Contemporary genetic technologies and female reproduction

The study of genetics is a rapidly expanding field in all areas of medicine, including fertility. This review focuses on the genetic aspects of a number of topics of importance in fertility medicine: ovarian function, primary ovarian insufficiency (POI) and poor response to ovarian stimulation, polycystic ovary syndrome (PCOS) and endometriosis. In addition, new technologies for the assessment of oocyte and embryo quality are reviewed, including gene expression profiling of cumulus cells, proteomics and metabolomics. The number of genes known to be expressed in the ovary and involved in reproductive function has increased dramatically in recent years. Increasing our understanding of the genes involved in ovarian ageing and poor response to ovarian stimulation may lead to improved clinical diagnosis and early detection, along with the development of new agents or technologies for use in fertility treatment. Although familial clustering of PCOS cases has been described, the mode of inheritance remains uncertain. Candidate genes for PCOS are discussed, although few studies have satisfied the rigorous criteria of population selection and size, repeatability and correction for multiple comparisons. Genetics also appear to have a role in complex reproductive diseases, such as endometriosis. A number of candidate gene families that may be differentially regulated in endometriosis have been identified. New developments in this field include the identification of a candidate population of endometrial stem cells, the study of which may provide new insights into this condition. Improving our knowledge of the genetics of the ovary and the genetic causes of infertility should lead to future improvements in fertility treatment. For instance, it is now recognized that the oocyte is not passive in the ovarian follicle but is a fundamental regulator of somatic cell differentiation and function. The cumulus–oocyte complex plays a central role in the regulation of folliculogenesis and is important for the maturation, reprogramming and fertilization of oocytes. Consequently, cumulus cell gene expression profiling is being explored as a new method for the assessment of oocyte competence and embryo developmental competence, which shows promise for the near future. Currently, IVF represents the only hope for many couples with fertility problems. Embryo assessment has an enormous impact on the success of IVF and, with moves towards single-embryo transfer (SET), assessment of embryo quality is of the utmost importance. Despite its evident importance, until recently, assessment of embryo quality has depended on subjective morphological assessment ( Assou et al ., 2011 ). Any objective method of embryo assessment that could determine its potential for implantation, as well as its health status, will increase success rates. With the recent expansion of the current knowledge and developments in this area, a thorough review of contemporary genetics and ‘omics’ technologies in human reproduction is timely.

The workshop and preparation of the manuscript were sponsored by an unrestricted educational grant from Merck Serono S.A., Geneva, Switzerland , an affiliate of Merck KGaA, Darmstadt, Germany .

Prior to the Fifth Evian Annual Reproduction (EVAR) Workshop Meeting, held on 16–17 April 2010, expert speakers prepared presentations based on published literature and current research. Presenters were asked to include comprehensive information using systematic literature search criteria. Combinations of the following keywords were used for PubMed searches: genes and fertility-associated pathologies, primary ovarian insufficiency, premature ovarian failure, genomics, proteomics, metabolomics, endometriosis, polycystic ovary syndrome (and PCOS), floating granulosa cell gene expression, cumulus cell gene expression and protein expression profiling. Following each presentation, a group discussion to reach joint conclusions on the topics covered was facilitated by the chairmen: Professors Fauser and Diedrich. The content of this report is based on the expert presentations and subsequent group discussions that took place during the workshop meeting. The discussions relating to each topic were complemented as necessary with key publications that were known to the authors and electronic literature searches. Articles of any type and published in English were permitted and were unlimited by the date of publication.

B.C.J.M. Fauser: developed the Fifth EVAR Workshop scientific programme, participated as a Chairman, was involved in drafting the article and revising it critically for important intellectual content and approved the final version of the manuscript. K. Diedrich: participated in the Fifth EVAR Workshop as a Chairman, was involved in drafting the article and revising it critically for important intellectual content and approved the final version of the manuscript. P. Bouchard, F. Domínguez, M. Matzuk, S. Franks, S. Hamamah, C. Simón, D. Ezcurra and C.M. Howles: participated in the Fifth EVAR Workshop, were involved in drafting the article and revising it critically for important intellectual content and approved the final version of the manuscript. P. Devroey: was involved in the planning of the Fifth EVAR Workshop and was involved in drafting the article and revising it critically for important intellectual content and approved the final version of the manuscript.

