Best Practice & Research Clinical Obstetrics and Gynaecology 26 (2012) 777788

Contents lists available at SciVerse ScienceDirect

Best Practice & Research Clinical

Obstetrics and Gynaecology
journal homepage: www.elsevier.com/locate/bpobgyn

In vitro fertilisation treatment and factors affecting success

Jack Yu Jen Huang, M.D., Ph.D., Zev Rosenwaks, M.D. *
The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College of Cornell University,
1305 York Avenue, 7th Floor, New York, NY, USA

Keywords:
assisted reproductive technology (ART)
in vitro fertilization (IVF)
ovarian reserve
follicle stimulating hormone (FSH)
anti-Mullerian hormone (AMH)
controlled ovarian hyperstimulation (COH)
intracytoplasmic sperm injection (ICSI)
embryo transfer
ovarian hyperstimulation syndrome (OHSS)

The efcacy of assisted reproductive technologies has improved

signicantly over the past decades. The main indications for in
vitro fertilisation include tubal obstruction, severe male-factor
infertility, severe endometriosis, ovulatory dysfunction, diminished ovarian reserve, and infertility of unexplained cause. In vitro
fertilisation has also become an effective treatment option for
couples wishing to undergo pre-implantation genetic diagnosis or
screening, and for those wishing to cryopreserve their oocytes or
embryos for preservation of fertility. The management of women
in late reproductive age poses a major challenge; the optimum in
vitro fertilisation treatment for poor responders remains elusive.
The success of in vitro fertilisation treatment can be optimised by
taking an individualised, patient-centered approach to controlled
ovarian hyperstimulation. Key components involve selection of an
appropriate controlled ovarian protocol, close-cycle monitoring,
adjustment of gonadotropin dosage to avoid hyper-response, and
individualised timing of human chorionic gonadotropin injection.
Future directions of assisted reproductive technologies include
development of non-invasive embryo selection methods, use of
transcriptomics, proteomics, metabolomics, and time-lapse
imaging technologies.
2012 Elsevier Ltd. All rights reserved.

Introduction
The efcacy of assisted reproductive technologies (ART) has improved signicantly since the rst
reports of successful pregnancies and live births after in vitro fertilisation (IVF) by Steptoe and
Edwards1,2 in 1978. In the USA, the live birth rate has increased from 38% to 48% among women under

* Corresponding author. Tel.: 1 646 9623745; Fax: 1 646 9620392.

E-mail address: zrosenw@med.cornell.edu (Z. Rosenwaks).
1521-6934/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bpobgyn.2012.08.017

