Abstract
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.
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.
Exome sequencing in 147 selected MA patients (discovery cohort and two validation cohorts).
We found strong evidence for five 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, nine patients carried pathogenic variants in seven 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.
Our findings contribute substantially to the development of a pre-testicular sperm extraction (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 nonobstructive azoospermia (NOA) and cancer predisposition, and between NOA and premature ovarian failure.
Expanding established tests, exome sequencing in crypto-/azoospermic men yielded 8.5% additional diagnoses. These predict the success rate of testicular sperm extraction. Thus, an analysis of ...validated genes in infertile men with crypto- or azoospermia will significantly improve the standard of care.
Crypto- and azoospermia (very few/no sperm in the semen) are main contributors to male factor infertility. Genetic causes for spermatogenic failure (SPGF) include Klinefelter syndrome and Y-chromosomal azoospermia factor microdeletions, and CFTR mutations for obstructive azoospermia (OA). However, the majority of cases remain unexplained because monogenic causes are not analysed.
To elucidate the monogenic contribution to azoospermia by prospective exome sequencing and strict application of recent clinical guidelines.
Since January 2017, we studied crypto- and azoospermic men without chromosomal aberrations and Y-chromosomal microdeletions attending the Centre of Reproductive Medicine and Andrology, Münster.
We performed exome sequencing in 647 men, analysed 60 genes having at least previous limited clinical validity, and strictly assessed variants according to clinical guidelines.
Overall, 55 patients (8.5%) with diagnostic genetic variants were identified. Of these patients, 20 (3.1%) carried mutations in CFTR or ADGRG2, and were diagnosed with OA. In 35 patients (5.4%) with SPGF, mutations in 20 different genes were identified. According to ClinGen criteria, 19 of the SPGF genes now reach at least moderate clinical validity. As limitations, only one transcript per gene was considered, and the list of genes is increasing rapidly so cannot be exhaustive.
The number of diagnostic genes in crypto-/azoospermia was almost doubled to 21 using exome-based analyses and clinical guidelines. Application of this procedure in routine diagnostics will significantly improve the diagnostic yield and clinical workup as the results indicate the success rate of testicular sperm extraction.
When no sperm are found in the semen, a man cannot conceive naturally. The causes are often unknown, but genetics play a major role. We searched for genetic variants in a large group of patients and found causal mutations for one in 12 men; these predict the chances for fatherhood.
Summary
This study examines spermatogonial numbers in testicular samples from 43 prepubertal patients undergoing haematopoietic stem cell transplantation (HSCT). High‐dose chemotherapy and/or ...radiation during HSCT can impact spermatogenesis requiring fertility preservation. Results show that 49% of patients have decreased and 19% severely depleted spermatogonial pool prior to HSCT. Patients with Fanconi anaemia exhibit significantly reduced spermatogonial numbers. Patients with immunodeficiency or aplastic anaemia generally present within the normal range, while results in patients with myelodysplastic syndrome or myeloproliferative neoplasm vary. The study emphasizes the importance of assessing spermatogonial numbers in patients with severe haematological diseases for informed fertility preservation decisions.
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.
High dietary salt (NaCl) is a known risk factor for cardiovascular pathologies and inflammation. High plasma Na
concentrations (high Na
) have been shown to stiffen the endothelial cortex and ...decrease nitric oxide (NO) release, a hallmark of endothelial dysfunction. Here we report that chronic high Na
damages the endothelial glycocalyx (eGC), induces release of inflammatory cytokines from the endothelium and promotes monocyte adhesion. Single cell force spectroscopy reveals that high Na
enhances vascular adhesion protein-1 (VCAM-1)-dependent adhesion forces between monocytes and endothelial surface, giving rise to increased numbers of adherent monocytes on the endothelial surface. Mineralocorticoid receptor antagonism with spironolactone prevents high Na
-induced eGC deterioration, decreases monocyte-endothelium interactions, and restores endothelial function, indicated by increased release of NO. Whereas high Na
decreases NO release, it induces endothelial release of the pro-inflammatory cytokines IL-1ß and TNFα. However, in contrast to chronic salt load (hours), in vivo and in vitro, an acute salt challenge (minutes) does not impair eGC function. This study identifies the eGC as important mediator of inflammatory processes and might further explain how dietary salt contributes to endothelialitis and cardiovascular pathologies by linking endothelial nanomechanics with vascular inflammation.
