Genetics plays a key role in the success or failure of human reproduction, both in normal fertility and in assisted conceptions. Our growing knowledge of the genetics of reproduction is furthering our understanding of the factors that regulate human conception and fertility treatment.
Embryologists and biologists have introduced genetics into assisted reproductive treatment (ART) with the aim of enhancing success, in terms of conception rates, live deliveries and healthy children. Genetic application has certainly played a vital role in alleviating inherited disorders through pre-implantation genetic diagnosis (PGD). However, we are still learning new ways where genetics can help us to improve outcomes in infertility and ART, through measures such as the application of pre-implantation genetic screening (PGS) and pharmacogenomics.
One of the ultimate aims for research and clinical practice is to decode the genomics of infertility and ART. But it is already becoming important to inform clinicians, biologists and scientists across the globe about strategies that are available and proven to translate into patient success.
With these aims in mind, nearly 250 delegates from 29 countries attended the 2015 Annual Conference in Reproductive Medicine, organized by EXCEMED. Entitled Decoding the genomics of infertility and ART, the conference had a highly innovative format that utilized a range of educational tools, to maximise learning and discussion.
Presentations and break-out sessions at the 2015 Annual Conference featured many approaches and emerging technologies that may help to improve patient outcomes. The conference format allowed delegates to express their views through debates, real-time surveys, discussions, electronic voting and via a question-wall system. The meeting also has a dedicated website and mobile application, which clinicians and researchers can continue to access after the event, to further their knowledge of many aspects of genomics in fertility management.
The objectives of the conference were to enable delegates to acquireknowledge of the key genetic aspects of human reproduction, to apply an understanding of human genetics to improve the management of infertility, and to be able to apply current evidence-based recommendations to achieve the best outcomes from ART.
PI3K–PTEN balance vital for maintaining follicular reserve
A woman’s reproductive lifespan is determined by the lifespan of her primordial follicules. Follicular reserve is maintained in the primordial follicle pool, through achieving a complex and carefully controlled balance between the release of phosphatidylinositol 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). The intra-oocyte PI3K pathway therefore has a two key roles: in follicular activation, but also in maintaining the survival of primordial follicles.
Describing data from murine models, Professor Kui Liu, (Gothenburg, Sweden) showed that inhibition of PTEN ‘releases’ the break, thereby triggering and enhancing follicular growth. Conversely, Professor Lui likened the release of PDK-1 (a signalling protein, indirectly regulated by the PI3K pathway) to ‘pushing the gas pedal’, thereby leading to premature death of primordial follicles.
In normal fertile conditions, optimal PI3K signalling ensures the correct balance between timely maturity and release of eggs. In conditions of infertility, inhibiting PTEN has promise in clinical practice, for triggering folicular growth. Different courses possible in the development of primordial follicles are shown in Figure 1.
Genetic tests for diagnosing infertility
A large body of evidence clearly shows the high prevalence of genetic causes of infertility, explained Professor Alberto Ferlin (Padua, Italy). Infertility has a genetic cause in at least 10–15% of affected men and up to 10% of affected women. Consequently, he recommended that all cases of male infertility are considered to have a genetic basis, until proven otherwise, with patients given counselling as appropriate.
Although a relatively small number of genetic tests are currently available (and recommended) in clinical practice, the genetics of infertility is a rapidly evolving area. Indeed, it is an area that clinicians need to be fully aware of, as technological developments continue and new tests emerge into daily practice.
Several genetic tests can already be included in the routine diagnostic workup of infertile men (Figures 2 and 3), but Professor Ferlin added that specific tests should be selected in the context of an individualized, comprehensive, clinical evaluation.
Genetic tests allow appropriate assistance of infertile couples, as they explore the causes of infertility but also assess the risk of transmitting specific genetic characteristics to any offspring.
The identification of genetic markers has led to a pharmacogenetic approach to FSH treatment of men with normogonadotrophic oligozoospermia. Polymorphisms in FSH receptor and FSHβ-chain (FSHB) genes are known to influence serum FSH levels and spermatogenesis. Professor Ferlin described preliminary reports that indicate a role for these polymorphisms in selecting patients for FSH treatment, and in predicting the response to such treatment.
