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.     

Inter‐individual variation of the urinary steroid profiles in Swedish and Norwegian athletes

The steroidal module of the Athlete Biological Passport (ABP) aims to detect doping with endogenous steroids, e.g. testosterone (T), by longitudinally monitoring several biomarkers. These biomarkers are ratios combined into urinary concentrations of testosterone and metabolically related steroids. However, it is evident after 5 years of monitoring steroid passports that there are large variations in the steroid ratios complicating its interpretation. In this study, we used over 11000 urinary steroid profiles from Swedish and Norwegian athletes to determine both the inter‐ and intra‐individual variations of all steroids and ratios in the steroidal passport. Furthermore, we investigated if the inter‐individual variations could be associated with factors such as gender, type of sport, age, time of day, time of year, and if the urine was collected in or out of competition. We show that there are factors reported in today's doping tests that significantly affect the steroid profiles. The factors with the largest influence on the steroid profile were the type of sport classification that the athlete belonged to as well as whether the urine was collected in or out of competition. There were also significant differences based on what time of day and time of year the urine sample was collected. Whether these significant changes are relevant when longitudinally monitoring athletes in the steroidal module of the ABP should be evaluated further.     

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.     

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.     

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.     

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.     

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.     

A novel mixed living high training low intervention and the hematological module of the athlete biological passport

Exposure to either natural or simulated hypoxia induces hematological adaptations that may affect the parameters of the Athlete Biological Passport (ABP). The aim of the present study was to examine the effect of a novel, mixed hypoxic dose protocol on the likelihood of producing an atypical ABP finding. Ten well‐trained middle‐distance runners participated in a “live high, train low and high” (LHTLH) altitude training camp for 14 days. The participants spent ?6 hr.d‐1 at 3000–5400 m during waking hours and ?10 h.d‐1 overnight at 2400–3000 m simulated altitude. Venous blood samples were collected before (B0), and after 1 (D1), 4 (D4), 7 (D7), and 14 (D14) days of hypoxic exposure, and again 14 days post exposure (P14). Samples were analyzed for key parameters of the ABP including reticulocyte percentage (Ret%), hemoglobin concentration ([Hb]), and the OFF‐score. The ABP adaptive model was administered at a specificity of 99% to test for atypical findings. We found significant changes in [Hb] and Ret% during the hypoxic intervention. Consequently, this led to ABP threshold deviations at 99% specificity in three participants. Only one of these was flagged as an “atypical passport finding” (ATPF) due to deviation of the OFF‐score. When this sample was evaluated by ABP experts it was considered “normal”. In conclusion, it is highly unlikely that the present hypoxic exposure protocol would have led to a citation for a doping violation according to WADA guidelines.     

Sensitivity and specificity of detection methods for erythropoietin doping in cyclists

Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti‐doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double‐blinded, placebo‐controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin β) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl‐PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl‐PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl‐PAGE and isoelectric focusing assays for the detection of erythropoietin abuse were 63.8% and 58.6%, respectively, with a false presumptive finding rate of 4.3% and 6%. None of the false presumptive findings tested positive in the confirmation analysis. Sensitivity was highest between 2 and 6 days after dosing, and dropped rapidly outside this window. Sensitivity of the ABP was 91.3%. Specificity of the urine assays was high; however, the detection window of rHuEPO was narrow, leading to questionable sensitivity. The ABP, integrating longitudinal data, is more sensitive, but there are still subjects that evade detection. Combining these methods might improve performance, but will not resolve all observed shortcomings.     

A multi‐parametric approach to remove the influence of plasma volume on the athlete biological passport during a Union Cycliste Internationale cycling stage race

Fluctuations in plasma volume (PV) present potential confounders within the concentration‐based markers of the haematological athlete biological passport (ABP). Here, a multi‐parametric approach involving a simple blood test is applied to the current ABP adaptive model in an attempt to remove the influence of PV expansion, induced by a cycling stage race. Blood samples were obtained from 29 professional cyclists (14 male, 15 female) before, during and after 4–5 consecutive days of racing. Whole blood was analysed in accordance with the World Anti‐Doping Agency ABP guidelines for haemoglobin ([Hb]) concentration and platelets. Serum and plasma were analysed for transferrin, albumin, calcium, creatinine, total protein and low‐density lipoprotein. PV variation (Z‐scores) was estimated using a multi‐parametric model (consisting of the biomarkers mentioned earlier) and compared against calculated variations in PV (measured via CO‐rebreathing). Significant reductions in [Hb] and the OFF‐score were observed in female cyclists after 3 and 4 days of racing, with accompanying increases in PV, which returned to baseline values 4 days post competition. Similarly, a significant increase in PV was observed in male cyclists after 3 and 5 days of racing. When individual estimations of PV variance were applied to the adaptive model, the upper and lower reference predictions for [Hb] and the OFF‐score were refined such that all outliers consistent with racing‐induced PV changes were removed. The PV model appears capable of reducing the influence of PV on concentration‐dependent markers during competition. This is an important step towards the inclusion of the PV correction in the ABP haematological module.     

