Genetic etiology and treatment of 'arrested type' spermatogenic failure

Harnessing the full potential of reproductive genetics and epigenetics for male infertility in the era of “big data”

Because of the common use of ICSI and the potential genetic aetiology of spermatogenic failure, concern has been raised about transmitting genetic disorders to ICSI offspring. However, to date, in only approximately 15% of all cases of spermatogenic failure, an underlying genetic cause can be identified. We have previously established an association between spermatogenic failure and chromosomal region 11p15. In this study, we set out to explore whether NALP14, a gene recently mapped to 11p15, has a function in spermatogenesis and whether mutations in NALP14 can cause spermatogenic failure. We applied two different multiple tissue northern (MTN) blots to determine tissue specificity of NALP14 and performed immunohistochemistry on human testis with anti-NALP14 antiserum. To determine imprinting status of NALP14, we tested the expression pattern of two single-nucleotide polymorphisms (SNPs) in human testis. Finally, we performed a mutation screen of the NALP14 gene in 157 men with azoospermia or severe oligozoospermia by direct sequencing; 158 normospermic men served as controls. NALP14 was, as are the three other genes in 11p15, exclusively expressed in testis. Within the testis, the NALP14 protein was mainly expressed in A dark spermatogonia, mid and late spermatocytes and spermatids. The mutation screen revealed five mutations that occurred only in the patient group. One of these unique mutations introduced an early stop codon in the NALP14 sequence, predicted to result in a severely truncated protein. Our data suggest that NALP14 has a function in spermatogenesis and that mutations in this gene might cause spermatogenic failure.

Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure

BackgroundAdvanced biological techniques have helped produce more insightful findings on the genetic etiology of infertility that may lead to better management of the condition. This review provides an update on genes predisposing to syndromic and nonsyndromic infertility.Main bodyThe review identified 65 genes linked with infertility and infertility-related disorders. These genes regulate fertility. However, mutational loss of the functions of the genes predisposes to infertility. Twenty-three (23) genes representing 35% were linked with syndromic infertility, while 42 genes (65%) cause nonsyndromic infertility. Of the 42 nonsyndromic genes, 26 predispose to spermatogenic failure and sperm morphological abnormalities, 11 cause ovarian failures, and 5 cause sex reversal and puberty delay. Overall, 31 genes (48%) predispose to male infertility, 15 genes (23%) cause female infertility, and 19 genes (29%) predispose to both. The common feature of male infertility was spermatogenic failure and sperm morphology abnormalities, while ovarian failure has been the most frequently reported among infertile females. The mechanisms leading to these pathologies are gene-specific, which, if targeted in the affected, may lead to improved treatment.ConclusionsMutational loss of the functions of some genes involved in the development and maintenance of fertility may predispose to syndromic or nonsyndromic infertility via gene-specific mechanisms. A treatment procedure that targets the affected gene(s) in individuals expressing infertility may lead to improved treatment.