He added that the effects of luteinizing hormone (LH), hCG and FSH are interrelated, therefore one should obtain a comprehensive picture in all patients who seek help for infertility.
Genetic technologies for PGS
Aneuploidy is the most common genetic abnormality occurring in humans, and its identification was discussed by several speakers at the Annual Meeting. For example, Dr Nathan Treff described how PGS now routinely involves the simultaneous evaluation of aneuploidy in all 24 chromosomes. He also reminded delegates that PGS is now often referred to as comprehensive chromosome screening (CCS).
The CCS technologies described by Dr Treff include metaphase comparative genomic hybridization (CGH), array CGH, single nucleotide polymorphism (SNP) arrays with quantitative or qualitative approaches, quantitative real time PCR (qPCR), and next-generation sequencing (NGS).
There are many differences between the technologies, explained Dr Treff, such as the amplification strategies used, and the levels of evidence for accuracy and clinical predictive values. He also reviewed evidence supporting the clinical application of such techniques to improve success rates in IVF.
Aneuploidy screening in ART: who will benefit and what is the evidence?
While it remains important to acknowledge that further studies are required to evaluate subsets of the infertile population, specific groups of women undergoing IVF may benefit from embryonic aneuploidy screening.
Professor Richard T. Scott (New Brunswick, NJ, USA) explained that CCS helps to achieve excellent delivery rates, together with improved safety and cost profiles for obstetric and neonatal outcomes (Figures 4 and 5). These benefits have the potential to greatly reduce the treatment burden when optimizing the pathway to healthy live birth.
Professor Scott described several paradigms for embryonic aneuploidy screening that have been validated for routine clinical application. He also demonstrated how these strategies can improve the selection of reproductively competent embryos.
Enhanced amplification strategies and the availability of reliable 24-chromosome testing platforms are key to the development of aneuploidy screening protocols. Professor Scott explained that these advances make it possible to attain reliable analysis of the genetic complement at several stages of embryonic development.
In addition, data from several randomized controlled trials demonstrate that embryonic aneuploidy screening enhances implantation and delivery rates, in couples undergoing ART. These technologies aid embryologists when selecting the embryos with the highest reproductive potential for transfer.
However, integrating aneuploidy screening into clinical practice impacts more than embryo selection in the laboratory. For example, to perform trophectoderm biopsy safely, extended culture is required. This means that, in centres without an on-site molecular genetics laboratory, vitrification of the embryos for transfer in a future cycle is mandatory.
CCS increases acceptance of SET
Perhaps the most important part of the enhanced implantation rates associated with CCS is its impact on decisions regarding the number of embryos to transfer, especially in the USA. Data from a randomized controlled trial has demonstrated that single embryo transfer (SET) following CCS attained the same delivery rate as the transfer of two unscreened embryos, said Professor Scott, decreasing the multiple pregnancy rate from 48% to 0%. He added that research shows that 80% of patients accept SET if CCS is undertaken as part of the reproductive-medicine programme.
Subsequent evaluations have demonstrated that PGS followed by SET results in dramatic reductions in preterm labour, low birth-weight infants (Figure 4), and neonatal intensive care unit admissions. In fact, the risk for these adverse obstetric outcomes returned to the equivalent risk for the general obstetric population, added Professor Scott.
CCS and SET have also had a beneficial impact on health care costs (Figure 5). While embryonic aneuploidy screening increases the costs of any given ART cycle, the enhanced outcomes offered by CCS and SET also result in an overall reduction in the number of treatments required to attain a live-infant delivery, said Professor Scott.
Poor ovarian response remains a difficult situation
Unfortunately, POR remains one of the most challenging tasks in reproductive medicine.
Professor Hakan Yarali (Ankara, Turkey) described the many strategies that are unproven, and the few that show some benefit, for people with POR. He cautioned delegates to remember that neither patient frustration, nor health care providers trying to offer something ‘different’, provides justification for using a medical procedure for POR in the absence of robust evidence.
