Summary Despite the high incidence of male infertility, only 30% of infertile men receive a causative diagnosis. To explore the regulatory mechanisms governing human germ cell function in normal and impaired spermatogenesis (crypto), we performed single-cell RNA sequencing (>30,000 cells). We find major alterations in the crypto spermatogonial compartment with increased numbers of the most undifferentiated spermatogonia (PIWIL4 + ). We also observe a transcriptional switch within the spermatogonial compartment driven by increased and prolonged expression of the transcription factor EGR4. Intriguingly, the EGR4-regulated chromatin-associated transcriptional repressor UTF1 is downregulated at transcriptional and protein levels. This is associated with changes in spermatogonial chromatin structure and fewer A dark spermatogonia, characterized by tightly compacted chromatin and serving as reserve stem cells. These findings suggest that crypto patients are disadvantaged, as fewer cells safeguard their germline’s genetic integrity. These identified spermatogonial regulators will be highly interesting targets to uncover genetic causes of male infertility.

BACKGROUND Human male infertility has a notable genetic component, including well-established diagnoses such as Klinefelter syndrome, Y-chromosome microdeletions and monogenic causes. Approximately 4% of all infertile men are now diagnosed with a genetic cause, but a majority (60–70%) remain without a clear diagnosis and are classified as unexplained. This is likely in large part due to a delay in the field adopting next-generation sequencing (NGS) technologies, and the absence of clear statements from field leaders as to what constitutes a validated cause of human male infertility (the current paper aims to address this). Fortunately, there has been a significant increase in the number of male infertility NGS studies. These have revealed a considerable number of novel gene–disease relationships (GDRs), which each require stringent assessment to validate the strength of genotype–phenotype associations. To definitively assess which of these GDRs are clinically relevant, the International Male Infertility Genomics Consortium (IMIGC) has identified the need for a systematic review and a comprehensive overview of known male infertility genes and an assessment of the evidence for reported GDRs. OBJECTIVE AND RATIONALE In 2019, the first standardised clinical validity assessment of monogenic causes of male infertility was published. Here, we provide a comprehensive update of the subsequent 1.5 years, employing the joint expertise of the IMIGC to systematically evaluate all available evidence (as of 1 July 2020) for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. In addition, we systematically assessed the evidence for all previously reported possible monogenic causes of male infertility, using a framework designed for a more appropriate clinical interpretation of disease genes. SEARCH METHODS We performed a literature search according to the PRISMA guidelines up until 1 July 2020 for publications in English, using search terms related to ‘male infertility’ in combination with the word ‘genetics’ in PubMed. Next, the quality and the extent of all evidence supporting selected genes were assessed using an established and standardised scoring method. We assessed the experimental quality, patient phenotype assessment and functional evidence based on gene expression, mutant in-vitro cell and in-vivo animal model phenotypes. A final score was used to determine the clinical validity of each GDR, across the following five categories: no evidence, limited, moderate, strong or definitive. Variants were also reclassified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines and were recorded in spreadsheets for each GDR, which are available at imigc.org. OUTCOMES The primary outcome of this review was an overview of all known GDRs for monogenic causes of human male infertility and their clinical validity. We identified a total of 120 genes that were moderately, strongly or definitively linked to 104 infertility phenotypes. WIDER IMPLICATIONS Our systematic review curates all currently available evidence to reveal the strength of GDRs in male infertility. The existing guidelines for genetic testing in male infertility cases are based on studies published 25 years ago, and an update is far overdue. The identification of 104 high-probability ‘human male infertility genes’ is a 33% increase from the number identified in 2019. The insights generated in the current review will provide the impetus for an update of existing guidelines, will inform novel evidence-based genetic testing strategies used in clinics, and will identify gaps in our knowledge of male infertility genetics. We discuss the relevant international guidelines regarding research related to gene discovery and provide specific recommendations to the field of male infertility. Based on our findings, the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing, or at least sequencing of the genes validated in this study, and expanding the patient groups for which genetic testing is recommended.

Purpose: Azoospermia affects 1% of men and it can be the consequence of spermatogenic maturation arrest (MA). Although the etiology of MA is likely to be of genetic origin, only 13 genes have been reported as recurrent potential causes of MA. Methods: Exome sequencing in 147 selected MA patients (discovery cohort and two validation cohorts). Results: We found strong evidence for 5 novel genes likely responsible for MA ( ADAD2 , TERB1 , SHOC1 , MSH4 , and RAD21L1 ), for which mouse knockout (KO) models are concordant with the human phenotype. Four of them were validated in the two independent MA cohorts. In addition, 9 patients carried pathogenic variants in 7 previously reported genes - TEX14 , DMRT1 , TEX11 , SYCE1 , MEIOB , MEI1 and STAG3 - allowing to upgrade the clinical significance of these genes for diagnostic purposes. Our meiotic studies provide novel insight into the functional consequences of the variants, supporting their pathogenic role. Conclusions: Our findings contribute substantially to the development of a pre-TESE prognostic gene panel. If properly validated, the genetic diagnosis of complete MA prior to surgical interventions is clinically relevant. Wider implications include the understanding of potential genetic links between NOA and cancer predisposition, and between NOA and premature ovarian failure.

Male infertility affects $7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.