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the age of 35 years treated with ART over the past decade.3 The European IVF-monitoring consortium
and the International Committee for Monitoring Assisted Reproductive Technology also observed
similar trends in ART success.4,5 Assisted reproductive technologies have also become more accessible.
In 2003, the number of ART cycles carried out worldwide exceeded over 1 million.5 To date, an estimated 3.75 million babies have been born worldwide using ART.6
The most common ART procedure is IVF, which involves controlled ovarian hyperstimulation (COH)
with gonadotropin administration to stimulate ovarian follicle development, followed by transvaginal
oocyte retrieval, fertilisation of the oocytes with sperm in vitro, culture of the resultant embryos, and
transfer of the embryo(s) to the recipient. An important innovation in ART is assisted fertilisation by
intracytoplasmic sperm injection (ICSI).7 Other modalities of ART include cryopreservation of gametes,
embryos, and ovarian tissue, pre-implantation genetic diagnosis (PGD) or screening (PGS), the use of
donor gametes, and gestational carriers.
In this chapter, the indications for IVF and factors affecting IVF success will be reviewed, along with
IVF treatment options for women of advanced maternal age. We will also provide an update on the
recent advancements in, and future directions of, ART.
Indications for in vitro fertilisation
In vitro fertilisation was rst reported as a treatment option for women with severe tubal disease.1,2
With improved efcacy after the introduction of gonadotropin stimulation and ICSI, the indications for
IVF have expanded to include infertility caused by severe male factor, diminished ovarian reserve,
ovulatory dysfunction, severe endometriosis, and infertility of unexplained cause. In vitro fertilisation
also provides a new means of preconception genetic diagnosis and preservation of fertility. In fact, IVF
is the most effective treatment option for couples with multi-factorial infertility problems.
Tubal factor
Tubal-factor infertility accounts for 30% of cases of female infertility.3 Tubal damage has classically
been associated with pelvic inammatory disease, most often with Chlamydia trachomatis and
gonorrhea infections. Other causes of tubal obstruction may be either intrinsic (e.g. ascending
salpingitis and salpingitis isthmica nodosa) or extrinsic (e.g. surgical sterilisation, endometriosis and
peritonitis) in origin.
Before the introduction of IVF, reconstructive tubal surgery was the only treatment option for
women with tubal obstruction. At present, IVF is the treatment of choice for women over the age of 35
years with signicant tubal disease and those with other co-existing infertility problems.8,9 In vitro
fertilisation is also indicated in women who remain infertile for 1 year after tubal surgery.
Male factor
Abnormal semen parameters may be a contributing factor in up to 40% of infertile couples.3,10 In
cases of severe oligospermia (fewer than 5 million motile sperm/ml), severe asthenospermia (less than
5% progressive motility), and severe teratospermia (less than 4% normal morphology based on strict
Kruger criteria), IVF, or a combination of IVF and ICSI, should be offered,11 as these semen parameters
are associated with poor success with articial insemination.12,13 For women with obstructive or nonobstructive azoospermia, IVF with ICSI are indicated to achieve fertilisation using surgically retrieved
spermatozoa from either microsurgical epididymal sperm aspiration14 or testicular sperm extraction.1416
Endometriosis
The incidence of endometriosis is reported to be in the range of 950% among women who
underwent laparoscopy for infertility evaluation.17,18 The exact pathophysiology of endometriosis and
its effects on fertility remain enigmatic. Proposed mechanisms of damage include distortion of adnexal
anatomy and adverse peritoneal environment characterised by increased inammatory cytokines and

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oxidative stress.19 This may interfere with follicular development,20 ovum pick up, fertilisation, and
embryo development.
Women with known or suspected moderate and severe endometriosis (stage IIIIV) may be
treated with either surgery or IVF.21 No prospective, randomised-controlled trial has compared the
efcacy of the two treatment modalities. Surgical treatment is preferred in women who are
symptomatic and those with endometriomas greater than 4 cm.22 In vitro fertilisation is indicated
in women with co-existing causes of infertility, such as tubal obstruction, advanced maternal age,
and abnormal semen parameters.22 A 3-month course of gonadotropin releasing hormone (GnRH)
agonist administered before starting IVF has been shown to improve the ongoing pregnancy
rate.23
Ovulatory dysfunction
Ovulatory dysfunction is a very common cause of female infertility, accounting for 25% of diagnoses.3 In this category, polycystic ovary syndrome (PCOS), characterised by the triad of polycystic
ovaries, oligo- or amenorrhoea, and clinical and biochemical signs of androgen excess, is most
common.24 Ovulatory dysfunction may also be a manifestation of other endocrinopathies, such as
thyroid disorders and hyperprolactinaemia. Other rare causes include congenital adrenal hyperplasia,
adrenal tumours, and androgen-secreting ovarian tumours.
Women with normogonadotropicnormogonadal ovulatory disorders (World Health Organization
group 2), including those with PCOS, can be successfully treated with ovulation induction combined
with timed intercourse or intrauterine insemination (IUI). Ovulation induction is usually successful
after treatment with clomiphene citrate,25 exogenous gonadotropins, aromatase inhibitors (e.g.
letrozole, anastrazole),26 or selective oestrogen receptor modulators (e.g. tamoxifen).27
Women with polycystic ovaries seen on ultrasound, irrespective of whether they harbor other
clinical features of PCOS, have an increased risk of over-responding to gonadotropin stimulation and for
developing ovarian hyperstimulation syndrome (OHSS). The risk of high-order multiple gestations is
also signicantly higher in women who have a high response to gonadotropin stimulation and who
undergo an IUI.28,29 In these settings, conversion to IVF represents a safe and effective alternative to an
IUI or to cycle cancellation.30 In vitro fertilisation is indicated for women who do not conceive after
conventional ovarian-induction treatments,31 and especially for couples with other co-existing infertility factors.
Unexplained infertility
Unexplained infertility is dened as the absence of an identiable cause of infertility despite
a thorough investigation demonstrating tubal patency, normal semen parameters, ovulation, normal
ovarian reserve, and a normal endometrial cavity. The incidence of unexplained infertility ranges from
1030%.32
Treatment options include expectant management, IUI, empiric treatment with clomiphene citrate
and IUI, clomiphene citrate combined with gonadotropins and IUI, and IVF. In vitro fertilisation is the
most effective treatment option for couples with unexplained infertility, resulting in the highest per
cycle pregnancy rate in the shortest time interval.33
An empirical treatment algorithm for couples with unexplained infertility typically involves three
cycles of clomiphene citrate and IUI, followed by three cycles of gonadotropin and IUI, and by IVF if the
patients remain unsuccessful. In a recent, prospective, randomised-controlled trial involving women
with unexplained infertility between the ages of 21 and 39 years, participants were randomised to
either an accelerated treatment algorithm (IVF after three unsuccessful clomiphene citrate and IUI
treatment cycles) or the conventional treatment algorithm (IVF after three unsuccessful clomiphene
citrate and IUI, and three unsuccessful gonadotropin and IUI treatment cycles). The time to pregnancy
was signicantly shorter in the accelerated arm compared with the conventional arm (hazard ratio
1.25; 95% CI, 1.00 to 1.56).34 The accelerated treatment algorithm was also more cost-effective,
compared with the conventional treatment group. Age seems to be the single most important determining factor of success.

