STUDY QUESTION:Which clinical definition of oocyte, zygote, and embryo maturation arrest (OZEMA) merits genetic investigation? SUMMARY ANSWER:The diagnosis OZEMA is considered when: only two mature oocytes are obtained from six cumulus-oocyte complexes (COCs), only one zygote is obtained from six metaphase II oocytes (MIIs), or no blastocysts are obtained from six two-pronuclei zygotes (2PNs). WHAT IS KNOWN ALREADY:OZEMA is a cover term for various defects, including oocyte abnormalities, fertilization failure, cleavage arrest, and abnormal embryo development, often associated with specific genetic variants. While some variants in specific genes are associated with distinct phenotypes, others commonly display variable expressivity across cycles and patients. Currently, there are no standardized cut-off values defining the OZEMA disorder. STUDY DESIGN, SIZE, DURATION:This is a pooled secondary analysis of aggregated data extracted from published studies. A PubMed search was conducted to identify peer-reviewed original studies in the human reporting genetically confirmed cases of OZEMA. Studies were included if patients had displayed OZEMA in at least two medically assisted reproduction (MAR) cycles. In total, clinical and genetic data were extracted from 132 publications, including 521 patients and 39 genes. PARTICIPANTS/MATERIALS, SETTING, METHODS:Clinical phenotypes from 132 publications were classified into oocyte maturation arrest (OMA), zona pellucida abnormality (ZPA), fertilization failure (FF), zygote arrest (ZA), early embryo arrest (EEA), mixed phenotypes, and other abnormalities. Data at both cycle and patient levels were evaluated using ratios corresponding to key developmental milestones (ratio of MII/COC, 2PN/MII, cleaved embryos/2PN, blastocysts/2PN). These ratios were compared with the expected values Vienna Consensus binomial modeling to assess whether the observed proportions were significantly lower than the established competency thresholds (P < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE:This review identified 39 OZEMA-associated genes. Analysis of 668 cycles from 253 patients carrying variants in 35 genes showed abnormal developmental outcomes in 93%, based on binomial modeling. Reliable diagnosis of OZEMA requires a sufficient number of COCs, MIIs, or 2PNs to distinguish true developmental impairment from normal biological variation. When these minimal thresholds are not reached, statistical power is insufficient to reliably confirm developmental arrest. Based on our findings, OZEMA is diagnosed when only two or fewer mature oocytes form from six COCs, one or no zygote forms from six MIIs, or no blastocysts form from six 2PNs. LIMITATIONS, REASONS FOR CAUTION:This study is limited by its retrospective design and reliance exclusively on published data from women selected for poor IVF outcomes and the presence of variants in candidate OZEMA genes, which may introduce publication bias and reporting heterogeneity. Therefore, this study does not report on genetic yield from women with normal IVF outcomes. Variability in clinical protocols and genetic testing methods, limited ancestral diversity, and missing data across studies could contribute to confounding factors. WIDER IMPLICATIONS OF THE FINDINGS:This study is an important step toward understanding the clinical and genetic spectrum of OZEMA, highlighting the variability of and overlap between affected subphenotypes. Establishing cut-off values against consensus oocyte and embryo developmental competency levels provides a benchmark for more accurate classification. Importantly, this represents the most comprehensive analysis of published OZEMA cases with genetic variants to date, strengthening the reliability of the proposed clinical thresholds. These findings may guide personalized treatment strategies and genetic counseling, ultimately improving outcomes in MAR. STUDY FUNDING/COMPETING INTEREST(S):No funding was received for this study. The authors declare no competing interests. REGISTRATION NUMBER:Not applicable.

