The X chromosome and male infertility

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Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model

Colorectal cancer (CRC), also known as colon cancer, is the third most common form of cancer worldwide in men and the second in women and is characterized by several genetic alterations, among them the expression of several genes. 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM) are procarcinogens commonly used to induce colon cancer in rats (DMH/AOM rat model). This rat model has been used to study changes in mRNA expression in genes involved in this pathological condition. However, a lack of proper detailed PCR primer design in the literature limits the reproducibility of the published data. The present study aims to design, optimize and validate the qPCR, in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, for seventeen genes commonly used in the DMH/AOM rat model of CRC (Apc, Aurka, Bax, Bcl2, β -catenin, Ccnd1, Cdkn1a, Cox2, Gsk3beta, IL-33, iNOs, Nrf2, p53, RelA, Smad4, Tnfα and Vegfa) and two reference genes (Actb or β - actin and B2m). The specificity of all primer pairs was empirically validated on agarose gel, and furthermore, the melting curve inspection was checked as was their efficiency (%) ranging from 90 to 110 with a correlation coefficient of r 2 > 0.980. Finally, a pilot study was performed to compare the robustness of two candidate reference genes

Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer

Combination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches. Imprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or "dynamic sites," also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of "dynamic" CpG sites. Distinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health. The online version contains supplementary material available at 10.1186/s13148-022-01417-1

A systematic review of the validated monogenic causes of human male infertility : 2020 update and a discussion of emerging gene-disease relationships

Altres ajuts: National Health and Medical Research Council (APP1120356); Netherlands Organisation for Scientific Research (918-15-667); Wellcome Trust (209451); German Research Foundation (DFG, CRU326); National Institutes of Health: Genomics of Spermatogenic Impairment (R01HD078641); Ministerio de Sanidad.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

From exome analysis in idiopathic azoospermia to the identification of a high-risk subgroup for occult Fanconi anemia

Purpose: in about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these "occult" FA cases is often a solid cancer and cancer treatment-related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing. - Methods: (1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test. - Results: ES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell-only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test. - Conclusion: we report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for "occult" FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance

A de novo paradigm for male infertility

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p -value = 1.00 × 10 −5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p -value = 5.01 × 10 −4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p -value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility. Germline de novo mutations can impact individual fitness, but their role in human male infertility is understudied. Trio-based exome sequencing identifies many new candidate genes affecting male fertility, including an essential regulator of male germ cell pre-mRNA splicing

The X chromosome and male infertility

The X chromosome is a key player in germ cell development, as has been highlighted for males in previous studies revealing that the mammalian X chromosome is enriched in genes expressed in early spermatogenesis. In this review, we focus on the X chromosome's unique biology as associated with human male infertility. Male infertility is most commonly caused by spermatogenic defects to which X chromosome dosage is closely linked; for example, any supernumerary X chromosome as in Klinefelter syndrome will lead to male infertility. Furthermore, because males normally only have a single X chromosome and because X-linked genetic anomalies are generally only present in a single copy in males, any loss-of-function mutations in single-copy X-chromosomal genes cannot be compensated by a normal allele. These features make X-linked genes particularly attractive for studying male spermatogenic failure. However, to date, only very few genetic causes have been identified as being definitively responsible for male infertility in humans. Although genetic studies of germ cell-enriched X-chromosomal genes in mice suggest a role of certain human orthologs in infertile men, these genes in mice and humans have striking evolutionary differences. Furthermore, the complexity and highly repetitive structure of the X chromosome hinder the mutational analysis of X-linked genes in humans. Therefore, we conclude that additional methodological approaches are urgently warranted to advance our understanding of the genetics of X-linked male infertility