Usefulness of serum androgen isotope ratio mass spectrometry (IRMS) to detect testosterone supplementation in women.

The detection of testosterone intake is facilitated by monitoring the urinary steroid profile in the athlete biological passport. This technique can be used with confidence to identify target samples for isotope ratio mass spectrometry. Regrettably, most research has been performed on male subjects resulting in a method that does not account for females' steroid concentration and/or variation. This study evaluates the usefulness of the carbon isotope ratio (CIR) in serum of female subjects. Two steroid sulphates are targeted in serum, androsterone and epiandrosterone. Both exhibit statistically significant depletion of their CIR after 10 weeks of daily (10 mg) transdermal testosterone administration. Of the 21 female subjects, samples from six individuals were identified as adverse analytical findings; additionally, four were found atypical considering the serum CIR. The urinary athlete biological passport was not sufficiently sensitive to identify target serum samples for isotope ratio mass spectroscopy. Of the six with a suspicious passport, only two could be confirmed using the serum CIR of androsterone and epiandrosterone. This study shows that CIR analysis in serum cannot be considered the sole confirmatory solution to detect testosterone doping in women due to low sensitivity. However, this analysis has the potential to be used as a complementary method in certain situations to confirm exogenous testosterone in women.

Detection of anabolic agents including selective androgen receptor modulators in samples outside of sport.

Selective androgen receptor modulators (SARMs) are prohibited by the World Anti-Doping Agency (WADA) since 2008. Similarly, to anabolic androgenic steroids (AAS), SARMs are detrimental to health not only in athletes but also in the general population. However, studies of the occurrence of SARMs outside of sport are scarce. Swedish healthcare samples from the Drugs of Abuse Laboratory at Karolinska were analyzed using WADA-accredited screening methods at the Doping Control Laboratory in Stockholm to estimate the frequency of SARM use outside of the WADA laboratories. Twenty (4%) of the male urine samples (n = 542) were positive for SARMs, whereas none of the analyzed female samples (n = 100) contained any SARMs. The top three SARMs found were LGD-4033 followed by RAD140 and ostarine. Two or more SARMs were found in >50% of the SARM-positive samples. AASs were identified in 40% of samples containing SARMs. A difference between genders was observed where 34% male and 7% female samples contained AAS. Many samples displayed testosterone/epitestosterone values indicative of testosterone intake, without presence of other AAS, and hence, there is a risk that these samples are being falsely reported as negative. Our results indicate that SARM use might be a concern outside of sport. Subsequently, in addition to AAS, the healthcare system should also be informed about SARM abuse and the associated adverse side effects.

Urinary Steroid Profile in Elite Female Athletes in Relation to Serum Androgens and in Comparison With Untrained Controls.

INTRODUCTION: In female athletes, the interpretation of doping tests is complex due to hormonal variations during the menstrual cycle and hormonal contraceptive use, both influencing the urinary steroid profile. Exercise is suggested to affect circulating steroid hormone levels, and in women, the urinary steroid profile differs between in competition testing and out of competition testing. No previous study has investigated the relationship between amount of exercise and the urinary steroid profile in female elite athletes. PURPOSE: To compare the urinary steroid profile between female Olympic athletes and age- and BMI-matched untrained controls, and to study the urinary steroid profile in relation to serum hormones and amount of exercise. METHODS: In this cross-sectional study conducted at the Women's Health Research Unit, Karolinska University Hospital, Stockholm, 94 female elite athletes and 86 untrained controls were included. Serum estrogens and testosterone and the urinary steroid profile were analyzed by liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry, respectively. Exercise hours/week were evaluated by questionnaire. RESULTS: Although serum steroid hormones were comparable between groups, the athletes demonstrated approximately 30% lower urinary steroid metabolites of testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstan-3α, 17ß-diol, and 5ß-androstan-3α, 17ß-diol compared to the controls. The urinary steroid metabolites correlated positively with serum steroid hormones. In the athletes, urinary steroid metabolites: androsterone (r s = -0.28, p = 0.007), epitestosterone (r s = -0.22, p = 0.034), 5αAdiol (r s = -0.31, p = 0.002) and testosterone (r s = -0.24, p = 0.026), were negatively correlated with amount of training (hours per week). CONCLUSION: The urinary concentrations of steroid metabolites were lower in elite athletes than in sedentary controls, although serum steroids were comparable between groups. Moreover, exercise time was negatively associated with the urinary concentrations. Our findings suggest alternative excretion routes of androgens in the athletes related to training.

[Increased availability needed for analysis of anabolic androgenic steroids outside of sports - proposal of a uniform clinical analysis method in Sweden]. / Ökad analys av anabola androgena steroider utanför idrotten behövs.

The abuse of anabolic androgenic steroids (AAS) outside of sports is a far greater societal problem than the abuse in sports. Therefore, there is an increasing need for a suitable clinical method for analysis of AAS in urine samples, but only three clinical laboratories in Sweden currently perform analyses of AAS outside of sports. There is a need for harmonization regarding which substances to be analyzed,  which analytes to measure and which concentration thresholds (¼cut-offs«) to us. Based on data from the three analyzing clinical laboratories, and data from the Swedish Customs Service and National Forensic Centre, a list of suggested substances, analytes and thresholds is presented. The proposed list allows detection of at least 95% of the positive samples outside of sports.

Disposition of Urinary and Serum Steroid Metabolites in Response to Testosterone Administration in Healthy Women.

CONTEXT: Little is known about how exogenous testosterone (T) affects the steroid profile in women. More knowledge would give the antidoping community keys as to how to interpret tests and detect doping. OBJECTIVE: This work aimed to investigate the steroid profile in serum and urine in young healthy women after T administration. METHODS: In a randomized, double-blind, placebo-controlled study, 48 healthy young women were assigned to daily treatment with T cream (10 mg) or placebo (1:1) for 10 weeks. Urine and blood were collected before and at the end of treatment. Serum steroids were analyzed with liquid chromatography-tandem mass spectrometry, and urine levels of T, epitestosterone (E), and metabolites included in the Athlete Biological Passport (ABP) were analyzed with gas chromatography-tandem mass spectrometry. RESULTS: In serum, T and dihydrotestosterone levels increased, whereas sex hormone-binding globulin and 17-hydroxyprogesterone decreased after T treatment as compared to placebo. In urine, T and 5α-androstanediol increased in the T group. The median T increase in serum was 5.0-fold (range, 1.2-18.2) and correlated to a 2.2-fold (range, 0.4-14.4) median increase in T/E in urine (rs = 0.76). Only 2 of the 24 women receiving T reached the T/E cutoff ratio of 4, whereas when the results were added to the ABP, 6 of 15 participants showed atypically high T/E (40%). In comparison, 22/24 women in the T group increased serum T more than 99.9% of the upper confidence interval of nontreated values. CONCLUSION: It seems that the T/E ratio is not sufficient to detect exogenous T in women. Serum total T concentrations could serve as a complementary marker of doping.

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.

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/5ßAdiol, 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.