertility Program, University Hospital
Case Medical Center, 11100 Euclid Ave, Cleveland, Ohio 44106, USA;
Tel: (216) 844 3317; Fax: (216) 201 4398; E-mail: [email protected],
[email protected], [email protected]
1573-4048/10 $55.00+.00
tested to determine whether the mutation or an unbalanced
chromosome is passed to the offspring. PGD usually is offered for three major disease groups: sex-linked disorders,
single-gene defects, and chromosomal disorders. The term
“pre-implantation genetic screening” (PGS) applies to
normal genetic parents whose embryos are screened for
aneuploidy.
Two main procedures are used in pre-implantation
genetic testing: Biopsy is the removal of a cell or material
from an embryo or blastocyst. This may include removing a
polar body, blastomeres, or cells from the trophectoderm.
After biopsy, diagnosis can be made by PCR for single-gene
disorders or FISH for chromosomal abnormalities.
To remove the materials from an oocyte or embryo, a
hole should be made in the zona pellucida of the oocyte/
embryo by micromanipulation using laser, a chemical
method such as acid Tyrode’s solution, or a mechanical
method such as a sharpened glass needle, called partial zona
dissection (PZD) [8]. The polar body, blastomere(s), or trophectoderm can be extracted using a small suction pipette or
by gently compressing the oocyte or embryo to extrude material through the opening.
Polar Body Biopsy
The oocyte extrudes polar bodies during meiosis. For
female inherited mutations or chromosomal disorders, genetic analysis can be performed on the first or, sometimes,
the second polar body. However it has limitation of providing maternal information only [9].
Cleavage Stage Biopsy
Cleavage stage biopsy is the most widely used technique,
usually involving biopsy of a single blastomere from day 3
embryos [2]. The advantage of cleavage stage biopsy is that
the genetic constitution of the embryo is complete, and the
timeframe for diagnosis is up to 72 hours (biopsy usually is
performed on day 3 and transfer on day 4, 5, or 6). The deci-
Table 1.
Sun et al.
Comparison of Pre-Implantation Genetic Diagnosis and Prenatal Diagnosis
Pre-Implantation Genetic Diagnosis
Prenatal Diagnosis
Samples
Polar body
Blastomere
Trophectoderm
Ultrasound
No sample
Maternal blood
Chorionic
Villous
Sampling (CVS)
Amniocentesis
Percutaneous
Umbilical Blood
Sampling
Testing Time
D0 or D1 after
oocyte retrieval
D3 after oocyte
retrieval
D5-6 after oocyte
retrieval
19-22w
First or second
trimester
8-11w
14-20w
Up to 18w
Invasive
Invasive
Invasive
Invasive
Non-invasive
Non-invasive
Invasive
Invasive
Invasive
Testing Method
FISH/PCR
FISH/PCR
FISH/PCR
Structural
Biochemical
Many
Many
Many
Cell Number
1 or 2
1 or 2
Up to 5
No
Many
Many
Many
Many
Advantages
Diagnosis before implantation, no need to abort
Non- invasive
More accurate than PGD
Limitations
Amplification failure, ADO, mosaicism
and self-correction.
Need to confirm by prenatal diagnosis
Indirect, no genetic diagnosis
If diagnosis is made, should be abortion
sion to remove one or two blastomeres depends on the quality of the embryo and the disease of interest.
Blastocyst Stage Biopsy
On day 5 after oocyte retrieval, if the embryos have developed to the blastocyst stage with presence of inner cell
mass and trophectoderm, some of the trophectoderm cells
can be removed from the embryo for analysis. The inner cell
mass that develops into the fetus is left undisturbed [10].
This method avoids a possible harmful effect from losing
cells in early day3 embryos. The trophectoderm biopsy
potentially can provide up to five cells for genetic analysis.
Fluorescence In Situ Hybridization (FISH)
FISH is employed for analysis of chromosomal disorders.
Cells are lysed after biopsy and their nuclei are fixed on a
glass slide individually with the cytoplasm dispersed. Chromosomal-specific DNA probes labeled with different fluorochromes can bind to the homologous denatured DNA sequences unique to each chromosome. The number of fluorescent signals of a particular color reflects the number of
copies of each of the chromosomes [11]. Sex determination
in cases of sex-linked disorders, chromosomal disorders, and
PGS for aneuploidy can be diagnosed by this method. Chromosomal disorders involve a variety of chromosomal rearrangements, including translocations, inversions, and deletions. Usually telomeric probes and breakpoint-related
probes specific to the loci site are employed [12]. A mother
who carries the abnormal X chromosome for an X-linked
disease has a 50% of chance of passing it to her son. If the
father is the one affected, the sons should be healthy, but the
daughters have a 100% risk of being carriers. Specific centromere probes are usually used for sexing the embryos.
Aneuploidy increases with maternal age and is one of the
most common causes for early pregnancy failure, recurrent
early pregnancy loss, and repeated failed IVF cycles despite
of transfer of high-quality embryos (based on morphology)
[13]. There are, however, no specific indications for PGS. It
has been proposed for patients at risk for increased prevalence of aneuploid embryos, including women of advanced
maternal age and those with a history of recurrent early
pregnancy loss, repeated IVF failure, or severe male factor
infertility. However, no prevailing evidence supports the use
of PGS to increase live birth rates as compared with control
groups where no PGS is performed. Therefore, PGS currently is not recommended by ASRM [14].
Polymerase Chain Reaction (PCR)
PCR is used to identify specific gene mutations, such
as cystic fibrosis, Tay-Sachs disease, sickle cell anemia,
and Huntington’s disease. A small portion of the genome
that contains the target gene of interest is amplified or replicated to provide a suitable sample for analysis. The blastomeres(s) are lysed and the gene amplified, along with a
gene from normal samples that is amplified simultaneously
as a control. Subsequently double stranded DNA gel electrophoresis is applied. A mismatch due to a genetic deletion or
substitution will result in a differential migration on the gel,
thus permitting diagnosis [15, 16]. Because only one to two
cells are amplified in PGD, the accuracy of PCR diagnosis
can be hampered by amplification failure, DNA contamination, and allele dropout [17].
Limitation of PGD/PGS Technique
Mosaicism is very frequent in in vitro generated embryos, meaning that normal blastomeres can co-exist with
abnormal ones [18]. The true proportions of normal and abnormal cells cannot be determined unless all of the cells are
analyzed. In some cases, embryos identified as aneuploid at
the cleavage stage were ‘‘self-corrected” at blastocyst stage
re-analysis [19]. This is one of the reasons that day 3 singlecell diagnosis has an inevitable false positive rate up to 10%
[20].
Future Prospects of PGD
Only up to five chromosomes can be enumerated simultaneously on a single biopsied cell by FISH. Although multi-
PGD and Prenatal Diagnosis
247
ple (3 to 4) rounds of FISH testing can be used, it cannot
screen all 24 chromosome pairs. This is achieved by comparative genomic hybridization (CGH) to get a complete 24
chromosome karyotype [21]. The limitation of CGH is that it
is time consuming, as the whole procedure takes 48 to 72
hours to complete. Whole-genome analysis using multiple
displacement amplification (MDA) sequencing with gene
chips is another new aspect of PGD testing. It can offer
greater accuracy, more information, and rapid throughout
testing in the near future [22].
Maternal Serum Alpha-Fetoprotein (MSAFP)
PRENATAL DIAGNOSIS
The trophoblast should produce beta-HCG when embryo
implantation occurs. It reaches a peak at 10 weeks of gestation, and then declines. Therefore, maternal serum beta-HCG
can be used to screen for fetal abnormalities, especially for
Down’s syndrome when coupled with MSAFP testing. An
elevated beta-HCG with a decreased MSAFP suggests
Down’s syndrome [30, 31].
Compared with the PGD procedure, prenatal diagnosis is
carried out after the pregnancy is established. Prenatal diagnostic tests can be categorized into non-invasive and invasive tests. Non-invasive diagnosis is achieved by using maternal blood plasma and medical imaging, including routine
obstetric ultrasound scan, echocardiography, and MRI,
which do not harm the fetus or mother. Alternatively, invasive diagnosis includes amniocentesis or chorionic villus
sampling for genetic analysis. These tests are conducted only
in pregnancies that are considered to be at high risk for
chromosomal abnormalities or after a PGD to confirm the
diagnosis.
Ultrasound
Ultrasound or ultrasonography is the most popular and
widely used technique in prenatal diagnosis. It is a noninvasive and cost-effective procedure that is considered
harmless to both the fetus and the mother. Fetal structural
malformations generated by an abnormal gene or chromosome may present, especially in the second trimester of gestation [23, 24]. Alternatively, fetal structural malformations
may arise during embryonic differentiation.
A limitation to ultrasound is that often subtle abnormalities may not be detected until later in pregnancy or not at all.
A good example of this is Down’s syndrome (trisomy 21)
where the morphologic abnormalities are usually not so remarkable, thereby evading detection by ultrasound. Often
only subtle appearance, such as nuchal thickening, is noted
[25]. Other sonographic markers include nuchal translucency
and nasal bone abnormality [26]. The finding of structural
malformations increases the likelihood of aneuploidy and
warrants genetic counseling and consideration of amniocentesis [26]. In recent years three-dimensional (3D) and fourdimensional (4D) ultrasound have been employed in prenatal
diagnosis. These methods can be used to evaluate visual facial features, central nervous system abnormalities, and
skeletal defects [27]. Fetal echocardiography can be performed at 18 to 23 weeks gestation to detect the presence of
heart defects in high-risk pregnancies. When this technique
is used with duplex or color flow Doppler, it can identify a
number of major structural cardiac defects and rhythm disturbances [28].
Biochemical Markers of Maternal Blood Plasma
Some alternative approaches, such as endocrine or
biochemical markers for detection of Down’s syndrome or
maternal serum beta hCG, should be considered, especially
for at-risk pregnancies.
The fetus can produce alpha-fetoprotein (AFP). Ordinarily, only a small amount of AFP can cross the placenta to the
mother's blood and amniotic fluid. In cases of a fetal neural
tube defect, however, more AFP escapes into the amniotic
fluid as well as into maternal serum. The MSAFP test has the
greatest sensitivity between 16 and 18 weeks gestation, but
can still be useful between 15 and 22 weeks gestation [29].
Maternal Serum Beta-HCG
Unconjugated Estriol
Combined evaluation of unconjugated estriol (uE3), betaHCG, and MSAFP in maternal plasma is called triple testing.
uE3 tends to be lower when Down’s syndrome is present and
in the presence of adrenal hypoplasia with anencephaly [31].
Inhibin-A
Inhibin-A is secreted by the placenta and the corpus luteum. An increased level of inhibin-A is associated with an
increased risk for trisomy 21. It is increasingly utilized with
the previous three markers, named quadruple testing when
performed together [31].
Pregnancy-Associated Plasma Protein A (PAPP-A)
Low levels of PAPP-A as measured in maternal serum
during the first trimester may be associated with fetal chromosomal anomalies [32].
Amniocentesis
Amniocentesis is an invasive procedure, usually performed between 14 and 20 weeks of gestation. A sample of
about 15 ml of amniotic fluid is obtained transabdominally
under ultrasound guidance. The amniotic fluid is sent for
biochemical and molecular biologic analysis, and the fetal
cells from amniotic fluid are cultured for chromosome analysis [32]. Risks of amniocentesis, although low, include fetal
loss and maternal Rh sensitization.
Chorionic Villus Sampling (CVS)
This procedure usually is performed between 8 and 11
weeks of gestation under ultrasound guidance. The sample of
placenta usually is obtained by a catheter through the cervix
or by alternative approaches, such as transvaginally and or
transabdominally. Similar to amniocentesis, cells from CVS
can be analyzed by a variety of techniques [32]. The major
advantage of CVS over amniocentesis is that the diagnosis is
performed at an earlier stage of gestation. The disadvantage
of CVS is that it is an invasive procedure which could induce
pregnancy loss. The risk of pregnancy loss is about 0.5 to
1%, which is higher than that of amniocentesis [32]. Rarely,
CVS also can be associated with limb defects of the fetus
[33]. The possibility of maternal Rh sensitization is present.
Sun et al.
Misdiagnosis may occur because of placental mosaicism and
sample contamination by maternal tissue [32].
More research hopefully will help us overcome these obstacles in the near future.
Percutaneous Umbilical Blood Sampling (PUBS)
KEY POINTS
PUBS, also named cordocentesis [34] is performed after
18 weeks of gestation. A needle is inserted into the umbilical
cord under ultrasound guidance, and a sample of 0.5-1 ml
fetal blood is collected from the umbilical vein for further
diagnosis. It is useful for evaluating fetal metabolism and
hematologic abnormalities.
Prenatal diagnosis consists of a range of non-invasive
and invasive testing that is used for diagnosing diseases or
conditions in a fetus prior to birth.
The optimal approach to prenatal diagnosis is to first refer the woman for genetic counseling and then to employ
non-invasive methods. In cases of abnormal screening results, invasive methods should be used to confirm the diagnosis. The use of PGD and prenatal genetic diagnosis have
been dramatically prevented genetic diseases and improved
the quality of life for many families.
EXPERT COMMENTARY
Although PGD is often regarded as an early form of
prenatal diagnosis, the nature of the PGD request often
differs from prenatal diagnosis, which is accomplished when
the mother is already pregnant. Some widely accepted
indications for PGD would not be acceptable for prenatal
diagnosis, such as screening for diagnosis of late-onset
disease or predisposition syndromes (like theBRCA1
mutation for breast cancer).
FISH is the most widely used method to determine an
embryo’s chromosomal constitution. The main problem with
cleavage embryo PGD is the high rate of mosaicism, up to
70% of analyzed embryos. It has been shown that 40% of the
embryos diagnosed as aneuploidy on day 3 turned out to
have euploid inner cell mass at day 6. The possibility of
discarding a viable embryo due to the limitation of the
technique following PGD on a day 3 embryo is a major
concern. One or two blastomeres are not representative of
the complete embryo. Biopsy at the blastocyst stage would
provide more accurate diagnosis as more cells can be
biopsied.
Currently, a large number of probes are available for
different segments of chromosomes; however, the limited
number of fluorochromes constrains the number of signals
that can be analyzed simultaneously. To overcome this
problem, up to three consecutive rounds of FISH can be
carried out on the same sample. Overall, 13 is the maximum
number of chromosomes that can be analyzed by FISH.
Whole-genome analysis using multiple displacement amplification (MDA) sequencing with gene chips would allow for
analysis of all 24 chromosomes.
Pre-implantation genetic diagnosis (PGD) refers to diagnostic procedures performed on oocytes, embryos, or blastocysts before implantation to enhance the transfer of a normal
embryo into the uterus and avoid selective pregnancy termination.
In FISH, the most widely used method for PGD, cells are
fixated on glass microscope slides and hybridized with
fluorescence DNA probes for analysis under the fluorescence
microscope.
Comparative genomic hybridization (CGH) is a
molecular cytogenetic method for genetic screening that
analyzes all 24 chromosomes to produce a map of DNA sequence copy number as a function of chromosomal location
throughout the entire genome.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
FIVE-YEAR REVIEW
Due to the limitation of FISH and PCR, we expect to
witness the entry of whole-genome analysis/CGH into the
mainstream of IVF to replace current PGD testing. Its preliminary application has been encouraging as more chromosomes can be can be analyzed with higher sensitivity and
accuracy. However, it takes longer to have results, which
may constrain fresh embryo transfer. Additionally, wholegenome analysis/CGH is not cost-effective for the patient.
[12]
[13]
[14]
KanYW, Dozy AM. Antenatal diagnosis of sickle-cell anaemia
by D.N.A. analysis of amniotic-fluid cells. Lancet 1978; 2: 9102.
Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Yspecific DNA amplification. Nature 1990; 344: 768-70.
Handyside AH, Pattinson JK, Penketh RJ, et al. Biopsy of human
preimplantation embryos and sexing by DNA amplication. Lancet
1989; 1: 347-9.
Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first
polar body: preconception genetic diagnosis. Hum Reprod 1990; 5:
826-9.
Handyside AH, Lesko JG, Tarin JJ, et al. Birth of a normal girl
after in vitro fertilization and preimplantation diagnostic testing for
cystic fibrosis. N Engl J Med 1992; 327: 905-9.
Delhanty JD, Griffin DK, Handyside AH, et al. Detection of Aneuploidy and chromosomal mosaicism in human embryos during
preimplantation sex determination by fluorescent in situ hybridization (FISH). Hum Mol Genet 1993; 2(8): 1183-5.
Schrurs BM, Winston RM, Handyside AH. Preimplantation diagnosis of aneuploidy using fluorescent in-situ hybridization: evaluation using a chromosome 18-specific probe. Hum Reprod 1993;
8(2): 296-301.
Nijs M, Camus M, Van Steirteghem AC. Evaluation of different
biopsy methods of blastomeres from 2-cell mouse embryos. Hum
Reprod 1988; 3(8): 999-1003.
Montag M, van der Ven K, Delacrétaz G, Rink K, van der Ven H.
Laser-assisted microdissection of the zona pellucida facilitates
polar body biopsy. Fertil Steril 1998; 69(3): 539-42.
Dokras A, Sargent IL, Ross C, Gardner RL, Barlow DH. Trophectoderm biopsy in human blastocysts. Hum Reprod 1990; 5(7): 8215.
Munné S, Weier HU, Stein J, Grifo J, Cohen J. A fast and efficient
method for simultaneous X and Y in situ hybridization of human
blastomeres. J Assist Reprod Genet 1993; 10(1): 82-90.
Scriven PN, Handyside AH, Ogilvie CM. Chromosome translocations: segregation modes and strategies for preimplantation genetic
diagnosis. Prenat Diagn 1998; 18(13): 1437-49. Review.
Brown S. Miscarriage and its associations.Semin Reprod Med
2008; 26(5): 391-400.
Practice Committee of the Society for Assisted Reproductive
Technology; Practice Committee of the American Society for
Reproductive Medicine. Preimplantation genetic testing: a Practice
Committee opinion. Fertil Steril 2007; 88(6): 1497-504.
PGD and Prenatal Diagnosis
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Navidi W, Arnheim N. Using PCR in preimplantation genetic
disease diagnosis. Hum Reprod 1991; 6(6): 836-49.
Lavery S. Preimplantation genetic diagnosis of haemophilia. Br J
Haematol 2009; 144(3): 303-7. Review
Ray PF, Handyside AH. Increasing the denaturation temperature
during the first cycles of amplification reduces allele dropout
from single cells for preimplantation genetic diagnosis. Mol Hum
Reprod 1996; 2: 213-8.
Baart EB, Martini E, van den Berg I, et al. Preimplantation genetic
screening reveals a high incidence of aneuploidy and mosaicism in
embryos from young women undergoing IVF. Hum Reprod 2006;
21: 223-33.
Li M, DeUgarte C, Surrey M, Danzer H, DeCherney A, Hill D.
Fluorescence in situ hybridization reanalysis of day-6 human
blastocysts diagnosed with aneuploidy on day 3. Fertil Steril 2005;
84: 1395-400. 20.
Munne S, Velilla E, Colls P, et al. Self-correction of chromosomally abnormal embryos in culture and implications for stem cell
production. Fertil Steril 2005; 84: 1328-34.
Wilton L. Preimplantation genetic diagnosis and chromosome
analysis of blastomeres using comparative genomic hybridization.
Hum Reprod Update 2005; 11(1): 33-41. Review.
Spits C, Sermon K. PGD for monogenic disorders: aspects of
molecular biology. Prenat Diagn 2009; 29(1): 50-6. Review.
Verrotti C, Caforio E, Gramellini D, Nardelli GB. Ultrasound
screening in second and third trimester of pregnancy: an update.
Acta Biomed 2007; 78(3): 229-32. Review
Tamsel S, Ozbek S, Demirpolat G. Ultrasound evaluation of fetal
chromosome disorders. Diagn Interv Radiol 2007; 13(2): 97-100.
Review
Crane JP, Gray DL. Midtrimester sonographically measured nuchal
skin fold thickness as a screening tool for Down syndrome. Obstet
Gynecol 1991; 77: 553.
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
249
Williamson RA, Weiner CP, Patil S, Benda J, Varner MW,
Abu-Yousef MM. Abnormal pregnancy sonogram: selective
indication for fetal karyotype. Obstet Gynecol 1987; 69: 15-20.
Kurjak A, Miskovic B, Andonotopo W, Stanojevic M, Azumendi
G, Vrcic H. How useful is 3D and 4D ultrasound in perinatal
medicine? J Perinat Med 2007; 35(1): 10-27.
Sklansky M, Miller D, Devore G et al. Prenatal screening for congenital heart disease using real-time three-dimensional echocardiography and a novel 'sweep volume' acquisition technique. Ultrasound Obstet Gynecol 2005; 25: 435-443.
Wang ZP, Li H, Hao LZ, Zhao ZT. The effectiveness of prenatal
serum biomarker screening for neural tube defects in second
trimester pregnant women: a meta-analysis. Prenat Diagn 2009;
29(10): 960-5.
Benn PA. Advances in prenatal screening for Down syndrome: I.
general principles and second trimester testing.Clin Chim Acta
2002; 323(1-2): 1-16. Review.
Wald NJ, Watt HC, Hackshaw AK. Intergrated screening for
Down's syndrome on the basis of tests performed during the first
and second trimesters. N Engl J Med 1999; 341: 461-467.
Alfirevic Z, Sundberg K, Brigham S. Amniocentesis and chorionic
villous sampling for prenatal diagnosis. Cochrame Database Syst
Rev 2003; 3: CD003252.
Burton BK, Schulz CJ, Burd LI. Limb anomalies associated
with chorionic villus sampling. Obstet Gynecol 1992; 79(5): 72630.
Daffos F, Capella-Pavlovsky M, Forestier F. Fetal blood sampling
during pregnancy with use of a needle guided by ultrasound:
a study of 606 consecutive cases. Am J Obstet Gynecol 1985;
153(6): 655-60.
250
James M. Goldfarb* and Nina Desai
Obstetrics and Gynecology, Cleveland Clinic Fertility Center, Cleveland, OH 44122, USA
Abstract: Multiple pregnancies have been and remain the most common and serious complication of assisted
reproductive technologies (ART). Prematurity is the major complication of multiple pregnancies although there are
other problems affecting the children and parents also increase significantly. The scope of the problem of ART and
multiple pregnancies is discussed as well as the specific issues involving children and parents. Progress in decreasing
ART multiple pregnancies and suggested steps to further decrease ART multiple pregnancies are also discussed.
Keywords: Multiple pregnancy, assisted reproductive technologies, in vitro fertilization, prematurity.
INTRODUCTION
Multiple pregnancies are the most common and serious
complication of assisted reproductive technologies (ART).
The significant stress of not being able to get pregnant can,
not infrequently, make both infertile couples and health care
providers forget the obvious --- the goal of ART is not
merely getting pregnant but having healthy children. Either
due to desperation for getting pregnant or lack of knowledge
regarding the morbidity of multiple pregnancies, couples
seem to lack appropriate concern about multiple pregnancies.
A survey by Goldfarb et al. of 77 women embarking on gonadotropin therapy or in vitro fertilization (IVF) showed that
95% of women preferred getting pregnant with triplets to not
getting pregnant, and 68% of women preferred getting pregnant with quadruplets to not getting pregnant [1]. Looking at
this issue in a different way, Ryan et al. [2] asked new infertility patients as to their preference for treatment outcomes.
More than 20% of women embarking on infertility treatment
preferred multiple pregnancies to a singleton pregnancy.
Very significantly, 4% of those favoring multiple pregnancies ranked quadruplets as their most desired outcome.
Based on these observations, the fact that couples undergoing IVF are often very aggressive regarding the number of
embryos to transfer is not surprising.
Besides the above described attitudes regarding multiple
pregnancies, many couples are of the philosophy that, because they have never been able to get pregnant at all, they
do not need to be worried about multiple pregnancies. Similarly, patients who failed to get pregnant with the transfer of
two embryos in a first IVF cycle often do not feel comfortable unless they transfer more embryos in a subsequent cycle. These understandable emotional issues, in combination
with the overwhelming financial investment for IVF, result
in higher than necessary multiple pregnancy rates in the
United States.
Further exacerbating this trend is the pressure on IVF
programs to report high success rates to stay competitive.
*Address correspondence to this Author at the Obstetrics and Gynecology,
Cleveland Clinic Fertility Center, 26900 Cedar Road Suite 220 South
Beachwood, Cleveland, OH 44122 USA; Tel: 216 839 2903;
Fax: 216 839 3194; E-mail [email protected]
1573-4048/10 $55.00+.00
While there is an increasing emphasis on the undesirability
of multiple pregnancies with IVF, success of IVF programs
still is undoubtedly judged predominantly by the “takehome-baby rate” which does not take into consideration the
significant financial costs and morbidity of multiple pregnancies.
SCOPE OF THE PROBLEM OF MULTIPLE PREGNANCIES WITH ART
Multiple pregnancies have increased greatly in the United
States since 1980. From 1980 to 1999 the overall multiple
pregnancy rate increased from 1.9% to 3.1% of all births, an
increase of approximately 60%. Numerically, most of the
increase was in twins. However, percentage-wise the increase in higher-order pregnancies (triplets or more) was
more than 400%, increasing from 37 to 193 per 100,000
births [3]. While a small part of the increase in multiple
pregnancy rates is due to other factors, such as increased
maternal age, the vast majority of the increase in multiple
pregnancies is due to infertility treatments. Infertility treatments involve more than ART and, indeed, ovulation induction, according to a review by Norwitz [4] may be responsible for twice as many high-order pregnancies than is ART.
The impact of ART, however, is extremely significant. In
2006, infants conceived with ART accounted for approximately 1% of the total births in the United States. However,
the proportion of twins and triplets or higher order multiples
resulting from ART were 17% and 38%, respectively [5].
