Pathogenic missense variation in PABPC1L/EPAB causes female infertility due to oocyte maturation arrest at the germinal vesicle stage.

Women undergoing controlled ovarian hyperstimulation prior to in vitro fertilization (IVF) are treated using various protocols to induce multiple follicular growths. Complete failure of all oocytes to mature during IVF cycles is rare; however, it is a known cause of primary female infertility. Recently, pathogenic variations in a few genes have been identified in women with oocyte maturation defects; however, the underlying genetic causes remain largely unknown.This study included a Turkish family comprising three sisters with recurring oocyte maturation arrest at the germinal vesicle stage after multiple ovarian stimulations. Exome sequencing revealed a homozygous missense variant (c.1037C>T, p.Ala346Val) in the EPAB gene (also known as PABPC1L) in all three affected sisters, which was either absent or heterozygous in the unaffected family members. Functional experiments confirming the pathogenicity of the variant were performed by transfecting HEK293T cells and demonstrated the instability and increased rate of proteolysis of the mutated PABPC1L/EPAB protein. The identified variant, located in the well-conserved fourth RNA recognition motif (RRM4), in silico 3D modelling suggested changes in the physical properties of the pathogenic variant of PABPC1L/EPAB. Our findings validate PABPC1L/EPAB as an essential genetic contributor to the oocyte maturation process in humans and have direct implications for the genetic counselling of patients and their family members.

Exome sequencing reveals novel causes as well as new candidate genes for human globozoospermia.

STUDY QUESTION: Can exome sequencing identify new genetic causes of globozoospermia? SUMMARY ANSWER: Exome sequencing in 15 cases of unexplained globozoospermia revealed deleterious mutations in seven new genes, of which two have been validated as causing globozoospermia when knocked out in mouse models. WHAT IS KNOWN ALREADY: Globozoospermia is a rare form of male infertility characterised by round-headed sperm and malformation of the acrosome. Although pathogenic variants in DPY19L2 and SPATA16 are known causes of globozoospermia and explain up to 70% of all cases, genetic causality remains unexplained in the remaining patients. STUDY DESIGN, SIZE, DURATION: After pre-screening 16 men for mutations in known globozoospermia genes DPY19L2 and SPATA16, exome sequencing was performed in 15 males with globozoospermia or acrosomal hypoplasia of unknown aetiology. PARTICIPANTS/MATERIALS, SETTING, METHOD: Targeted next-generation sequencing and Sanger sequencing was performed for all 16 patients to screen for single-nucleotide variants and copy number variations in DPY19L2 and SPATA16. After exclusion of one patient with DPY19L2 mutations, we performed exome sequencing for the 15 remaining subjects. We prioritised recessive and X-linked protein-altering variants with an allele frequency of <0.5% in the population database GnomAD in genes with an enhanced expression in the testis. All identified candidate variants were confirmed in patients and, where possible, in family members using Sanger sequencing. Ultrastructural examination of semen from one of the patients allowed for a precise phenotypic characterisation of abnormal spermatozoa. MAIN RESULTS AND ROLE OF CHANCE: After prioritisation and validation, we identified possibly causative variants in eight of 15 patients investigated by exome sequencing. The analysis revealed homozygous nonsense mutations in ZPBP and CCDC62 in two unrelated patients, as well as rare missense mutations in C2CD6 (also known as ALS2CR11), CCIN, C7orf61 and DHNA17 and a frameshift mutation in GGN in six other patients. All variants identified through exome sequencing, except for the variants in DNAH17, were located in a region of homozygosity. Familial segregation of the nonsense variant in ZPBP revealed two fertile brothers and the patient's mother to be heterozygous carriers. Paternal DNA was unavailable. Immunohistochemistry confirmed that ZPBP localises to the acrosome in human spermatozoa. Ultrastructural analysis of spermatozoa in the patient with the C7orf61 mutation revealed a mixture of round heads with no acrosomes (globozoospermia) and ovoid or irregular heads with small acrosomes frequently detached from the sperm head (acrosomal hypoplasia). LIMITATIONS, REASONS FOR CAUTION: Stringent filtering criteria were used in the exome data analysis which could result in possible pathogenic variants remaining undetected. Additionally, functional follow-up is needed for several candidate genes to confirm the impact of these mutations on normal spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: Our study revealed an important role for mutations in ZPBP and CCDC62 in human globozoospermia as well as five new candidate genes. These findings provide a more comprehensive understanding of the genetics of male infertility and bring us closer to a complete molecular diagnosis for globozoospermia patients which would help to predict the success of reproductive treatments. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by The Netherlands Organisation for Scientific Research (918-15-667); National Health and Medical Research Council of Australia (APP1120356) and the National Council for Scientific Research (CONICET), Argentina, PIP grant 11220120100279CO. The authors have nothing to disclose.

Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer : a multicentre randomized controlled trial.

Despite excellent published results, the lack of well-designed, multicentre, randomized clinical trials results in an absence of general consensus on the efficacy of autologous endometrial cells coculture (AECC) in Assisted Reproductive Technology (ART). An open, multicentre, prospective, randomized controlled trial was designed to compare the pregnancy rate (PR) after the transfer of one blastocyst on day 5 after AECC to the transfer of one embryo on day 3 (control group). Patients were women aged 18 to 36, undergoing an ART cycle with no more than 1 embryo transfer failure. Sample size was calculated at 720 for a superiority trial involving an intermediate analysis at 300 patients. We present the results of the intermediate analysis that resulted in the study ending considering the observed difference. Three hundred thirty nine patients were randomized: 170 in the AECC group and 169 in the control group. The clinical PR per transfer was 53.4% with AECC and 37.3% in the control group (p=0.025). The quality of embryos was improved with AECC. These results suggest that implementation of the AECC technique to a large number of In-Vitro Fertilization (IVF) centres could lead to a substantial improvement in the proportion of successful assisted reproduction. The study was supported by the Laboratoires Genévrier, France.

Assisted oocyte activation overcomes fertilization failure in globozoospermic patients regardless of the DPY19L2 status.

STUDY QUESTION: Does DPY19L2 status influence intracytoplasmic sperm injection (ICSI) outcomes with or without assisted oocyte activation (AOA)? SUMMARY ANSWER: DPY19L2 mutations have no major impact on ICSI outcomes in globozoospermic patients. WHAT IS KNOWN ALREADY: Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking an acrosome. Recently, it has been shown that DPY19L2 mutations can be found in a vast majority of, but not all, globozoospermic patients (66.7%). These patients suffer from primary infertility due to a sperm-related oocyte activation deficiency secondary to the absence of an acrosome that can be overcome by the application of AOA. STUDY DESIGN, SIZE, DURATION: Cohort study, retrospective, 34 patients, 83 cycles. MATERIALS, SETTING, METHODS: Clinical and biologic data were collected from 29 patients mutated for DPY19L2 and 5 non-mutated patients. In total, 35 ICSI cycles using AOA and 48 conventional ICSI cycles were included in the analysis. Patients were divided into groups according to whether or not they were mutated for DPY19L2 and whether or not they received AOA. MAIN RESULTS AND THE ROLE OF CHANCE: Regardless of the presence of a DPY19L2 mutation, the fertilization rates with AOA are restored to normal when compared with conventional ICSI in our cohort of globozoospermic patients. Also, when performing ICSI plus AOA, both mutated and non-mutated cases have similar positive hCG rates, ongoing pregnancy rates and live birth rates per transfer. On the contrary, the fertilization rate in globozoospermic patients using conventional ICSI is correlated with the presence of a DPY19L2 mutation, with slightly better, although still very low, fertilization rates in patients carrying a DPY19L2 mutation. Nevertheless, when performing conventional ICSI, both mutated and non-mutated cases have similar very low positive hCG rates, ongoing pregnancy rates and live birth rates per transfer. LIMITATIONS: A limitation of this study is the low number of included non-mutated cases. WIDER IMPLICATIONS OF THE FINDINGS: We propose a pathway for the clinical management of globozoospermic patients depending on the phenotype that includes several diagnostic and therapeutic steps. STUDY FUNDING/COMPETING INTEREST(S): None.

Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest.

Mest (also known as Peg1), an imprinted gene expressed only from the paternal allele during development, was disrupted by gene targeting in embryonic stem (ES) cells. The targeted mutation is imprinted and reversibly silenced by passage through the female germ line. Paternal transmission activates the targeted allele and causes embryonic growth retardation associated with reduced postnatal survival rates in mutant progeny. More significantly, Mest-deficient females show abnormal maternal behaviour and impaired placentophagia, a distinctive mammalian behaviour. Our results provide evidence for the involvement of an imprinted gene in the control of adult behaviour.

Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization.

Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only.

Creation of a registry for human embryonic stem cells carrying an inherited defect: joint collaboration between ESHRE and hESCreg.

