Concerns About Serum Androgens Monitoring During Testosterone Replacement Treatments in Hypogonadal Male Athletes: A Pilot Study

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ORIGINAL RESEARCH—ENDOCRINOLOGY Concerns About Serum Androgens Monitoring During Testosterone Replacement Treatments in Hypogonadal Male Athletes: A Pilot Study jsm_2600

873..886

Luigi Di Luigi, MD,* Paolo Sgrò, MD,* Antonio Aversa, MD,† Silvia Migliaccio, MD,* Serena Bianchini, BLT,* Francesco Botrè, PhD‡, Francesco Romanelli, MD,† and Andrea Lenzi, MD† *Unit of Endocrinology, Department of Health Sciences, University of Rome “Foro Italico,” Rome, Italy; †Department of Experimental Medicine, University of Rome “Sapienza,” Rome, Italy; ‡Department of Management, University of Rome “Sapienza”—Anti-Doping Laboratory of the Italian Federation of Sport Medicine (FMSI–CONI), Rome, Italy DOI: 10.1111/j.1743-6109.2011.02600.x

ABSTRACT

Introduction. A well-tailored testosterone replacement treatment (TRT) in male hypogonadal athletes plays a pivotal role to restore physiological performances, to reduce health risks, and to guarantee the ethic of competition. Few studies evaluated individual androgens profiles during TRT in trained individuals. Aim. The aim of this article was to verify the efficacy in restoring eugonadal serum and urinary androgens profiles after testosterone enanthate (TE) and gel (TG) administration. Methods. Ten male Caucasian-trained volunteers affected by severe hypotestosteronemia (<8 nmol/L) were included. Serum androgens and urinary testosterone metabolites were evaluated, in the same subjects, before and weekly for 5 weeks after both a single intramuscular TE injection (250 mg) and during a daily administration of TG (50 mg/die of testosterone), respectively. Main Outcome Measures. The main outcome measures of this article were serum total testosterone (TT), dihydrotestosterone (DHT), calculated free and bioavailable testosterone (cFT, cBioT), 17-b-estradiol, and urinary glucuronide testosterone metabolites. Results. Supraphysiological TT concentrations were observed in 50% of our volunteers until 7 days after TE and in the 4% of total samples after TG. Serum DHT was high both after TE (all volunteers on day 7 and 50% on day 14) and during TG (32% of total samples). A relatively low number of samples showed normal cFT and cBioT both after TE and TG (20–44%, respectively). Urinary metabolites were related to the type of treatment and to serum androgens profile and resulted in the normal ranges from 15% to 60% of total samples. Conclusion. Besides well-known variations of mean serum TT, we showed a high percentage of serum and urinary samples with abnormal androgens, being TG safer than TE. We conclude that monitoring TRT with TT only may be inaccurate because of abnormal fluctuations of other circulating androgens. Further studies to identify the appropriate markers of eugonadism during TRT are highly warranted both in athletes and in non-athletes. Di Luigi L, Sgrò P, Aversa A, Migliaccio S, Bianchini S, Botrè F, Romanelli F, and Lenzi A. Concerns about serum androgens monitoring during testosterone replacement treatments in hypogonadal male athletes: a pilot study. J Sex Med 2012;9:873–886. Key Words. Testosterone; DHT; Free Testosterone; Bioavailable Testosterone; Doping; Sport

Introduction

I

n the general population, males with low testosterone concentrations are commonly submitted to a testosterone replacement treatment © 2012 International Society for Sexual Medicine

(TRT), if a clinical and biochemical diagnosis of hypogonadism (i.e., due to classical diseases affecting the hypothalamus-pituitary-gonadal axis) is established and if there are no contraindications [1]. In hypogonadal males engaged in competitive J Sex Med 2012;9:873–886

874 sports or in heavy non-competitive physical activities, an adequate TRT plays a pivotal role in restoring physiological and functional conditions and in reducing health risks during either physical activity or rest [2,3]. After puberty, endogenous testosterone largely modifies body composition in males and influences the physiology of all the organs and tissues involved in different motor behaviors (e.g., walking, working, practicing physical exercise, and sports) and in the physiological short- and longterm mechanisms of adaptation to exercise-related stress [4–8]. Also, because of the observed linear relationship between testosterone levels and leg press strength, thigh, and quadriceps muscle volume [6], a biologically normal serum total testosterone (TT) concentration is fundamental in athletes to guarantee both a physiological exercise performance and a safe sport participation. Because of the multiple genomic and nongenomic effects of testosterone, and even though evidence-based criteria does not exist, untreated athletes with hypotestosteronemia are theoretically exposed to further specific risks for health and for their biologically normal physical performances, when compared with sedentary hypogonadal individuals [9,10]. For example, they are at increased risk of osteoporotic fractures in case of falling or trauma (e.g., cyclists, combat sports), of cardiovascular accidents related to high exercise strain, of worsened sport-related anemia, and of reduced stress hormones balance (e.g., vs. cortisol). In addition, testosterone deficiency alters the endocrine-metabolic and neuromuscular adaptations to exercise, reduces muscles strength, aggressiveness in competition and proteins resynthesis during recovery, and increases the risk of overtraining. On these bases, and because we observed that the history of clinical symptoms of hypogonadism may be inaccurate to diagnose testosterone deficiency in trained individuals [3], we believe that TRT should be considered in all athletes with low serum testosterone due to true hypogonadism (e.g., not related to anabolic androgens abuse), independent of the presence of classical symptoms of hypogonadism. True hypogonadal athletes should receive testosterone at all ages, if there are no contraindications [2,11,12], and after receiving a therapeutic use exemption (TUE) by the respective antidoping organization. In fact, since the early 1970s, testosterone and other androgens are prohibited in athletes. Only recently has the complete ban of androgens makes an exception for athletes with androgen deficiency due to wellJ Sex Med 2012;9:873–886

Di Luigi et al. established diagnostic entities, like pituitary or testicular diseases [7,13–15]. Adequately tailoring and monitoring TRT in male athletes is essential to reduce health risks, to guarantee physiological physical performances, and to respect the ethics of sport [2,12,16–18]. This is of great importance because both low androgens concentrations are associated to specific consequences for health and reduced performance, and long-term supraphysiological androgens concentrations may increase the risk for side effects and provide an unfair advantage for performance because of the dose-dependent effects of androgens on muscle mass, strength, and central nervous system (CNS) [2,6,12,19,20]. In the general population, it is recommended to tailor TRT to maintain serum TT concentration into the midnormal range [11]. Furthermore, it is suggested to monitor injectable testosterone esters (such as enanthate or cypionate) or gel administration by assessing serum TT midway between two consecutive testosterone esters injections or at any time after patient has been on treatment with the gel for at least 1 week, respectively [11]. It is our opinion that the recommended strategies (i.e., evaluated hormones and timing of evaluation) for monitoring TRT with such testosterone preparations during dose titration cannot permit per se the exact identification of the true serum androgens profiles. In fact, besides a concomitant voluntary abuse with testosterone (e.g., doping) during TRT, hypogonadal individuals might attain unwanted, and very often unrevealed, abnormal TT serum concentrations and/or fluctuations, depending upon the type of testosterone preparations, doses, and timing of monitoring [19,21–24]. In addition, even if also dihydrotestosterone (DHT)—a biologically active metabolite of testosterone that is prohibited because of its anabolic and psychotropic effects—may increase after testosterone administration and induce health concerns, it is not clear why it is not routinely monitored during TRT. Furthermore, whereas in the future a possible doping control on random serum and urine samples may be used to suspect both a not well-tailored TRT or an abuse with testosterone, few studies evaluated the risk to detect high serum androgens concentrations and the associated urinary profile’s characteristics during different TRT [19]. The aim of this short-term exploratory open label investigative study was to verify serum androgens and urinary testosterone metabolites profiles after a single administration of a long half-life injectable testosterone ester preparation, one of the most com-

