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 diagnostic strategies rely on invasive procedures such as testicular sperm extraction, underscoring the urgent need for reliable, non-invasive alternatives. 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 establish a molecular framework for the non-invasive classification of azoospermia subtypes and provide novel insights into the metabolic disruptions underlying male infertility.