Non-invasive proteomic profiling of seminal plasma extracellular vesicles facilitates precise molecular stratification in non-obstructive azoospermia

Non-obstructive azoospermia (NOA) constitutes the most severe and phenotypically heterogeneous manifestation of male infertility, currently lacking robust non-invasive molecular biomarkers for precise disease stratification and prognostic assessment. In this study, we performed comprehensive deep proteomic profiling of seminal plasma-derived extracellular vesicles (SPEV) to systematically elucidate their molecular signatures, explore etiologic heterogeneity, and evaluate their potential for clinical translation in NOA management. The study cohort comprised 173 seminal plasma samples, including 66 from normozoospermic subjects (NS) and 107 from patients diagnosed with NOA. Proteomic analysis was executed via an integrated workflow incorporating rapid DSPE (1, 2-distearoyl- sn - glycero -3-phosphoethanolamine) bead-based enrichment, solid-phase-enhanced sample preparation (SP3), and data-independent acquisition mass spectrometry (DIA-MS). The SPEV proteome effectively discriminated between NOA and NS. Subsequent analyses revealed that distinct etiologic subtypes of NOA share a conserved core proteomic signature while retaining subgroup-specific molecular characteristics. Leveraging tissue-specific annotation, we identified etiology-dependent biomarkers, which were subsequently validated via Western blotting. Furthermore, unsupervised clustering stratified idiopathic NOA (iNOA) into two novel proteomic clusters. Notably, within subcohorts possessing comprehensive clinical data, SPEV proteomic profiles delineated molecular variations correlated with testicular histopathological phenotypes. For sperm-retrieval prediction, the SPEV-based model demonstrated discriminative ability in the internal test set, yielding an area under the receiver operating characteristic curve (AUC) of 1.00, with a sensitivity of 100.0% and a specificity of 85.7%. Collectively, these findings indicate that SPEV proteomic profiling constitutes a promising non-invasive liquid biopsy strategy for NOA, offering preliminary evidence to support molecular stratification and facilitating potential clinical translation.