Seminal plasma cell-free DNA
Research Question: Does seminal plasma cell-free DNA (sp-cfDNA), analyzed by fragmentomics from low-coverage whole-genome sequencing, carry biologically informative signals that are related to sperm count in the ejaculate and can distinguish between clinical groups? Design: A cross-sectional exploratory study based on 40 men undergoing infertility evaluation. Participants were stratified by sperm count into normozoospermia (n=9), oligozoospermia (n=8), cryptozoospermia (n=8), and azoospermia (n=15; 12 non-obstructive, 2 obstructive). Extraction and sequencing of sp-cfDNA were performed using single-stranded DNA libraries with low-coverage paired-end WGS (~30M reads/sample; ~0.4× nuclear genome vertical coverage). Analyses included sp-cfDNA concentration, fragmentation characteristics (fragment length and end motifs), and microbial DNA content. Results: Sp-cfDNA concentration followed sperm count across the groups, with the lowest levels observed in azoospermic men (P < 0.05), while sequencing coverage revealed mitochondrial DNA abundance to be greatest and most variable in this group. End-motif profiling showed global fragmentation differences across groups (PERMANOVA R²=0.21, P = 0.005), and 141 motifs significantly distinguished azoospermic from normozoospermic men, providing moderate separation (silhouette width=0.48; ARI=0.42). Microbial cfDNA was consistently detectable, comprising on average ~0.02% of sequences (range 0.001–0.5%), with azoospermic men carrying the highest number of identifiable taxa. Conclusions: In this exploratory study, low-coverage WGS of seminal plasma cfDNA captured multiple molecular features, including concentration, mitochondrial content, fragmentation profiles, and microbial DNA, that differed between groups defined by sperm count. These findings highlight sp-cfDNA fragmentomics as a potential source of biologically informative signals for further investigation in male infertility.