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    Asian J Androl 2008; 10 (3): 403-415

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- Review -

Androgen abuse in sports

David J. Handelsman1, Alison Heather2

1ANZAC Research Institute and Department of Andrology, Concord Hospital, Sydney, NSW 2139, Australia
2Heart Research Institute, University of Sydney, Sydney, NSW 2050, Australia

Abstract

Androgens remain the most effective and widely abused ergogenic drugs in sport. Although androgen doping has been prohibited for over 3 decades with a ban enforced by mass spectrometric (MS)-based urine testing for synthetic and exogenous natural androgens, attempts continue to develop increasingly complex schemes to circumvent the ban. A prominent recent approach has been the development of designer androgens. Such never-marketed androgens evade detection because mass spectrometry relies on identifying characteristic chemical signatures requiring prior knowledge of chemical structure. Although once known, designer androgens are readily detected and added to the Prohibited List. However, until their structures are elucidated, designer androgens can circumvent the ban on androgen doping. To combat this, in vitro androgen bioassays offer powerful new possibilities for the generic detection of unidentified bioactive androgens, regardless of their chemical structure. Another approach to circumvent the ban on androgen doping has been the development of indirect androgen doping, the use of exogenous drugs to produce a sustained increase in endogenous testosterone (T) production. Apart from estrogen blockers, however, such neuroendocrine active drugs mostly provide only transient increases in blood T. Finally the ban on androgen doping must allow provision for rare athletes with incidental, proven androgen deficiency who require T replacement therapy. The Therapeutic Use Exemption mechanism makes provision for such necessary medical treatment, subject to rigorous criteria for demonstrating a genuine ongoing need for T and monitoring of T dosage. Effective deterrence of sports doping requires novel, increasingly sophisticated detection options calibrated to defeat these challenges, without which fairness in sport is tarnished and the social and health idealization of sporting champions devalued. (Asian J Androl 2008 May; 10: 403_415)

Keywords: androgen; doping; sport; testosterone; bioassay

Correspondence to: Prof. David J. Handelsman, ANZAC Research Institute, University of Sydney and Department of Andrology, Concord Hospital, Sydney, NSW 2139, Australia.
Tel: +61-2-9767-9100 Fax: +61-2-9767-9101
E-mail: djh@anzac.edu.au
Received 2007-12-29 Accepted 2008-01-02

DOI: 10.1111/j.1745-7262.2008.00406.x


1 Introduction

Hormones remain the most effective performance enhancing drugs used illicitly in elite sports. Androgens and erythropoietin (Epo) have proven efficacy for enhancing muscular strength and physical endurance whereas ergogenic effects of other prohibited hormones, such as growth hormone (GH), insulin, corticotrophins and glucocorticoids, remain less clearly established. The latter uncertainty reflects the longstanding difficulty in establishing a firm scientific basis for which chemicals should be prohibited in sports. According to the World Anti-doping Agency (WADA) Code, chemicals are prohibited if they conform to at least two of the three criteria: likely to enhance sports performance, represent a health risk or violate the spirit of sport [1]. However, definitive ergogenic testing of pharmaceutical or nutraceutical agents is costly and unattractive to commercial sponsors as the product would be either prohibited or proved ineffective. In addition, for hormonal drugs such testing involves potential safety, logistic and ethical problems for clinical trials administering high doses without potential therapeutic benefit to healthy volunteers. Yet despite the difficulties, history shows that such direct evidence is indispensable. For example, even a technically high quality meta-analysis reached the wrong conclusion that androgens did not enhance muscular strength in healthy men [2], a misguided conclusion that undermined credibility of deterrence programs among target androgen abusers who were convinced otherwise by experience. This erroneous conclusion resulted from the fact that all prior evidence was confined to studies using only low androgen doses. This anomaly was only rectified by the landmark randomized placebo-controlled prospective studies of Bhasin, which overcame formidable ethical hurdles to prove unequivocally that supraphysiological testosterone (T) doses enhance muscle mass and strength in healthy men [3]. Although equally direct evidence for ergogenic effects of high dose Epo in healthy athletes is not available, there is strong inferential evidence from clinical trials that Epo improves aerobic performance based on its increasing circulating hemoglobin (and thereby oxygen delivery to muscle) in patients with renal [4_6] or cardiac [7, 8] failure as well as human athletes [9, 10] and horses [11]. The potential direct ergogenic effects of GH on muscle and of acute high dose glucocorticoids on mood and motivation remain no more than plausible speculations while definitive studies are lacking. Indeed, clinical experience suggests that prolonged and/or high dose use of GH, glucocorticoids or (non-banned) thyroid hormones (thyroxine, tri-iodothytronine) are likely to have detrimental effects on muscle and sports performance. This review examines the current status and highlights key ongoing issues in the detection and deterrence of androgen doping in sports, still the most widely abused class of ergogenic drugs.