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Fertility preservation
In recent years, individuals with cancer and oncologists have been increasingly aware of the effect of
cancer treatment on fertility.35 Women at risk of premature ovarian failure owing to gonadotoxic
chemotherapy or radiation treatment should be offered the possibility of fertility preservation. Women
receiving gonadotoxic treatment for autoimmune disorders, such as systemic lupus erythematosus,
and those at risk of premature ovarian failure caused by chromosomal anomalies, such as mosaic
Turner syndrome, may also benet from fertility preservation.36,37
The only strategy of female fertility preservation recognised by the American Society of Clinical
Oncology and the American Society of Reproductive Medicine is cryopreservation of embryos.38,39
Other fertility-preservation procedures, such as cryopreservation of oocytes and ovarian tissues and
in vitro maturation, have enjoyed increasing success.4042
Pre-implantation genetic diagnosis and screening
Pre-implantation stage embryos can be screened for aneuploidy, genetic diseases, and inherited
chromosome abnormalities, enabling transfer of genetically normal, euploid embryo(s).43,44 A more indepth discussion on pre-implantation genetic diagnosis and pre-genetic screening will be provided in
Chapter 8.
Factors affecting in vitro fertilisation success
Although many attribute IVF success to innovations in the ART laboratory, we believe that the
success of IVF treatment can be optimised by taking an individualised, patient-directed approach in the
management of women undergoing ovarian hyperstimulation. Key components involve selection of
the appropriate COH protocol and gonadotropin dosage, close monitoring of follicular growth and
serum E2 levels, adjustment of gonadotropin dosage to avoid hyper-response, and individualised
timing of human chorionic gonadotropin (hCG) injection. We believe this approach to COH monitoring
improves oocyte and embryo quality, pregnancy and implantation rates, and, most importantly,
minimises the risk of complications, namely OHSS Figs. 13.
Selection of an appropriate controlled ovarian hyperstimulation protocol
A signicant clinical challenge in determining the optimal COH protocol is predicting and managing
variability between women. The central question when designing an IVF protocol is whether the
woman will likely have a good or poor response to exogenous gonadotropin stimulation. We believe
the appropriate IVF protocol should be determined on the basis of an aggregate of patient characteristics and ovarian reserve assessment.
Predictive factors of ovarian response include patient characteristics, such as age, parity, reproductive history, body mass index, and prior response to ART treatment. Endocrine markers of
ovarian reserve, including basal follicle stimulating hormone (FSH), oestradiol (E2), inhibin B, and,
more recently, anti-Mllerian hormone (AMH) levels,4551 are also useful in distinguishing good and
poor responders. Ultrasonographic assessments of the ovary, including antral follicle count (AFC),
ovarian volume, and ovarian blood ow, have also been extensively evaluated as potential markers
of ovarian reserve.52 Dynamic evaluation of ovarian reserve, including clomiphene citrate challenge
testing, GnRH agonist stimulation testing, and exogenous FSH ovarian test, may be helpful.5355 A
recent meta-analysis56 concluded that all the dynamic ovarian reserve tests have limited predictive
values.
Anti-Mllerian hormone and AFC seem to be the two most accurate predictors of ovarian response
to COH.50,57 Advantages of AMH over AFC and other endocrine and sonographic markers of ovarian
reserve include consistent serum levels throughout the menstrual cycle and minimal cycle-to-cycle
variability.58 Anti-Mllerian hormone has been shown to be an accurate marker of both hyper- and
poor ovarian response to gonadotropin stimulation. A recent meta-analysis57 found that AMH, using
cut-offs ranging from over 1.59 ng/ml to over 7.00 ng/ml, has an estimated sensitivity of 82% and