Insufficient evidence
Systematic reviews and meta-analyses of POR studies suggest there is insufficient evidence to recommend most of the treatments proposed to improve pregnancy rates, said Professor Hakan Yarali. He added that although the Bologna criteria for defining POR represents a step forward (Figure 6), it is far from perfect. Between eight and 13 subgroup patient populations fulfil these criteria, he explained. Such populations may be inhomogeneous, and may therefore be associated with different live birth rates following IVF, thus confounding any study findings.
COS protocols
Long GNRH agonist and GnRH antagonist protocols are the most effective options for women with POR; the short GnRH agonist regimen is less effective. In addition, the GnRH-antagonist protocol may reduce treatment burden and benefits in terms of compliance, added Professor Yarali.
Increasing the FSH dose does not overcome POR, although oestrogen or combined oral contraceptive pill priming in the preceding luteal phase may contribute to follicular synchronization and hence increase the number of mature follicles in women at risk of POR. However, explained Professor Yarali, there is insufficient evidence to comment on improved outcome with such priming.
Addition of recombinant human luteinizing hormone (r-hLH) to recombinant human follicle-stimulating hormone (r-hFSH) in COS may be beneficial for women with POR. Professor Yarali described the findings of a recent meta-analysis, enrolling 40 randomized controlled trials (6443 patients), in which significantly more oocytes were retrieved and significantly higher clinical pregnancy rates (risk ratio = 1.30) were attained with r-hFSH plus r-hLH versus r-hFSH alone.
Small sample sizes
There may be a positive effect with the use of growth hormone (GH) supplementation in patients with POR; however, studies have been small scale, weak and heterogeneous, and larger, more robust trials are needed.
Treatment with transdermal testosterone prior to IVF was reported to improve clinical outcomes for patients with POR undergoing IVF, in three RCTs. However, the results should be interpreted with caution, due to the small sample sizes and heterogeneity across the studies. Evidence does not support the routine use of dehydroepiandrosterone (DHEA) as an adjuvant in patients with POR undergoing IVF. Furthermore, potential side effects of long-term androgen supplementation in women seeking fertility have not been widely addressed.
Reduce the treatment burden
Reduced treatment burden should be a primary objective in patients with POR. In this context, natural cycle IVF with/without minimal stimulation is cost-effective and patient friendly. Oocyte/embryo accumulation has been recently advocated to improve pregnancy rates and decrease dropout rates in women with POR who undergo ART; it can be achieved either with conventional/minimal stimulation with an interval of at least one cycle or immediately after oocyte retrieval (double stimulation).
Professor Yarali reminded delegates that patients with POR are not homogeneous with regards to pregnancy prospects. Female age and number of oocytes retrieved will modulate the chances for pregnancy in current and subsequent cycles.
GnRH antagonist profile: good compliance, good safety profile in hyper-responders
The utilization of GnRH antagonists in controlled ovarian stimulation (COS) protocols is increasing. Such strategies offer clear advantages, in terms of reducing the incidence of severe ovarian hyperstimulation syndrome (OHSS), said Professor Raoul Orvieto (Tel Hashomer, Israel).
The most common protocols for COS incorporate GnRH-agonist and antagonist co-treatment, mainly to prevent a premature rise in LH levels. But to improve fertility outcomes with this approach, different modifications of luteal support are being applied. One of the most promising approach is the 1500 IU hCG luteal rescue, which aims to reduce (rather than eliminate) severe early OHSS, without compromising safety or efficacy outcomes.
The risk of OHSS in an individual can be predicted by the following combination, explained Professor Orvieto:
• E2 level >6000 pg/ml on day of hCG administration and >30 retrieved oocytes – associated with an 80% chance of developing severe OHSS
• E2 levels <3500 pg/ml and/or <20 oocytes – almost negligible risk of OHSS
Professor Orvieto discussed the different strategies that aim to eliminate the occurrence of severe OHSS, which involve clinical observations, GnRH-antagonist protocols, GnRH agonist trigger, and luteal rescue protocols.
Time-lapse technology, not yet a replacement for PGS/CCS
Evidence for time-lapse parameters in genetic and chromosomal health is very much at a preliminary level. However, the technique causes no harm, and may help patients at low risk of genetic abnormalities who are unable, or unwilling, to undergo more invasive forms of PGS/CCS.