2 Androgen doping

Androgen abuse appears to have started as an epidemic among national elite athletes with an epicenter in Eastern European countries in the 1950_1960s [12]. This timing coincides with both the post-World War II (WWII) pharmaceutical industry boom, the golden age of steroid chemistry that produced the oral contraceptive and anti-inflammatory synthetic glucocorticoids, as well as the early years of the Cold War. This historical intersection of industrial means, unscrupulous operators and political goals shaped the emergence of androgen abuse as a convenient short-cut tool by which socio-politically dysfunctional Eastern bloc countries could gain quick ascendancy through symbolic victories over their Western political rivals, a challenge quickly reciprocated by athletes from the advanced non-communist countries. This escalating bidding war culminated in national sports doping programs operated covertly by Eastern European communist governments. These organized programs of unscrupulous cheating mixed competitive fraudulence with callous ruination of athletes welfare for national political goals. Of these, only the East German program, with its dire consequences for athletes health has so far been fully disclosed [13]. Subsequently, androgen abuse has become an established endemic in countries where the population is sufficiently affluent to support this variant of drug abuse. Once entrenched in the community, androgen abuse spreads beyond elite sports to non-sporting users with recreational, cosmetic and occupational motivations for body-building. The International Olympic Committee (IOC) recognized the issues of sports doping by releasing its first list of prohibited drugs in 1967 following deaths of competitive cyclists using stimulants. Androgen abuse was prohibited in 1974 with implementation soon after of detection testing to deter use of prohibited drugs in competition.

3 Direct androgen doping

Pharmacological doses of exogenous androgens can enhance muscle mass and strength in athletes regardless of androgen type, gender or age. This inevitably improves performance in power and explosive effort sports, although the influence on more skill-dependent sports remains speculative. The IOC's WADA now enforces the banning of androgens through urine screening tests using mass spectrometry (MS) to detect even traces of synthetic androgens or exogenous administration of natural androgens. Androgens remain the class of drugs most widely detected in sports doping control labs screening urine specimens from international competitions, including the Olympics [14]. For example, in 2006, among the 34 international WADA-accredited doping control laboratories nearly half of the positive findings were due to androgens, three times the frequency of the next category of prohibited agents. The most easily detected are synthetic androgens whose distinctive, non-natural chemical signatures on MS are readily cataloged, in principle. This prohibited list banned all available marketed or easily manufactured androgens, and the highly sensitive detection of even trace amounts for months after last administration maintains a strong disincentive to their use. This effective suppression of androgen doping is confirmed by the now persistently low rate (< 2%) of positive tests in urine specimens from major international competitions [14]. The quest to exploit androgen doping has, therefore, shifted to seeking still undetectable (because unidentified and, therefore, unspecified) synthetic designer androgens. These mostly exploit archival knowledge of steroid chemistry stemming from the 1960_1970s now existing only in obscure patent specifications. These are often low potency and/or toxic, second tier synthetic androgens that were considered unsuitable for commercialization in the heyday of legitimate synthetic androgen development programs.

4 Natural androgen doping

One approach to circumventing the ban on androgen doping has been to use pharmaceutical forms of the two potent natural androgens, T or dihydrototestosterone (DHT). Detecting illicit exogenous use of these natural androgens requires distinguishing between endogenous and exogenously administered forms of the same steroid. Broadly, two approaches are used to achieve this distinction. One is to measure the natural androgen (T or DHT) together with a co-secreted precursor steroid that is neither a T precursor or metabolite. Although administering exogenous T increases its circulating and excreted levels, the levels of the endogenous co-secreted product remain the same (because T cannot be converted to that co-secreted product) or diminished (if the exogenous androgen dose is sufficiently high to suppress the pituitary-testicular axis and Leydig cell steroid secretion). The most convenient co-secreted Leydig cell product is the 17 epimer of T, epitestosterone (EpiT), which is excreted in urine as a glucuronide conjugate [15] and is not formed by endogenous metabolism of administered T. Despite evidence for weak anti-androgenic effects in some non-human species, EpiT is considered biologically inactive as an androgen in humans [15]. Although EpiT is produced at only approximately 3% of the whole body T production rate resulting in circulating levels in adults of 10%_20% of blood T levels, EpiT is more rapidly cleared into urine as a glucuronide conjugate so that the urinary excretion rates of T and EpiT are similar, making the urinary T to EpiT (T/E) ratio approximately 1. As a result, administration of exogenous T or DHT can be detected by increases in the T/E [16, 17] or DHT to EpiT (DHT/E) [18_20] ratio in urine.

