Non-invasive azoospermia subtyping via a seminal plasma extracellular vesicle-based SERS platform

Azoospermia is a leading cause of male infertility. Clinically, it is classified into two subtypes: obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). Accurate subtyping is critical for guiding treatment decisions. However, current diagnostics rely on invasive testicular biopsy, which poses several limitations, including procedural risks, patient discomfort, sampling bias, and the possibility of inconclusive results. To provide a non-invasive alternative, we developed SPER (Seminal Plasma Extracellular Vesicle-based Raman Classifier), a liquid biopsy platform that integrates a nanomixing-enhanced microfluidic chip for extracellular vesicle (EV) enrichment with surface-enhanced Raman scattering (SERS) for EV surface protein profiling. SPER quantifies three target proteins-ECM1, LDH, and cAMP-on EV surfaces, with detection limits of 190, 440, and 520 EV/mL, respectively. Combined with logistic regression modeling, SPER distinguishes NOA from OA with 100 % accuracy. This SPER platform offers a rapid, reliable, and non-invasive solution for clinical azoospermia subtyping.