Urinary proteomic signatures of exogenous testosterone exposure following oral testosterone undecanoate...

Existing approaches for evaluating exogenous testosterone exposure rely primarily on steroid profiling, which provides limited insight into downstream biological responses and may be affected by substantial inter-individual variability. Here, we applied a time-resolved urinary proteomics strategy to characterize molecular alterations following controlled oral testosterone undecanoate (TU) administration in humans. Urine samples collected from 10 healthy male volunteers before and after TU administration were analyzed using data-independent acquisition (DIA)-based proteomics. A total of 3379 proteins were retained for quantitative analysis. Differential abundance analysis revealed pronounced time-dependent proteomic alterations at all post-administration time points, with 536, 412, 780, and 293 differentially abundant proteins identified at 4 h, 24 h, 48 h, and 72 h, respectively. Functional enrichment and protein-protein interaction analyses indicated coordinated changes involving extracellular matrix organization, membrane-associated signaling, immune-related pathways, and metabolic processes. Integrated analyses identified TGFβ1, CAV1, and ITGB2 as the most consistently altered candidate proteins. Exploratory receiver operating characteristic analysis showed that TGFβ1 displayed promising discriminatory performance between baseline and early post-administration samples (AUC = 0.850). Semi-quantitative Western blotting further supported the detectability and directional alterations of selected candidate proteins in urine. These findings demonstrate that urinary proteomics can capture systemic molecular responses associated with exogenous testosterone exposure beyond conventional steroid measurements and provide a preliminary basis for future biomarker development in anti-doping research.