Intra-individual stability of longitudinal urinary steroid profiles in Swedish athletes

The steroid module of the athlete biological passport (ABP) aims to detect doping with endogenous steroids by longitudinally monitoring epitestosterone (E), testosterone (T), and four metabolically related steroids and their ratios. There are large variations in the urinary levels of the androgen metabolites due to genetic polymorphisms, drug use, menstrual cycle, and other factors. In this study, we aimed to increase our understanding of the natural, within-individual variations of the established ABP markers in males and females over time, looking at samples collected both in and out-of-competition (IC/OOC). Urinary steroid profiles from 323 Swedish athletes, with at least five samples per athlete, were extracted from ADAMS together with information on type of sport, IC/OOC, and time of day. Data were analyzed using coefficient of variation (CV%) to examine within-subject variability and linear mixed effects models to estimate within-subject change in the metabolites over time. The metabolites and ratios expressed higher individual CV% in females (23–56) than in males (18–39). Samples taken OOC showed larger intra-individual variations than samples collected IC for most of the ABP metabolites in both sexes. The median concentrations were higher IC for some metabolites, particularly testosterone being 52% higher among females. Time of day influenced the intra-individual variation of the urinary steroid profile with decreases in androgen metabolites over time, if measured in evening versus daytime. These findings can aid in the testing strategies and interpretation of the steroidal module of ABP.     

Pregnancy greatly affects the steroidal module of the Athlete Biological Passport

Concentrations of urinary steroids are measured in anti‐doping test programs to detect doping with endogenous steroids. These concentrations are combined into ratios and followed over time in the steroidal module of the Athlete Biological Passport (ABP). The most important ratio in the ABP is the testosterone/epitestosterone (T/E) ratio but this ratio is subject to intra‐individual variations, especially large in women, which complicates interpretation. In addition, there are other factors affecting T/E. Pregnancy, for example, is known to affect the urinary excretion rate of epitestosterone and hence the T/E ratio. However, the extent of this variation and how pregnancy affect other ratios has not been fully evaluated. Here we have studied the urinary steroid profile, including 19‐norandrosterone (19‐NA), in 67 pregnant women and compared to postpartum. Epitestosterone was higher and, consequently, the T/E and 5αAdiol/E ratios were lower in the pregnant women. Androsterone/etiocholanolone (A/Etio) and 5αAdiol/5βAdiol, on the other hand, were higher in the first trimester as compared to postpartum (p<0.0001 and p=0.0396, respectively). There was no difference in A/T during pregnancy or after. 19‐NA was present in 90.5% of the urine samples collected from pregnant women. In this study, we have shown that the steroid profile of the ABP is affected by pregnancy, and hence can cause atypical passport findings. These atypical findings would lead to unnecessary confirmation procedures, if the patterns of pregnancy are not recognized by the ABP management units.     

Variability of the urinary and blood steroid profiles in healthy and physically active women with and without oral contraception

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17β-diol/5ß-androstane-3α,17β-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.     

Urinary steroid profile in relation to the menstrual cycle

The interpretation of the steroidal module of the Athlete Biological Passport (ABP) in female athletes is complex due to the large variation of the endogenous urinary steroids. The menstrual cycle seems to be one of the largest confounders of the steroid profile. The duration of the different phases in the menstrual cycle differs between women and is difficult to predict only by counting days after menstruation. Here, we have determined the follicle, ovulation, and luteal phases, by assessing the menstrual hormones in serum samples collected from 17 healthy women with regular menses. Urine samples were collected three times per week during two consecutive cycles to measure the urinary steroid concentrations used in the ABP. The metabolite that was mostly affected by the menstrual phases was epitestosterone (E), where the median concentration was 133% higher in the ovulation phase compared to the follicle phase (p < 0.0001). The women with a large coefficient of variation (CV) in their first cycle also had a large CV in their second cycle and vice versa. The inter-individual difference was extensive with a range of 11%–230% difference between the lowest and the highest T/E ratio during a cycle. In conclusion, E and ratios with E as denominator are problematic biomarkers for doping in female athletes. The timing of the sample collection in the menstrual cycle will have a large influence on the steroid profile. The results of this study highlight the need to find additional biomarkers for T doping in females.     

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

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.     

