Stratified profiling of azoospermia: Differentiating histological subtypes with seminal plasma metabolic signatures

Non-obstructive azoospermia (NOA), characterized by impaired spermatogenesis and the complete absence of sperm in the ejaculate, represents one of the most severe forms of male infertility. Current approaches for definitive histological characterization and sperm retrieval often require invasive testicular procedures, underscoring the need for reliable, non-invasive methods for preoperative stratification. In the present study, we performed untargeted metabolomic profiling of human seminal plasma to identify biomarker panels capable of stratifying azoospermia subtypes through a stepwise approach. We identified distinct metabolite biomarker panels comparing NOA vs. obstructive azoospermia (OA), Sertoli cell-only syndrome vs. other NOA subtypes, and hypospermatogenesis vs. maturation arrest. Additionally, cross-species comparative analysis with high-resolution metabolomic data from purified mouse testicular cell populations implicated these biomarkers in specific germ cell stages and metabolic transitions during spermatogenesis. These findings provide a preliminary molecular framework for the non-invasive classification of azoospermia subtypes and warrant validation in larger independent cohorts.