The T/E ratio test is now in routine use for sports doping control. The threshold for declaring a positive test is a compromise balancing false positives with false negative tests. Although the original threshold of 6 is high enough that false positives are uncommon, the recent lowering to 4 has led to many more false positive tests. Problems arise due to the existence of individual, environmental and population genetic differences influencing the T/E ratio. For example, rare individual athletes whose T/E ratio naturally exceeds 6, partly attributable to low endogenous EpiT production, have been studied in detail [21, 22]. Recent studies have identified a genetic polymorphism that systematically influences population T/E ratios, explaining the systematically lower T/E ratios in Asian populations. A genetic deletion leading to functional inactivation of the major hepatic androgen glucuronidation enzyme, uridine diphosphate glucuronyl transferase 2B17 (UGT2B17), has a major influence on urinary T/E ratio [23]. Originally identified as a polymorphism of a minor histocompatibility antigen without known functional significance [24], the UGT2B15 deletion occurs approximately seven times more frequently in Asian [23] and five times less frequently in African-American [25] compared with Caucasian populations. The use of urinary EpiT measurement in the T/E ratio has also led to attempts to mask the use of natural androgens by co-administration of large amounts of EpiT [26]. Such EpiT doping, banned as a masking technique, is detected by its characteristic urinary steroid metabolite patterns [27]. These limitations of the T/E ratio as a criterion to detect exogenous T doping suggest that the T/E ratio threshold criterion might be more accurately and reliably deployed in serial, within-subject testing using Bayesian adaptive thresholds [28, 29].

A second approach to identifying exogenous administration of naturally occurring androgens has been the development of isotope ratio mass spectrometry (IRMS) [22, 30]. Adapted from stable isotope MS techniques widely used in metabolism, pharmacological and forensic applications [31_34], this more complex testing requires specialized steps and equipment in order to quantify stable carbon isotope (C13/C12) ratios. These differ between endogenously produced steroids and plant sterols because the latter, which forms the common base material for all commercial androgen synthesis, have characteristically lower C13/C12 ratios, resulting from the lower heavy isotope (C13) enrichment during photosynthetic fixation of carbon dioxide [35]. This more laborious test currently forms the ultimate proof for distinguishing endogenous from exogenous natural androgens, including T, DHT, dehydroepiandrosterone (DHEA) and nandrolone. Doping with other naturally occurring androgens, such as DHEA [36_38] and nandrolone [39], is readily detected by measurement of corresponding metabolite ratios. Moreover, DHEA doping might also be suspected with an otherwise unexplained abnormally increased T/E ratio [40].

5 Designer androgens

The most recent challenge in androgen doping has been the development of designer androgens. These are synthetic androgens purposely developed to evade detection by the conventional urine MS-based doping tests for the closed list of specified steroids [41]. Most designer androgens originate from 1960_1970s pharmaceutical industry androgen discovery and synthesis programs largely geared towards identifying a purely "anabolic steroid". Once the futility of that quest was recognized, leaving behind only the vivid but meaningless term, androgen synthesis programs were abandoned with the loss of in-house steroid chemistry knowledge then relegated to obscure, wide patent specifications. Decades later, however, some re-emerged as designer androgens selected from the vast stock of never marketed androgens for being undetectable by WADA-approved testing. The first illicit designer androgen identified, norbolethone, had undergone limited clinical testing in the 1960s [42], but was never marketed, probably because of its hepatotoxicity as an 17-alkylated androgen. Its discovery in 2002, based on suspicion of an unknown androgen in an athlete's urine, led rapidly to its chemical structure being deduced and a detection method developed [43]. Soon after, in 2003, tetrahydrogestrinone (THG) was identified as the first designer androgen never previously described [44]. Manufactured by a simple, one-step reduction of a commercially available but seldom used progestin, gestrinone, THG is a potent androgen and progestin [45, 46] with additional non-specific effects on other steroid receptors [47]. In 2005, a third designer androgen was identified as desoxymethyltestosterone (DMT, Madol) [48, 49], yet another synthetic androgen patented in the 1960s but never marketed. Subsequently, other designer androgens appearing on the market include 1-testosterone [50, 51] and a variety of other steroids [52] marketed illicitly on the internet, usually in nutraceutical food supplements. Much of this illicit androgen industry arose from 1994 US legislation facilitating the sale of steroid precursors (prohormones) as nutraceutical food supplements. It continues despite amending 2004 legislation reclassifying specified synthetic androgens as drugs rather than food supplements [53]. Although such reclassification restricts sales of the specified androgens, the almost infinite variety of structural modifications of the steroid backbone limits the effectiveness of this legal tightening. The sheer diversity of the structural modifications compatible with androgenic bioactivity in a steroid or its in vivo metabolites indicates that, although MS-based methods are powerful specific and sensitive detection methods, they have insuperable technical difficulties identifying androgens of unknown structure [54].

