Molecular stratification of sperm donors: Raman spectroscopy of seminal plasma.

Seminal plasma (SP), comprising 95% of ejaculate volume, modulates sperm function through proteins, metabolites, and lipids that reflect the state of male reproductive system. This study applied Raman microspectroscopy to compare molecular profiles of SP from selected sperm donors, normozoospermic men, and patients with pathological semen parameters according to WHO criteria. Spectral decomposition revealed major contributors associated with tyrosine, spermine phosphate hexahydrate (SPH), protein-rich material, and other metabolites. Donor samples exhibited stronger spatial heterogeneity with more frequent tyrosine- and SPH-related spectra indicative of favorable biochemical homeostasis, while pathological samples showed peak intensities consistent with oxidative stress and metabolic impairment. The most discriminatory Raman bands were located at 625, 850, and 1667 cm-1, allowing 90% sensitivity and specificity for pathology detection and 80% true recognition of selected donors. The distinct spectral patterns between the groups demonstrate that Raman spectroscopy of SP can both confirm sperm degradation captured by routine semen analysis and reveal donor-specific molecular differences possibly associated with improved cryotolerance and implantation rates. These findings support the potential of Raman-based molecular profiling as a complementary tool for donor selection, quality control in sperm banking, and advanced semen assessment in assisted reproduction.