ABSTRACT

Testosterone, together with its bioactive metabolites dihydrotestosterone and estradiol, determines the development and maintenance of male sexual differentiation and the characteristic mature masculine features. Defects in androgen action at various epochs of life produce characteristic clinical features. From an outline of the biochemistry and physiology of androgen action, the pathophysiology of defects in androgen action are derived and defined. The pharmacology of testosterone and its applications to replacement therapy for pathological hypogonadism as well as for pharmacological androgen therapy based on using either testosterone or synthetic androgens is described.

INTRODUCTION

An androgen, or male sex hormone, is defined as a substance capable of developing and maintaining masculine characteristics in reproductive tissues (notably the genital tract, secondary sexual characteristics, and fertility) and contributing to the anabolic status of somatic tissues. Testosterone together with its potent metabolite, dihydrotestosterone (DHT), are the principal androgens in the circulation of mature male mammals. Testosterone has a characteristic four ring C18 steroid structure and is synthesized mainly by Leydig cells, located in the interstitium of the testis between the seminiferous tubules. Leydig cell secretion creates a very high local concentration of testosterone in the testis as well as a steep downhill concentration gradient into the bloodstream maintaining circulating testosterone levels which exert characteristic androgenic effects on distant androgen sensitive target tissues. The classical biological effects of androgens are primarily mediated by binding to the androgen receptor, a member of the steroid nuclear receptor superfamily encoded by a single gene located on the X chromosome, which then leads to a characteristic patterns of gene expression by regulating the transcription of an array of androgen responsive target genes. This physiological definition of an androgen in the whole animal is now complemented by a biochemical and pharmacological definition of an androgen as a chemical that effectively competes with testosterone binding to the androgen receptor (1) to stimulate post-receptor functions in isolated cells or cell-free systems. In addition, non-genomic mechanisms of androgen action involving rapid, membrane-mediated nontranscriptional processes in the cytoplasm have been described but not yet fully characterized (2-4).

Testosterone is used clinically at physiologic doses for androgen replacement therapy and, at typically higher doses, testosterone or synthetic androgens based on its structure are also used for pharmacologic androgen therapy. The principal goal of androgen replacement therapy is to restore a physiologic pattern of androgen exposure to all tissues. Such treatment is usually restricted to the major natural androgen, testosterone, and aims to replicate physiological circulating testosterone levels and the full spectrum (including pre-receptor androgen activation) of endogenous androgen effects on tissues and recapitulating the natural history of efficacy and safety. Pharmacologic androgen therapy exploits the anabolic or other effects of androgens on muscle, bone, and other tissues as hormonal drugs that aim to modify the natural history of the underlying disorder and are judged on their efficacy, safety, and relative cost effectiveness like other therapeutic agents. Insight into the physiology of testosterone is a prequisite for understanding and making most effective use of androgen pharmacology (5, 6).

TESTOSTERONE PHYSIOLOGY

Biosynthesis

Testosterone is synthesized by an enzymatic sequence of steps from cholesterol (7, 8) (Figure 1) within the 500 million Leydig cells located in the interstitial compartment of the testis between the seminiferous tubules, which constitutes approximately 5% of mature testis volume (see Endotext, Endocrinology of Male Reproduction, Chapter entitled Endocrinology of the Male Reproductive System and Spermatogenesis, for details) (9). The cholesterol is predominantly formed by de novo synthesis from acetate, although preformed cholesterol either from intracellular cholesterol ester stores or extracellular supply from circulating low-­density lipoproteins also contributes (8). Testosterone biosynthesis involves two multifunctional cytochrome P-450 complexes involving hydroxylations and side-chain scissions (cholesterol side-chain cleavage [CYP11A1 or P450scc which produces C20 and C22 hydroxylation and C20,22 lyase activity] and 17-hydroxylase/17,20 lyase [CYP17A1 or P450c17 which hydroxylates the C17 and then excises two carbons (20 & 21) coverting a 21 to a 19 carbon structure]) together with 3 and 17b-hydroxysteroid dehydrogenases and ∆^4,5 isomerase (8). The highly tissue-selective regulation of the 17,20 lyase activity (active in gonads but inactive in adrenals) independently of 17-hydroxylase activity (active in all steroidogenic tissues) is a key branch-point in steroidogenic pathways. Both activities reside in a single, multifunctional protein with the directionality of pathway flux determined by enzyme co-factors, notably electron supply from NADPH via the P450 oxidoreductase (POR), a membrane-bound flavoprotein serving diverse roles as a reductase, and cytochrome b₅ (10, 11). In addition, some extragonadal biosynthesis of testosterone and dihydrotestosterone from circulating weak adrenal androgen precursor DHEA within specific tissues has been described (12); however, the net contribution of adrenal androgens to circulating testosterone in men is minor (13, 14) though it makes a much larger proportional contribution to circulating testosterone in women (15, 16).

FIGURE 1. Pathways of testosterone biosynthesis and action. In men, testosterone biosynthesis occurs almost exclusively in mature Leydig cells by the enzymatic sequences illustrated. Cholesterol originates predominantly by the de novo synthesis pathway from acetyl CoA with luteinizing hormone regulating the rate limiting step, the conversion of cholesterol to pregnenolone within mitochondria, while remaining enzymatic steps occur in smooth endoplasmic reticulum. The 5 and 4 steroidal pathways are on the left and right, respectively. Testosterone and its androgenic metabolite, dihydrotestosterone, exert biological effects directly through binding to the androgen receptor and indirectly through aromatization of testosterone to estradiol, which allows action via binding to the estrogen receptor (ER). The androgen and ERs are members of the steroid nuclear receptor superfamily with highly homologous structure differing mostly in the C-terminal ligand binding domain. The LH receptor has the structure of a G-protein linked receptor with its characteristic seven transmembrane spanning helical regions and a large extracellular domain which binds the LH molecule. LH is a dimeric glycoprotein hormone consisting of an  subunit common to other pituitary glycoprotein hormones and a  subunit specific to LH. Most sex steroids bind to sex hormone binding globulin (SHBG) which binds tightly and carries the majority of testosterone in the bloodstream.

Testicular testosterone secretion is principally governed by luteinizing hormone (LH) through its regulation of the rate-limiting conversion of cholesterol to pregnenolone within Leydig cell mitochondria by the cytochrome P-450 cholesterol side-chain cleavage enzyme complex located on the inner mitochondrial membrane. Cholesterol supply to mitochondrial steroidogenic enzymes is governed by proteins including sterol carrier protein 2 (17). This facilitates cytoplasmic transfer of cholesterol to mitochondria together with steroidogenic acute regulatory protein (18) and translocator protein (19), which govern cholesterol transport across the mitochondrial membrane. All subsequent enzymatic steps are located in the Leydig cell endoplasmic reticulum. The high testicular production rate of testosterone creates both high local concentrations (up to 1 μg/g tissue, ~100 times higher than blood concentrations) and rapid turnover (200 times per day) of intratesticular testosterone (20); however, the precise physical state in which such high concentrations of intratesticular testosterone and related steroids exist in the testis remains to be clarified.

Secretion

Testosterone is secreted at adult levels during three epochs of male life: transiently during the first trimester of intrauterine life (coinciding with masculine genital tract differentiation), during early neonatal life as the perinatal androgen surge (with still undefined physiologic significance), and continually after puberty to maintain virilization. The dramatic somatic changes of male puberty are triggered by the striking increases in testicular secretion of testosterone, rising ~30-fold over levels which prevail in pre-pubertal children and in women or castrate men originating from extra-testicular sources. After middle age, there are gradual decreases in circulating testosterone as well as increases in gonadotrophin and sex hormone–binding globulin (SHBG) levels (21, 22) with these trends being absent till late old age among men who remain in excellent health (23, 24) but exaggerated by the coexistence of chronic illness (22, 25-27). In addition there are temporal trends including increasing prevalence of obesity (28-30)and artefactual method-specific changes in testosterone immunoassays that deviate from reference mass spectrometry-based measurements (31, 32). These age-related changes from the accumulation of chronic disease states are functionally attributable to impaired hypothalamic regulation of testicular function (33-36), as well as Leydig cell attrition (9) and dysfunction (37-39) and atherosclerosis of testicular vessels (40). As a result, the ageing hypothalamic-pituitary-testicular axis progressively increasingly operates with multi-level functional defects that, in concert, lead to reduced circulating testosterone levels during male ageing (41, 42).

Testosterone, like other lipophilic steroids secreted from steroidogenic tissues, leaves the testis by diffusing down a concentration gradient across cell membranes into the bloodstream, with smaller amounts appearing in the lymphatics and tubule fluid. After male puberty, over 95% of circulating testosterone is derived from testicular secretion with the remainder arising from extragonadal conversion of precursors with negligible intrinsic androgenic potency such as dehydroepiandrosterone and androstenedione. These weak androgens, predominantly originating from the adrenal cortex, constitute a large circulating reservoir of precursors for conversion to bioactive sex steroids in extragonadal tis­sues including the liver, kidney, muscle, and adipose tissue. Unlike in women where adrenal androgens are the major source of biologically active androgen precursors, endogenous adrenal androgens contribute negligibly to direct virilization of men (13) and residual circulating and tissue androgens after medical or surgical castration have minimal biologic effect on androgen-sensitive prostate cancer (43). Conversely, however, adrenal androgens make a proportionately larger contribution to the much lower circulating testosterone concentrations in children and women (~5% of men) in whom blood testosterone is derived approximately equally from direct gonadal secretion and indirectly from peripheral interconversion of adrenal androgen precursors (15, 16). Exogenous dehydroepiandrosterone at physiologic replacement doses of 50 mg/day orally (15) is incapable of providing adequate blood testosterone for androgen replacement in men but produces dose-dependent increases in circulating estradiol in men (44, 45) and hyperandrogenism in women (14).

Hormone production rates can be calculated from either estimating metabolic clearance rate (from bolus injection or steady-state isotope infusion using high specific-activity tracers) and mean circulating testosterone levels (46, 47) or by estimation of testicular arteriovenous differences and testicular blood flow rate (48). These methods give consistent estimates of a testosterone production rate of 3 to 10 mg/day using tritiated (49, 50) or nonradioactive deuterated (51)tracers with interconversion rates of approximately 4% to dihydrotestosterone (DHT) (50, 52) and 0.2% to estradiol (53) under the assumption of steady-state conditions (hours to days). These steady-state methods are a simplification that neglects diurnal rhythm (54, 55), episodic fluctuation in circulating testosterone levels over shorter periods (minutes to hours) entrained by pulsatile LH secretion (56) and postural influence on hepatic blood flow (49). The major known determinants of testosterone metabolic clearance rate are circulating SHBG concentration (57), diurnal rhythmn (51) and postural effects on hepatic blood flow (49, 51). Major genetic influences on circulating testosterone levels mediated via changes in SHBG (58-61) and other mechanisms (50) have been described as well as environmental (28, 29, 51) factors.

Transport

Testosterone circulates in blood at concentrations greater than its aqueous solubility by binding to circulating plasma proteins. The most important is SHBG, a high affinity but low capacity binding protein (62), and other low affinity binding proteins include albumin, corticosteroid binding globulin (63) and a₁ acid glycoprotein (64). Testosterone binds avidly to circulating SHBG, a homodimer of two glycoprotein subunits each comprising 373 amino acids with 3 glycosylation sites, 2 N-linked and 1 O-linked and containing a single high-affinity steroid binding site (65). The two binding sites in the homodimer display dynamic, co-operative binding affinities upon sequential binding of an androgen (62). The affinity of SHBG for binding testosterone is subject to genetic polymorphisms (66) but is not altered by acquired liver disease (67). It remains unknown as to whether it is influenced by other chronic diseases or pregnancy (when circulating levels increase). SHBG is secreted into the circulation by human, but not rodent, liver as well as into the seminiferous tubules of the testis by rodent, but not human, Sertoli cells where it is known as testicular androgen-binding protein (68), and by the placenta where it may contribute to the rise in blood SHBG during pregnancy (69). As a product of hepatic secretion, circulating SHBG levels are particularly influenced by first-pass effects on the liver of oral drugs including sex steroids. Circulating SHBG (and thereby total testosterone) concentrations are characteri­stically decreased (androgens, glucocorticoids) or increased (estrogens, thyroxine) by supraphysiologic hormone concentrations at the liver such as produced by oral administration (first pass effects) or by high-dose parenteral injections of androgens. In contrast, endogenous sex steroids and parenteral (non-oral) administration, which maintain predominantly physiologic circulating hormone concentrations (transdermal, depot implants), have minimal effects on blood SHBG levels (70, 71) (72). Other modifiers of circulating SHBG levels include up-regulation by acute or chronic liver disease and androgen deficiency and down-regulation by obesity, protein-losing states (65), non-alcoholic fatty liver disease (73, 74) and, rarely, genetic SHBG deficiency(75-77). Under physiologic conditions, 60% to 70% of circulating testosterone is SHBG bound with the remainder bound to lower affinity, high-capacity binding sites (albumin, a₁ acid glycoprotein, corticosteroid binding protein) and 1% to 2% remaining non-protein bound.