Human embryonic stem cells (hESCs), derived from human blastocysts, hold a great promise for regenerative medicine, drug development and basic research in developmental biology. Moreover, hESC lines that carry a clinically relevant inherited defect, monogenic or chromosomal, present an important tool for research into the pathophysiology of these diseases. The hESC registry (hESCreg) was started up in 2007 in order to register all stem cell lines derived in Europe (www.hescreg.eu). Because of the special nature of the hESC lines that carry an inherited disease, they are of particular interest to researchers outside the assisted reproductive technologies or stem cell fields, for instance, those involved in regenerative medicine and in medical and human genetics. To reach these researchers, and to better disseminate the information on the cell lines, a concerted action of the hESCreg together with ESHRE's Special Interest Groups in Reproductive Genetics and Stem Cells was initiated. This mini-review is a first report that will be followed by yearly reports of new lines, not unlike the reports from the Preimplantation Genetic Diagnosis Consortium or the European IVF Monitoring.

Diversity of endogenous epitopes bound to MHC class II molecules limited by invariant chain.

The invariant chain (Ii) binds nascent major histocompatibility complex (MHC) class II molecules, blocking peptide binding until the complex dissociates in the endosomes. This may serve to differentiate the MHC class I and II antigen presentation pathways and enable class II molecules to efficiently bind peptides in the endosomes. This hypothesis was addressed by probing spleen cells from a combination of knock-out and transgenic mice with a large panel of T cell hybridomas. The Ii molecule blocked the presentation of a range of endogenously synthesized epitopes, but some epitopes actually required Ii. Thus, the influence of Ii on presentation does not follow simple rules. In addition, mice expressing Ii were not tolerant to epitopes unmasked in its absence, a finding with possible implications for autoimmunity.

[Genetic aspects of male infertility: From bench to clinic]. / Aspect génétique de l'infertilité masculine : de la recherche à la clinique.

OBJECTIVES: The objective of our manuscript is to review the current state of research on the genetics of male infertility, highlighting the genetic abnormalities that can lead to non-syndromic male infertility and genetic testing proposed to patients. It is intended primarily for clinicians and biologists of reproductive medicine. METHODS: A comprehensive review of the scientific literature available on PubMed was conducted using keywords related to male infertility and genetics. Since the first genes related to non-syndromic male infertility were identified after the 2000s, bibliographic research was conducted after this date. RESULTS: Thirty-three genes have been identified as responsible for non-syndromic male infertility. The evolution of techniques based on whole genome analysis has allowed the development of more successful methods in the identification of new genes and mutations inducing an infertility phenotype. Through this article, we propose, by concrete examples, a clinical approach for genetic tests considering the semen analysis alterations. CONCLUSIONS: The identification and characterization of these genes and the mutations responsible for certain infertility phenotypes allow better management and better treatment for patients as well as a better understanding of the physiopathological mechanisms of human gametogenesis.

What next for preimplantation genetic screening? More randomized controlled trials needed?

The recent debate on preimplantation genetic screening (PGS) has raised questions about its routine use in clinical practice. It has been suggested that the most effective way to resolve the debate about the usefulness of PGS is to perform more well-designed and well-executed randomized controlled trials (RCTs). However, in view of the lack of evidence for the effectiveness of PGS and the accumulating evidence for its harmfulness, it is our opinion that it is unethical to perform additional RCTs for the indication advanced maternal age using cleavage stage biopsy.

ESHRE PGD consortium data collection VII: cycles from January to December 2004 with pregnancy follow-up to October 2005.

The seventh report of the ESHRE PGD Consortium is presented documenting cycles collected for the calendar year 2004 and follow-up of the pregnancies and babies born subsequent to these cycles up to October 2005. Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported. For data collection VII, 45 centres have participated, reporting on 3358 cycles to oocyte retrieval (OR), 679 pregnancies and 528 babies born. Five hundred and fifty nine OR were reported for chromosomal abnormalities, 113 OR for sexing for X-linked diseases, 520 OR for monogenic diseases, 2087 OR for PGS, and 79 OR for social sexing. Data VII is compared with the cumulative data for data collections I-VI.

Recent developments in genetics and medically assisted reproduction: from research to clinical applications.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Fine-tuning of MHC class II gene expression in defined microenvironments.

Strict control of major histocompatibility complex class II gene expression is essential for proper functioning of the immune system. Recent transgenic mouse studies have revealed an intricate fine-tuning of class II gene transcription in microenvironments such as the germinal centers and thymic cortex and medulla.