While an increased number of dizygotic multiples are
expected with ovulation induction and ART, the number of
monozygotic twins is also increased with ART. An increased
number of monzygotic twins also has been reported with
ovulation induction. Derom et al. [6] reported a 1.2% incidence of monozygotic twinning after ovulation induction
versus the generally accepted 0.4% incidence in the general
population [7]. All these incidences may be understated to
some degree since diamniotic-dichorionic monozygotic gestations can easily be overlooked [8].
Ovulation induction itself may be one factor causing the
increased incidence of monozygotic twins in ART, but it is
not the only one. It seems the incidence of monozygotic
twins is higher in ART than in ovulation induction, especially when ART involves blastocyst transfer. Milki et al. [7]
found monozygotic twinning occurred in 5.6% of pregnancies conceived after blastocyst transfer compared with 2%
after cleavage-stage transfer. Further exacerbating this problem is the finding of Milki and others [7-9] that the incidence
of monozygotic twinning seems to be highest when more
than one gestational sac is present, thus leading to a relatively higher rate of triplets than twins caused by monozygotic twinning. Another negative factor regarding monozygotic twinning associated with ART is the possibility that the
incidence of monoamniotic twinning, and all it associated
problems, may be higher with ART than with natural
monozygotic twinning [10].
PROBLEMS ASSOCIATED WITH MULTIPLE PREGNANCIES
Prematurity
Clearly the major cause of morbidity with multiple pregnancies is prematurity. The relative risk of birth at gestational age less than 32 weeks is increased 7-, 23-, and 40fold for twins, triplets, and quadruplets, respectively. Fully
one-third of triplets and two-thirds of quadruplets will be
born at less (and often considerably less) than 32 weeks [11].
Approximately 25% of twins, 75 % of triplets, and essentially 100% of quadruplets or more will be admitted to a
neonatal intensive care unit. According to Newman and
Luke, the average days in the NICU average 18, 30, and 58
for twins, triplets, and quadruplets, respectively [12].
Small for Gestational Age
In addition to prematurity, multiple births increase the
number of small for gestational age (SGA) babies, and the
incidence seems to increase with gestational age. Alexander
et a.l in reviewing United States live births from 1991 to
1995 found 28% of twins were SGA by 34 weeks and 40%
were SGA by 37 weeks. They found that 50% of triplets
were SGA by 35 weeks gestation [13].
Cerebral Palsy and other Handicaps
Of all the sequelae of prematurity, cerebral palsy seems
to be the most studied. Pharoah and Cooke reported in1996
the rate of cerebral palsy was increased 5.5-fold for twins
and almost 20-fold for triplets [14] Similar numbers were
reported by Peterson et al. in 1993 [15].
Other handicaps also clearly are increased by multiple
births. Luke and Keith [16] using data from the US Congress, Office of Technology Assessment, found that overall,
handicaps were increased by 39% in twins and by 97% in
triplets. Severe handicaps were increased by 30% and 71%,
respectively.
Birth defects are also increased by multiple gestations.
Monozygotic twins are twice as likely as dizygotic twins to
be born with congenital malformations. In one study, the risk
of significant congenital anomalies among triplets was 5%
[17].
Infant Mortality
Infant deaths (deaths at less than 1 year of age) are not as
commonly discussed as are the other sequelae of multiple
251
pregnancies. Not surprisingly, however, infant death statistics are just as impressive. Keith and Luke [16], using Vital
Statistics of the United States, 1998, calculated the number
of infant deaths to be 6-fold higher in twins and 17- fold
higher in triplets compared to singletons. The perinatal mortality for triplets, including all intrauterine demises and deliveries occurring after 20 weeks, is greater than 10% in
many studies [17].
Maternal Complications
Maternal morbidity, while of significantly less magnitude
than that of the babies, is nevertheless a consideration.
Essentially all maternal complications of pregnancy are
increased by the occurrence of multiple pregnancies. In addition to premature labor, complications include preeclampsia,
gestational diabetes, anemia, postpartum hemorrhage, and
even the relatively rare acute fatty liver of pregnancy [17].
Parenting Issues
The psychological impact of multiple pregnancies has
been well described. Cook et al. [18] evaluated parents of
IVF twins and reported that these couples found parenting to
be not as satisfying as they expected. Furthermore, they
found that parents of IVF twins seem to be more stressed
than were parents of naturally conceived twins.
FINANCIAL COSTS
While not as important as the morbidity caused by multiple pregnancies, the financial costs of multiple pregnancies
also are overwhelming. Callahan et al. [10] evaluated the
births at a Boston hospital from 1986 to 1991 and found that
added costs of multiple pregnancies were approximately
$38,000 and $110,000, respectively, for twins and higherorder pregnancies. Goldfarb et al. [19] estimated the added
cost (above normal costs for a singleton delivery) per woman
delivered of pregnancies from an IVF program during the
years 1991 and 1992. Estimated added costs included the
cost of the IVF procedure, admissions for premature labor,
time off of work, and, very importantly, costs for ongoing
care of premature infants. The authors found the added cost
per delivery of IVF patients was approximately $39,000 for
single and twin deliveries versus approximately $343,000 for
triplet and quadruplet pregnancies.
STEPS TO MINIMIZE THE PROBLEM OF ARTASSOCIATED MULTIPLE PREGNANCIES
There is little debate as to the significance of the problems resulting from multiple pregnancies and ART. There is,
conversely, a great deal of debate as to the best way to
minimize the problem. Issues such as patients’ rights to decide on the number of embryos and trade off between higher
pregnancy rates and higher multiple pregnancy rates result in
controversy over embryo transfer policy. Legislation in some
European countries strictly limits the embryo transfers to two
embryos for most patients.
The American Society of Reproductive Medicine has
established guidelines for number of embryos to transfer in
an IVF cycle, the most recent of which was published in
2008 [20]. These guidelines recommend, for example, the
Table 1.
Goldfarb and Desai
Recommended Limits on the Numbers of Embryos to Transfer
Cleavage-Stage Embryos
Age
Age
Age
Age
Prognosis
<35
35-37
38-40
>40
Favorable
1-2
2
3
5
All others
2
3
4
5
Blastocysts
Age
Age
Age
Age
Prognosis
<35
35-37
38-40
>40
Favorable
1
2
2
3
All others
2
2
3
3
See text for more complete explanations. Justification for transferring more than the recommended number of embryos should be clearly documented in the patient’s medical record.
Favorable = First cycle of IVF, good embryo quality, excess embryos available for cryopreservation, or previous successful IVF cycle. ASRM Practice Committee Guidelines on
number of embryos transferred. Fertil Steril 2008.
transfer of one embryo for younger patients with a “favorable prognosis” (Table 1). Many are strongly advocating
even stricter guidelines because of the feeling that the goal of
ART should be a healthy singleton birth. and the fact that
increasingly good success rates can be achieved by transferring a single embryo, particularly at the blastocyst stage [21].
Of course, as stated earlier, even a single embryo transfer,
particularly at the blastocyst stage, does not completely
eliminate multiple pregnancies.
Several questions arise regarding how insurance coverage influences the number of embryos transferred. Reynolds’s et al. [22, 23] showed a lower incidence of transfer
of three or more embryos in insured versus non-insured
states. However, the effects of insurance coverage on multiple pregnancies were modest. Many feel a substantial effect
on multiple pregnancies would occur only if insurance coverage was combined with mandated limits regarding number
of embryos to transfer.
Another way to decrease the number of multiple pregnancies is to revise the way program success is assessed. In
the United States the “take-home-baby rate” is the standard
by which a program is judged; the multiple pregnancy rate of
a program is much more rarely examined. Embryo implantation rate, a very good indicator of the quality of an IVF program, has been incorporated into the Society of Assisted
Reproductive Technologies (SART) reporting system but is
not prominently displayed. The CDC does not include implantation rates in its report to the public.
Despite the still significant barriers to decreasing multiple pregnancies, considerable progress has been made in
decreasing triplet births. The SART 2007 report shows a
triplet birth rate of less than 2% compared with almost 8% 8
years earlier. Twin pregnancy rates, however, remain
unchanged at more than 30% for patients under 38 years old.
Clearly there is no simple answer to the problem of further decreasing ART multiple pregnancies. Efforts on many
fronts are necessary. Insurance coverage with a reasonable
limitation to the number of embryos transferred combined
with a SART and CDC reporting system that emphasize implantation rates and/ or singleton pregnancy rates would be
very helpful. Also, very importantly, educating our patients
as to the risks of multiple pregnancies is imperative. We
must not forget, our goal is not to get patients pregnant but
for them to have a healthy family.
EXPERT COMMENTARY
The purpose of this article was to bring attention to the
increase in multiple pregnancies caused by assisted reproductive technologies and to increase awareness of the significant health and financial costs of multiple pregnancies.
Couples and health care workers involved with ART often
tend to focus on pregnancy rates and often do not give as
much consideration to trying to decrease the incidence of
multiple pregnancies. Efforts that include guidelines by the
American Society of Reproductive Medicine on maximum
number of embryos to transfer have been successful in
decreasing high-order multiple pregnancies from ART.
However, as yet, these efforts have not been successful in
decreasing the incidence of ART twins.
FIVE-YEAR VIEW
In the next 5 years it is anticipated there will be increasing emphasis on a singleton birth as the goal of ART. Acceptance of the importance of this goal will be accomplished
through several avenues:
• Education of physicians and patients regarding the health
risks of twins.
• Changes in the reporting of ART success rates that will
incentivize ART programs to strive for singleton pregnancies.
• Further improvements in embryo culture methods resulting in better implantation rates per embryo.
• Better methods of evaluating embryos to increase ability
to choose the best embryos for transfer.
• More stringent guidelines from professional groups, insurance companies, and possibly government regarding
maximum number of embryos to transfer.
[6]
[7]
KEY ISSUES
[8]
• Multiple pregnancies are the most common and serious
complication of assisted reproductive technologies
(ART).
[9]
• The major cause of morbidity with multiple pregnancies
is prematurity, but other complications, including small
for gestational age, also are increased with multiple pregnancies.
[10]
[11]
• There has been good success in reducing the number of
high-order multiple pregnancies (triplets or more) resulting from ART, but little progress has been made in reducing the number of twins.
[12]
• A combination of actions is needed to decrease the number of twins resulting from ART. These actions include
acceptance by patients and doctors that the goal of ART
should be a singleton pregnancy.
[14]
[13]
[15]
ACKNOWLEDGEMENT
Most of this article was published in Clinical Reproductive Medicine and Surgery [edited by] Tommaso Falcone,
William W. Hurd. Copyright 2007 Mosby, Inc., an affiliate
of Elsevier, Inc. Pages 601- 603.
[2]
[3]
[4]
[5]
Goldfarb J, Kinzer DJ, Boyle M, et al. Attitudes of in vitro
fertilization and intrauterine insemination couples toward multiple
gestation pregnancy and multifetal pregnancy reduction. Fertil
Steril 1996; 65: 815.
Ryan GL, Zhang SH, Dokras A, et al. The desire of infertile
patients for multiple births. Fertil Steril 2004; 81: 500.
Mathews TJ, MacDorman MF, Menacker F. Infant mortality statistics from the 1999 period linked birth/infant death data set. Nat Vital Stat Rep 2002; 50: 1.
Norwitz ER. Multiple pregnancy: Trends past, present and future.
Infertil Reprod Med Clin North Am 1998; 9: 351.
Morbidity and Mortality Weekly Report. Assisted Reproductive
Technology Surveillance – United States, 2006. Department of
Health and Center for Disease Control. Surveillance Summaries.
June 12, 2009; 58: No.SS-5.
[17]
[18]
REFERENCES
[1]
[16]
[19]
[20]
[21]
[22]
[23]
253
Derom C, Vlietinck R, Derom R, et al. Increased monozygotic
twinning rate after ovulation induction. Lancet 1987; 1: 1236.
Milki AA, Jun SH, Hinckley MD, et al. Incidence of monozygotic
twinning with blastocyst transfer compared to cleavage-stage transfer. Fertil Steril 2003; 79: 503.
Machin G, Bamforth F, Innes M, et al.Some perinatal characteristics of monozygotic twins who are dichorionic. Am J Med Genet
1995; 81: 995.
Su LL. Monoamniotic twins: Diagnosis and management. Acta
Obstet Gynecol Scand 2002; 81: 995.
Callahan TL, Hall JE, Ettner SL, et al. The economic impact of
multiple-gestation pregnancies and the contribution of assisted
reproduction techniques to their incidence. N Engl J Med 1998;
339-573.
Martin JA, Halimton BE, Venture SJ, et al. Births: Final data for
2001. Nat Vital Stat Rep 2002; 51: 104.
Newman RB, Luke B. Neonatal and post-neonatal considerations.
In: RB Newman, B Luke Eds. Multifetal Pregnancy. Philadelphia,
Lippincott Williams & Wilkins, 2000; p. 243.
Alexander G, Kogan M, Martin J. What are the fetal growth
patterns of singletons, twins and triplets in the USA? Clin Obstet
Gynecol 1998; 41: 115.
Govaerts I, Devreler F, Delbaere A, et al. Short-term medical
complications of 1500 oocyte retrievals for in vitro fertilization
and embryo transfer. Eur J Obstet Gynecol Reprod Biol 1998; 77:
239.
Peterson B, Nelson K, Watson L, et al. Twins, triplets and cerebral
palsy in births in Western Australia in the 1980’s. Br Med J 1993;
307: 1239.
Luke B, Keith LG. The contribution of singletons, twins and
triplets to low birthweight, infant mortality and handicap in the
United States. J Reprod Med 1992; 37: 661.
Devine PC, Malone FD, Athanassiou A, et al. Maternal and neonatal outcome of 100 consecutive triplet pregnancies. AM J Perinatol
2001; 18: 225-35.
Cook R, Bradley S, Golombok S. A preliminary study of parental
stress and child behaviour in families with twins conceived by in
vitro fertilization. Hum Reprod 1998; 13: 3244.
Goldfarb JM, Austin C, Lisbona, H, et al. Cost-effectiveness of in
vitro fertilization. Obstet Gynecol 1996; 87: 18.
ASRM Practice Committee. Guidelines on number of embryos
transferred. Fertil Steril 2008; 90(Supp l3): 799-813.
Healey D. Damaged babies from assisted reproductive technologies. Focus on the BESST (birth emphasizing a successful singleton at term) outcome. Fertil Steril 2004; 81: 512.
Reynolds MA, Schieve LA, Jeng G, et al. Does insurance coverage
decrease the risk for multiple births associated with assisted reproductive technology? Fertil Steril 2003; 80: 16.
Reynolds MA, Schieve LA, Peterson HB. Insurance is not a magic
bullet for the multiple birth problem associated with assisted reproductive technology. Fertil Steril 2003; 80: 32.
254
Metabolomic Profiling for Selection of the Most Viable Embryos in
Clinical IVF
George A. Thouas and David K. Gardner*
Department of Zoology, The University of Melbourne, Parkville, Victoria 3010, Australia
Abstract: Along with standard morphological indicators of preimplantation embryo development, information gained
from novel “Omics” platforms is providing more detailed functional characterizations of embryo phenotype. Since
embryo metabolism is a critical driver of development and implantation, it is proposed that analysis of the embryo
metabolome may reveal several physiologically relevant markers. Metabolomics analysis is currently showing significant
promise in this context, providing a systematic method of screening for low molecular weight metabolic by-products,
in isolated cell extracts and biological fluids. Correlations between in vitro development and pregnancy outcomes have
already been described after retrospective data comparison with metabolomic profiles. This article summarizes progress
and current findings of metabolomic analysis as a new and complimentary technology for screening embryo cohorts in
clinical IVF, to facilitate prognostic selection of a single embryo for transfer.
Keywords: Implantation rate, metabolomics, pregnancy, preimplantation embryo, viability.
INTRODUCTION
Analysis of blastomere morphology and in vitro growth
rate of the preimplantation embryo have been the key indicators of embryo quality in clinical IVF laboratories for over
30 years [1-5]. However, morphological evaluation remains
a subjective metric and consequently there is an ongoing
need for more systematic quantitative assays of the biochemical and molecular processes within embryos that
underlie these phenotypes. Over the five day period between
fertilization and blastocyst formation, the preimplantation
embryo relies on energy generation from oxidative metabolism of carbohydrates and amino acids [6], with the
notable shift from three-carbon to six-carbon sugars at Day 3
and an increase in aerobic glycolysis at the blastocyst stage
[7, 8]. This latter change in metabolism is attributed to both
an increased demand for biosynthesis, consistent with an
increased growth trajectory and in preparation for implantation [9]. Drawing from this data, human embryo culture
media formulations have been improved significantly,
which in turn has led to increased success rates of in vitro
embryo production, cryopreservation survival and posttransfer pregnancy outcomes. Improvements in laboratory
conditions have also led to the ability to culture human
embryos to the blastocyst stage [10]. One advantage of
such an approach is the ability to apply alpha-numeric grading systems, capable of grading both the trophectoderm and
inner cell mass, which have been shown to be of value in
blastocyst selection for transfer [5].
Although blastocyst transfer has been adopted for several
groups of patients, a significant number of transfers are
performed on either day 2 or 3 of development [11]. At
these early stages of development there is, perhaps, a greater
*Address correspondence to this author at the Department of Zoology, The
University of Melbourne, Parkville VIC 3010, Australia;
Tel: +61-3-8344-6259; Fax: +61-3-8344-7909;
1573-4048/10 $55.00+.00
need for systematic assays of embryo quality and prospective
viability, considering that the cleavage stages tend to be
morphologically similar. To this end, application of emerging “Omics” technologies, benchmarked by functional
genomics (whole genome analysis, transcriptomics) and
proteomics [12] are starting to provide innovative and
clinically amenable technologies for obtaining functional
biomolecular profiles that correlate with embryo development. In combination with detailed bioinformatics sorting
and statistical analysis of datasets, fine differences between
profiles of individual embryos can be detected using an array
of methods [13].
METABOLOMICS
SYSTEM
AS
A
CLINICAL
BIOASSAY
Overview of Analytical Principles
Metabolomics profiling is emerging as a highly useful
adjunct to standard clinical diagnostics, yielding a wealth of
phenotypic information. Like other “Omics” techniques,
metabolomics relies on the chemical purification of cell
extracts or biological fluids, followed by spectroscopic
analysis [14]. In some cases, spectroscopy can be performed
directly without the need for sample purification, such as the
case of buffers with low levels of secreted metabolites. Also,
the purification method itself can yield sufficient information
on fractions based on molecular weights and charge distributions on molecules. For example, chromatographic methods
rely on separation of different fractions based on their binding affinity with a porous matrix through which the sample
flows, very similar to the way different molecular weight
DNAs are separated using electric fields in gel electrophoresis. While specific extraction protocols are required for
isolating unique cellular fractions (e.g., DNA, protein, lipids
and polysaccharides) the method of spectroscopic analysis
of those fractions varies dramatically, depending on the
chemical properties of the molecular constituents.
Metabolomic Profiling for Selection of the Most Viable Clinical IVF
Briefly, spectroscopy relies on probing a solid or fluid
sample with a specific range of optical or non-visible wavelengths, followed by analysis of the resultant energy output
to obtain an absorbance profile. Different absorbance patterns correlate with the abundance and chemical structure of
the complete set of molecules in the sample. For example,
non-visible infrared wavelengths (greater than visible region,
>800 nm) interfere with the vibrational frequency of chemical bonds, providing information about the abundance and
type of bonding between individual atoms and side groups in
the sample. Shorter wavelengths tend to interfere with higher
energy bonds, such as atomic-scale associations. There are
some exceptions, which could be regarded as both purification and spectroscopic methods in one. For example, mass
spectrometry (MS) uses high energy electrons and other
chemical methods to ionize samples into their constituent
atoms, providing a distribution map of all atoms in the sample. On the other hand, nuclear magnetic resonance (NMR)
spectroscopy uses very low energy waves to detect single
elements such as hydrogen protons, using proton movement
to reveal precise information on organisation and structure.
In general, the complex chemical structure of molecules
and abundance of elements can be quantitated using combinations of chemical purification and spectroscopy.
Biospectroscopy as a Clinical Diagnostic Tool
Overall, there are several terms that describe the approach adopted in the adaptation of these spectroscopic techniques to biological systems (sometimes termed “biospectroscopy”) – the term itself refers to the nature of the energy
profiles obtained from samples as “spectra”, since they are
typically over a specific wavelength range, and have a characteristic pattern. Chemical extraction of whole cells or biological fluids can yield a spectral representation (profile) of
the entire metabolome, often termed a metabolic “fingerprint” [15, 16], as distinct from a metabolic “footprint”,
which can refer to subsets of extracellular metabolites
produced by cells or tissues [16]. For simplicity, we will
use the term “metabolomic profile”, in the same context as
the term for the applications described in this article. Generation of either type of profile, usually over an arbitrary range
(spectrum) of wavelengths, is generally classified as a
“bottom-up” (discovery driven) or “top-down” (hypothesis
driven) exercise [17]. The same spectroscopic approaches
can be used in both contexts, such as in generation of
distributions of broad compositional data for basic comparisons (e.g., control versus treatment), or analysis of specific
elemental abundance (eg specific metabolic responses to
a drug).
For biomedical applications, profiles of spectral data
obtained from diseased versus healthy biological samples
forms the basis of a diagnostic assay, as has been demonstrated successfully for cancer diagnosis using Fouriertransformed infrared spectroscopy (FTIR) and Raman spectroscopy directly on preserved histological specimens [18].
In conjunction with these comparative assessments is the
need for further computer-based statistical processing (commonly termed “chemometrics”) which can standardize and
increase the power of the comparisons to detect minute differences in the profiles. A similar approach has been used
255
extensively in functional genomics, where whole genome
maps of changes in gene expression can be related to single
genes based on differential optical fluorescence of base pairs,
and later fed into gene databases. In this way, a bottom-up
approach can rapidly yield candidates for further top down
studies [19], such as in the systematic search for biomarkers
of cancer cells [20].
Metabolomics: Analysis of Metabolite Presence and
Abundance
Specific types of spectroscopy are amenable for screening of the largely non-protein pool of approximately 2500 or
more small molecule metabolites (<800 kDa) that constitute
the metabolome, synthesized endogenously within the cytoplasm, and often secreted from cells [15]. Metabolomics has
been used to great success in plant biology, such as for assessments of metabolism relative to environmental stress or
chemical toxicity, as recently demonstrated in an intricate
biochemical pathway analysis [21]. In relation to clinical
bioassays, metabolomics screening has proven useful diagnostically, in the search for metabolic biomarkers of tumour
phenotype [22], as well as in treatment, for assessing pharmacotherapy outcomes in vivo [23]. In combination with
metabolome and biochemical pathway databases [24, 25],
it is now possible to use metabolomics data to piece together
entire intracellular pathways, a powerful approach in any
clinical application.
Common spectroscopic techniques used in metabolomics
include gas chromatography/mass spectrometry (GCMS) and
liquid chromatography/mass spectrometry (LCMS). GCMS
and LCMS have become particularly useful for metabolite
screening due to their ability to separate low molecular
weight polar molecules and organic compounds, a criterion
that satisfies most metabolites [15]. Atomic scale techniques
such as NMR, tandem mass spectrometry (MS/MS), time-offlight mass spectrometry (TOF/MS) and other combinations
are also amenable to metabolites, based on their ability
to further discriminate organic compounds by charge versus
molecular weight.
Aside from cost and time expenditure, the main limitations on the choice of platform to use clinically include the
molecular weight range of interest, the yield of data versus
the amount or availability of sample, and the extent of sample degradation following sample processing such as solvent
and heat treatments [26]. Conversely, while combinatorial
methods like GCMS and LCMS may reduce the yield
and require more starting sample, there is an improvement in
purity.
APPLICATION OF METABOLOMICS TO ASSISTED
REPRODUCTION
In a similar manner to the systematic application of
genomics [27], transcriptomics [28] and proteomics [12]
analysis, traditional metabolomics approaches are becoming
widely used as a diagnostic tool in assisted reproduction for
two main purposes: (i) the determination of aetiological
biomarkers of infertility, and (ii) the analysis of gamete and
embryo quality, for prognostic screening to assist in the
treatment of infertility using IVF and other clinical labora-
tory interventions. In the first instance, information gained
from metabolomic analysis of sperm samples has revealed
potential links to biochemical factors associated with poor
sperm quality in infertile men, including reactive oxygen
species [17]. Similarly, oocyte specific markers may be predictive of subsequent post-fertilization survival of oocytes
based on factors found in follicular fluid [29]. In the second
instance, several techniques have been adopted for the selection of embryos based on metabolomic profiles [30] which
will be discussed further in the following section.
EMBRYO SELECTION USING METABOLOMICS
General Considerations
From a research perspective, metabolomics would seem a
logical progression in the development of viability assays
given the wealth of information in the field of preimplantation embryo metabolism. Much of this data has been obtained through the use of microfluorimetry (MF) to quantify
metabolic rates and exchange of specific metabolites from
individual embryos in vitro [31-33]. This approach has revealed key findings including a strong correlation between
glucose metabolism and morphological grade of human
blastocysts [34]. A dramatic improvement in pregnancy
outcomes following the transfer of blastocysts selected
according to their metabolic efficiency, using rodent models
has also been shown [35, 36].
Methods to screen embryo physiology and genetics are
generally classified as “invasive” or “non-invasive” [6], and
“direct” or “indirect” [37] depending on the degree of manual interference with the embryo and its culture microenvironment. Invasive assay ranges from whole or partial
embryo analysis (eg. extracts from whole embryos, biopsied
blastomeres or polar bodies). Non-invasive assay involves
sampling of spent culture medium used to incubate live embryos, and remains the most ideal approach for clinical IVF
embryo assessment. Advantages of non-invasive sampling
include its close correlation with morphological parameters
of individual embryos, its high sensitivity and the potential
for performance of multiple assays on the same embryo as it
develops in vitro, to obtain a kinetic profile of metabolism.
