Screening and identification of salivary biomarkers for assessing the effects of exogenous testosterone administration on HPG and HPA axes and ECS

The contents of steroids and endocannabinoids along with the ratios between them would be candidate biomarkers for sensitively and comprehensively assessing the role of testosterone in regulating the activities of the hypothalamus-pituitary-gonadal (HPG) axis, the hypothalamus-pituitaryadrenal (HPA) axis and endogenous cannabinoid system (ECS). However, previous studies mostly used the contents rather than the ratios as biomarkers. This study aimed to systematically screen and identify sensitive biomarkers from 21 candidates including both the contents of nine steroids and one endocannabinoid and their ratios in saliva. Three screening criteria were whether there were intergroup differences, time-dependent changes and considerable relative stability during a 4-h period after exogenous testosterone administration. This study used LC-APCI⁺-MS/MS to determine the salivary levels of the candidate biomarkers on 62 male healthy undergraduates who were divided into testosterone administration and placebo control groups. The results revealed that salivary testosterone, androstenedione, DHEA and the ratios of testosterone to estradiol and AEA, and of cortisol to testosterone and DHEA were sensitive biomarkers for assessing the effects of testosterone administration on the three neuroendocrine systems because they all showed significant intergroup differences and time-dependent changes and good relative stability. Salivary cortisol, cortisone and the ratios of testosterone to androstenedione and DHEA and of androstenedione to estrone, and of cortisol to cortisone, androstenedione and AEA might be suitable biomarkers because they met only two of the three criteria, but needed to be validated in the future. The rest biomarkers were unsuitable because they mostly showed no significant intergroup differences, blunt time-dependent changes and poor relative stability.