Whole genome sequencing approach for the detection of rare protein-altering variants associated with familial male infertility

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

We explain environmental and genetic factors determining male genetic conditions and infertility and evaluate the significance of environmental stressors in shaping defensive responses, which is used in the diagnosis and treatment of male infertility. This is done through the impact of external and internal stressors and their instability on sperm parameters and their contribution to immunogenetic disorders and hazardous DNA mutations. As chemical compounds and physical factors play an important role in the induction of immunogenetic disorders and affect the activity of enzymatic and non-enzymatic responses, causing oxidative stress, and leading to apoptosis, they downgrade semen quality. These factors are closely connected with male reproductive potential since genetic polymorphisms and mutations in chromosomes 7, X, and Y critically impact on spermatogenesis. Microdeletions in the Azoospermic Factor AZF region directly cause defective sperm production. Among mutations in chromosome 7, impairments in the cystic fibrosis transmembrane conductance regulator CFTR gene are destructive for fertility in cystic fibrosis, when spermatic ducts undergo complete obstruction. This problem was not previously analyzed in such a form. Alongside karyotype abnormalities AZF microdeletions are the reason of spermatogenic failure. Amongst AZF genes, the deleted in azoospermia DAZ gene family is reported as most frequently deleted AZF. Screening of AZF microdeletions is useful in explaining idiopathic cases of male infertility as well as in genetic consulting prior to assisted reproduction. Based on the current state of research we answer the following questions: (1) How do environmental stressors lessen the quality of sperm and reduce male fertility; (2) which chemical elements induce oxidative stress and immunogenetic changes in the male reproductive system; (3) how do polymorphisms correlate with changes in reproductive potential and pro-antioxidative mechanisms as markers of pathophysiological disturbances of the male reproductive condition; (4) how do environmental stressors of immunogenetic disorders accompany male infertility and responses; and (5) what is the distribution and prevalence of environmental and genetic risk factors.

What is the impact of undiagnosed microdeletion and microduplication syndromes (MMS) for men with idiopathic low sperm count? Among idiopathic male infertility, ∼2% of cases harbour known disease-causing microdeletions and duplications linked to clinically well-established syndromes, representing ∼2.5-fold higher prevalence than in the general population. While infertility affects up to 10% of men, a substantial proportion of cases remain with no identifiable underlying cause. Recurrent submicroscopic losses or gains cause MMS, some of which also impact reproductive phenotypes, including cryptorchidism and reduced fertility. This retrospective study investigated the proportion of undiagnosed MMS among idiopathic male infertility cases. Patients with unexplained low total sperm counts (TSC; defined as ≤39 million sperm per ejaculate) were recruited to the ESTonian ANDrology (ESTAND) cohort at the Andrology Clinic of Tartu University Hospital (AC-TUH) in Estonia. A total of 504 men were included in the analysis, and the study capitalized on available whole-exome sequencing (WES) data to explore large (>500 kb) chromosomal deletions and duplications. Copy number variant (CNV) calling was executed on the WES dataset, followed by a stringent, custom-developed filtering pipeline that retained only high-confidence CNVs larger than 500 kb. Candidate CNVs were validated by chromosomal microarray analysis (CMA) or whole-genome sequencing (WGS). Prevalence of identified MMS-linked deletions and duplications in the ESTAND cohort was compared to general population literature data. A total of nine patients (1.8%) carried losses and gains linked to clinically well-characterized MMS-recurrent microdeletions at 16p11.2 (two cases), 2q13-14.1, and 15q13.2-13.3, and microduplications at 22q11.21 (three cases), 16p11.2, and 8p23.1. The total burden of MMS among infertile men was ∼2.5-fold higher compared to the general population (P = 0.01, χ2 test). Cryptorchidism was a novel shared feature among all individuals with 16p11.2 rearrangements, suggesting a potential role in disrupting testicular development. Three subjects with MMS-linked microduplications, but none with a microdeletion, had achieved biological fatherhood. An oligozoospermia case (TSC 1.92 × 106/ej.) with 16p11.2 duplication had a naturally conceived child in youthhood. For two men carrying 22q11.21 duplication (TSC 0 and 4.2 × 106/ej., respectively), implementation of ARTs-ICSI with or without preceding testicular sperm aspiration-resulted in successful conception and childbirth. Evidence for a plausible link to male gonadal development and function has been reported for MAZ and KCTD13 at 16p11.2, and LZTR1 at 22q11.21. As an additional finding, a novel ∼3.8 Mb microduplication at 3p25.1 was identified in an oligozoospermia patient and his azoospermic son, conceived naturally at the age of 28 years. This region encompasses a triplosensitive gene, NR2C2, linked to affected meiosis and oligozoospermia in mouse models. WES may not detect all structural variations, such as inversions or balanced translocations. As some MMS CNVs were only identified in singleton cases (8p23.1 duplication, 2q13-14.1 and 15q13.2-13.3 deletions), the link to male infertility could not be clarified. Further data are needed about the novel large 3p25.1 microduplication to understand its effect on spermatogenesis. The yield of identified MMS in idiopathic cases with low sperm counts (∼2%) was close to the carriership of Y-chromosome microdeletions, tested in routine infertility workup. Early identification of MMS can inform genetic counselling regarding congenital health risks to the patient and future offspring and options for decision-making in ART. This study was supported by the Estonian Research Council (Grant number PRG1021 to M.L.). The authors declare that they have no conflict of interest in relation to the data in this paper. N/A.