The main disadvantage lies in the fact that it remains an indirect measure of embryo performance in an in vitro system,
typically based on substrate consumption and metabolite
release rather than metabolic rate, and that until recently
such analyses were only feasible in a handful of laboratories
world-wide.
Similar considerations apply to metabolite screening using metabolomics. In terms of purification yield, regardless
of the degree of sample invasiveness, there are major limitations due to the amount of embryo (picogram) or medium
(microlitre) starting material, dictating that the analytical
technique be sufficiently sensitive and miniaturized, making
MS and small scale optical spectroscopies (Raman, NIR and
FTIR spectroscopy) good candidate technologies. By the
same token, purity is less of an issue with medium samples,
making them highly amenable to single-step methods, compared to combinatorial ones [5]. While indirect screening
may as yet not provide a complete diagnostic profile of
metabolism, it may provide sufficient information to make
discriminations in spectral differences that are non-specific
Thouas and Gardner
or broad. Furthermore, direct methods such as optical
biospectroscopy are particularly rapid, providing metabolomic profiles in minutes, which can later be quantified
statistically as necessary.
Beyond the technical hurdles are considerations such
as how metabolomic information integrates with data on
human embryos obtained from existing Omics platforms
which have led to the postulation of a “secretome” [12, 38]
and embryo-specific “implantome” [39], which could make
the basis of an embryo “selectome” (see Fig. (1)). How the
human embryo metabolome ultimately overlaps with these
subsets of information and forms a universal selectome is
unclear at this stage, although the schema proposed represents a simplified view of possible classifications. These
classifications are likely to become more complicated and
integrated as more data is obtained and related to pregnancy
outcomes. In relation to a selectome, the basis of choosing
a profile feature based on non-specific changes remains to
be fully justified, since metabolism itself is an associative
physiological indicator of the embryo and does not consider
maternal factors that play a role in implantation and ongoing
pregnancy. Also, during early cleavage stages, it may not be
feasible to use metabolomics data as a prognostic marker of
how a blastocyst is expected to implant [10, 13]. Therefore
the metabolomics profiles should be considered in tandem
with proven endpoints at specific stages to improve clinical
efficacy.
Specific Metabolomic Methods used in Embryo Selection
Several analytical methods have been used successfully
to obtain metabolomic profiles of preimplantation human
embryos (summarized in Table 1), [13, 17, 40]. High performance liquid chromatography (HPLC) has been used noninvasively to analyse global amino acid exchange by single
human embryos [41-43]. Of these, the study by Brison and
colleagues demonstrates that embryos with profiles showing
selective changes in three specific amino acids were the most
viable after transfer [42]. Most recently, Stokes and
colleagues show that metabolic rates extrapolated from the
HPLC amino acid profiles of single embryos were highly
predictive of embryo survival to Day 5 following Day 3
cryopreservation [43], in confirmation of the original report
by Houghton and colleagues [41]. In terms of the attainment
of embryo viability with progressive development of
embryos toward Day 5, these findings provide close correlation to the stage-specific changes in embryo metabolism
already understood from MF studies. Using LCMS, as a
combinatorial MS approach with higher resolution than
HPLC alone for amino acid analysis, it has recently been
shown that significant differences in amino acid uptake
occur between viable and non-viable human blastocysts [44],
in parallel with increased glucose uptake as measured using
MF (data not shown).
Like MS, NMR spectroscopy has become one of the
most widely used metabolic tools at the elemental and
atomic level [15], particularly for hydrogen detection, making it extremely useful for metabolite screening. Proton
NMR spectroscopy has been used to demonstrate significant
correlations between uptake of a subset of six metabolites in
single embryos with pregnancy outcomes after transfer [48],
257
Fig. (1). Diagrammatic representation of “Omics” interactions in the preimplantation embryo. The embryo protein pool (proteome)
results from the stage-specific translation of de novo or stored transcripts (transcriptome), from the genome. A functional proteomic subset
that regulates metabolism (metabolic proteome) affects the metabolite pool (the metabolome), some of which are released and form part of
the secretome. As part of the hypothetical embryo implantome/selectome, such metabolomic markers may prove useful as embryo viability
indicators for non-invasive diagnostic assay.
Table 1.
Summary of Metabolomics Methods Analysis of Viable Embryos in Clinical IVF
Method
Purification
Optical Emission
Direct or Indirect
Clinical Analysis
References1
MF
MS
HPLC
LCMS
IR
Raman
NMR
N
N
Y
Y
N
N
N
Y
N
N
N
N
Y
N
I, D
I, D
I
I
I
I
I
Yes
Yes
Yes
Yes
Yes
Yes
Yes
[7, 8]
[12]
[42, 43]
[44]
[45, 46]
[45, 47]
[48]
MF: microfluorimetry; MS: mass spectrometry; HPLC: High Performance Liquid Chromatography; LCMS: Liquid Chromatography followed by Mass Spectrometry;
IR: infra-red spectroscopy (includes near-infrared and Fourier-transformed infrared, NMR: nuclear magnetic resonance.
1
All references are cited in the text.
although further studies are required to validate these preliminary findings. Other combinatorial techniques such as
MS/MS and GCMS may become more feasible based on the
increased sensitivity of MS signatures from fractionated
samples further sorted into known metabolite subsets.
Another innovation in non-invasive metabolomics analysis has been the implementation of variants of optical spectroscopy including FTIR, Raman and Near Infrared (NIR)
methods, which deal with emission profiles beyond the visible end of the electromagnetic spectrum, as discussed earlier
[13]. These methods have previously dominated biospectroscopy, because of their efficiency, sensitivity and amenability to milli- and micro-scale volumes. Seli and colleagues
were the first to report comparisons of metabolomic embryo
profiles obtained from spent culture media using all three
techniques non-invasively, and found strong correlations
between pregnancy outcomes [45]. More recently, Vergouw
and colleagues [46] showed similar correlations between
NIR spectra and pregnancy outcomes, with a somewhat
lower accuracy than the previous study, despite having a
tenfold larger patient cohort. It remains to be determined
whether such inter-laboratories variations are clinically
significant, and whether the choice of chemometrics based
on the outcomes of one study are suitable for universal
applications.
Raman spectroscopy is slightly different from FTIR and
NIR methods, in relation to its use of visible light excitation
and scattering, and its lack of interference from water,
making it ideal for biological systems for invasive and noninvasive analysis [49]. While Scott and colleagues showed
similar results to the described infrared spectroscopy studies
in terms of significant correlation between metabolomic
profiles and preganancy outcomes, the method is somewhat
limited due to its notoriously low detection sensitivity [47].
Undoubtedly, further optimization in terms of choice of
specific metabolomic screening methods using these popular
optical methods will require validation across several laboratories, to generate a larger pool of data towards a global
selectivity index for embryos.
General Considerations for Selecting the Optimal
Embryo
In terms of the biological relationship between embryo
metabolism and implantation potential, metabolomics profiles represent a potential wealth of information that can be
drawn from, in the search for both selective and diagnostic
biomarkers of embryo health. As a selection tool, Raman and
infrared spectroscopy may provide a practical clinical solution as a non-invasive, bench-top approach. Prospective
studies will determine the accuracy and reproducibility of
such methods. Important considerations include the need
to relate this metabolomic data to implantation markers
that have arisen from other Omics platforms, such as
TOF/MS proteomics and single embryo metabolite analysis
using MF and LCMS [34, 44], as well as to global datasets
appearing in new metabolome and pathway databases [24,
25]. While selecting an appropriate tool may be as difficult
as using one to select the most viable embryo, it is heartening that such an array of innovative analytical biotechnology is currently available, and being tailored to this single
objective.
EXPERT COMMENTARY
Metabolomics is perceived as an important diagnostic
tool in clinical IVF, with the potential to calculate embryo
viability for the purposes of screening prior to transfer or
cryopreservation. Regardless of how adequately the metabolomic data is resolved with morphological and developmental scores, sufficient information can allow embryos of
like morphology to be discriminated and selected. Affordability of the new techniques is likely to improve, as they
become more readily available and tailored to clinical IVF,
and as they integrate with other cost effective and efficient
technologies such as chip-based analysis [50]. This latter
approach has the further advantage of increased analytical
sensitivity. Regardless of which technique is adopted, some
specific requirements include that:
Thouas and Gardner
• The technology is reproducible, rapid and provides
quantitative data that is complementary to other patient
specific laboratory data.
• The technology interfaces easily within the context of
working ART laboratories.
• The methods can discriminate between samples from
different patient subpopulations in the context of their
individual clinical diagnosis.
• The methods can distinguish variation within a patient’s
cohort of embryos with similar developmental rates and
morphologies, but different implantation outcomes.
FIVE-YEAR VIEW
As an embryo selection technique, metabolomics screening will form an integral part of ART laboratories. This will
improve the ability to determine the functional metabolic
phenotype of an embryo as a key indicator of viability. A 5
to 15% increase in the efficiency of clinical IVF laboratories
to generate pregnancies based on this information would not
be an unrealistic outcome.
Beyond embryo selection, there is also the potential to
analyse a range of patient specific material, including sperm
extracts and purified sperm samples, testicular biopsies,
follicle fluid, endometrial tissue and fluids, ovarian biopsies
and cumulus cell extracts. The same methods may also be
useful retrospectively in the determination of unexplained
recurrence of pregnancy loss by analysis of maternal serum
or urine. Overall, a holistic approach in correlating metabolomics profiles within and between patients will be a
continued requirement, both prospectively in the context of
laboratory indications, as well as retrospectively in relation
to clinical and pathological indications.
KEY ISSUES
• Metabolomics is one of an emerging set of technologies
that are becoming important diagnostic tools in clinical
ART, for the detection of abundance and type of metabolites in biological samples as correlates of a functional or
physiological state.
• As an “Omics” technique, metabolomics integrates with
existing bioinformatics platforms, providing global information on functional phenotypes of cells and tissues of
interest.
• Metabolomic profiling, combined with statistical data
analysis, can be performed non-invasively on spent
embryo culture medium samples, making it suitable for
single embryo diagnosis of implantation potential.
• An increase in the sensitivity of embryo viability detection can be expected, which will complement existing
morphological criteria, especially among embryos of like
morphology; this will improve the ability to choose a
single viable embryo for transfer.
• Potential improvements to patient outcomes include
incremental increases in pregnancy rate and foetal normality following single embryo transfer.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
Edwards RG, Fishel SB, Cohen J, et al. Factors influencing
the success of in vitro fertilization for alleviating human infertility.
J In Vitro Fert Embryo Transf 1984; 1: 3-23.
Cummins JM, Breen TM, Harrison KL, Shaw JM, Wilson LM,
Hennessey JF. A formula for scoring human embryo growth rates
in in vitro fertilization: its value in predicting pregnancy and in
comparison with visual estimates of embryo quality. J In Vitro Fert
Embryo Transf 1986; 3: 284-95.
Van Royen E, Mangelschots K, De Neubourg D, et al. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 1999; 14: 2345-9.
Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft
WB. Blastocyst score affects implantation and pregnancy outcome:
towards a single blastocyst transfer. Fertil Steril 2000; 73: 11558.
Sakkas D, Gardner DK. Evaluation of embryo quality: new
strategies to facilitate single embryo transfer. DK Gardner, A
Weissman, CM Howles, Z. Shoham Eds. Textbook of Assisted
Reproductive Technologies: Laboratory and clinical perspectives.
3rd ed. Informa Healthcare, London, UK. 2009; pp. 241-54.
Leese HJ. Metabolism of the preimplantation mammalian embryo.
Oxf Rev Reprod Biol 1991; 13: 35-72.
Conaghan J, Hardy K, Handyside AH, Winston RM, Leese HJ.
Selection criteria for human embryo transfer: a comparison of
pyruvate uptake and morphology. J Assist Reprod Genet 1993; 10:
21-30.
Gott AL, Hardy K, Winston RM, Leese HJ. Non-invasive measurement of pyruvate and glucose uptake and lactate production by
single human preimplantation embryos. Hum Reprod 1990; 5: 1048.
Gardner DK, Pool TB, Lane M. Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and
viability. Semin Reprod Med 2000; 18: 205-18.
Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J,
Hesla J. A prospective randomized trial of blastocyst culture
and transfer in in-vitro fertilization. Hum Reprod 1998; 13: 343440.
Gardner DK, Balaban B. Choosing between day 3 and day 5
embryo transfers. Clin Obstet Gynecol 2006; 49: 85-92.
Katz-Jaffe MG, Schoolcraft WB, Gardner DK. Analysis of protein
expression (secretome) by human and mouse preimplantation
embryos. Fertil Steril 2006; 86: 678-85.
Brison DR, Hollywood K, Arnesen R, Goodacre R. Predicting
human embryo viability: the road to non-invasive analysis of the
secretome using metabolic footprinting. Reprod Biomed Online
2007; 15: 296-302.
Kitteringham NR, Jenkins RE, Lane CS, Elliott VL, Park BK.
Multiple reaction monitoring for quantitative biomarker analysis in
proteomics and metabolomics. J Chromatogr B Analyt Technol
Biomed Life Sci 2009; 877: 1229-39.
Fiehn O. Metabolomics--the link between genotypes and phenotypes. Plant Mol Biol 2002; 48: 155-71.
Hollywood K, Brison DR, Goodacre R. Metabolomics: current
technologies and future trends. Proteomics 2006; 6:4716-23.
Deepinder F, Chowdary HT, Agarwal A. Role of metabolomic
analysis of biomarkers in the management of male infertility.
Expert Rev Mol Diagn 2007; 7: 351-58.
Kendall C, Isabelle M, Bazant-Hegemark F, et al. Vibrational
spectroscopy: a clinical tool for cancer diagnostics. Analyst 2009;
134: 1029-45.
Shai RM. Microarray tools for deciphering complex diseases. Front
Biosci 2006; 11: 1414-24.
Camp RL, Neumeister V, Rimm DL. A decade of tissue microarrays: progress in the discovery and validation of cancer biomarkers.
J Clin Oncol 2008; 26: 5630-7.
Roessner U, Patterson JH, Forbes MG, Fincher GB, Langridge
P, Bacic A. An investigation of boron toxicity in barley using
metabolomics. Plant Physiol 2006; 142: 1087-101.
Kim YS, Maruvada P, Milner JA. Metabolomics in biomarker
discovery: future uses for cancer prevention. Future Oncol 2008; 4:
93-102.
Chung YL, Griffiths JR. Using metabolomics to monitor anticancer
drugs. Ernst Schering Found Symp Proc 2007: 55-78.
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
259
Wishart DS, Knox C, Guo AC, et al. HMDB: a knowledgebase
for the human metabolome. Nucleic Acids Res 2009; 37: D60310.
Frolkis A, Knox C, Lim E, et al. SMPDB: The Small Molecule
Pathway Database. Nucleic Acids Res 2010; 38(Database
issue): D480-7.
Canelas AB, ten Pierick A, Ras C, et al. Quantitative evaluation of
intracellular metabolite extraction techniques for yeast metabolomics. Anal Chem 2009; 81: 7379-89.
Goto T, Holding C, Daniels R, Salpekar A, Monk M. Gene expression studies on human primordial germ cells and preimplantation
embryos. Ital J Anat Embryol 2001; 106: 119-27.
Zhang P, Zucchelli M, Bruce S, et al. Transcriptome profiling
of human pre-implantation development. PLoS One 2009; 4:
e7844.
Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M,
Rinaudo P. Follicular fluid content and oocyte quality: from single
biochemical markers to metabolomics. Reprod Biol Endocrinol
2009; 7: 40.
Botros L, Sakkas D, Seli E. Metabolomics and its application for
non-invasive embryo assessment in IVF. Mol Hum Reprod 2008;
14: 679-90.
Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse
ova and preimplantation embryos. J Reprod Fertil 1984; 72: 913.
Gardner DK, Leese HJ. Assessment of embryo metabolism and
viability. In: Trounson A, Gardner DK, Eds, Handbook of In Vitro
Fertilization Boca Raton: CRC Press; 1999: pp. 347-72.
Gardner DK. Noninvasive metabolic assessment of single cells.
Methods Mol Med 2007; 132: 1-9.
Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive
assessment of human embryo nutrient consumption as a measure of
developmental potential. Fertil Steril 2001; 76: 1175-80.
Gardner DK, Leese HJ. Assessment of embryo viability prior to
transfer by the noninvasive measurement of glucose uptake. J Exp
Zool 1987; 242: 103-5.
Lane M, Gardner DK. Selection of viable mouse blastocysts prior
to transfer using a metabolic criterion. Hum Reprod 1996; 11:
1975-8.
Barnett DK, Bavister BD. What is the relationship between the
metabolism of preimplantation embryos and their developmental
competence? Mol Reprod Dev 1996; 43: 105-33.
Katz-Jaffe MG, McReynolds S, Gardner DK, Schoolcraft WB. The
role of proteomics in defining the human embryonic secretome.
Mol Hum Reprod 2009; 15: 271-7.
Dominguez F, Pellicer A, Simon C. The human embryo proteome.
Reprod Sci 2009; 16: 188-90.
Singh R, Sinclair KD. Metabolomics: approaches to assessing
oocyte and embryo quality. Theriogenology 2007; 68(Suppl 1):
S56-62.
Houghton FD, Hawkhead JA, Humpherson PG, et al. J. Noninvasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002; 17: 999-1005.
Brison DR, Houghton FD, Falconer D, et al. Identification of
viable embryos in IVF by non-invasive measurement of amino acid
turnover. Hum Reprod 2004; 19: 2319-24.
Stokes PJ, Hawkhead JA, Fawthrop RK, et al. Metabolism of
human embryos following cryopreservation: implications for the
safety and selection of embryos for transfer in clinical IVF. Hum
Reprod 2007; 22: 829-35.
Gardner DK, Wale PL, Collins R, Lane M. Viable human
blastocyts exhibit a different amino acid utilization profile
than those which fail to implant. Hum Reprod 2009; 24: 146
(O-115).
Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, Burns DH.
Noninvasive metabolomic profiling of embryo culture media using
Raman and near-infrared spectroscopy correlates with reproductive
potential of embryos in women undergoing in vitro fertilization.
Fertil Steril 2007; 88: 1350-7.
Vergouw CG, Botros LL, Roos P, et al. Metabolomic profiling by
near-infrared spectroscopy as a tool to assess embryo viability: a
novel, non-invasive method for embryo selection. Hum Reprod
2008; 23: 1499-504.
Scott R, Seli E, Miller K, Sakkas D, Scott K, Burns DH. Noninvasive metabolomic profiling of human embryo culture media using
[48]
Thouas and Gardner
Raman spectroscopy predicts embryonic reproductive potential: a
prospective blinded pilot study. Fertil Steril 2008; 90: 77-83.
Seli E, Botros L, Sakkas D, Burns DH. Noninvasive metabolomic
profiling of embryo culture media using proton nuclear magnetic
resonance correlates with reproductive potential of embryos in
women undergoing in vitro fertilization. Fertil Steril 2008; 90:
2183-9.
[49]
[50]
Ellis DI, Goodacre R. Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy.
Analyst 2006; 131: 875-85.
Urbanski JP, Johnson MT, Craig DD, Potter DL, Gardner DK,
Thorsen T. Noninvasive metabolic profiling using microfluidics for
analysis of single preimplantation embryos. Anal Chem 2008; 80:
6500-7.
261
Kathryn D. Coyne, Amr Kader and Ashok Agarwal*
Center for Reproduction Ob-Gyn & Women’s Health Institute and Glickman Urological & Kidney Institute Cleveland
Clinic, Cleveland, OH 44195, USA
Abstract: Fertility preservation options for women are currently only routinely offered to patients who face iatrogenic
fertility loss, and most options are considered experimental. The most common modalities for female fertility preservation
are embryo cryopreservation, oocyte cryopreservation, and ovarian tissue cryopreservation, the later two of which remain
in the experimental arena. Natural fertility loss is affecting women as significantly as premature fertility loss. Increasing
cancer survival and the modern reproductive trend of delaying childbearing are indications for the need and demand for
fertility preservation. Advances in the field are necessary to respond to this demand and include superior cryopreservation
techniques and fertility preservation technologies, customized guidelines, comprehensive care plans, and availability of
more cost-efficient procedures. The obstacles to creating a standard of care for fertility preservation are as broad as the
field itself. Lack of patient awareness, limited physician experience and knowledge, inadequate counseling, costs, and
ethical issues are some examples of the many challenges to establishing a standard of care. With continued research and
multidisciplinary collaboration, a higher quality of care may be provided to a larger patient population who wishes to
maximize their fertility potential in the future.
Keywords: Fertility preservation, standard of care, treatment options, challenges, assisted reproductive technology (ART),
patient expectations.
CURRENT STATUS OF FERTILITY PRESERVATION
The technologies of fertility preservation include all
methods and efforts to maintain the ability to reproduce even
after natural or iatrogenic loss of fertility. The many methods
to preserve fertility for women include gonadal protective
measures during potentially harmful therapeutic treatments
and cryopreservation of the ovary, oocyte, or embryo. Of the
cryopreservation options for females, embryo cryopreservation is the only form currently accepted as an established
practice [1]. Oocyte and ovarian tissue cryopreservation are
still considered experimental forms of preservation due to
the less than satisfactory outcomes of cryopreserved oocytes
in assisted reproductive techniques (ARTs) and the short
lifespan of ovarian tissue transplantation grafts [2]. Additionally, this option is offered mainly for patients who face
iatrogenic loss from treatments related to cancer and other
diseases. Fertility preservation is less available for patients
who wish to delay their childbearing years at the risk of becoming sterile.
Currently there is an increased demand for a broader
range of options and availability of treatment for both cancer
survivors and healthy individuals [3, 4]. Translational research and improvements in methods and technologies in the
last decade have provided a diversity of potential fertility
preservation options; however, at present these options remain limited and suboptimal [5]. Continued research and
success present the need to establish standards of care for the
many available fertility preservation techniques.
*Address correspondence to this author at the Center for Reproduction ObGyn &Women’s Health Institute and Glickman Urological & Kidney Institute 9500 Euclid Avenue, Desk A19.1Cleveland, OH 44195 USA;
Tel: 216-444-4402; Fax: 216-445-6049; E-mail: [email protected]
1573-4048/10 $55.00+.00
NATURAL AND PREMATURE FERTILITY LOSS
Natural fertility loss occurs for all women with advancing age. For most women, menopause occurs during their
mid-50s; however, fecundity occurs some 10 years before
the onset of menopause [6]. A natural atresia of oocytes over
the years accounts for the decrease in fertility potential. As
the quantity and quality of oocytes declines as age advances,
the possibility of conceiving a healthy child decreases.
Premature fertility loss occurs before the naturally occurring time and may be induced by gonadotoxic treatments
necessary for cancer and other diseases, from genetic causes
such as Turner’s syndrome [7], or from environmental
hazards. With premature fertility loss women not only lose
their reproductive potential, but, in addition, the cessation of
ovarian function induces menopause symptoms, including
osteoporosis, which may have greater consequences when
endured at a younger and more active age [6].
The most common forms of cancer that a female may
suffer during her reproductive years that require gonadotoxic
treatment are breast and cervical cancer [3, 8]. Most diagnoses of these cancers occur before the age of 35, and many
others occur during the prepubertal and adolescent years,
including hematologic malignant conditions, sarcomas,
central nervous system lesions, renal cancer, and bone cancer
[4]. Autoimmune diseases that require similar chemotherapeutic treatments include rheumatoid arthritis, systemic
lupus erythematosus, steroid-resistant glomerulonephritis,
inflammatory bowel disease, and pemphigus vulgaris [3, 8].
Treatment for these diseases may wreak havoc on the
female reproductive system. Chemotherapy, radiation therapy
of the pelvic region, gonadotoxic medication, and surgical
incisions of or near the gonads or reproductive system
produce a high risk for induced fertility loss. Among women
who undergo combination chemotherapies, 20-50% under
the age of 20 and 80-90% over the age of 25 will present
with amenorrhea after treatment [9]. Radiation may cause
apoptosis to occur, decreasing the amount of primordial
follicles available to mature and resulting in premature
ovarian failure (POF) [3, 10-12]. Gonadotoxic drugs such
as alkylating agents arrest cell proliferation and may also
induce POF [3, 9].
INDICATIONS FOR DEMAND FOR FERTILITY
PRESERVATION
In the last decade cancer treatment and ART have made
significant advances and achieved higher success rates than
ever [13]. The goal of cancer treatment is first and foremost
survival, yet the inevitable gonadotoxity of the treatment
harms the potential for survivors to genetically parent their
own children. Until an effective treatment for cancer can be
developed to target tumor cells specifically, sterility will
remain a possible consequence of successful cancer treatment [14]. Many patients undergoing treatment are nulliparous but desire to have children in the future.
With the increase in cancer survival more attention is
being given to the patient’s quality of life post-treatment
[15]. Current survival rates for patients with Hodgkin’s disease, lymphoma, and leukemia are as high as 90% [5], and of
10,700 children diagnosed with cancer in 2008, approximately 80% of them were expected to survive [4]. Additionally, the incidence of cancer in the adolescent and young
adult population has increased steadily over the last 25 years
[13]. Thus, as more patients are undergoing treatment and
more of those patients are living longer, fertility preservation
must become an integral part of holistic cancer treatment.
Coyne et al.
bryos [19]. The Practice Committee is prepared to reconsider
its position on oocyte cryopreservation with continued supportive evidence.
NECESSARY ADVANCES IN THE FIELD
Fertility preservation strategies emerged from techniques
used for infertility treatment, yet the development of specific
protocols and technologies is necessary for this advancing
and distinct field of medicine [20]. The complexity and
broadness of the field of fertility preservation requires that
its technology and research be particular and organized.
Novel techniques have been developed that are promising,
yet challenges remain to applying these techniques routinely
and successfully [21]. Research findings must be applied in
protocol to improve the efficacy of the current strategies, as
well as to develop new methods and tools [15].