Transfer of hydrophobic steroids into tissues is presumed to occur passively according to physicochemical partitioning between the hydrophobic protein binding sites on circulating binding proteins, the hydrophilic aqueous extracellular fluid and the lipophilic cellular plasma membranes. According to the free hormone hypothesis (78-80), recently restated and updated (62), the free (non-protein bound) fraction of testosterone is the most biologically active with the loosely protein-bound testosterone constituting a less accessible but mobilizable fraction, with the largest moiety tightly bound to SHBG constituting only an inactive reservoir. The free hormone hypothesis derived from now outdated 1970’s pharmacological theory on the mechanism of drug-drug interactions as due to mutual protein binding displacement; however, this theory is long superseded in molecular pharmacology by well-established physiological mechanisms such as cytochrome P450 enzyme induction, drug transporter activity and cognate mechanisms unrelated to binding to circulating proteins (81). As the free and/or bioavailable fractions would also have enhanced access to sites of testosterone inactivation by degradative metabolism that terminates androgen action, the free fractions may equally be considered the most evanescent and least active so that the net biological significance of the free or bioavailable fractions remains unclear and undermines a theoretical basis for the free hormone hypothesis. Furthermore empirical evidence indicates that, rather than being biologically inert, SHBG participates actively in cellular testosterone uptake via specific SHBG membrane receptors, uptake mechanisms and signaling via G protein and cyclic AMP (82-86). These mechanisms include the megalin receptor, a multi-valent low-density lipoprotein endocytic receptor located on cell surface membranes that can mediate receptor-mediated cellular uptake of SHBG loaded with testosterone by endocytosis (87, 88) and might influence tissue androgen action (89, 90). Consequently, lacking a physiological basis for the free hormone hypothesis (91) and with empirical evidence in its favor scarce and speculative,  it is refuted by intensive, prospective clinical evaluation (92). Hence, the biological significance of partitioning circulating testosterone into these derived fractions remains to be firmly established and its clinical application is unknown or possibly misleading. Furthermore, direct measurement of free testosterone requires laborious, manual methods only available in research or specialist pathology laboratories. Where available, they are costly and lack any external quality control programs or validated reference ranges. As a result, calculations purporting to replicate dialysis-based measurements are often substituted for direct measurements. These formulae come in two different formats – equilibrium binding equations requiring assumptions on testosterone binding stoichiometry and arbitrary plug-in binding affinity estimates (Sodergard (93), Vermeulen (94), Zakharov (95)) or assumption-free empirical methods (Ly(96, 97), Nanjee-Wheeler (98)) calibrated directly to dialysis-based laboratory measurements. Direct comparison has proven that empirical equations are more accurate compared with laboratory dialysis-based measurements (95, 96, 99, 100). Furthermore, calculations of free testosterone using any formula do not contribute significant to mortality or morbidity prognosis for older men’s health beyond accurate measurement of serum testosterone by liquid chromatography-mass spectrometry (92).

Measurement

Measuring blood testosterone concentration is an important part of the clinical evaluation of androgen status and for confirming a clinical and pathological diagnosis of androgen deficiency. The circulating testosterone concentration is a surrogate measure for whole body testosterone production rate and the inferred impact of androgens on tissues. However, the reliance on a spot measurement of blood testosterone concentration neglects changes in the whole body metabolic clearance rate as well as other factors influencing net androgen effects at tissue levels. These include the efficiency of blood testosterone transfer into adjacent tissues during capillary transit as well as pre-receptor, receptor and post-receptor factors influencing the testosterone activation, inactivation and action in that tissue. Circulating testosterone levels are also dynamic and feature distinct circhoral and diurnal rhythms. Circhoral LH pulsatility entrains some pulsatility in blood testosterone levels (36) although the buffering effects of the circulating steroid-binding proteins dampens the pulsatility of blood testosterone concentrations. This is illustrated by comparison with the strikingly pulsatile patterns of circulating testosterone in rodents which lack hepatic SHBG gene expression thereby having no circulating SHBG to buffer testosterone fluctuations (101, 102). Diurnal patterns of morning peak testosterone levels and nadir levels in the mid-afternoon are evident in younger and healthy older men (54) but lost in some ageing men (55). Consequently, it is conventional practice to standardize testosterone measurements to morning blood samples on at least 2 different days.

The advent of steroid radioimmunoassay in the 1970’s made it feasible to measure blood testosterone concentrations affordably with speed and sensitivity. However, cross-reacting steroids and non-specific matrix effects are limitations on modern direct (non-extraction) testosterone immunoassays relative to the high specificity of mass spectrometry-based methods, the reference method (103). In the next decades, the steep rise in demand for testosterone measurements in clinical practice and research led to method simplications to integrate steroid immunoassays into automated immunoassay platforms. These changes, notably eliminating preparative solvent extraction and chromatography as well as introducing bulky non-authentic tracers, undermine the specificity of unextracted testosterone immunoassays (104), particularly at the low circulating testosterone levels such as in women and children (105). Even at the higher testosterone concentrations in men, commercial testosterone immunoassays demonstrate wide discrepancies due to method-specific bias (32). New generation, bench-top mass spectrometers with higher sensitivity and throughput now overcome these limitations of testosterone immunoassays.

Assays to measure blood “free” testosterone levels directly in serum samples have been developed using tracer reference methods of equilibrium dialysis (106, 107) or ultrafiltration (108, 109) or calculated various formulae based on immunoassay measurement of total testosterone and SHBG (93, 110). Similarly, another derived testosterone measure, bioavailable testosterone, is defined as the non-SHBG bound testosterone (in effect the combination of albumin-bound plus unbound testosterone) and can also be measured directly or calculated by a formula from total testosterone and SHBG and albumin measurements. Some estimates of free testosterone, notably the direct analog assay (111, 112) and the free testosterone index (113) are invalid for use in men. As measurement of “free” or “bioavailable” testosterone is laborious, calculational formulae with limited validation (93, 110, 114) have been widely used; however, these estimates for “free” (115-117) or “bioavailable”(118, 119) testosterone are not accurate in large scale evaluation. Overall, the clinical utility of various derived (“free”, “bioavailable”) measures of testosterone arising from the unproven free hormone hypothesis remain to be established; consequently, they have minimal involvement in consensus clinical guidelines for diagnosis and management of androgen deficiency.

Metabolism

After testicular secretion, a small proportion of testosterone undergoes activation to two bioactive metabolites, estradiol and DHT, whereas the bulk of secreted testosterone undergoes inactivation by hepatic phase I and II metabolism to inactive oxidized and conjugated metabolites for urinary and/or biliary excretion (figure 2) (120).

FIGURE 2. Pathways of Testosterone Action. In men, most (>95%) testosterone is produced under LH stimulation through its specific receptor, a heptahelical G-protein coupled receptor located on the surface membrane of the steroidogenic Leydig cells. The daily production of testosterone (5-7 mg) is disposed along one of four major pathways. The direct pathway of testosterone action is characteristic of skeletal muscle in which testosterone itself binds to and activates the androgen receptor. In such tissues there is little metabolism of testosterone to biologically active metabolites. The amplification pathway is characteristic of the prostate and hair follicle in which testosterone is converted by the type 2 5 reductase enzyme into the more potent androgen, dihydrotestosterone. This pathway produces local tissue-based enhancement of androgen action in specific tissues according to where this pathway is operative. The local amplification mechanism was the basis for the development of prostate-selective inhibitors of androgen action via 5 reductase inhibition, the forerunner being finasteride. The diversification pathway of testosterone action allows testosterone to modulate its biological effects via estrogenic effects that often differ from androgen receptor mediated effects. The diversification pathway, characteristic of bone and brain, involves the conversion of testosterone to estradiol by the enzyme aromatase which then interacts with the ERs  and/or . Finally, the inactivation pathway occurs mainly in the liver with oxidation and conjugation to biologically inactive metabolites that are excreted by the liver into the bile and by the kidney into the urine.

The amplification pathway converts ~4% of circulating testosterone to the more potent, pure androgen, DHT (50, 52). DHT has higher binding affinity to (121) and 3-10 time greater molar potency in transactivation (122-124) of the androgen receptor relative to testosterone. Testosterone is converted to the most potent natural androgen DHT by the 5a-reductase enzyme that ­originates from two distinct genes (I and II) (125). Type 1 5a-­reductase is expressed in the liver, kidney, skin, and brain, whereas type 2 5a-reductase is characteristically expressed strongly in the prostate but also at lower levels in the skin (hair follicles) and liver (125). Congenital 5a-reductase deficiency due to mutation of the type 2 enzyme protein (126) leads to a distinctive form of genital ambiguity causing undermasculinization of genetic males, who may be raised as females, but in whom puberty leads to marked virilization including phallic growth, normal testis development and spermatogenesis (127) and bone density (128) as well as, occasionally, masculine gender reorientation (129). Prostate development remains rudimentary (130) and sparse body hair without balding is characteristic (131). This remarkable natural history reflects the dependence of urogenital sinus derivative tissues on strong expression of 5a-reductase as a local androgen amplification mechanism for their full development. This amplification mechanism for androgen action was exploited in developing azasteroid 5a-reductase inhibitors (132). As the type 2 5a-reductase enzyme results in over 95% of testosterone entering the prostate being converted to the more potent androgen DHT (133), blockade of that isoenzyme (the expression of which is largely restricted to the prostate) confines the inhibition of testosterone action to the prostate (and other urogenital sinus tissue derivatives) without blocking extra-prostatic androgen action. DHT circulates at ~10% of blood testosterone concentrations, due to spillover from the prostate (134, 135) and nonprostatic sources (136). Whereas genetic mutations disrupting type 2 5a-reductase produce disorders of urogenital sinus derived tissues in men and mice (137), genetic inactivation of type 1 5a-reductase has no male phenotype in mice and no mutations of the human type 1 enzyme have been reported. Whether this reflects the type I enzyme having an unexpected phenotype or an evolutionarily conserved vital function, remains unclear. A third 5a-reductase enzyme (type 3, SRD5A3) has been described (138) but is widely expressed in human tissues, lacks steroidal 5a-reductase activity and has other roles in fatty acid metabolism (139).

An important issue is whether eliminating intraprostatic androgen amplification by inhibition of 5a-reductase can prevent prostate disease. Two major randomized, placebo-controlled studies of men at high risk of (but without diagnosed) prostate cancer have both shown that oral 5a reductase inhibitors (finasteride, dutasteride) reduced the incidence of low-grade prostate cancer as well as prevalence of lower urinary tract symptoms from benign prostate hyperplasia(140, 141). The Prostate Cancer Prevention Trial (PCPT) was a major 10-year chemoprevention study randomizing 18,882 men over 55 years of age without known prostate disease to daily treatment with 5 mg finasteride (inhibitor of type 2 5a reductase) or placebo observed a cumulative 25% reduction after 7 years of treatment in early stage, organ-confined, low-grade prostate cancer. Another study randomized over 8231 men aged 50-75 years with serum PSA <10 ng/mL and negative prostate biopsy to either daily treatment with 0.5 mg dutasteride (inhibitor of both type 1 and 2 5a reductases) or placebo for 4 years observed a 23% reduction in incidence of biopsy-proven prostate cancer. Although neither study was designed to determine mortality benefit, both showed no reduction in higher grade, but still organ-confined, cancers. Although this stage selectivity may be explained by diagnostic biases due to drug effects on prostate size and histology (142, 143), registration for chemoprevention of prostate cancer was refused by FDA (144). Overall, the evidence indicates that 5α-reductase inhibition reduces the incidence of early stage, screen-detected and organ-confined prostate cancers but there is insufficient evidence that such treatment reduces mortality from advanced (metastatic) prostate cancer. Whether or not preventive use of prostatic 5a-reductase inhibition in men with high prostate cancer risk proves warranted, novel synthetic androgens refractory to 5a-reductive amplification may have advantages for clinical development.

The diversification pathway of androgen action involves testosterone being converted by the enzyme aromatase to estradiol (145) to activate estrogen receptors (ERs) (also see Endotext, Endocrinology of Male Reproduction, Chapter entitled Estrogens and Male Reproduction). Although this involves only a small proportion (~0.2%) of testosterone output, the higher molar potency of estradiol (~100-fold higher vs testosterone) makes aromatization a potentially important mecha­nism to diversify androgen action via ER-mediated effects in tissues where aromatase is expressed. The diversification pathway is governed by the cytochrome P-450 enzyme (CYP19) aromatase (145, 146). In eugonadal men, most (~80%) circulating estradiol is derived from extratesticular aromatization (53). The biological importance of aromatization in male physiology was first recognized in the early 1970’s (147) when the local conversion of testosterone to estradiol within the neural tissues was identified and subseqeuntly shown to have an important role in mediating testosterone action, including negative feedback as well as activational and organisational effects, on the brain (148). More recently the importance of local aromatization in testosterone action has been reinforced by the striking developmental defects in bone and other tissues of men and mice with genetic inactivation of aromatase, leading to complete estrogen deficiency due to genetic inactivation of the aromatase (149). This phenotype is also strikingly similar to that of a man (150) and mice (150) with genetic mutations inactivating ERa. Furthermore, men with aromatase deficiency treated with exogenous estradioll or other estrogens also demonstrated significant bone maturation. By contrast, genetic inactivation of ERb has no effect on male mice (151) and no human mutations have been reported. Aromatase expression in tissue such as bone (152) and brain (148) may influence development and function by variation in aromatization that modulates local tissue-specific androgen action. By contrast other tissues, like mature liver and muscle, express little or no aromatase. Nevertheless, despite the importance of aromatization for male bone physiology, other observations indicate that androgens acting via androgen receptors have important additional direct effects on bone. These include the greater mass of bone in men despite very low circulating estradiol concentrations compared with young women (153), the failure of androgen insensitive rats lacking functional androgen receptors but normal estradiol and ERs to maintain bone mass of normal males (154)and the ability of nonaromatizable androgens to increase bone mass in estrogen-deficient women (155, 156). Testosterone action on bone and in the brain cannot be accounted for solely as a prohormone for local estradiol production (and action via estrogen receptors a and/or b) and androgen receptor mediated effects are required to manifest the full spectrum of testosterone effects on bone (157, 158) and in the brain (159). Conflicting evidence is available about the need for aromatization to mediate the effects of testosterone on male sexual function. One study using aromatase inhibitor-induced estrogen deficiency showed partial dependence (160) whereas another using DHT-induced estrogen deficiency showing no requirement of male sexual function for aromatization (161). Further studies are needed to fully understand the significance of aromatization in maintaining androgen action in mature male animals (162).