To explore genetic basis leading to meiotic disruption in human gametogenesis via exome sequencing. This study included three consanguineous families with well-defined infertility phenotypes. Exome sequencing was performed for the index case in family 1 and for the trio (index with parents) in the other two families. Sanger sequencing was used for confirmation and family segregation analysis. Exome sequencing revealed homozygous loss-of-function variations in SPIDR, TOP6BL, and RAD51AP2 in families 1, 2, and 3, respectively. Segregation in individual families revealed that the parents were carriers, as were the fertile siblings in families 1 and 2. All three genes function in double-strand break formation or repair, identified variants may therefore impair, potentially preventing its completion and contributing to infertility in the index cases. Gene-disease relationships (GDR) were re-evaluated due to the addition of new patients and/or variants in the literature. Our findings provide additional evidence for the role of SPIDR, TOP6BL, and RAD51AP2 as genetic contributors to human infertility due to meiotic errors. For patients with a similar phenotype, genetic screening could be recommended, and the identification of pathogenic variations might help avoid unsuccessful fertility treatments. Additionally, in patients with molecular defects in DNA repair genes, chromosomal instability may increase the risk of cancer; therefore, long-term follow-up by a multidisciplinary team is recommended.

Objective To improve fertility outcomes of infertility associated CCDC188 variations in humans affected by Acephalic Spermatozoa Syndrome. Design Case report. Subjects A 20-year-old man from a consanguineous family applied to our fertility clinic. Semen analysis revealed 100% acephalic spermatozoa, preventing sperm concentration assessment. Exome-based gene panel analysis identified a novel pathogenic homozygous nonsense variation in CCDC188. Exposure Based on CCDC188-null mice demonstrating the presence of a few intact spermatozoa in the testis (while 100% acephalic in the epididymis) and recently reported poor ICSI outcome with fresh semen, ICSI using fresh testicular sperm was proposed. Main Outcome Measures Molecular genetic results, fertilization outcomes, biochemical pregnancy. Results Conventional testicular sperm extraction TESE was performed on the day of the oocyte retrieval. Microscopic examination revealed a few intact motile spermatozoa at 2 sites. 13 cumulus-oocyte complexes were retrieved, 11 mature oocytes were injected with motile testicular sperm, and artificial oocyte activation was applied. 2 oocytes were fertilized normally and developed into high-quality embryos, which were cryopreserved. Frozen embryo transfer in a natural cycle resulted in a biochemical pregnancy. Conclusion TESE combined with ICSI may represent a potential therapeutic approach in infertility related to CCDC188 variations. Accurate clinical phenotyping and complementary genetic analysis are crucial for developing patient-specific therapeutic strategies in medically assisted reproduction.