Androgens Profile in Treated Male Hypogonadism monly used and abused testosterone preparation (e.g., mainly for their availability and low cost), and during the administration of a newer TRT modality (e.g., testosterone gel [TG]) in trained males affected by severe hypotestosteronemia. In particular, we aimed to evaluate both the probability of establishing drug-related abnormal androgens profiles and the possible relationships between serum and urinary androgens profiles during such treatments. Subjects and Methods

Subjects Ten male Caucasian hypogonadal volunteers practicing noncompetitive physical activities were included in the study; five were affected by primary hypogonadism (e.g., three of them had bilateral anorchia and two a bilateral orchitis) and five by secondary hypogonadism (e.g., congenital, idiopathic), and their mean serum TT at diagnosis was 2.91 ⫾ 2.32 nmol/L. All of them were already treated with intramuscular (i.m.) testosterone esters (250 mg every 3 weeks) and were considered well treated (inclusion criteria) because of normal serum TT (e.g., 14.1–24.5 nmol/L) as measured midway between two consecutive injections [11]. At inclusion, the hypogonadal volunteers had the following anthropometric characteristics as mean ⫾ standard deviation (SD): age 31.3 ⫾ 7.5 years, height 176.8 ⫾ 7.4 cm, weight 73.4 ⫾ 10.8 kg, and body mass index 27.4 ⫾ 4.8 kg/m2. The other inclusion criteria were: diagnosis of severe hypotestosteronemia (serum TT <8 nmol/ L), diagnosed according to clinical, hormonal, and instrumental criteria, and absence of positive history for diseases contraindicating testosterone administration, and of other factors influencing the experimental evaluations (e.g., diseases, assumption of drugs or supplements, and so forth) [25–27]. University Ethical Committee’s approval and written informed consent were obtained. Treatments After 8 weeks of pharmacological washout, all volunteers first received a single i.m. injection containing testosterone enanthate (TE: 250 mg; Testo-Enant; Geymonat, Italy). Then, after 5 weeks from TE administration, they started a daily application (e.g., at 08.00 am) of TG (50 mg/ die of testosterone; Testogel; Bayer, Schering Healthcare, Milan, Italy) for 5 weeks. Each volunteer was his own control, receiving both TE and TG. The volunteers maintained their usual lifestyle throughout all the study.

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Blood Samples Collections Immediately before starting both TE and TG administration, morning blood samples for serum hormones evaluations were collected from a forearm vein, under standardized conditions after an overnight fasting, between 07.30 and 08.30 am and after 30 minutes in resting conditions (day 1). Then, morning blood samples were collected on days 7, 14, 21, 28, and 35 after or during TE and TG administration, respectively. Blood samples were immediately placed on ice until centrifugation (800 ¥ g) and serum separation. Serum samples were frozen at -40°C until analyzed. Urine Samples Collections Immediately before starting both TE and TG administration, morning clean-catch urine samples were collected (50 mL) after an overnight fast, from 06.00 to 07.00 am (day 1). Subsequently, morning urine samples were collected on days 7, 14, 21, 28, and 35 after or during TE and TG administration, respectively. Samples were frozen at -40°C until analyzed. Blood and Urine Samples Analysis Serum TT and sex hormone-binding globulin (SHBG) were measured by immunoradiometric assay using commercial kits (Spectria ORION, Espoo, Finland). Serum concentrations of DHT and 17-b-estradiol (E) were measured by radioimmunoassay (RIA) using commercial RIA kits (Diagnostic System Laboratory, Webster, TX, USA). All samples were analyzed in duplicate; the intra- and inter-assays coefficients of variation were: 3.8% and 4.8% for TT, 3.7% and 6.9% for DHT, 5.3% and 8.1% for E, and 5.3% and 3.3% for SHBG, respectively. The sensitivity of the methods was: 0.1 nmol/L for TT, 0.014 nmol/L for DHT, 17.3 pmol/L for E, and 1.3 nmol/L for SHBG. The serum concentrations of calculated free testosterone (cFT) and calculated bioavailable testosterone (cBioT), which includes free testosterone plus testosterone weakly bound to albumin, were calculated from TT and SHBG concentrations, assuming an albumin concentration of 4.3 g/ dL, by using published equations [28,29]. The free androgen index (FAI) was also calculated [FAI (nmol/nmol) = (TT/SHBG) ¥ 100]. Serum DHT/TT and TT/E ratios were calculated by serum DHT/TT (nmol/nmol) and TT/E (nmol/ nmol). J Sex Med 2012;9:873–886

876 Urine samples were analyzed for free and glucuronide androgen metabolites: testosterone (T), epitestosterone (EpiT), androsterone (A), etiocholanolone (Et), 11b-hydroxyandrosterone (11OH-A), 11b-hydroxyetiocholanolone (11-OH-E), 5a-androstane-3a,17b-diol (5aAD3a17b), 5bandrostane-3a,17b-diol (5bAD3a17b), via gas chromatography–mass spectrometry (Agilent 5890/5973, Agilent Technologies SpA, Cernusco sul Naviglio, Milan, Italy) as previously described [30]. The urinary metabolites concentration (C) per sample was corrected to a specific gravity of 1.020 [C1020 = (1.020 - 1)/(specific gravitysample 1) · Csample]. Urinary T/EpiT, 5aAD3a17bl/ 5bAD3a17b, and A/Et ratios were calculated by respective urinary metabolites ratios (ng/ng). The serum hormones and proteins reference ranges reported by manufacturers for male individuals (20–40 years) were as follows: TT, 8.2– 34.6 nmol/L; DHT, 2.24–3.85 nmol/L; E, 50–240 pmol/L; and SHBG, 16–61 nmol/L. The intralaboratory reference ranges for absolute and calculated testosterone fractions, evaluated on age matched healthy active male individuals, were: TT, 12.9–27.7 nmol/L; SHBG, 14–50 nmol/L; cFT, 0.30–0.58 nmol/L; cBioT, 6.9–14.1 nmol/L; and FAI, 33–101.

