Genome-wide fragmentomics of seminal-plasma cell-free DNA by low-coverage WGS reveals signatures linked to spermatogenic activity.

Seminal plasma cell-free DNA (sp-cfDNA) originates from germ and somatic cells of the male reproductive tract, yet its genome-wide features are not well defined. In this exploratory study, low-coverage whole-genome sequencing with single-stranded library preparation was applied to sp-cfDNA from 40 men classified by sperm concentration. sp-cfDNA yield decreased with sperm count and was lowest in azoospermia, which also showed elevated and variable mitochondrial DNA levels. Fragment-end motif composition differed between groups, and 141 motifs distinguished azoospermia from normozoospermia. Microbial cfDNA represented a minor but consistent fraction, highest in azoospermic samples. Chromosome-level coverage patterns were preserved even at shallow depth, enabling basic cytogenomic assessment. Together, these results indicate that low-coverage WGS reveals mitochondrial, fragmentomic, and microbial signatures reflecting spermatogenic activity and provides a foundation for future applications of cfDNA analysis in male infertility research.