[Huntington disease: presymptomatic testing, prenatal diagnosis, preimplantation genetic diagnosis experience]. / Maladie de Huntington: l'expérience du test présymptomatique, du diagnostic prénatal et préimplantatoire.

Presymptomatic testing for Huntington disease has been available for 15 years. The possibility of determining the genetic status of an at-risk person for the disorder which runs in his or her family raises questions because of the absence of preventive treatments. In addition, being carrier does not allow to determine when the disease starts and how it will evolve, impairing the possibilities of planning the future. A pluridisciplinary approach to predictive testing with care before, during and after the test taking into account the medical, social and psychological aspects of the disease is good practice. At the present time, only a minority of at-risk individuals request presymptomatic testing and almost 50% do not pursue until the results. The consequences of the test may be harmful, more frequently after an unfavorable than after a favorable result. Motivations and the outcome in terms of request for prenatal testing after a carrier result are known today and the number or prenatal testing remains very limited. Preimplantation genetic testing is an alternative for couples who knows or do not their own genetic status. We report our experience in two French centres: Paris for presymptomatic and prenatal testing and Strasbourg for preimplantation diagnosis.

[Embryo donation in France: practice and difficulties. Strasbourg's experience]. / L'accueil d'embryon en France: pratiques et difficultés. L'expérience de Strasbourg.

OBJECTIVE: The aim of this study was to present the situation of embryo donation in France and around the world, to expound the difficulties of its practice and the results obtained in our centre 3 years after the introduction of this procedure. PATIENTS AND METHODS: Embryo donation in France is controlled by implemented decrees published between 1999 and 2004. The couples, who have stored frozen embryos since at least two years, were contacted for a pluridisciplinary medical consultation. The indication of embryo donation was evaluated for the recipients through a pluridisciplinary approach. RESULTS: Among the interviewed couples, 16.7% have chosen embryo donation but only half of them have completed the procedure (6% of the couples with frozen embryos). The main indications for embryo donation were a double sterility, unexplained genetic disease, ART failures (poor fertilization or bad embryo quality) and oocyte donation when the delay was too long for the couples. The pregnancy rate was 28.6% after the 21 first embryo transfers. DISCUSSION AND CONCLUSION: The results of embryo donation confirm the international experience both considering the poor number of donated embryos, medical indications and results. Embryo donation has its place among ART techniques, but one should not ignore the general debate on ethical questions raised by this procedure.

Recent developments in genetics and medically-assisted reproduction: from research to clinical applications†‡.

Two leading European professional societies, the European Society of Human Genetics and the European Society for Human Reproduction and Embryology, have worked together since 2004 to evaluate the impact of fast research advances at the interface of assisted reproduction and genetics, including their application into clinical practice. In September 2016, the expert panel met for the third time. The topics discussed highlighted important issues covering the impacts of expanded carrier screening, direct-to-consumer genetic testing, voiding of the presumed anonymity of gamete donors by advanced genetic testing, advances in the research of genetic causes underlying male and female infertility, utilisation of massively-parallel sequencing in preimplantation genetic testing and non-invasive prenatal screening, mitochondrial replacement in human oocytes, and additionally, issues related to cross-generational epigenetic inheritance following IVF and germline genome editing. The resulting paper represents a consensus of both professional societies involved.

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: teratozoospermia.

Teratozoospermia is characterized by the presence of spermatozoa with abnormal morphology in sperm. This condition is frequently associated with infertility and intracytoplasmic sperm injection (ICSI) is frequently used as the treatment of choice. However, the use of ICSI has created consequential debate concerning the genetic risk for the offspring. Fluorescence in situ hybridization technique (FISH), allowing the specific identification of human chromosomes in sperm nuclei, has been used to study chromosome abnormalities in sperm from men with teratozoospermia and a normal karyotype. In this review, we present studies that have tried to determine if men with a normal blood karyotype but suffering from teratozoospermia present a higher aneuploidy frequency. The literature is limited to three forms of teratozoospermia. The first group consists of "polymorphic teratozoospermia", where a majority of spermatozoa display more than one type of abnormality. In this case, only a slight increase in aneuploidy frequency is observed, which cannot be differentiated from the results observed in oligo-astheno-teratozoospermia (OAT). The second group, named "globozoospermia", is characterized by round spermatic heads, absence of acrosome and disorganization of mid-piece and tail. In this case, some studies have shown a significant, but moderate, increase in the aneuploidy frequency for acrocentrics and sex chromosomes. The aneuploidy frequency remains low, also ICSI can be proposed to these patients, but few successes occur. The third group consists of "enlarged head teratozoospermia", where almost all spermatozoa have an enlarged head, multiple tail and abnormal acrosome. In this case a very high level of missegregation is observed, leading to nearly 100% aneuploidy. In this particular group, ICSI must be refuted, and patients have to be redirected to other possibilities, like sperm donation.