Statistical Analysis Results are expressed as absolute values and as mean ⫾ SD. Serum and urine hormonal parameters and calculated respective ratios (DHT/TT, TT/E, FAI, T/EpiT, 5aAD3a17bl/5bAD3a17b, A/Et), evaluated before and after treatments, were analyzed by anova for repeated measures to detect significant effects of two main factors: treatment (TE and TG) and time (before and after treatment). When a significant F-ratio (P < 0.05) was observed, the presence of significant differences was investigated using adequate post hoc tests. A level of 0.05 was selected for statistical significance. All other presented analyses were based on descriptive statistics. At an individual level, to evaluate the number of samples within the normal ranges (e.g., as percent of samples at each time points and as percent of total samples), we compared the serum hormones concentrations both with the manufacturer’s reference ranges (for TT and DHT) and with our laboratory references ranges (for TT, cFT, cBioT, and FAI) and our urinary profiles with the existing reference ranges for glucuronide metabolites of testosterone in a Caucasian athletic population, as reported by Van Renterghem et al. [31]. The concentrations of J Sex Med 2012;9:873–886

Di Luigi et al. urinary metabolites of testosterone has been also evaluated by grouping the urinary samples based on the respective serum TT and DHT concentrations (normal, high, low) both after TE and during TG administration. As reported in tables and figures, because of the more common interval of administration of TE during TRT, for some statistical evaluations, we considered the samples collected during the first 3 weeks after TE administration and all the samples collected during TG administration. The statistical software SPSS (version 18.0 for Windows; SPSS Inc., Chicago, IL, USA) was used.

Results

Clinical symptoms of hypogonadism (loss of libido, erectile dysfunction, hot flushes, reduced vigor, weakness, etc.) have been observed at low levels of intensity in almost all volunteers during the last weeks of washout periods; none withdrew in the study because of severe symptoms of hypogonadism and drug-related side effects. For all evaluated parameters, no differences between pretreatments (day 1) mean serum samples hormones concentrations were observed (Table 1). As expected, both TE and TG administration induced modifications in many of the evaluated or calculated serum and urinary parameters. All results are reported in Tables 1–4 and Figures 1 and 2.

Serum TT As expected, mean serum TT concentration significantly increased with respect to pretreatment values both after TE and during TG administration (Table 1). The mean TT concentrations were in the normal range only on day 14 after TE administration and all time points during TG administration. The probability to find a sample containing serum TT within the normal manufacturer’s range was from 50% to 100% of samples at different time points (70% on total samples) during the first 3 weeks after TE administration (Table 2). Particularly, after TE administration serum, TT concentrations were significantly above the upper limit of the manufacturer’s reference range in the 5/10 subjects on day 7, within the normal reference range in 10/10 subjects on day 14, and clearly below the reference range (i.e., hypoandrogenism) in 5/10 subjects on day 21 and in all of subjects on day 28 and 35, respectively (Table 2, Figure 1).

2.5 ⫾ 1.9 2.0; 0.8–6.9 23.7 ⫾ 7.2 27.3; 15.0–33.4 0.21 ⫾ 0.49 0.04; 0.01–1.44 1.2 ⫾ 0.9 0.9; 0.3–3.3 10.9 ⫾ 7.1 8.1; 2.5–24.4 0.4 ⫾ 0.3 0.3; 0.1–1.1 102.3 ⫾ 24.5 97.1; 39.2–163.1 0.19 ⫾ 0.03 0.16; 0.12–0.23 2.9 ⫾ 2.2 2.3; 0.9–8.0 26.7 ⫾ 24.4 15.4; 8.6–80.3

2.9 ⫾ 1.6 2.5; 1.0–5.6 23.4 ⫾ 6.6 22.5; 16.3–32.1 0.07 ⫾ 0.04 0.05; 0.02–0.14 1.6 ⫾ 1.0 1.1; 0.4–3.4 13.7 ⫾ 8.7 11.3; 3.2–30.1 0.58 ⫾ 0.38 0.44; 0.11–1.27 115.9 ⫾ 44.5 111.2; 44.0–177.7 0.18 ⫾ 0.05 0.19; 0.11–0.25 3.5 ⫾ 2.0 3.1; 1.1–6.9 27.2 ⫾ 14.8 19.8; 16.0–51.8 12.9 ⫾ 7.1**1†† 12.0; 2.5–23.9 22.6 ⫾ 8.6 28.0; 15.8–40.5 0.31 ⫾ 0.20*1†† 0.26; 0.04–0.56 7.4 ⫾ 4.8**1†† 6.1; 0.9–13.3 60.3 ⫾ 42.1**1†† 41.4; 6.1–119.4 3.2 ⫾ 1.9**1†† 3.6; 0.3–6.4 162.6 ⫾ 35.1*1 157.1; 65.1–211.2 0.23 ⫾ 0.06*1 0.23; 0.12–0.31 16.1 ⫾ 9.0**1†† 15.6; 2.8–30.4 76.5 ⫾ 47.6*1†† 55.0; 36.9–155.7

35.8 ⫾ 9.8**1,14,21,28,35 35.6; 24.3–55.5 24.8 ⫾ 8.1 26.0; 14.8–38.5 0.99 ⫾ 0.3**1,14,21,28,35 0.93; 0.61–1.55 23.8 ⫾ 7.1**1,14,21,28,35 23.4; 14.3–36.3 162.5 ⫾ 62.5**1,14,21,28,35 154.9; 76.5–278.2 7.90 ⫾ 2.29**1,14,21,28,35 7.45; 5.10–12.52 229.7 ⫾ 82.1*28,35**1 212.9; 114.5–396.5 0.22 ⫾ 0.01*1,14,21 0.22; 0.20–0.23 43.7 ⫾ 12.1**1,14,21,28,35 43.0; 29.4–68.0 168.4 ⫾ 56.8*28,35**1,14,21 162.6; 96.5–256.9

Day 7

14.4 ⫾ 8.1**1 14.7; 3.1–26.5 22.5 ⫾ 6.3 25.6; 16.1–33.6 0.36 ⫾ 0.23*1 0.34; 0.05–0.65 8.5 ⫾ 5.5**1 8.1; 1.3–15.3 69.4 ⫾ 43.0**1 57.1; 9.2–132.7 3.2 ⫾ 2.0**1 3.3; 0.4–6.7 148.6 ⫾ 24.8*1 137.2; 43.6–182.4 0.21 ⫾ 0.03**1 0.22; 0.15–0.25 17.6 ⫾ 10.1**1 18.4; 3.5–33.3 94.4 ⫾ 73.7*1 63.8; 44.0–259.5

13.7 ⫾ 3.5**1,7,21,28,35 13.4; 9.6–19.8 24.0 ⫾ 7.0 23.6; 12.9–36.3 0.33 ⫾ 0.12**1,7,21,28,35 0.32; 0.20–0.56 7.8 ⫾ 2.8**1,7,21,28,35 7.6; 4.8–13.1 64.4 ⫾ 29.8*21**1,7,28,35 61.5; 29.8–106.1 3.59 ⫾ 0.72**1,7,21,28,35 3.42; 2.31–4.70 168.6 ⫾ 65.2*1 162.6; 75.6–262.5 0.26 ⫾ 0.04*7,35**1 0.28; 0.20–0.31 17.3 ⫾ 4.1**1,7,21,28,35 17.4; 11.9–23.9 89.9 ⫾ 31.2**1,7,21,28,35 93.5; 51.3–149.7