What is the load and profile of hereditary cancer-linked germline variants in infertile compared to fertile men? This study showed almost 5-fold enrichment of disease-causing findings in hereditary cancer genes in infertile compared to fertile men (6.9% vs 1.5%, P = 2.3 × 10-4). Epidemiological studies have revealed that men with low sperm count have a 2-fold higher risk of developing cancer during their lifetime. Our recent study observed a 4-fold increased prevalence of cancer in men with monogenic infertility compared to the general male population (8% vs 2%). Shared molecular etiologies of male infertility and cancer have been proposed. This retrospective study analyzed germline likely pathogenic and pathogenic (LP/P) variants in 157 hereditary cancer genes in 522 infertile and 323 fertile men recruited to the ESTonian ANDrology (ESTAND) cohort. All study participants (n = 845) had been recruited and phenotyped at an Andrology Clinic. Identification of LP/P variants in the cancer gene panel was performed from an exome sequencing dataset generated for the study cohort. All variants passed an automated filtering process, final manual assessment of pathogenicity, and experimental confirmation using Sanger sequencing. Retrospective general health records were available for 36 out of 41 (88%) men with LP/P findings. Infertile men presented a nearly 5-fold higher load of LP/P findings (36 of 522 cases, 6.9%) compared to fertile subjects (5 of 323, 1.5%; odds ratio (OR) = 4.7, 95% CI 1.81-15.5; P = 2.3 × 10-4) spanning over 24 hereditary cancer genes. The prevalence of findings was not significantly different between azoospermic and oligozoospermic cases. There was also no enrichment of findings in men with a history of cryptorchidism. By the time of the study, six men carrying hereditary cancer variants had been diagnosed with a tumor. Family members affected with cancer had been documented for 10 of 14 cases with available pedigree health data.Nearly half of the infertile men with LP/P findings (17 out of 36) carried variants in genes belonging to the Fanconi anemia (FA) pathway involved in the maintenance of genomic integrity in mitosis and meiosis, repair of DNA double-stranded breaks, and interstrand crosslinks. Overall, FA-pathway genes BRCA2 (monoallelic) and FANCM (biallelic) were the most frequently affected loci (five subjects per gene).LP/P findings in pleiotropic genes linked to human development and hereditary cancer (TSC1, PHOX2B, WT1, SPRED1, NF1, LZTR1, HOXB13) were identified in several patients with syndromic phenotypes. Four cryptorchid infertile men were carriers of MLH1, MSH2, and MSH6 variants implicated in Lynch syndrome. Future studies will reveal whether this observation is a by chance or replicable finding.Most hereditary cancer genes with LP/P variants show high expression in one or more testicular cell types, and mouse models for 15 of 24 affected genes have been reported to exhibit male sub- or infertility. These data support shared genetic etiology of impaired spermatogenesis and cancer. A significantly increased fraction of cancer-linked variants in infertile compared to fertile men could also explain the reported high prevalence of cancer as a comorbidity in male infertility. All hereditary cancer-linked variants identified in this study have been submitted to the National Center for Biotechnology Information (NCBI) ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). All recruited participants were of white European ancestry and living in Estonia. Thus, the results might not apply to other ethnic groups. Due to the young age of study participants (median age 34.4 years), the true incidence of cancer during lifetime could not be assessed. As retrospective clinical data were not available for all men, it was not possible to evaluate all possible genotype-phenotype links. The absence of genetic data from family members precluded the assessment of the hereditary nature of the variants or their potential de novo occurrence. Infertility affects about 7-10% of men worldwide. In this study, one in 15 men with spermatogenic failure carried germline LP/P variants in hereditary cancer genes. As exome sequencing is gradually entering the molecular diagnostics setup in andrology, analyzing hereditary cancer-linked variants in the workup of infertile men will offer additional clinical benefits. Male factor infertility is typically diagnosed in men in their 30s, often before the onset of cancer or its symptoms. Early knowledge of germline predisposition to cancer enables timely screening and multidisciplinary management options, potentially improving the prognosis. The study data provide support for the shared monogenic etiologies of hereditary cancer and spermatogenic failure. This study was funded by the Estonian Research Council grant PRG1021 (M.L. and M.P.). The authors declare no conflicts of interest.

Infertility affects 15% couples attempting pregnancy and in 40-50% of these cases the male partner has qualitative or quantitative abnormalities of sperm production. Microdeletions in the azoospermia factor (AZF) region on the long arm of the Y chromosome are known to be associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb and AZFc) which are critical for spermatogenesis and are recurrently deleted in infertile males. Semen analysis was carried out on one hundred and twenty five infertile males with oligozoospermia and azoospermia. Cytogenetic analysis was done for all the cases and in all cytogenetically normal cases (n = 83) microdeletion analysis was carried out on DNA extracted from peripheral blood using PCR. The sequence tagged sites (STS) primers sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc) were used for each case. Eight of the eighty three cases (9.63%) showed deletion of at least one of the STS markers. Correlation of phenotype with microdeletion was done in each case to determine any phenotype association with deletion of particular AZF locus. Based on the present study, the frequency of microdeletion in the Indian population is 9.63%. This study emphasizes the need for PCR analysis for determining genetic aetiology in cases with idiopathic severe testiculopathy.

Chromosomal Abnormality and Y Chromosome Microdeletion in Chinese Patients with Azoospermia or Severe Oligozoo-spermia

Acephalic spermatozoa syndrome (ASS) is one of the most severe spermatogenic failures of all infertility in men. The cognition of ASS has experienced a tortuous process. Over the past years, with the in-depth understanding of spermatogenesis and the emergence of new genetic research technologies, the unraveling of the genetic causes of spermatogenic failure has become highly active. From these advances, we established a genetic background and made significant progress in the discovery of the genetic causes of ASS. It is important to identify pathogenic genes and mutations in ASS to determine the biological reasons for the occurrence of the disease as well as provide genetic diagnosis and treatment strategies for patients with this syndrome. In this review, we enumerate various technological developments, which have made a positive contribution to the discovery of candidate genes for ASS from the past to the present. Simultaneously, we summarize the known genetic etiology of this phenotype and the clinical outcomes of treatments in the present. Furthermore, we propose perspectives for further study and application of genetic diagnosis and assisted reproductive treatment in the future.