Dr Natalia Basile (Madrid, Spain) made these comments as she described how time-lapse systems have revolutionized some practices in the IVF laboratory. Such technologies have solved the problem of applying static and subjective scoring systems to evaluate dynamic processes such as embryo development.
Semi- or fully automatic time-lapse systems allow uninterrupted surveillance of the embryos and the detection and quantification of various events (e.g. pronuclear formation, syngamy, embryo cleavage, compaction and blastocyst formation). New kinetic markers associated with higher implantation potential have also been proposed, and the safety of these systems has been validated. Consequently, the effects of different intrinsic and extrinsic factors on kinetic markers can now be analysed at the earliest stages of embryo development.
Some studies have also used time-lapse technology to analyse and compare morphokinetic behaviour of chromosomally normal and abnormal embryos. Specific markers related to early and late stages of development have been found to differ between chromosomally normal and abnormal embryos, giving rise to algorithms that may increase the probability of selecting chromosomally normal embryos in a noninvasive way.
However, results can be contradictory and some authors suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for CCS. Consequently caution is advised in clinical practice. The selection of embryos through time-lapse technology should not be considered as a replacement for more traditional methods of PGS until large multicentre studies are undertaken to elucidate the possible relationship between morphokinetics and embryo euploidy. However, especially considering the noninvasive nature of time-lapse technology, there is no harm in utilizing this approach as an embryo-selection tool for patients with a good prognosis who are not indicated for CCS, or for patients with a history of at least two early implantation failures or early pregnancy losses (or women ≥ 38 years of age, or men with severe male factor infertility) (Figure 7). Time-lapse technology is also suitable for those who do not wish to undergo any form of PGS. In these situations, a clear benefit is gained with morphokinetic screening and selection using a defined algorithm.
Clinical application of mitochrondrial replacement techniques
In February 2015, the United Kingdom Parliament passed regulations that permit mitochondrial replacement techniques to be translated into clinical treatment strategies. Procedures can legally be performed in the UK from October 2015.
Professor Alison Murdoch (Newcastle upon Tyne, UK), provided an overview of the genetic role of mitochondria. She described how mitochondrial replacement techniques have the potential to reduce the transmission of abnormal mitochondria. Such methods are pronuclear transfer, using eggs from two women (Figure 8), spindle transfer, and polar body transfer.
Mitochondrial diseases may place heavy burden on affected families, causing severe disability and early death. Mitochondria are passed through the female line only, via the cytoplasm in the eggs. Thus, it is theoretically possible to prevent transmission of abnormal mitochondria by replacing them with normal mitochondria before implantation. There is no evidence or theoretical indication that mitochondrial abnormalities are related to infertility.
The physiology of mitochondria is complex, and currently it is often difficult to predict inheritance patterns, said Professor Murdoch.
Research involving murine models and abnormal human embryos provides further support for mitochondrial replacement technology, and ongoing studies are being undertaken using donated eggs from healthy women.
Mitochondria provide >90% of cellular energy and are vital for normal function. There are numerous copies of mitochondria in each cell, and if some of these are abnormal, the individual may develop mitochondrial disease. Mutation load varies between organs and even between oocytes, but loads >70% are considered to be at high risk of mitochondrial disease development.
Professor Murdoch also discussed the scientific, ethical and regulation hurdles faced by those working in, or with an interest in, the application of mitochondrial transfer techniques in clinical or research situations. Quoting directly from the publication entitled Novel techniques for the prevention of mitochondrial DNA disorders: an ethical review (available at www.nuffieldbioethics.org), Professor Murdoch said that if these novel techniques are adequately proven to be acceptably safe and effective treatments, it would be ethical for families to use them, if they wish to do so and have been offered an appropriate level of information and support.
Epigenetics and imprinting in extended embryo culture: is the child’s health affected?
The emerging fields of epigenetics and genomic imprinting provide stunning and totally unanticipated, discoveries regarding how our ancestors contribute to every aspect of human health and behaviour. Such discoveries fundamentally alter ideas that inheritance is almost exclusively the result of DNA transmission, both in terms of nuclear and mitochondrial DNA.