Day 14

13.4 ⫾ 10.4**1 11.5; 2.3–38.6 21.9 ⫾ 6.5 26.3; 15.5–32.8 0.33 ⫾ 0.27*1 0.26; 0.04–0.90 7.5 ⫾ 6.6**1 6.2; 0.7–21.3 59.1 ⫾ 38.9**1 44.3; 8.0–112.0 3.2 ⫾ 2.5*1 2.7; 0.2–8.6 171.3 ⫾ 52.1*1 165.9; 79.0–216.5 0.22 ⫾ 0.06*1 0.22; 0.11–0.33 16.6 ⫾ 13.0**1 14.5; 2.5–45.3 82.3 ⫾ 75.8*1 46.6; 39.4–260.2

7.5 ⫾ 2.4*28,35**1,7,14 7.5; 3.7–10.4 24.3 ⫾ 6.4 23.8; 15.5–33.7 0.17 ⫾ 0.07*,35**1,7,14 0.17; 0.07–0.27 3.7 ⫾ 2.1*1,7,14 3.9; 0.1–6.3 34.6 ⫾ 17.2**1,7*14,35 30.8; 11.4–61.9 1.92 ⫾ 0.69*28**1,7,14,35 1.77; 0.82–2.94 160.8 ⫾ 62.1**1 155.3; 80.0–246.3 0.25 ⫾ 0.04*7,35**1 0.23; 0.21–0.32 9.5 ⫾ 3.0*28**1,7,14,35 9.3; 4.5–13.3 50.7 ⫾ 19.5*1,28**7,14 43.2; 34.8–94.6

Day 21

13.4 ⫾ 5.9**1†† 14.0; 2.7–21.0 21.8 ⫾ 6.3 26.7; 16.2–33.1 0.33 ⫾ 0.17†† 0.34; 0.05–0.59 7.8 ⫾ 4.0**1†† 8.1; 1.2–13.9 61.7 ⫾ 36.4**1† 58.5; 9.8–122.6 3.2 ⫾ 1.7**1† 2.9; 0.3–5.3 163.0 ⫾ 44.0*1 142.5; 69.6–220.4 0.22 ⫾ 0.04**1 0.22; 0.14–0.28 16.7 ⫾ 7.6**1†† 17.0; 3.0–26.4 88.4 ⫾ 69.7*1 74.0; 32.0–253.0

5.4 ⫾ 1.5*21**1,7,14 5.6; 3.1–7.3 23.9 ⫾ 5.8 26.1; 15.6–31.1 0.12 ⫾ 0.04*7,14 0.12; 0.06–0.18 2.8–1.0**1,7,14 2.8; 1.6–4.4 25.0 ⫾ 11.7**1,7,14 22.9; 11.5–44.1 1.29 ⫾ 0.45*21,35**1,7,14 1.23; 0.70–2.17 136.1 ⫾ 54.0*7 131.1; 75.62–227.24 0.23 ⫾ 0.03**1 0.23; 0.18–0.29 6.6 ⫾ 1.9*21**1,7,14 6.9; 3.8–9.4 43.3 ⫾ 14.9*1,7,21**14 41.9; 24.0–62.5

Day 28

13.0 ⫾ 10.0**1†† 9.7; 5.6–36.1 21.1 ⫾ 5.3 25.4; 17.5–35.0 0.33 ⫾ 0.33† 0.24; 0.11–1.15 7.8 ⫾ 7.9 5.6; 2.7–27.0 61.1 ⫾ 65.2 42.6; 19.7–206.5 3.0 ⫾ 2.1**1† 2.4; 1.1–7.7 152.4 ⫾ 21.8*1 110.4; 45.3–198.5 0.21 ⫾ 0.03*1 0.22; 0.18–0.30 16.0 ⫾ 12.1*1 12.3; 6.9–43.9 66.6 ⫾ 45.6*1 55.7; 20.4–169.3

4.3 ⫾ 1.5*21**7,14 3.7; 2.0–6.6 24.0 ⫾ 6.8 24.4; 16.4–35.4 0.09 ⫾ 0.03*21**7,14 0.09; 0.04–0.14 2.2 ⫾ 0.8**1,7,14 2.2; 1.0–3.4 18.5 ⫾ 7.4*21**1,7,14 19.4; 7.7–31.0 0.87 ⫾ 0.32*1,28**7,14,21 0.91; 0.28–1.35 116.8 ⫾ 54.2*7 119.3; 39.6–206.3 0.20 ⫾ 0.04*14,21 0.19; 0.13–0.25 5.2 ⫾ 1.8**1,7,14,21 4.6; 2.3–7.7 43.4 ⫾ 20.6*7**14,21 36.7; 17.9–79.2

Day 35

*P ⱕ 0.05 and **P ⱕ 0.01 vs. other respective days (1, 7, 14, 21, 28, and 35); †P ⱕ 0.05, ††P ⱕ 0.01 TG vs. TE Hormones and respective ratios have been evaluated before (day 1) and weekly for 5 weeks (from day 7 to day 35) after a single i.m. administration of testosterone enanthate (TE: 250 mg) (A) and during testosterone gel administration (TG: 50 mg/die) (B) in male hypogonadal volunteers (N = 10) TT = total testosterone; SHBG = sex hormone-binding globulin; cFT = calculated free testosterone; cBioT = calculated bioavailable testosterone; FAI = free androgen index (TT/SHBG ¥ 100); E = 17-bestradiol; DHT = dihydrotestosterone

TT/E

DHT+TT

DHT/TT

E

DHT

FAI

cBioT

cFT

SHBG

(B) TT

TT/E

DHT+TT

DHT/TT

E

DHT

FAI

cBioT

cFT

SHBG

(A) TT

Day 1

Table 1 Morning absolute and calculated serum androgens (nmol/L), 17-b-estradiol (pmol/L), and SHBG (nmol/L), concentrations and respective ratios before and after testosterone administration (mean ⫾ SD/median; min-max)

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Table 2 Percentage of serum samples with absolute and calculated androgens concentrations within the manufacturer’s (m) or laboratory (lab) reference ranges (% NR) % NR after TE vs. % NR during TG

TT (m) TT (lab) DHT (m) cFT (lab) cBioT (lab) FAI (lab)

Day 7

Day 14

Day 21

Day 28

Day 35

Total§

50*14,28,35 vs. 80 40 vs. 50 0**14 vs. 40† 0**14 vs. 50† 0**14 vs. 40 0*21**14 vs. 40

100*7**21,28,35‡ vs. 80 60*28,35**21 vs. 50 60*28**7 vs. 10 60*21**7,28,35 vs. 20† 70*21**7,28,35 vs. 20† 70**7,35 vs. 60

50*28,35**14‡‡ vs. 60‡ 0**14 vs. 30*28 20 vs. 40 30*14 vs. 10 20*14 vs. 10*28 40*7,35 vs. 60

0*7,21**14 vs. 90†† 0*14 vs. 70*21†† 0*14 vs. 50† 0**14 vs. 50† 0**14 vs. 60*21,35†† 20 vs. 70

0*7,21**14 vs. 70†† 0*14 vs. 40 0*14 vs. 20 0**14 vs. 10 0**14 vs. 10*28 0*21**14 vs. 70†