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Fig. 1. Gonadotropin ovarian stimulation protocol with gonadotropin releasing hormone antagonists. Pre-treatment with oestradiol,
gonadotropin releasing hormone antagonist or the oral contraceptive pill is dependent on patient characteristics. Dotted line
represents theoretical follicle-stimulating hormone levels. AFC, antral follicle count; AMH, anti-Mllerian hormone; BMI, body mass
index; E2, oestradiol; FSH, follicle stimulating hormone; GnRH, gonadotropin releasing hormone; hCG, human chorionic gonadotropin; hMG, human menopausal gonadotrophins; LH, luteinising hormone; OCP, oral contraceptive pill; US, ultrasound.

specicity of 76% in predicting excessive ovarian response. A recent prospective study of poor
responders found an AMH level of less than 0.99 ug/L to be associated with 100% sensitivity and 73%
specicity in predicting poor response.59 None of the ovarian reserve markers, however, possesses
adequate sensitivity or specicity for predicting pregnancy outcomes.49

Fig. 2. Clomiphene citrate and letrozole gonadotropin ovarian stimulation protocol. Dotted line represents theoretical folliclestimulating hormone levels. AFC, antral follicle count; AMH, anti-Mllerian hormone; BMI, body mass index; CC, clomiphene
citrate; E2, oestradiol; FSH, follicle stimulating hormone; GnRH, gonadotropin releasing hormone; hCG, human chorionic gonadotropin; hMG, human menopausal gonadotrophins; LH, luteinising hormone; US, ultrasound.

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Fig. 3. Modied natural cycle in vitro fertilisation protocol. E2, oestradiol; hCG, human chorionic gonadotropin; hMG, human
menopausal gonadotrophins; GnRH, gonadotropin releasing hormone; US, ultrasound.

Normal responders
Normal responders are characterised by favourable prognostic factors, including, but not limited to,
young maternal age (less than 35 years), normal body mass index, adequate ovarian reserve (AFC
between 6 and 10), normal basal FSH and E2 levels (FSH < 10 mIU/ml, E2 < 75 IUpg/ml), short duration
of subfertility, a history of previous live birth, and previous successful IVF treatment.60
Commonly used COH protocols include the mid-luteal, GnRH agonist down regulation or long
protocol61 and, more recently, the GnRH antagonist COH protocol,62 which is also known as the short
protocol.
High responders
The management of women who are at risk of developing an exaggerated response to COH
represents a formidable challenge. An important consideration is the prevention of OHSS. Women with
polycystic ovaries on ultrasound, even in the absence of other clinical features of PCOS, are at greater
risk of developing OHSS63,64; the incidence of OHSS has been reported to be as high as 30% in this
patient population.6567 Other known risk factors for OHSS include young age, lean body weight, and
a history of OHSS. In women undergoing COH treatment, high gonadotropin doses, high absolute levels
(greater than 3000 pg/ml), and rapidly rising E2 levels also represent risk factors for the development of
OHSS.68
Strategies for the prevention of OHSS include identifying patients at risk, individualisation of COH
protocols, and judicious use of gonadotropins. In this context, the aim of the COH is to decrease ovarian
response, ideally to develop ve to 15 follicles, while maintaining an E2 level of less than 3000 pg/ml.
Two effective COH protocols for high responders are the oral contraceptive pill GnRH agonist dual
suppression protocol69 and the GnRH antagonist protocol in combination with GnRH agonist ovulatory
trigger.7073
Poor responders
Diminished ovarian reserve is relatively common among women undergoing IVF, with reported
prevalence ranging from 1025%.3,74 The variations in the prevalence rate can be attributed to a lack of
a universally accepted denition of what constitutes a poor response. Most denitions of poor response
are based on arbitrary levels of peak serum E2 or oocyte yield. Poor responders have also been dened
by age over 40 years,75 elevated basal FSH levels (over 10 to 15 mIU/L),7578 previous cycle cancellation,

