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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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