70‡‡ vs. 78‡‡ 33.3 vs. 48 23.3 vs. 46 20 vs. 44 23.4 vs. 44 40 vs. 62

*P ⱕ 0.05 and **P ⱕ 0.01 vs. other respective days (1, 7, 14, 21, 28, and 35); †P ⱕ 0.05, ††P ⱕ 0.01 TG vs. TE; ‡P ⱕ 0.05, ‡‡P ⱕ 0.01 vs. lab §Evaluated during 3 weeks after TE administration (n = 30) and during 5 weeks of TG administration (N = 50) Measurements of % NR have been evaluated from serum samples collected weekly for 5 weeks after a single i.m. administration of testosterone enanthate (TE: 250 mg) and during testosterone gel administration (TG: 50 mg/die) in male hypogonadal volunteers (N = 10) TT = total testosterone; DHT = dihydrotestosterone; cFT = calculated free testosterone; cBioT = calculated bioavailable testosterone; FAI = free androgen index (TT/SHBG ¥ 100)

During TG administration, serum TT concentrations were in the manufacturer’s reference range from 60% to 90% of samples at different time points (78% on total samples) (Table 2), being the majority of abnormal samples because of low TT concentrations in the same two subjects (i.e., probably because of reduced individual gel absorption or for a relatively low TG dose), and only two samples (e.g., 2/50) showed elevated serum TT levels (Figure 1). Serum TT concentration was significantly higher on day 7 after TE compared with TG administration and, as expected, was significantly higher on days 28 and 35 during TG administration compared with TE. The comparison with our laboratory reference range for TT evidenced a lower number of samples in the normal range, with respect to the manufacturer’s range comparison, both after TE and during TG administration (Table 2).

Serum DHT Mean serum DHT concentration significantly increased as compared with pretreatment values both after TE and during TG administration (Table 1). The mean DHT concentrations were within the normal range at all time points after TE and during TG administration, respectively. The probability to find a sample containing serum DHT within the normal range was from 0% to 50% of samples at different time points (23% on total samples) during the first 3 weeks after TE administration (Table 2). Particularly, after TE administration, serum DHT concentrations were above the upper limit in 10/10 subjects on day 7 and in 5/10 subjects on day 14, and normal or below of the manufacturer’s reference range from day 21 to day 35, respectively (Table 2, Figure 1). J Sex Med 2012;9:873–886

During TG administration, serum DHT concentrations were within the normal range from 10% to 40% of samples at different time points (26% on total samples) during 5 weeks of TG administration (Table 2); elevated serum DHT levels were observed in 16/50 samples and low DHT levels in 16/50 samples (Figure 1).

Serum Calculated Hormones As expected, mean cFT, cBioT, and FAI increased significantly both after TE and during TG administration; serum cFT, cBioT, and FAI were significantly higher on day 7 after TE compared with TG administration (Table 1). It is of interest that compared with our laboratory reference ranges, cFT and cBioT were considered normal in 60–70% of our volunteers only on day 7 after TE, particularly on day 7 after TE, all volunteers showed high serum cFT and cBioT; on day 14, about 20% of them have low values and 20% have high values, and from day 21 to day 35, quite all volunteers showed low cFT and cBioT values (Table 2, Figure 2). During 5 weeks of TG administration, about 44% of total samples showed normal cFT and cBioT values; furthermore, whereas the majority of samples outside the reference range values showed low cFT and cBioT levels, in few samples (5/50), high values have been also observed (Table 2, Figure 2). Urinary Testosterone Metabolites Profiles The evaluated glucuronide metabolites of testosterone differently increased after TE and during TG administration, also depending on serum TT and DHT profiles (Table 3). Particularly, the observed differences in the evaluated urinary testosterone metabolites concentrations were mainly related to serum TT concentrations (normal, high,

40.7 ⫾ 24.4**pt 28.9 ⫾ 12.2**pt 2,053.8 ⫾ 1,473.0 2,050.8 ⫾ 1,214.7 1,312.3 ⫾ 669.1 1,330.6 ⫾ 998.1 4.1 ⫾ 1.6 5.2 ⫾ 2.5 329.7 ⫾ 316.7 733.2 ⫾ 327.7*pt 149.3 ⫾ 194.8 219.4 ⫾ 380.0*pt 74.7 ⫾ 47.9 78.5 ⫾ 51.0*pt 170.0 ⫾ 115.3**pt 109.9 ⫾ 77.9 11.3 ⫾ 6.5**pt 6.8 ⫾ 4.6**pt† 0.4 ⫾ 0.1 0.7 ⫾ 0.2 1.8 ⫾ 0.9 1.7 ⫾ 0.6

Pt

8.5 ⫾ 4.7 7.8 ⫾ 4.2 1,379.8 ⫾ 1,291.5 1,061.4 ⫾ 1,172.7 1,009.5 ⫾ 827.4 812.0 ⫾ 790.5 5.2 ⫾ 2.5 5.8 ⫾ 2.6 516.6 ⫾ 590.9 371.8 ⫾ 276.3 166.6 ⫾ 253.1 237.3 ⫾ 310.9 38.9 ⫾ 22.1 37.7 ⫾ 17.1 53.9 ⫾ 23.8 58.5 ⫾ 30.3 1.8 ⫾ 1.2 1.4 ⫾ 0.5 0.8 ⫾ 0.4 0.7 ⫾ 0.3 1.4 ⫾ 0.5 1.4 ⫾ 0.6

c TTN, DHT– (n = 4, 5) 40.5 ⫾ 30.5**pt 27.1 ⫾ 18.4*c 2,263.6 ⫾ 1,867.3 2,328.3 ⫾ 1,720.9 771.2 ⫾ 455.2 1,245.9 ⫾ 515.5 4.2 ⫾ 0.96 5.8 ⫾ 2.6 181.8 ⫾ 198.1 800.2 ⫾ 445.2 15.6 ⫾ 22.0 44.6 ⫾ 19.9 73.7 ⫾ 27.8 63.9 ⫾ 48.3 99.3 ⫾ 66.0 75.3 ⫾ 46.7 9.1 ⫾ 5.2**pt 4.7 ⫾ 2.9**pt 0.83 ⫾ 0.3 0.78 ⫾ 0.18 2.7 ⫾ 0.8*pt 1.6 ⫾ 0.7

b TTN, DHT+ (n = 9, 14) 74.7 ⫾ 52.1**pt 27.8 ⫾ 15.0**pt† 2,224.9 ⫾ 796.4 1,899.3 ⫾ 1,282.5 1,660.4 ⫾ 734.8 1,050.2 ⫾ 632.7 5.0 ⫾ 2.5 4.8 ⫾ 2.3 664.6 ⫾ 695.4 671.5 ⫾ 359.3 149.9 ⫾ 188.6 135.6 ⫾ 176.2*pt 156.5 ⫾ 74.5*a**pt 71.1 ⫾ 33.5*pt†† 331.9 ⫾ 201.5**pt 98.4 ⫾ 74.7†† 17.3 ⫾ 12.2**pt 7.0 ⫾ 4.2**pt† 0.5 ⫾ 02 1.5 ⫾ 2.5 1.4 ⫾ 0.3 1.8 ⫾ 0.5