Testosterone is metabolized to inactive metabolites in the liver, kidney, gut, muscle, and adipose tissue. Inactivation is predominantly by hepatic oxidases (phase I metabolism), notably cytochrome P-450 3A family (163) leading ultimately to oxidation of most oxygen moieties followed by hepatic conjugation to glucuronides (phase II metabolism), which are rendered sufficiently hydrophilic for renal excretion. Uridine diphospho (UDP) glucuronosyl transferase (UGT) enzymes UGT2B7, UGT2B15 and UGT2B17 catalyze most phase II metabolism (glucuronidation) of testosterone with 2B17 being quantitatively the most important (164). A functional polymorphism of UGT 2B17, a deletion mutation several times more frequent in Asian than European populations (165), explains the concordant population difference in testosterone to epitestosterone (T/E) ratio (165), a World AntiDoping Agency-approved urine screening test for testosterone doping in sport, which constitutes an ethnic differential, false negative in surveillance for exogenous testosterone doping (166).

The metabolic clearance rate of testosterone is reduced by increases in circulating SHBG levels (57) or decreases in hepatic blood flow (e.g. posture) (49) or liver function. Theoretically, drugs that influence hepatic oxidase activity could alter metabolic inactivation of testosterone, but empirical examples of sufficient magnitude to influence clinical practice are rare. Rapid hepatic metabolic inactivation of testosterone leads to both low oral bioavailability (167, 168) and short duration of action when injected parenterally (169). To achieve sustained androgen replacement, these limitations dictate the need to deliver testosterone via parenteral depot products (e.g., injectable testosterone esters, testosterone implants, transdermal testosterone) or oral delivery systems that either bypass hepatic portal absorption (buccal (170, 171), ­sublingual (170, 172), gut lymphatic (173)) or use synthetic androgens with substituents rendering them resistant to first pass hepatic inactivation (174).

Regulation

During sexual differentiation in early intrauterine life, the testosterone required for masculine sexual differentiation is secreted by fetal Leydig cells. The regulation of this fetal Leydig cell testosterone secretion appears to differ between species. Higher primate and equine placenta secrete a chorionic gonadotropin during early fetal life (175) that may drive fetal human Leydig cell steroidogenesis (176) at the relevant time. By contrast, in subprimate mammals male sexual differentiation occurs without expression of any placental gonadotropin and prior to the time when pituitary gonadotropin secretion starts so that fetal Leydig cell testosterone secretion may be autonomous of gonadotropin stimulation during fetal development of most mammalian species (177).

Puberty is a complex series of maturational events originating during fetal life but completing only during adolescence. Recent genetic studies have enlightened the understanding of the physiology of puberty and its timing without yet identifying the ultimate trigger that initiates puberty. All components of the hypothalamo-pituitary testicular (HPT) axis are established during fetal life and its first activation is during the neonatal period manifested as a transient surge lasting several months in circulating testosterone (“mini-puberty”) reaching adult male levels and creating androgen imprinting in non-reproductive tissues. Following the neonatal surge, the HPT axis activity becomes quiescent during a decade of childhood under the restraint of mechanisms revealed by recent genetic studies to involve activities of at least four genes. These studies have identified premature (precocious) onset of puberty arising from inactivating mutations of makorin ring finger protein 3 (MKRN3) (178) and delta-like homolog 1 (DLK1) (179) as well as activating mutations of kisspeptin (KISS1) and its receptor (KISSR1) (180) indicating that these genes are involved in the mechanism of the childhood restraint of puberty. Alleviation of this restraint mechanism at the end of childhood unleashes the hormonal cascade of puberty. Although the ultimate trigger remains elusive, its activation initiates the timely onset of mature patterns of pulsatile GnRH secretion. This involves a complex and still poorly understood integration of a cascade of over 50 genes/proteins (180, 181). It is known that monogenic mutations in GnRH receptor, kisspeptin (KISS1 and its receptor), neurokinin B (TAC3 and its receptor), FGF8 or FGF17 and their FGFR1 receptor, prokineticin-2 (PROK) and its receptor (PROKR2) as well as compound heterozygotes and oligogenic inheritance of combinations of known mutated alleles disrupt the normal timing of puberty. Variants include reversible forms of gonadotropin deficiency, and this growing spectrum of underlying genetic susceptibilities probably overlaps and accounts for the variability in timing of puberty onset in the community.

Pulsatile hypothalamic GnRH secretion is the final common pathway driving pituitary gonadotrophin secretion that leads to testicular growth and maturation resulting in completion of spermatogenesis and steroidogenesis to produce adult male circulating testosterone concentrations. This results in testicular growth, the earliest and most salient external manifestation of male puberty, being initiated between 9 and 14 years of age with earlier onset defined as precocious and later onset as delayed puberty. The timing of puberty is under strong but complex genetic control with strong patterns detected in familial, twin and community studies (182-187). Disorders in this complex mechanism are relatively common with delayed puberty evident in 2% of adolescents although precocious puberty is rare with a frequency between 1:5000-10,000 involving only 10-20% in males (181, 188).

The still mysterious suprahypothalamic process initiating puberty involves a developmental clock and multiple permissive processes (189, 190) that lift the central neuroendocrine restraint on the final common pathway that drives reproductive function in the mature male – the episodic secretion of gonadotropin-releasing hormone (GnRH) from hypothalamic neurons (56). Various explanatory theories including the gonadostat, somatometer (191), neurally-driven changes in GABAergic inhibition and glutaminergic stimulation (192), triggering by kisspeptin-1 secretion and activating its receptor GPR54 (193, 194) and epigenetic factors (195) are proposed to explain the restraint and resurgence of the hypothalamic GnRH pulse generator without a comprehensive picture having yet emerged (190). Hypothalamic GnRH neurons are functional at birth but, after the perinatal androgen surge, remain tonically suppressed during infantile life. Puberty is initiated by a maturation process that awakens the dormant hypothalamic GnRH neurons to unleash mature cirhoral patterns of pulsatile GnRH secretion which in turn entrains pulsatile LH secretion from pituitary gonadotropes. Initially this resurgence of pulsatile GnRH and LH secretion occurs mainly during sleep (196) but eventually extends throughout the day with a persisting association with an underlying diurnal rhythmn. The timing and tempo of male puberty is under tight genetic control, encompassing nutrition influences on body weight and composition (185), with a correspondingly growing number of genetic causes of delayed puberty identified (197). Environmental factors that optimize growth (eg high socio-economic status with better nutrition and health care) may explain secular trends to earlier puberty with increased statural growth (198, 199) whereas claims that exposure to hormonally active chemical pollution contributes to earlier puberty (200) remain speculative (201) and not supported by available evidence in boys (202, 203). Very large UK population-based studies, using voice breaking as a self-reported marker of male puberty, show that both early and late male puberty are associated with a wide-range of adverse health outcomes (204, 205).

After birth, testicular testosterone output is primarily regulated by the pulsatile pattern of pituitary LH secretion. This is driven by the episodic secretion of GnRH from hypothalamic neurons into the pitu­itary portal bloodstream providing a direct short circuit route to pituitary gonadotropes. Under this regular by intermittent GnRH stimulation, pituitary gonadotrophs secrete LH in high amplitude pulses at ~60-90 min intervals with minimal intervening LH secretion between pulses with the net effect that circulating LH levels are distinctly pulsatile. This pulsatile pattern of trophic hormone exposure maintains Leydig cell sensitivity to LH to maintain mature male patterns of testicular testosterone secretion (206).

LH stimulates Leydig cell steroidogenesis via increasing substrate (cholesterol) availability and activating rate-limiting steroidogenic enzyme and cholesterol transport proteins (8, 18). LH is a dimeric glycoprotein consisting of an asubunit common to the other glycoprotein hormones (human chorionic gonadotropin (hCG), follicle-stimulating hormone, and thyrotropin-stimulating hormone) and a b subunit providing distinctive biologic specificity for each dimeric glycoprotein hormone by dictating its specific binding to the LH/hCG rather than the folliclle-stimulating hormone or thyroid stimulating hormone receptors (207, 208). These cell surface ­receptors are highly homologous members of the heptahelical, G protein–linked family of membrane receptors. LH receptors are located on Leydig cell surface membranes and use signal transduction mechanisms involving primarily cyclic adenosine monophosphate as well as calcium as second messengers to cause protein kinase–­dependent protein phosphorylation and DNA transcription, ultimately resulting in testosterone secretion (209). Functionally, hCG is a natural, long-acting analogue of LH because they both bind to the same LH/CG receptor and their b subunits are nearly identical except that hCG has a C-terminal extension of 31 amino acids containing four O-linked, sialic acid–capped carbohydrate side chains. These glycosylation differences confer greater resistance to degradation, which prolongs circulating residence time and biologic activity compared with LH (210, 211), a feature that has been exploited to engineer longer acting analog of other circulating hormones such as FSH (212), TSH (213) and erythropoietin (214).

Additional fine tuning of Leydig cell testosterone secretion is provided by paracrine factors originating within the testis (215). These include cytokines, inhibin, activin, follistatin, prostaglandins E₂ and F_2a, insulin-like and other growth factors as well as still uncharacterized factors secreted by Sertoli cells. LH also influences testicular vascular physiology by stimulating Leydig cell secretion of vasoactive and vascular growth factors (216).

Testosterone is a key element in the negative testicular feedback cycle through its inhibition of hypothalamic GnRH and, consequently, pituitary gonadotropin secretion. Such negative feedback involves both testosterone effects via androgen receptors as well as aromatization to estradiol within the hypothalamus (217, 218). These culminate in reduction of GnRH pulse frequency in the hypothalamus together with reductions in amplitude of LH pulses due to both reduced GnRH quantal secretion as well as gonadotropin response to GnRH stimulation (206). By contrast, the small proportion of estradiol in the bloodstream that is directly secreted from the testes (~20%) means that cirulating estradiol is under minimal physiological regulation and unlikely to be a major influence on negative feedback regulation of physiological gonadotropin secretion in men.

Action

Androgen action involves pre-receptor, receptor and post-receptor mechanisms that are centered on the binding of testosterone (or an analog) to the androgen receptor. Testosterone undergoes pre-receptor activation by conversion to potent bioactive metabolites, DHT and estradiol. The steroidogenic enzyme 5a-reductase has two isozymes, types 1 and 2, which form a local androgen amplification mechanism converting testosterone to the most potent natural androgen, DHT (219). The two isozymes have different chromosomal location and distinct biochemical features but are homologous genes (125). They are structurally and functionally unrelated to a third 5a-reductase (SRD5A3) which may have physiological role in fatty acid rather than steroidal biochemistry (138, 139). This local androgen amplification mechanism is exemplified in urogenital sinus derived tissues, notably external and internal genitalia and the prostate, which characteristically express high levels of 5a-reductase type 2 (125). Other tissues such as nongenital skin and liver express 5a-reductase type 1.

The other form of pre-receptor androgen activation is conversion of testosterone to estradiol by the enzyme aromatase (220) which diversifies androgen action by facilitating effects mediated via ERs (221). Consequently, while DHT may be considered a pure androgen because its bioactivity is solely mediated via AR, testosterone has a wider spectrum of action which includes diversification by aromatization and ER mediated effects. These pre-receptor mechanisms provide testosterone with a versatile and subtle range of regulatory mechanisms prior to receptor mediated effects, depending on the balance between direct AR mediated vs indirect actiational and/or ER mediated mechanisms. In addition, tissues vary in their androgenic thresholds and dose-response characteristics to testosterone and its bioactive metabolites.

The role of aromatization in androgen action was originally identified by the 1970s in the brain (222) whereby local expression of the aromatase enzyme within brain regions leads to local production of estradiol to mediate testosterone effects selectively in that region via ER and not AR mechanisms. Subsequently, the importance of aromatization to androgen action on bone was identified through investigations of inactivating mutations in ERα in men (223) and mice (150, 224). The role of aromatization in the estrogen-mediated effects of testosterone action is clearly shown by studies of Finkelstein et al who use the paradigm of complete suppression of endogenous testosterone production by administration of a depot GnRH analog with a range of doses of add-back testosterone without and with an aromatase inhibitor (anastrozole), the latter to investigate the effects of selective estrogen deficiency (225). These studies showed that aromatization was important in mediating testosterone effects in reducing fat mass and sexual function but not on muscle mass or strength. However,in another study using a different design, a high dosage of the non-aromatizable androgen dihydrotestosterone (vs. placebo) to induce selective complete estrogen deficiency in healthy men, demonstrated complete preservation of sexual function (161). Testosterone effects on bone involve dual mediation via indirect mechanisms, via aromatization to estradiol and ER-mediated effects, as well as via direct AR-mediated effects (226). In male mice, aromatization of testosterone must occur locally within bone as circulating estradiol levels are too low to activate ERs ; however, the role of local vs circulating estradiol effects on male bone remain to be clarified. A much wider role of estrogen action in male health is now identified (227). In that light the off-label use of aromatase inhibitors carries the risk of adverse effects on brain (manifest as sexual dysfunction), fat, and bone.

ANDROGEN RECEPTOR

The androgen receptor is required for masculine sexual differentiation and sexual maturation that ultimately leads to development of a mature testis capable of supporting spermatogenesis and testosterone production that form the basis for male fertility. The human androgen receptor is specified by a single X chromosome encoded gene located at Xq11-12 that specifies a protein of 919 amino acids (1), a classical member of the large nuclear receptor superfamily (228) which includes receptors for the 5 mammalian steroid classes (androgen, estrogen, progesterone, glucocorticoid, mineralocorticoid) as well as for thyroid hormones, retinoic acid and vitamin D and numerous orphan receptors where the ligand was originally not identified (229). Androgen receptor expression is not confined to reproductive tissues and it is ubiquitously expressed, athough levels of expression and androgen sensitivity of non-reproductive tissues vary.

The androgen receptor gene has 8 exons specifying a protein of 919 amino acids with the characteristic structure of mammalian steroid receptors. It has an N-terminal domain (NTD) that specifies a long transactivating functional domain (exon 1), a middle region specifying a DNA-binding domain (DBD) consisting of two zinc fingers (exons 2 and 3) separated by a hinge region from the C-terminal ligand binding domain (LBD) which specifies the steroid binding pocket (exons 4 to 8).