Study question : Do variants in HSP90AA1 cause human male infertility? Summary answer: Variants in HSP90AA1 appear as a possible autosomal dominant cause of human male infertility. What is known already : Male infertility is a highly heterogeneous condition, with so far over 300 genes described in this context. HSP90AA1 appears as a promising candidate gene for human male infertility, because the gene is highly conserved between species and knock-out of Hsp90aa1 in mice results in male-specific infertility due to azoospermia without further health implications. Study design, size, duration : We screened >2,500 infertile men for possibly pathogenic variants in HSP90AA1 and created a mouse line harbouring the homozygous missense variant c.605G>A p.(Arg202Lys). Participants/ materials, setting, methods : Phenotypes of men with identified variants were determined based on semen analysis and testicular histology. Pathogenicity of detected variants was assessed using AlphaMissense and a mouse model. Male fertility of the mutant mouse line was analysed via plug-matings, histology and immunofluorescence staining (IF). Expression of HSP90AA1 in testicular tissue was assessed by IF. Main results and the role of chance : The mode of inheritance (MOI) in mice is autosomal recessive but the constraint metrics (oe-score = 0.2, pLI = 1) and in silico prediction suggest that HSP90AA1 is an autosomal dominant gene in humans. We therefore screened for both, heterozygous and biallelic variants in exome sequencing data of infertile men. While we did not detect any biallelic loss-of-function variants, we identified the homozygous missense variant c.605G>A p.(Arg202Lys) in an azoospermic man as a promising variant. This variant is extremely rare and affects a highly conserved amino acid. However, male homozygous mice with this variant are fertile with no differences in litter size and testicular size or histology, making it unlikely that this variant is the cause of the man′s azoospermia. We therefore focused on heterozygous possibly pathogenic variants in HSP90AA1 and found a heterozygous frameshift variant in an azoospermic man with hypospermatogenesis as well as four heterozygous missense variants, predicted to affect protein function in azoo- or cryptozoospermic men. Large scale data : N/A Limitations, reasons for caution : Our findings suggest a dominant MOI but currently cannot fully prove this. To further clarify the MOI and ultimately improve clinical validity of HSP90AA1 replication of our findings in independent cohorts of infertile men as well as segregation analyses are required. Wider implications of the findings : While most human male infertility genes follow an autosomal recessive MOI, HSP90AA1 might be one of the few autosomal dominant infertility genes in humans. Differences in the MOI between humans and mice are also known from well-established infertility genes such as DMRT1 . Study funding/ competing interest(s) : This work was supported by a German Research Foundation (DFG) fellowship (award WY 215/1-1 to MJW), the DFG-sponsored Clinical Research Unit ′Male Germ Cells′ (CRU326, project 329621271 to FT), and Wellcome Trust funding (225237 to DOC). This work was supported by funding for the Wellcome Discovery Research Platform for Hidden Cell Biology (226791). ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by a German Research Foundation (DFG) fellowship (award WY 215/1-1 to MJW), the DFG-sponsored Clinical Research Unit Male Germ Cells (CRU326, project 329621271 to FT), and Wellcome Trust funding (225237 to DOC). This work was supported by funding for the Wellcome Discovery Research Platform for Hidden Cell Biology (226791). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol was approved by the respective local ethics committees: MERGE cohort Muenster (2010-578-f-S) and Giessen (26/11); Strasbourg (CPP 09/40 WAC 2008-438 1W DC 200 9I 002), and Yeni Yuzyıl University, Scientific, social and noninterventional health sciences research ethics committee, Istanbul, Turkey (approval no: 2019/08). All persons gave written consent compliant with local requirements and all experiments were performed in accordance with the criteria set by the Declaration of Helsinki. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

In recent years, an increasing number of genes associated with male and female infertility have been identified. The genetics of infertility is no longer limited to the analysis of karyotypes or specific genes, and it is now possible to analyse several dozen infertility genes simultaneously. Here, we present the diagnostic activity over the past two years including 140 patients (63 women and 77 men). Targeted sequencing revealed causative variants in 17 patients, representing an overall diagnostic rate of 12.1%, with prevalence rates in females and males of 11% and 13%, respectively. The gene-disease relationship (GDR) was re-evaluated for genes due to the addition of new patients and/or variants in the actual study. Five genes changed categories: two female genes (MEIOB and TBPL2) moved from limited to moderate; two male genes (SOHLH1 and GALNTL5) moved from no evidence to strong and from limited to moderate; and SEPTIN12, which was unable to classify male infertility, was reclassified as limited. Many infertility genes have yet to be identified. With the increasing integration of genetics in reproductive medicine, the scope of intervention extends to include other family members, in addition to individual patients or couples. Genetic counselling consultations and appropriate staffing will need to be established in fertility centres. Trial registration number: Not applicable.

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells. piRNAs are small RNA molecules found primarily in the testes of mice and men. Stallmeyer et al. demonstrate that variants in specific genes can disrupt the formation of piRNAs, impairing spermatogenesis and causing human male infertility.