101.2 ⫾ 61.3**pt,a 11.4 ⫾ 3.0 3,031.1 ⫾ 1,054.9*pt 1,212.0 ⫾ 112.2 3,082.9 ⫾ 1,603.9*pt,a 737.2 ⫾ 264.3 8.8 ⫾ 2.3*pt,b**a 2.8 ⫾ 2.5 1,522.1 ⫾ 1,022.3*pt,a 524.1 ⫾ 60.8 246.2 ⫾ 204.6 31.1 ⫾ 1.1 214.6 ⫾ 112.8*a**pt 40.4 ⫾ 20.2 420.0 ⫾ 184.6*a**pt 38.5 ⫾ 7.9 10.5 ⫾ 5.5**pt 5.9 ⫾ 4.2**pt 0.5 ⫾ 0.05 1.0 ⫾ 0.3 1.0 ⫾ 0.2 1.7 ⫾ 0.4

d TT+, DHT+ (n = 5, 2)

17.9 ⫾ 8.8**pt,a,b,d 19.6 ⫾ 11*e 1,151.4 ⫾ 871.6*b,d 1,686.9 ⫾ 1,276.0 896.2 ⫾ 735.8*b,d 1,781.1 ⫾ 1,582.3† 4.8 ⫾ 4.1 4.2 ⫾ 3.1 420.4 ⫾ 462.6**d 424.5 ⫾ 209.6*a 154.5 ⫾ 180.3*pt 281.2 ⫾ 119.8*b**c 48.4 ⫾ 22.2**b,d 92.1 ⫾ 84.2† 88.3 ⫾ 41.3*pt,a**b,d 138.3 ⫾ 113.2 5.3 ⫾ 3.8*a,d**pt,b 6.8 ⫾ 6.1*pt 0.6 ⫾ 0.2 0.6 ⫾ 0.1 1.8 ⫾ 1.9 1.1 ⫾ 0.2**a,b

e TT–, DHT– (n = 21, 11)

*P ⱕ 0.05 and **P ⱕ 0.01 vs. other groups (pt, a, b, c, d, and e); †P ⱕ 0.05, ††P ⱕ 0.01 TG vs. TE; n = number of samples TE, number of samples TG All parameters have been evaluated on urine and blood samples (n = 50 + 50) collected before and weekly for 5 weeks after a single i.m. administration of testosterone enanthate (TE: 250 mg) (n = 50) and during testosterone gel administration (TG: 50 mg/die) (n = 50) in male hypogonadal volunteers (n = 10). Urinary concentrations are normalized for urinary specific gravity of 1.020 T = testosterone; EpiT = epitestosterone; A = androsterone; Et = etiocholanolone, 11-OH-A = 11b-hydroxyandrosterone; 11-OH-E = 11b-hydroxyetiocholanolone; 5aAD3a17b = 5a-androstane-3a,17b-diol; 5bAD3a17b = 5bandrostane-3a,17b-diol

A/Et

5aAD3a17bl/5bAD3a17b

T/EpiT

5bAD3a17b

5aAD3a17b

11-OH-E

11-OH-A

EpiT

Et

A

T

a TTN, DHTN (N = 11, 18)

Table 3 Urinary concentrations (means ⫾ SD) of glucuronide metabolites of testosterone (ng/mL) and respective ratios before (pretreatment, pt) and after testosterone enanthate (up) and during testosterone gel (down) administration grouped (a, b, c, d, and e) for serum total testosterone (TT) and dihydrotestosterone (DHT) concentrations (N = normal; + = high; - = low), respectively

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Table 4 Percentage of urinary samples with glucuronide metabolites of testosterone within the reported reference ranges (% NR) for athletes (from Van Renterghem et al. [31]) total and grouped (a, b, c, d, and e) for serum total testosterone (TT) and dihydrotestosterone (DHT) concentrations (N = normal; + = high; - = low), respectively % NR after TE vs. % NR during TG

T A Et EpiT 11-OH-A 11-OH-E 5aAD3a17b 5bAD3a17b T/EpiT 5aAD3a17bl/5bAD3a17b A/Et

a TTN, DHTN (n = 11, 18)

b TTN, DHT+ (n = 9, 14)

c TTN, DHT– (n = 4, 5)

d TT+, DHT+ (n = 5, 2)

e TT–, DHT– (n = 21, 11)

Total§

86*b,c,e**d vs. 69*e 43 vs. 38 57 vs. 38 0 vs. 0 43 vs. 62 14 vs. 15*e 71*b,d vs. 38 71*b,d vs. 62 43*b vs. 0† 86 vs. 23*e†† 29 vs. 85†

25 vs. 50 88*c**e vs. 25†† 75*e vs. 33 0 vs. 0 25 vs. 58 25 vs. 8*e 13*e vs. 42 13*e vs. 42 0 vs. 0 63 vs. 42 38 vs. 67

0 0 50 0 0 0 50 50 0 50 0

0 vs. 0 75*e vs. 0 25 vs. 0 0 vs. 0 50 vs. 100 50 vs. 0 0*e vs. 100*e† 0**e vs. 0 0 vs. 0 100 vs. 0† 50 vs. 100

43 24 33 10 33 24 62 76 24 52 48

43.3 vs. 56 53.3 vs. 46 50 vs. 46 0 vs. 0 26.6 vs. 62† 16.6 vs. 36 33.3 vs. 50 40 vs. 46 0 vs. 2 63.3 vs. 50† 56.7 vs. 74††

vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.

50 50 75*e 0 50 0 25 50 50 25 50

vs. vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.

22 22 11 0 33 56 22†† 22†† 11 67 67

*P ⱕ 0.05 and **P ⱕ 0.01 vs. other groups (a, b, c, d, and e); †P ⱕ 0.05, ††P ⱕ 0.01 TG vs. TE §Evaluated during 3 weeks after TE administration (n = 30) and during 5 weeks of TG administration (n = 50) Measurements of % NR have been evaluated from urine samples collected weekly for five weeks after a single i.m. administration of testosterone enanthate (TE: 250 mg) (n = 50) and during testosterone gel administration (TG: 50 mg/die) (n = 50) in male hypogonadal volunteers (n = 10) T = testosterone; EpiT = epitestosterone; A = androsterone; Et = etiocholanolone, 11-OH-A = 11b-hydroxyandrosterone; 11-OH-E = 11b-hydroxyetiocholanolone; 5aAD3a17b = 5a-androstane-3a,17b-diol; 5bAD3a17b = 5b-androstane-3a,17b-diol

or low) and as observed in the group TT-normal/ DHT-high, probably also to the serum DHT (e.g., vs. TT-normal/DHT-normal for 5aAD3a17b) and to the type of treatment (e.g., mainly for urinary T, 5aAD3a17b, and 5bAD3a17b) (Table 3). The concentrations of evaluated urinary parameters resulted within the normal reference ranges [31] in about 30–60% of total urinary samples, and for some parameters, the great relative percentage urine samples with normal concentrations were observed only when serum was TT-high/DHThigh. Depending on respective serum androgens profile, differences were between TE and TG administration for some urinary metabolites concentrations (e.g., for A, 11-OH-A, 5aAD3a17b, 5bAD3a17b, T/EpiT, 5aAD3a17b/5bAD3a17b, and A/Et) were observed (Table 4). Only for urinary EpiT ratio, quite all samples showed low concentrations, independently from serum androgens concentrations (Table 4). Discussion

Few investigations evaluated the prevalence of true hypogonadism or the effects of TRT in male athletes, and no cross-sectional or longitudinal trials evaluated the consequences of untreated testosterone deficiency or of a not well-tailored TRT on athletes’ health and/or performance [3,32]. The majority of clinical studies on TRT have been performed in sedentary males and focused on moniJ Sex Med 2012;9:873–886

toring specific clinical target and/or mean serum hormones variations (e.g., TT, LH, etc.) after testosterone administration [13,24,33–37]. Very few clinical data on individual absolute androgens profiles during TRT exist, because of the great interest for individual androgens profile upon pharmacological loading, with different testosterone preparations, coming from sport-linked organizations faced with detecting testosterone abuse in healthy and in hypogonadal athletes [19,38–40].