The NTD (exon 1) is relatively long comprising over half (535/919) the overall length of the AR. It has the least conserved sequence compared with other steroid receptors with a flexible and mobile tertiary structure harbouring a transactivation domain (AF-1) that interacts with AR co-regulator proteins and target genes (230). Its loose, naturally disordered structure (231) also contains three homopolymeric repeat sequences (glutamine, glycine, proline) with the most important being the CAG triplet (glutamine) repeat polymorphism (232). The less variable glycine (usually 24 residues) and proline (9 residues) repeat polymorphisms have little apparent independent pathophysiological significance although linkage dysequilibrium between the glutamine and glycine repeat polymorphisms requires haplotype analysis for interpretation (232). Among healthy people, where the glutamine repeat polymorphism has alleles of lengths between 5 and 35 (population mean ~21), the length of the glutamine repeat is inversely proportional to AR transcriptional efficiency so that this polymorphism dictates genetic differences between individuals in the androgen sensitivity of their target tissues (233). This genetic variation in in vivo tissue androgen sensitivity is proven experimentally (234) but, although modest in magnitude, influences physiological responses to endogenous testosterone in prostate size (235) and erythropoeisis (236) in carefully controlled studies. Wider epidemiological implications of population variation in the genetic androgen sensitivity as specified by the polyglutamine repeat have been studied in a variety of potentially androgen sensitive disorders (reviewed in (232)) including reproductive health disorders and hormone dependent cancers in men and women as well as non-gonadal disorders where there are significant gender disparities in prevalence. In men these include prostate (237) and other male preponderant cancers (liver, gastrointestinal, head & neck), prostate hypertrophy, cryptorchidism and hypospadias, male infertility (238)whereas in women they include reproductive health disorders (polycystic ovary syndrome, premature ovarian failure, endometriosis, uterine leiomyoma, preeclampsia) and hormone dependent cancers (breast, ovary, uterus). In addition, studies have also examined risks of obesity and cardiovascular disease, mental and behavioural disorders including dementia, psychosis, migraine, and personality disorders (232). However, as in many large-scale genetic association studies (239), the findings remain mostly inconsistent reflecting methdological limitations notably in recruitment, participation and publication bias as well as multiple hypothesis testing all of which tend to inflate spurious associations.

Remarkably, the pathological elongation of the polygutamine (CAG triplet) repeat to lengths of over 37 cause a neurodegenerative disease, Spinal Bulbar Muscular Atrophy (SBMA, also known as Kennedy’s syndrome). This is a form of late-onset, slow progressing but ultimately fatal motor neuron disease (240), one of several late-onset neurodegenerative polyglutamine repeat disorders (241). Although the extreme length of the polyglutamine repeat does determine mild androgen resistance, these men usually have normal reproductive function including fertility and virilization prior to diagnosis in mid-life (242). Furthermore, since complete androgen receptor inactivation in humans and other mammals does not cause motor neuron disease and female carriers are protected from symptomatic neurodegeneration, SBMA represents a toxic gain-of-function involving pathological protein aggregates of the mutant AR (243) like other genetic polyglutamine repeat neurodegenerative diseases do with other proteins (244). Surprisingly, transgenic mouse models of SBMA suggest that testosterone deprivation by medical castration using a GnRH agonist may slow progression of neuropathy (243) and that genetic (245) or pharmacological (246)administration of IGF-I may slow disease progression. However the first major clinical trial of leuprolide, a GnRH analog, failed to demonstrate neuromuscular benefit in swallowing (247) and further studies of selected subgroups and therapeutic targets are warranted (248, 249).

The DBD (exons 2 and 3) consists of ~70 amino acids with a high proportion of basic amino acids including eight cysteines distributed as two sets of 4 cysteineseach forming a zinc cooordination center for a single zinc atom, thereby creating two zinc fingers. The DBD is highly conserved between steroid receptors reflecting its tightly defined function of forming the two zinc fingers that bind to DNA by intercalating between its grooves. The first zinc finger (exon 2) is directly involved with the major DNA groove of the androgen response element through a proximal (P-box) region whereas the second zinc finger (exon 3) is also responsible for enhancing receptor dimerization through its distal (D-box) region.

The hinge region (first half of exon 4) of ~40 amino acids between the DBD and LBD is considered a flexible linker region but may have additional functions involving interactions with DNA (nuclear localization, androgen response element) and protein (AR dimerization, co-regulators) which influence AR transcriptional activity.

The LBD (mid-exon 4 to 8) of AR comprises ~250 amino acids which specify a steroid binding pocket which creates the characteristic high affinity, stable and selective binding of testosterone, DHT and synthetic androgens. While the LBD’s overall architecture is broadly conserved among nuclear receptors, the AR sequence diverges significantly to ensure the specificity of binding from other steroid classes and their different cognate ligands. Structural studies of the AR’s LBD shows it has similar tertiary conformation as other steroid receptors (most closely resembling PR) with 12 stretches of a helix interspersed with short b pleated sheets. The most C terminal helix 12 seals the binding pocket and influences whether a bound ligand acts as an agonist or antagonist as well as forming a hydrophobic surface for binding of co-regulator proteins that modify transcriptional activity of the androgen target genes. The LBD also participates in receptor dimerization, nuclear localization and transactivation via its activation function (AF-2) domain.

The AR has a predominantly nuclear location in androgen target cells regardless of whether bound to its ligand or not, unlike other steroid receptors which are more often evenly distributed between cytoplasm and nucleus when not bound to their cognate ligands. Androgen binding to the C-terminal LBD causes a conformational change in the androgen receptor protein and dimerization to facilitate binding of the ligand-loaded receptor to segments of DNA featuring a characteristic palindromic motif known as an androgen-response element, located in the promoter regions of androgen target genes. Ligand binding leads to shedding of heat shock proteins 70 and 90 that act as a molecular chaperone for the unliganded androgen receptor (250). Specific binding of the dimerized, ligand-bound androgen receptor complex to tandem androgen-response elements initiates gene transcription so that the androgen receptor acts as a ligand-activated transcription factor. Androgen receptor transcriptional activation is governed by a large number of coregulators (251, 252) whose tissue distribution and modulation of androgen action remain incompletely understood.

Androgen Insensitivity

Mutations in the androgen receptor are relatively common with over 1000 different mutations recorded by 2012 (253)in the McGill database (http://androgendb.mcgill.ca/) making androgen insensitivity the most frequent form of genetic hormone resistance. As the androgen receptor is an X chromosomal gene, functionally significant AR mutations are effectively expressed in all affected males because they are hemizygous. By contrast, women bearing these mutations (including the obligate heterzygote mothers of affected males) are silent carriers without any overt phenotype because they have a balancing allele as well as their circulating testosterone levels never rise to post-pubertal male levels sufficient to activate AR mediated effects.

Germline AR mutations produce a very wide spectrum of effects from functionally silent polymorphisms to androgen insensitivity syndromes that display phenotypes proportionate to the impairment of AR function and, thereby, the degree of deficit in androgen action (1). These clinical manifestations extend from a complete androgen insensitivity syndrome (CAIS, formerly known as testicular feminization) which produces a well developed female external phenotype in a spectrum spanning across all grades of undervirilized male phenotype to, at the other extreme, a virtually normal male phenotype. The severity of androgen insensitivity can be categorized most simply as complete, partial and mild although a more detailed 7 stage Quigley classification based on degree of hypospadias, phallic development, labioscrotal fusion and public/axillary hair is also described (1, 232). The degree of urogenital sinus derivative development together with testis descent provide clinical clues to the degree of androgen sensitivity. In addition, somatic androgen receptor mutations, notably generated during androgen deprivation treatment of prostate cancer (254), result in generation and expression of mutations and splice variants of the androgen receptor in a form of accelerated molecular evolution which may result in resistance to androgen effects and/or efficacy of androgen deprivation treatment (255).

CAIS due to completely inactivating AR mutations results in a 46XY individual with a hormonally active testis that secretes abundant testosterone but which cannot activate AR-mediated action so no male internal or external genitalia or somatic features develop. However, testosterone aromatization to estradiol is unimpeded, leading to the development of normal female somatic features including breast and external genital development after puberty. The population prevalence of CAIS is estimated to be at least 1:20,000 male births or 1-2% among female infants with inguinal hernia (1). The typical presentation of CAIS is a relatively tall, normally developed girl with delayed puberty and/or primary amenorrhea. The clinical features usually include well developed breasts, hips and female fat pattern deposition, acne-free facial complexion with minimal axillary and pubic hair with testes located within an inguinal hernia or in the abdominal cavity. The uterus and fallopian tubes are absent and the vagina is short and blind ending reflecting unimpeded effects of testicular AMH secretion causing regression of Mullerian structures including the upper third of the vagina. Earlier diagnosis is increasingly possible where a prenatal 46XY karyotype is discrepant from a female phenotype on ultrasound or at birth or among female infants presenting with inguinal hernia (256). The family history may be informative with infertile maternal (but not paternal) aunts consistent with an X-linked inheritance. Laboratory investigations of post-pubertal individuals show elevated blood LH, SHBG (at adult female levels) and testosterone (at adult male levels) prior to gonadectomy. The androgen sensitivity index, the product of LH and testosterone concentrations, is elevated (257). These features reflect high amplitude and frequency LH pulses due to the absence of effective negative androgenic feedback on the hypothalamus as well as the increased LH drive to maintain high-normal male levels of testicular testosterone secretion. In untreated individuals, failure to suppress blood SHBG with short-term, high dose androgen administration may be useful confirmation of androgen resistance (258, 259). After gonadectomy, blood LH and FSH increase to castrate levels but are partially suppressed by estradiol replacement therapy.

Long-term management includes (a) reinforcing female gender identity with counseling to cope with eventual infertility and acceptance of the genetic diagnosis, (b) post-pubertal gonadectomy to prevent the risk of gonadoblastoma (especially if the gonad is impalpable) but allowing the completion of puberty balanced against the low risks of tumour at that age and of unwanted virilization due to any residual AR function or mosaicism (260), and (c) post-gonadectomy estrogen replacement therapy to maintain bone density, breast development and quality of life. Long-term bone density is often subnormal for age due not only to the deficit in androgen action but also inadequate post-gonadectomy estrogen replacement, often resulting from suboptimal adherence to medication (128, 261-263). Although the long-term outcomes for AR mutations based on large prospective studies of a consistent management approach remain very limited, the clinical outcomes for individuals with CAIS reared as females are reported as successful (264, 265) although some gender role and psychosexual functional outcomes remain suboptimal (266-268).

Partial androgen insensitivity syndrome (PAIS) is characterized by a full range of external genital virilization and breast development from female to male phenotype, reflecting the functional severity of the AR mutation. A simple clinical guide to the severity of the deficit in AR function is provided by the level of testis descent and phallic development. PAIS was originally recognised under a variety of eponymously named syndromes (Reifenstein, Gilbert-Dreyfus, Lubs, Rosewater) and only more recently clearly distinguished from other developmental disorders of 46XY individuals with incomplete virilization especially those due to steroidogenic enzyme defects. Severe forms of PAIS with minimal AR function produce a predominantly female phenotype with clitoromegaly whereas PAIS with mutations displaying more functional AR are characterized by a male phenotype with various grades of labioscrotal formation (varying from minimal posterior partial labial fusion to labioscrotal fusion and bifid, ruggose scrotum) and hypospadias (urinary orofice ranging from perineal aperture to hypospadia with meatus at locations along penile shaft to the corona), micropenis and gynecomastia, each in inverse proportion to the AR function. These features have been combined into a External Masculinization Score (EMS) ranging from 0 (female) to 12 (male) based on degree of scrotal fusion, phallic development, location of urethral meatus and testis descent each scored 0-3 (269). The biochemical finding in PAIS are similar to those of CAIS but with a wide spectrum of severity from mildly virilized, predominantly female to an undervirilized male phenotype. The increase in blood LH and testosterone are less severe and consistent but the androgen sensitivity index (257) may help confirm the diagnosis of androgen resistance. Unlike CAIS, which usually presents during adolescence with failure of puberty, PAIS usually presents at birth with ambiguous genitalia requiring a crucial and decisive clinical judgement on sex of rearing to be made rapidly. The expert pediatric endocrinologist must balance the need for early genital surgery and vicarious decision-making against the risk of possible subsequent regret by the affected individual as an adult. This makes for inevitably complex, difficult and contentious choices as the available systematic prospective evidence from long-term follow-up of sex or rearing is still limited. Most intersex individuals due to PAIS, especially those with an EMS of 4 or more (270), are raised as males (269). Genital surgery for hypospadias is often required and usually uncertainty remains about the adequacy of the potential for post-pubertal virilization due to either endogenous or exogenous testosterone. If pubertal progression is inadequate, exogenous testosterone may be useful but higher than usual dosage may be required to get satisfactory effects. Long-term follow-up of PAIS raised as males has shown apparently adequate psychosexual function despite phallic underdevelopment, limited somatic virilization and dissatisfaction with outcomes by some patients as adults (268, 271). For those to be reared as females, the management is similar to that for CAIS and involves early genital surgery and pre-pubertal gonadectomy to prevent unwanted virilization.

Mild androgen insensitivity (MAIS) is the most minor form of androgen insensitivity dsplaying near-normal male phenotype with only subtle changes in hair patterns relative to family norms (less body and facial hair, absence of temporal recession or balding) and/or minor defects restricted to spermatogenesis alone. The blood LH and testosterone concentrations are usually but not always elevated although the androgen sensitivity index, the product of serum LH and testosterone concentrations, is more consistently raised. In common with mutations in many other genes, making a clear distinction between the most minor grades of clinical pathology and a silent, functionally insignificant polymorphism is challenging and depends on reproducing experimentally the functional consequences of the mutation in an authentic biological system. Ideally such verification is performed in vivo (eg in genetically modified mouse models) but, as this is laborious and expensive, it is rarely undertaken. The functional verification of putative mutations is usually undertaken by either in silico prediction of functional effects of structural protein changes from sequence data or in vitro studies of cultured cells or cell-free systems aiming to characterize protein functions. Nevertheless, although informative, the biological fidelity of these surrogate endpoints relative to the in vivo effects on androgen action may remain questionable.