Male infertility research and clinical advances had vast progress in the last few decades. Strong research evidence underpinned the concepts of oxidative stress (OS)-mediated male reproductive disruptions, which bear answers to several cases of idiopathic male infertility. Antioxidant treatment held the prime solution for OS-mediated male infertility. But excess use of antioxidants is challenged by the research breakthrough that reductive stress also predisposes to male infertility, resolutely instituting that any biological extremes of the redox spectrum are deleterious to male fertility. Superfluity of reducing agents may hinder essential oxidation mechanisms, affecting physiological homeostasis. These mechanisms need to be explicated and updated time and again to identify the fine thread between OS-mediated male infertility treatment and induction of reductive stress. This chapter thus presents the evidence-based concepts pertaining to the antioxidants actions to combat OS-induced male infertility, the mechanism of induction of reductive stress and its impact on male reproduction.

Male infertility is a complex multifactorial disease affecting approximately 10% of couples who want to have children. Some cases of infertility can be explained by genetic factors. Septins are members of the GTPase superfamily, which are involved in diverse biological processes including morphogenesis, compartmentalization, cytokinesis, and apoptosis. The septin 12 gene, SEPT12, is expressed exclusively in post-meiotic male germ cells and is considered as a critical gene for spermatogenesis. In this study, we evaluated 200 patients with non-obstructive azoospermia and detected mutations of 25 spermatogenesis-associated genes by targeted exome sequencing. We report a missense SEPT12 variant, c.673C>A/p.Gln225Lys, in an infertile man with non-obstructive azoospermia. The variation was located inside the GTPase domain and had a SIFT score of 0.02 (<0.50) and was considered to be ‘probably damaging’ by PolyPhen. This case may provide clues to help establish the relationship between SEPT12 gene alterations and some cases of idiopathic male infertility. The role of this variant should thus be investigated further.

Introduction: Rhabdomyosarcoma (RMS) is among the most frequently occurring tumors in Li-Fraumeni syndrome patients and is often observed in patients with a strong family history of cancer. Recent studies suggest that at least 10% of children with cancer harbor an underlying pathogenic germline mutation, although the frequency in children with RMS is not well characterized. In this study we sought to determine the rate of likely pathogenic germline mutations in rhabdomyosarcoma patients, unselected for family history. Methods: Of 275 RMS patients enrolled on one clinical trial (COG ARST0531), whole-genome sequencing was performed on blood-derived DNA of an initial set of 50 using the Illumina HiseqX to an average sequencing depth of 44.9X [34.2-58.4]. Single nucleotide variants (SNVs) and Indels were called and filtered to select only those occurring in exons or splicing regions. Variants were further filtered to include only those with a frequency of &amp;lt;0.5% in the ExAC database and lying in one of 99 genes identified as a cancer predisposition gene in the COSMIC Cancer Gene census. Nonsynonymous SNVs were required to be predicted as deleterious by one or more of Polyphen, mutation assessor or sift. Results: Across the 50 initial samples, a total of 290 variants ([0-17], median of 5) passed the described filters. Notably, no point mutations were identified in TP53 in the initial 50 samples. Two samples contained variants annotated as pathogenic in ClinVar - a frameshift deletion in CHEK2 and a rare SNP in MUTYH. Also notable was a potentially pathogenic frameshift deletion in MSH6 that lacked previous annotation. Conclusions: Our preliminary analysis suggests a lower incidence of causative TP53 mutations in RMS than previously suggested. Whole genome sequencing of the remaining samples in the cohort along with analysis of copy number and structural variants is ongoing. Statistical analysis of the entire cohort of 275 patient samples will allow for the most complete characterization of the germline genomic landscape of rhabdomyosarcoma to date. This information will better inform clinical surveillance and management parameters for RMS patients and families. Citation Format: Nicholas Light, Philip Lupo, Javed Khan, Joshua Schiffman, Douglas Hawkins, Adam Shlien, David Malkin. Whole genome sequencing of rhabdomyosarcoma germline cohort identifies low frequency of pathogenic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-223. doi:10.1158/1538-7445.AM2017-LB-223