B.C.J.M. Fauser has received fees and grant support from the following companies: Andromed, Ardana, Ferring Pharmaceuticals, Genovum, Glycotope, Merck Serono, MSD, Organon, Pantarhei Bioscience, Philips, PregLem, Schering, Schering-Plough, Serono and Wyeth. C.S. is an International Scientific Advisor for Merck Serono S.A., Geneva, Switzerland, an affiliate of Merck KGaA, Darmstadt, Germany. D. Ezcurra and C.M. Howles are employees of Merck Serono S.A., Geneva, Switzerland, an affiliate of Merck KGaA, Darmstadt, Germany.

Endometriosis is an oestrogen-dependent condition that is characterized by the presence of endometrium-like tissue at ectopic sites such as the pelvic peritoneum and ovaries (Fig.  3 ). Pelvic pain and infertility are the most common features of endometriosis. There are several proposed hypotheses to explain the pathogenesis of endometriosis ( Sasson and Taylor, 2008 ); the most widely accepted theory is that of retrograde menstruation. The mechanisms involved in the development of an endometriotic lesion in the pelvic peritoneum may include attachment, invasion into the mesothelium, and survival and proliferation of ectopic endometrial cells. Figure 3 A theoretical model of the development of endometriosis. A theoretical model of the development of endometriosis. The basic underlying cause of endometriosis is likely to be multifactorial and involves interplay between several factors. The pathogenesis of endometriosis may, for example, involve retrograde menstruation in the context of an abnormal immune response and a genetic predisposition to developing endometriotic lesions; this may possibly occur after exposure to an unidentified environmental factor ( Sasson and Taylor, 2008 ). The study of endometriosis is complicated by a number of factors: these include the presence of different cell types within endometriotic lesions and the involvement of different pathogenic mechanisms in the formation of distinct types of endometriotic lesion (such as peritoneal, ovarian and rectovaginal) ( Nisolle and Donnez, 1997 ). In addition, it may also be difficult to distinguish between cause and effect: differences in eutopic endometrium between patients with and without endometriosis, or in eutopic versus ectopic endometrium, may be the cause of this pathological condition or the result of another causative factor. Although endometriosis can be treated surgically, recurrence of endometriotic lesions can occur. GnRH agonists and long-cycle oral contraceptives can also be used to manage the condition by suppressing ovulation and inducing a pseudo-menopausal state. Although many potential new agents, such as aromatase inhibitors, progesterone antagonists and immunomodulatory drugs, have been investigated, there is still a lack of suitable alternative pharmacological treatments ( Guo, 2008 ; Huang, 2008 ; Vercellini et al ., 2009 ). A better understanding of the pathogenesis of endometriosis may help to identify new pharmacological targets and facilitate the development of new treatments. It has been proposed that endometriosis results from a series of multiple hits within target genes, in a mechanism similar to the development of cancer ( Bischoff and Simpson, 2004 ). The initial mutation may be either somatic or heritable. Endometriosis has long been recognized as having a familial association; data are consistent with a complex genetic basis ( Malinak et al ., 1980 ; Simpson et al ., 1980 ). Gene expression microarray studies have identified a number of candidate gene families that may be differentially regulated in endometriosis ( Kao et al ., 2003 ; Wu et al ., 2006 ; Burney et al ., 2007 ; Hull et al ., 2008 ; Ohlsson Teague et al ., 2009 ). A search for genetic polymorphisms associated with susceptibility to endometriosis has focused mainly on genes involved in inflammation, steroid hormone regulation, metabolism, biosynthesis, detoxification, vascular function and tissue remodelling ( Tempfer et al ., 2009 ). However, some polymorphisms have been investigated in only a single or a limited number of studies and, in some cases, conflicting results have been obtained. The majority of polymorphisms studied have, therefore, been found not to be associated with endometriosis. An evaluation of SNPs in 22 miRNAs that were differentially expressed in paired ectopic and eutopic endometria in women with endometriosis, however, identified two SNPs that were significantly associated with endometriosis-related infertility and disease severity: Wolf–Hirschhorn syndrome candidate gene 1 (WHSC1) alleles and solute carrier family 22, member 23 (SLC22A23) haplotypes ( Zhao et al ., 2011 ). Two studies have shown differential expression of miRNAs in microarray analysis of eutopic and ectopic endometrial tissues ( Pan et al ., 2007 ; Ohlsson Teague et al ., 2009 ). Although eight miRNAs were differentially expressed in both studies, the direction of dysregulation was not in agreement for any of these ( Ohlsson Teague et al ., 2010 ). A recent study using next-generation sequencing reported 22 dysregulated miRNAs (10 up-regulated miRNAs and 12 down-regulated miRNAs) in endometriomas compared with eutopic endometrium; miR-29c was implicated as a key up-regulated miRNA in endometriosis ( Hawkins et al ., 2011b ). It is likely that specific miRNAs have a function in the pathophysiology of endometriosis, and the differences in results could be due to many factors, including the menstrual cycle phase at the time of biopsy. The putative roles of miRNAs in endometriosis and other reproductive diseases are described extensively in a recent review ( Hawkins et al ., 2011a ). A recent GWAS has identified a strong association signal for endometriosis at locus 7p152 ( Painter et al ., 2011 ). This locus is located in an intergenic region upstream of two plausible candidate genes, NFE2L3 and HOXA10 . Single molecular alterations in endometriosis may result in differential regulation of hormone metabolism ( Attar and Bulun, 2006 ). The alterations may arise through epigenetic mechanisms such as DNA methylation. An example is transcriptional activation of SF-1 ( NR5A1 ) in endometriotic stromal cells by hypomethylation of DNA ( Xue et al ., 2007 ). The presence of SF-1 in endometriosis and its absence in the endometrium are determined primarily by the methylation of its promoter. In endometriotic lesions, the presence of SF-1 may contribute to increased oestrogen production ( Bulun et al ., 2009 ). Stem cells located in the basal layer of the endometrium are thought to be responsible for cyclical tissue regeneration. Studies have been performed to isolate and characterize this cell population ( Kato et al ., 2007 , 2010 ; Cervelló et al ., 2010 ; Gargett and Masuda, 2010 ; Masuda et al ., 2010 ). Such studies have identified a population of candidate endometrial stem cells in human endometrium; the cells demonstrate multipotency and are different from other endometrial cell types. These cells have been termed side population cells (SP cells) as they exhibit a side population phenotype upon staining with Hoechst dye and flow cytometric analysis. SP cells have been demonstrated to proliferate and differentiate into various endometrial cell types in vitro and have unique angiogenic and migratory properties ( Masuda et al ., 2010 ). Functional proof-of-concept studies have shown that human endometrium can be reconstructed in immunodeficient mice upon injection with human endometrial SP cells ( Cervelló et al ., 2010 ). It has been suggested that the retrograde menstruation of SP cells and subsequent implantation onto the surface of ectopic sites are responsible for the establishment of endometriotic lesions ( Sasson and Taylor, 2008 ; Masuda et al ., 2010 ). It has also been hypothesized that an initial genetic mutation may cause aberrant behaviour of a subpopulation of endometrial stem cells; clonal expansion of such cells may then occur and result in development of endometriosis ( Gargett et al ., 2009 ). It is also conceivable that peritoneal cells could undergo de-differentiation back to endometrial cells, which take on an altered activity ( Gargett et al ., 2009 ). Studies of endometrial stem cells have provided a new focus for endometriosis research. Research on this subpopulation of endometrial cells could be of particular value; the findings may further our understanding of the pathogenesis of endometriosis and provide new therapeutic approaches.

Currently, many couples with fertility problems resort to IVF or ICSI when other treatments fail. Problems related to multiple pregnancies and poor embryo implantation are among the factors that may limit the success of IVF. The improvement of in vitro techniques, with sequential or co-culture systems, and subsequent reduction in the number of embryos transferred per cycle have ameliorated problems related to multiple pregnancy. However, implantation rates in IVF have not improved in the last decade ( Nygren et al ., 2006 ; Andersen et al ., 2008 ). Any objective procedure that could assess the oocyte competence and potential for embryo implantation and development should increase success rates in assisted reproductive technology (ART). In the study of oocyte competence, in recent years, the cumulus cell complex that surrounds and connects with the oocyte has received renewed attention. In a mouse model, this complex has been shown to play a central role in ovulation, and the capacity of the oocytes to support cumulus gene expression has been shown to be linked to the oocyte's developmental competence ( Russell and Robker, 2007 ). Cumulus gene expression profiling therefore represents a possible means of identifying reliable biomarkers for oocyte quality and competence. It may also be useful to identify biomarkers for embryo development prediction. Many studies suggest that cumulus cells: (i) coordinate follicular development with oocyte maturation, (ii) provide