All types of mutations have been reported in the AR gene including disruption of the reading frame by deletions, insertions, splice site interruption and frame-shift which usually produce major interference with function as well as the more common single base substitutions with effects ranging from nil to complete functional inactivation. In addition, mutation can produce less common mechanisms of interrupting AR function such as inefficient translation, unstable protein, or aberrant translational start sites all leading to reduced expression of functional AR protein. Mutations occur throughout the AR gene, probably at random; however, those reported are distributed unevenly because the most important functional regions of the gene are sensitive to even minor changes in sequence whereas the more variable regions may tolerate sequence changes without functional consequences. Over 90% of known mutations are single base substitutions which have pathophysiological consequences when they change the amino acid sequence in the functionally critical DBD or LBD regions whereas sequence changes in other regions may not alter AR function thereby constituting silent polymorphisms. For example, despite forming more than half the AR sequence, few functionally important mutations are reported in the NTD (exon1). Those described in exon 1 mostly represent major disruptions of the AR protein due to creation of a premature stop codon, a major deletion or frame shift mutation causing mistranslation onward from exon 1 whereas point mutations are more likely to constitute functionally insignificant (silent) polymorphisms. Mutations in the LBD, comprising ~25% of AR sequence, constitute the majority (~60%) of reported mutations whereas mutations in the DBD, representing ~7% of AR sequence, constitute ~14% of cases (272). The functional effects of these two types of mutations generally differ in that LBD mutations demonstrate various degrees of reduced affinity and/or loosened specificity of ligand binding characteristics whereas DBD mutations demonstrate normal ligand binding but reduced or absent receptor binding to DNA. The profusion of AR mutations has created numerous experiments of Nature with multiple different mutations involving the same amino acid with the physiological consequences depending generally on how conservative is the amino acid substitution. Nevertheless, there are exceptions to such categorization with mutations in regions other than the DBD or LBD sometimes unexpectedly affecting DNA or ligand binding properties presumably through physical interaction effects in the tertiary structure of the AR in its 3 dimensional topography.

The familial occurrence of androgen insensitivity due to X-linked inheritance of mutated AR makes carrier detection and prenatal genetic diagnosis feasible. A carrier female has a 50% chance of having a child bearing the mutant AR allele so they would be either a carrier female or an affected male and 50% of her fertile daughters will also be carriers. A specific mutation detection test needs to be established usually involving PCR-based genotyping for point mutations athough other mutational mechanisms may require more complex genotyping methods. For prenatal genetic diagnosis now usually applied to chorionic villus samples, the genetic diagnosis must be rapid, reliable and efficient. However, accurate genetic counselling relies on the a consistent and predictable phenotype for any specific genotype. This is usually, but not invariably, true for AR mutations as the clinical manifestations for the same mutation are usually consistent in CAIS with rare exceptions (273) whereas for PAIS the phenotype may vary even within a single family with significant implications for sex of rearing and/or need for genital surgery so that skilled genetic counselling is essential (274). Discrepancies in the fidelity of phenotype within families, or between unrelated individual bearing the identical mutation, is relatively common in PAIS and may be attributable to somatic mosaicism (275) or the effect of modifier genes that influence androgen action such as 5a reductase (276). An exotic, complex DNA breakage repair slippage mechanism has also been described to produce mutiple mutations within a single family (277). Wider population genetic screening for AR mutations is not currently cost-effective because, despite diminishing costs for increasingly facile genetic testing, the large number of different mutations featuring diverse mechanisms and variable phenotype which still mostly predict a normal life expectancy but a diminished quality of life that is difficult to cost or cure (278).

Acquired androgen insensitivity during life can arise either through postnatal somatic or germline AR mutations or by non-genetic, non-receptor mechanisms that hinder androgen action. Among overt cases of androgen insensitivity, ~30% are absent in the mother’s germline so must arise as a de novo mutation in the postnatal maternal germline (275) or in the fetal germline soon after fertilization (279). Somatic AR mutations, arising de novo postnatally in the stem cell pool of repopulating cells, are theoretically possible but have not been reported. Somatic AR mutations are relatively common in prostate cancer usually arising in late stage disease palliatively treated by androgen deprivation when AR mutations and functional splice variants are reported (255). The switch of highly androgen dependent prostate cancer cells to an androgen deplete milieu may encourage clonal selection of androgen insensitive sublines to proliferate in the terminal stage of the disease. Genetic instability of prostate cancer cells may also contribute to this process although somatic AR mutations are rare in other cancers such as liver (280) or breast (281) cancer in the absence of androgen deprivation. Somatic AR mutation in prostate cancer cells are responsible for the paradoxical anti-androgen withdrawal syndromes observed with non-steroidal (flutamide, bicalutamide, nilutamide) or steroidal (cyproterone, megestrol) (282, 283) treatment. In this state, anti-androgen withdrawal or switch-over (283) produces remission of worsening disease attributable to the occurrence of a de novo AR mutation in prostate cancer cells which alters ligand specificity turning the non-steroidal antiandrogens into AR agonists (255, 284). The LNCaP prostate cell line widely used in cancer cell biology research harbors a mutated AR (T877A) which occurs relatively frequently in prostate cancer metastases and can cause the flutamide withdrawal syndrome (285). Since the Nobel prize-winning discovery in the 1940’s of androgen deprivation as palliative treament of advanced prostate cancer (286), targeting of AR in the treatment of prostate cancer has focused on surgical or medical castration to eliminate AR’s cognate endogenous ligand, testosterone. After transient remission following castration, however, prostate cancers resume growth in the apparently androgen independent terminal, treatment resistant stage of the disease. Although castration eliminates the major (>95%) contribution to overall androgen synthesis, ongoing production of androgens from other tissues expressing steroidogenic enzymes, such as the adrenal (287) and prostate tumors (288), has been proposed to explain the late development of apparent androgen independence. Extensive clinical trials of maximum androgen blockade which aims to more thoroughly ablate androgen action by adding anti-androgens to castration, however, have produced only minimal improvement in survival (289), possibly due to antiandrogens countering the deleterious initial “flare” effect of superactive GnRH analogs used for medical castration. A more effective approach has been the development of abiraterone, a rationally designed, mechanism-based inhibitor of CYP17A1 (17-hydroxylase/17,20 lyase) incorporating a 16-17 double bond to inhibit 17-hydroxylation. Abiraterone has proven effective and well-tolerated in treatment of late stage, apparently androgen independent prostate cancer (290) although the blockade of glucocorticoid and mineralocorticoid synthesis requires adrenal replacement therapy. In addition, newer androgen receptor blockers also provided promising new therapeutic approaches especially for castration-resistent advanced prostate cancer (291).

Acquired androgen insensitivity may occur without AR mutations by mechanisms such as drugs including non-steroidal (flutamide, bicalutamide, nilutamide) and steroidal (cyproterone acetate), drugs that block part of testosterone activation such as 5a reductase inhibitors (finasteride, dutasteride) or estrogen antagonists or aromatase inhibitors. In addition, drugs may have physiological effects or pharmacological actions that oppose various steps in androgen action such as LH and FSH suppression by estrogens or progestins or that cause an increase in circulating SHBG which may influence testosterone transfer from blood into tissues to produce a functional phenocopy of androgen insensitivity.

Acquired androgen insensitivity in various disease states is reported with hormonal findings reflecting impeded androgen action which may be reversible with alleviation of the underlying disease. The disease-related mechanisms that impede androgen action vary but the most frequent is increase in hepatic SHBG secretion due to the underlying disease and/or its drug treatments that impede androgen action by reducing testosterone transport from blood to tissues as part of its overall reduction in metabolic clearance rate of testosterone. For example, in hyperthyroidism, increased blood LH and testosterone concentrations with clinical features of androgen deficiency (292) are mediated by increased circulating SHBG due to thyroid hormone-induced hepatic SHBG secretion (293) whereas in hypothyroidism the reduced blood testosterone and SHBG are rapidly corrected by thyroid hormone repacement therapy (292). In epilepsy, anticonvulsant-induced increase in hepatic SHBG secretion appears to be a common denominator in the near ubiquitous reproductive endocrine abnormalities in men with epilepsy (294). The relative contributions of impaired tissue transfer of testosterone, reduced testosterone metabolic clearance rate (295) or direct anti-androgenic effects of valproate (296) remain to be clarified. A similar mechanism of disease- and/or drug-induced increases in hepatic SHBG secretion may explain apparent acquired androgen insensitivity, often reversible with alleviation of the underlying disease, in various other conditions such as gluten enteropathy (297, 298), Wilson’s disease (299), relapsed acute intermittent porphyria (300), acute alcoholism (301), chronic liver disease and transplantation (67, 302).

PHARMACOLOGY OF ANDROGENS

Indications for Androgen Therapy

Androgen therapy can be classified as physiologic replacement or pharmacologic therapy according to the dose, type of androgen, and objectives of treatment. Androgen replacement therapy aims to restore tissue androgen exposure in androgen-deficient men due to pathological hypogonadism (disorders of the reproductive system) to levels comparable with those of eugonadal men. Using the natural androgen testosterone and a dose limited to one that maintains blood testosterone levels within the eugonadal range, androgen replacement therapy aims to restore the full spectrum of androgen effects while replicating the efficacy and safety experience of eugonadal men of similar age. Androgen replacement therapy is unlikely to prolong life because androgen deficiency, whether due to castration (303-307) or biological disorder (308) has minimal effect in shortening life expectancy (309). As an alternative, pharmacologic androgen therapy uses androgens without restriction on androgen type or dose but aims to produce androgen effects on muscle, bone, brain, or other tissues. In such pharmacological treatment, regardless of androgen status, an androgen is used therapeutically to exploit the anabolic or other effects of androgens on muscle, bone, and other tissues as hormonal drugs in various non-reproductive disorders. Such pharmacological androgen therapy is neither constrained to using the natural androgen, testosterone, nor it is limited to physiological replacement doses or their equivalent. Rather, it is judged on its efficacy, safety, and relative cost-effectiveness for that specific indication just as any other hormonal or xenobiotic non-hormonal therapeutic drug. Many older uses of pharmacologic androgen therapy are now considered second-line therapies as more specific treatments are developed (310). For example, erythropoietin has largely supplanted androgen therapy for anemia due to marrow or renal failure and improved first-line drug treatments for endometriosis, osteoporosis and advanced breast cancer have similarly relegated androgen therapy to a last resort while newer mechanism-based agents in development for hereditary angioedema may displace 17a-alkylated androgens (311, 312). Nevertheless in many clinical situations, pharmacological androgen therapy remains a cost-effective option with a long-established efficacy and safety profile.

Androgen Replacement Therapy

The sole unequivocal clinical indication for testosterone treatment is in replacement therapy for androgen deficient men suffering from pathological disorders of their reproductive system (hypothalamus, pituitary, testis) that prevent the testes from producing sufficient testosterone supply to meet the body’s usual needs. Establishing a pathological basis for androgen replacement therapy requires identifying well-defined disorders of the hypothalamus, pituitary or testis which have a known and clearly defined pathological basis. These disorders can, and often do, lead to persistent testosterone deficiency either due to disorders of the testis, where damaged Leydig cells cannot produce sufficient testosterone, or disorders of the hypothalamus and/or pituitary, where impaired pituitary luteinizing hormone (LH) secretion reduces the sole driving force to testosterone production by Leydig cells.

The principal goal of androgen (testosterone) replacement therapy is to restore a physiologic pattern of net tissue androgen exposure in androgen deficient men whose damaged reproductive systems are unable to secrete adequate testosterone to levels comparable with those of eugonadal men. This treatment uses only the natural androgen, testosterone, aimed at restoring a physiologic pattern of androgen exposure using a dose limited to that which maintains blood testosterone levels within the eugonadal range. Such treatment aims to restore the full spectrum of androgen effects when endogenous testosterone production fails due to pathological disorders of the reproductive system (testicular-hypothalamic-pituitary axis). This requires restricting replacement therapy to the major natural androgen, testosterone, which aims to not only replicate physiological circulating testosterone levels but also to provide testosterone’s two bioactive metabolites, DHT and estradiol, so that all 3 bioactive sex steroids are available to androgen target tissues. Synthetic androgens are unsuitable because they are incapable of metabolism to the more potent 5α reduced metabolites or being aromatized to estrogens. The overall goal of such replacement therapy is to replicate the efficacy and safety experience of eugonadal men of similar age by recreating the full spectrum of endogenous natural androgen effects on tissues so as to recapitulate the natural history of efficacy and safety of endogenous testosterone.

The prevalence of male hypogonadism requiring androgen repacement therapy in the general community can be estimated from the known prevalence of Klinefelter’s syndrome (15.6 per 1000 male births in 33 prospective birth survey studies (313)) because Klinefelter syndrome accounts for 25-35% of men requiring androgen replacement therapy. The estimated prevalence of ~5 per 1000 men in the general community makes androgen deficiency the most common hormonal deficiency disorder among men. Although life expectancy is not reduced by castration as an adult (303-307) or only minimally (~2 years) shortened (308) by life-long androgen deficiency, the hormonal deficit causes preventable morbidity and a suboptimal quality of life (313). Due to its variable and often subtle clinical features, androgen deficiency remains significantly underdiagnosed, thus denying sufferers simple and effective medical treatment with often striking benefits. Only ~20% of men with Klinefelter ­syndrome characterized by the highly distinctive tiny (<4 mL) testes, are diagnosed during their lifetime (314) indicating that most men go through life without a single pelvic examination by any medical professional in stark contrast to the usual expectation of reproductive health care for women.

