View metadata, citation and similar papers at core.ac.uk brought to you by CORE ORIGINAL RESEARCH ARTICLE published:provided 10 April by 2014 Frontiers - Publisher Connector doi: 10.3389/fendo.2014.00050 The impact of genetics and hormonal contraceptives on the steroid profile in female athletes 1† 1 † 2 1 1 Jenny J. Schulze , Jenny E. Mullen * , Emma Berglund Lindgren , Magnus Ericsson , Lena Ekström and 2 Angelica Lindén Hirschberg 1 Division of Clinical Pharmacology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden 2 Department of Women’s and Children’s Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Edited by: The steroid module of the Athlete Biological Passport, the newest innovation in doping test- Russell J. Borski, North Carolina State ing, is currently being finalized for implementation. Several factors, other than doping, can University, USA affect the longitudinal steroid profile. In this study, we investigated the effect of hormonal Reviewed by: contraceptives (HC) as well as the effect of three polymorphisms on female steroid profiles Moshe Finel, University of Helsinki, Finland in relation to doping controls. The study population consisted of 79 female elite athletes Honoo Satake, Suntory Institute for between the ages of 18 and 45. HC were used by 32% of the subjects. A full urinary steroid Bioorganic Research, Japan profile was obtained using World Anti-Doping Agency accredited methods. In addition all Kaiping Yang, University of Western Ontario, Canada subjects were genotyped for copy number variation of UGT2B17 and SNPs in UGT2B7 and *Correspondence: CYP17. Subjects using HC excreted 40% less epitestosterone as compared to non-users Jenny E. Mullen, Division of Clinical (p D 0.005) but showed no difference in testosterone excretion. When removing individuals Pharmacology, Department of homozygous for the deletion in UGT2B17, the testosterone to epitestosterone (T/E) ratio Laboratory Medicine, Karolinska was 29% higher in the HC group (p D 0.016). In agreement with previous findings in men, Institutet, Karolinska University Hospital, C1:68, Huddinge, copy number variation of UGT2B17 had significant effect on female urinary testosterone Stockholm 141 86, Sweden excretion and therefore also the T/E ratio. Subjects homozygous for the T allele of CYP17 e-mail: [email protected] showed a lower urinary epitestosterone concentration than the other CYP17 genotypes. It †Jenny J. Schulze and Jenny E. is of great importance that the athlete’s steroidal passport can compensate for all possible Mullen have contributed equally to normal variability in steroid profiles from women. Therefore, considering the large impact this work. of HC on female steroid profiles, we suggest that the use of HC should be a mandatory question on the doping control form. Keywords: doping in sports, hormonal contraceptives, genetic polymorphism, UGT2B17, CYP17, testosterone doping, epitestosterone,T/E ratio INTRODUCTION The deletion polymorphism is much more common in East Asian Detection of doping with endogenous steroids, such as testos- populations as compared to Caucasians and Africans (12). There terone, has been and continues to be a challenge. To overcome are also individuals that have naturally high T/E ratios due to the problem of separating testosterone doping from endogenous decreased excretion of epitestosterone. In males, part of this low testosterone the ratio between the glucuronides of testosterone epitestosterone excretion can be explained by a promoter polymor- and epitestosterone (T/E) is used. This T/E ratio was introduced in phism in the CYP17 gene (13), resulting in 64% higher T/E ratios doping tests with an authorized upper limit of 4. Interestingly, the in men homozygous for the T allele (14). However, this polymor- mean T/E ratio in Caucasian men is approximately 1 (1), whereas phism in relation to epitestosterone excretion has not been studied in Asians, the mean ratio is considerably lower (2). in women. Glucuronidation of androgens by UDP-glucuronosyltransferases Women show a greater individual variation in T/E ratio than (UGTs), i.e., phase II metabolism, is the major route for andro- men due to concentrations near the detection limit for the method gen inactivation and excretion (3–6). UGT2B7 has been identified of analysis as well as variations during the menstrual cycle (15). as the enzyme responsible for epitestosterone conjugation (7, 8) Furthermore, the use of hormonal contraceptives (HC) has been whereas testosterone is a poor substrate for this enzyme. Testos- suggested to suppress the production of epitestosterone and thus terone is mainly conjugated by UGT2B17 and to a minor extent by lead to an increase in the T/E ratio (16). In Sweden, a study has UGT2B15. We have recently shown that testosterone glucuronida- shown that almost half of female elite athletes are using HC, a tion activity in the