Subject-based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports

Doping with natural steroids can be detected by evaluating the urinary concentrations and ratios of several endogenous steroids. Since these biomarkers of steroid doping are known to present large inter-individual variations, monitoring of individual steroid profiles over time allows switching from population-based towards subject-based reference ranges for improved detection. In an Athlete Biological Passport (ABP), biomarkers data are collated throughout the athlete's sporting career and individual thresholds defined adaptively. For now, this approach has been validated on a limited number of markers of steroid doping, such as the testosterone (T) over epitestosterone (E) ratio to detect T misuse in athletes. Additional markers are required for other endogenous steroids like dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA). By combining comprehensive steroid profiles composed of 24 steroid concentrations with Bayesian inference techniques for longitudinal profiling, a selection was made for the detection of DHT and DHEA misuse. The biomarkers found were rated according to relative response, parameter stability, discriminative power, and maximal detection time. This analysis revealed DHT/E, DHT/5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol as best biomarkers for DHT administration and DHEA/E, 16α-hydroxydehydroepiandrosterone/E, 7β-hydroxydehydroepiandrosterone/E and 5β-androstane-3α,17β-diol/5α-androstane-3α,17β-diol for DHEA. The selected biomarkers were found suitable for individual referencing. A drastic overall increase in sensitivity was obtained. The use of multiple markers as formalized in an Athlete Steroidal Passport (ASP) can provide firm evidence of doping with endogenous steroids.     

Evaluation of longitudinal steroid profiling with the ADAMS adaptive model for detection of transdermal,intramuscular, and subcutaneous testosterone administration

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites in order to identify samples suspicious for the use of synthetic forms of endogenous anabolic androgenic steroids (EAAS). Samples identified by the module may then be confirmed by isotope ratio mass spectrometry (IRMS) to establish clearly the exogenous origin of testosterone and/or metabolites in the sample. To examine the detection capability of the steroidal ABP model, testosterone administration studies were performed with various doses and three routes of administration – transdermal, intramuscular, and subcutaneous with 15 subjects for each route of administration. Urine samples were collected before, during, and after administration and steroid profiles were analyzed using the steroidal ABP module in ADAMS. A subset of samples from each mode of administration was also analyzed by IRMS. The steroidal ABP module was more sensitive to testosterone use than population‐based thresholds and with high dose administrations there was very good agreement between the IRMS results and samples flagged by the module. However, with low dose administration the ABP module was unable to identify samples where testosterone use was still detectable by IRMS analysis. The testosterone/epitestosterone (T/E) ratio was the most diagnostic parameter for longitudinal monitoring with the exception of low testosterone excretors for whom the 5α‐androstane‐3α, 17β‐diol/epitestosterone (5αAdiol/E) ratio may provide more sensitivity.     

Impact of hormonal contraceptives on urinary steroid profile in relation to serum hormone changes and CYP17A1 polymorphism

To detect doping with endogenous steroids, six urinary steroids are longitudinally monitored in the athlete biological passport (ABP). These steroids include testosterone, etiocholanolone, androsterone, 5α‐androstane‐3α,17β‐diol, 5β‐androstane‐3α,17β‐diol, and the testosterone isomer epitestosterone. It is known that the intake of hormonal contraceptives may interfere with the ABP biomarkers. A previous study showed that athletes using hormonal contraceptives (HCs) display lower urinary epitestosterone concentrations than non‐using athletes. In this study, we analyzed the urinary steroid profile prior to and three months after administration of an oral HC including levonorgestrel and ethinylestradiol (n = 55). The urinary concentrations of all the ABP metabolites decreased after three months, with epitestosterone showing the largest decline (median 6.78 to 3.04 ng/mL, p?0.0001) followed by 5α‐androstane‐3α,17β‐diol (median 23.5 to 12.83 ng/mL, p?0.0001), and testosterone (median 5.32 to 3.66, p?0.0001). Epitestosterone is included in two of the five ratios in the ABP (T/E and 5αAdiol/E), and consequently these ratios increased 1.7‐fold (range 0.27 to 8.50) and 1.26‐fold (range 0.14 to 5.91), respectively. Some of these changes may mimic the changes seen after administration of endogenous steroids leading to atypical findings. Notably, even though participants used the same contraceptive treatment schedule, the HC‐mediated epitestosterone change varied to a large extent (median 0.43‐fold, range 0.06 to 6.5) and were associated with a functional T?C promoter polymorphism in CYP17A1. Moreover, the epitestosterone changes correlated with HC‐induced testosterone and gonadotropins changes in serum, indicating that urinary epitestosterone reflects the androgen load in HC‐using women.     