The testis has two physiological functions, spermatogenesis and steroidogenesis, either of which can be impaired independently, resulting in infertility or androgen deficiency, respectively, so the term hypogonadism is inherently ambiguous. However, hypogonadism of any cause may require androgen replacement therapy if the deficit in endogenous testosterone production is sufficient to cause clinical and biochemical manifestations of androgen deficiency. Androgen deficiency is a clinical diagnosis with a characteristic presentation and underlying pathological basis in hypothalamus, pituitary or testis disorder, and confirmed by blood hormone assays (see other Endotext chapters for details). The clinical features of androgen deficiency vary according to the severity, chronicity, and epoch of life at presentation. These include ambiguous genitalia, microphallus, delayed puberty, sexual dysfunction, infertility, osteoporosis, anemia, flushing, muscular ache, lethargy, lack of stamina or endurance, easy fatigue, or incidental biochemical diagnosis. For each androgen deficient man, his leading clinical symptoms of androgen deficiency are distinctive, reproducible and corresponds to a specific blood testosterone threshold for any individual but both the symptom(s) and threshold vary between men (315). Because the underlying disorders are mostly irreversible, lifelong treatment is usually required. Androgen replacement therapy can rectify most clinical features of androgen deficiency apart from defective spermatogenesis (316). When fertility is required in gonadotropin-deficient men, spermatogenesis can be initiated by treatment with pulsatile GnRH (317) (if pituitary gonadotroph function is intact (318)) or gonadotropins (319) to substitute for pituitary gonadotropin secretion (320) (see also Endotext, Endocrinology of Male Reproduction, Hypogonadotropic Hypogonadism (HH) and Gonadtropin Therapy). The short half-life of LH would require multi-daily injections rendering it unsuitable for gonadotrophin therapy (321). Instead practical gonadotropin therapy uses hCG, a placental heterodimeric glycoprotein which has a much longer duration of action allowing it to be administered every two or three days. The chorionic gonadotropin hCG consists of an identical a subunit as LH (also the same as in FSH and TSH) combined with a distinct b subunit that is highly homologous to the LH b subunit except for a C terminal extension of 22 amino acids which includes four O-linked sialic acid-capped, carbohydrate side chains. This C terminal extension markedly prolongs the circulating half-life of hCG relative to LH thereby making it a naturally occuring long-acting LH analog. Both endogenous LH and hCG act on the Leydig cell LH/hCG receptor to stimulate endogenous testosterone production. Pharmaceutical hCG, originally purified from pregnancy urine and more recently its recombinant form, can be administered 2-3 times weekly for several months. Where spermatogenesis remains persistently suboptimal, recombinant FSH may subsequently be added (319). Once fertility is no longer required and any pregnancy has passed the 1^st trimester, androgen replacement therapy usually reverts to the simpler and cheaper use of testosterone while preserving the ability subsequently to reinitiate spermatogenesis by gonadotropin replacement (319). The potential value of hCG therapy in gonadotropin-deficient adolescents to produce timely testis growth replicating physiologic puberty (322), rather than reliance on exogenous testosterone which leaves a dormant testis but remains standard management, has yet to be fully evaluated (323, 324).

The extension of testosterone replacement therapy to men with partial, subclinical or compensated androgen deficiency states remain of unproven value (figure 3). Biochemical features of Leydig cell dysfunction, notably persistently elevated LH with low to normal levels of testosterone constituting a high LH/testosterone ratio are observed in aging men (325-327), in men with testicular dysfunction associated with male infertility (328), or after chemotherapy-induced testicular damage (329-332). Although such features may signify mild androgen deficiency, substantial clinical benefits from testosterone replacement therapy remain to be demonstrated (333, 334). Furthermore, testosterone administration may have deleterious effects on spermatogenesis so that its potential adverse effect on men’s fertility must be considered with regard to their marital and fertility status.

Hormonal male contraception can be considered a form of androgen replacement therapy because all currently envisaged regimens aiming to suppress spermatogenesis (and thereby endogenous testosterone production) by inhibiting gonadotropin secretion, use testosterone either alone or with a progestin or a GnRH antagonist (335) (see also Endotext, Endocrinology of Male Reproduction, Male Contraception). As a consequence, exogenous testosterone is required to replace endogenous testosterone secretion.

FIGURE 3. The Hypothalamo-Pituitary Testicular Axis in Health and Intrinsic and Extrinsic Diseases. Schematic representation of the hypothalamo-pituitary-testicular (HPT) system in health (left panel) and in disease (right panel). Note in the right panel the distinction between organic disorders of the reproductive system causing pathologcal hypogonadism leading to male infertility and/or androgen deficiency and, alternatively, non-reproductive disorders which lead to an adaptive hypothalamic response to systemic non-reproductive disorders. While these non-reproductive disorders may lead to a reduced serum testosterone, that is neither a testosterone deficiency state nor warrants testosterone treatment without convincing evidence of safety and efficacy.

Many important questions and opportunities remain for pharmacologic androgen therapy in nongonadal disease, but careful clinical trials are essential for proper evaluation (336). Recent well designed placebo-controlled clinical studies of pharmacologic androgen therapy in chronic disease have been reported. In men with severe chronic obstructive pulmonary disease it produces modest increases in muscle mass and strength with improved quality of life but no effect on underlying lung function (389-391) whereas oral megestrol administration had similar effects despite marked suppression of blood testosterone levels (392). Similarly, although in an observational study chronic heart failure is associated with lower blood testosterone that is proportional to the decrease in cardiac function and which predicts survival (393), a placebo-controlled prospective study of testosterone administration showed improvement in effort-dependent exercise capacity but not in left ventricular function or survival (394). This discrepancy suggests that the lowered blood testosterone is the consequence of a non-specific adaptive reaction of the reproductive hormonal axis to chronic disease (ontogenic regression (395)) rather than a detrimental effect susceptible to being overcome by androgen supplementation. Both testosterone and its non-aromatizable derivative nandrolone, produce increased bone density in men with glucocorticoid-induced osteoporosis with minimal short-term side-effects (396, 397). The best opportunities for future evaluation of adjuvant use of androgen therapy in men with nongonadal disease include steroid-induced osteoporosis; wasting due to AIDS or cancer cachexia; and chronic respiratory, rheumatologic, and some neuromuscular diseases. In addition, the role of pharmacologic androgen therapy in recovery and/or rehabilitation after severe catabolic illness such as burns, critical illness, or major surgery are promising (398) but requires thorough evaluation because detrimental effects may occur (399). Future studies of adjuvant androgen therapy require high-quality clinical data involving randomization and placebo controls as well as finding the optimal dose and authentic clinical, rather than surrogate, end points.

FIGURE 4. Serum Testosterone Across the Lifespan in Men and Women. Serum testosterone, SHBG & calculated "free" testosterone in males and females over the lifetime as determined in >100,000 consecutive blood samples from a single laboratory. After Handelsman et al. Ann Clin Biochem 2016.

Testosterone Treatment for Male Ageing

The prospect of ameliorating male ageing by androgen therapy has long been of interest and recently has been the subject of many observational and short-term interventional controlled ­clinical trials. The consensus from population-based cross—sectional (325, 326) and longitudinal studies (27, 400, 401) is that circulating testosterone concentrations fall by up to ~1% per annum from mid-life onward, an age-related decline that is accelerated by the presence of concomitant chronic disease (401) and associated with decreases in tissue androgen levels (402, 403) as well as numerous co-morbidities of male ageing (327, 404) (figure 4). Numerous cross-sectional and longitudinal observational studies show that low blood testosterone is associated with greater all-cause and/or cardiovascular mortality summarized in multiple meta-analyses (405-412). An observational study of older war veterans reported testosterone treatment was associated with better survival (413); however, bias in the non-randomized design allowing for preferential treatment of healthier men with testosterone may explain those findings (414). However, as observational studies cannot ascribe causality it remains likely that such reductions in blood testosterone may be a consequence rather than a cause of the increased mortality.

 

Interventional studies have remained too small and short-term to resolve this dilemma. Definitive evidence as to whether androgen treatment ameliorates age-related changes in bodily function and improves quality of life requires high quality, randomized placebo-controlled clinical trials using testosterone (415), DHT (416, 417) or hCG (418) or synthetic androgens (419); however, so far the only consistent changes observed in well controlled studies of at least 3 months duration have been small increases in lean (muscle) and decreases in fat mass.

 

The best available summary evidence from meta-analyses indicates no or only inconsistent benefits in bone (420, 421), muscle (422), sexual function (423, 424) and detrimental effects on cardiovascular disease/risk factors (405-412) and polycythemia (425). As a result, the 2004 Institute of Medicine report (426) recommended a priority to acquire more convincing, target-defining feasibility evidence to justify a large-scale clinical trial to weight potential benefits against risks of accelerating cardiovascular and prostate disease.

 

Arising from that recommendation, the NIH-funded series of inter-related ‘Testosterone Trials’ reported their main result in which 790 older men (>65 years), mostly obese, hypertensive, ex-smokers (ie men with “andropause/lowT” but not pathological hypogonadism), treated daily with testosterone for one year showed a modest improvement in sexual function compared with placebo (427). The improvement in sexual function, about 1/3 increase over baseline sexual activity, waned during the year’s treatment and there was no concomitant improvement in either vitality or physical activity compared with placebo. The benefit in sexual function was less robust than the effects of PDE5 inhibitors (427) and of uncertain clinical significance, insufficient to warrant initiating testosterone treatment of older men (428). These findings do not materially change the unfavorable balance of evidence for testosterone treatment for functional causes of a low serum testosterone in the absence of pathological hypogonadism. Although these results fail to meet the 2004 mandate of the Institute of Medicine (now National Academy of Medicine) for sufficient short-term efficacy to warrant public funding of a large scale efficacy trial, the FDA has mandated an industry-funded safety study (TRAVERSE) to investigate major adverse cardiovascular events involving 6000 patients with a low serum testosteronbe but no pathological hypogonadism randomized to daily testosterone vs placebo gel treated for up to 5 years.

 

The major hypothetical population risk from androgen therapy for male ageing remains increased cardiovascular disease (309) as was proven unexpectedly by the WHI study for the risks of estrogen replacement for menopause (429). Cardiovascular disease has earlier onset and greater severity in men resulting in a 2-3-fold higher age-specific risks of cardiovascular death compared with women (430). The male disadvantage in cardiovascular disease has a complex pathogenesis with androgens having apparently beneficial effects including in regulating cardiac ion channel fluxes that dictate QT interval length, cardiac ventricular repolarization and lesser risk of arrthymia (431-439) as well as angiogenesis (440, 441) which must be integrated with other apparently deleterious effects (309, 442). Prospective observational data remains conflicting, with low blood testosterone predicting subsequent cardiovascular death in some (443, 444) but not other (445-447) studies. Testosterone therapy for older, frail men may increase adverse cardiovascular events (355), side-effects that may be under reported (448) in previous studies not reporting such hazards (449). Observational data linking cardiovascular disease with low blood testosterone levels may however be the consequence of non-specific effects of chronic cardiovascular disease and/or confounding effects by major cardiovascular risk factors, like diabetes and obesity. The latter interpretations are supported by Mendelian randomization studies which report only non-causal relationships (450, 451) albeit with important methodological caveats (452).

 

Similarly, for the more feared but quantitatively less significant late-life prostate diseases, although their androgen dependence is well established, it is also known that life-long androgen deficiency (Klinefelter’s syndrome) reduces risk of fatal prostate cancer (453) and prevailing endogenous testosterone levels in healthy men do not predict risk of subsequent prostate cancer (454, 455).

 

These epidemiological observations are consistent with either circulating testosterone levels being a biomarker, a non-specific barometer of ill-health, or else that restoring circulating testosterone to eugonadal levels could reduce age-related cardiovascular and prostate disease (the “andropause” hypothesis, also known as “LowT” or”late-onset hypogonadism”). Independent critical analyses have concluded that it is not valid to extrapolate the features of pathological hypogonadism in younger men to older men with possibly age-related hypogonadism (456). Nor did a comprehensive meta-analysis identify any valid basis for testosterone treatment of such older men (457). Decisive testing of these alternatives requires an adequately powered, placebo-controlled, prospective, randomized clinical trial (426). As the decisive safety and efficacy evidence on testosterone supplementation for male ageing remains distant, interim clinical guidelines have been developed by academic and professional societies (458-461) ostensibly aiming to restrain the unproven testosterone prescribing which nevertheless escalated over recent decades in Australia (462), Europe (463, 464) and most dramatically in North America (465-468). Consequently, more recent clinical guidelines have curbed the tacit promotion of testosterone precsribing for men without pathological hypogonadism (469, 470).

 

At present, testosterone treatment ­cannot be recommended as routine treatment for male ageing (see also Endotext, Endocrinology of Male Reproduction, Age-Related Changes in the Male Reproductive Axis). Nevertheless, androgen replacement therapy may be used cautiously even in older men with pre-existing pathological pituitary-gonadal disorders causing androgen deficiency if contraindications such as prostate cancer are excluded.

 

Androgen Misuse and Abuse

 

Misuse of androgens involves medical prescription without a valid clinical indication and outside an approved clinical trial, and androgen abuse is the use of androgens for nonmedical purposes. Medical misuse includes prescribing androgens for male infertility (471) or sexual dysfunction in men without androgen deficiency (423) where there are no likely benefits or as a tonic for non-specific symptoms in older men (“male menopause”, ‘andropause”, “late-onset hypogonadism”) (426) or women (368) where safe and effective use is unproven. Although there is no exact boundary defining overuse, mass marketing and promotion to fend off ageing in the absence of reliable evidence are hallmarks of systemic misuse of androgens. Androgens have a mystique of youthful virility making them ideal for manipulative marketing to the wealthy, worried well as they grow older.