Male infertility is a complex and polygenic reproductive disease. 10-15% of the males are affected by idiopathic infertility conditions. Acetylcholine (ACh), a major neurotransmitter has been reported to play a non-neuronal role as well. Acetylcholinesterase (AChE) is the primary ACh hydrolyzing enzyme whose over or lower expression influence the availability of ACh for physiological roles. The purpose of the study was to find the possible impact and association of acetylcholinesterase, ACHE gene variant rs 17228602, and pro-inflammatory cytokines in clinically diagnosed infertile males. The study includes clinically diagnosed fifty non-infertile (control) and forty-five infertile males. Whole blood AChE enzymatic activity was measured. Genotyping of rs17228602 was carried out from peripheral blood by standard molecular methods. Pro-inflammatory cytokines were determined by the ELISA method. AChE enzyme was found to be significantly elevated in infertile than non-infertile males. ACHE SNP rs17228602 had shown significant association in dominant model (odd ratio = 0.378, 95% CI = 0.157-0.911, p-value 0.046). Pro-inflammatory cytokine IL-1β was notably increased with statistical significance (p ≤0.05) in male infertile patients. The study concludes and speculates that AChE plays role in the pathogenesis of male infertility through the modulation of inflammatory pathways. Further studies in this direction may resolve the idiopathic cases of male infertility. Other variants of ACHE and the association of miRNA for the regulation of AChE in male infertility are suggested for further insight.

Plakophilin-2 (PKP2) is the most prevalent mutant gene causing arrhythmogenic cardiomyopathy (ACM) and PKP2 carriers are prone to develop ventricular arrhythmic events. The objective of this study is to use integrated analysis of whole genome sequencing (WGS) and transcriptome sequencing (RNAseq) to identify deep intronic and/or coding variants that cause aberrant splicing events in ACM patients, and hence, to test the hypothesis that recessive variants in PKP2 may lead to early-onset ACM with severe heart failure. We performed WGS and RNAseq in 27 heart transplanted ACM patients. By integrated analysis of WGS/RNAseq, we discovered that two patients with PKP2 variants were affected in recessive pattern. One patient had aberrant splicing arising from two intronic variants that led to exon skipping and exon retention. We screened three additional recessive PKP2 variants in 47 non-heart transplanted ACM patients. We compared the clinical characteristics of recessive PKP2 (n = 5) and heterozygous PKP2 carriers (n = 18), and found that recessive PKP2 variant carriers all had early-onset ACM with left ventricular dysfunction. We examined truncating PKP2 variants in explanted hearts and confirmed that truncated PKP2 was not translated. Moreover, the morphology of intercalated disc in recessive PKP2 variants carriers was similar to normal heart suggesting little intercalated disc remodelling. By using combined implementation of WGS RNAseq, we were able to demonstrate that recessive variants in PKP2 may contribute to early-onset ACM with severe heart failure. These findings may play a role in risk stratification of ACM based on genetic testing in clinical practice.

Novel association of PhosphoSerine PHosphatase (PSPH) gene mutations with male infertility identified through whole exome sequencing of South Indians