TRT Monitoring in Male Hypogonadal Athletes In the present study, we partially confirmed some already known variations of mean serum androgens concentrations upon the administrations of TE and TG in hypogonadal males. At individual level, we showed that it is really difficult to mimic physiological androgens rhythm by using these preparations. Furthermore, we also confirmed that, at individual level, it is inaccurate to identify the presence of a normal and stable androgens profile during TRT only by evaluating serum TT, as actually recommended. This is of great importance for male athletes, where it is highly recommended to reproduce a true and stable physiological androgenization. Even if, according to the common strategy for monitoring TRT, all of our volunteers resulted substantially well treated (e.g., for past history and because of normal serum TT in all volunteers on day 14 after TE and in the 60–80% of total samples during TG) or needing a TG dose’s

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Androgens Profile in Treated Male Hypogonadism A 60

Serum Total Testosterone (nmol/L)

55 50 45 40 35 30 25 20 15 10 5 0 0

1

2

3

4

5 6 7 8 9 Serum Dihydrotestosterone (nmol/L)

10

11

12

13

0

1

2

3

4

5 6 7 8 9 Serum Dihydrotestosterone (nmol/L)

10

11

12

13

B 60

Serum Total Testosterone (nmol/L)

55 50 45 40 35 30 25 20 15 10 5 0

Figure 1 Serum total testosterone (TT) and dihydrotestosterone (DHT) concentrations (absolute values) evaluated (A) every 7 days for 3 weeks after a single i.m. administration of testosterone enanthate (250 mg) (dark circles, N = 30) and (B) for 5 weeks during daily administration of testosterone gel (50 mg/die) (dark squares, N = 50) in male hypogonadal volunteers (N = 10). The horizontal and vertical areas indicate the manufacturer’s reference ranges for serum TT and DHT.

increase (Table 2; Figure 1), a high number of them (e.g., or of their serum samples) showed supraphysiological serum testosterone (e.g., TT, cFT, and cBioT) and/or DHT concentrations, both after TE and during TG administration.

Furthermore, 3 weeks after TE (e.g., usually before a successive injection), 50% of our athletes resulted hypogonadal. Because TE is commonly used in 3- or 4-weeks interval, our data suggest that intervals longer than 2 weeks, while bearing J Sex Med 2012;9:873–886

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Serum Calculated Free Testosterone (nmol/L)

A 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 Serum Calculated Bioavailable Testosterone (nmol/L)

30

32

34

36

38

B 60

Serum Total Testosterone (nmol/L)

55 50 45 40 35 30 25 20 15 10 5 0 0

40

80

120

160 200 240 280 Serum 17-β-Estradiol (pmol/L)

320

360

400

440

Figure 2 (A) Serum calculated free testosterone (cFT) and calculated bioavailable testosterone (cBioT) concentrations (absolute values) and (B) total testosterone (TT) and 17-b-estradiol (E) evaluated every 7 days for 3 weeks after a single i.m. administration of testosterone enanthate (250 mg) (dark circles, N = 30) and for 5 weeks during daily administration of testosterone gel (50 mg/die) (open squares, N = 50) in male hypogonadal volunteers (n = 10). The horizontal and vertical areas indicate the laboratory reference ranges for serum cFT and cBioT and the manufacturer’s reference ranges for serum TT and E.

the risk of achieving supraphysiological levels, are often not sufficient to maintain eugonadal testosterone concentrations, this being responsible of possible unwanted too large fluctuations in energy level, mood, and performance [2]. J Sex Med 2012;9:873–886

Nieschlag et al. have already pointed out that men who receive TE every 3 weeks have either supra or subphysiological TT levels, and supraphysiological TT concentrations are reported from 10 hours to 3–4 days after a single adminis-

Androgens Profile in Treated Male Hypogonadism tration of 200–250 mg of TE [21,22,41]. Even if our results were partially predictable [41], they are of great interest for doping concerns in hypogonadal athletes. In fact, we highlight the observed high probability (e.g., at least 50%) to find out at an individual level a random serum sample with supraphysiological TT level until 7 days after TE administration compared with the 4% of total samples during fixed dose TG administration at the doses we used. Consequently, based on our data, a great percentage of hypogonadal athletes assuming 250 mg of TE every 3 weeks may be hyperandrogenic for serum TT for one-third of the period between two consecutive TE administration (i.e., at the common, 3-weeks interval of administration) and for about 120 days per year, and even more if also DHT is considered. In fact, the probability to find out an hyperandrogenic serum sample for DHT resulted higher both after TE (e.g., in all volunteers on day 7 and in 50% of them on day 14) and during TG (e.g., in 32% of total samples); we also highlight that for some of our volunteers the of TG dose and/or absorption were low, based on the observed low serum TT concentration (Figure 1). On this basis, we believe that it is not easy to titrate TRT to obtain stable and eugonadal serum TT without major fluctuations and/or elevations in DHT concentrations in all hypogonadal athletes treated with TE or TG. We might also hypothesize the possible use of low doses of 5a-reductase inhibitors, to reduce both the conversion of testosterone to DHT and, indirectly, also the doses of administered exogenous testosterone. However, this is only a very speculative hypothesis, because we do not know if this co-treatment could be really useful, safe, and ethical. Finally, we also highlight that even if aromatization of testosterone to E during TRT is traditionally related to the extent of adipose tissue, which in trained athletes is often low (e.g., unfortunately we did not evaluate body composition), our data showed both the presence of active aromatization of both TE and TG that it is likely to occur also at muscle site or at testicular level [42] and a large fluctuation of serum E and TT/E ratio only after TE administration (Table 1). In addition, no differences for TT/E ratio between subjects affected by primary and secondary hypogonadism were observed (unpublished results). As already reported [2], when necessary (e.g., difficulty in dose tailoring with testosterone esters

883 or gel) and/or possible (e.g., drugs and/or money availability, and so forth), the best way to generate a more physiological and long-life stable androgens profile (e.g., normal serum TT and DHT) may be to start TRT with newer testosterone preparations (e.g., parenteral testosterone undecanoate, subdermal testosterone implants) [43–45] or delivery systems (e.g., devices releasing variable amounts of testosterone), particularly when individualized dose titration are necessary. Based on our results, the concepts of testosterone dose tailoring and monitoring during TRT in hypogonadal individuals should be probably reevaluated, particularly for athletes. In fact, we have both clinical and ethical concerns about the presence of often unrevealed abnormal circulating androgens concentrations during TRT. Because of the possible short-term effects of supraphysiological doses of testosterone and DHT, and also to the fact that serum TT fluctuations are necessary to induce some physiological effects of testosterone, we cannot exclude that also a transitory hyperandrogenism may influence health and/or exercise performance [8]. Unfortunately, we actually do not know whether the commonly observed side effects of TRT (e.g., increased hematocrit and prostate growth) could be also related to the presence of too often unrevealed and prolonged transitory abnormally high circulating TT and DHT concentrations (also for high cFT and cBioT?) during different TRT. Furthermore, it is not known whether the observed fluctuation and/or abnormal (e.g., low or high) levels of circulating androgens after TE and TG could negatively or positively affect athlete’s physical adaptations (e.g., neurological, muscular, metabolic) and performance [46].