A new class of non-steroidal androgens has recently been developed [55]. These chemicals, originally developed by structure-activity guided variation of the structure of the non-steroidal anti-androgen bicalutamide, now comprise various subclasses of arylpropanilimide and quinoline derivatives. They are partial, tissue-selective androgen receptor (AR) agonists, unable to be aromatized and, therefore, manifesting only part of the full spectrum of androgenic effects of T. Often referred to by the marketing term specific AR modulator (SARM), these drugs are in clinical development but not yet marketed and seem highly likely to be abused in sports although no positive tests are yet reported. An MS-based doping control method to detect arylpropionamide SARM in urine has been reported [56] and further methods will be required for this growing class of new synthetic androgens.

6 In vitro androgen bioassays

An unusually versatile possibility for detection of novel androgens is the catch-all approach of using highly sensitive in vitro androgen bioassays. These in vitro steroid bioassays were originally developed during the last decade using the availability of the steroid receptors and molecular biological tools to powerfully amplify their biological activity in various host cells. Developed originally to detect minute quantities of hormonally active chemicals in the environment, these in vitro steroid bioassays have the potential to provide a generic form of detection for illicit androgens in doping control regardless of whether their structure is known or unknown.

The original methods to measure androgens were in vivo bioassays, typically using castrated animals. These methods evolved from the classical testicular transplantation experiments of John Hunter (18th century) and Arnold Berthold (1849) showing that transplanting a cock's testis maintained its comb, a distinctive sexually dimorphic characteristic [57]. The adaptation of androgen bioassays to using castrated rats in the 1930s [58, 59] was of pivotal importance to the Nobel-prize winning isolation and characterization of testosterone from urine in 1935. With some technical refinements, the Hershberger androgen bioassay [60] remains an important method in pharmacology and toxicology [61, 62]. Subsequently, the advent of steroid radioimmunoassay around 1970 revolutionized androgen measurement by facilitating highly sensitive and faster, high throughput methods. More recently, mass spectrometric techniques to measure steroids grew in importance for their ultimate specificity, although they have remained limited by their sensitivity and quantitative reproducibility in comparison to radioimmunoassay. Both radioimmunoassay and MS require structural knowledge of the steroid. By contrast, androgen bioassays measure the androgenic potency of a sample that activates AR without any requirement for information about the chemical nature of the agonist. Most recently the advent of in vitro androgen bioassays using cultured host cells instead of whole animals combines the virtues of the original whole animal bioassays, the generic detection of androgens via their bioactivity without regard to chemical structure, with the streamlined features (high throughput, reproducibility, precision, sensitivity) of bench techniques.

7 Principles of in vitro androgen bioassays

The fundamental design of an in vitro androgen bioassay exploits the natural signaling pathway of androgens. Androgens exert their cellular effects by diffusing into cells, binding to the AR, which triggers the translocation of AR into the nucleus where it binds to an androgen response element (ARE) [63, 64] in the presence of cofactors [65]. The DNA-bound liganded receptor drives transcription of the target gene, leading to new protein synthesis and altered cell functioning. A host cell line is chosen for convenience from among yeast or certain mammalian cell lines that express no background endogenous AR. These cells then have AR introduced through an expression vector and a convenient reporter vector in order to measure AR activity. The reporter vector encodes a minimal promoter driven by a suitable hormone response element that, together, drive the expression of a reporter enzyme or protein (e.g. luciferase, secreted alkaline phosphatase, β-galactosidase or green fluorescent protein) so that enzyme activity can be readily measured according to the nature of the read-out. Similar in vitro steroid bioassays have been developed for all other major classes of human steroid receptors in the superfamily of nuclear receptors, such as progestins, estrogen, glucocorticoids and mineralocorticoids.

In vitro androgen bioassays measure directly and unequivocally the androgenic bioactivity of any tested steroid. This is advantageous compared with simpler AR binding assays, which cannot distinguish between agonists and antagonists as well as being less sensitive [66]. Conversely, this is also a limitation in that it cannot account for in vivo conversion of a parent compound to more or less potent androgens. For example, T bioactivity in vivo is augmented by its conversion to the more potent androgen DHT and, conversely, a non-androgenic steroid with androgenic metabolites formed in vivo (e.g. tibolone) would have its androgenic activity in vivo underestimated by the in vitro AR bioactivity. Similarly, a potent androgen in vitro might be ineffective in vivo if it is metabolized too rapidly to reach its tissue targets [67].