Male infertility is a widespread health issue globally, frequently remaining untreated due to stigmatization and the challenges in diagnosis and treatment. This review presents a comprehensive update on the literature covering key aspects such as causes, diagnostic techniques, and treatment options for managing male factor infertility. It includes an in-depth analysis of global infertility data, using resources from the World Health Organization, Web of Science, Google Scholar, Elsevier, Medline, PubMed, and Scopus databases to gather relevant articles on male infertility. A total of 41 articles from 2000-2023 were reviewed. High throughput techniques, along with sophisticated assays, are being employed for accurate diagnosis. Surgical procedures such as testicular sperm extraction, vasovasostomy, vasoepididymostomy, sperm retrieval techniques, and non-surgical procedures including sclerotherapy, gonadotropin-releasing hormone therapy, and antiestrogens are available to treat infertile males. Additionally, in recent years, flavonoids have been extensively explored for their antioxidant, anti-inflammatory, immune-stimulating, anti-apoptotic, anticarcinogenic, anti-allergic, and antiviral activities. These properties of flavonoids are being investigated for their potential to address biological mechanisms underlying anomalies such as spermatogenesis disturbance and sperm quality decline. This review serves as a comprehensive guide to better understand the etiologies and treatment modalities of male factor infertility, ultimately facilitating affected individuals in making informed reproductive choices.

Study question How do novel GPX4 mutations contribute to idiopathic male infertility, and what are the potential therapeutic candidates for ferroptosis regulation identified through computational docking techniques? Summary answer Novel GPX4 mutations are implicated in idiopathic male infertility among Indian Bengali-speaking men and advanced Computational docking has identified potential therapeutic candidates for ferroptosis regulation. What is known already Genetic underpinnings of male infertility display diverse results across populations. Significant risk factors include Y chromosomal microdeletions, sex chromosome variations, and autosomal gene polymorphisms. Ethnic disparities exist in the association between CAG repeat polymorphism in the androgen receptor gene and male infertility. Indian cohorts have demonstrated notable findings related to glutathione-S-transferase genes. Spermatogenesis, dependent on iron, involves pathways regulated by glutathione peroxidase 4 (GPX4). Elevated iron levels impair spermatogenesis through oxidative stress, though Sertoli cells mitigate these effects. GPX4 mutations impact ferroptosis, sperm count and motility. Identifying therapeutic candidates through molecular docking enhances GPX4 activity, offering promising infertility treatments. Study design, size, duration This population-based case-control study included 218 cases of idiopathic male infertility and 235 age-matched fertile controls, conducted between March 2018 and April 2023. The study aimed to elucidate the significant association between GPX4 gene mutations and spermatogenic failure. Subsequently, we focused on identifying potent therapeutic molecules with enhanced binding affinity to the conserved domain of the gene through molecular structure and pathway interaction analysis which might address idiopathic male infertility in the targeted population. Participants/materials, setting, methods Samples were collected from the Institute of Reproductive Medicine, Kolkata. DNA was isolated and PCR amplified, followed by Sanger sequencing of the GPX4 gene. BLAST comparisons, statistical tests, and in silico analyses were performed to interpret variations and their potential effects. Molecular docking of 111 ferroptosis-inhibiting molecules from PubChem with the GPX4 enzyme from the RCSB Protein Data Bank aimed to identify potential therapeutic candidates, shedding light on drug discovery and personalized medicine. Main results and the role of chance Upon sequencing the GPX4 gene, three substitution variants (OQ927973 C&amp;gt;G, OQ939947 G&amp;gt;T, and OR381683 G&amp;gt;A) and three indel mutations (two insertions: OR003916 insT, OR381684 insG; one deletion: OR037273 delA) were identified. Statistical analysis showed these novel variants were highly significant. The substitutions revealed increased odds ratios: OQ927973 (20.4, p = 0.0002), OQ939947 (3.955, p = 0.056), and OR381683 (7.5, p &amp;lt; 0.0001). The insertions indicated higher odds ratios: OR003916 (26.905, p &amp;lt; 0.0001) and OR381684 (5.077, p = 0.0012). The deletion OR037273 exhibited a 9.651-fold increase (p = 0.0003). Odds ratios with 95% CI were calculated using Fisher’s exact test. A two-tailed p-value &amp;lt;0.02 was considered statistically significant after Bonferroni correction. In silico analyses predicted these mutations' association with spermatogenic failure. We tested 111 compounds with a binding affinity threshold of -6 kcal/mol. Among the 64 synthetic compounds, 21 met the criteria, including Keap1-NRF2-IN3, Irbesartan, Sertraline, and Rifampicin, with Keap1-NRF2-IN3 achieving the highest initial docking score (-7.4 kcal/mol). Redesigning and modifying these ligands improved binding affinities. Six Rifampicin models were evaluated for suitability. Phytochemicals like 7,8-dihydroflavone and Baicalein also showed significant binding affinities. Molecular Dynamics simulations using CABS-flex2.0 confirmed the top protein-ligand complexes' stability, with Root Mean Square Fluctuation and B-factor values indicating stable interactions throughout the simulation. Limitations, reasons for caution The origin of the novel mutations remains elusive, and the findings do not provide mechanistic insights into how de novo mutations impair spermatogenesis. Furthermore, the small sample size may limit the generalizability of the results, necessitating validation through studies in diverse ethnic populations. Wider implications of the findings This study sheds light on unexplained infertility in Indian Bengali-speaking men, aiding in the management of their condition. It also supports personalized medicine and genetic screening, and uses bioinformatics analyses to pave the way for drug discovery and future mechanistic research. Trial registration number No