For women undergoing cancer treatment, bridging the
treatment gap between the oncologists and the fertility specialist is imperative [13]. By establishing a multidisciplinary
team that includes oncologists, fertility and oncology nurses,
social workers, reproductive endrocrinology and infertility
specialists, embryologists, and researchers, a specific treatment plan may be established that provides the highest
possibility for success [13]. The members of the team that
provide fertility preservation care for the female cancer
patient must work together and keep the patient and the
center of their focus; refer to Fig. (1). The goal is to provide
a multidisciplinary treatment plan for the female cancer
patient that maintains the greatest survival potential,
preserves fertility, and offers the best quality of life with the
least long-term suffering post-treatment [4, 22].
Concurrently with increased cancer survival, the modern
reproductive trend of delaying childbearing necessitates a
greater need for fertility preservation. Women all over the
world, especially in developed countries, are choosing to
defer pregnancy in pursuit of academic or occupational
achievements or for lack of a suitable partner [7, 9, 16, 17].
With advancing reproductive age the ability to conceive
greatly decreases due to the naturally accelerating atresia of
primordial follicles that occurs in the late 30s and early 40s
of a woman’s lifetime [9, 12]. During this time the possibility of spontaneous abortion and aneuploidy increases [9, 12].
Cryopreservation of oocytes at a maternal age of 35 years or
younger for fertility preservation may provide the potential
for women to reproduce even after the biological clock has
run down [17].
The positive risk-benefit ratio for the cryopreservation of
oocytes to be electively self-donated to healthy individuals at
a later date calls for an increased availability of these services for all women who desire its potential benefits [18]. As
oocyte cryopreservation has been considered experimental,
the Practice Committee of the American Society for Reproductive Medicine did not recommend it to postpone reproductive aging and asserts that it should be offered only in an
experimental setting and with the approval of an institutional
review board [2]. The Practice Committee recently purported
that oocyte cryopreservation appears promising and may
assist in reducing the rising number of cryopreserved em-
Fig. (1). The members of the multidisciplinary team that provide
fertility preservation care for the female cancer patient must work
together and keep the patient and the center of their focus.
With a cohesive and knowledgeable team, an individualized care plan based on case-specific motives, situations,
age, disease, and other necessary medical treatments can be
established for each patient [11, 23]. Counseling is a fundamental aspect of a standard of care because it ensures that the
woman may make the most informed decisions based on an
understanding of realistic expectations and after weighing
the risk-benefit ratio [2, 9, 10, 24].
An established standard of care may allow more women
who might desire the benefits of fertility preservation to utilize the services [5]. The standard must also be flexible to
adjust to new technologies and discoveries, as well as to
adapt to each patient in her specific socio-economic case.
With increased awareness, success, and use of fertility preservation with a standard of care, the different options may
become more clinically available and affordable for those
who desire to maximize their fertility potential later in life.
MOST COMMON MODALITIES FOR FERTILITY
PRESERVATION
1. Embryo Cryopreservation
As the only established form of cryopreservation, embryo
cryopreservation is the most commonly utilized option based
on its predictable and reproducible success [5, 9, 25, 26].
The human embryo is highly resistant to cryoinjury and thus
has a high cryosurvival rate [12]. Cryopreserved embryos
produce pregnancy rates similar to those of fresh embryos
[3]. With embryo cryopreservation, stimulation is required to
retrieve mature oocytes from the woman to be fertilized and
then frozen until the embryo is ready to be transferred to the
uterus in attempt for pregnancy. The stimulation cycle requires time that may delay cancer treatment and also may
induce ovarian hyperstimulation syndrome (OHSS).
Depending on the time allowed for stimulation, multiple
cycles may be utilized to preserve many embryos for multiple attempts. The number of oocytes necessary can be estimated based on fertilization, cryosurvival, and implantation
rates for the woman’s age, along with consideration of the
woman’s desired family size.
From an economic perspective, the most expensive aspect of treatment is the stimulation protocol for retrieval of
mature oocytes. The cost of medication may constitute half
the total cost and is dependent on age as higher doses of
stimulation are required and more attempts are necessary
with increasing age [27]. If multiple stimulation cycles are
desired to maximize the potential for achieving multiple
pregnancies, the costs increase exponentially.
In long-term studies of children born from cryopreserved
embryos, no negative effects have been reported on cognitive, psychomotor, or neurodevelopmental outcomes [22].
2. Oocyte Cryopreservation
Oocyte cryopreservation remains in the experimental
arena; however, recent promising and repeated successes
have moved this method closer to becoming established [17].
Oocytes may be retrieved in the mature or immature stage of
development. Hormonal stimulation is required to retrieve
mature oocytes. This is the traditional method; however, as
one of the largest and most complex human cells, the mature
oocyte is particularly susceptible to cryoinjury [7]. Cryopreservation techniques cause a dissemblance of the meiotic
spindle, depolymerization of chromosomes, and a hardening
of the zona pellucida [12, 25, 26]. With novel techniques
such as vitrification to evade ice crystallization and the use
263
of intracytoplasmic sperm injection (ICSI) to bypass a hardened zona pellucida, these complications may be resolved
[26].
Immature oocyte retrieval requires little or no stimulation, and the smaller, less developed oocyte is less susceptible to cryoinjury [7, 28]. For fertilization to occur, the immature oocyte must be matured through in vitro maturation
(IVM), which unfortunately has low success rates [7, 11,
29]. If optimized, this option would allow immediate
retrieval with no delay in treatment and elimination of the
risk of OHSS that could be induced through stimulation of
mature oocytes [3].
Vitrification of oocytes has been proven to increase success in oocyte cryopreservation, producing reliable results
and excellent obstetric and perinatal outcomes over the last
decade [30]. Additionally, recent data suggests the repolymerization of the meiotic spindle upon thawing of the mature
oocyte [7]. No known congenital or developmental deficits
were reported from any child born from fertilized cryopreserved oocytes [28].
Utilizing immature oocytes decreases costs by eliminating the need for stimulation [17]. Cryopreservation of
oocytes also reduces ethical dilemmas with children and
women with no partner at the time of preservation. With
improved techniques, this option is likely to become the
most routine and reliable fertility preservation method within
the next three to five years [25], and a universal vitrification
protocol is expected to be established [7].
3. Ovarian Tissue Cryopreservation
Ovarian tissue is the definitive source of female fertility,
and the ability to preserve and restore the endocrine and reproductive function of the ovary after removal and cryopreservation would be an ideal option if optimized [3, 31].
Ovarian tissue may be harvested immediately, as no stimulation is required, so cancer treatment would not be delayed
[3]. Gonadal tissue may be retrieved and cryopreserved as a
whole ovary with its vascular pedicle, as cortical strips,
fragments, or as isolated primordial follicles [11, 23].
Primordial follicles in the ovarian cortex are more resistant
to cryoinjury than the mature oocyte [12]. The tissue may be
autotransplanted to the woman later in either an orthotopic or
heterotopic location [1, 9]. Autotransplantation of the whole
ovary orthotopically has the advantage of immediately resuming blood flow, thus avoiding ischemic damage; pregnancy also may occur spontaneously with this method [1, 9].
Transplantation to a heterotopic location presents a higher
risk of ischemic damage, and this method relies completely
on ART to achieve pregnancy [1, 31].
The risks involved with ovarian tissue cryopreservation
include the risks of a surgical procedure, as well as the possibility of reintroducing malignant cells upon autotransplantation [11, 25]. This option should not be offered to patients
with ovarian cancer [3], and it should be utilized with caution for other cancer patients, especially if the cancer has
metastasized [4]. Cancer recurrence as a result of ovarian
tissue grafts after autotransplantation has not been reported
since the technique’s introduction [32].
Despite current reports of success, evidence of reproducible results is still limited, and a standard protocol has yet
to be established [12, 23]. Most cases evidence only a short
longevity of the ovarian grafts and produce low success rates
[1], and a definitively superior location for autotransplantation has not been decided [33]. Only seven live births have
been reported to date [34], yet this still may provide some
hope for young girls as this is the only option suitable for
pre-pubertal girls undergoing treatment [11, 12, 25].
The most significant advances in cryopreservation
over the next several years most likely will occur in ovarian
tissue cryopreservation [25]. Advances may result from
innovations in surgical technologies and research in tissue
engineering to promote better uptake of ovarian grafts and
reduced loss of primordial follicles from ischemic damage
[11, 23].
OBSTACLES TO FERTILITY PRESERVATION
Obstacles to fertility preservation include, but are not
limited to, lack of patient awareness, limited physician experience and knowledge, inadequate counseling about fertility
preservation options, the costs of the procedures involved,
and ethical dilemmas.
The patient’s own lack of awareness of the option for
fertility preservation is the first challenge for ensuring fertility preservation for all those who may desire it. For patients
who have been told about fertility preservation at the time of
diagnosis, looking beyond their own survival can be difficult. These patients may not adequately assess the importance of fertility preservation before treatment, especially if
it causes a delay in treatment.
Secondly, the physician’s own lack of knowledge relating to fertility preservation options constitutes another hurdle
in the effort to avoid sterility. An oncologist may not refer a
patient to a fertility specialist because of the risk of delaying
treatment, the patient’s limited resources, or feeling that a
discussion of fertility preservation is inappropriate in the
midst of fighting a disease [35]. The vast majority of studies
examining the discussion and referral of fertility preservation
among oncologists and patients indicate that less than half of
eligible patients are presented with this information [36].
Discussion is more common than an actual referral to a
reproductive endocrinologist, and, according to a survey of
oncologists, patients may be willing to sacrifice more in survival than would the physician [37]. A recent study determined that with supportive resources available, the time required for stimulation of mature oocytes for cryopreservation
does not significantly extend the period of time between diagnosis and the start of chemotherapy [38].
It is the physician’s role to be knowledgeable about the
options and adequately present fair information to the patient
or couple, or at least to refer the patient to a more expert specialist [39]. This is often the best solution as many techniques involved in fertility preservation are only performed
at specialized centers and the oncologist may have limited
access to the most current developments in the field.
Discussion and management of preserving reproductive
potential are challenging aspects of care, and inadequate
Coyne et al.
counseling results from the lack of a multidisciplinary approach to providing fertility preservation [40]. Information
about actual clinical success rates, outcomes, and risks must
be imparted to the patient so that she may make an informed
decision founded in practical expectations and an understanding of the risks and benefits [41]. In a study on the experience of receiving fertility preservation options around
the time of diagnosis, women were likely to report low levels
of comprehension regarding the physiological impact of
treatment as well as express distress from lack of services
available [40]. The findings of this study suggest that young
women may cope with information about fertility preservation alongside cancer diagnosis if there is sufficient professional and familial support [40].
Professional counseling is vital as much of the information that patients may find in the media may be biased or
scientifically unfounded [41]. Specifically with healthy
women who elect to cryopreserve oocytes, misunderstanding
the actual potential can cause emotional devastation if they
are unable to conceive later in life [41].
Another significant obstacle to fertility preservation is the
high cost of services and the lack of insurance coverage. In
the United States, there are currently no states that offer insurance for fertility preservation for cancer patients, although
15 states have laws for insurance coverage of IVF and/or
other ART services [35]. Loss of fertility is an inevitable
sequela of cancer treatment and as such may justify the coverage of fertility preservation by insurance. Especially with
cancer treatment versus normal infertility services, the patient may not have the time to attain the necessary monetary
resources required to undergo fertility preservation. The
costs may deter both cancer patients and healthy patients
alike until these services can be made more affordable.
Many ethical dilemmas currently present obstacles to
achieving fertility preservation. For young girls and females
without a partner at the time of preservation, embryo cryopreservation may not be an acceptable modality if the female
is unwilling to use a donor sperm. In the case of children, it
may be difficult to accurately gain informed consent or even
for a parent to make an informed decision [22]. The use of
oocytes for cryopreservation avoids this ethical issue, yet
oocyte cryopreservation remains experimental and is not
offered routinely [10]. Ovarian tissue cryopreservation also
avoids the dilemma concerning donor sperm, yet it is also
experimental, and the faint risk of reintroducing malignant
cells should be considered.
In the case of cryopreserved embryos, the future fate of
unused embryos presents a dilemma. The options of disposition of the embryo include using all the embryos in an attempt to conceive, donating embryos to another couple or to
research, discarding the embryos, and continuing cryopreserved storage indefinitely [26]. Couples often have difficulty resolving this issue before undergoing IVF [42] as the
outcome of the treatment is indeterminate.
With optimized outcomes and safe procedures involved
with oocyte preservation, arguments against elective selfdonation of oocytes are unjustified on clinical or ethical
grounds [18, 29]. A person is free to choose when and
whether to reproduce if medical and technological means
exist to execute that choice. With this principal of reproductive autonomy, oocyte cryopreservation for the healthy individual represents a legitimate use of physician and medical
resources [41]. Recognizing the medical insufficiencies and
the limitations of current fertility preservation technologies,
balancing realistic expectations with the desire to conceive
and the right to maximize the potential to do so is a wise
approach.
EXPERT COMMENTARY
The aim of this article is to elucidate the necessity for and
the challenges to establishing a standard of care for fertility
preservation. Currently embryo cryopreservation is the only
routinely performed procedure for fertility preservation, yet
oocyte and ovarian tissue cryopreservation are promising
modalities that would increase the ease and availability of
fertility preservation for a more diverse patient population.
These experimental options have the potential to avoid the
risks associated with hormonal stimulation and/or a delay in
cancer treatment and to eliminate the ethical dilemmas of
embryo cryopreservation, including embryo disposition and
use of donor sperm in the case of children or single women.
These options also allow healthy patients to maximize their
potential to achieve pregnancy later in life. The techniques
and methods of fertility preservation evolved from the use of
ART for infertile patients, yet the circumstances involved
with preservation solicit the need for specific standards, research, and multidisciplinary care. Fertility preservation is
emerging as a distinct and specialized field of reproductive
medicine, necessitating further research and standards of
practice that may lead to better patient care.
• Increased cancer survival and cancer diagnoses in the
adolescent and young adult population, as well as the
modern reproductive trend of delaying childbearing,
indicate a growing need for fertility preservation.
• For women, embryo cryopreservation is the only
established technique, and options for oocyte and ovarian
tissue cryopreservation are still considered experimental.
• The standardization of oocyte and ovarian tissue modalities of cryopreservation would increase the availability of
fertility preservation for a greater patient population
while reducing risks and eliminating ethical issues.
• Necessary advances in the field include improved
cryopreservation techniques and fertility preservation
technologies, comprehensive care plans and customized
guidelines, and the availability of more cost-efficient
procedures.
• Obstacles to creating a standard of care consist of lack of
patient awareness, limited physician experience with new
modalities, inadequate counseling, the costs of procedures involved, and ethical dilemmas.
• Continued collaborative efforts among disciplines will
assist in greater utilization of research discoveries
in clinical applications and support multidisciplinary
standards of care for fertility preservation patients.
REFERENCES
[1]
[2]
FIVE-YEAR REVIEW
Attention to and demand for fertility preservation have
increased significantly in the past few years. This was
compelled by the increase in cancer survival and the modern
reproductive trend of delaying childbearing, and indicated by
the boom in research and collaborative efforts. Of specific
note, the founding of the International Fertility Preservation
Society points to the worldwide importance of preserving
the potential to genetically parent one’s own child [20].
This collaborative effort allows clinical and research experts
to work together to develop standards of care that provide
minimal risk and greater efficiency. In addition, public
education should be improved to confer objective and
analytical information about current advances in technologies, the lack of evidence of long-term outcomes, and the
ethical dilemmas a patient faces [5]. With the advancement
of technology and establishment of a standard of care, the
cost of procedures and treatment may fall, allowing more
patients to take advantage of these benefits with greater ease
and accessibility.
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
KEY POINTS
• Fertility preservation is the preservation of the potential
to genetically parent a child beyond natural fertility and
despite iatrogenic loss through the means of ART.
• Fertility loss occurs naturally with age or prematurely
from gonadotoxic treatment, genetic disorders, or
environmental hazards.
265
[12]
[13]
Bedaiwy MA, Shahin AY, Falcone T. Reproductive organ transplantation: advances and controversies. Fertil Steril 2008; 90(6):
2031-55.
Practice Committee of Society for Assisted Reproductive,
Technology and Medicine. Practice Committee of American
Society for Reproductive, Essential elements of informed consent
for elective oocyte cryopreservation: a Practice Committee opinion.
Fertil Steril 2008; 90(5 Suppl ): S134-5.
Georgescu, Elena S, Goldberg, et al. Present and future fertility
preservation strategies for female cancer patients. Obstet Gynecol
Surv 2008; 63(11): 725-32.
Jeruss JS, Woodruff TK. Preservation of fertility in patients with
cancer. N Engl J Med 2009; 360(9): 902-11.
Lamar CA, DeCherney AH. Fertility preservation: state of the
science and future research directions. Fertil Steril 2009; 91(2):
316-9.
Frank O, Bianchi PG, Campana A. The end of fertility: age,
fecundity and fecundability in women. J Biosoc Sci 1994; 26: 34968.
AbdelHafez F, Desai N, Ali MY, et al. Oocyte cryopreservation:
aÂ technical and clinical update. Expert Rev Obstet Gynecol 2009;
4(4): 443-54.
Falcone T, Bedaiwy MA. Fertility preservation and pregnancy
outcome after malignancy. Curr Opin Obstet Gynecol 2005; 17(1):
21-6.
Lobo RA. Potential options for preservation of fertility in women.
N Engl J Med 2005; 353(1): 64-73.
Bedaiwy MA, Mohamed A, Falcone T. Fertility preservation in
cancer patients. Women's Health 2006; 2(3): 479-89.
Donnez J, Martinez-Madrid B, Jadoul P, Langendonckt AV,
Demylle D, Dolmans M. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006; 12(5): 519-35.
Maltaris T, Beckmann MW, Dittrich R. Review. Fertility
preservation for young female cancer patients. In vivo (Athens,
Greece) 2009; 23(1): 123-30.
Nagel K, Cassano J, Wizowski L, Neal MS. Collaborative multidisciplinary team approach to fertility issues among adolescent
and young adult cancer patients. Int J Nurs Pract 2009; 15(4): 3117.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
Coyne et al.
Revel A, Revel-Vilk S. Pediatric fertility preservation: is it time to
offer testicular tissue cryopreservation? Mol Cell Endocrinol 2008;
282(1-2): 143-9.
Chevalier N, Dewailly D, Fenichel P. Oncofertility: a new focus in
women health-care. Ann Endocrinol (Paris) 2009; 70(Suppl 1):
S33-41.
Bredkjaer, H.E. and J.G. Grudzinskas, Cryobiology in human
assisted reproductive technology. Would Hippocrates approve?
Early Pregnancy (Online) 2001; 5(3): 211-3.
Molloy D, Hall BA, Illbery M, Irving J, Harrison KL. Oocyte
freezing: timely reproductive insurance? Med J Aust 2009; 190(5):
247-9.
Rybak EA, Lieman HJ. Egg freezing, procreative liberty, and ICSI:
the double standards confronting elective self-donation of oocytes.
Fertil Steril 2009; 92(5): 1509-12.
ASRM Practice Committee response to Rybak and Lieman:
elective self-donation of oocytes. Fertil Steril 2009; 92(5): 15134.
Gosden RG. Fertility preservation: definition, history, and prospect.
Semin Reprod Med 2009; 27(6): 433-7.
Moffat R, De Geyter C, von Wolff M. New strategies for fertility
preservation in young women with cancer. Ther Umsch 2009;
66(12): 831-8.
Pauli SA, Berga SL, Shang W, Session DR, et al. Current status of
the approach to assisted reproduction. Pediatr Clin North Am 2009;
56(3): 467-88 Table of Contents.
Chang HJ, Suh CS. Fertility preservation for women with
malignancies: current developments of cryopreservation. J Gynecol
Oncol 2008; 19(2): 99-107.
Goodwin PJ, Options for preservation of fertility in women. N Engl
J Med 2005; 353(13): 1418-20; author reply 1418-20.
Bagchi A, Woods EJ, Critser JK. Cryopreservation and
vitrification: recent advances in fertility preservation technologies.
Expert Rev Med Devices 2008; 5(3): 359-70.
Bankowski BJ, Lyerly AD, Faden RR, Wallach EE. The social
implications of embryo cryopreservation. Fertil Steril 2005; 84(4):
823-32.
Bouwmans CA, Lintsen BM, Eijkemans MJ, Habbema JD, Braat
DD, Hakkaart L. A detailed cost analysis of in vitro fertilization
and intracytoplasmic sperm injection treatment. Fertil Steril 2008;
89(2): 331-41.
Varghese AC, Nagy ZP, Agarwal A. Current trends, biological
foundations and future prospects of oocyte and embryo
cryopreservation. Reprod Biomed Online 2009; 19(1): 126-40.
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
Dondorp WJ, De Wert GM. Fertility preservation for healthy
women: ethical aspects. Hum Reprod (Oxford, England) 2009;
24(8): 1779-85.
Schoolcraft WB, Keller JL, Schlenker T. Excellent embryo quality
obtained from vitrified oocytes. Reprod Biomed Online 2009; 9(6):
820-3.
Varghese AC, du Plessis SS, Falcone T, Agarwal A. Cryopreservation/
transplantaion of ovarian tissue and in vitro maturation of follicles
and oocytes: Challenges for fertility preservation. Reprod Biol
Endocrinol 2008; 6:47.
Oktay K, Rodriguez-Wallberg K, Schover L. Preservation of
fertility in patients with cancer. N Engl J Med 2009; 360(25): 2681
author reply 2682-3.
Oktay KH. Options for preservation of fertility in women. N Engl J
Med 2005; 353(13): 1418-20; author reply 1418-20.
Donnez J, Dolmans MM. Cryopreservation of ovarian tissue: an
overview. Minerva Med 2009; 100(5): 401-13.
Quinn GP, Vadaparampil ST, Bell-Ellison BA, Gqede CK,
Albrecht TL. Patient–physician communication barriers regarding
fertility preservation among newly diagnosed cancer patients. Soc
Sci Med 2008; 66(3): 784-9.
Quinn GP, Vadaparampil ST, Lee J, et al. Physician referral for
fertility preservation in oncology patients: a national study of
practice behaviors. J Clin Oncol 2009; 27(35): 5952-7.
Forman EJ, Anders CK, Behera MA. A nationwide survey of
oncologists regarding treatment-related infertility and fertility
preservation in female cancer patients. Fertil Steril 2009; 90: S2678
Baynosa J. Timing of breast cancer treatments with oocyte retrieval
and embryo cryopreservation. J Am Coll Surg 2009; 209(5): 6037.
Leblanc E, Narducci F, Ferron G, Querleu D. Indications and
teaching of fertility preservation in the surgical management of
gynecologic malignancies: European perspective. Gynecol Oncol
2009; 114(2 Suppl): S32-6.
Crawshaw MA, Sloper P. Male and female experiences of having
fertility matters raised alongside a cancer diagnosis during the
teenage and young adult years. Eur J Cancer Care (Engl), 2009;
18(4): 381-90.
Harwood, K., Egg freezing: a breakthrough for reproductive
autonomy? Bioethics 2009; 23(1): p. 39-46.
Nachtigall RD, Becker G, Friese C, Butler A, Mac Dougall K.
Parents' conceptualization of their frozen embryos complicates the
disposition decision. Fertil Steril 2005; 84(2): 431-4.
267
Jashoman Banerjee1,*, Mona H. Mallikarjunaiah1 and John M. Murphy2
1
University of Toledo Medical Center, Toledo, OH 43614, USA; 2University of Toledo Medical Center, Fertility Center,
Toledo Hospital, Toledo, OH 43614, USA
Abstract: The existing quantity of follicles and their response to stimulation at a particular age predicts the ovarian
reserve. In vitro fertilization is emerging as a common method of treatment of infertility. This technique is not 100%
accurate and puts the patient and the couple in both physical and financial burden. It is important to predict the outcome of
a cycle of IVF to counsel a patient prior to proceeding. The ovarian reserve is the main functional component which might
guide the outcome. There may be many factors affecting ovarian reserve but the age of the patient is the most important
one. Various markers have been utilized to determine ovarian reserve in women which are endocrine, radiological or
dynamic tests. No perfect marker has been identified yet but some have proven to be better than the other. This review
will discuss the different methods of testing ovarian reserve and the current research that might help the clinician to
predict outcome prior to initiating IVF.
Keywords: Ovarian reserve, anti mullerian hormone, antral follicle count, basal follicle stimulating hormone, ovarian
volume.
INTRODUCTION
The ovarian reserve describes the functional potential of
the ovary at a particular age reflecting the quantity and quality of existing oocytes. The human ovary undergoes a steady
decline of follicles with advancing age. This decline is
marked after the age of 30. Women are currently deferring
their reproductive functions for pursuing career goals. More
women are adopting in vitro fertilization (IVF) as an option
to conceive at an older age. Extensive utilization of IVF has
also encouraged women with cancer at younger ages to preserve their ovaries for future reproductive functions. Hence
existing ovarian reserve during diagnosis may help provide
information regarding outcomes. In case of oocyte donors
ovarian reserve testing may help individualize laboratory
protocols and help improve cycle cancellation rates. Since a
considerable percentage of women are adopting IVF, which
is an expensive modality of treatment with lower outcomes
at ages above 40, it is also expected from physicians to counsel patients regarding outcomes and success rates. Ovarian
reserve testing may serve as a useful guide for physicians to
give their patients an estimate of outcome and expectation.
Various biochemical and radiological parameters have
been used to measure ovarian reserve. But the most important factor is the patient’s age at initiation of treatment.