8 Yeast cell-based bioassays

Crucial aspects of an in vitro androgen bioassay include ease of use, a high throughput platform, sensitivity and selectivity. For these considerations, the first androgen bioassay, and then many others subsequently, were established in the laboratory yeast strain, Saccharomyces cerevisiae [68_72] (Table 1). These are advantageous as yeast grow rapidly at low cost relative to mammalian cell culture and might be less susceptible to non-specific interference from human biological fluid matrix effects.

A widely used yeast in vitro androgen bioassay was developed by transforming S. cerevisiae cells with an AR expression vector encoding human AR cDNA together with a reporter vector harboring two tandem ARE sites upstream of the β-galactosidase reporter gene [68]. For this assay, aliquots of yeast culture are exposed to pure androgens for 24 h before cells are harvested, lysed, and β-galactosidase activity measured, making for an approximately 5 h long experimental protocol. This assay has proven versatile with a sensitivity (ED50) for T and DHT of 4.7 nmol/L and 3.5 nmol/L, respectively. We used this yeast in vitro bioassay to show that THG is a potent androgen and progestin [45]. Illustrating its versatility, this in vitro androgen bioassay can be adapted to detecting anti-androgenic activity by screening for chemicals that interfere with the full activation of the AR by testosterone. Using this approach we discovered that valproate, previously unrecognized as an anti-androgen, had strong AR antagonist activity [73].

The common yeast androgen bioassay, while robust, reliable and reproducible, requires pre-assay cell preparation, long incubation times and cell lysis steps, which could ideally be simplified. One simplification uses a bioluminescent assay with a reporter vector expressing firefly luciferase, controlled by two tandem repeat ARE sites, so that read-out signals are detectable directly from cells after adding D-luciferin substrate, avoiding additional steps and allowing the full assay to be completed within a day [74]. This bioassay had slightly lower sensitivity for T and DHT (EC50 10 nmol/L and 5.5 nmol/L, respectively). A more recent development involves the use of yeast-integrating plasmids where the hAR cDNA and ARE-yEGFP reporter construct cDNA are stably integrated into the yeast genome. This assay also allows a direct read-out, making for a very rapid assay protocol completed within approximately 4 h [72]. However, this assay is approximately 10-fold less sensitive for T and DHT (EC50: 50 nmol/L and 2.3 nmol/L), although the explanation for the unusual discrepancy between the sensitivity for T and DHT is unclear. A hybrid approach, bioassay guided MS, combining the versatility of the yeast-based in vitro androgen bioassay with the specificity of the MS-based assay, has also been described [75].

A completely different approach to a yeast-based in vitro androgen bioassay has been modeled on the yeast two-hybrid assay. This somewhat complicated approach involves transforming yeast cells with an expression vector that expresses the hinge-ligand domain (hLBD) of AR fused to the DNA binding domain of LexA (a transcription factor) and a second expression vector that express the ASCI protein (a coactivator of AR) fused to the activation domain, B42 (that interacts and activates LexA) [70]. In addition to the two expression vectors, the yeast cell harbors the lacZ reporter plasmid under the control of the LexA promoter. In the presence of an androgen, the AR/LexA fusion protein will interact with the ASC1/B42 fusion protein, leading to activation of LexA by B42 and subsequent transcription of lacZ to produce β-galactosidase. Sensitivity of this bioassay is also less than the conventional AR/ARE-based yeast androgen bioassay, with the EC50 reported to be 15 nmol/L for T and 4.8 nmol/L for DHT.

9 Mammalian cell-based bioassay

Several different mammalian cell bioassays for detecting androgens have been developed (Table 2). Among the first was transient transfection involving three different plasmids [76]. In practice, this approach is too complex and inconsistent to be an effective tool for doping control screening. More practical systems feature stably transfected human cell lines with a hormone sensitive read-out system comprising an AR expression vector and an ARE-driven or MMTV-driven luciferase reporter vector. Several systems with varying success have been reported [77_82]. The first was developed in CHO-K1 cells with reported EC50 of 0.04 nmol/L for T, which provided a major improvement in sensitivity compared with yeast-based androgen bioassays [78]. However, CHO-K1 cells had measurable 5a-reductase, 17β-hydroxysteroid reductase, and 3α-hydroxysteroid activities so that test steroids could be both activated and inactivated prior to measurement, leading to an unacceptable loss in fidelity for an analytical AR bioassay. Transferring the same assay system into a different host (U2-OS) cells of bone origin lacking most steroidogenic enzymes except 17β-hydroxysteroid reductase, resulted in reduced sensitivity (EC50 0.66 nmol/L), which remained approximately 10-fold more sensitive than yeast-based assays [83]. Subsequently, another variation using a HEK293 host cell with minimal steroidogenic enzyme activity and no endogenous AR expression was also highly sensitive (EC50 0.1 nmol/L) and able to detect T levels as low as 150 pmol/L [82].

