Analysis of copy number variation in men with non-obstructive azoospermia.

BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

Bi-allelic variants in DNA mismatch repair proteins MutS Homolog MSH4 and MSH5 cause infertility in both sexes.

STUDY QUESTION: Do bi-allelic variants in the genes encoding the MSH4/MSH5 heterodimer cause male infertility? SUMMARY ANSWER: We detected biallelic, (likely) pathogenic variants in MSH5 (4 men) and MSH4 (3 men) in six azoospermic men, demonstrating that genetic variants in these genes are a relevant cause of male infertility. WHAT IS KNOWN ALREADY: MSH4 and MSH5 form a heterodimer, which is required for prophase of meiosis I. One variant in MSH5 and two variants in MSH4 have been described as causal for premature ovarian insufficiency (POI) in a total of five women, resulting in infertility. Recently, pathogenic variants in MSH4 have been reported in infertile men. So far, no pathogenic variants in MSH5 had been described in males. STUDY DESIGN, SIZE, DURATION: We utilized exome data from 1305 men included in the Male Reproductive Genomics (MERGE) study, including 90 males with meiotic arrest (MeiA). Independently, exome sequencing was performed in a man with MeiA from a large consanguineous family. PARTICIPANTS/MATERIALS, SETTING, METHODS: Assuming an autosomal-recessive mode of inheritance, we screened the exome data for rare, biallelic coding variants in MSH4 and MSH5. If possible, segregation analysis in the patients' families was performed. The functional consequences of identified loss-of-function (LoF) variants in MSH5 were studied using heterologous expression of the MSH5 protein in HEK293T cells. The point of arrest during meiosis was determined by γH2AX staining. MAIN RESULTS AND THE ROLE OF CHANCE: We report for the first time (likely) pathogenic, homozygous variants in MSH5 causing infertility in 2 out of 90 men with MeiA and overall in 4 out of 902 azoospermic men. Additionally, we detected biallelic variants in MSH4 in two men with MeiA and in the sister of one proband with POI. γH2AX staining revealed an arrest in early prophase of meiosis I in individuals with pathogenic MSH4 or MSH5 variants. Heterologous in vitro expression of the detected LoF variants in MSH5 showed that the variant p.(Ala620GlnTer9) resulted in MSH5 protein truncation and the variant p.(Ser26GlnfsTer42) resulted in a complete loss of MSH5. LARGE SCALE DATA: All variants have been submitted to ClinVar (SCV001468891-SCV001468896 and SCV001591030) and can also be accessed in the Male Fertility Gene Atlas (MFGA). LIMITATIONS, REASONS FOR CAUTION: By selecting for variants in MSH4 and MSH5, we were able to determine the cause of infertility in six men and one woman, leaving most of the examined individuals without a causal diagnosis. WIDER IMPLICATIONS OF THE FINDINGS: Our findings have diagnostic value by increasing the number of genes associated with non-obstructive azoospermia with high clinical validity. The analysis of such genes has prognostic consequences for assessing whether men with azoospermia would benefit from a testicular biopsy. We also provide further evidence that MeiA in men and POI in women share the same genetic causes. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation sponsored Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326), and supported by institutional funding of the Research Institute Amsterdam Reproduction and Development and funds from the LucaBella Foundation. The authors declare no conflict of interest.

Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest.

STUDY QUESTION: Are sequence variants in the stromal antigen 3 (STAG3) gene a cause for non-obstructive azoospermia (NOA) in infertile human males? SUMMARY ANSWER: Sequence variants affecting protein function of STAG3 cause male infertility due to meiotic arrest. WHAT IS KNOWN ALREADY: In both women and men, STAG3 encodes for a meiosis-specific protein that is crucial for the functionality of meiotic cohesin complexes. Sequence variants in STAG3 have been reported to cause meiotic arrest in male and female mice and premature ovarian failure in human females, but not in infertile human males so far. STUDY DESIGN, SIZE, DURATION: The full coding region of STAG3 was sequenced directly in a cohort of 28 men with NOA due to meiotic arrest. In addition, a larger group of 275 infertile men that underwent whole-exome sequencing (WES) was screened for potential STAG3 sequence variants. Furthermore, meiotic spreads, immunohistochemistry, WES and population sampling probability (PSAP) have been conducted in the index case. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 28 infertile but otherwise healthy human males who underwent Sanger sequencing of the full coding region of STAG3. Additionally, WES data of 275 infertile human males with different infertility phenotypes have been screened for relevant STAG3 variants. All participants underwent karyotype analysis and azoospermia factor (AZF) screening in advance. In the index patient, segregation analysis, WES data, PSAP, lab parameters, testis histology and nuclear spreads have been added to suplort the findings. MAIN RESULTS AND THE ROLE OF CHANCE: Two compound-heterozygous variants in STAG3 (c.[1262T>G];[1312C>T], p.[(Leu421Arg)];[(Arg438Ter)]) have been found to cause male infertility due to complete bilateral meiotic arrest in an otherwise healthy human male. Compound heterozygosity was confirmed by Sanger sequencing of the parents and the patient's brother. Other variants which may affect spermatogenesis have been ruled out through analysis of the patient's WES data and application of the PSAP pipeline. As expected from Stag3 knockout-mice meiotic spreads, germ cells did not develop further than zygotene and showed drastic chromosome aberrations. No rare variants in STAG3 were found in the 275 infertile males with other phenotypes. Our results indicate that STAG3 variants that negatively affect its protein function are a rare cause of NOA (<1% of cases). LIMITATIONS, REASONS FOR CAUTION: We identified only one patient with compound-heterozygous variants in STAG3 causing NOA due to meiotic arrest. Future studies should evaluate STAG3 variants in larger cohorts to support this finding. WIDER IMPLICATIONS OF THE FINDINGS: Identification of STAG3 sequence variants in infertile human males should improve genetic counselling as well as diagnostics and treatment. Especially before testicular sperm extraction (TESE) for ICSI, STAG3 variants should be ruled out to prevent unnecessary interventions with frustrating outcomes for both patients and clinicians. STUDY FUNDING/COMPETING INTEREST(S): This work was carried out within the frame of the German Research Foundation (DFG) Clinical Research Unit 'Male Germ Cells: from Genes to Function' (CRU326). Work in the laboratory of R.J. is supported by a grant of the European Union H2020 program GermAge. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.