energy substrates for oocyte meiotic resumption, (iii) regulate oocyte transcription, (iv) promote nuclear and oocyte molecular maturation, (v) stimulate amino acid transport and sterol biosynthesis, (vi) promote glycolysis and (vii) provide protection for the oocyte ( Sugawara et al ., 1997 ; Elvin et al ., 1999b , 2000 ; Varani et al ., 2002 ; Sutton et al ., 2003 ; Pangas et al ., 2004 ; Su et al ., 2004 ; Eppig et al ., 2005 ; Diaz et al ., 2007 ; Gilchrist et al ., 2008 ). The vital supporting role of cumulus cells during in vivo and in vitro maturation has led many groups recently to focus research interest on cumulus cells. Isolated cumulus cells are relatively homogeneous with almost no contamination with other cells, whereas isolated granulosa cells, despite meticulous efforts, generally contain theca and blood cells: this is due to the methods employed for obtaining these cells. In addition, with the advent of the functional genomics and proteomics era, it has become possible to identify the transcriptome and proteome of cumulus cells using high-throughput technologies, such as microarray ( Assou et al ., 2006 ) and high-resolution two-dimensional protein electrophoresis ( Hamamah et al ., 2006 ). The current concept is that cumulus cells may constitute a reliable model for understanding oocyte quality and ovarian hyperstimulation protocol efficiency and may indirectly predict oocyte aneuploidy, embryo development and pregnancy outcomes ( McKenzie et al ., 2004 ; Zhang et al ., 2005 ; Feuerstein et al ., 2007 ; van Montfoort et al ., 2008 ). Cumulus cells are typically discarded during classical IVF and ICSI. These cells are easily accessible and plentiful, which makes them an ideal material to use for the assessment of oocyte quality and embryo development potential. Thus, analysis of gene expression in cumulus cells may provide an indirect indication of the microenvironment in which the oocyte matures, and will help embryologists to better assess embryo quality. Currently, the most common ways of evaluating embryo quality non-invasively are by examining parameters such as morphology and cleavage rate. However, these approaches are subjective and lack precision. Several groups have used microarray technologies, reverse transcriptase PCR (RT–PCR) and quantitative RT–PCR analyses to study the association between cumulus cell gene expression profiles and oocyte competence, embryo quality and pregnancy outcome (Table  III ) ( McKenzie et al ., 2004 ; Cillo et al ., 2007 ; Feuerstein et al ., 2007 ; Assou et al ., 2008 , 2010 ; van Montfoort et al ., 2008 ; Anderson et al ., 2009 ; Hamel et al ., 2010 ; Wathlet et al ., 2011 ). In a new and indirect approach for predicting embryo quality and pregnancy outcome, gene expression signatures have been identified recently by Assou et al . (2008 , 2010 ) using transcriptomic data of cumulus cells. This non-invasive approach is based on the level of expression of potential biomarkers in cumulus cells (indicators of successful pregnancy) to assess the potential and quality of the embryo. Table III Association of cumulus cell gene expression with embryo quality and pregnancy outcomes. Cumulus cell origin Approaches Biomarkers Outcome Reference Individual oocytes Microarray (50 chips) Including PCK1, BCL2L11, NFIB and others Predict embryo and pregnancy outcomes Assou et al . (2008 ) Individual oocytes Microarray (16 chips) CCND2, CXCR4, GPX3, HSPB1, DVL3, DHCR7, CTNND1, TRIM28 Negatively associated with oocyte competence van Montfoort et al . (2008 ) Individual oocytes RT–PCR HAS2, GREM1 Positively associated with oocyte developmental competence Cillo et al . (2007 ) Individual oocytes RT–PCR STAR, AREG, CX43, PTGS2, SCD1, SCD5 Negatively associated with oocyte competence Feuerstein et al . (2007 ) Individual oocytes qRT–PCR HAS2, PTGS2, GREM1 Positively associated with oocyte competence and embryo development McKenzie et al . (2004 ) Individual oocytes qRT–PCR GREM1, BDNF Positive and negative predictors of embryo quality, respectively Anderson et al ., (2009 ) Mural granulosa cells and individual oocytes qRT–PCR PGK1, RGS2, RGS3, CDC42 Associated with pregnancy Hamel et al . (2010 ) Individual oocytes qRT–PCR SDC4, PTGS2, VCAN, activated leucocyte cell adhesion molecule, GREM1, TRPM7, ITPKA Predict embryo development and pregnancy Wathlet et al . (2011 ) Modified with permission from Assou et al . (2010 ). AREG, amphiregulin; BCL2L11, BCL-like protein 11; BDNF, brain-derived neurotrophic factor; CCND2, cyclin D2; CDC42, cell division cycle 42; CTNND1, catenin delta 1; CX43, connexin 43; CXCR4, chemokines receptor 4; DHCR7, 7-dehydrocholesterol reductase; DVL3, dishevelled dsh homolog 3; GPX3, glutathione peroxidase; GREM1, gremlin 1; HAS2, hyaluronic acid synthase 2; HSPB1, heatshock 27 kDa protein 1; ITPKA, inositol 1,4,5-trisphosphate 3-kinase A; NFIB, nuclear factor 1B; PCK1, phosphoenolpyruvate carboxykinase 1; PGK1, phosphoglycerate kinase 