Background
Infertility affects around 15% of all couples worldwide and is increasingly linked to variants in genes specifically expressed in the testis. Well‐established causes of male infertility ...include pathogenic variants in the genes TEX11, TEX14, and TEX15, while few studies have recently reported variants in TEX13B, TEX13C, FAM9A (TEX39A), and FAM9B (TEX39B).
Objectives
We aimed at screening for novel potential candidate genes among the human TEX (“testis expressed”) genes as well as verifying previously described disease associations in this set of genes.
Materials and methods
To this end, we screened the exome sequencing data of 1305 men, including 1056 crypto‐ and azoospermic individuals, and determined cell‐specific expression by analyzing testis‐specific single‐cell RNA sequencing data for genes with identified variants. To investigate the overarching role in male fertility, we generated testis‐specific knockdown (KD) models of all 10 orthologous TEX genes in Drosophila melanogaster.
Results
We detected rare potential disease‐causing variants in TEX10, TEX13A, TEX13B, TEX13C, TEX13D, ZFAND3 (TEX27), TEX33, FAM9A (TEX39A), and FAM9B (TEX39B), in 28 infertile men, of which 15 men carried variants in TEX10, TEX27, and TEX33. The KD of TEX2, TEX9, TEX10, TEX13, ZFAND3 (TEX27), TEX28, TEX30, NFX1 (TEX42), TEX261, and UTP4 (TEX292) in Drosophila resulted in normal fertility.
Discussion
Based on our findings, the autosomal dominant predicted genes TEX10 and ZFAND3 (TEX27) and the autosomal recessive predicted gene TEX33, which all three are conceivably required for germ cell maturation, were identified as novel potential candidate genes for human non‐obstructive azoospermia. We additionally identified hemizygous loss‐of‐function (LoF) variants in TEX13B, TEX13C, and FAM9A (TEX39A) as unlikely monogenic culprits of male infertility as LoF variants were also found in control men.
Conclusion
Our findings concerning the X‐linked genes TEX13B, TEX13C, and FAM9A (TEX39A) contradict previous reports and will decrease false‐positive reports in genetic diagnostics of azoospermic men.
Abstract
STUDY QUESTION
What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility?
SUMMARY ...ANSWER
Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected.
WHAT IS KNOWN ALREADY
The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear.
STUDY DESIGN, SIZE, DURATION
Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2.
PARTICIPANTS/MATERIALS, SETTING, METHODS
For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein’s cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined.
MAIN RESULTS AND THE ROLE OF CHANCE
This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients’ testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation.
LIMITATIONS, REASONS FOR CAUTION
Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism.
WIDER IMPLICATIONS OF THE FINDINGS
In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility.
STUDY FUNDING/COMPETING INTEREST(S)
This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit ‘Male Germ Cells: from Genes to Function’ (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG (‘Emmy Noether Programme’ project number 464240267). The authors declare no conflict of interest.
TRIAL REGISTRATION NUMBER
N/A.
The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of ...interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co‐expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single‐cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole‐exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell‐specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.
Implications for a novel role for WWC2 in spermatogenesis and male fertility.
An increasing number of genes are being described in the context of non-syndromic male infertility. Linking the underlying genetic causes of non-syndromic male infertility with clinical data from ...patients is important to establish new genotype-phenotype correlations. This process can be facilitated by using universal nomenclature, but no standardized vocabulary is available in the field of non-syndromic male infertility. The International Male Infertility Genomics Consortium aimed at filling this gap, providing a standardized vocabulary containing nomenclature based on the Human Phenotype Ontology (HPO). The "HPO tree" was substantially revised compared with the previous version and is based on the clinical work-up of infertile men, including physical examination and hormonal assessment. Some causes of male infertility can already be suspected based on the patient's clinical history, whereas in other instances, a testicular biopsy is needed for diagnosis. We assembled 49 HPO terms that are linked in a logical hierarchy and showed examples of morphological features of spermatozoa and testicular histology of infertile men with identified genetic diagnoses to describe the phenotypes. This work will help to record patients' phenotypes systematically and facilitate communication between geneticists and andrologists. Collaboration across institutions will improve the identification of patients with the same phenotypes, which will promote the discovery of novel genetic causes for non-syndromic male infertility.
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