Genetic alterations in poor-quality individually selected sperm highlight candidate biomarkers for male subfertility

Abstract Whole-genome sequencing (WGS) of morphology-selected sperm pools provides an exploratory approach for investigating genomic features associated with variable sperm quality. In this study, pooled DNA from 1500 individually selected spermatozoa per sample was analyzed from paired high-quality (HQ; motile, normal morphology) and poor-quality (PQ; immotile, abnormal morphology) sperm populations obtained from the same ejaculate of six male partners undergoing infertility treatment. A multi-step filtering and annotation pipeline incorporating American College of Medical Genetics and Genomics (ACMG) criteria, population allele frequencies, tissue-specific expression, and predicted protein effects identified 42 unique high-confidence variants affecting 32 genes across all samples, all consistent with homozygous genotypes in the corresponding individuals as inferred from pooled sperm DNA. Across descriptive comparisons, PQ sperm pools frequently showed numerically higher counts across several variant categories, including structural alterations, splice-related annotations, and coding variant annotations, although none of these differences reached statistical significance. Because HQ and PQ pools were derived from the same individuals, these observations were interpreted descriptively rather than as evidence of distinct inherited variant burden between sperm quality groups. A frameshift insertion in FOXO6, detected in a single PQ sample, represented the most notable exploratory candidate variant, while recurrent variants such as NPIPB15 p.Tyr301Cys and ANKRD36C p.Gly311Asp were observed across multiple samples. When considered together with semen characteristics and pregnancy outcomes, the data support the possibility that poor-quality sperm may be associated with altered genomic integrity, particularly at the level of structural instability, while also highlighting recurrent candidate variants for further study. These findings support the value of broad genomic profiling of morphology-selected sperm pools as an exploratory strategy for investigating male subfertility and identifying candidate variants for future validation.