Seminal plasma cfDNA fragmentomics landscape delineates male infertility subtypes

Background While cell-free DNA (cfDNA) fragmentomics has transformed liquid biopsy applications in prenatal screening and oncology, its potential in male reproductive health remains uncharted. Methods Through integrated whole-genome sequencing and jagged end sequencing (Jag-Seq) coupled with non-CpG methylation analysis, we established the first fragmentomic atlas of seminal plasma (SP) cfDNA from 18 healthy donors, with 20 plasma cfDNA samples. And we applied this method to 33 infertility cases (14 varicocele / 19 non-obstructive azoospermia), to obtain disease-specific characteristics. ROC curve analysis was employed to study the potential diagnostic ability for these two diseases. Results Size distribution profiling showed SP cfDNA enrichment in short fragments (< 150bp) with bimodal distribution (151bp main peak/110bp subpeak), contrasting with plasma's sharp 166-bp peak pattern ( P  < 0.001). Motif analysis identified SP-specific patterns: elevated AAAA-end motif frequency and A-base preference at positions − 2 to -4. And SP showed higher jagged end index based on Jag-Seq ( P  < 0.0001). For disease, varicocele exhibited 7 different frequency motifs and longer jagged end length while non-obstructive azoospermia demonstrated higher methylation level at CH sites. Translating these findings to clinical contexts, we developed a ROC curve analysis integrating all fragmentomic signatures, achieving 83% accuracy in distinguishing varicocele and 87% accuracy in distinguishing non-obstructive azoospermia. Conclusions This research highlights the distinct cfDNA profiles in SP and demonstrates the potential of cfDNA metrics as biomarkers for diagnosing male infertility subtypes, and the disease-specific cfDNA dynamics offering new avenues for non-invasive diagnostic tools in reproductive medicine.