Abstract Female infertility is a complex issue affecting a significant number of women. In the United States, among married women aged 15–49 years with no prior births, approximately one in five (19%) were unable to get pregnant after one year of trying. In addition, approximately one in four (26%) women in this group had difficulty getting pregnant or carrying a pregnancy to term. For women under 35 years of age, infertility is defined as a lack of success within one year, while for women aged 35 and older, it is within six months. Various factors, such as genetic, endocrine, physiological, anatomical, and immunological irregularities within the reproductive system, can affect a woman's chances of achieving pregnancy and delivering a healthy child. Among these factors, oocyte maturation is an important prerequisite for successful fertilization and subsequent embryonic development. In Medically Assisted Reproduction (MAR), the meiotic maturation of oocytes is induced by human chorionic gonadotropin (hCG) injection, which mimics the natural endogenous luteinizing hormone (LH) surge during the menstrual cycle. Although it is common for a few oocytes to remain immature despite ovarian stimulation and hCG administration, generally, immature oocytes have the competency to spontaneously mature in vitro. However, oocyte maturation is a sophisticated process involving multiple genes or protein molecules. Therefore, pathogenic variations in the genomic sequence or functional defects in any molecule that affects meiosis may lead to impaired oocyte maturation. While complete failure of all oocytes to mature in vivo is extremely rare, understanding the molecular aetiology of oocyte maturation is crucial for addressing infertility issues related to this process and improving fertility outcomes. The transition from the cleavage to the blastocyst stage often represents a significant bottleneck, with nearly 10% of fertilized eggs arrested at the cleavage stages. Developmental arrest of the preimplantation embryo is characterized by the cessation of cellular division for at least 24 h. Various factors originating from both embryonic and parental sources contribute to arrest of early stage embryo development. The genetic roots of developmental arrest were examined by considering both the parental causes of infertility and factors within the embryo. Examination revealed shared elements, regardless of the source of origin. Chromosomal abnormalities, abnormal preimplantation development, and monogenic variations have been reported to cause embryonic developmental arrests. Recently, deficiencies in oocyte maturation and embryo development have been categorized as part of the oocyte/zygote/embryo arrest (OZEMA) phenotype in the Online Mendelian Inheritance in Man (OMIM) database. A latest extensive review of monogenic causes associated with female infertility identified 23 genes with a total of 27 gene disease relationships (GDRs) for the OZEMA phenotype. Seventeen GDRs were classified as having at least moderate evidence of involvement in OZEMA, in addition to ten GDRs with less substantial evidence. Several additional genes were subsequently identified. This presentation will provide an overview of gene defects linked to the OZEMA phenotype, discuss the current status and future prospects of pre-MAR screening of females with OZEMA, and assess a novel workflow for modern genetic diagnosis to maximize the benefit of the patient.

Objective The ultimate treatment management of NOA involves testicular sperm extraction; however, in almost half of the men with NOA, no sperm can be retrieved. DNA defects such as double-strand breaks (DSBs) can disturb meiotic recombination and cause infertility in men due to complete early meiotic arrest. To date, few genes participating in DSB formation and/or repair have been associated with infertility in humans. However, the relationship between the failure of the meiotic DSB process and human infertility remains poorly understood. We aim to identify new genetic causes responsible for the nonobstructive azoospermia (NOA) with meiotic arrest by using exome sequencing Materials and Methods This case-control study was conducted on two Turkish consanguineous families, comprising infertile men with NOA. Testicular histopathology results showed meiotic arrest at the spermatid stage. Karyotypes were normal and no Y chromosome microdeletion was detected in the affected men. Ethical approval was obtained from the Comité de Protection de la Personne (CPP) of Strasbourg University Hospital.Saliva samples from index cases and both parents were collected after obtaining written informed consent. Exome sequencing was performed using the GenomEast Platform (IGBMC, Strasbourg, France). Detected variants were annotated and ranked by VaRank (v1.4.3). Results Exome sequencing revealed homozygous pathogenic variations in two genes namely C11orf80 (also known as TOPBBL) and RAD51AP2. The first gene is related to meiotic DNA DSB formation, while the latter is associated with DSB repair in the respective families. Meiosis could not be completed in either situation, resulting in spermatogenesis arrests at the pachytene stage of meiosis 1. Two recent reports described four NOA men from two families presenting homozygous truncating variants in C11orf80. Similarly, homozygous or compound heterozygous loss-of-function variations in RAD51AP2 were previously identified in four men diagnosed with idiopathic NOA. Since the identification of both genes were after the latest systematic review, they were neither scored norvalidated for the male infertility. The gene-disease relationship (GDR) was evaluated by including published patients and variants as well as results from the actual study. C11orf80 and RAD51AP2 were scored as 13 and 14 respectively with a classification of “strong” for both genes. Conclusion Our findings support the role of C11orf80 and RAD51AP2 as genetic contributors to human infertility by causing meiotic arrest during spermatogenesis in men. Discussion The main limitation of our study is the small number of cases included. However, it provides fresh data regarding on NOA phenotypes associated with meiotic arrest. For men with a similar phenotype, genetic screening for C11orf80 and RAD51AP2 could be recommended, and the identification of pathogenic variations might help to avoid unsuccessful testicular biopsy procedures.