1; PTGS2, prostaglandin-endoperoxide synthase 2; qRT–PCR, quantitative real-time PCR; RGS2, regulator of G-protein signalling 2; RGS3, regulator of G-protein signalling 3; RT–PCR, real-time PCR; SCD1, stearoyl-co-enzyme A desaturase 1; SCD5, stearoyl-co-enzyme A desaturase; SDC4. syndecan 4; STAR, steroidogenic acute regulatory protein; TRIM28, tripartite motif-containing 28; TRPM7, transient receptor potential cation channel, subfamily M, member 7; VCAN, versican. Association of cumulus cell gene expression with embryo quality and pregnancy outcomes. Modified with permission from Assou et al . (2010 ). AREG, amphiregulin; BCL2L11, BCL-like protein 11; BDNF, brain-derived neurotrophic factor; CCND2, cyclin D2; CDC42, cell division cycle 42; CTNND1, catenin delta 1; CX43, connexin 43; CXCR4, chemokines receptor 4; DHCR7, 7-dehydrocholesterol reductase; DVL3, dishevelled dsh homolog 3; GPX3, glutathione peroxidase; GREM1, gremlin 1; HAS2, hyaluronic acid synthase 2; HSPB1, heatshock 27 kDa protein 1; ITPKA, inositol 1,4,5-trisphosphate 3-kinase A; NFIB, nuclear factor 1B; PCK1, phosphoenolpyruvate carboxykinase 1; PGK1, phosphoglycerate kinase 1; PTGS2, prostaglandin-endoperoxide synthase 2; qRT–PCR, quantitative real-time PCR; RGS2, regulator of G-protein signalling 2; RGS3, regulator of G-protein signalling 3; RT–PCR, real-time PCR; SCD1, stearoyl-co-enzyme A desaturase 1; SCD5, stearoyl-co-enzyme A desaturase; SDC4. syndecan 4; STAR, steroidogenic acute regulatory protein; TRIM28, tripartite motif-containing 28; TRPM7, transient receptor potential cation channel, subfamily M, member 7; VCAN, versican. As cumulus cells respond to stimulation protocols with a distinct gene expression profile and are sensitive to changes in environmental conditions, it may be possible to identify the genes that are expressed in the follicular environment. The individual characterization of gene expression may provide important insights for the evaluation of the impact of ovarian hyperstimulation protocols used during IVF. Using fertilized and non-fertilized oocytes resulting from specific ovarian hyperstimulation protocols, potential differences in the protein expression profiles of cumulus cells have been investigated ( Hamamah et al ., 2006 ). In cumulus cells from either fertilized or non-fertilized oocytes, the greatest degree of similarity (more than 85%) was in the protein expression profiles. The analysis of protein expression profiles of cumulus cells from follicles obtained with the same ovarian hyperstimulation protocol showed a strong correlation between protein expression profiles whether or not embryos had been produced successfully. More than 80% of proteins were expressed similarly between cumulus cells from fertilized versus unfertilized oocytes from a cycle in a single patient that used a long GnRH protocol with human menopausal gonadotrophin (HP-hMG). In contrast, when cumulus cell protein expression profiles were analysed in oocytes with the same outcome from the same patient, but originating from different ovarian hyperstimulation protocols, a significant difference was seen in the protein expression profiles. Comparing protein expression profiles in cumulus cells from fertilized oocytes from the same patient, but after GnRH agonist long protocols with HP-hMG compared with those with recombinant FSH, only 55% of the proteins showed similar mobility and expression levels. Comparison of two groups of patients indicated that dissimilarities in protein pattern between patients become very high, even when comparing the same stimulation protocol and oocyte fertilization outcome. These data from protein expression profiling of human cumulus cells suggest that there may be a correlation between the synthesis of specific cumulus cell proteins and the maturity and fecundity of the oocyte. The study reveals three important new findings with respect to human cumulus cell biology: (i) human cumulus cells have robust metabolic activity, (ii) the overall protein expression profiles are highly similar between cumulus cells from oocytes obtained using the same ovarian hyperstimulation protocol and (iii) there are significant differences in protein expression between cumulus cells from oocytes obtained under two different ovarian hyperstimulation protocols, even when the outcomes are the same ( Hamamah et al ., 2006 ). Cumulus cells play a major role in the control of oocyte metabolism and, therefore, it is likely that malfunction of these cells might play a role in PCOS. Recently, the gene expression profile of cumulus cells isolated from patients with PCOS has been studied ( Kenigsberg et al ., 2009 ). The different gene expression patterns of cumulus cells from lean and obese women with PCOS support the idea that in the two types of patient, the condition may have different pathophysiologies. Furthermore, in recent years, several groups have initiated research aimed at identifying non-invasive biomarkers of