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.     

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.     

Stable carbon isotope ratio profiling of illicit testosterone preparations – domestic and international seizures

Gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) is now established as a robust and mature analytical technique for the doping control of endogenous anabolic androgenic steroids in human sport. It relies on the assumption that the carbon isotope ratios of naturally produced steroids are significantly different to synthetically manufactured testosterone or testosterone prohormones used in commercial medical or dietary supplement products. Recent publications in this journal have highlighted the existence of black market testosterone preparations with carbon isotope ratios within the range reported for endogenous steroids (i.e. δ¹³C ≥ ?25.8 ‰). In this study, we set out to profile domestic and international law enforcement seizures of illicit testosterone products to monitor the prevalence of ‘enriched’ substrates – which if administered to human subjects would be considered problematic for the use of current GC‐C‐IRMS methodologies for the doping control of testosterone in sport. The distribution of δ¹³C values for this illicit testosterone sample population (n = 283) ranged from ?23.4 ‰ to ?32.9 ‰ with mean and median of ?28.6 ‰ – comparable to previous work. However, only 13 out of 283 testosterone samples (4.6 %) were found to display δ¹³C values ≥ ?25.8 ‰, confirming that in the vast majority of cases of illicit testosterone administration, current GC‐C‐IRMS doping control procedures would be capable of confirming misuse. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Temporal changes in physiology and haematology in response to high‐ and micro‐doses of recombinant human erythropoietin

There is evidence to suggest athletes have adopted recombinant human erythropoietin (rHuEPO) dosing regimens that diminish the likelihood of being caught by direct detection techniques. However, the temporal response in physiology, performance, and Athlete Biological Passport (ABP) parameters to such regimens is not clearly understood. Participants were assigned to a high‐dose only group (HIGH, n  = 8, six rHuEPO doses of 250 IU/kg over two weeks), a combined high micro‐dose group (COMB, n  = 8, high‐dose plus nine rHuEPO micro‐doses over a further three weeks), or one of two placebo control groups who received saline in the same pattern as the HIGH (HIGH‐PLACEBO, n  = 4) or COMB (COMB‐PLACEBO, n  = 4) groups. Temporal changes in physiology and performance were tracked by graded exercise test (GXT) and haemoglobin mass assessment at baseline, after high dose, after micro‐dose (COMB and COMB‐PLACEBO only) and after a four‐week washout. Venous blood samples were collected throughout the baseline, rHuEPO administration, and washout periods to determine the haematological and ABP response to each dosing regimen. Physiological adaptations induced by a two‐week rHuEPO high‐dose were maintained by rHuEPO micro‐dosing for at least three weeks. However, all participants administered rHuEPO registered at least one suspicious ABP value during the administration or washout periods. These results indicate there is sufficient sensitivity in the ABP to detect use of high rHuEPO doping regimens in athletic populations and they provide important empirical examples for use by anti‐doping experts. Copyright © 2017 John Wiley & Sons, Ltd.     

Studies on the minor metabolite 6a‐hydroxy‐androstenedione for doping control purposes and its contribution to the steroid profile

Recent publications have shown that the concentrations of minor metabolites such as formestane and 6a‐hydroxy‐androstenedione (6aOHADION) are import parameters, capable of increasing the specificity and efficiency of steroid abuse screening. The importance of such minor metabolites has been recognized for some time, but setting up concentration thresholds is not that straightforward with a single quadrupole gas chromatograph mass spectrometer (GC‐MS) because of the low concentrations; this is especially the case for 6aOH‐ADION. The main aim of this study was to propose a concentration threshold above which the detected 6aOH‐ADION is considered suspicious and isotope ratio mass spectrometry (IRMS) is recommended. Routine doping control samples (2128) from athletes that entered our lab and were not found suspicious for the intake of any doping substance were used to determine the baseline concentrations of 6a‐OH‐ADION. For this purpose, the more sensitive gas chromatography‐tandem mass spectrometry (GC‐MS/MS) was used, capable of quantifying these low concentrations with high reliability. A urinary concentration threshold of 5 ng/mL was set. Concentrations above this threshold are considered suspicious and are forwarded to IRMS for confirmation in routine practice. In addition, an IRMS method was developed, capable of determining the 13C value of 6aOH‐ADION. If a urine sample has an elevated 6aOH‐ADION concentration and normal 13C values for the traditional IRMS target compounds, we are still able to check the 13C value of 6aOH‐ADION. Six excretion studies were executed to stress the applicability of the threshold by visualizing the concentration and δ13C value time profiles of 6aOH‐ADION. Copyright © 2014 John Wiley & Sons, Ltd.     