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.

Abstract Study question What is the diagnostic yield of custom designed gene panel for patients with premature ovarian insufficiency (POI)? Summary answer The diagnostic yield of our POI gene panel (POIGP) is 7.3% What is known already POI is a specific female syndrome with a high clinical and genetic heterogeneity. It is characterized by a premature exhaustion of the ovarian function and infertility and affects approximately 1% of women. POI can be related to genetic factors which include chromosomal abnormalities, FMR1 premutation and rare variants in numerous genes. The advent of high throughput sequencing methods has led to the identification of an increasing number of variants implicated in the development of POI over the last decades. However, POI etiologies still remain undetermined in the majority of cases. Study design, size, duration An observational analytic cohort study of 150 patients presenting idiopathic POI (normal karyotype, absence of FMR1 premutation, absence of adrenal and/or ovarian antibodies) recruited prospectively at three Belgian academic and university hospitals between 2016 and 2021. Participants/materials, setting, methods Patients were included if they experienced POI, as defined by ESHRE guidelines on POI (2016). POI genes included in the panel were selected from PubMed using different key words mainly premature ovarian insufficiency, gonadal dysgenesis, hypergonadotropic hypogonadism, ovarian failure and genetics. The panel included 156 genes, variants were filtered based on allele frequency (≤1%) in latest available population databases and classified according to ACMG/AMP (American College of Medical Genetics/Association for Molecular Pathology) guidelines 2015. Main results and the role of chance Our analysis revealed a potential causative variant for 11 patients in the following genes: MEIOB, BMP4, CFTR, FANCA, FSHR, FANCG, MLH1, MRPS22 and STARD9. This means that the diagnostic yield of our POI gene panel (POIGP) is 7.3%. Patients were mainly Caucasian (63%), North African (17%) and sub-Saharan African (13%). They presented primary amenorrhea in 14.7% of cases. Consanguinity and/or a family history of POI or early menopause in 28% of cases. Mean patient’s age (years) at POI diagnosis was 28.9± 8.5 (mean ± SD). The overall mean coverage was 229X, and more than 95% of the target exome was represented with more than 30-fold coverage. Limitations, reasons for caution The present study was limited to monogenic etiologies of POI, potential oligogenic causes have not been searched. Functional studies and/or family segregation were not performed for the identified variants. Wider implications of the findings Our findings show the importance of targeted next generation sequencing in clinical practice and highlight the limit of our current genetic knowledge in the field of POI. A regular update of genes included in POIGP will improve its diagnostic yield. Trial registration number P2016/196/CCB B406201628264

The World Health Organization (WHO) defines infertility as the inability to conceive within 12 months despite regular unprotected intercourse, a condition that concerns about 10–15% of couples globally. Infertility is considered as primary or secondary depending on whether a couple has experienced a prior pregnancy or not.