10 Applications of in vitro AR bioassays to biological fluids

The practical application of in vitro AR bioassays to measure androgen bioactivity in human serum samples remains problematic, especially for the precise and reproducible quantitation required for doping control [76, 78, 82, 84_89]. The most extensive studies using a transient transfection system report serum T levels systematically lower than levels measured by a reliable radioimmunoassay [76], a disparity attributable to some or all of the presence of binding proteins (notably SHBG), cross-reactivity from structurally related free and conjugated steroids and non-specific matrix effects. In one study, serum with high levels of the major binding proteins SHBG led to markedly reduced recovery of T compared with non-human serum lacking SHBG [89]. In another study, pre-assay organic solvent extraction of serum samples, which releases hydrophobic steroids from their binding proteins as well as removing hydrophilic steroid conjugates, gives values similar to those measured by radioimmunoassay of the same solvent extracts of serum [82]. However, the relative contributions to the non-quantitative measurements in human serum samples of the presence of binding proteins versus other non-specific matrix effects remain unclear. A preliminary analysis of using a yeast-based AR bioassay to qualify T in human serum samples indicates a good correlation with immunoassay [90]; however, detailed validation of agreement between assays is lacking. There are no studies evaluating the use of human urine samples in any in vitro AR bioassay.

In summary, in vitro androgen bioassays represent an interesting option for generic detection of androgens, including novel designer steroids of unknown structure if the quantitative aspects of their application to human biological fluids can be resolved satisfactorily. At present, none of the available in vitro androgen bioassays could be used with the validity and reliability required for doping control. The ideal in vitro androgen bioassay would be rapid, simple, sensitive, precise, reproducible and inexpensive. Among the variety of choices for host cell and read-out systems, present knowledge suggests that mammalian cell-based bioassays are more sensitive than yeast-based bioassays, with reported sensitivities ranging from 0.002_30.000 nmol/L for T and 0.04_3.00 nmol/L for DHT, compared with approximately 5 nmol/L for yeast-based AR bioassays. The wide variation in sensitivity is attributable to experimental factors: notably, stable versus transient transfection approaches, and endogenous expression of AR and steroidogenic enzymes (5α-reductase, aromatase, 3α- and 17β-hydroxysteroid dehydrogenase) and their co-factors. Conversely, the lack of AR or steroidogenic enzyme expression in yeast cell-based bioassays might offer greater fidelity, speed and affordability. Both yeast and mammalian cell bioassays suffer from non-specific serum matrix effects and are likely to require pre-assay sample extraction without greatly increased sensitivity so that the added serum in the bioassay has insignificant effects on its validity. Future developments of in vitro androgen bioassays therefore need to concentrate on improving sensitivity and overcoming matrix effects to ensure the reliability and validity required for a doping control test.

11 Indirect androgen doping

Various indirect strategies have also been developed to circumvent the ban on direct androgen doping. These focus on increasing endogenous T and thereby aiming to exploit the myotrophic effects of T but bypassing the specific testing regimens for known synthetic androgens including exogenous T.

Indirect androgen doping methods aim to stimulate luteinizing hormone (LH)-dependent Leydig cell T biosynthesis and secretion. The two main indirect androgen doping approaches have been either: (i) direct stimulation by administration of exogenous LH or hCG, a placental glycoprotein and long-acting natural analog of LH; or (ii) by indirect stimulation by increasing endogenous LH [91]. This is analogous to the indirect Epo doping strategies designed to evade the bans on Epo and its analogs, such as emulating Epo effects by autologous or heterologous blood doping or manipulating habitual oxygen exposure to increase endogenous Epo through altitude living/training or hypoxic conditioning.

Endogenous LH secretion is tightly regulated by hypothalamic GnRH secretion [92]. The distinctive feature of GnRH physiology is that its intermittent secretion in brief bursts from hypothalamic neurons into the pituitary portal system is both necessary and sufficient to entrain the physiological pattern of blood LH levels, characteristically highly pulsatile with peaks at 60_90 min intervals [93, 94]. Administration of exogenous GnRH in any non-pulsatile fashion rapidly desensitizes pituitary gonadotrophs and suppresses their secretion of LH, which only recovers when intermittent GnRH administration is restored [93, 94]. Consequently, replicating physiological pulsatility by intermittent delivery of brief bursts of GnRH is a sine qua non to maintain physiological circulating LH and T levels. For example, in men who are gonadotrophin deficient as a result of GnRH deficiency, restoration of physiological circulating LH and T levels is only achieved by intermittent GnRH administration, typically administered by mechanical pumps worn around the clock and delivering timed, small GnRH doses at 60_90 min intervals [95, 96]. Although not directly tested, this cumbersome treatment if it was superimposed on normal GnRH physiology in eugonadal men would likely suppress, rather than enhance, endogenous LH and T secretion. Similarly, sustained non-physiological stimulation of pituitary gonadotrophes by superactive GnRH agonists in older men with prostate cancer cause a transient "flare" lasting 5_10 days during which time LH and T are moderately elevated [97_102] before the onset of the profound and sustained inhibition of LH and T secretion, now used clinically to maintain medical castration for hormone dependent cancers [103]. Hence, although exogenous GnRH and its superactive analogs can transiently increase endogenous LH levels by direct stimulation of pituitary gonadotrophs, such pharmacological (non-pulsatile) GnRH exposure cannot sustain supraphysiological LH and T levels in men. GnRH and its analogs are not prohibited by WADA as sports doping agents.