Evaluation of longitudinal steroid profiles from male football players in UEFA competitions between 2008 and 2013

Testosterone and related compounds are the most recurrent doping substances. The steroid profile, consisting of the quantification of testosterone and its metabolites, has been described as the most significant biomarker to detect doping with pseudo‐endogenous anabolic steroids. The steroidal module of the Athlete Biological Passport (ABP) was launched by the World Anti‐Doping Agency (WADA) in 2014. To assess the value of introducing the module to its anti‐doping programme, the Union of European Football Associations (UEFA) decided to analyze retrospectively the steroid profile data of 4195 urine samples, collected from 879 male football players and analyzed in 12 WADA‐accredited laboratories between 2008 and mid‐2013. This study focused on the evaluation of T/E ratios. The coefficient of variation (CV) and the adaptive model were the two statistical models used to study the longitudinal follow‐up. A CV of 46% was determined to be the maximal natural intra‐individual variation of the T/E when the sequence consisted of single data points analyzed in different laboratories. The adaptive model showed some profiles with an atypical T/E sequence and also enabled an estimate of the prevalence of external factors impacting the T/E sequences. Despite the limitations of this retrospective study, it clearly showed that the longitudinal and individual follow‐up of the T/E biomarker of the players is a good tool for target testing in football. UEFA has therefore decided to implement the steroidal module of the ABP from the start of the next European football season in September 2015. Copyright © 2015 John Wiley & Sons, Ltd.     

Codeine influences the serum and urinary profile of endogenous androgens but does not interact with the excretion rate of administered testosterone

Today's doping tests involve longitudinal monitoring of urinary steroids including the testosterone glucuronide and epitestosterone glucuronide ratio (T/E) in an Athlete Biological Passport (ABP). The aim of this study was to investigate the possible influence of short‐term use of codeine on the urinary excretion of androgen metabolites included in the steroidal module of the passport prior to and after the co‐administration with testosterone. The study was designed as an open study with the subjects being their own control. Fifteen healthy male volunteers received therapeutic doses of codeine (Kodein Meda) for 6 days. On Day 3, 500 mg or 125 mg of testosterone enanthate (Testoviron®‐Depot) was administered. Spot urine samples were collected for 17 days, and blood samples were collected at baseline, 3, 6, and 14 days after codeine intake. The circulatory concentration of total testosterone decreased significantly by 20% after 3 days' use of codeine (p = 0.0002) and an atypical ABP result was noted in one of the subjects. On the other hand, the concomitant use of codeine and testosterone did not affect the elevated urinary T/E ratio. In 75% of the individuals, the concentration of urinary morphine (a metabolite of codeine) was above the decision limit for morphine. One of the participants displayed a morphine/codeine ratio of 1.7 after codeine treatment, indicative of morphine abuse. In conclusion, our study shows that codeine interferes with the endogenous testosterone concentration. As a result, the urinary steroid profile may lead to atypical findings in the doping test.     

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 5β‐ androstan‐3α,17β‐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.     

Multivariate interpretation of the urinary steroid profile and training‐induced modifications. The case study of a Marathon runner

The steroidal module of the athlete biological passport (ABP) introduced by the World Anti‐Doping Agency (WADA) in 2014 includes six endogenous androgenic steroids and five of their concentration ratios, monitored in urine samples collected repeatedly from the same athlete, whose values are interpreted by a Bayesian model on the basis of intra‐individual variability. The same steroid profile, plus dihydrotestosterone (DHT) and DHEA, was determined in 198 urine samples collected from an amateur marathon runner monitored over three months preceding an international competition. Two to three samples were collected each day and subsequently analyzed by a fully validated gas chromatography–mass spectrometry protocol. The objective of the study was to identify the potential effects of physical activity at different intensity levels on the physiological steroid profile of the athlete. The results were interpreted using principal component analysis and Hotelling's T² vs Q residuals plots, and were compared with a profile model based on the samples collected after rest. The urine samples collected after activity of moderate or high intensity, in terms of cardiac frequency and/or distance run, proved to modify the basal steroid profile, with particular enhancement of testosterone, epitestosterone, and 5α‐androstane‐3α,17β‐diol. In contrast, all steroid concentration ratios were apparently not modified by intense exercise. The alteration of steroid profiles seemingly lasted for few hours, as most of the samples collected 6 or more hours after training showed profiles compatible with the “after rest” model. These observations issue a warning about the ABP results obtained immediately post‐competition.     

Changes in blood parameters after intramuscular testosterone ester injections – Implications for anti‐doping

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the ‘haematological module’ of the athlete biological passport (ABP) used by the World Anti‐Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo‐controlleddouble‐blind design. Urine samples and blood samples were collected twice pre‐treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post‐injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®‐treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope‐ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF‐Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3–10 days after injection. From an anti‐doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow‐up testing and substantiating likely misuse of testosterone.     