Commonly used serum markers are basal follicle stimulating
hormone (FSH) on day 3 of the cycle, inhibin –B, and serum
anti-mullerian hormone (AMH). Radiological parameters
include estimating the ovarian volume and antral follicle
count (AFC). Some dynamic tests have been used like the
clomiphen citrate challenge test (CCCT), GnRH agonist
stimulation test (GAST) and exogenous FSH ovarian reserve
*Address correspondence to this author at the University of Toledo Medical
Center, Department of Obstetrics and Gynecology, 3000, Arlington Avenue,
Toledo, OH-43614, USA; Tel: (419)383-4590; Fax: (419)383-3090;
E-mails: [email protected], [email protected]
1573-4048/10 $55.00+.00
test (EFORT), but these have fallen out of favor. This article
will focus on current methods of ovarian reserve testing,
their application as a guide in fertility preservation. We will
discuss the major parameters namely age, day 3 FSH, inhibin-B, AMH, AFC, ovarian volume and dynamic testing and
their advantages and disadvantages in predicting poor
outcomes after in vitro fertilization.
Age
An increasing incidence of sub fertility in the past 3 - 4
decades has been due to the delay in child-bearing as result
of changing societal shift. This postponing has been attributed to the changing career priorities, work force equality
and widely available birth control. Many studies conducted
on populations in whom birth control measures have not
been used, show that optimal fertility occurs from 18 to 30
years, followed by declining fertility after 31 years and sterility at a mean age of 41 years [1, 2].
The normal reserve of ovarian follicles in an embryo at
the age of 8 weeks and 5 months has been reported to be
600,000 and 7 106 oocytes respectively [3, 4]. At birth, this
reduces to 1 106 and at the beginning of menarche are
around 300,000 follicles [5]. Although, one egg is released in
a month by the process of ovulation, a thousand additional
eggs are lost during each cycle, due to follicular atresia. During midlife, there is an acceleration of follicle degeneration,
when the numbers fall less than 25,000. The onset of menopause is heralded when the follicles reach approximately
1000 in number [6].
It is known that some women will not be able to conceive
in their thirties, while others conceive in their forties. This
may be partly explained by the above mathematical model,
which gives more importance to the critical number of follicles, rather than the age, in determining the onset of menopause. However, others consider that ovarian reserve is not
merely a function of follicular quantity but also the quality of
the oocytes, and is dependent upon a complex, dynamic
process [7, 8].
Epidemiological studies have reported on the relationship
between increased maternal age and increased incidence of
chromosomal abnormalities [9, 10]. Several mechanisms
have been proposed to explain the increased risk of these
abnormalities with increased maternal age: impaired perifollicular microcirculation, abnormalities in follicular development resulting from the aging of the somatic cells surrounding the oocytes and hormonal imbalances [11]. Although the
major contributing factor to the age related decline in female
fecundity is the gradual but constant degradation of cohesins
and other factors holding the four chromatids together during
the metaphase, recently it has been proposed that shortening
of telomeres may have important role [12].
Bukman et al. have used prognostic models in patients
with subfertility for predicting response to the chances of
pregnancy [13]. To determine the prognostic values of different tests, they have described a ratio, termed, Likelihood
ratio (LR). This ratio, for a positive test, describes the
chances of having a positive test while having the disease,
compared to the chances of having a positive test and being
healthy. A test with high LR could be considered appropriate
for screening for patients with diminished ovarian reserve.
In a subgroup of population receiving ART, age was
found to be poor predictor for pregnancy than basal serum
FSH levels in some studies, although LR could not be calculated [14, 15]. In others, though calculated LR was low, age
was considered to be a predictor for pregnancy rate [16-18].
Age was considered to be an important prognostic factor for
pregnancy rate, among the general infertile population, for
women with normal ovarian reserve. However, in the same
group, women with poor ovarian reserve, were considered to
have a poor prognosis, independent of age [19].
In summary, age is held to be a better predictor of ART
outcome than basal FSH levels in younger women, and
seems to affect egg quality more than quality [20]. Women
younger than 35 years can still have a favorable IVF outcome, even with elevated basal FSH levels [21].
Day 3 FSH
This is the most commonly used test to assess ovarian
reserve. It is an indirect estimate of the ovarian reserve.
Normally, in women with reproductive potential, FSH values
on the 3rd day of cycle are expected to be below 10 mIU/ml.
If however, there is a reduction in the oocytes pool, due to
the reduced feedback from estrogens and inhibins, there is
corresponding increase in FSH [22, 23]. Thus, it is important, before analyzing FSH levels, that, corresponding estradiol levels (normally, low on day 3) are also assessed. In a
patient with infrequent menstruation, an FSH level and estrogen level can be measured at random and is valid if the
estrogens levels are low.
A majority of the data in the literature with regards to day
3 serum FSH concentration with pregnancy rates relates to
subgroup of population on ART cycles [15, 17, 20, 24, 25].
No similar studies have been performed in women of the
general infertile population. There was a better response to
Banerjee et al.
ovulation induction, demonstrated by the number of mature
oocytes, in patients with low basal FSH concentrations. Hall
et al. reported age as a better predictor for IVF outcome than
basal FSH levels while others [26] reported basal FSH to be
the main predictor for pregnancies in patients with high 3
day FSH concentration (> 20 mIU/ml) as none of these patients had pregnancies. A combination of early follicular
FSH and age has been said to be better than age alone in
predicting outcome in women undergoing IVF [20].
A significant limiting factor for interpreting the values of
day 3 FSH serum values relates to the intercycle fluctuations,
which are greater in the presence of high basal concentrations [27] although, a low inter-cycle variability was found
in patients with normal basal FSH levels [28]. In addition,
interpretations of multiple cycles are also controversial, although it is recommended that fertility estimates may be
more reliable if based on the highest FSH concentration and
not the lowest. The specific FSH assay used in any laboratory can also affect the boundary between normal and abnormal results. [29, 30] FSH concentrations predict low fertility when its values are high, but do not accurately predict
fertility when normal. Finally, the finding of normal FSH
concentrations in older women with poor ovarian reserve
emphasizes the importance of looking beyond age and FSH.
Inhibin B
Inhibin A and B are both released by the granulose cells
of 2 – 5 mm ovarian follicle. They are dimeric peptides
composed of an alpha (A) subunit (Inhibin A) and beta (B)
subunit (Inhibin B) [17]. Inhibin A seems to be secreted by
the dominant follicle, since it increases just after the rise in
estradiol concentration during the late follicular phase and
thus may mark follicular maturity [17, 31]. Inhibin B is
possibly secreted by the developing cohort of follicles in a
cycle, since its concentration rises across the luteal-follicular
transition and peaks in the mid-follicular phase [32]. So,
Inhibin B may indicate the number or quality of developing
follicles [17, 31].
Early follicular inhibin B correlates inversely with early
follicular FSH levels in perimenopausal women and has been
promoted as an early indicator of poor ovarian reserve [33,
34]. It has been demonstrated that women with a low cycle
day 3 inhibin B concentrations show a poorer response to
ovulation induction than do women with a high day 3 inhibin
B concentration [34].
Hofmann et al. investigated inhibin B concentration during clomiphen citrate challenge test in women with normal
and poor ovarian reserve and reported that women with poor
ovarian reserve had lower inhibin B concentrations than the
former [35]. Decreased inhibin B concentrations were also
found in women with poor ovarian reserve, despite having
non-elevated FSH concentrations, suggesting that a fall in
inhibin B concentration might be an earlier marker for limited ovarian reserve than was elevated FSH concentration
[34]. Inhibin B concentration is also found to be a better
predictor for cancellation than age [15], although its value
is still questioned [36]. Although, inhibin B might provide
an additional tool for analyzing ovarian reserve, more data
is necessary in order to establish normal ranges in clinical
practice.
269
Anti-Mullerian Hormone
Ovarian Volume
AMH is a dimeric glycoprotein of the transforming
growth factor –alfa family which plays a major role in male
sex differentiation. In the female ovary it is expressed as
early as 32 weeks of gestation from the granulosa cells [37].
Its role is primarily related to inhibition of progression of
primordial follicles to primary follicles as evidenced by animal studies [38]. It can also influence follicle dominance and
recruitment [39]. It reduces responsiveness of follicles to
FSH in dominance selection [40].
It is a well known fact that ovaries get smaller with ageing. This may also correspond to the number of follicles in
them [56, 57]. Various ultrasound modalities are used to
measure ovarian volume. Three dimensional ultrasound with
power Doppler angiography is the most recent type. This
method has advantages over color Doppler of being more
sensitive and the three dimension feature adds to the details
and accurate measurement of volume and blood flow [58].
There has been no definite cutoff established by measurement of the ovarian volume as it is an age dependant measurement. Kupesic et al. in a prospective study in women undergoing IVF found statistically significant differences in
total ovarian volume (TOV) in women of different age
groups utilizing three dimensional power Doppler ultrasound
[59]. Another group of researchers did find declining ovarian
volumes in women in perimenopausal age groups when
compared to women of younger age and calculated a 0.2 cm3
decline with increasing age [60].
Role of AMH to determine ovarian response have been
extensively studied. It has been noted that AMH levels decline throughout the reproductive age and is almost non detectable after menopause [41]. This same group of researchers also noted that the AMH levels correlate well with antral
follicle count (AFC) and that day 3 serum AMH also declines as reproductive age advances [41]. The level of AMH
shows a steady decline over time compared to other markers
both in women <35 yrs and >40 [42]. Due to this characteristic of lower levels corresponding to age, many researchers
have suggested that AMH levels may predict onset of menopause [43, 44].
Role of AMH in ovarian reserve testing has been studied
in IVF setting in predicting number of oocytes available
for retrieval. The first study was done by Seifer et al. who
proposed that a higher day 3 AMH corresponded to more
oocytes retrieved [45]. Other groups established this above
fact including predicting number of embryos for IVF but
failed to provide information on oocyte quality and fertility
outcomes [46]. Silberstein et al. had demonstrated that
higher AMH levels during HCG administration could predict
better embryo scores [47]. In patients susceptible to develop
ovarian hyperstimulation syndrome (OHSS), serum AMH
may be used as a predictive marker as does estradiol levels
and follicle numbers [48-50].
AMH levels may also be used as markers of treatment
and progression in patients with polycystic ovarian syndrome (PCOS). Since AMH has significant role in primordial follicle transition and recruitment, it is hypothesized that
abnormal AMH may hamper maturation of follicle and ultimately ovulation in PCOS patients. The serum level of AMH
is 2 to 3 fold greater in PCOS compared to normal patients
[51, 52]. Studies have suggested that in absence of ultrasonographic aid, in patients with oligoamenorrhoea and
hyperandrogenism, serum AMH levels can help diagnose
PCOS [53].This can also serve as a prognostic indicator of
recovery of menstrual function after weight loss treatment
[54].
AMH estimation is technically advantageous secondary
to its very low intra and inter assay variability [55]. Other
advantages are its comparability to antral follicle count
(AFC) and capacity to predict menopause as mentioned
above. But there are certain disadvantages. Serum AMH fails
to determine the exact cohort of follicles 2-5 mm in size
where smaller follicles may also secrete AMH [51]. Besides
the cost of estimation, the variability in levels in a single
cycle has not been studied yet and its clinical application
may be limited [46].
Though the measurement of ovarian volume is quite accurate, the main question is whether it has any prognostic
role in determining outcomes of assisted reproduction techniques. Some studies documented that women with ovarian
volumes <3 cm3 had higher cancellation rates [61] and there
may be a correlation with success of assisted reproduction
cycles but not significant for pregnancy outcomes [62, 63].
A prospective cohort study in women undergoing IVF correlated IVF parameters with mean ovarian volumes and failed
to establish a cutoff volume which could predict pregnancy
outcome [64]. It has also been documented that ovarian
volume estimation has similar sensitivity and specificity to
basal FSH in predicting menopause [65].
It is currently evident that though ovarian volume
measurement may indicate available oocytes at a particular
age group but may not be good in providing pregnancy
outcomes after IVF.
Total Antral Follicle Count
Total antral follicle count during the follicular phase is a
sonographic parameter of estimating ovarian reserve and an
excellent marker of chronological age [66]. Scheffer et al.
found that the antral follicle pool declines at 4.8% per year
before 37 years and at a rate of 11.7% after the age of 37
[66].
Ultrasound guidance to measure the follicles have also
been recommended. Antral follicles measuring 2-10 mm in
diameter measured by transvaginal ultrasound are the normal
standard [67, 68]. Some studies have also predicted that diameters less than 5 mm may result in no pregnancy [69, 70]
and some group has suggested that practically the range 2-10
mm actually represent the smaller follicle pool of 2-6 mm
that has a better representation of the ovarian reserve compared to those that are larger [71]. Inter observer and biological variations in measurement might be the only disadvantage of AFC [72]. Technically most commonly used
methods utilize 2D or 3D measurements but recent research
has postulated that Sono-AVC (Sono – Automated Volume
Calculation) technique may reduce inter and intra observer
variations [67].
Various studies have postulated that AFC is still the best
test to predict fertility outcomes after IVF. A standardized
AFC measurement is superior to basal FSH, inhibin-B in
measuring ovarian response after stimulation [72]. AFC has
also been studied to be a superior predictor of poor outcome
when compared to basal FSH [73] and ovarian volume [74]
in two meta-analysis. It has been also documented to be superior when compared with clomiphen citrate challenge test,
inhibin-B, and ovarian volume in predicting poor outcome
[75].
A retrospective study of 278 patients undergoing IVF
established that an AFC of 11 or more is a better predictor of
live birth after in vitro fertilization and embryo transfer
(IVF-ET) and is a good number to counsel patients about
success rates [76]. Combination of other parameters with
AFC has also been studied. A day 7 follicle count with a
basal AFC together had a high positive predictive value and
was a better predictor of cycle cancellation in a retrospective
study of 91 IVF cycles [77].
AFC and serum AMH have been documented to be
strong predictor of poor ovarian response after IVF having
similar capacity individually though combining them did not
augment the predictive value in a prospective study of 135
women undergoing first IVF. They found that AFC has a
sensitivity of 93% and a specificity of 88% while AMH had
that of 100% and 73% respectively. This group also documented a cutoff of 10 for AFC and 0.99 ng/ml for AMH
[78].
As discussed above AFC is a great marker of chronological age [66]. Relationship of AFC with menopause has also
been studied [56, 79]. Maternal age of menopause and declining AFC may indicate genetic trait in patients. Women
with a lower maternal age of menopause had low AFC and a
slower decline in the number as documented in a recent cross
sectional study [79].
Dynamic Testing
These are a set of tests which utilizes exogenous stimuli
to stimulate folliculogenesis and measure hormone levels
and or antral follicle counts to get an indirect indication of
the nature of ovarian response. These tests namely the exogenous FSH ovarian reserve test (EFORT), clomiphen citrate challenge test (CCCT) and GnRH agonist stimulation
test (GAST) are rarely used today since individual parameters like AFC and or serum AMH levels are better indicators.
Exogenous FSH Ovarian Reserve Test (EFORT)
This test can be used to predict ovarian reserve in patients prior to undergoing IVF treatment. It was documented
that if estradiol levels increased above 30 pg/ml after administration of 300IU of exogenous FSH on day 3 of the
cycle, the pregnancy and implantation rates were better [80].
A baseline FSH was also measured. Some studies also recommended measuring inhibin-B levels after EFORT as a
guide for ovarian reserve testing and possibly a better predictor compared to clomiphen citrate challenge test (CCCT)
[81]. Recent studies utilizing EFORT to predict pregnancy
outcomes are currently lacking both in general and subfertile
population.
Banerjee et al.
GnRH Agonist Stimulation Test (GAST)
This test is performed by administering supraphysiologic
doses of GnRH agonist and measuring change in levels of
estradiol from day 2 to day 5 of the cycle. An increase in
these levels is considered a good response. Controversies
exist based on earlier and newer studies in patients undergoing assisted reproduction regarding the benefit of this test.
Some groups did not find this to be useful [23, 82] whereas
others thought the opposite [83, 84]. Hendriks et al. compared day 3 AFC and inhibin –B with the GAST and did not
find it to be superior and in fact it could not predict ongoing
pregnancy outcomes well. Thus GAST has fallen out of favor compared to serum AMH, AFC or inhibin-B estimation.
Clomiphen Citrate Challenge Test (CCCT)
100 mg of clomiphen citrate is administered orally from
days 5 to 9 after estimating basal FSH on day 2 or 3 of a
cycle. If an increase of FSH is found on day 10 by more than
two standard deviations from normal the test is declared as
abnormal. Jain et al. published a meta-analysis including 12
studies for basal FSH (6296 patients) and 7 studies for
CCCT (1352 patients). Their outcome was to predict pregnancy. They concluded that both basal FSH and CCCT had
equal capacity to predict achievable pregnancy though a
normal test was not useful but an abnormal test was more
confirmatory for failure to achieve pregnancy [85].
CONCLUSION
Infertility is a growing problem and age has been a main
factor. Ovarian reserve declines with age and so does the
success of pregnancy after IVF. Various parameters have
been studied to predict outcomes after IVF which may reduce cancellation rates and develop protocols in infertility
clinics to achieve better success rates. Though routine testing
for ovarian reserve is not recommended, individualization of
cases may indicate the need to do so. Serum basal FSH,
AMH and AFC are still the most common parameters utilized due to evidenced based studies advocating their superiority. Dynamic testing has fallen out of favor due to advancements in estimation of endocrine markers and ultrasonography. Though studies have attempted to define cut off
values for each parameter larger population based studies are
required to get a standard definition for each. Various factors
may influence the estimation of these markers which need to
be considered. In clinical practice each patient need to be
evaluated individually to utilize the best prognostic indicator
of their reproductive outcome. Along with development
of predictive markers of ovarian reserve testing, assisted
reproduction techniques also require further development to
improve their outcome.
REFERENCES
[1]
[2]
[3]
[4]
te Velde ER, Pearson PL. The variability of female reproductive
ageing. Hum Reprod Update 2002; 8: 141-54.
Wood JW. Fecundity and natural fertility in humans. Oxf Rev
Reprod Biol 1989; 11: 61-109.
Block E. Quantitative morphological investigations of the follicular
system in women; variations at different ages. Acta Anat (Basel)
1952; 14: 108-23.
Baker TG. A quantitative and cytological study of germ cells in
human ovaries. Proc R Soc Lond B Biol Sci 1963; 158: 417-33.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
Faddy MJ, Gosden RG. A mathematical model of follicle dynamics
in the human ovary. Hum Reprod 1995;10: 770-5.
Faddy MJ, Gosden RG, Gougeon A, Richardson SJ, Nelson JF.
Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992; 7: 1342-6.
Ranieri DM, Serhal P. Ovarian reserve: A simple mathematical
problem?Hum Reprod 2000; 15: 1423-4.
te Velde ER. Disappearing ovarian follicles and reproductive ageing. Lancet 1993; 341: 1125-6.
Sherman SL, Freeman SB, Allen EG, Lamb NE. Risk factors for
nondisjunction of trisomy 21. Cytogenet Genome Res 2005; 111:
273-80.
McFadden DE, Friedman JM. Chromosome abnormalities in human beings. Mutat Res 1997; 396: 129-40.
Johnson NP, Bagrie EM, Coomarasamy A, et al. Ovarian reserve
tests for predicting fertility outcomes for assisted reproductive
technology: The International Systematic Collaboration of Ovarian
Reserve Evaluation protocol for a systematic review of ovarian reserve test accuracy. BJOG 2006; 113: 1472-80.
Keefe DL, Marquard K, Liu L.The telomere theory of reproductive
senescence in women. Curr Opin Obstet Gynecol 2006; 18: 280-5.
Bukman A, Heineman MJ. Ovarian reserve testing and the use of
prognostic models in patients with subfertility. Hum Reprod Update 2001; 7: 581-90.
Cahill DJ, Prosser CJ, Wardle PG, Ford WC, Hull MG. Relative
influence of serum follicle stimulating hormone, age and other factors on ovarian response to gonadotrophin stimulation.Br J Obstet
Gynaecol 1994; 101: 999-1002.
Balasch J, Creus M, Fabregues F, et al. Inhibin, follicle-stimulating
hormone, and age as predictors of ovarian response in in vitro fertilization cycles stimulated with gonadotropin-releasing hormone
agonist-gonadotropin treatment. Am J Obstet Gynecol 1996; 175:
1226-30.
Sharif K, Elgendy M, Lashen H, Afnan M. Age and basal follicle
stimulating hormone as predictors of in vitro fertilisation outcome.
Br J Obstet Gynaecol 1998; 105: 107-12.
Hall JE, Welt CK, Cramer DW. Inhibin A and inhibin B reflect
ovarian function in assisted reproduction but are less useful at predicting outcome. Hum Reprod 1999; 14: 409-15.
Magarelli PC, Pearlstone AC, Buyalos RP. Discrimination between
chronological and ovarian age in infertile women aged 35 years and
older: predicting pregnancy using basal follicle stimulating hormone, age and number of ovulation induction/intra-uterine insemination cycles. Hum Reprod 1996; 11: 1214-9.
Scott RT, Jr., Hofmann GE. Prognostic assessment of ovarian
reserve. Fertil Steril 1995; 63: 1-11.
Toner JP, Philput CB, Jones GS, Muasher SJ. Basal folliclestimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril 1991; 55: 784-91.
Chuang CC, Chen CD, Chao KH, et al. Age is a better predictor of
pregnancy potential than basal follicle-stimulating hormone levels
in women undergoing in vitro fertilization. Fertil Steril 2003; 79:
63-8.
Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB.A
systematic review of tests predicting ovarian reserve and IVF
outcome. Hum Reprod Update 2006; 12: 685-718.
Winslow KL, Toner JP, Brzyski RG, et al. The gonadotropinreleasing hormone agonist stimulation test--a sensitive predictor of
performance in the flare-up in vitro fertilization cycle. Fertil Steril
1991; 56: 711-7.
Bancsi LF, Huijs AM, den Ouden CT, et al. Basal folliclestimulating hormone levels are of limited value in predicting ongoing pregnancy rates after in vitro fertilization. Fertil Steril 2000; 73:
552-7.
Ebrahim A, Rienhardt G, Morris S, et al. Follicle stimulating hormone levels on cycle day 3 predict ovulation stimulation response.
J Assist Reprod Genet 1993; 10: 130-6.
Martin JS, Nisker JA, Tummon IS, et al. Future in vitro fertilization pregnancy potential of women with variably elevated day 3
follicle-stimulating hormone levels. Fertil Steril 1996; 65: 1238-40.
Schipper I, de Jong FH, Fauser BC. Lack of correlation between
maximum early follicular phase serum follicle stimulating hormone
concentrations and menstrual cycle characteristics in women under
the age of 35 years. Hum Reprod 1998; 13: 1442-8.
Scott RT, Jr., Hofmann GE, Oehninger S, Muasher SJ. Intercycle
variability of day 3 follicle-stimulating hormone levels and its ef-
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
271
fect on stimulation quality in in vitro fertilization. Fertil Steril
1990; 54: 297-302.
Scheffer GJ, Broekmans FJ, Dorland M, et al. Antral follicle counts
by transvaginal ultrasonography are related to age in women with
proven natural fertility. Fertil Steril 1999; 72: 845-51.
Taieb J, Olivennes F, Birr AS, et al. Comparison of day 3 FSH
serum values as determined by six different immunoassays. Hum
Reprod 2002; 17: 926-8.
Santoro N, Adel T, Skurnick JH. Decreased inhibin tone and increased activin A secretion characterize reproductive aging in
women. Fertil Steril 1999; 71: 658-62.
Danforth DR, Arbogast LK, Mroueh J, et al. Dimeric inhibin: a
direct marker of ovarian aging. Fertil Steril 1998; 70: 119-23.
Burger HG, Cahir N, Robertson DM, et al. Serum inhibins A and B
fall differentially as FSH rises in perimenopausal women. Clin Endocrinol (Oxf) 1998; 48: 809-13.
Seifer DB, Scott RT, Jr., Bergh PA, et al. Women with declining
ovarian reserve may demonstrate a decrease in day 3 serum inhibin
B before a rise in day 3 follicle-stimulating hormone. Fertil Steril
1999; 72: 63-5.
Hofmann GE, Danforth DR, Seifer DB. Inhibin-B: the physiologic
basis of the clomiphene citrate challenge test for ovarian reserve
screening. Fertil Steril 1998; 69: 474-7.
Corson SL, Gutmann J, Batzer FR, et al. Inhibin-B as a test of
ovarian reserve for infertile women. Hum Reprod 1999; 14: 281821.
Rajpert-De Meyts E, Jorgensen N, Graem N, et al. Expression of
anti-Mullerian hormone during normal and pathological gonadal
development: association with differentiation of Sertoli and granulosa cells. J Clin Endocrinol Metab 1999; 84: 3836-44.
Durlinger AL, Gruijters MJ, Kramer P, et al. Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the
mouse ovary. Endocrinology 2001; 142: 4891-9.
Weenen C, Laven JS, Von Bergh AR, et al. Anti-Mullerian hormone expression pattern in the human ovary: potential implications
for initial and cyclic follicle recruitment. Mol Hum Reprod 2004;
10: 77-83.
van Rooij IA, Broekmans FJ, te Velde ER, et al. Serum antiMullerian hormone levels: a novel measure of ovarian reserve.
Hum Reprod 2002; 17: 3065-71.
de Vet A, Laven JS, de Jong FH, Themmen AP, Fauser BC. Antimullerian hormone serum levels: a putative marker for ovarian
aging. Fertil Steril 2002; 77: 357-62.
van Rooij IA, Tonkelaar I, Broekmans FJ, et al. Anti-mullerian
hormone is a promising predictor for the occurrence of the menopausal transition. Menopause 2004; 11: 601-6.
Sowers MR, Eyvazzadeh AD, McConnell D, et al. Anti-mullerian
hormone and inhibin B in the definition of ovarian aging and
the menopause transition. J Clin Endocrinol Metab 2008; 93: 347883.
Tehrani FR, Solaymani-Dodaran M, Azizi F. A single test of antimullerian hormone in late reproductive-aged women is a good
predictor of menopause. Menopause 2009; 16: 797-802.
Seifer DB, MacLaughlin DT, Christian BP, Feng B, Shelden RM.
Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 2002; 77: 468-71.