Abstract Study question Is there a risk factor association or a monogenic relationship between FMR1 premutation and developing Fragile X-associated primary ovarian insufficiency (FXPOI)? Summary answer There is an association between FMR1 premutation and FXPOI rather than a monogenic relationship, which is highly dependent on ethnicity. What is known already Among the 40 genes involved in primary ovarian insufficiency (POI), identified in our recent systematic review (Van Der Kelen et al., 2022), the FMR1 premutation is considered as the most common cause of POI. A premutation in the FMR1 gene is defined as a CGG trinucleotide repeat length between 55 and 200 in the 5′ untranslated region. The term FXPOI is used for women with premutation in FMR1 who have a loss of normal function of the ovaries before the age of 40. There is conflicting evidence about the relationship between POI and length of premutation FMR1 alleles. Study design, size, duration Curated publications identified in PubMed and Web of Science on genetics of human female infertility and sex development by using the MESH terms, key words and inclusion/exclusion criteria described in our comprehensive systematic review were subjected to screening for “FMR1 gene.” The articles included cover a period from 1988 to the 1st of November 2021. Participants/materials, setting, methods A total of 161 publications in PubMed and 61 unique publications in Web of Science were identified, and subsequently screened for triplet expansion, primary ovarian insufficiency, and/or early menopause. Of these, 56 papers were selected, excluding publications exclusively on males as well as studies on non-human species, pediatric cases, reviews, and expression studies. The study and control groups repeat numbers, ethnicity, and conclusions in the article are listed. Main results and the role of chance Expansion in CGG trinucleotide repeat length happens at female meiosis and the risk for expansion increases with an increasing number of CGG repeats. There is an intermediate zone between 45 and 55 CGG repeats when the allele may be stable or unstable. Among 56 publications, 54 describing FMR1 triplet repeat size in women with POI, 34 were studying only the women with POI (observational) while 20 were including also control group (comparative). 2 publications were meta-analyses in which 13 and 11 case control studies were included respectively, 5 studies being in common. POI-associated premutations showed a wide range of repeat sizes. Women carrying midsize range repeats (>70-<100) potentially have a higher risk for POI compared to the general population. In recent decades, population-based screenings have indicated that FMR1 premutations are not as prevalent in women with ovarian insufficiency as previous estimates have suggested, but they still represent a substantial cause of POI. When present, the number of AGG interruptions and the size of uninterrupted CGG repeats are directly correlated with the ovarian reserve. No increased risk of POI associated with a premutation was reported among populations of non-European descent, such as the Han Chinese, Indian, and Jordan populations. Limitations, reasons for caution Available reports are difficult to compare because the ethnicity and number of patients analyzed, availability of clinical data and the quality of results are different in each study. Additionally, an effect of X-inactivation in POI women with premutation was not studied. Wider implications of the findings Owing to the low penetrance and a molecular mechanism that has not yet been fully elucidated, the clinical utility of FMR1 screening in women with reduced ovarian reserve needs further investigation before clinical implication. Trial registration number N/A

STUDY QUESTION Can the analysis of a large Turkish consanguineous family via whole exome sequencing (WES) identify novel causative genetic variation responsible for nonobstructive azoospermia (NOA) characterized by arrest at primary spermatocyte stage? SUMMARY ANSWER WES analysis revealed a homozygous nonsense variant in HORMAD1 in three affected brothers of a Turkish family. WHAT IS KNOWN ALREADY Studying patient cohorts in small or large consanguineous families using high-throughput sequencing allows the identification of genetic causes of different pathologies, including infertility. Over the last two decades, a number of genes involved in human male infertility have been discovered, but only 14 genes have been identified as being at least moderately linked to isolated NOA or oligozoospermia in men. STUDY DESIGN, SIZE, DURATION The study included a Turkish family comprising three brothers with NOA. Two brothers had a normal karyotype, normal hormonal levels and no Yq microdeletion. The testicular histopathology analysis revealed the complete arrest of spermatogenesis at the primary spermatocyte stage. PARTICIPANTS/MATERIALS, SETTING, METHODS We recruited a consanguineous Turkish family where parents were first-degree cousins and had seven children; three sons who had NOA, two sons who were fertile and two daughters for whom no information was available. Saliva samples from the index patient, his two affected brothers, parents and two nonaffected brothers (seven samples in total) were collected. Prior to WES, the index patient underwent targeted genetic testing using an infertility panel, which includes 133 infertility genes. No pathogenic variations were identified. WES was then performed on the DNA of the seven family members available. Bioinformatics analysis was performed using an in-house pipeline. Detected variants were scored and ranked, and copy number variants were called and annotated.The consequences of mutation on protein expression and localization were investigated by cell transfection followed by immunofluorescence or immunoblotting. MAIN RESULTS AND THE ROLE OF CHANCE WES revealed a homozygous nonsense variant chr1:150675797G>A; HORMAD1 (NM_032132.5): c.1021C>T, p.Gln341* in exon 13, which was confirmed in all three affected brothers. HORMAD1 encodes the HORMA domain-containing protein 1. The parents as well as the two fertile brothers were carriers of this variant. This variant may lead to the production of a truncated protein lacking the nuclear localization signal; therefore, human cells were transfected with the wild-type and mutated form, in fusion with green fluorescent protein. Immunoblotting experiments confirmed the production of a truncated HORMAD1 protein, and immunofluorescence microscopy revealed that the mutated protein displayed cytoplasmic localization while the wild type protein located to the nucleus. Altogether, our findings validate HORMAD1 as an essential genetic factor in the meiotic process in human. LIMITATIONS, REASONS FOR CAUTION According to one scoring system used to evaluate the clinical validity of male infertility genes, this study would classify HORMAD1 as displaying limited clinical evidence of being involved in male infertility. However, such a score is the maximum possible when only one family is analyzed and the addition of one patient showing a pathogenic or likely pathogenic variant would immediately change this classification to 'moderate'. Thus, this report should prompt other researchers to screen patients with NOA for this genetic variant. WIDER IMPLICATIONS OF THE FINDINGS Identification of new genetic factors involved in the human meiosis process will contribute to an improvement of our knowledge at the basic level, which in turn will allow the management of better care for infertile patients. Since Hormad1-/- knock-out female mice are also infertile, HORMAD1 could also be involved in human female infertility. Our findings have direct implications for the genetic counseling of patients and their family members. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by Fondation Maladies Rares (High Throughput Sequencing and Rare Diseases-2018, 'GenOmics of rare diseases'). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