Discordant genotyping results using DNA isolated from anti‐doping control urine samples

The UGT2B17 gene deletion polymorphism is known to correlate to urinary concentration of testosterone‐glucuronide and hence this genotype exerts a large impact on the testosterone/epitestosterone (T/E) ratio, a biomarker for testosterone doping. The objective of this study was to assess if DNA isolated from athletes’ urine samples (n = 713) obtained in routine doping controls could be targeted for genotyping analysis for future integration in the athlete's passport. A control population (n = 21) including both urine and blood DNA was used for genotyping concordance test. Another aim was to study a large group (n = 596) of authentic elite athletes in respect of urinary steroid profile in relation to genetic variation. First we found that the genotype results when using urine‐derived DNA did not correlate sufficiently with the genotype obtained from whole blood DNA. Secondly we found males with one or two UGT2B17 alleles had higher T/E (mean 1.63 ± 0.93) than females (mean 1.28 ± 1.08), p˂0.001. Unexpectedly, we found that several male del/del athletes in power sports had a T/E ˃1. If men in power sport exert a different urinary steroid profile needs to be further investigated. The other polymorphisms investigated in the CYP17A1, UGT2B7 and UGT2B15 genes did not show any associations with testosterone and epitestosterone concentrations. Our results show that genotyping using urine samples according to our method is not useful in an anti‐doping setting. Instead, it is of importance for the anti‐doping test programs to include baseline values in the ABP to minimize any putative impact of genotype. Copyright © 2016 John Wiley & Sons, Ltd.     

Sensitivity of doping biomarkers after administration of a single dose testosterone gel

Micro‐doping with testosterone (T) is challenging to detect with the current doping tests. Today, the methods available to detect T are longitudinally monitoring of urine biomarkers in the Athlete Biological Passport (ABP) and measuring the isotopic composition of excreted biomarkers to distinguish the origin of the molecule. In this study, we investigated the detectability of a single dose of 100 mg T gel in 8 healthy male subjects. We also studied which biomarkers were most sensitive to T gel administration, including blood biomarkers. The ABP successfully detected T gel administration in all 8 subjects. The most sensitive ratio was 5αAdiol/E, however, all ratios showed atypical findings. Isotope ratio mass spectrometry (IRMS) was performed on 5 subjects and only 2 met all the criteria for a positive test according to the rules set by the World Anti‐Doping Agency (WADA). The other 3 showed inconclusive results. Other markers that were affected by T gel administration, not used for this detection today, were serum dihydrotestosterone (DHT) and T as well as reticulocyte count and percentage in whole blood. miRNA‐122 was not significantly affected by the single T dose. A single dose of 100 mg T gel is possible to detect with today's doping tests. Since a single dose of T gel has an impact on some hematological biomarkers, access to both modules of the ABP when evaluating the athletes' profiles will increase the possibility to detect micro‐doses of T. In addition, serum DHT and T may be a useful addition to the future endocrine module of the ABP.     

Studies of athlete biological passport biomarkers and clinical parameters in male and female users of anabolic androgenic steroids and other doping agents

The use of anabolic androgenic steroids (AAS) and other performance enhancing substances can change over time, so there is a need to constantly update what substances are used and can be detected. Six women and 30 men anabolic androgenic steroid users were recruited who filled out an anonymous questionnaire about their use of performance enhancing substances during the past year. Sampling took place on a single occasion and included blood and urine collection. Our aim was to identify which doping agents can be detected in men and women self‐reporting AAS use. The first choice of substances differed between men (testosterone) and women (oxandrolone). The use of growth hormones was reported among men (10%) and women (50%). Growth hormone releasing factors/secretagogs were reported by about ~ 20% in both genders. Nandrolone was the most frequently detected anabolic androgenic steroid even in those who did not report use in the past year. Of the current male testosterone users, 82% exhibited testosterone/epitestosterone (T/E) ratios of > 4. Men with current testosterone use displayed 4‐fold and 6‐fold higher median T/E, respectively, when compared with recent and previous testosterone users (P = 0.0001). Dermal testosterone use in women (n = 2) was not associated with a T/E ratio of > 4, but with supra‐physiological total serum testosterone concentrations. Changes in gonadotropins and hematological parameters were associated with the time of the last anabolic androgenic steroid intake in men, whereas in women these biomarkers were within the normal range. This highlights gender specific differences and indicates the need for additional biomarkers in female athletes.     