Therefore, indirect androgen doping requires a sustained increased in endogenous LH secretion. In turn, this requires manipulating physiological regulatory systems governing pulsatile hypothalamic GnRH secretion. Factors known to enhance endogenous hypothalamic GnRH secretion include: (i) neurotransmitters acting on excitatory glutamate, GABA, noradrenergic, galanin and/or NPY receptor systems; (ii) neuropeptides such as kisspeptin and its analogs acting as GPR54 agonists [104] and opioid peptides or analogs acting as μ opioid antagonists [105, 106]; and (iii) blockers of sex steroid negative feedback such as anti-androgens and estrogen blockers like anti-estrogens or aromatase inhibitors.

Furthermore, although drugs that acutely stimulate endogenous GnRH secretion through specific neurotransmitter or neuropeptide mechanisms can produce short-term or transient elevation of LH [106_110], attenuation over time and nonspecificity of their effects mean they do not maintain a sustained increase in endogenous T production. Drugs such as μ opioid antagonists (naloxone, naltrexone and nalmephene) are not prohibited by WADA.

12 Therapeutic use exemptions for testosterone

Under the WADA Code's strict liability rule, a positive urine doping test is an anti-doping code violation regardless of intent, fault, negligence or knowing use. The complete ban on androgens has to make allowance for men with androgen deficiency due to well established pituitary or testicular disorders. Such men have a valid clinical need for T replacement therapy without which they could suffer from long-term consequences of androgen deficiency, such as delayed or impaired pubertal development, reduced bone or muscle mass and strength, sexual dysfunction, depression and metabolic consequences. Therefore, there is a need to make provision for androgen deficient male athletes to have approved and supervised T replacement therapy that still allows them to compete fairly in elite sport.

As classical androgen deficiency has a prevalence of approximately 1:200 (excluding age-related androgen deficiency or "andropause") [111], coincidental androgen deficiency among athletes will be regularly, if infrequently, observed. Where it occurs in non-power sport athletes, androgen deficiency or replacement therapy might be of little significance for performance. However, among power sport athletes androgen status has significant influence on performance so the decision to grant a Therapeutic Use Exemption (TUE) requires careful judgment and particular attention to ensuring excessive T doses are not allowed.

Under WADA rules, athletes with a valid medical reason for using a prohibited substance may apply to their national antidoping organization affiliates for a TUE. Evaluation for a TUE requires expert medical assessment to establish: (i) a valid medical indication for T replacement therapy; and (ii) an acceptable regimen for T use, including dosage and monitoring. For this system to remain above reproach, the justification for a TUE for T should be approved by an independent, medical expert unaware of the identity of the athlete or his or her treating doctor.

Establishing a valid clinical diagnosis of androgen deficiency requires identifying clearly the type and cause of the underlying pathology leading to sustained, permanent decrease in endogenous T production. The clinical diagnosis requires biochemical confirmation by appropriate assays of circulating reproductive hormones (LH, follicle stimulating hormone [FSH], T and sex hormone-binding globulin [SHBG]), but blood hormone assays alone are not sufficient to establish a full diagnosis. The clinical diagnosis includes defining a cause of primary (testicular or hypergonadotrophic) or secondary (hypothalamic or pituitary [pre-testicular], hypogonadotrophic) hypogonadism. Common causes of primary hypogonadism include Klinefelter's syndrome, bilateral anorchidism or orchidectomy (e.g. tumor, torsion, cryptorchidism) and other severe bilateral testicular disorders (orchitis, infiltrative and injury). The causes of secondary hypogonadism include pituitary tumors, such as prolactinoma, and their treatment by surgery and/or radiotherapy, panhypopituitarism, and idiopathic hypogonadotropic hypogonadism, including Kallmann's syndrome, hemochromatosis or other infiltrative hypothalamic-pituitary disorders. In cases of iatrogenic hypogonadism (orchidectomy, pituitary surgery, radiotherapy) details of surgery, pathology, imaging and/or radiotherapy must to be provided.