chromosome imbalance. Some investigators have detected characteristic transcriptional changes in the cumulus cells attached to aneuploid oocytes ( Wells et al ., 2008 ). This suggests that genes involved in the meiotic process in human oocytes are regulated by genes expressed in cumulus cells. Accurate selection of the most appropriate embryo for SET has been a major aim for reproductive specialists in recent years. To date, the only universally used method to score the potential development and implantation capability of the human embryo is morphological assessment. Therefore, there is a need for new adjunctive technologies for determining the best embryo for transfer and improvement of implantation rates. Successful embryo implantation requires endometrial receptivity, the development of a viable embryo and adequate bi-directional communication between the blastocyst and endometrium. The advent of the era of high-throughput ‘omic’ methodologies (genomics, transcriptomics, proteomics and metabolomics) has facilitated the study of such processes. Some of these technologies may provide non-invasive methods for embryo evaluation in the future. Preimplantation genetic screening (PGS) can be used during early stages of embryo development to assess aneuploidy. In some countries, PGS is used to maximize the chances of a successful pregnancy in certain patient subpopulations, such as those of advanced maternal age, or women with a history of recurrent implantation failure or miscarriage. However, the use of PGS is associated with a number of potential drawbacks. Standard PGS methods utilize a Day-3 biopsy, in which one or two blastomeres are removed from the embryo, followed by FISH to detect chromosomal abnormalities. Therefore, the loss of embryos and a reduction in embryo quality are possible. Furthermore, there is a high rate of mosaicism in Day-3 embryos (which might lead to an incorrect diagnosis), and the survival of embryos after blastomere removal and subsequent cryopreservation has been found to be severely reduced ( Joris et al ., 1999 ; Platteau et al ., 2006 ). Several comprehensive chromosome analysis methods are now available, including aCGH, quantitative PCR and SNP microarrays ( Johnson et al ., 2010 ). aCGH evaluates aneuploidy of each pair of chromosomes and results can be available within 24 h. Combined with single blastomere biopsy on Day-3 embryos, aCGH has been shown to be robust, with only 2.9% of embryos with no results, and associated with low error rates (1.9%) ( Gutiérrez-Mateo et al ., 2011 ). Unlike SNP arrays, aCGH does not require prior testing of parental DNA; therefore, advanced planning and careful scheduling are unnecessary. A short CGH method has also been shown to detect more chromosomal aneuplodies than FISH and has been used successfully to achieve pregnancy ( Rius et al ., 2011 ). Over the last decade, microarray studies have characterized the gene expression profile of the human endometrium in different physiological states, such as during decidualization, the window of implantation, endometriosis, the normal menstrual and stimulated cycles, and endometrial cancer ( Horcajadas et al ., 2007 ; Haouzi et al ., 2009a , b ). However, gene expression profiling studies of human embryos are limited, due to legal and ethical issues. Data on early mammalian development have been provided mainly by murine studies ( Tanaka et al ., 2000 ; Hamatani et al ., 2004 ; Wang et al ., 2004 ; Jeong et al ., 2006 ). Gene expression profile studies in cumulus and granulosa cells have been described earlier and are summarized in Table  III . Such studies have identified candidate biomarkers for oocyte quality and competence ( Cillo et al ., 2007 ; Hamel et al ., 2008 ), early embryo development ( McKenzie et al ., 2004 ; van Montfoort et al ., 2008 ; Anderson et al ., 2009 ) and embryo quality and pregnancy outcome ( Assou et al ., 2008 ; Hamel et al ., 2010 ). Furthermore, in mice, blastocyst gene expression profiles correlate with outcome, including successful implantation and pregnancy loss ( Parks et al ., 2011 ). miRNA expression in human blastocysts is different in transferable blastocysts from infertile patients (male factor infertility and those from women with PCOS) compared with those from donor fertile controls, and it has been suggested that an association of aberrant miRNA profiles may exist with human infertility ( McCallie et al ., 2010 ). Transcriptomic studies may provide valuable information that could lead to the identification of biomarkers. For a comprehensive review, refer to Assou et al . (2011) . Although not as developed as the genomics/transcriptomics methodologies, proteomic analysis of mammalian embryos is also emerging as a powerful assessment tool ( Katz-Jaffe et al ., 2005 ; Shankar et al ., 2005 ). Analysis of the proteome of individual human embryos has the potential to provide novel biomarkers of good embryo development and implantation potential ( Katz-Jaffe et al ., 2006a ). Furthermore, by using bioinformatics to create networks linking proteins found to be differentially regulated between biological samples, functional pathways can be identified ( Dominguez et al ., 2010 ). However, analysis of the proteome in a research setting requires extraction of proteins from lysed blastocysts, which limits the use of this technology in routine embryo assessment. Analysis of the proteins contained in the surrounding embryo culture medium may provide a non-invasive method of using proteomic techniques to identify novel biomarkers of embryo viability ( Katz-Jaffe et al ., 2006b ). Domínguez et al. have used protein array analysis to characterize pooled spent sequential culture media prior to SET and to compare it with control media. A lower abundance of CXCL13, stem cell factor and macrophage-stimulating protein--α, but a higher abundance of soluble TNF receptor 1 was detected in the sequential culture media containing a blastocyst relative to the control ( Domínguez et al ., 2008 ). Furthermore, blastocysts that went on to successfully implant were found to have a relatively lower abundance of CXCL13 and granulocyte-macrophage colony-stimulating factor in their culture media than those that did not implant; these proteins may be markers of successful implantation ability ( Domínguez et al ., 2008 ). Domínguez et al. have also used co-culture studies to explore the role of the endometrium in implantation. This technique involves endometrial biopsy, followed by separation and culture of the endometrial epithelial cells (EEC). A total of 32 proteins were found to be up- or down-regulated in blastocysts that went on to successfully implant and were co-cultured with EEC relative to those cultured in sequential media; of these proteins, interleukin 6 (IL-6) was found to be the most abundantly secreted in the EEC culture. Furthermore, the IL-6 concentration in sequential media from blastocysts that implanted was significantly lower than that of blastocysts that did not implant ( Dominguez et al ., 2010 ). The proteomics of endometrial receptivity have also been studied by analysis of endometrial biopsies taken during the pre-receptive and receptive phases of the menstrual cycle ( DeSouza et al ., 2005 ; Domínguez et al ., 2009 ; Haouzi et al ., 2009a , b ). Two proteins found to be differentially expressed in pre-receptive and receptive endometria were stathmin 1 and annexin A2 (down- and up-regulated in the receptive endometrium, respectively) ( Domínguez et al ., 2009 ). Support for the involvement of these proteins in endometrial receptivity was provided by the pre-receptive pattern of stathmin 1 and annexin A2 expression observed when an intrauterine device was used to model the non-receptive endometrium ( Domínguez et al ., 2009 ). In previous studies, a quantitative approach for proteomic assessment using isotope-coded affinity tags, affinity purification and online tandem mass spectrometry has been used to study differences between proliferative and secretory endometria ( DeSouza et al ., 2005 ). Only five proteins with significant differential expression were found; the glutamate NMDA receptor subunit zeta 1 precursor and FRAT1 were of greatest interest. Endometrial receptivity has also been studied by other groups using non-invasive techniques. Boomsma et al . (2009 ) have used a multiplex immunoassay to analyse endometrial secretions, aspirated immediately prior to embryo transfer during IVF. Using this approach, cytokine profiles predictive of implantation and clinical pregnancy have been identified ( Boomsma et al ., 2009 ). The data obtained provide support for associations between MCP-I and IP-10 levels with implantation, and IL-1β and TNF-α levels with clinical pregnancy ( Boomsma et al ., 2009 ). Recent studies have investigated discriminating signatures between individual euploid and aneuploid blastocysts. Microdrops of spent IVF culture medium from individual blastocysts of transferable quality were processed and analysed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to determine a blastocyst secretome fingerprint. Each individual blastocyst was then subjected to CGH for comprehensive analysis of all chromosomes ( Fragouli et al ., 2008 ). Of the 14 aneuploid blastocysts analysed, nine had a single chromosomal aneuploidy and five had chaotic changes involving more than two chromosomes. Secretome fingerprints from individual blastocysts identified protein signatures that allowed discrimination between euploid and aneuploid chromosomal constitutions ( Katz-Jaffe et al ., 2008 ). Metabolites represent the end products of cell regulatory processes and can be used to study the response of biological systems to genetic, nutritional and environmental influences; the metabolome (i.e. the complete set of small molecule metabolites found within a biological sample) therefore provides a good indicator of cellular activities. High-resolution