Removal of the influence of plasma volume fluctuations for the athlete biological passport and stability of haematological variables in active women taking oral contraception

The haematological module of the athlete biological passport (ABP) monitors longitudinal haematological variations that could be indicative of blood manipulation. This study applied a multi-parametric model previously validated in elite cyclists to compare inferred and actual PV variations, whereas the potential influence of the oral contraceptive pill (OCP) cycle on the ABP blood biomarkers and plasma volume (PV) in 14 physically active women taking OCPs was also investigated. Blood and serum samples were collected each week for 8 weeks, and the ABP haematological variables were determined according to the World Anti-Doping Agency guidelines. Transferrin (sTFN), ferritin (FERR), albumin (ALB), calcium (Ca), creatinine (CRE), total protein (TP) and low-density lipoprotein (LDL) were additionally computed as ‘volume-sensitive’ variables in a multivariate analysis to determine individual estimations of PV variations. Actual PV variations were indirectly measured using a validated carbon monoxide rebreathing method. We hypothesised ABP markers to be stable during a standard OCP cycle and estimated PV variations similar to measured PV variations. Measured PV variations were in good agreement with the predictions and allowed to explain an atypical passport finding (ATPF). The ABP biomarkers, Hbmass and PV were stable over 8 weeks. Significant differences occurred only between Week 7 and Week 1, with lower levels of haemoglobin concentration ([Hb]), haematocrit (HCT) and red blood cell count (RBC)(−4.4%, p < 0.01; −5.1%, p < 0.01; −5.2%, p < 0.01) and higher levels of PV at week 7 (+9%, p = 0.05). We thus concluded that estimating PV variations may help interpret individual ABP haematological profiles in women.     

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration

Although the introduction by the World Anti‐Doping Agency (WADA) of the steroid module of the athlete biological passport (ABP) marked an important step forward in the screening of testosterone (T) misuse, it still remains one of the most difficult challenges in doping control analysis. The urinary determination of alternative markers has been recently reported as a promising tool for improving the screening of T oral administration. However, their evaluation for other, commonly used, administration routes is still required. The main goal of this study is the evaluation of the potential of 2 groups of metabolites (cysteinyl conjugated and glucuronoconjugated) after transdermal and intramuscular administration of T. Their suitability was evaluated in individuals with both low basal (L‐T/E) and medium basal (M‐T/E) values of T/E. In this Part I, we evaluated the urinary excretion profile of these 2 groups of T metabolites after the administration of 3 doses of T gel to 12 volunteers (6 L‐T/E and 6 M‐T/E) for 3 consecutive days. For this purpose, 9 different concentration ratios (5 cysteinyl conjugated and 4 glucuronoconjugated markers) were studied. Both, the intra‐individual variability and the detection windows (DW) obtained by each ratio were evaluated. Cysteinyl conjugates showed a general low intra‐individual variability and DWs that were shorter than any other tested marker. Despite the relatively large intra‐individual variability, the DWs reached by glucuronoconjugates (2–3 days) were similar to those obtained by markers currently included in the ABP. Overall; this evaluation advises for the introduction of additional glucuronoconjugated markers in the screening of transdermal T administration.     

Altitude training causes haematological fluctuations with relevance for the Athlete Biological Passport

The impact of altitude training on haematological parameters and the Athlete Biological Passport (ABP) was evaluated in international‐level elite athletes. One group of swimmers lived high and trained high (LHTH, n = 10) for three to four weeks at 2130 m or higher whereas a control group (n = 10) completed a three‐week training camp at sea‐level. Haematological parameters were determined weekly three times before and four times after the training camps. ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (RET%), OFF score and the abnormal blood profile score (ABPS) were calculated using the Bayesian model. After altitude training, six swimmers exceeded the 99% ABP thresholds: two swimmers exceeded the OFF score thresholds at day +7; one swimmer exceeded the OFF score threshold at day +28; one swimmer exceeded the threshold for RET% at day +14; and one swimmer surpassed the ABPS threshold at day +14. In the control group, no values exceeded the individual ABP reference range. In conclusion, LHTH induces haematological changes in Olympic‐level elite athletes which can exceed the individually generated references in the ABP. Training at altitude should be considered a confounding factor for ABP interpretation for up to four weeks after altitude exposure but does not consistently cause abnormal values in the ABP. Copyright © 2014 John Wiley & Sons, Ltd.