 

ANDROGEN MISUSE: PATTERNS OF TESTOSTERONE PRESCRIPTION

Despite the absence of any new approved indications beyond the treatment of pathological classical hypogonadism, testosterone prescribing has displayed major and progressive increases especially since 2000 (472). The market for testosterone prescribing has increased 100-fold from $18 million in the late 1980’s (465) to $1.8 billion in this decade (468). In Australia, for example, where a national health scheme provides accurate prescription data, striking differences between states, increasing use of costlier newer products and partially effective regulatory curbs on unproven testosterone prescribing were reported (462, 473). Similar increases are also described in the USA (465, 467), Switzerland (464) and UK (463). A 2013 study of international trends in testosterone prescribing based on per capita sales of testosterone usage and pooling all products into standardized testosterone usage estimates (per person per month), showed testosterone usage increased in every world region and for 37 of 41 countries surveyed over the 11 years (2000-11) (468). The increases were most striking in North America where they rose 40-fold in Canada and 10-fold in the USA over only a decade. Other estimates from the US confirm an increase in testosterone prescribing although with a lower increase when based on selective sources such as private insurance databases (467, 474, 475) or the Veterans Administration (VA) system (476). These lower increases underestimate the national usage, indicating the efficacy of formulary or other restrictions constraining unjustified testosterone prescribing but implies much greater increases in testosterone usage outside those populations served by private medical insurance and the VA system.

 

The increased testosterone prescribing appears to be primarily for older men and driven by clinical guidelines that endorse testosterone prescribing for age-related low circulating testosterone concentration (459, 477, 478), commonly referred to as “LowT” or “late-onset hypogonadism”. The major factors driving these increases include direct-to-consumer advertising as part of a broad spectrum of pharmaceutical promotional activities as well as permissive clinical prescribing guidelines from professional and single-issue societies. The latter have, in concert, tacitly encouraged, facilitated, and promoted increased off-label testosterone prescribing, bypassing the requirement for high quality clinical evidence of safety and efficacy. Prescribing guidelines that systematically eliminated the fundamental distinction between pathological hypogonadism and functional causes of a low circulating testosterone have significantly contributed to legitimizing epidemic-like increases in testosterone prescription overuse based upon highly inflated incidence data for “hypogonadism” (468).

 

The known prevalence of pathological androgen deficiency (~0.5% of men (479)) equates to a figure of ~15 defined monthly doses per year per 1000 population. Population linkage registry data from the UK (453) and Denmark (314)prove severe under-diagnosis of Klinefelter’s syndrome, the most frequent cause of pathological AD. Nevertheless, it is highly unlikely that recent steep increases in testosterone prescribing and use can be attributed to rectifying the under-diagnosis of KS, which is generally diagnosed in young male adults. Not only does total testosterone prescribing in some countries exceed the maximal amount that could be attributed to pathological AD, there is no evidence the diagnosis of KS has increased in recent years (473, 480).

 

By contrast, the estimated population prevalence of “andropause” among older men is up to 40% (481) or more usually claimed in the range 10-25% (326, 482, 483) with even the lowest estimates of 2-3% (484) representing major (5-100 fold) increases in potential market size over pathological hypogonadism.

 

ANDROGEN ABUSE

 

(see also Endotext, Endocrinology of Male Reproduction Section, Performance Enhancing Hormone Doping in Sport)

Androgen abuse originated in the 1950s as a product of the Cold War (485) whereby communist Eastern European countries could develop national programs to achieve short-term propaganda victories over the West in Olympic and international sports (486). This form of cheating was readily taken up by individual athletes seeking personal rewards of fame and fortune in elite competitive sports. Over decades, androgen abuse has become endemic in developed countries with sufficient affluence to support drug abuse subcultures. Androgen abuse is cultivated by underground folklore among athletes and trainers, particularly in power sports and body building, promoting the use of so-called “anabolic steroids” to enhance personal image and sports performance. A lucrative illicit industry is fostered through wildly speculative underground publications promoting the use of prodigious androgen doses in combination (“stacking”) and/or cycling regimens. The myotrophic benefits of supraphysiologic androgen doses in eugonadal men were long doubted (347) in the belief that alleged performance gains were attributable to placebo responses involving effects of motivation, training and diet, This belief was overturned by a randomized, placebo-controlled clinical study showing decisively that supraphysiologic testosterone doses (600 mg testosterone enanthate weekly) for 10 weeks increases muscular size and strength (346). In well-controlled studies of eugonadal young (487) and older (350) men, testosterone shows strong linear relationships of dose with muscular size and strength throughout and beyond the physiologic range. The additional dose-dependent increases in erythropoesis (352) and mood (488) may also enhance the direct myotrophic benefits of supraphysiologic androgen dose. While these studies prove the unequivocal efficacy of supraphysiological androgen dosage to increase muscle size and strength even in eugonadal men, the specific benefits for skilled athletic performance depend on the sport involved with greatest advantages evident in power sports. The overall safety of the sustained supraphysiological androgen exposure in these settings remains undefined, notably for cardiovascular and prostate disease as well as psychiatric sequelae (489).

 

Progressively, the epidemic of androgen abuse has spread from elite power athletes so that the majority of abusers are no longer athletes but recreational and cosmetic users wishing to augment body building or occupational users working in security-related professions (490). A remarkable meta-analysis of 271 papers reporting prevalence of androgen abuse within various populations comprising 2.8 million people (490), deduced a lifetime (“ever use”) prevalence of 6.4% for males and 1.6% for females with higher rates for recreational sports (18.4%), athletes (13.4%), prisoners (12.4%), drug users (8.0%), high school students (2.3%) compared with non-athletes (1.0%). As an illicit activity, the extent of androgen abuse in the general community is difficult to estimate, although point estimates of prevalence are more feasible in captive populations such as high schools. The prevalence of self-reported lifetime (“ever”) use is estimated to be 66 in the United States (491), 58 in Sweden (492), 32 in Australia (493), and 28 in South Africa (494) per 1000 boys in high school, with a much lower prevalence among girls. Predictors of androgen abuse in high schools are consistent across many cultures include truancy, availability of disposable income, minority ethnic or migrant status and there is significant overlap with typical features of adolescent abuse of other drugs. Voluntary self-report of androgen abuse understates prevalence of drug use among weight lifters (495) and prisoners (496, 497).

 

Abusers consume androgens from many sources including veterinary, inert, or counterfeit preparations, obtained mostly through illicit sales by underground networks with a small proportion obtained from compliant doctors. Highly sensitive urinary drug screening methods for detection of natural and synthetic androgens, standardized by the Word AntiDoping Agency (WADA) for international and national sporting bodies as a deterrent, has contributed to the progressive elimination of known androgens from elite sporting events. The persistent demand for androgens as the most potent known ergogenic drugs has led to the production in unlicensed laboratories of illicit designer androgens such as norbolethone (498), tetrahydrogestrinone (499, 500) and dimethyltestosterone (501) custom-developed for elite professional athletes to evade doping detection. The rapid identification of these designer androgens has meant that they have been seldom, if ever, used (502). Corresponding legislation has also been introduced by some governments to regulate clinical use of androgens and to reduce illicit supply of marketed androgens. While overall, the community epidemic of androgen abuse driven by user demands shows little signs of abating (503, 504), rigorous detection is reducing demand in elite sports and similar trends have been reported in the long running Monitoring the Future Project (http://www.monitoringthefuture.org/) whereby self-reported androgen abuse peaked in US high schools around 2000 and is now abating.

 

Androgen abuse is associated with reversible depression of spermatogenesis and fertility (505-509), gynecomastia (510), hepatotoxicity due to 17a-alkylated androgens (511), HIV and hepatitis from needle sharing (512-516) although the infectious risks are lower than among other iv drug users due to less needle and syringe sharing (517), local injury and sepsis from injections (518, 519), overtraining injuries (520), rhabdomyolysis (521), popliteal artery entrapment (522), cerebral (523) or deep vein thrombosis and pulmonary embolism (524), cerebral hemorrhage (525), convulsions (526) as well as mood and/or behavioral disturbances (527, 528). The medical consequences of androgen abuse for the cardiovascular system have been reviewed (529-533), but only few anecdotal reports are available relating to prostate diseases (534-536). However, for both, long-term consequences of androgen abuse based on anecdotal reporting are likely to be significantly underestimated due to underreporting of past androgen use and non-systematic follow-up. Few well controlled prospective clinical studies of the cardiovascular (537, 538) or prostatic (350, 487, 539) effects of high dose androgens have been reported. Most available clinical studies consist of non-randomized, observational comparisons of androgen users compared with non or discontinued users (540-552). However, such retrospective observational studies suffer from ascertainment, participation and other bias so that important unrecognized determinants of outcomes may not be measured. Given the low community prevalence of androgen abuse, well designed, sufficiently powerful retrospective case-control studies are required to define the long-term risks of cardiovascular and prostate disease (553). The best available evidence ­suggests elite athletes have longer life expectancy due to reduced cardiovascular disease (554, 555). This benefit, however, is least evident among power athletes, the group with highest likelihood of past androgen abuse, a finding confirmed by a small study finding a greater than 4-fold increase in premature deaths (from suicide, cardiovascular disease, liver failure and lymphoma) among 62 former power athletes compared with population norms (556). More definitive studies are required, but, at present, largely anecdotal information suggests that serious short-term medical danger is limited considering the extent of androgen abuse, that androgens are not physically addictive (557, 558) and that most androgen abusers eventually discontinue drug use. After cessation of prolonged use of high-dose androgens, recovery of the hypothalamic-pituitary-testicular axis may be delayed for months and up to 2 years (559), creating a transient gonadotropin and androgen deficiency withdrawal state (560-563). This may lead to temporary androgen deficiency symptoms and/or oligozoospemia and infertility that eventually abate without requiring additional hormonal treatments. Although hCG can induce spermatogenesis (507, 564), like exogenous testosterone, it further delays recovery of the reproductive axis and perpetuates the drug abuse cycle (565). There remains anecdotal evidence from experienced observers that prolonged hypothalamic-pituitary suppression by high dose exogenous androgens may not always be fully reversible after even a year off exogenous androgens, resembling the incomplete reversibility of GnRH analog suppression of circulating testosterone in older men after cessation of prolonged medical castration for prostate cancer (566, 567). An educational program intervention had modest success in deterring androgen abuse among secondary school footballers (568) and more effective interventions to prevent and/or halt androgen abuse capable of overcoming strong contrary social incentives of fame and fortune are yet to be defined.

Practical Goals of Androgen Replacement Therapy

 

The goal of androgen replacement therapy is to replicate the physiologic actions of endogenous testosterone, usually for the remainder of life as the pathological basis of hypogonadism is usually irreversible disorders of the hypothalamus, pituitary or testis. This requires rectifying the deficit and maintaining androgenic/anabolic effects on bone (153, 569), muscle (349), blood-forming marrow (352, 570), sexual function (71, 571), and other androgen-responsive tissues. The ideal product for long-term androgen replacement therapy should be a safe, effective, convenient, and inexpensive form of testosterone with long-acting depot properties providing steady-state blood testosterone levels due to reproducible, zero-order release kinetics. Androgen replacement therapy usually employs testosterone rather than synthetic androgens for reasons of safety and ease of monitoring. The aim is to maintain physiologic testosterone levels and resulting tissue androgen effects. Synthetic steroidal and non-steroidal androgens are likely to lack the full spectrum of testosterone tissue effects due to local amplification by 5a reductase to DHT and/or diversification to act on ERa by aromatization to estradiol. The practical goal of androgen replacement therapy is therefore to maintain stable, physiologic testosterone levels for prolonged periods using convenient depot testos­terone formulations that facilitate compliance and avoid either supranormal or excessive fluctuation of androgen levels. The adequacy of testosterone replacement therapy is important for optimal outcomes (572) as suboptimal testosterone regimens, whether due to inadequate dosage or poor compliance, produce suboptimal bone density (573-575) compared with maintenance of age-specific norms achieved with adequate testosterone regimens (572, 576). Differences in testosterone-induced bone density according to type of hypogonadism (577) may be attributable to delay in onset and/or suboptimal testosterone dose in early onset androgen deficiency (578, 579) leading to reduced peak bone mass achieved in early manhood. Similarly, the severity of the androgen deficiency also predicts the magnitude of the restorative effect of testosterone replacement with greatest effects early in treatment of severe androgen deficiency (569, 572) whereas only minimal effects are evident for testosterone treatment of mild androgen deficiency (333, 334). The potential for individual tailoring of testosterone replacement dose according to an individual’s pharmacogenetic background of androgen sensitivity has been proposed by a study showing that the magnitude of the prostate growth response to exogenous testosterone in androgen deficient men is inversely related to the CAG triplet (polyglutamine) repeat length in exon 1 of the androgen receptor (235). However, this polyglutamine repeat is inversely related to ambient blood testosterone levels (580) consistent with the reciprocal relationship between repeat lengths and AR transactivational activity. Hence this polymorphism is only a weak modulator of tissue androgen sensitivity. Whether the magnitude of this pharmacogenetic effect is sufficiently large and significantly influences other androgen-sensitive end points will determine whether this approach is useful in practice.

Pharmacologic Features of Androgens

 

The major features of the clinical pharmacology of testosterone are its short circulating half-life and low oral bioavailability, both largely attributable to rapid hepatic conversion to biologically inactivate oxidized and glucuronidated excretory metabolites. The pharmaceutical ­development of practical testosterone products has been geared to overcoming these limitations. This has led to the development of parenteral depot formulations (injectable, implantable, transdermal), or products to bypass the hepatic portal system (sublingual, buccal, gut lymphatic absorption, transdermal) as well as orally active synthetic androgens that resist hepatic degradation (120, 581).