The field of reproductive genetics has undergone significant advancements with the completion of the Human Genome Project and the development of high-throughput sequencing techniques. This has led to the identification of numerous genes involved in both male and female infertility, revolutionizing the diagnosis and management of infertility patients. Genetic investigations, including karyotyping, specific genetic tests, and high-throughput sequencing, have become essential in determining the genetic causes of infertility. Moreover, the integration of genetics into reproductive medicine has expanded the scope of care to include not only affected individuals or couples but also their family members. Genetic consultations and counselling play a crucial role in identifying potentially affected relatives and offering tailored therapy and the possibility of fertility preservation. Despite the current limited therapeutic options, an increasing understanding of genotype-phenotype correlations in infertility genes holds promise for improved treatment outcomes. The availability of genetic diagnostic tools has reduced the number of idiopathic infertility cases by providing accurate aetiological diagnoses. The transition from research to clinical practice in reproductive genetics requires the establishment of genetic consultations and data warehousing systems to provide up-to-date information on gene-disease relationships. Overall, the integration of genetics into reproductive medicine has brought about a paradigm shift, emphasizing the familial dimension of infertility and offering new possibilities for personalized care and family planning.

BACKGROUND:As in other domains of medicine, high-throughput sequencing methods have led to the identification of an ever-increasing number of gene variants in the fields of both male and female infertility. The increasing number of recently identified genes allows an accurate diagnosis for previously idiopathic cases of female infertility and more appropriate patient care. However, robust evidence of the gene-disease relationships (GDR) allowing the proper translation to clinical application is still missing in many cases.OBJECTIVE AND RATIONALE:An evidence-based curation of currently identified genes involved in female infertility and differences in sex development (DSD) would significantly improve both diagnostic performance and genetic research. We therefore performed a systematic review to summarize current knowledge and assess the available GDR.SEARCH METHODS:PRISMA guidelines were applied to curate all available information from PubMed and Web of Science on genetics of human female infertility and DSD leading to infertility, from 1 January 1988 to 1 November 2021. The reviewed pathologies include non-syndromic as well as syndromic female infertility, and endocrine and reproductive system disorders. The evidence that an identified phenotype is caused by pathogenic variants in a specific gene was assessed according to a standardized scoring system. A final score (no evidence, limited, moderate, strong, or definitive) was assigned to every GDR.OUTCOMES:A total of 45 271 publications were identified and screened for inclusion of which 1078 were selected for gene and variant extraction. We have identified 395 genes and validated 466 GDRs covering all reported monogenic causes of female infertility and DSD. Furthermore, we present a genetic diagnostic flowchart including 105 genes with at least moderate evidence for female infertility and suggest recommendations for future research. The study did not take into account associated genetic risk factor(s) or oligogenic/polygenic causes of female infertility.WIDER IMPLICATIONS:We have comprehensively reviewed the existing research on the genetics of female infertility and DSD, which will enable the development of diagnostic panels using validated genes. Whole genome analysis is shifting from predominantly research to clinical application, increasing its diagnostic potential. These new diagnostic possibilities will not only decrease the number of idiopathic cases but will also render genetic counselling more effective for infertile patients and their families.