nuclear magnetic resonance (NMR) has been used to analyse the metabolic profile of follicular fluid samples from oocyte donors and has identified metabolites that could be useful as biomarkers of the follicular maturation state ( Pinero-Sagredo et al ., 2010 ). NMR spectroscopy has also been used for compositional analysis of other areas of the reproductive tract, including cervical mucus, ovarian tissue, fallopian tubes and uterine matter ( Baskind et al ., 2011 ). Incorporation of such techniques into female fertility research may be valuable for understanding subfertility and for predicting outcomes of assisted conception treatments ( Baskind et al ., 2011 ). Metabolomic technology is being used in the development of a new method for aneuploidy detection. This approach is based on the assumption that an embryo with a missing or extra chromosome may have modified metabolism. Given the drawbacks of the use of PGS to detect chromosomal abnormalities, alternative methods would be of value. Analysis of the global metabolome of the embryo (using a combination of ultra-performance liquid chromatography and mass spectroscopy) to analyse low-molecular-weight molecules in spent culture media allows study of the embryo without invasive techniques (unlike PGS). By correlating metabolomic profiles with the results of subsequently performed PGS, it has been possible to differentiate between embryos with normal and abnormal chromosome numbers, and even classify embryos according to their chromosomal anomaly ( Sanchez-Ribas et al ., 2008 ). Although this technique may be limited by the low quantity of metabolites in spent culture media, preliminary results are promising and encourage further development of this non-invasive tool for embryo assessment. Aneuploidy screening represents a unique and valuable application of metabolomics. Analysis of the metabolomic profile of embryo culture media using infra-red techniques is also being explored as an embryo assessment strategy ( Seli et al ., 2007 , 2010 ; Scott et al ., 2008 ). As with any ART procedure, the easier the technique, the more likely that it can be used to produce reliable results in routine procedures in ART clinics. Most ART laboratories have, or have easy access to, enzyme-linked immunosorbent assay platforms for routine hormone testing. However, few laboratories have sophisticated equipment for performing quantitative PCR or mass spectrometry. If the cost of this equipment falls over time, or as more focused assays become available, one or more of these technologies may become used routinely in clinical practice. Another possibility is that as procedures become routine and as key genes, proteins or small molecules are identified that correlate with pregnancy outcomes, clinical samples (cumulus cells, media from embryo cultures or blastomeres) could be collected and stored in specialized solutions or containers, and testing could be performed at a commercial facility. Such a process could allow cost-effective performance of the assays and standardization of the technologies. In addition, this practice could allow selection of high-quality oocytes based on cumulus cell profiles after the oocytes have been vitrified. Under such circumstances, there would not be immediate time pressures to obtain the results and oocytes could be assessed before transfer in a subsequent cycle. In the future, it is likely that a combination of multiple technologies will be used with the goal of obtaining a healthy newborn on the first attempted embryo transfer.

In conclusion, our knowledge of ovarian genetics is rapidly expanding and this review provides an important update of those genes identified to have a role in infertility, particularly POI. To date, the search for a candidate gene for PCOS has produced limited positive findings despite involvement of numerous genes. In the field of endometriosis, it has been proposed that the condition results from a series of multiple hits within target genes and gene expression microarray studies have identified a number of candidate gene families that may be differentially regulated. The members of the Fifth EVAR Workshop Group agreed that the way forward in advancing the knowledge of genes involved in reproduction and fertility-associated pathologies was through GWAS involving large numbers of patients. As GWAS are expensive to conduct, it was suggested that pooling of data from different research groups may be the way forward. Future challenges for genomics and genetic screening of patients include identification of high-throughput strategies and how to apply findings to patients being seen in the ART clinic. The identification of high-quality embryos in ART remains a priority in order to select those with high potential development and implantation capability for transfer. Gene expression profiling of cumulus cells and proteomic and metabolomic technologies show promise for non-invasive oocyte and embryo assessment.