Smeenk JM, Sweep FC, Zielhuis GA, et al. Antimullerian hormone
predicts ovarian responsiveness, but not embryo quality or pregnancy, after in vitro fertilization or intracyoplasmic sperm injection. Fertil Steril 2007; 87: 223-6.
Silberstein T, MacLaughlin DT, Shai I, et al. Mullerian inhibiting
substance levels at the time of HCG administration in IVF cycles
predict both ovarian reserve and embryo morphology. Hum Reprod
2006; 21: 159-63.
Lee TH, Liu CH, Huang CC, et al. Serum anti-Mullerian hormone
and estradiol levels as predictors of ovarian hyperstimulation syndrome in assisted reproduction technology cycles. Hum Reprod
2008; 23: 160-7.
Nakhuda GS, Chu MC, Wang JG, Sauer MV, Lobo RA. Elevated
serum mullerian-inhibiting substance may be a marker for ovarian
hyperstimulation syndrome in normal women undergoing in vitro
fertilization. Fertil Steril 2006; 85: 1541-3.
Tremellen KP, Kolo M, Gilmore A, Lekamge DN. Anti-mullerian
hormone as a marker of ovarian reserve. Aust N Z J Obstet Gynaecol
2005; 45: 20-4.
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
Banerjee et al.
Cook CL, Siow Y, Brenner AG, Fallat ME. Relationship between
serum mullerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and
normal women. Fertil Steril 2002; 77: 141-6.
Broekmans FJ, Knauff EA, Valkenburg O, et al. PCOS according
to the Rotterdam consensus criteria: Change in prevalence among
WHO-II anovulation and association with metabolic factors. BJOG
2006; 113: 1210-7.
Pigny P, Jonard S, Robert Y, Dewailly D. Serum anti-Mullerian
hormone as a surrogate for antral follicle count for definition of the
polycystic ovary syndrome. J Clin Endocrinol Metab 2006; 91:
941-5.
Moran LJ, Noakes M, Clifton PM, Norman RJ. The use of antimullerian hormone in predicting menstrual response after weight
loss in overweight women with polycystic ovary syndrome. J Clin
Endocrinol Metab 2007; 92: 3796-802.
Laven JS, Mulders AG, Visser JA, et al. Anti-Mullerian hormone
serum concentrations in normoovulatory and anovulatory women
of reproductive age. J Clin Endocrinol Metab 2004; 89: 318-23.
Giacobbe M, Mendes Pinto-Neto A, Simoes Costa-Paiva LH,
Martinez EZ. The usefulness of ovarian volume, antral follicle
count and age as predictors of menopausal status. Climacteric
2004; 7: 255-60.
Faddy MJ, Gosden RG. A model conforming the decline in follicle
numbers to the age of menopause in women. Hum Reprod 1996;
11: 1484-6.
Jarvela IY, Sladkevicius P, Tekay AH, Campbell S, Nargund G.
Intraobserver and interobserver variability of ovarian volume, grayscale and color flow indices obtained using transvaginal threedimensional power Doppler ultrasonography. Ultrasound Obstet
Gynecol 2003; 21: 277-82.
Kupesic S, Kurjak A, Bjelos D, Vujisic S. Three-dimensional ultrasonographic ovarian measurements and in vitro fertilization outcome are related to age. Fertil Steril 2003; 79: 190-7.
Oppermann K, Fuchs SC, Spritzer PM. Ovarian volume in pre- and
perimenopausal women: a population-based study. Menopause
2003; 10: 209-13.
Sharara FI, McClamrock HD. The effect of aging on ovarian volume measurements in infertile women. Obstet Gynecol 1999; 94:
57-60.
Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel
outcome predictor for assisted reproduction. Fertil Steril 1995; 64:
1167-71.
Lass A, Skull J, McVeigh E, Margara R, Winston RM. Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophin for in-vitro
fertilization can predict poor response. Hum Reprod 1997; 12: 2947.
Frattarelli JL, Levi AJ, Miller BT, Segars JH. Prognostic use of
mean ovarian volume in in vitro fertilization cycles: a prospective
assessment. Fertil Steril 2004; 82: 811-5.
Flaws JA, Langenberg P, Babus JK, Hirshfield AN, Sharara
FI.Ovarian volume and antral follicle counts as indicators of menopausal status. Menopause 2001; 8: 175-80.
Scheffer GJ, Broekmans FJ, Looman CW, et al. The number of
antral follicles in normal women with proven fertility is the best reflection of reproductive age. Hum Reprod 2003; 18: 700-6.
Deb S, Jayaprakasan K, Campbell BK, et al. Intraobserver and
interobserver reliability of automated antral follicle counts made
using three-dimensional ultrasound and SonoAVC. Ultrasound Obstet Gynecol 2009; 33: 477-83.
Sills ES, Alper MM, Walsh AP. Ovarian reserve screening in infertility: practical applications and theoretical directions for research.
Eur J Obstet Gynecol Reprod Biol 2009; 146: 30-6.
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. Use of the
antral follicle count to predict the outcome of assisted reproductive
technologies. Fertil Steril 1998; 69: 505-10.
Chang MY, Chiang CH, Chiu TH, Hsieh TT, Soong YK. The antral
follicle count predicts the outcome of pregnancy in a controlled
ovarian hyperstimulation/intrauterine insemination program. J Assist Reprod Genet 1998; 15: 12-7.
Haadsma ML, Bukman A, Groen H, et al. The number of small
antral follicles (2-6 mm) determines the outcome of endocrine
ovarian reserve tests in a subfertile population. Hum Reprod 2007;
22: 1925-31.
Bancsi LF, Broekmans FJ, Eijkemans MJ, et al. Predictors of poor
ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 2002; 77: 32836.
Hendriks DJ, Mol BW, Bancsi LF, Te Velde ER, Broekmans FJ.
Antral follicle count in the prediction of poor ovarian response and
pregnancy after in vitro fertilization: a meta-analysis and comparison with basal follicle-stimulating hormone level. Fertil Steril
2005; 83: 291-301.
Hendriks DJ, Kwee J, Mol BW, te Velde ER, Broekmans FJ. Ultrasonography as a tool for the prediction of outcome in IVF patients:
a comparative meta-analysis of ovarian volume and antral follicle
count. Fertil Steril 2007; 87: 764-75.
Kwee J, Elting ME, Schats R, McDonnell J, Lambalk CB. Ovarian
volume and antral follicle count for the prediction of low and hyper
responders with in vitro fertilization. Reprod Biol Endocrinol 2007;
5: 9.
Maseelall PB, Hernandez-Rey AE, Oh C, et al. Antral follicle
count is a significant predictor of livebirth in in vitro fertilization
cycles. Fertil Steril 2009; 91: 1595-7.
Durmusoglu F, Elter K, Yoruk P, Erenus M. Combining cycle day
7 follicle count with the basal antral follicle count improves the
prediction of ovarian response. Fertil Steril 2004; 81: 1073-8.
Jayaprakasan K, Campbell B, Hopkisson J, Johnson I, RaineFenning N. A prospective, comparative analysis of anti-Mullerian
hormone, inhibin-B, and three-dimensional ultrasound determinants of ovarian reserve in the prediction of poor response to controlled ovarian stimulation. Fertil Steril 93: 855-64.
Rosen MP, Johnstone EB, Gillham SJ, et al. Is antral follicle count
a genetic trait? Menopause 2010; 17:109-13.
Fanchin R, de Ziegler D, Olivennes F, et al. Exogenous follicle
stimulating hormone ovarian reserve test (EFORT): a simple and
reliable screening test for detecting 'poor responders' in in-vitro fertilization. Hum Reprod 1994; 9: 1607-11.
Dzik A, Lambert-Messerlian G, Izzo VM, et al. Inhibin B response
to EFORT is associated with the outcome of oocyte retrieval in the
subsequent in vitro fertilization cycle. Fertil Steril 2000; 74: 11147.
Padilla SL, Bayati J, Garcia JE. Prognostic value of the early serum
estradiol response to leuprolide acetate in in vitro fertilization. Fertil Steril 1990; 53: 288-94.
Ranieri DM, Quinn F, Makhlouf A, et al. Simultaneous evaluation
of basal follicle-stimulating hormone and 17 beta-estradiol response to gonadotropin-releasing hormone analogue stimulation: an
improved predictor of ovarian reserve. Fertil Steril 1998; 70: 22733.
Hendriks DJ, Broekmans FJ, Bancsi LF, et al. Single and repeated
GnRH agonist stimulation tests compared with basal markers of
ovarian reserve in the prediction of outcome in IVF. J Assist Reprod Genet 2005; 22: 65-73.
Jain T, Soules MR, Collins JA. Comparison of basal folliclestimulating hormone versus the clomiphene citrate challenge test
for ovarian reserve screening. Fertil Steril 2004; 82: 180-5.
273
Bruno Salle1,* and Jacqueline Lornage2
1
2
Service de Médecine de la Reproduction, Hospices Civils de Lyon, Université Claude Bernard Lyon I, Lyon, France;
Unité INSERM 846, Cellules Souches & Cerveau, Lyon, France
Abstract: Ovarian transplantation may be the future of fertility preservation. Although ovarian fragments graft is known
to restore fertility, but a large number of primordial follicles are lost during the neo vacularisation period and the life span
of the transplant is usually short. Whole ovary transplantation may restore ovarian function immediately and during long
time. Nevertheless results of whole transplantation are still disappointing. Only one gestation in animal had been reported.
Keywords: Whole ovary, vitrification, transplantation, gestation, delivery.
INTRODUCTION
Many girls and young women have been or will be
treated for cancer. Depending on the type of treatment, early
menopause can occur. Thus, preserving female fertility is a
major issue that concerns most reproductive medicine teams
worldwide. One fertility preserving option that is being
studied is ovarian tissue cryopreservation followed by
implantation.
In one type of cryopreservation procedure, ovarian tissue
fragments are taken from the patient and frozen. Indeed,
many pregnancies and deliveries have been obtained in a
number of species after freezing and implantation of ovarian
tissue fragments [1]. Freezing cortical fragments enables a
large number of primary follicles to be stored with relative
ease due to the thinness of the tissue [2]. Cryprotectants can
easily penetrate the thinned cortex to protect the primary
follicles [3]. The first human live birth using this procedure
was reported by Jacques Donnez in 2007 [4]. Since then,
many grafts have been performed in humans, and the number
of births published or under publication is estimated at
around 13 [5].
Ovarian tissue grafting, however, poses many problems.
The functional lifespan of the grafts is sometimes short,
requiring repeated grafting. For many women who choose to
undergo in vitro fertilization after the ovarian freeze/thaw
procedure, the results are poor. The follicles may be empty,
and oocytes can be abnormal or immature, thereby greatly
reducing the rates of pregnancy by transfer [6].
Whole Ovary Cryo-Preservation (WOCP) with vascular
anastomosis, on the other hand, offers what may be a better
alternative. With WOCP, the entire follicular reserve is
preserved and transferred, and revascularization after
implantation is almost immediate. WOCP could thus restore
ovarian function entirely and over the long term.
WOCP with autograft has been performed in small
mammals such as the rat and mouse with relative success for
*Address correspondence to this author at the Service de Médecine de la
Reproduction, Hospices Civils de Lyon 59 Boulevard Pinel 69677 Bron
Cedex, Bron, France; Tel: 00 33 4 27856188; Fax: 00 33 4 72 12 94 14;
1573-4048/10 $55.00+.00
several years. With such small ovaries, the freezing technique
is straightforward and the cryoprotectants penetrate easily. In
larger species such as sheep and humans, however, the
process is more technically challenging. The human ovary is
composed of various types of tissues, such as vessels
(arteries and veins), the ovarian medulla and follicles.
Cryoprotectants do not always completely penetrate these
tissues. We have shown using VS4 as cryoprotectant
solution that VS4 perfused ovarian cortex fragments did
not vitrify but formed ice crystal around in 18 % [14]. The
surgical demands are also considerable. When transplanting
ovarian fragments, surgery is quick and simple and can be
performed using laparoscopy. WOCP, on the other hand,
requires termino-terminal arteriovenous anastomosis, which
is technically challenging. The difficulty lies in the microscopic size of the vessels and the length of the operation.
Additionally, laparotomy is required.
Arav [8] was the first to report results on WOCP, which
was performed in 8 ewes. Cyclic progesterone secretion was
documented in only 3 of the ewes and then only between 24
and 36 months after surgery. Oocytes were harvested from of
the 2 ewes, and MRI found intact ovaries with functioning
vessels in the 3 same ewes. In 2003, Bedaiwy demonstrated
the feasibility of performing WOCP. But, only 27% of the
whole ovary transplants had normal vascularization at 10
days [9]. In 2008, the same team recorded follicular survival
rates in whole ovaries and in tissue fragments that had been
frozen and transplanted in female patients—the rates were
identical. Imhoff reported gestation and birth following
WOCP in one ewe: the gestation occurred 2 years after
transplantation. The results of Imhoff study suggest that
recovery of ovarian function can be slow [10].
We have performed 12 ovarian transplantations in ewes
(Fig. 1): 6 of the transplantations used vitrified-thawed
ovarian fragments (Fig. 2), and 6 used fresh whole ovary (the
right ovary was immediately attached to the left pedicle after
the left ovary was removed). Follicle survival at 1 year was
near zero in the vitrification group, with, obviously, no
pregnancies whereas in the slow cooling group, ovarian
function recurred in 5 of 10 ewes, and 2 gestations were
documented, leading to healthy live birth [11, 12]. At the
time of this writing (unpublished data), we have also
transplanted 12 ewes using a slow-freezing protocol in 6 and
a vitrification protocol in the other 6. In both groups, cyclic
steroid secretion has occurred, and gestations are expected
for 2010.
Fig. (1). End to end anastomosis of the ovary.
Salle and Lornage
IS WHOLE OVARY FREEZING WORTH CONTINUING?
When comparing the results of slow tissue freezing with
those of WOCP, the evidence suggests that the former is
more advantageous, as witnessed by the live births obtained
in animals and humans. Only one animal gestation and birth
has been obtained with WOCP; an additional 2 were
reported after fresh ovary grafting. The advantages expected
from WOCP have yet to be confirmed. Functional recovery,
including hormonal secretion, is no faster, which may
be explained by the time needed for primary follicles to
enter their growth phase and reach maturity. Primary
follicle destruction rates are the same in both cases, but the
mechanisms differ. In WOCP the problem lies in the
difficulty of freezing primary follicles under the cortex or
with damage to subcortical microvascularization. The choice
between slow cooling and vitrification favors slow cooling.
New vitrification solutions such as VM3, however, could
improve outcomes. It is still too early to say that the future
for ovary grafting lies with WOCP. Many further studies
will be needed to improve both vitrification / freezing and
surgical protocols. In our opinion, the future of ovarian
vitrification lies not in transplantation so much as in ex
corporis whole ovary culture, which could provide oocytes
ad libitum for egg donation or cloning.
REFERENCES
[1]
[2]
[3]
[4]
[5]
Fig. (2). Vitrified entire ovary before thawing.
WHY VITRIFICATION?
Deciding whether to use slow freezing or vitrification is
tricky [13]. In the literature, most teams have performed
WOCP using a slow-freezing protocol, including the ewe
studies that resulted in gestations. We consider vitrification
better suited to WOCP because of the variety of tissues that
must be preserved. Using a vitrification solution called VS4,
we achieved a 78% follicle survival rate--a rate that is no
different than a fresh ovary follicle count. We demonstrated,
however, that the cooling speed did not result in complete
vitrification, which may account for the failure of transplantation using VS4 [14]. VM3 is a new vitrification solution containing so-called “super-iced molecules”, and it seems
to yield much better results. Most ewes transplanted using
VM3 have shown restored ovarian function (unpublished
data). This is in line with Onions’ theory according to which
follicle loss may be due not only to large-vessel thrombosis
but also to cryodamage to subcortical ovarian microvascularization [15].
[6]
[7]
[8]
[9]
[10]
[11]
[12]
Gosden RG, Baird DT, Wade JC, et al. Restoration of fertility to
oophorectomized sheep by ovarian autografts stored at -196
degrees C. Hum Reprod 1994; 9(4): 597-603.
Salle B, Demirci B, Franck M, et al. Long-term follow-up of
cryopreserved hemi-ovary autografts in ewes: pregnancies, births,
and histologic assessment. Fertil Steril 2003; 80: 172-77.
Demirci B, Lornage J, Salle B, et al. Follicular viability and
morphology of sheep ovaries after exposure to cryoprotectant and
cryopreservation with different freezing protocols. Fertil Steril
2001; 75(4): 754-62
Donnez J, Dolmans MM, Demylle D, et al. Livebirth after
orthotopic transplantation of cryopreserved ovarian tissue. Lancet
2004; 364(9443): 1405-10.
Meirow D, Levron J, Eldar-Geva T, et al. Pregnancy after
transplantation of cryopreserved ovarian tissue in a patient with
ovarian failure after chemotherapy. N Engl J Med 2005; 353: 31821.
Dolmans MM, Donnez J, Camboni A, et al. IVF outcome in
patients with orthotopically transplanted ovarian tissue. Hum
Reprod 2009; 24(11): 2778-87.
Revel A, Elami A, Bor A, et al. Whole sheep ovary cryopreservation and transplantation. Fertil Steril 2004; 82: 1714-5.
Arav A, Revel A, Nathan Y, et al. Oocyte recovery, embryo
development and ovarian function after cryopreservation and
transplantation of whole sheep ovary. Hum Reprod 2005; 20: 35549.
Bedaiwy MA, Hussein MR, Biscotti C, et al. Cryopreservation of
intact human ovary with its vascular pedicle. Hum Reprod 2006;
21(12): 3258-69.
Imhof M, Bergmeister H, Lipovac M, et al. Orthotopic
microvascular reanastomosis of whole cryopreserved ovine ovaries
resulting in pregnancy and live birth. Fertil Steril 2006; 85 (suppl. 1):
1208-15.
Courbiere B, Odagescu V, Baudot A, et al. Cryopreservation of the
ovary by vitrification as an alternative to slow-cooling protocols.
Fertil Steril 2006; 86: 1243-51.
Courbiere B, Caquant L, Mazoyer C, et al. Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification. Fertil Steril 2009; 91(6): 2697706.
[13]
[14]
Fahy GM, MacFarlane DR, Angell CA, et al. Vitrification as an
approach to cryopreservation. Cryobiology 1984; 21: 407-26.
Baudot A, Courbiere B, Odagescu V, et al. Towards whole sheep
ovary cryopreservation. Cryobiology 2007; 55: 236-48.
[15]
275
Onions VJ, Webb R, McNeilly AS, et al. Ovarian endocrine profile
and long-term vascular patency following heterotopic autotransplantation of cryopreserved whole ovine -ovaries. Hum Reprod
2009; 24(11): 2845-55.
276
Ahmed Nasr1 and Mohamed Ali Bedaiwy1,2,*
1
2
Department of Obstetrics and Gynecology, Women Health Center, Assiut University, Assiut, 71111, Egypt;
Department of Obstetrics and Gynecology University Hospitals Case Medical Center, Cleveland, OH 44106, USA
Absract: A wide variety of fertility preservation options in women is available; however, most of the currently available
strategies are still experimental and do not guarantee subsequent fertility. The only established method is in vitro fertilization with embryo cryopreservation prior to cancer therapy. Other proposed strategies to preserve fertility in women with
cancer include: storage of frozen ovarian tissue or the whole ovary for future transplantation, storage isolated follicles
for in vitro growth and maturation and ovarian transposition before radiotherapy. The effectiveness of ovarian protection
during chemotherapy with GnRH analogs is yet to be shown.
Keywords: Embryo cryopreservation, ovarian transplantation, chemotherapy, radiotherapy, premature ovarian failure.
INTRODUCTION
Female fertility can be adversely influenced by cancer
therapies that affect the ovary and uterus. At the ovarian
level, pelvic irradiation and chemotherapy can decrease
ovarian reserve and result in ovarian failure. Moreover, pelvic irradiation can adversely affect the uterus, increasing the
subsequent risk of obstetrical complications such as intrauterine fetal growth restriction and preterm birth [1]. A number of potential options are currently available, however, to
help preserve a woman's future fertility, thanks to a better
understanding of the intricate mechanisms governing human
reproduction. They are: [1] storage of frozen embryos, [2]
storage of frozen ovarian tissue or the whole ovary for future
transplantation, [3] storage of frozen ovarian tissue or isolated follicles for in vitro growth and maturation, [4] ovarian
transposition before radiotherapy, [5] ovarian protection during chemotherapy with either GnRH analogs or antiapoptotic
agents (e.g., sphingosine-1-phospate), [6] uterine transplant
after successful cancer treatment where a functional uterus
no longer exists, and [7] in vitro fertilization (IVF) with embryo cryopreservation prior to cancer therapy.
Of those, only the last one--IVF followed by embryo
cryopreservation—is considered a clinically proven option
for women requiring cancer treatment [2]. The remaining
strategies are considered experimental largely due to
unproven efficacy or lack of adequate long-term follow-up.
In this review, the consequences of irradiation and
chemotherapy on ovarian function and fertility as well as
current and future fertility preservation strategies in female
cancer patients will be discussed.
EVOLUTION OF HUMAN OOGENESIS
By the end of the first trimester of pregnancy, the human
ovaries are almost fully formed. By the seventh week of
*Address correspondence to this author at the Department of Obstetrics
and Gynecology Case Western Reserve University School of Medicine
Cleveland, OH 44106-5034, USA; Tel: (216) 235-3061; Fax: 216-844-3796;
1573-4048/10 $55.00+.00
intrauterine life, primordial germ cells reach their final destination in the gonadal ridge after migrating from the yolk sac
endoderm. They differentiate into oogonia and eventually
into primary oocytes by mitosis. Before the twelfth week,
some primary oocytes enter their first meiotic division,
which becomes arrested in the dictyotene stage of prophase
I. This process resumes with the first ovulation. Contrary to
spermatogenesis, oogenesis in humans does not continue
after birth. The maximum number of primary oocytes (6 to 7
million) is attained at 20 weeks of intrauterine life. That
number falls dramatically to 400,000 at birth and approximately 200,000 at the time of puberty [3,4]. Oocyte reserve
continues to diminish both in quantity and quality throughout
the remaining life cycle.
A multitude of endocrine, paracrine, autocrine and intracrine factors govern the fate of each ovarian follicle [5].
During the menstrual cycle, follicles mature in a series of
stages (primordial, primary, secondary and tertiary) in a
process called folliculogenesis which is independent of gonadotropin stimulation. The vast majority of follicles undergo atresia after differentiation into tertiary follicles with
an antral cavity. During any ovarian cycle, one or two follicles mature to the preovulatory stage as a result of gonadotropin-induced folliculogenesis [5]. Throughout reproductive life, the main source of ovarian estrogens remains
the mature Graafian follicle [6]. Many genes, hormones,
proteins and factors are responsible for governing the intricate process of folliculogenesis in humans [7-14]. Intrafollicular communication between the cells controls follicular dynamics; exposure to cancer therapy frequently results
in the death of many neighboring cells, too [6, 7, 12, 13].
Traditionally, it has been well established that the germline
cells (primordial follicles0 are non renewable. This concept
was challenged by 2 recent reports [15, 16] stating that
germline cells may be renewed locally [15] or from bone
marrow stem cells [16]. However, this new and exciting
findings were not reproduced by other groups and it was
challenged itself by the opinion of some other experts in the
field [17-19].
ANTICANCER THERAPY-INDUCED PREMATURE
OVARIAN FAILURE (POF)
POF Induced by Chemotherapy
Premature ovarian failure (POF) occurs after exposure
to many anticancer drugs. Unfortunately, chemotherapyinduced ovariotoxicity is almost always an irreversible
insult. After treatment with ovariotoxic drugs, the ovary exhibits a spectrum of histological changes ranging from decreased numbers of follicles to absent follicles to fibrosis
[20]. The variable effects of different anticancer regimens on
ovarian functions have been reported by many other investigators [21, 22].
The incidence of chemotherapy-induced POF can not be
precisely estimated because of the multifactorial nature of
the insult. However, a number of factors can increase the
risk:
•
The cumulative dose.
•
The patient’s age at the start of treatment. POF increases
with advancing age owing to progressive age-dependent
follicular depletion.
•
The degree of gonadotoxicity. This varies from one
chemotherapeutic drug to another. Cell cycle specificity
of the chemotherapeutic agent majorly determines the
magnitude of ovarian injury; cell cycle–nonspecific
chemotherapeutic agents are more ovariotoxic than cell
cycle–specific ones. Alkylating agents are the most
gonadotoxic of all cell cycle–nonspecific anticancer
drugs. Cyclophosphamide, a commonly treatment for
breast cancer, is the most ovariotoxic of its group. Given
the fact that breast cancer is the most common malignancy in women, most cases of chemotherapy-induced
ovariotoxicity are induced by cyclophosphamide. Patient
age and cumulative dose most strongly influence the POF
rate in those taking this drug [20]. Less than 50% of
women treated with cyclophosphamide who are younger
than 30 years develop POF. On the other hand, approximately 60% of women between the ages of 30 and 40
years will develop POF and hypergonadotropic amenorrhea [20].
Consequently, the risk of POF is not the same in all patients receiving multiagent gonadotoxic chemotherapy.
Women with the highest risk for developing POF are those
who receive high-doses of alkylating agents with pelvic irradiation. On the other hand, young women with Hodgkin’s
disease treated with multiagent chemotherapy and radiation
to a field that does not include the ovaries will mostly remain
fertile, although usually for a shorter period of time than that
of age-matched controls [23]. In one case report, a young
woman treated with repeated courses of ifosfamide combined with pelvic irradiation for Ewing’s sarcoma of the pelvis who developed POF was able to achieve a spontaneous
pregnancy [24].