Unwanted Hyperandrogenism and Anti-Doping During TRT in Hypogonadal Athletes Testosterone is voluntary abused by healthy athletes not only for its proteo-anabolic properties (e.g., long-term effects) but also for its acute effects on CNS, neuromuscular system, and metabolisms [6,8]. Consequently, besides the wellknown long-term effects on muscles (e.g., hypertrophy, increased strength), also an unwanted transitory hyperandrogenism (e.g., hyper-TT and/or hyper-DHT) during TRT may further improve athlete’s performance. The onset of action and time course of testosterone action on different target organs are poorly investigated [47]. For example, when an athlete’s behavior and cognition are concerned, a transitory increase of J Sex Med 2012;9:873–886

884 androgenization degree may reduce the sense of fatigue, increase aggressiveness, and ameliorate cognitive processes during competition. Consequently, during authorized TRT with TE, as actually tailored, hypogonadal athletes are expected to benefit from transitory hyperandrogenism. This is mainly based on anecdotal reports, because it is not demonstrated that soon reaching supraphysiological androgen levels during competition may yield in the short-term to favorable muscular performances [47]. When anti-doping is concerned, exogenous testosterone abuse in healthy athletes may be suspected for urinary T/EpiT ratio higher than a proper cutoff value, and it is confirmed by evaluating the 13C/12C ratio with gas chromatographycombustion-isotope ratio mass spectrometry and/or by specific laboratory reports on longitudinal urinary steroids profiles and expert’s data analyses [48]. However, actually, no data are available on possible strategies for suspecting and confirming testosterone misuse or abuse during TRT in male hypogonadal athletes. A specific monitoring strategy and the institution of a specific biological passport for treated hypogonadal athletes could be very useful in anti-doping activity in treated hypogonadal athletes [49]. Even if, theoretically, hypogonadal active individuals, competitive, and not competitive athletes, at all ages are probably numerous in the world, there are few competitive true hypogonadal athletes treated with testosterone who are exposed to doping controls [3]. However, based on our results, we can hypothesize that a relatively high percentage of worldwide hypogonadic athletes (e.g., and also non-athletes) may involuntary “live and work” for many days with inappropriate serum TT and /or DHT concentrations during a lifelong TRT with the testosterone ester or gel used in the present study. Consequently, if in the future the evaluation of serum TT and DHT during a random serum doping control is introduced by anti-doping organizations, the risk of suspecting a testosterone misuse or abuse in this population could be really high. In addition, the use of more sophisticated delivery systems that would allow easier dose-titration controls for testosterone are necessary. Our study was not designed to study metabolic pathways or to identify a procedure to confirm doping with testosterone during TRT with TE and TG. Based on the observed relationships between serum and urinary androgens profiles (Tables 3 and 4), we suggest that the future J Sex Med 2012;9:873–886

Di Luigi et al. methods to detect testosterone abuse during TRT should be based on the parallel evaluation of both serum androgens and specific urinary metabolites of testosterone, whose respective cutoffs should be identified for each type of testosterone preparation, timing of doping control, and serum androgens profile. In fact, besides the relatively low number of samples, we observed significant differences in the urinary concentrations of some testosterone metabolites, both between TE and TG administration and between urinary samples grouped for serum TT and DHT concentrations (Table 3). Being our urinary metabolites of testosterone within the reported reference ranges for healthy athletes only from the 15% to 60%, different TRT related reference ranges for each urinary testosterone metabolite should be identified, only when TRT is tailored to obtain normal serum TT and DHT concentrations. Conclusion

Considering the results of this study, we believe that in hypogonadal male athletes it is incorrect to monitor a TRT only by evaluating serum TT. In addition, it is necessary to identify and standardize the optimal target for serum androgens profile (e.g., in terms of TT, cFT, cBioT, and DHT concentrations) both to guarantee health status and physiological performances and to adequately manage doping controls to suspect and confirm a possible testosterone abuse during TRT. Waiting for further clinical investigations, the optimal target for a correct TRT in athletes should be to induce a long-life stable eugonadal androgens profile throughout an individualized welltailored treatment; this means to avoid as much as possible also transitory hypo or hyperandrogenisms. Because few studies are available on genetic differences in individual testosterone metabolism and androgen receptors sensitivity during TRT in athletes [50], the optimal testosterone formulation would allow each athlete to titrate the dose, in order to guarantee at least both physiological and stable serum TT and DHT concentrations. Furthermore, from a practical point of view, it is advisable to document this target before a TUE request, by evaluating serum TT and DHT at different time points during the first doses titration, depending by different available specific testosterone formulations, doses, and frequency of administration [2,13]. Because of some limitations of our study (e.g., reduced number of volunteers, lack of evaluations

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Androgens Profile in Treated Male Hypogonadism of long-term effects on health and performance, lack of comparison with other TRT, and so forth), related to the difficulty in finding and evaluating for a long period hypogonadal competitive athletes, further and larger experimental and clinical evaluations are highly warranted on this issues, by studying also newer TRT modalities, both in athletes and in non-athletes.

4

5

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Acknowledgment

This study was supported by a grant of Italian Ministry of Health—Commission for Vigilance on Doping and Athletes’ Health. Corresponding Author: Luigi Di Luigi, MD, Unit of Endocrinology, Department of Health Sciences, University of Rome “Foro Italico,” Rome 00135, Italy. Tel: +390636733563; Fax: +390636733231; E-mail: [email protected] Conflict of Interest: None.

Statement of Authorship

Category 1 (a) Conception and Design Luigi Di Luigi (b) Acquisition of Data Antonio Aversa; Serena Bianchini; Francesco Botrè; Silvia Migliaccio; Francesco Romanelli; Paolo Sgrò (c) Analysis and Interpretation of Data Antonio Aversa; Luigi Di Luigi; Francesco Romanelli; Paolo Sgrò

Category 2

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8

9 10 11

12 13 14

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(a) Drafting the Article Luigi Di Luigi (b) Revising It for Intellectual Content Luigi Di Luigi; Andrea Lenzi

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Category 3

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(a) Final Approval of the Completed Article Luigi Di Luigi; Paolo Sgrò; Antonio Aversa; Silvia Migliaccio; Serena Bianchini; Francesco Botrè; Francesco Romanelli; Andrea Lenzi

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