Androgen insensitivity syndrome results from a mutation in androgen receptor gene. It has wide spectrum of phenotypic presentations. Mild androgen insensitivity syndrome (MAIS) is the milder form, present as undervirilized male syndrome. In some instances, the only observed abnormality appears to be male infertility; therefore, MAIS could explain some cases of idiopathic male infertility. Here we describe a case of MAIS in a 38 years old male who presented with azoospermia and infertility.

Microdeletions in the azoospermia factor region on the long arm of Y chromosome are associated with spermatogenic failure. There are many markers for the diagnosis of Y chromosome microdeletion analysis, but in routine practice only a limited set of markers can be tested. The objectives of this study were to determine the frequency of Y chromosome microdeletion in idiopathic cases of male infertility in India, to attempt genotype-phenotype correlation, and to evaluate whether markers to be tested for diagnosis of Y chromosome microdeletion should be ethnicity specific. Microdeletions in the Y chromosome were analyzed in 200 infertile males. The six sequence tag site (STS) markers prescribed by the European Academy of Andrology (EAA) were used initially. Patients in whom no deletions were detected by use of these markers were tested by markers selected from other studies from India. The STS markers prescribed by EAA detected deletions in only 6 (3%) of 200 infertile males. However, markers selected from previous Indian studies showed deletions in an additional 15 (7.5%) of infertile males. Overall, Y chromosome microdeletions were observed in 21 (10.5%) of 200 patients. Of these, 13 were cases of azoospermia and 8 were cases of severe oligospermia. The markers prescribed by EAA alone are not suitable for the diagnosis of Y chromosome microdeletions in infertile males. The protocol for identification of Y chromosome microdeletions in cases of nonobstructive azoospermia/severe oligospermia would have to include a different set of STS markers.

Objectives: In human, olfactory receptor (ORs) gene polymorphism regulate testicular development and physiological functions during spermatogenesis. However, the etiopathology of OR gene in male infertility is still restricted that required further studies. Basic mechanism involves the identification of peripheral signals based on specific chemoreceptors site that are expressed on the surface of mature spermatozoa inside the female reproductive tract. These receptors belong to G-protein couple receptors (GPCR) of superfamily and the roles of ORs gene linked to sperm chemotaxis through chemokinesis. In the present study, we have studied the role of Olfactory receptor family 2 subfamilies AT member 4 (OR2AT4) and Olfactory receptor family 9 subfamily G member 1/9 (OR9G1/9) genes polymorphism in clinically diagnosed azoospermic patients. Materials and Methods: Systematically, GTG banded karyotypes were developed using short term lymphocyte cultures, fluorescence in situ hybridization using SRY probe and PCR based microdeletion of Y-chromosome was characterized using STS marker (SY255). Whole exome sequencing (WES) based analysis was performed in azoospermic patients to identify the involvement of de-novo mutation in OR2AT4 and OR9G1/OR9G9 genes mutation followed by bioinformatics tools used for the analysis to identify changes in respective amino acids. Results: OR2AT4 showing missense mutation after substitution of nucleotide (G→A) followed by lack of change in amino acid (Gln→Gln), but in OR9G1/G9 gene showing changes of substitution of nucleotide thymidine into adenine (T→A), whereas valine is replaced by glutamine (Val→ Glu), similarly thymidine is substituted by cysteine (T→C), after decode phenylalanine is replaced by leucine (Phe→Leu). Further, 3D protein helical structure was developed with the help of bioinformatics on the basis of amino acids coded protein and compare with mutated amino acid or truncated protein residues with the help of iTASSER server. Conclusion: Present study demonstrate the role of OR2AT4 and OR9G1/9 gene polymorphism elucidates the functional role of protamine in ectopic receptors followed by increasing “risk” of developing male infertility that leads the clinical condition defined as non-obstructive azoospermia.

O-29: Defective recombination: The role of MLH3 in infertile men