POF INDUCED BY RADIOTHERAPY
Among young women of reproductive age, radiotherapy
is standard treatment for many genital as well as extragenital
cancers including cervical, vaginal, and anorectal cancers,
some germ cell tumors, Hodgkin’s disease, and central nerv-
277
ous system (CNS) tumors. Pelvic radiotherapy can not only
damage the ovaries but also the uterus. The patient’s age,
total dose of radiation reaching the ovaries and the number
of sessions needed to deliver the total dose are the prominent
factors that determine both the extent and duration of ovarian
damage. This is quite similar to the impact of chemotherapy.
Fractionating the total dose of radiotherapy significantly
reduces the extent of ovarian damage [25]. Single-dose
radiation is more toxic than fractionated therapy. The
approximate threshold for radiation-induced ovarian failure
is 300cGy to the ovaries; only 11% to 13% of women developed POF at doses below 300cGy versus 60% to 63% at
doses above that threshold [26]. Standard pelvic irradiation
to the ovaries will consistently result in POF; this is compounded by co-administered chemotherapy [27, 28].
Ionizing radiation is particularly deleterious to ovarian
follicles, resulting in radiation-induced DNA damage, ovarian atrophy and a significant depletion of ovarian follicles
[29]. Oocytes exhibit a rapid onset of pyknosis, disruption of
the nuclear envelope, chromosome condensation, and cytoplasmic vacuolization. Within four to eight weeks after exposure to radiation, serum levels of FSH and LH progressively rise while serum E2 levels fall [29, 30]. Radiotherapy
results in a dose-dependent reduction in the follicular pool
[30]. Fifty percent of the oocyte population is destroyed by a
radiation dose < 2 Gy (LDL50 < 2 Gy) [31].
CHEMOTHERAPY/RADIOTHERAPY-INDUCED POF:
DIAGNOSIS AND PREDICTION
The diagnostic and prognostic implications of predicting
POF in cancer patients can not be overemphasized.
Researchers are currently looking for a biomarker that can
reliably predict chemotherapy- or radiotherapy-induced POF
with satisfactory sensitivity and specificity. Among those
potential markers are anti-Mullerian hormone (AMH), serum
FSH, inhibin B and basal antral follicle counts (AFC).
AMH is produced by the granulosa cells of virtually all
types of follicles, from the primary to early antral stages.
Growing follicles progressively lose their ability to produce
AMH, which is why peripheral AMH concentrations decrease during ovarian stimulation. Unlike all other hormonal
biomarkers, which are dependent on the stage of follicular
development, AMH is independent of FSH, LH, and inhibin
levels.
Serum FSH levels were elevated in cancer survivors with
regular menstrual cycles whereas AMH levels were lower
than those of age-matched controls [32]. Despite the smaller
ovarian volume in cancer patients, AFC were similar between cancer survivors and controls [32]. In prepubertal girls
receiving anticancer chemotherapy, serum levels of inhibin
B transiently decreased. Consequently, serum FSH, coupled
with serum inhibin B, was proposed as a potential biomarker
of the ovariotoxic effects of cancer chemotherapy in prepubertal girls [33].
Basal AFC has been used extensively alone or with other
markers to predict POF. Measurement of serum AMH levels
could be a quantitative and possibly a qualitative biomarker
of granulosa cell health and activity [34]. For prepubertal
girls undergoing sterilizing cancer therapy, AMH may have
promising prospects.
WHEN IS FERTILITY PRESERVATION INDICATED
IN WOMEN?
Breast cancer is the most common malignancy seen during the reproductive years that requires an immediate fertility-preserving intervention. One of every seven breast cancer
patients is younger than forty years [35, 36]. Another common malignancy seen in younger women that requires fertility-preserving intervention is cervical cancer. Of the 13 000
new cervical cancer cases diagnosed in the United States
each year, 50% are estimated to occur in women younger
than 35 years of age [37]. Moreover, owing to the peculiar
anatomic location of the cervix, fertility preservation is even
more challenging in those cases.
Table 1.
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The indications of fertility preservation in women currently include ovariotoxic radiotherapy/chemotherapy for
cancer as well as for the treatment of a multitude of nonmalignant conditions including systemic lupus erythematosus, acute glomerulonephritis, and Behcet’s disease. On the
other hand, many other patients with extra-genital cancers
are candidates for fertility-preserving procedures including
those with hematopoietic cancers such as leukemias and
lymphomas, musculoskeletal cancers such as Ewing’s sarcoma
and osteosarcoma, neuroblastomas, and Wilms’ tumor. Women
receiving chemotherapy while undergoing bone marrow
transplantation or umbilical cord stem-cell transplantation
are potential candidates as well as patients with nongynecologic cancers including lymphomas, sarcomas and
colorectal carcinoma. Some of the salient indications of
fertility preservation in women are summarized in Table 1.
Situations Necessitating Fertility Preservation in Women
1. NON-MALIGNANT CONDITIONS TREATED WITH OVARIOTOXIC RADIOTHERAPY OR CHEMOTHERAPY:
A-MULTI-SYSTEM DISORDERS:
Acute glomerulonephritis
Behcet’s disease
Systemic lupus erythematosus (SLE)
B-TRANSPLANTATION PROCEDURES:
Umbilical cord stem-cell transplantation (SCT)
Bone marrow transplantation (BMT)
2. MALIGNANCIES TREATED WITH OVARIOTOXIC RADIOTHERAPY OR CHEMOTHERAPY:
A-ADULT CANCERS:
1. GYNECOLOGIC CANCERS:
Cancer of the cervix
Breast cancer
2. NON-GYNECOLOGIC CANCERS:
Sarcomas
Lymphomas
Colorectal cancer
B-CHILDHOOD MALIGNANCIES:
1. HEMATOPOIETIC CANCERS:
Lymphomas
Leukemias
2. MUSCULOSKELETAL CANCERS:
Ewing’s sarcoma
Osteosarcoma
3. OTHER CANCERS:
Wilms’ tumor
Neuroblastomas
3. BILATERAL OOPHORECTOMY FOR BENIGN OVARIAN TUMORS, ENDOMETRIOSIS, OR PROPHYLAXIS.
STRATEGIES FOR FERTILITY PRESERVATION
A spectrum of potentially promising strategies has been
assessed for fertility preservation. Although the currently
available armamentarium encompasses an ever expanding
list, most of those are more experimental than practical.
Moreover, none has been tested in a prospective randomized
controlled trial (RCT).
PHARMACOLOGICALLY-BASED PROTECTION
The theoretical rationale behind pharmacologically-based
protection came from the intriguing observation that the
premenarchal ovary is less sensitive to the effects of ovariotoxic agents. Consequently, researchers have studied pretreatment with a gonadotropin-releasing hormone (GnRH)
agonist, as well as many other medications that suppress the
hypothalamic pituitary ovarian axis, in an endeavor to maintain the prepubertal ovarian quiescence during chemotherapy
[38, 39]. Meanwhile, a direct ovarian effect has also been
postulated [38, 39]. However, theory and practice do not
always meet. The few observational studies addressing this
issue have come to conflicting conclusions. Blumenfeld and
colleagues reported a diminished frequency of POF in patients who received a GnRH agonist prior to chemotherapy
in their excellent analysis of all studies on GnRH agonist
use. Most of those studies, however, were limited by short
follow-up period in the GnRH group and the retrospective
nature of the control groups [40]. Other investigators [41,
42] have come to similar conclusions. On the other hand, a
prospective controlled study found that GnRh analogues
were not effective in the prevention of POF [43]. That
particular study however was limited by a small sample size.
Patients and oncologists strongly advocate GnRH use;
oncologists are satisfied with starting cancer therapy without
avoidable delays caused by standard ART protocols, and
patients find it simple. On the other hand, counter-arguments
downplaying the use of GnRH agonists are centered on the
fact that FSH suppression will not protect the primordial
follicles constituting the ovarian reserve. The debate on the
effectiveness of GnRH agonists will continue until it is settled by the results of prospective randomized controlled trials
that are sufficiently powered. Empirical use of other suppressive drugs such as combined oral contraceptives (COCs)
or progestins has not been successful in preventing chemotherapy/radiotherapy-induced ovarian damage.
279
more effective than medial transposition--with the latter, the
ovaries are sutured posteriorly to the uterus and shielded
during treatment [46, 47].
Lateral ovarian transposition can be performed during
staging laparotomy for Hodgkin’s disease (which is not customary nowadays) and during radical hysterectomy for cervical cancer [48]. Laparoscopic ovarian transposition could
be considered a valuable option before starting radiotherapy.
Moreover, immediate postoperative radiotherapy could be
initiated, which could furthermore help prevent POF if the
ovaries were to migrate back to the irradiation field [28, 49,
50]. Moreover, the same anesthetic can be used for laparoscopic ovarian transposition as that used to insert a
brachytherapy device in cases of cervical or vaginal cancers
treated by brachytherapy [51]. It can also be performed on an
outpatient basis in patients with Hodgkin’s disease, leading
to a better cosmetic effect and earlier recovery with minimal
cost and discomfort.
Following laparoscopic ovarian transposition, almost all
women with Hodgkin’s disease (stage I and stage II) treated
with radiation alone or with minimal chemotherapy retain
their ovarian function and fertility [28]. Laparoscopic ovarian transposition is not a complication-free procedure, however. POF is still a concern, and symptomatic ovarian cysts
may develop as well. There are many potential causes of
ovarian failure following ovarian transposition [52], and they
are summarized in Table 2. Although COCs can help suppress cyst formation, the underlying etiology remains largely
obscure [53].
Table 2.
Potential Causes of Ovarian Failure Following
Ovarian Transposition Before Radiotherapy
A-VASCULAR INSULT:
1. Jeopardizing ovarian vessels
2. Radiation-mediated injury of the vascular pedicle
B-ANATOMICALLY-MEDIATED MECHANISMS:
1. Transposed ovaries not moved far enough
2. Ovarian migration back to original position following use of
absorbable sutures
TRANSPOSITION OF THE OVARIES (OOPHOROPEXY)
ASSISTED
(ART)
Another possible fertility preservation option for women
scheduled to undergo sterilizing ovariotoxic radiotherapy is
surgery in which the ovaries are moved away from the intended irradiation field. This technique helps curtail ovarian
radiation exposure in patients with low intestinal and genitourinary malignancies and in those with Hodgkin’s disease
receiving pelvic irradiation. The radiation dose to the ovaries
is reduced to approximately 5% to 10% following transposition [44]. Given an initial dose of 4,500 cGy, the dose to
each transposed ovary is 126 cGy for intracavitary radiation,
135–190 cGy for external radiation therapy and 230–310
cGy with para-aortic lymph node inclusion in irradiation
[45]. Lateral transposition of the ovaries was shown to be
In patients who wish to proceed to fertility preservation,
ART is probably the most commonly adopted policy.
REPRODUCTIVE
TECHNOLOGIES
Oocyte Cryopreservation
In unmarried women and those without a male partner,
freezing mature or immature oocytes may be the only practically option. A multitude of factors come into play to determine the efficacy of oocyte cryopreservation, given the complex structural nature of the ovary. In contrast to preimplantation embryos, subcellular organelles in the oocytes are far
more complex and perhaps more sensitive to thermal injury
[54, 55]. Oocytes can be cyropreserved for years and still
retain their reproductive potential. Moreover, the duration
of oocyte cryopreservation does not seem to interfere with
oocyte survival; a number of pregnancies have been reported
several years after oocyte cryopreservation using liquid
nitrogen [56].
In its recent evaluation of the currently available
evidence, the ASRM practice committee concluded that
oocyte cryopreservation is associated with a limited number
of established pregnancies and deliveries resulting from
cryopreserved oocytes. However, no increases in chromosomal abnormalities, birth defects, or developmental deficits
have been noted so far among offspring. It also states that
oocyte cryopreservation should still be considered an
experimental technique that should only be performed under
an investigational protocol approved by an internal review
board (IRB) [57].
Despite an unacceptably low initial pregnancy rate per
vitrified–thawed oocyte, recent technical modifications have
resulted in improved oocyte survival, fertilization, and pregnancy rates. Intriguingly, pregnancy rates are steadily increasing, whether slow-freeze or vitrification methods are
used [58]. Therefore, cryopreservation of oocytes has its
place not only as an additional procedure in conventional
IVF but also as a viable option for fertile women at risk of
losing their fertility. The results of larger prospective controlled clinical trials focusing on efficacy and long-term
safety of oocyte cryopreservation are needed. Oocyte cryopreservation can only be considered to have practical value
when an adequate number of births is achieved and followed-up. Until then, it remains basically an experimental
procedure that can only be carried out under strict surveillance [59].
Embryo Cryopreservation
In 2005, embryo cryopreservation was described by the
ASRM as the only established evidence-based and clinically
valuable fertility preservation option for women with a male
partner. It is presumably the most effective means of fertility
preservation in women undergoing ovarian stimulation regimens for IVF, and it offers them a satisfactory chance of
success. Delivery rates per embryo transfer have been reported by the society for assisted reproduction technology
(SART), using cryopreserved embryos, to be 31.8% for
women under 35 years of age [60]. Post-thaw embryo survival rates range between 35% and 90%, and implantation
rates range between 8% and 30%; cumulative pregnancy
rates have been reported to be more than 60% [61, 62].
Consequently, embryo cryopreservation is considered the
fertility preservation option with the best outcome. Moreover, long-term follow up data on the outcome of children
born from these procedures are reassuring and confirm that
the procedure is safe. A number of limitations do, however,
exist. Firstly, it is unsuitable for prepubertal, adolescent and
unmarried females and women without a partner. Secondly,
ovarian stimulation protocols in IVF inevitably lead to extremely high estradiol levels, which may be dangerous for
women with estrogen-sensitive tumors such as breast cancer.
The typical time interval between breast cancer surgery and
initiation of chemotherapy is six weeks. In lieu of the standard protocol, a short flare protocol is adopted, which usu-
Nasr and Bedaiwy
ally requires less time to achieve follicle recruitment [63].
Conventional controlled ovarian hyperstimulation (COH)
produces exceptionally high levels of estrogen, potentially
affecting the overall prognosis [64]. Therefore, some centers
offer unstimulated natural-cycle IVF, where a single oocyte
is aspirated. Cancellation rates are, however, high, and pregnancy rates very low (7.2% per cycle and 15.8% per embryo
transfer) [65, 66].
The nonsteroidal antiestrogen tamoxifen [66] and the
aromatase P450 inhibitor letrozole [67] were used as potential alternatives for ovulation induction in the breast cancer
patient. Oktay and colleagues reported, in their prospective
controlled study comparing tamoxifen to letrozole as ovarian
stimulation agents in breast cancer patients, that when combined with low-dose FSH, both drugs had a better outcome
than when tamoxifen was used alone. Letrozole was preferred to tamoxifen because its use was accompanied with
significantly lower peak serum estradiol levels, which is a
potential asset for breast cancer patients [67]. They further
compared the safety and success of ovarian stimulation with
letrozole and tamoxifen in breast cancer patients undergoing
IVF to cryopreserve their embryos for fertility preservation
and reported similar cancer recurrence rates between those
who underwent COH and those who did not [68].
CRYOPRESERVATION AND OVARIAN TISSUE
Transplantation
Ovarian tissue cryopreservation and transplantation have
emerged as promising experimental fertility preservation
procedures in women at risk of losing their reproductive capacity. Given the fact that ovarian tissue transplantation is an
autotransplantation rather than transplantation between two
genetically distinct humans, immunosuppression is unnecessary. In contrast, long-term immunosuppression is required
after transplantation of reproductive organs between genetically distinct humans. Cryopreservation of ovarian tissue is,
therefore, an indispensible prerequisite to the potential autotransplantation process, allowing preserved germ cells to be
replaced after completion of the ovariotoxic cancer therapy.
Owing to their smaller size and lack of follicular fluid, better
survival is expected to occur with primordial follicles occupying the ovarian cortical strips [69].
Animal models have provided invaluable information
about transfer methods. Sheep ovaries, which resemble human ovaries, have been used as a model for ovarian tissue
cryopreservation and transplantation by Gosden and colleagues [70]. Survival and endocrine activity of the follicular
apparatus as well as pregnancy and delivery after transplantation of cryopreserved-thawed ovarian cortical strips have
been successfully reported [70, 71].
MINIMIZING ISCHEMIC DAMAGE IN OVARIAN
TISSUE TRANSPLANTATION
In some human ovarian transplants where nonvascularized grafts were used, an initial ischemic insult was noted,
which was postulated to be, at least in part, responsible for
the limited lifespan of ovarian function. Therefore, it is vital
to reduce the duration of ischemia and provide immediate
revascularization of the grafts to prolong their longevity and
help preserve their function for as long a duration as possible. To minimize ischemic damage of the grafts, Almodin
and associates described a novel technique in which frozenthawed fragments of one ovary were injected into the cortex
of the remaining sterile ovary. In that experimental trial, radiotherapy was administered to ewes to induce ovarian failure in one ovary while fragments of the other ovary were
kept frozen. Consequently, injection of the thawed fragments
of the frozen ovary inside the cortex of the remaining irradiated ovary was carried out adopting a ‘‘sowing’’ procedure,
which did require suturing. Rams were used to impregnate
the ewes six months after grafting. By this novel procedure,
they documented that prevention of ischemic damage was
successful via intracortical grafting of the germinative ovarian tissue [72]. Transplantation of ovarian tissue into angiogenic granulation tissue during the process of wound healing
was improvised to help prevent the initial ischemic insult to
oocytes. The duration of ischemia was reduced by 24 hours
thereby significantly increasing the healthy primordial follicular pool and the reperfused area of the transplanted
grafts. Two days after transplantation, the graft became revascularized (functional blood vessels were seen) and functional integrity retuned [73].
INTRODUCTION OF
ANIMAL MODELS
VASCULAR
GRAFTS
IN
As a potential way of avoiding ischemic damage to the
graft, vascularized grafts have been successfully developed.
With these grafts, there is immediate revascularization of the
grafted ovarian tissue. The principle of vascularized grafting
was challenging in two distinctive perspectives, despite its
theoretical plausibility. First, the whole ovary along with the
vascular pedicle must be cryopreserved, which is technical
challenging.Second, the graft is reattached using microvascular anastomosis, which is a time-consuming and skilldemanding procedure. Reproductive surgeons are, therefore,
required to be skillful at microvascular anastomosis; experience can readily be obtained from vascular and plastic surgeons with their vast experience in microsurgery on small
vessels.
The ‘ischemia time’ is defined as the duration of time in
which the grafted ovarian tissue can withstand ischemia
without resultant ischemic tissue damage. Large ovarian cortical strips were reported to withstand an ischemic insult for
variable durations [74] without inducing significant histological or molecular changes [75]. Transplanting an intact
fresh ovary together with its vascular pedicle using microvascular reanastomosis has been successfully performed.
As with other tissue grafting procedures, if the vascular graft
was technically feasible and successful, survival of the ovarian graft was assured [76].
Preservation of ovarian function after grafting is all important. Restoration of ovarian function after transplantation
of a cryopreserved intact sheep ovary with its vascular pedicle has been documented [77]. Consequently, transplantation
of fresh as well as cryopreserved-thawed (C-T) intact ovaries
to heterotopic sites was established as a relatively simple,
easily accessible, and technically feasible procedure with a
short operative time [77]. Moreover, the intricate procedure
of orthotopic autotransplantation of an intact frozen-thawed
281
ovary to the upper genital tract using conventional microvascular principles has also been documented in rats [78].
A number of different techniques of autotransplantation
of an intact fresh or frozen-thawed ovary together with its
vascular pedicle using microvascular anastomosis in animal
models were recently published. Special attention was directed towards identifying potential heterotopic locations
containing adequate blood vessels that can be used to host
and vascularize human ovarian grafts [79]. Moving one step
further, contralateral orthotopic autotransplantation of cryopreserved whole ovaries with microanastomosis of the ovarian vascular pedicle has been successfully performed with
very promising results, emphasizing the feasibility, practicality and reproducibility of that principle. Intriguingly, luteal
function was demonstrated in four sheep, and delivery of a
healthy lamb less than two years after transplantation following spontaneous intercourse has recently been reported.
Unfortunately, histological verification of ovarian tissue 18
to 19 months after transplantation has shown that the life
span of the graft was short, and the follicular survival rate in
the grafted ovaries was only 1.7% to 7.6% [80]. Despite a
functioning vascular anastomosis and fertility, the grafts still
had a limited survival that could only be maintained for a
finite length of time. Apart from decreased graft longevity,
the concept of whole organ cryopreservation became established as a promising new popular fertility preserving option.
In the same context, recent models for cryopreservation of
an intact uterus [81] as well as a whole rabbit ovary have
been documented [82].
THE USE OF VASCULARIZED GRAFTS IN HUMAN
TRIALS
A number of human trials using cryopreserved autotransplanted ovarian cortical strips have been carried out, based
on previous successes with animal models using C-T ovarian
cortical strips with reported follicular survival, preservation
of endocrine function as well as restoration of fertility [70,
71].
Many techniques can be adopted to make use of the
cryopreserved ovarian tissue: transplantation back into the
host; in vitro maturation (IVM) of primordial follicles, and
xenografting into a host animal. Considering the first principle, cryopreserved ovarian tissue may be transplanted back
into patient, with the obvious limitation of reintroducing
cancer in malignancies known to preferentially involve the
ovaries such as leukemias and possibly breast cancer. Ovarian tissue strips are removed from the women and kept frozen in small strips prior to chemotherapy using novel techniques. When pregnancy is desired, the ovarian tissue strips
are retransplanted into the patient in an orthotopic or heterotopic site. Given the fact that such ovarian tissue grafts are
avascular, any ischemic insult to the transplanted tissue
would invariably result in irreversible loss of the whole
growing follicle population together with a significant number of primordial follicles.
Three different surgical techniques of transplanting ovarian cortical strips have been improvised by Oktay and associates [83]. Those are orthotopic transplantation into the pelvis and heterotopic transplantation either into the arm or abdominal wall. In one study, the orthotopic transplant stopped
functioning nine months after transplantation [83]. In the
heterotopic transplant, however, function was preserved and
resulted in the generation of a four-cell stage embryo that
was transferred without pregnancy occurring. Multiple ovarian stimulation cycles were undertaken before this one embryo was obtained [84].
A 32-year-old woman from Belgium treated for Hodgkin’s lymphoma was reportedly the first lady to give birth to
a baby after successful orthotopic autotransplantation of
cryopreserved ovarian tissue obtained prior to the start of
ovariotoxic chemotherapy. Despite developing chemotherapy-induced POF, the reimplantation of her ovarian tissue
was successful in resuming ovulatory activity five months
later. She became pregnant 11 months after retransplantation
by natural fertilization and gave birth to a healthy baby 7
years after ovarian tissue banking [85].
Another pregnancy was reported in a woman with nonHodgkin’s lymphoma in which a refined IVF stimulation
protocol was used following orthotopic autotransplantation
of cryopreserved-thawed ovarian cortical strips [86]. Some
critics were doubtful as to the exact site of origin of the oocytes that resulted in the two pregnancies in the abovementioned reports. The reason for such skepticism was based on
reports of documented spontaneous pregnancies in women
with POF after repeated courses of ovariotoxic radiotherapy
and/or chemotherapy and the fact that the tissue transplanted
to the native ovary does not rule out the chance of resumption of native ovarian activity [24].
A recognized complication of ovarian tissue cryopreservation and transplantation is loss of a considerable part of the
follicular apparatus during the initial post-transplantation
ischemic insult, which can seriously limit its use. After
transplantation, almost 70% of follicles are lost. Freezing is
not the major cause of such follicular loss, though [70, 87,
88]. Therefore, ovarian tissue freezing has been recommended only for women under the age of 35 [83]. Proposed
sites of ovarian tissue transplantation with and without vascular anastomosis were reported previously by us and others.
HETEROLOGOUS OVARIAN TRANSPLANTATION
Silber and colleagues reported on the success of ovarian
transplantation from a healthy fertile 24-year-old woman to
her monozygotic twin sister, who had suffered from POF at
the young age of 14. Monozygotic twins with discordant
ovarian function have provided the basis of this work. Via
minilaparotomy, ovarian cortical tissue was transplanted
from the fertile sister to her sterile twin sister. Resumption of
normal menstrual cyclicity was witnessed three months after
transplantation, with a consequent drop of serum gonadotropin levels back to normal levels. During the second cycle,
she conceived, and the pregnancy progressed uneventfully
until delivery of a healthy female baby at 38 weeks’ gestation [89]. Such an outstanding success only helps to emphasize the concept of transplanting large segments of ovarian
tissue between monozygotic twins without the need for immunosuppression.
The same group recently reported on ovarian cortex
transplantation in a cohort of seven sets of twins discordant
for POF in which ovarian cortical tissue was transplanted
Nasr and Bedaiwy
from the twin with normal ovarian function to the sister with
POF. Similar to their previous reports, folliculogenesis, hormonal functions, and menses were restored in all recipients.
Five spontaneous pregnancies were documented in this cohort [90, 91]. Donnez and associates [92] have recently reported on a successful ovarian allograft between two nonidentical twins. Ovarian cortical tissue from the donor sister
who had already been the donor of bone marrow for a transplant was used. The recipient had received chemotherapy,
total body radiation, and bone marrow transplantation. The
recipient developed spontaneous cycles after receiving the
transplant. Moreover, two oocytes and two embryos were
obtained [90, 92]. However, in most patients with an intact
immune system, the potential for acute graft rejection and
risks of long-term immunosuppressive complications in the
mother such as infection and obstetrical complications may
seriously limit its use [90]. Following documented ovarian
function in a small number of cases following both orthotopic and heterotopic transplantation of thawed ovarian cortical strips, the ASRM practice committee recommended that
the procedures of ovarian tissue cryopreservation or transplantation are to be regarded as experimental and should be
performed only under IRB surveillance [57].
Del Priore et al. performed uterine extirpation during a
multiorgan retrieval from a cadaver and demonstrated the
technical feasibility in eight donors. A number of vascular
pedicles were utilized including the ovarian, uterine, or internal iliac vessels. Serial histology sections throughout the
period of cold ischemia, taken every 15 to 30 minutes,
showed no significant change over 12 hours of cold ischemia. They concluded that the human uterus can be obtained
from local organ donor networks using existing protocols
[93]. Since a multitude of methodological and technical difficulties are associated with this procedure, the technique of
human uterus transplantation is more experimental than
practical at this point.