The Probenecid‐story – A success in the fight against doping through out‐of‐competition testing

The effectiveness of doping control in sport has been improved continuously during the past 50 years. One of the major steps forward was the introduction of unannounced and targeted out‐of‐competition testing in order to control the misuse of anabolic‐androgenic steroids (AAS), mainly during the end of the 1980s. It also led to the misuse of masking agents in case a surprise control was performed. Athletes tried to be “prepared”, when the doping control officer showed up. The disclosure of the masking agent probenecid in 1987 is a perfect example of a memorable finding, of a suspected and purported case of performance manipulation. Probenecid and its metabolites were identified in five urine samples collected from Norwegian athletes in an out‐of‐competition test, while they were staying and training in the USA. Probenecid is a drug that reduces the urinary excretion of AAS from the body. It was the first time that it had showed up in a doping control sample. The athletes were sanctioned for hampering the analysis of their urine sample, although probenecid was not yet specified on the Prohibited List. Its detection was the result of a successful collaboration of laboratories and investigative diligence and enthusiasm following up suspicious observations in the actual samples. Immediately afterwards probenecid was added to the Prohibited List for 1988 as well as including the manipulation of doping control samples.     

The effect of athletes` hyperhydration on the urinary ‘steroid profile’ markers in doping control analysis

The urinary ‘steroid profile’ in doping control analysis is a powerful tool aimed at detecting intra‐individual deviations related to the abuse of endogenous steroids. Factors altering the steroid profile include, among others, the excessive fluid intake leading to low endogenous steroids concentrations compared to an individual's normal values. Cases report the use of hyperhydration by athletes as a masking method during anti‐doping urine sample collection. Seven healthy physically active non‐smoking Caucasian males were examined for a 72‐hour period using water and a commercial sports drink as hyperhydration agents (20 mL/kg body weight). Urine samples were collected and analyzed according to World Anti‐Doping Agency (WADA) technical documents. Although, significant differences were observed on the endogenous steroid concentrations under the studied hyperhydration conditions, specific gravity adjustment based on a reference value of 1.020 can eliminate the dilution induced effect. Adjustment methods based on creatinine and urinary flow rate were also examined; however, specific gravity was the optimum method in terms of effectiveness to adjust concentrations close to the baseline steroid profile and practicability. No significant effect on the urinary steroid ratios was observed with variability values within 30% of the mean for the majority of data. Furthermore, no masking on the detection ability of endogenous steroids was observed due to hyperhydration. It can be concluded that any deviation on the endogenous steroid concentrations due to excessive fluid intake can be compensated by the specific gravity adjustment and therefore, hyperhydration is not effective as a masking method on the detection of the abuse of endogenous steroids.     

Re-evaluation of combined ((ES/EG)/(TS/TG)) ratio as a marker of testosterone intake in men

To detect doping with pseudo-endogenous anabolic steroids in sports, a urinary steroid profile with glucuronidated plus unconjugated androgens is used. In addition to analyze androgen glucuronide metabolites, it can be of interest to also include sulfate metabolites in the urinary steroid profile. The combined ratios of epitestosterone sulfate/epitestosterone glucuronide to the ratios of testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) have previously been investigated as a complementary biomarker for testosterone doping. In this restudy, the aim was to evaluate this biomarker in a larger study sample population. A single dose of 500-mg testosterone enanthate was administered to 54 healthy male volunteers. Urine was collected prior to (Day 0) administration and throughout 15 days and analyzed for the sulfate and glucuronide conjugates of testosterone and epitestosterone. The results show that the combined ratio increased to a larger extent than the traditional T/E ratio in all subjects. This increase was independent on UGT2B17 gene polymorphism. Moreover, a delayed peak of the combined ratio was observed in ~60% of the participants. The results confirm that complementary analyses of the sulfate metabolites may be a useful approach to detect testosterone doping in men.     

Evaluation of blood parameters by linear discriminant models for the detection of testosterone administration

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites to identify samples suspicious for the use of synthetic forms of Endogenous Anabolic Androgenic Steroids (EAAS). Multiple recent studies have suggested that monitoring of blood parameters may provide enhanced detectability of exogenous testosterone administration. Transdermal and intramuscular testosterone administration studies were carried out in 15 subjects, and the effect on blood steroidal levels, hematological parameters, and gonadotropins was evaluated. Serum testosterone and dihydrotestosterone levels increased while gonadotropin levels were suppressed after administration. A modest increase in reticulocytes was also observed. The blood parameters that were responsive to the administrations were combined into several linear discriminant models targeting both administration (on) and washout (off) phases. The models were effective in detecting the large dose intramuscular administration but were less successful in the detection of the lower dose transdermal application. The blood profiling models may provide complementary value but do not appear to be substantially more advantageous than longitudinal urinary profiling.