Intercellular bridges (ICBs) are critical in intercellular communication, coordinated cellular development, and the equilibration of cytoplasmic contents between germ cells during vertebrate spermatogenesis. Mammalian TEX14 is specifically expressed in the testes and is a pivotal component of ICBs. It is indispensable for proper spermatogenesis; its deficiency causes meiotic arrest at the pachytene stage of meiotic prophase I, resulting in male infertility with non-obstructive azoospermia (NOA) in murine models. However, the specific effects of TEX14 deficiency on spermatogenesis remain poorly understood. In this study, we identified a novel compound heterozygous mutation in TEX14 (c.802A>T, p.S268C and c.1021C>T, p.R341*) in a patient with NOA. Functional analyses demonstrated that these mutations disrupted TEX14 synthesis. This led to spermatogenic failure characterized by meiotic arrest at the pachytene stage and impaired ICB assembly in the testes harboring the mutations. Our findings provide robust evidence that pathogenic biallelic TEX14 mutations are recurrent genetic etiologies of NOA in humans.

Overall, approximately 11% of men attending infertility clinics suffer unexplained oligo- or azoospermia. Cytogenetic observations of loss of the distal portion of the Y chromosome long arm (Yq) were found to be associated with disrupted spermatogenesis. The existence of a gene locus involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was thus postulated. It is suggested that microdeletions, or mutations, at the AZF locus could result in impaired spermatogenesis in chromosomally normal men. In order to test this hypothesis we have carried out Y chromosome genetic screening of 100 oligo- or azoospermic 46XY patients. We have also assessed phenotype/genotype relationships in those patients whose infertility has an underlying genetic aetiology. Patients were screened by polymerase chain reaction (PCR) with a set of Y chromosome-specific sequence tagged sites (STS) for submicroscopic deletions of their Y chromosome. Our results show that as many as 8% of cases of unexplained male infertility may have an underlying genetic aetiology related to microdeletions in two specific regions of the Y chromosome. Positive results from such a screen will be important when deciding the suitability of a patient for assisted conception schemes such as intracytoplasmic sperm injection.

Asthenozoospermia, characterized by diminished sperm motility, shows substantial genetic heterogeneity. Although many familial cases of asthenozoospermia have been documented, the genetic etiology for most affected males remains unknown. This study used clinical and molecular approaches to characterize a hereditary form of asthenozoospermia. Two patients born to consanguineous parents exhibited impaired sperm motility. Whole-genome sequencing (WGS), followed by Sanger sequencing, was performed for molecular diagnosis and was coupled with three-dimensional protein modeling to characterize the identified variant. WGS revealed that both affected brothers carried a pathogenic variant (c.580C>T; p.Gln194*) in dynein heavy chain domain 1 (DNHD1). Homozygous variants in DNHD1 have previously been linked to spermatogenic failure 65 (Online Mendelian Inheritance in Man [OMIM] 619712). Three-dimensional protein modeling demonstrated significant alterations in DNHD1 secondary structure that could contribute to loss-of-function. These findings support a role for DNHD1 in maintaining the structural and functional integrity of sperm. Accurate molecular diagnosis may facilitate genetic counseling and inform clinical management.

Non-obstructive azoospermia (NOA) represents the most severe form of male infertility; however, its genetic etiology remains largely elusive. MCM9 is crucial for DNA damage repair in mammalian somatic cells, playing a key role in regulating both homologous recombination (HR) and mismatch repair (MMR) pathways. In mice, MCM9 deficiency leads to spermatogenic failure characterized by progressive germ cell depletion and impaired HR repair. However, the underlying mechanism remains unclear in humans. Our study identified two novel homozygous loss-of-function (LoF) mutations in MCM9 in two unrelated NOA patients presenting with Sertoli cell-only syndrome (SCOS). The absence of testicular MCM9 confirmed the pathogenicity of these LoF mutations. Furthermore, diminished HR-mediated DNA repair capacity observed in HEK293T cells, either lacking MCM9 or overexpressing mutant MCM9 plasmids, highlighted the deleterious impact of these LoF mutations on HR repair. Additionally, the confirmed interaction between human testicular MCM9 and both MSH2 and MLH1, alongside findings that human MCM9 is predominantly expressed in spermatogonial stem cells and spermatogonia, provides compelling evidence for the involvement of the MCM9-mediated MMR pathway in maintaining genomic integrity and supporting the viability and proliferation of spermatogonia in humans. Given the poor outcomes of microdissection testicular sperm extraction (micro-TESE) observed in both probands, we propose that biallelic LoF mutations in MCM9 may serve as non-invasive molecular biomarkers for predicting micro-TESE failure. These findings enhance our understanding of the genetic basis of human NOA, particularly SCOS, and provide valuable insights for genetic counseling and fertility guidance tailored to these patients.