IN VITRO MATURATION OF OOCYTES (IVM)
The rationale behind in vitro maturation (IVM) is the fact
the primordial follicles obtained from the frozen-thawed
ovarian cortical strips can be allowed to mature in vitro.
However, this procedure is still under investigation and will
only become available in the future. In severe combined immune deficiency mice (SCID), transplantation studies demonstrated follicular maturation and completion of meiosis I
in preparation for ovulation and potential fertilization [94].
Concerns have been raised pertaining to technical problems
and potential viral infection transmission. To avoid spreading malignant cells, ovarian tissue culture with in vitro follicle maturation can be performed. Isolated follicle culture
from the primordial stage has been tried, given the fact that
the primordial cells represent >90% of the total follicular
pool and by virtue of their ability to withstand cryoinjury
[95]. However, isolated primordial follicles do not mature
properly in culture [96]. Originally described for patients
with polycystic ovary syndrome (PCOS), IVM of antral follicles may have applications for cancer patients. Unfortunately, its implementation is still being studied. IVM of
oocytes could be considered a fertility preservation strategy
as well; it is a safe and effective treatment offered in some
fertility centers for assisted reproduction. Potential advantages include avoidance of ovarian stimulation with expensive, and at times dangerous, gonadotropins, side effects of
the medications, and risks such as ovarian hyperstimulation
syndrome (OHSS).
Although primary candidates for IVM of oocytes have
classically been PCOS patients with multiple antral follicles,
the spectrum of IVM indications is expanding to include
women with primarily poor-quality embryos in repeated
cycles and poor responders to stimulation. Two new applications for IVM, especially in women with cancer who are
undergoing ovariotoxic therapy, are oocyte donation and
fertility preservation. It is combined with oocyte vitrification
in younger women without partners needing this treatment
for fertility preservation. Clinical pregnancy rates per cycle
in women choosing IVM are related to their age; approximately 38% for infertility treatment up to the age of 30 and
around 50% in recipients of IVM egg donation.
The issue of clinical applicability and practical value of
IVM as a fertility preservation strategy is still unsettled
[97,98]. Xu et al. improvised a method using tissue engineering principles for the culture of immature ovarian follicles
followed by fertilization of oocytes in vitro [99]. This methodology is a great step forward in the search for new fertility
preservation options in female cancer patients [99-101]. An
additional strategy of fertility preservation, which combines
ovarian tissue cryobanking with retrieval of immature oocytes from excised ovarian tissue, followed by IVM and vitrification has recently been described [102]. Recently, the
first healthy baby was delivered following retrieval of immature oocytes in a natural menstrual cycle, followed by IVM
and cryopreservation of the oocytes by vitrification [103].
This provides proof-of-principle evidence that the novel fertility preservation strategy of immature oocyte retrieval,
IVM, and vitrification of oocytes can lead to a successful
pregnancy and healthy live birth [103].
collection, optimal tissue size, choice of cryoprotectants and
cryopreservation protocols, and possible IVM of oocytes for
human ovarian tissue.
In addition, research is needed to enhance the revascularization process with the goal of minimizing the follicular
loss that occurs after tissue grafting. Appropriate current
experimental indications primarily focus on providing an
alternative for women who will immediately face near term
medical therapies that clearly threaten their future fertility
[57]. In humans, it is essential to generate active research to
fill the currently available gaps and to optimize the results of
cryopreservation and ovarian tissue transplantation. Of particular importance is the ideal transplantation site, which
should be easily accessible via a simple and minimally invasive surgical procedure. Graft establishment, survival, and
long-term function depend on adequate perfusion ensured by
a generous blood supply to the recipient site. Development
of new cryopreservation strategies, novel cryoprotectants,
and new transplantation techniques tailored to decrease
ischemia, particularly the use of vascularized grafts, are
needed [76-78].
In IVM, future research is necessary to define the prerequisites of adequate follicular growth and maturation and to
decipher the exact role of supporting theca and granulosa
cells in this process.
KEY ISSUES
1.
There is increasing need for fertility preservation in
women for a number of benign conditions and malignant diseases.
2.
Exposure to ovariotoxic chemotherapy and/or radiotherapy can seriously jeopardize future female fertility.
3.
Despite the multiplicity of potential fertility preservation strategies, only few are of practical significance
and the majority remains experimental.
4.
Only in vitro fertilization followed by embryo
cryopreservation is considered a clinically proven
option for women requiring cancer treatment.
5.
There is generally insufficient time available to allow
for ovarian stimulation, oocyte retrieval, and embryo
freezing for women who are scheduled to receive
ovariotoxic chemotherapy and/or radiotherapy.
6.
Despite being largely experimental, ovarian tissue
cryopreservation and oocyte cryopreservation are
promising avenues for future fertility preservation in
women.
7.
In a small number of cases, ovarian function has been
reported following both orthotopic and heterotopic
transplantation of thawed ovarian cortical strips.
8.
Ovarian tissue cryopreservation and transplantation
and oocyte cryopreservation are to be envisaged as
experimental procedures that can only be performed
under institutional review board (IRB) guidance.
9.
In IVF, improved oocyte survival, oocyte fertilization, and pregnancy rates from frozen-thawed oocytes
EXPERT COMMENTARY
Most of the currently available strategies to preserve fertility in women are still experimental and do not guarantee
subsequent fertility. The only established method in women
is IVF with embryo cryopreservation prior to cancer therapy.
Other proposed strategies to preserve fertility in women with
cancer include: [1] storage of frozen embryos, [2] storage of
frozen ovarian tissue or the whole ovary for future transplantation, [3] storage of isolated follicles for in vitro growth and
maturation, [4] and ovarian transposition before radiotherapy. The effectiveness of ovarian protection during chemotherapy with GnRH analogs is yet to be shown.
FIVE-YEAR VIEW
Development of more successful strategies for fertility
preservation among cancer survivors remains a challenging
problem. Currently, ovarian tissue cryopreservation and
transplantation are considered experimental. Future research
recruiting larger numbers of patients may help determine
whether acceptable longevity can be achieved with both pelvic and forearm ovarian cortical transplant procedures and
whether fertility can be reliably restored. Research should
focus on better defining patient suitability, methods of tissue
283
are the result of dramatic achievements in various
laboratory techniques.
10.
To date, no increase in chromosomal abnormalities,
birth defects, or developmental deficits was witnessed
in pregnancies resulting from cryopreserved oocytes.
11.
Fertility preservation is a new application for IVM,
especially in women with cancer who are undergoing
ovariotoxic therapy. Tissue engineering principles for
the culture of immature ovarian follicles followed by
fertilization of oocytes in vitro is an emerging new
technology for fertility preservation in female cancer
patients.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Signorello LB, Cohen SS, Bosetti C, et al. Female survivors of
childhood cancer: preterm birth and low birth weight among their
children. J Natl Cancer Inst 2006; 98(20): 1453-61.
Ethics Committee of the American Society for Reproductive
Medicine. Fertility preservation and reproduction in cancer
patients. Fertil Steril 2005; 83(6): 1622-8.
Block E. A quantitative morphological investigation of the follicular system in newborn female infants. Acta Anat (Basel) 1953;
17(3): 201-6.
Forabosco A, Sforza C, De Pol A, Vizzotto L, Marzona L, FerrarioVF. Morphometric study of the human neonatal ovary. Anat Rec
1991; 231(2): 201-8.
Gougeon A. Regulation of ovarian follicular development in
primates: facts and hypotheses. Endocr Rev 1996; 17(2): 121-55.
Fauser BC, Van Heusden AM. Manipulation of human ovarian
function: physiological concepts and clinical consequences. Endocr
Rev 1997; 18(1): 71-106.
Wandji SA, Srsen V, Voss AK, Eppig JJ, Fortune JE. Initiation
in vitro of growth of bovine primordial follicles. Biol Reprod 1996;
55(5): 942-8.
Oktay K, Briggs D, Gosden RG. Ontogeny of follicle-stimulating
hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 1997; 82(11): 3748-51.
Ahmed CE, Dees WL, Ojeda SR. The immature rat ovary is innervated by vasoactive intestinal peptide (VIP)-containing fibers and
responds to VIP with steroid secretion. Endocrinology 1986;
118(4): 1682-9.
Ezoe K, Holmes SA, Ho L, et al. Novel mutations and deletions of
the KIT (steel factor receptor) gene in human piebaldism. Am J
Hum Genet 1995; 56(1): 58-66.
Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM.
Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol 1999; 13(6): 1035-48.
Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk
MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 1996; 383(6600): 531-5.
Elvin JA, Yan C, Wang P, Nishimori K, Matzuk MM. Molecular
characterization of the follicle defects in the growth differentiation
factor 9-deficient ovary. Mol Endocrinol 1999; 13(6): 1018-34.
Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM.
The bone morphogenetic protein 15 gene is X-linked and expressed
in oocytes. Mol Endocrinol 1998; 12(12): 1809-17.
Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL. Germline stem
cells and follicular renewal in the postnatal mammalian ovary. Nature 2004; 428(6979): 145-50.
Johnson J, Bagley J, Skaznik-Wikiel M, et al. Oocyte generation in
adult mammalian ovaries by putative germ cells in bone marrow
and peripheral blood. Cell 2005; 122(2): 303-15.
Gosden RG. Germline stem cells in the postnatal ovary: is the
ovary more like a testis? Hum Reprod Update 2004; 10(3): 193-5.
Telfer EE. Germline stem cells in the postnatal mammalian ovary:
a phenomenon of prosimian primates and mice? Reprod Biol
Endocrinol 2004; 2: 24.
Telfer EE, Gosden RG, Byskov AG, et al. On regenerating the
ovary and generating controversy. Cell 2005; 122(6): 821-2.
Manger K, Wildt L, Kalden JR, Manger B. Prevention of gonadal
toxicity and preservation of gonadal function and fertility in young
Nasr and Bedaiwy
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
women with systemic lupus erythematosus treated by cyclophosphamide: the PREGO-Study. Autoimmun Rev 2006; 5(4): 269-72.
Schilsky RL, Sherins RJ, Hubbard SM, Wesley MN, Young RC,
DeVita VT. Long-term follow up of ovarian function in women
treated with MOPP chemotherapy for Hodgkin’s disease. Am J
Med 1981; 71(4): 552-6.
Blumenfeld Z, Avivi I, Linn S, Epelbaum R, Ben-Shahar M, Haim
N. Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a gonadotrophin-releasing
hormone agonist in parallel to chemotherapy. Hum Reprod 1996;
11(8): 1620-6.
BFS. MWGcbt. A strategy for fertility services for survivors of
childhood cancer. Hum Fertil 2003; 6: A1-40.
Bath LE, Tydeman G, Critchley HO, Anderson RA, Baird DT,
Wallace WH. Spontaneous conception in a young woman who had
ovarian cortical tissue cryopreserved before chemotherapy and
radiotherapy for a Ewing’s sarcoma of the pelvis: case report. Hum
Reprod 2004; 19(11): 2569-72.
Meirow D, Nugent D. The effects of radiotherapy and chemotherapy on female reproduction. Hum Reprod Update 2001; 7(6): 53543.
Husseinzadeh N, Nahhas WA, Velkley DE, Whitney CW, Mortel
R. The preservation of ovarian function in young women undergoing pelvic radiation therapy. Gynecol Oncol 1984; 18(3): 373-9.
Gaetini A, De Simone M, Urgesi A, et al. Lateral high abdominal
ovariopexy: an original surgical technique for protection of the
ovaries during curative radiotherapy for Hodgkin_s disease. J Surg
Oncol 1988; 39(1): 22-8.
Williams RS, Littell RD, Mendenhall NP. Laparoscopic
oophoropexy and ovarian function in the treatment of Hodgkin disease. Cancer 1999; 86(10): 2138-42.
Meirow D, Schenker JG, Rosler A. Ovarian hyperstimulation
syndrome with low oestradiol in non-classical 17 alphahydroxylase,17,20-lyase deficiency: what is the role of oestrogens?
Hum Reprod 1996; 11(10): 2119-21.
Gosden RG, Wade JC, Fraser HM, Sandow J, Faddy MJ. Impact of
congenital or experimental hypogonadotrophism on the radiation
sensitivity of the mouse ovary. Hum Reprod 1997; 12(11): 2483-8.
Wallace WH, Thomson AB, Kelsey TW. The radiosensitivity of
the human oocyte. Hum Reprod 2003; 18(1): 117-21.
Bath LE, Wallace WH, Shaw MP, Fitzpatrick C, Anderson RA..
Depletion of ovarian reserve in young women after treatment for
cancer in childhood: detection by anti-Mullerian hormone, inhibin
B and ovarian ultrasound. Hum Reprod 2003; 18(11): 2368-74.
Crofton PM, Thomson AB, Evans AE, et al. Is inhibin B a potential
marker of gonadotoxicity in prepubertal children treated for cancer? Clin Endocrinol (Oxf) 2003; 58(3): 296-301.
Feyereisen E, Mendez Lozano DH, Taieb J, Hesters L, Frydman R,
Fanchin R. Anti-Mullerian hormone: clinical insights into a promising biomarker of ovarian follicular status. Reprod Biomed Online
2006; 12(6): 695-703.
Weir HK, Thun MJ, Hankey BF, et al. Annual report to the nation
on the status of cancer, 1975-2000, featuring the uses of surveillance data for cancer prevention and control. J Natl Cancer Inst
2003; 95(17): 1276-99.
Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ.
Cancer statistics, 2003. CA Cancer J Clin 2003; 53(1): 5-26.
Waggoner SE. Cervical cancer. Lancet 2003; 361: 2217-25.
Chiarelli AM, Marrett LD, Darlington G. Early menopause and
infertility in females after treatment for childhood cancer diagnosed
in 1964-1988 in Ontario, Canada. Am J Epidemiol 1999; 150(3):
245-54.
Tangir J, Zelterman D, Ma W, Schwartz PE. Reproductive function
after conservative surgery and chemotherapy for malignant germ
cell tumors of the ovary. Obstet Gynecol 2003; 101(2): 251-7.
Blumenfeld Z, Dann E, Avivi I, Epelbaum R, Rowe JM. Fertility
after treatment for Hodgkin’s disease. Ann Oncol 2002;
13(Suppl.1): 138-47.
Pereyra Pacheco B, Mendez Ribas JM, Milone G, et al. Use of
GnRH analogs for functional protection of the ovary and preservation of fertility during cancer treatment in adolescents: a preliminary report. Gynecol Oncol 2001; 81(3): 391-7.
Fox KR Ball JE, Mik R, Moore HC. Prevention of chemotherapy
associated amenorrhea (CRA) with leuprolide in young women
with early stage breast cancer (Abstract). Proc Ann Soc Clin Oncol
2001; 1(20): 25a.
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
Waxman JH, Ahmed R, Smith D, et al. Failure to preserve fertility
in patients with Hodgkin’s disease. Cancer Chemother Pharmacol
1987; 19(2): 159-62.
Howell SJ, Shalet SM. Fertility preservation and management of
gonadal failure associated with lymphoma therapy. Curr Oncol Rep
2002; 4(5): 443-52.
Covens AL, van der Putten HW, Fyles AW, et al. Laparoscopic
ovarian transposition. Eur J Gynaecol Oncol 1996; 17(3): 177-82.
Hadar H, Loven D, Herskovitz P, Bairey O, Yagoda A, Levavi H.
An evaluation of lateral and medial transposition of the ovaries out
of radiation fields. Cancer 1994; 74(2): 774-9.
Howard FM. Laparoscopic lateral ovarian transposition before
radiation treatment of Hodgkin disease. J Am Assoc Gynecol
Laparosc 1997; 4(5): 601-4
Anderson B, LaPolla J, Turner D, Chapman G, Buller R. Ovarian
transposition in cervical cancer. Gynecol Oncol 1993; 49(2): 20614.
Treissman MJ, Miller D, McComb PF. Laparoscopic lateral ovarian transposition. Fertil Steril 1996; 65(6): 1229-31.
Yarali H, Demirol A, Bukulmez O, Coskun F, Gurgan T.
Laparoscopic high lateral transposition of both ovaries before pelvic irradiation. J Am Assoc Gynecol Laparosc 2000; 7(2): 237-9.
Clough KB, Goffinet F, Labib A, et al. Laparoscopic unilateral
ovarian transposition prior to irradiation: prospective study of 20
cases. Cancer 1996; 77(12): 2638-45.
Feeney DD, Moore DH, Look KY, Stehman FB, Sutton GP. The
fate of the ovaries after radical hysterectomy and ovarian transposition. Gynecol Oncol 1995; 56(1): 3-7.
Chambers SK, Chambers JT, Holm C, Peschel RE, Schwartz PE.
Sequelae of lateral ovarian transposition in unirradiated cervical
cancer patients. Gynecol Oncol 1990; 39(2): 155-9.
Magistrini M, Szollosi D. Effects of cold and of isopropylNphenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980; 22(2): 699-707.
Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium
during mouse oocyte cryopreservation. Biol Reprod 1998; 59(2):
395-400.
Porcu E, Fabbri R, Damiano G, Fratto R, Giunchi S, Venturoli S.
Oocyte cryopreservation in oncological patients. Eur J Obstet Gynecol Reprod Biol 2004; 113 (Suppl. 1): S14-16.
Ovarian tissue and oocyte cryopreservation. Fertil Steril 2008; 90:
S241-6.
Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a
meta-analysis. Fertil Steril 2006; 86(1): 70-80.
Jain JK, Paulson RJ. Oocyte cryopreservation. Fertil Steril 2006;
86(Suppl 4): 1037-46.
Assisted reproductive technology in the United States: 1998 results
generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil
Steril 2002; 77(1): 18-31.
Wang JX, Yap YY, Matthews CD. Frozen-thawed embryo transfer:
influence of clinical factors on implantation rate and risk of multiple conception. Hum Reprod 2001; 16(11): 2316-19.
Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron
microscopy grids after induced collapse of the blastocoele. Hum
Reprod 2003; 18(1): 137-9.
Meniru GI, Craft I. In vitro fertilization and embryo cryopreservation prior to hysterectomy for cervical cancer. Int J Gynaecol
Obstet 1997; 56(1): 69-70.
Pena JE, Chang PL, Chan LK, Zeitoun K, Thornton MH 2nd, Sauer
MV. Supraphysiological estradiol levels do not affect oocyte and
embryo quality in oocyte donation cycles. Hum Reprod 2002;
17(1): 83-7.
Pelinck MJ, Hoek A, Simons AH, Heineman MJ. Efficacy of natural cycle IVF: a review of the literature. Hum Reprod Update 2002;
8(2): 129-39.
Oktay K, Buyuk E, Davis O, Yermakova I, Veeck L, Rosenwaks Z.
Fertility preservation in breast cancer patients: IVF and embryo
cryopreservation after ovarian stimulation with tamoxifen. Hum
Reprod 2003; 18(1): 90-5.
Oktay K, Buyuk E, Akar Z, Rosenwaks N, Libertella N. Fertility
preservation in breast cancer patients: a prospective controlled
comparison of ovarian stimulation with tamoxifen and letrozole for
embryo cryopreservation. Fertil Steril 2004; 82(2): s1(Abstract).
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
285
Oktay K. Further evidence on the safety and success of ovarian
stimulation with letrozole and tamoxifen in breast cancer patients
undergoing in vitro fertilization to cryopreserve their embryos for
fertility preservation. J Clin Oncol 2005; 23(16): 3858-9.
Mazur P. The role of intracellular freezing in the death of cells
cooled at supraoptimal rates. Cryobiology 1977; 14(3): 251-72.
Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility
to oophorectomized sheep by ovarian autografts stored at -196
degrees C. Hum Reprod 1994; 9(4): 597-603.
Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG.
Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at 196 C. Endocrinology 1999;
140(1): 462-71.
Almodin CG, Minguetti-Camara VC, Meister H, Ceschin AP,
Kriger E, Ferreira JO. Recovery of natural fertility after grafting of
cryopreserved germinative tissue in ewes subjected to radiotherapy.
Fertil Steril 2004; 81(1): 160-4.
Israely T, Nevo N, Harmelin A, Neeman M, Tsafriri A. Reducing
ischaemic damage in rodent ovarian xenografts transplanted into
granulation tissue. Hum Reprod 2006; 21(6): 1368-79.
Jeremias E, Bedaiwy MA, Nelson D, Biscotti CV, Falcone T.
Assessment of tissue injury in cryopreserved ovarian tissue. Fertil
Steril 2003; 79(3): 651-3.
Hussein MR, Bedaiwy MA, Falcone T. Analysis of apoptotic
cell death, Bcl-2, and p53 protein expression in freshly fixed and
cryopreserved ovarian tissue after exposure to warm ischemia. Fertil Steril 2006; 85 (Suppl. 1): 1082-92.
Jeremias E, Bedaiwy MA, Gurunluoglu R, Biscotti CV, Siemionow
M, Falcone T. Heterotopic autotransplantation of the ovary with
microvascular anastomosis: a novel surgical technique. Fertil Steril
2002; 77(6): 1278-82.
Bedaiwy MA, Jeremias E, Gurunluoglu R, et al. Restoration of
ovarian function after autotransplantation of intact frozenthawed
sheep ovaries with microvascular anastomosis. Fertil Steril 2003;
79(3): 594-602.
Wang X, Chen H, Yin H, Kim SS, Lin Tan S, Gosden RG.
Fertility after intact ovary transplantation. Nature 2002; 415(6870):
385.
Bedaiwy MA, Falcone T. Harvesting and autotransplantation of
vascularized ovarian grafts: approaches and techniques. RBM Online 2007; 14(3): 360-71.
Imhof M, Bergmeister H, Lipovac M, Rudas M, Hofstetter G,
Huber J. Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth. Fertil
Steril 2006; 85(Suppl. 1): 1208-15.
Dittrich R, Maltaris T, Mueller A, et al. Successful uterus cryopreservation in an animal model. Horm Metab Res 2006; 38(3): 1415.
Chen CH, Chen SG, Wu GJ, Wang J, Yu CP, Liu JY. Autologous
heterotopic transplantation of intact rabbit ovary after frozen
banking at 196 degrees C. Fertil Steril 2006; 86(Suppl. 4): 105966.
Sonmezer M, Oktay K. Fertility preservation in female patients.
Hum Reprod Update 2004; 10(3): 251-66.
Oktay K, Buyuk E, Veeck L, et al. Embryo development after
heterotopic transplantation of cryopreserved ovarian tissue. Lancet
2004; 363(9412): 837-40.
Donnez J, Dolmans MM, Demylle D, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;
364(9443): 1405-10.
Meirow D, Levron J, Eldar-Geva T, et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian
failure after chemotherapy. N Engl J Med 2005; 353(3): 318-21.
Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG.
Isolation and characterization of primordial follicles from fresh and
cryopreserved human ovarian tissue. Fertil Steril 1997; 67(3): 4816.
Aubard Y. Ovarian tissue graft: from animal experiment to practice
in the human. Eur J Obstet Gynecol Reprod Biol 1999; 86(1): 1-3.
Silber SJ, Lenahan KM, Levine DJ, et al. Ovarian transplantation
between monozygotic twins discordant for premature ovarian failure. N Engl J Med 2005; 353(1): 58-63.
Bedaiwy MA, Shahin AY, Falcone T. Reproductive organ transplantation: advances and controversies. Fertil Steril 2008; 90(6):
2031-55.
[91]
[92]
[93]
[94]
[95]
[96]
Nasr and Bedaiwy
Silber SJ, Gosden RG. Ovarian transplantation in a series of
monozygotic twins discordant for ovarian failure. N Engl J Med
2007; 356: 1382-4.
Donnez J, Dolmans MM, Pirard C, et al. Allograft of ovarian
cortex between two genetically non-identical sisters: Case Report.
Hum Reprod 2007; 22(10): 2653-9.
Del Priore G, Stega J, Sieunarine K, Ungar L, Smith JR. Human
uterus retrieval from a multi-organ donor. Obstet Gynecol 2007;
109(1): 101-4.
Gook DA, Edgar DH, Borg J, Archer J, Lutjen PJ, McBain JC.
Oocyte maturation, follicle rupture and luteinization in human
cryopreserved ovarian tissue following xenografting. Hum Reprod
2003; 18(9): 1772-81.
Smitz JE, Cortvrindt RG. The earliest stages of folliculogenesis in
vitro. Reproduction 2002; 123(2): 185-202.
Abir R, Fisch B, Nitke S, Okon E, Raz A, Ben Rafael Z. Morphological study of fully and partially isolated early human follicles.
Fertil Steril 2001; 75(1): 141-6.
[97]
[98]
[99]
[100]
[101]
[102]
[103]
Rao GD, Chian RC, Son WS, Gilbert L, Tan SL. Fertility preservation in women undergoing cancer treatment. Lancet 2004;
363(9423): 1829-30.
Rao GD, Tan SL. In vitro maturation of oocytes. Semin Reprod
Med 2005; 23(3): 242-7.
Xu M, Kreeger PK, Shea LD, Woodruff TK. Tissue-engineered
follicles produce live, fertile offspring. Tissue Eng 2006; 12: 273946.
Brännström M, Milenkovic M. Advances in fertility preservation
for female cancer survivors. Nat Med 2008; 14: 1182 - 4.
Kim SS. Fertility preservation in female cancer patients: current
developments and future directions. Fertil Steril 2006; 85: 1-11.
Huang JY, Tulandi T, Holzer H, Tan SL, Chian RC. Combining
ovarian tissue cryobanking with retrieval of immature oocytes followed by in vitro maturation and vitrification: an additional strategy of fertility preservation. Fertil Steril 2008; 89: 567-72.
Chian RC, Gilbert L, Huang JY, et al. Live birth after vitrification
of in vitro matured human oocytes. Fertil Steril 2009; 91: 372-6.

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