Urinary steroid profile in females - the impact of menstrual cycle and emergency contraceptives.

Today's doping tests involving longitudinal monitoring of steroid profiles are difficult in women. Women have more complex hormonal fluctuations than men and commonly take drugs such as hormonal contraceptives that are shown to affect biomarkers used in these doping tests. In this study, we followed six women's urinary steroid profile during one menstrual cycle, including both glucuronides and sulfate conjugated fractions. Additionally, we studied what happens to the steroidal module of the Athlete Biological Passport (ABP) after administration of an emergency contraceptive (levonorgestrel, NorLevo®). The study shows that there are large individual variations in all metabolites included in the ABP and that the administration of emergency contraceptives may lead to suspicious steroid profile findings in the ABP. Urinary epitestosterone concentration increased during the menstrual cycle, leading to a decrease in the testosterone/epitestosterone ratio. The ratios followed in the ABP varied widely throughout the menstrual cycle, the coefficient of variation (CV) ranging from 4 to 99%. There was a 3-fold decrease in epitestosterone 24 h post administration of the emergency contraceptive pill and androsterone, etiocholanolone, and 5ß- androstan-3α,17ß-diol concentrations decreased about 2-fold. When analyzed with the ABP software, one of the six women had an atypical profile after taking the emergency contraceptive. Furthermore, we could not find any alterations in excretion routes (i.e., if the metabolites are excreted as glucuronide or sulfate conjugates) during the menstrual cycle or after administration of emergency contraceptive, indicating no direct effect on phase II enzymes. Copyright © 2016 John Wiley & Sons, Ltd.

Dose-dependent testosterone sensitivity of the steroidal passport and GC-C-IRMS analysis in relation to the UGT2B17 deletion polymorphism.

The newly implemented Steroid Module of the Athlete Biological Passport has improved doping tests for steroids. A biomarker included in this passport is the urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratio, a ratio greatly affected by a deletion polymorphism in UGT2B17. Suspect urine doping tests are further analyzed with gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the origin of the androgen. In this study, we investigated the sensitivity of the steroidal module and the IRMS analysis, in subjects administered with three doses of testosterone enanthate (500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4. On the other hand, using the athlete biological passport and IRMS analysis, all three doses could be detected to a high degree of sensitivity. The concentrations of all steroids included in the steroidal module were dose dependently increased, except for epitestosterone which decreased independent of dose. The decrease in epitestosterone was significantly associated with circulatory levels of testosterone post dose (rs =0.60 and p=0.007). In conclusion, these results demonstrate that administration of a single dose of 125-500 mg testosterone enanthate could be detected using the athlete biological passport, together with IRMS. Since IRMS is sensitive to testosterone doping independent of UGT2B17 genotype, also very small changes in the steroidal passport should be investigated with IRMS.

The impact of genetics and hormonal contraceptives on the steroid profile in female athletes.

The steroid module of the Athlete Biological Passport, the newest innovation in doping testing, is currently being finalized for implementation. Several factors, other than doping, can affect the longitudinal steroid profile. In this study, we investigated the effect of hormonal contraceptives (HC) as well as the effect of three polymorphisms on female steroid profiles in relation to doping controls. The study population consisted of 79 female elite athletes between the ages of 18 and 45. HC were used by 32% of the subjects. A full urinary steroid profile was obtained using World Anti-Doping Agency accredited methods. In addition all subjects were genotyped for copy number variation of UGT2B17 and SNPs in UGT2B7 and CYP17. Subjects using HC excreted 40% less epitestosterone as compared to non-users (p = 0.005) but showed no difference in testosterone excretion. When removing individuals homozygous for the deletion in UGT2B17, the testosterone to epitestosterone (T/E) ratio was 29% higher in the HC group (p = 0.016). In agreement with previous findings in men, copy number variation of UGT2B17 had significant effect on female urinary testosterone excretion and therefore also the T/E ratio. Subjects homozygous for the T allele of CYP17 showed a lower urinary epitestosterone concentration than the other CYP17 genotypes. It is of great importance that the athlete's steroidal passport can compensate for all possible normal variability in steroid profiles from women. Therefore, considering the large impact of HC on female steroid profiles, we suggest that the use of HC should be a mandatory question on the doping control form.