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poor response to clomiphene citrate challenge testing,53 prolonged duration of COH,79 increased daily
and total gonadotropin ampoules used (over 44),78,80 and when less than three to ve oocytes are
retrieved.81
Recently, a European Society of Human Reproduction and Embryology consensus working group
dened poor ovarian response as having at least two of the following three features: (1) advanced
maternal age ( 40 years) or any other risk factor for diminished ovarian reserve; (2) previous
history of poor ovarian response (fewer than three oocytes retrieved with a conventional COH
protocol); and (3) an abnormal ovarian reserve test (AFC less than ve to seven follicles or AMH
more than 0.51.1 ng/ml).82
Given the relationship between advanced maternal age and the decline in fertility,83 it is not
surprising that age is the most important determining factor of success in women undergoing IVF.
Although ART may overcome infertility in younger women, it does not reverse the age-dependent
decline in fertility.84 Advanced maternal age is associated with a decline in the number of oocytes
retrieved, embryos available for transfer, and embryo quality,8587 ultimately resulting in lower
implantation,87 pregnancy, and live birth rates.3 A strong correlation also exists between advanced
maternal age and the incidence of chromosomal anomalies.88 The risk of miscarriage approaches 50%
among women over the age of 40 years.89
The management of women in late reproductive age remains a major challenge. In fact, among
women undergoing IVF for various indications, patients with diminished ovarian reserve seem to have
the worst prognosis.3 In vitro fertilisation treatment among older women is often associated with low
fecundity and poor clinical outcomes. These women also have a high no-start rate owing to abnormally
elevated baseline serum FSH levels and high cycle cancellation rates owing to poor response.
A variety of COH protocols have been published for treating poor responders. Commonly used COH
protocols include Luteal E2 patch and GnRH antagonist protocol,90 co-are and micro-are protocols,9193 clomid and letrozole protocols,9496 and modied natural cycles.97 The optimum IVF treatment strategy remains elusive.98
Timing and dose of human chorionic gonadotropin
The timing of hCG injection should be individualised on the basis of several factors, including
leading follicle diameter, E2 level, prior cycle response and embryo quality, and the type of COH
protocol. In normal responders who are undergoing an IVF cycle for the rst time, hCG is usually
administered when two or more follicles reach a size of 17 mm or larger in diameter. Ideally, the E2
level should be greater than 400 pg/ml for 3 days.
In women with a history of retrieval of mostly immature oocytes, consideration should be given to
continue COH until the follicles attain 1920 mm in diameter before triggering with hCG. Similarly, in
women who are treated with clomiphene citrate or letrozole COH protocols, hCG injection is usually
given when follicles reach 1920 mm. Each clinic, however, should establish their ideal criteria for hCG
administration based on their methods of ultrasound follicular assessment. In women with poor oocyte
or embryo quality, especially in the presence of a high proportion of polyspermic fertilisation after IVF,
one should be wary of oocyte post-maturity. In such circumstances, the woman may benet from
administering the ovulatory trigger at smaller lead follicle diameters in the subsequent cycles. Another
important parameter for administrating hCG is a plateau or doubling of E2 levels on consecutive days
once the leading follicle has exceeds 16 mm in average diameter.
Future of in vitro fertilisation
An area of ART in particular that has undergone dramatic improvements in recent years is noninvasive methods of embryo selection. Selection of embryos with optimal implantation potential is
of great importance in the eld of ART. Current embryo selection relies on morphological appearance
and possesses limited sensitivity in predicting implantation and pregnancy. Biomarkers of embryo
developmental potential urgently need to be identied, as they will enhance embryo selection,
improve the efcacy of single embryo transfer, and reduce the risk of multiple gestations. In recent
years, the development of non-invasive methods of embryo selection using transcriptomic, proteomic,
and metabolomic technologies has generated much research interest. Transcriptomics involves

