Effects of Microbial Activity on Testosterone and Its Major Metabolites in Urine Samples: An Artificial Intervention Study

Artificially cultured bacterial solutions derived from urine were used to simulate urine degradation. Bacterial activity primarily induced the hydrolysis of glucuronide‐conjugated steroids during the early stage, followed by steroid transformation and redistribution among conjugated forms as degradation progressed. By separating and analyzing free and glucuronide‐conjugated steroid fractions, the free‐to‐total ratios of the diols (5αAdiol and 5βAdiol) were demonstrated to be sensitive indicators of degradation progression, particularly in low‐testosterone (T)–excreting populations where the T free / T total ratio may be difficult to determine. In the early stage of degradation, the concentrations of steroids in the Urinary Markers of the Steroidal Module of the Athlete Biological Passport (ABP‐USM) were not significantly affected, even when a small proportion of free steroids was detected. However, as the degradation process advanced, the degradation markers currently used by WADA (the 5 α AND/A and 5 β AND/Etio ratios) increased rapidly. Once these ratios exceeded 0.1, the concentrations of both free and glucuronide‐conjugated steroids underwent notable changes, which may potentially affect the interpretation of the ABP‐USM markers.