Genetic Alterations in Poor-Quality Individually Selected Sperm Reveal a Potential Biomarker for Male Subfertility

Objective: To combine whole-genome sequencing (WGS) of individually selected spermatozoa with detailed semen parameters and IVF outcomes in order to identify genetic variants associated with poor sperm quality and subfertility. Design: Experimental genomic analysis of individually isolated single sperm cells. Setting: University research institute and IVF center. Patients: Twelve sperm samples from male partners of infertile couples, consisting of six high-quality (HQ; motile with normal morphology) and six poor-quality (PQ; immotile with abnormal morphology) samples. Intervention(s): From each sample, 1,500 sperm cells were individually selected (18,000 in total) and analyzed by WGS. A multi-step filtering and annotation pipeline was applied, integrating ACMG criteria, population allele frequencies, tissue-specific expression, and predicted protein effects. Main Outcome Measure(s): Identification and characterization of coding, splice-site, and protein-altering variants in HQ and PQ sperm, and assessment of their potential relevance to IVF outcomes. Results: WGS identified 22 high-confidence variants across all samples, all present in the homozygous state. PQ sperm showed a consistent increase in total variant burden, structural alterations, splice-site disruptions, and predicted damaging coding variants compared with HQ sperm, although statistical significance was limited by sample size. A frameshift insertion in FOXO6, found only in one PQ sample, represented the only variant with potential pathogenic relevance. Several variants appeared in multiple samples, such as NPIPB15 p.Tyr301Cys and ANKRD36C p.Gly311Asp, while others were unique to either the HQ or PQ group. When combined with clinical findings and IVF outcomes, the data suggest that PQ sperm tend to carry a greater number of variants that could affect function. Conclusion: Poor-quality sperm showed a modestly higher burden of coding and splice-site variants, including those predicted to interfere with protein function. These findings point to underlying genomic differences between morphologically normal and abnormal sperm and demonstrate the value of single-sperm WGS for identifying molecular features linked to reduced sperm quality and subfertility.