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quantitative analysis of messenger RNA. An example is transcriptomic proling of cumulus cells using
microarray to identify potential biomarkers of oocyte quality and embryo development.99 Recent
advances in proteomic analysis using mass spectrometry, high-performance liquid chromatography,
and protein microarray have enabled identication of amino acids and proteins within limited
amounts of biological uids.100 Analysis of spent culture media revealed signicant correlations
between amino acid and protein turnover with embryonic developmental potential and clinical
pregnancy and live birth rates.101,102 Metabolomic analysis involves the use of spectrophotometric
assays, such as Raman and near-infrared (NIR), to detect cellular metabolites in the surrounding
environment (also known as secretome or exometabolome).103 Metabolomic proling of spent embryo
culture media using Raman and NIR seemed to be two promising approaches for predicting the
reproductive potential of embryos.104106 A recent prospective, randomised-controlled trial, however,
failed to show any benet of NIR spectroscopy in predicting pregnancy outcomes.107 Non-invasive
imaging of human embryos has also been evaluated as a potential predictor of embryonic development. Using time-lapse imaging analysis and gene-expression proling, cytokinetic imaging parameters were shown to predict blastocyst formation, with 93% sensitivity and specicity.108
Conclusion
The accessibility to ART and the efcacy of IVF treatment have improved signicantly over the past
decades. The main indications for IVF include tubal obstruction, severe male factor infertility, severe
endometriosis, ovulatory dysfunction, diminished ovarian reserve, and infertility of unexplained cause.
In vitro fertilisation has also become an effective treatment option for couples wishing to undergo PGD for
inherited genetic disorders or PGS for aneuploidy, and for those wishing to cryopreserve their oocytes or
embryos for preservation of fertility. The management of women in late reproductive age remains a major
challenge. The optimum IVF treatment for poor responders is still elusive. The success of IVF treatment can
be optimised by using an individualised, patient-centered approach to COH. Key components involve
selection of appropriate COH protocols, careful cycle monitoring, adjustment of gonadotropin dosage to
avoid hyper-response, and individualised timing of hCG injection. Future emphasis in IVF research should
lead to the development of non-invasive methods for embryo selection to optimise single embryo
implantation and avoiding multiple pregnancies. Moreover, further elucidation of the effect of ovarian
controlled hyperstimulation on endometrial receptivity will also lead to optimal IVF success.

Practice points
 Indications for IVF include infertility caused by tubal obstruction, severe male factor,
diminished ovarian reserve, ovulatory dysfunction, severe endometriosis, and infertility of
unexplained cause. In vitro fertilisation also provides a new mean of preconception genetic
diagnosis and preservation of fertility.
 Women between the ages of 21 and 39 years with unexplained infertility should proceed to
IVF after three unsuccessful clomiphene citrate and treatment cycles. This approach is more
cost-effective and associated with shorter time to pregnancy.
 Key factors affecting success of IVF treatment include selection of the appropriate controlled
ovarian hyperstimulation protocol and gonadotropin dosage, close monitoring of follicular
growth and serum E2 levels, adjustment of gonadotropin dosage to avoid hyper-response,
and individualised timing of hCG injection.
 AMH and AFC seem to be the two most accurate predictors of ovarian response to controlled
ovarian hyperstimulation.
 The management of women in late reproductive age remains a major challenge. Commonly
used COH protocols for poor responders include luteal E2 patch and GnRH antagonist protocol,
co-are and micro-are protocols, clomiphene citrate and letrozole protocols, and modied
natural cycles. The optimum IVF treatment strategy for poor responders remains to be elusive.

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Research agenda
 Development of accurate, non-invasive biomarkers of embryo developmental potential will
enhance embryo selection, improve the efcacy of single embryo transfer, and reduce the risk
of multiple gestations.
 The combination of day-5 blastocyst trophectoderm biopsy and chromosomal analysis using
molecular diagnostic technologies, including single nucleotide polymorphism, comparative
genomic hybridisation microarray, and quantitative polymerase chain reaction, improves the
accuracy of PGS and represents one approach to enhance embryo selection.
 A better understanding of the effect of ovarian controlled hyperstimulation on endometrial
receptivity will improve the efcacy of IVF treatment.

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