Abstract Study question What is the relevance to offer panels of infertility genes as a diagnostic tool for infertile patients? Summary answer Our results demonstrate the interest of custom designed panel to define the genetic etiology of infertile couple in up to 26% of cases. What is known already The etiology of infertility is very heterogeneous, and due to the complexity of the reproductive process, standard fertility examinations fail to identify an etiology in 15 to 30% of infertile couples. It is believed that about half of these cases could be explained by a genetic defect. Until recently, routine genetic tests in the field of infertility were restricted to karyotyping, Yq microdeletion, CFTR mutation screening and FMR1 gene screening. The improvement of high throughput sequencing (HTS) technologies has considerably modified the discovery of the genetic causes of diseases allowing the development of new genetic diagnostic tools. Study design, size, duration Since 2016, we have set up, within the genetic diagnostic service of the Strasbourg University Hospital (HUS), a gene panel assay of preselected genes for the genetic diagnosis of male and female non-syndromic infertility. Since 2016, reflecting the high level of the research in this field, we have upgraded our panel two times. Initially, our panel included 16 infertility genes, then 51. We are now offering a panel of 133 infertility genes. Participants/materials, setting, methods Three different infertility gene panels were used to analyse 205 infertile cases; 62 females and 143 males. Agilent-SureSelectQXT Target Enrichment system was used to prepare libraries from genomic DNA. Sequencing was performed on Illumina NextSeq 550 with 2x150bp reads. Variant analysis was achieved using VaRank tool. One control DNA with known genotype was used in the first run of each design for the quality control. Identity of each patient was confirmed by independent Taqman technology. Main results and the role of chance Our first custom designed infertility panel (V1) contained 16 genes; one female and 11 male samples were tested, one positive male sample was identified and resulted in a diagnostic yield of 8.3% (1/12). The second version of the panel (V2), encompassed 51 genes, 22 female and 91 male samples were analysed, allowing a diagnostic yield of 6.1% [7/113; diagnostic rate for female was 9% (2/22), diagnostic rate for male was 5.5% (5/91)]. The third version of our panel (V3) contains 133 infertility genes and so far the diagnostic yield is 26.3% (21/80). Among 80 samples analysed, 39 of them were female for whom we got a diagnostic rate of 17.9% (7/39) and 41 were male for whom we got a diagnostic rate of 34.1% (14/41). Concerning V2 panel, we analysed 69 azoospermic patients for whom the diagnosis was only based on semen analysis and we identified only 2 pathogenic variants (2.8%, 2/69). For the V3 panel, the analysis of 30 azoospermic samples among which 26 diagnosed via testicular biopsy and pathology report, leaded to a diagnostic yield of 33.3% (10/30). These results underline the importance of well-defined clinical data in the field of genetics of infertility. Limitations, reasons for caution The main limitation is the number of patients’ analysed so far. In order to fully challenge the clinical interest of such diagnostic tool, a larger group of patients needs to be analysed. Wider implications of the findings The precise molecular diagnostic is important for adapting the best treatment and counsel not only to patients but to their spouse and relatives. This study allows us to decipher the prevalence of mutations in identified genes for a specific infertility phenotype on our cohort of infertile patients. Trial registration number not applicable