The clinical diagnosis of age-related androgen deficiency is more complex. Delayed puberty might be due to constitutional or familial factors or extreme, sustained intensive exercise in an adolescent; however, T therapy is rarely justified for such inherently reversible states. Making the distinction in an adolescent with delayed puberty between such cases of transient androgen deficiency and a diagnosis of permanent gonadotrophin deficiency requiring a timely start of permanent T therapy requires a specialist physician with experience in adolescent endocrinology. At the other end of the age spectrum, the diagnosis of age-related hypogonadism ("andropause") remains an ill-defined clinical condition and not an acceptable medical diagnosis warranting a TUE. This is rarely, if ever, an issue for elite sport at international level. A TUE for T treatment is not acceptable for female athletes.

The clinical diagnosis of androgen deficiency is confirmed biochemically by immunoassays of blood T, LH and FSH performed by an accredited laboratory. These tests should ideally be undertaken on two different mornings, both prior to starting T treatment and interpreted by an experienced endocrinologist. Dynamic tests (e.g. GnRH or hCG stimulation tests) are no longer required for the routine diagnosis of androgen deficiency, although they may be part of the clinical evaluation of delayed puberty.

A variety of T products (Table 3) may be approved for replacement therapy under a TUE, including T in gel, patch, buccal adhesive or implant products or as injectable T esters in an oil vehicle. A standard dose regimen should be specified and monitored to verify approved usage. Although the causes of classical androgen deficiency are nearly always irreversible and require life-long T replacement therapy [111], a TUE is subject to regular review to confirm well-controlled therapy within specified guidelines, including verification of approved dosage. Synthetic androgens are not permitted on safety grounds as most are 17-alkylated androgens with unacceptable class-specific hepatotoxicity with numerous safer alternatives available.

Maintaining fairness in sport when a TUE is approved for T requires ongoing monitoring to confirm compliance with, while not exceeding, the specified T dosage. This is essential as excessive T dosage provides an unfair pharmacological advantage because of the dose-proportional effects of T on muscle mass and strength. Monitoring of a TUE for T requires: (i) establishing an approved T dose regimen (Table 1); (ii) maintaining a register of T prescriptions and administrations by medical personnel; (iii) review of compliance and requests to change dose or regimen; and (iv) random, unannounced blood tests.

An athlete with an approved TUE for T should be responsible for maintaining an up-to-date register that records all parenteral administrations (injections, implants) by a health professional. The register should record every administration of an injection or implant, including dose, date, place, name and signature of health professional administering the treatment. Athletes should not self-inject T products. If the athlete is responsible for his or her own medication (e.g. transdermals), he or she must maintain a record of all filled T prescriptions. Failure to maintain or provide for inspection on demand can be considered equivalent to having misused T during that period.

Review of an approved TUE for T will rarely require any change in dose or regimen. The rare instances of short-term T treatment in adolescent boys with delayed puberty require regular (i.e. annual) re-evaluations by an experienced adolescent endocrinologist. In routine clinical practice, T replacement therapy rarely, if ever, requires dose changes after establishing an individual's optimal dosage for a new T product. Ongoing monitoring is usually focused on maintaining adequate compliance and identifying incidental or product-specific adverse effects [111]. The T dosage must be decided by at least one doctor with appropriate endocrinology experience and any variations to the approved TUE condition should be approved in writing based on appropriate evidence from the supervising specialist.

Monitoring by regular, random and unannounced blood tests is an objective method to verify that the athlete does not exceed the approved T dosage. Blood samples are taken for serum T, LH and FSH and must be carefully interpreted in the light of knowledge of the time interval between blood sampling and last T dose administered and of the pharmacokinetics of the T product being used. For parenteral administration this should be obtained from the register and for self-administered non-parenteral products, from the athlete.

13 Conclusion

The effective detection and deterrence of androgen doping in sports remains an ongoing challenge. However, for every new challenge, steady scientific progress has continued to overcome the efforts to cheat using drugs in competition. Effective progress so far includes the virtual elimination of marketed androgens from elite competitions as indicated by the rarity (< 2%) of positive urine tests when such steroids are easily detectable for long periods after use. In response to the elimination of the most effective and available androgens from competition, new doping schemes, including indirect doping and doping with natural or designer androgens, have been tried, but suitable detection methods have not been established and further tests are in development. Finally, a rational approach has been developed to allow athletes with incidental gonadal disorders to compete fairly while preventing unjustified use or overdosage of T. The application of the effective methods that have largely eliminated drug cheating in competition to out-of-competition testing represents the next frontier where a positive outcome can be anticipated if the logistic challenges can be overcome. Although elimination of androgen doping in sports remains a worthy but still distant goal, the practical creation of hurdles that are insurmountable for all but the most desperate can now be considered achievable.

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