Tissue-specific expression of retinoic acid receptor isoform transcripts in the mouse embryo.

The three murine retinoic acid receptor (RAR) genes each contain two distinct promoters which give rise to protein isoforms differing in their N-terminal regions. This study used in situ hybridization to describe the expression patterns of RARalpha1, RARalpha2, RARbeta1/3, RARbeta2/4, RARgamma1 and RARgamma2 isoform transcripts during mouse embryogenesis. RARalpha1 transcripts are widely distributed, with the exception of the central nervous system. Highest expression is found in developing muscle, pituitary gland and various epithelia. On the other hand, RARalpha2 is essentially expressed along the spinal cord up to the hindbrain 7th rhombomere and in the 4th rhombomere, pons and developing basal ganglia (corpus striatum and pallidum). RARbeta2/4 transcripts account for most of the previously described RARbeta expression features being expressed specifically, or more prominently than RARbeta1/3, in foregut endoderm and its derivatives, olfactory and periocular mesenchyme, urogenital region, proximal limb bud mesenchyme and later within interdigital regions. RARbeta1/3 is more prominently expressed in the developing heart outflow tract mesenchyme, intervertebral disks, midgut loop mesenchyme and umbilical vessel walls. RARbeta1/3 and RARbeta2/4 are coexpressed in the developing corpus striatum. They exhibit, however, distinct dorsoventral distributions along the spinal cord and caudal hindbrain. RARgamma2 is the RARgamma isoform expressed at high levels in the caudal neural groove at embryonic day 8.5. At later stages, both RARgamma isoforms are essentially coexpressed, although the progressive restriction of RARgamma1 transcripts to craniofacial or limb precartilaginous condensations appears to precede that of RARgamma2.

[Preimplantation genetic diagnosis of monogenic diseases]. / Le diagnostic génétique préimplantatoire des maladies monogéniques.

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis allowing the detection of genetic diseases on IVF embryos before their transfer into the uterus and before the pregnancy. The aim of this procedure is to obtain unaffected or carrier embryos in order to avoid the burden of termination of pregnancy after prenatal diagnosis for couples at risk of transmitting particularly severe genetic disorders to their offspring. For monogenic diseases, PGD is most often based on single blastomere amplification by polymerase chain reaction (PCR). More than a decade after the first births, the possibilities of diagnosis for monogenic diseases have considerably increased. As for molecular biology and conventional diagnosis, the technologies and strategies for PGD are continually improved, with for instance introduction of fluorescent PCR or multiplex amplification. In this review, we describe several approaches for PGD of monogenic diseases, followed by an overview of the French practice, particularly in our lab.

[Abnormal bacterial colonisation of the vagina and implantation during assisted reproduction]. / Colonisation bactérienne vaginale anormale et implantation en assistance médicale à la procréation.

OBJECTIVE: To evaluate the efficiency of our treatment of vaginal infection for couples included in an IVF program. PATIENTS AND METHODS: Microbiologic screening of vaginal flora and semen has been performed one month prior to in vitro fertilization for 951 couples in 2000. Antibiotic treatment was prescribed in case of positive culture. RESULTS: Positive microbial growths were observed from endocervical and vaginal cultures in 218 women (22.9%). The clinical pregnancy rate was 30.29% in the group of patients without growth and 30.27% in the group with positive microbial growth. The implantation rate was significantly diminished in case of bacterial growth: 14.6 compared to 19.3% (P <0.02) for sterile endocervical culture. Five main bacterial species were found at the cervical level: Candida albicans (69 cases), Ureaplasma urealyticum (49 cases), Gardnerella vaginalis (43 cases), Streptococcus B or D (24 cases) and Escherichia coli (22 cases). Positive cultures from both vagina and semen were observed for 77 couples whose clinical pregnancy rate was 19.5 vs 36.2% in case of vaginal infection alone (P <0.01) with a spontaneous miscarriage rate of 46.7 compared to 17.6% (P <0.01). DISCUSSION AND CONCLUSION: Endocervical microorganisms, even treated with adapted antibiotics, may affect embryonic implantation. Positive culture from both female and male partner may enhance this negative effect. In this case, the best strategy would be to cancel the IVF treatment.