Abstract
PURPOSE: To systematically investigate the genetic architecture of severe male infertility in Indian men, with a specific focus on chromosomal abnormalities and the contribution of de novo variants. METHOD: We recruited 247 infertile males between 2021 and 2024 presenting with severe quantitative and qualitative sperm defects. All patients underwent karyotyping and Y chromosome microdeletion STS-PCR. A single molecule molecular inversion probe-based targeted sequencing assay covering 39 male infertility genes was performed in 120 patients, while whole exome sequencing (WES) was conducted in 48 patients using a duo/trio-based approach to enable segregation and de novo variant detection. RESULT: Gonosomal aneuploidies were observed in 3/247 patients (1.2%, 95% CI 0.3-3.5%) and causal AZF microdeletions in 8/247 (3.2%, 95% CI 1.4-6.3%). Targeted sequencing identified pathogenic/likely pathogenic (P/LP) variants in 4/120 patients (3.3%, 95% CI 0.9-8.3%), with additional CFTR variants in 3 patients where parental DNA was unavailable for phasing. WES yielded P/LP variants in 4/48 patients (8.3%, 95% CI 2.3-19.9%) affecting PMFBP1, DNAH1, and AR genes, confirmed via segregation analysis. No de novo or copy number variants were confirmed as causative, though several candidate genes were prioritised. Sequencing-based approaches provided an additional ~ 6-8% diagnostic yield, with the overall diagnostic rate reaching 7.7% (19/247; 95% CI 4.7-11.8%). CONCLUSION: Sequencing-based strategies, particularly family-based trio WES, significantly enhance diagnostic yield beyond current guideline-recommended tests, with data supporting their adoption as first-tier investigations for severe male infertility. This represents India's largest cohort-based genomic study on male infertility to date. Larger family-based cohorts will be essential to delineate the contribution of de novo variants to male infertility genetics.