Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle

Abstract

In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers, was investigated for the monitoring of a 28-day T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17β-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the 'blood steroid profile' represents a powerful complementary approach to the 'urinary module' and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration.

Detection of testosterone microdosing in healthy females

“…This may be due to the stratum corneum reaching steady state within days as it forms a dermal barrier and depot mediating T delivery from transdermal systems into the systemic circulation 68,69 . In this study, the serum T displayed a marked decline over 14 days after end of T treatment confirming that serum T measurement is highly sensitive but has a limited window for detecting exogenous T use in women 22,24,25 . This interpretation is also consistent with findings after injection of a long‐acting T undecanoate to transgender and hypogonadal men whereby serum T demonstrated a steep peak and decline with more stable findings observed in the urine steroid profile 70 .…”

“…Whereas potent negative feedback operates in men whose circulating T and E derive almost exclusively from the testis, such negative T feedback is less operative in women whose much lower circulating T arises from three sources—adrenal, ovarian, and extraglandular conversion of precursors—none subject to equally potent and specific negative T feedback as in men. This raises doubt about the applicability of the urine T/E ratio to detect T doping in women 22–25 . Additionally, menstrual cycle variation and hormonal contraceptive use may also impact on the urine steroid profile 24,26–28 .…”

“…[22][23][24][25] Additionally, menstrual cycle variation and hormonal contraceptive use may also impact on the urine steroid profile. 24,[26][27][28] Finally, these limitations have led to development of serum steroid profiling to complement and extend the repertoire of antidoping tests. 22,[29][30][31][32][33] As there are no clearly established indications for T treatment in healthy women, 34,35 most research studies of T administration in women have aimed to replicate endogenous T production rates (0.3 mg/day) 36 for treatment of female sexual dysfunction.…”

The use of RNA‐based 5'‐aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses

“…Moreover, AUC was higher than 0.75 and thus comparable to other anti-doping markers. 19,20 By contrast, SLC4A1 mRNA levels were not significantly altered by microdosing, and an ROC curve analysis confirmed poor specificity at a sensitivity of ≥50% (Figure 2). Hypoxic training performed under natural (altitude training) or artificial conditions (hypoxic rooms with decreased oxygen content) is a legal way to stimulate erythropoiesis in order to increase endurance performance.…”

Carbon isotope ratios of endogenous steroids found in human serum—method development, validation, and reference population-derived thresholds

“…These Published in the topical collection Recent Trends in (Bio)Analytical Chemistry with guest editors Antje J. Baeumner and Günter Gauglitz. results were corroborated by clinical trials encompassing lowdose administrations of T to female volunteers [7,8]. In addition to T concentrations alone, methods for the determination of a comprehensive subset of serum steroids have been developed in the context of sports drug testing [9][10][11].…”