Epigenetics and genomic imprinting also open new areas of research and questioning for infertility medicine. In the closing Keynote Lecture of the 2015 Annual Meeting, Professor Gerald Phillip Schatten (Pittsburgh, USA), considered the state of knowledge and areas of concern regarding the role of epigenetics and imprinting in extended embryo culture.
He posed the question as to whether epigenetics and imprinting might affect the health of a child born following assisted conception: a point that he envisioned as particularly appropriate in light of the conference’s objectives to “decode the genomics of infertility and ART.”
Professor Schatten used the following key definitions:
Genomic imprinting – the epigenetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner. If the allele inherited from the father is imprinted, it is silenced, and only the allele from the mother is expressed.
Epigenetic change – a regular, natural occurrence that can be influenced by several factors including age, environment/lifestyle, and disease state. For example, transmission of maternal stress has been shown in controlled animal studies but also in studies of women (and their offspring) involved in genocide events.
Epigenetic modifications – can manifest as commonly as the manner in which cells terminally differentiate to end up as skin cells, liver cells or brain cells. For example, there is evidence that maternal diabetes, obesity and famine may each lead to specific health effects in the offspring (Figure 9).
Beyond genomics, epigenetic modifications add complexities for clinicians and researchers working in reproductive medicine, particularly in extended embryo culture. Although many unknowns remain, Professor Schatten explained that it is theoretically possible that some ART procedures may affect methylation status of imprinted regions in the DNA, leading to imprinting disorders in offspring.
However, the low prevalence of imprinting disorders makes it challenging to perform conclusive studies, to investigate this adequately. He called for further studies, in large registries, to better determine whether innovative ART procedures have any impact on the occurrence of such disorders. He also spoke of the vital need for patient counselling, conducted in line with evidence-based findings, in order to properly evaluate the benefits of any procedure versus any safety or other risks.miRNA analysis offers noninvasive aneuploidy screening
In countries where embryo biopsy is legally restricted, miRNA analysis show promise as a non-invasive option to evaluate ovarian failure and improve pregnancy outcome through euploid embryo selection. Professor Sarmir Harmamah (Montpellier, France) made these comments and added that miRNAs are involved in the regulation of mammalian embryo development.
Global miRNA expression profiling suggests that miRNA synthesis and degradation coexist dynamically during preimplantation embryo development. Many miRNAs are expressed in developing mammalian embryos and hESCs, including miR-320, miR-92a, let-7a and miR-146b. Data also indicate that deregulated miRNA expression in the embryo is associated with human infertility, and that the embryo miRNA expression profile varies according to its chromosomal make-up and sex.
As miRNAs have been detected in the culture medium following release by cells grown in culture, it is now possible to quantify the embryonic miRNAs released in the medium in order to monitor embryo health during preimplantation in vitro culture.
PTEN inhibitor therapy for POI
In clinicial studies, PTEN inhibitor therapy has been used to treat women with premature ovarian insufficiency (POI).
Professor Liu explained the experimental technique, in which strips of ovarian tissue from patients with POI were treated with PTEN inhibitors, then autografted beneath the serosa of fallopian tubes.
After detection of antral follicles, patients received follicle-stimulating hormone (FSH), which was followed by human chorionic gonadotropin (hCG), when preovulatory follicules were identified. Professor Liu showed a picture of an infant born following this pioneering therapeutic and surgical technique
Be aware of empirical technologies
One of the key take-home messages from the 2015 Annual Conference was is for clinicians and scientists working in ART to be aware of the empirical nature of many current techniques.
Due largely to a lack of robust scientific data, it can be difficult to provide objective, evidence-based information that establishes which techniques work best, in specific patient groups.
In particular, in his scientific summary of the conference, Professor Johannes LH Evers (Maastricht, The Netherlands), urged delegates not to translate a clinical trial finding that shows a high correlation in a group, with the same finding having high predictive value for an individual patient. There is an urgent need for more well-designed research studies to be undertaken, to provide better guidance when counselling individuals about IVF and ART.