liver is significantly higher in men homozygous frequency comparable to the general population of the same age for the insertion (ins/ins) of UGT2B17 than in women with the group (17). HC consist of a progesterone derivative (progesto- same genotype (9). gens) or a combination of a progestogen and synthetic or nat- We have also shown that the ethnic disparity in the T/E ratio is ural estrogen. Their main mechanism of action is inhibition of strongly associated with a deletion polymorphism in the UGT2B17 ovulation. Both progesterone and estrogen are negative regula- gene (10). Individuals homozygous for this deletion (del/del) may tors of the hypothalamic–pituitary–gonadal (HPG) axis, meaning not reach a T/E ratio of 4 when doped with testosterone (11). that an increase in these sex hormones results in a decrease in www.frontiersin.org April 2014 | Volume 5 | Article 50 | 1 Schulze et al. Contraceptives, genetics, and testosterone doping gonadotropin releasing hormone (GnRH), luteinizing hormone unconjugated [typically <8% of glucuronide fraction (22)] plus (LH), and follicle stimulating hormone (FSH). the glucuronide conjugated fraction. The effect of urine dilution In 1994, it was shown that between-subject variation can was adjusted for by normalizing the samples to a specific gravity be removed in doping tests by using a series of measurements of 1.020. obtained from the same individual (18). Six years later the con- cept of the Athlete Biological Passport (ABP) was proposed. The COPY NUMBER ANALYSIS OF UGT2B17 ABP uses a longitudinal approach where an individual’s previ- DNA was extracted from the whole blood samples using QIAamp® ous results are logged and compared to the new results (19). The DNA Blood Mini kit (Qiagen). Triplicates of 10 ng/mL DNA- hematological module of the ABP,used to detect blood doping, has samples were used to genotype for the UGT2B17 deletion poly- successfully been in use since 2009, while the module for steroid morphism as we have previously described (14). This genotyp- doping is currently being finalized for implementation. ing was achieved by using 7500 Fast Real-Time PCR systems In this study, we investigated how three polymorphisms and with albumin as internal control as described by Schaeffeler HC affect the steroid module of the ABP. Previously, there has et al. (23). only been a few studies investigating the effect of genetic variation and HC on female steroid profiles in relation to doping controls. UGT2B7 AND CYP17 SNP ANALYSES With the implementation of the steroid module of the ABP, the Genotyping assays from Applied Biosystems (Foster City, CA, 268 sensitivity of doping tests has improved. The improved sensitivity USA) were used to genotype for UGT2B7 H Y and the T > C requires increased knowledge of how external factors influence the substitution in the promoter region of CYP17A1. For CYP17 a steroid profile. More knowledge in this area is of great importance premade 20× genotyping mix was used (c_2852784_30) with 2× for correct interpretation of female steroid profiles using the ABP TaqMan Universal PCR Master mix and 1 mL DNA sample for a program. total of 15 mL. For the UGT2B7 genotyping a mix was prepared with 2× TaqMan Universal PCR Master Mix, 0.6 mM forward and MATERIALS AND METHODS reverse primer (forward: AGC TGA CGT ATG GCT TAT TCG AA, STUDY POPULATION reverse: GGG TTT GGC AGG TTT GCA), 0.1 mM of both probes The study population included 57 female Olympic level athletes each containing a different allele (FAM-TTC AGT TTC CAT ATC who planned to participate in the up-coming summer or win- CAC TCT-MGB, VIC-TTC AGT TTC CTC ATC CAC TCT-MGB) ter Olympic Games (either Olympic team members or members and 3 mL DNA sample per 15 mL reaction. Thermal cycling fol- within the programs of the Swedish Olympic Committee). The lowed a protocol for activation at 95°C for 10 min; 40 cycles of mean age of these subjects was 26 years with a range from 18 to denaturation at 96°C for 15 s, annealing/elongation at 60°C for 45. Information about HC use was collected and a written consent 1 min, and lastly a hold at 4°C. was obtained from all subjects. In addition, 22 elite female athletes, whom have given consent DATA EVALUATION for research on their blood and urine samples in their anony- As most of the data were not normally distributed,the median with mous doping control forms, were included in the study. The range is given for all data, even in the few cases where the data medical information on the doping control form was used to were normally distributed. Statistical analyses were performed gather information on hormonal contraceptive use. according to distribution with Student’s two-tailed t-test or the Out of the 79 female athletes, 25 (32%) were using HC. Mann–Whitney U -test using GraphPad PRISM 6.0® (GraphPad, Two women were using progestogen only contraceptives (birth San Diego, CA, USA).