 

Androgens are defined pharmacologically by their binding and activation of the androgen receptor (1). Testosterone is the model androgen featuring a 19-carbon, four-ring steroid structure with two oxygens (3-keto, 17b-hydroxy) including a ∆⁴ nonaromatic A ring. Testosterone derivatives have been developed to enhance intrinsic androgenic potency, prolong duration of action, and/or improve oral bioavailability of synthetic androgens. Major ring structural modifications of testosterone include 17b-esterification, 19-nor-methyl, 17a-alkyl, 1-methyl, 7a-methyl, and D-homoandrogens. Most synthetic androgens are 17a-alkylated analogs of testosterone developed to exploit the fact that introducing a one (methyl) or two (ethyl, ethinyl) carbon group at the 17a position of the D ring allowed for oral bioactivity by reducing hepatic oxidative degradative metabolism. In 1998 the first nonsteroidal androgens, modified from nonsteroidal aryl propionamide antiandrogen structures, were reported (582) followed by quinoline, tetrahydroquinoline and hydantoin derivatives (583).

 

The identification of a single gene and protein for the androgen receptor in 1988 (584-586) explains the physiologic observation that, at equivalent doses, all androgens have essentially similar effects (587). The term “anabolic steroid” was invented during the post-WWII golden age of steroid pharmacology to define an idealized androgen lacking virilizing features but maintaining myotrophic properties so that it could be used safely in chidren and women. Although this quest proved illusory and was abandoned after all industry efforts failed to identify such a hypothetical synthetic androgen, the obsolete term “anabolic steroid” persists mainly as a lurid descriptor in popular media despite continuing to make a false distinction where there is no difference. Better understanding of the metabolic activation of androgens via 5a-reduction and aromatization in target tissues and the tissue-specific partial agonist/antagonist properties of some synthetic androgens may lead to more physiological concepts of tissue-specific androgen action (“specific androgen receptor modulator”) governed by the physiological processes of pre-receptor androgen activation as well as post-receptor interaction with co-regulator proteins analogous to the development of synthetic estrogen partial agonists with tissue specificity (“specific estrogen receptor modulator”) (588). The potential for new clinical therapeutic indications of novel tissue-selective androgens in clinical development remain to be fully evaluated (589).

 

Formulation, Route, and Dose

 

UNMODIFIED TESTOSTERONE

Testosterone Implants

 

Implants of fused crystalline testosterone provide stable, physiologic testosterone levels for as long as 6 months after a single implantation procedure (590). Typically, four 200-mg pellets are inserted under the skin of the lateral abdominal wall or hip using in-office minor surgery and a local anesthetic. No suture or antibiotic is required, and the pellets are fully biodegradable and thus do not require removal. This old testosterone formulation (591) has excellent depot properties, with testosterone being absorbed by simple dissolution from a solid reservoir into extracellular fluid at a rate governed by the solubility of testosterone in the extracellular fluid resulting in a standard 800 mg testosterone dose releasing ~5 mg per day (592) replicating the testosterone production rate in healthy eugonadal men (49-51, 593). The long duration of action makes it popular among younger androgen-deficient men as reflected by a high continuation rate (594). The major limitations of this form of testosterone administration are the cumbersome implantation procedure and extrusion of a single pellet after ~5% of proce­dures. Extrusions are more frequent among thin men undertaking vigorous physical activities (594) but surface washing (595), antibiotic impregnation (596) or varying the site of implantation or track geometry (597) do not reduce extrusion rate. Other side effects such as bleeding or infection are rare (<1%) (594). Recent studies using a smaller (75 mg) implant reproduce these features although requiring administration of a larger number of pellets (598-600). Despite its clinical advantages and popularity, this simple, non-patented ­technology has limited commercial marketing appeal and, consequently, is not widely available apart from compounding chemists and niche manufacturers (598).

Transdermal Testosterone

 

Delivery of testosterone across the skin has long been of interest (174). More recently products delivering testosterone via adhesive dermal patches and gels have been developed to maintain physiologic testosterone levels by daily application. The first transdermal patch was developed for scrotal application where the thin, highly vascular skin facilitates steroid absorption (601, 602) and scrotal patches showed long-term efficacy (603) including minimal skin irritation (604, 605). However, their large size, need for shaving and ­disproportionately high increase blood DHT levels due to 5a-reduction of testosterone during transdermal passage led to the development of a smaller non-scrotal patch (606) effective for long-term use (607). For non-scrotal patches, the smaller size and application to less permeable non-scrotal (trunk, proximal limb) skin limit testosterone absorption. Although this can be enhanced by heating (608), in practice this required inclusion of absorption enhancers that cause skin irritation (604, 605) of varying severity (609). Although skin irritation may be reduced by topical corticosteroid cream (610), the majority of users experience some skin reaction with ~25% having to discontinue due to dermal intolerance (394).

 

Dermal testosterone (611) or DHT (612, 613) gels developed in Europe are now more widely available as topical gels (571, 614-619), solution (620) or a cream (72). They must be applied daily on the trunk or axilla, and the volatile hydroalcoholic gel base evaporates rapidly with a short-lived stinging sensation but is relatively nonirritating to the skin so there are few discontinuations for adverse skin reactions (571, 621). Transdermal testosterone delivery depends on a small fraction (typically <5%) of testosterone applied to the skin in the dermal gel or solution transfering into the skin where it forms a secondary reservoir in the stratum corneum. From this depot, testosterone is gradually released into the circulation by diffusion down a concentration gradient into the blood stream. A novel and well accepted form of transdermal testosterone delivery is application of a testosterone cream to the sccrotum taking advantage of the thin, highly vascular scrotal skin which demonstrates an 8-fold greater permeability to testosterone than truncal skin (622). As a large amount of testosterone remains on the skin after topical application, transfer of testosterone by direct skin contact is a risk for an intimate partner (623-625) or children (626-631). Serum testosterone concentrations are increased in non-dosed female partners making direct skin contact with men using transdermal testosterone products (632). Creating a physical barrier such as using a testosterone transdermal patch (633) or covering the application site with clothing (632, 634) reduces this risk. Washing off excess gel from the application site after a short time (<30 min) may reduce the risk of transfer (632, 634, 635) but also reduces effective testosterone absorption in some (636) but not all (637) studies. Unlike transdermal patches, topical gel or solutions have considerable misuse and abuse potential.

Testosterone Microspheres

 

Suspensions of biodegradable microspheres, consisting of polyglycolide-lactide matrix similar to absorbable suture material and laden with testosterone, can deliver stable, physiologic levels of testosterone for 2 to 3 months after intramuscular injection (638, 639). Subsequent findings (640) suggest that the practical limitations of microsphere technology such as loading capacity, large injection volumes, and batch variability may be overcome.

Oral Testosterone

 

Finely milled testosterone (167, 641) or testosterone suspended in an oil vehicle (642, 643) have low oral bioavailability requiring high daily doses (200–400 mg) to maintain physiologic testosterone levels. Such a heavy androgen load causes prominent hepatic enzyme induction (644) without hepatotoxicity (645). Although effective in small studies (646), oral testosterone is not commercially available and little used. Sufficiently high oral testosterone doses (400-900 mg daily) also reduce serum SHBG (647) which may explain the concomitant acceleration of testosterone metabolism (167, 646, 648).

 

Buccal and Sublingual Testosterone

 

Buccal or sublingual delivery of testosterone is an old technology (170) designed to bypass the avid first-pass hepatic metabolism of testosterone that is inevitable with the portal route of absorption. Once absorbed into the general circulation, however, testosterone is rapidly inactivated in accord with its short circulating half-time. Revivals of this technology include testosterone in a sublingual cyclodextrin formulation (649) and in a buccal lozenge (171, 650). The multiple daily dosing required to maintain physiologic testosterone levels are drawbacks for long-term androgen replacement using such products, and their effectiveness and acceptability remain to be established. Like all transepithelial (nonparenteral) testosterone delivery ­systems, disproportionate amounts of testosterone undergo 5a-reduction during local absorption, resulting in higher blood DHT levels than those in eugonadal men (651). Because intraprostatic DHT is produced locally within the prostate and unlikely to be affected by changes in circulating DHT levels as well as the fact that prostate ­diseases remain rare among androgen ­deficient men receiving androgen replacement therapy, the higher blood DHT levels appear to pose no real risk of accelerating prostate disease (652).

 

TESTOSTERONE ESTERS

FIGURE 5. Schematic overview of the pharmacology of testosterone esters. Testosterone is esterified through the 17 β hydroxyl group with fatty acid esters of different aliphatic or other chain length which is a biologically inactive pro-drug. The esterified testosterone in an oil vehicle is injected deeply into a muscle forming a local drug depot from which the testosterone ester is released at a slow rate determined by its physico-chemical partitioning according to the testosterone ester’s hydrophobicity. Once the testosterone ester exits the depot and enters the extracellular fluids, it is rapidly hydrolyzed by ubiquitous non-specific esterases thereby releasing the testosterone into the general circulation.

Injectable Testosterone

The most widely used testosterone formulation for many decades has been intramuscular injection of testosterone esters (figure 5), formed by 17b-esterification of testosterone with fatty acids of various aliphatic and/or aromatic chain lengths, injected in a vegetable oil vehicle (653). This depot product relies on retarded release of the testosterone ester from the oil vehicle injection depot because esters undergo rapid hydrolysis by ubiquitous esterases to liberate free testosterone into the circulation. The pharmacokinetics and pharmacodynamics of androgen esters is therefore primarily determined by ester side-chain length, volume of oil vehicle, and site of injection via hydrophobic physicochemical partitioning of the androgen ester between the hydrophobic oil vehicle and the aqueous extracellular fluid (654).

 

The short 3-carbon aliphatic ester side-chain of testosterone propionate gives the product a brief duration of action requiring injections of 25 to 50 mg at 1-2 day intervals for effective testosterone replacement therapy. In contrast, the 7-carbon side-chain of testosterone enanthate has a longer duration of action so that it is used at doses of 200 to 250 mg per 10 to 14 days for androgen replacement therapy in hypogonadal men (655-657) and has been for decades the most widely used form of testosterone used in replacement therapy. Other testosterone esters (cypionate, cyclohexane carboxylate) have simillar pharmacokinetics making them pharmacologically equivalent to testosterone enanthate (658). Similarly, mixtures of short- and longer acting testosterone esters also have essentially the same pharmacokinetics of the longest ester.

 

Longer acting testosterone esters, testosterone buciclate and undecanoate, intended to provide depot release over months rather than weeks, have been developed. Testosterone buciclate (trans-4-n-butyl cyclohexane carboxylate) is an insoluble testosterone ester in an aqueous suspension that produces prolonged testosterone release due to steric hindrance of ester side-chain hydrolysis slowing the liberation of unesterified testosterone. Although the buciclate ester produces blood testosterone levels in the low-normal physiologic range for up to 4 months after injection in nonhuman primates (659) as well as hypogonadal (660) and eugonadal (661) men, product development has not progressed. Injectable testosterone undecanoate, an ester of an 11 carbon aliphatic fatty acid, in an oil vehicle provides a longer (~12 weeks) duration of action (662-664) now widely marketed as a long-acting injectable depot testosterone product. Due to its limited solubility in the castor oil vehicle, testosterone undecanoate is administered as a 1000 mg dose in a large (4 mL) injection volume at 12 week intervals after the first and one 6 week loading dose or multiple loading doses (665) or in the USA, 750 mg in 3ml volume administered at the start of treatment and then 4 and subsequently at 10 weekly intervals. Once available, the rapid uptake of long-acting injectables show that they are very popular among younger hypogonadal men whereas transdermal products are more suited for older men in case of need to rapidly discontinue testosterone treatment such as after diagnosis of prostate cancer. The relatively long duration of action is also well suited to male hormonal contraception either alone in Chinese men (666) or as part of an androgen-progestin combination (667-669). For treatment of androgen deficiency, although its longer duration of action entails fewer injections with advantages for convenience and compliance, the efficacy and safety does not differ from that of the shorter acting testosterone enanthae (670).

 

Although testosterone esters in oil vehicle are approved for im injection, they can be effectively used by subcutaneous injection (671) in their marketed formulation or via a pre-filled autoinjector (672). There is increasing use of subcutaneous injections (1 ml) of medium acting testosterone esters (cypionate or enanthate) in oil vehicle especially for masculinizing female to male transgender (673-675). However, the sc use of larger volume (4 ml) for injecting testosterone undecanoate is feasible but less popular than im injection (676).

Oral Testosterone Undecanoate

 

Oral testosterone undecanoate, a suspension of the ester in 40-mg oil-filled capsules, is administered as 160 to 240 mg in two or more doses per day (677). The hydrophobic, long aliphatic chain ester in a castor oil/propylene glycol laurate vehicle favors preferential absorption into chylomicrons entering the gastrointestinal lymphatics and largely bypassing hepatic first-pass metabolism (173). Oral testosterone undecanoate is not absorbed under fasting conditions but is taken up when ingested with food (678) containing a moderate amount (at least 19 gm) of fat (679). Although oral testosterone undecanoate produces a disproportionate increase in serum DHT which is unaffected by concomitant administration of an oral 5 α reductase inhibitor (680); such modest increases in circulating DHT would have no impact on prostate size (681) or apparent risk of prostate cancer (454, 682) presumably because DHT of extra-prostatic origin fails to increase intra-prostatic DHT concentrations (683). Its low oral bioavailability (684) and short duration of action requiring high and multiple daily doses of testosterone lead to only modest clinical efficacy compared with injectable testosterone esters (657, 685). Widely marketed, it may cause gastrointestinal intolerance but has otherwise well established safety (682). A new formulation of oral testosterone undecanoate was approved for US marketing in 2019 (686) over four decades after its introduction in Europe (687) to close the gap in the market for a safe non-hepatotoxic oral androgen. Its limitations in efficacy, notably its capricious bioavailability, make it a second choice (657), unless parenteral therapy is best avoided (e.g., bleeding disorders, anticoagulation) or a low dose, as for induction of male puberty, must be provided (688, 689) as a better option than the hepatotoxic alkylated androgen, oxandrolone (690).

 

SYNTHETIC ANDROGENS

Synthetic androgens include both steroidal and non-steroidal androgens. Synthetic steroidal androgens, most developed by 1970, comprise categories of 17a-alkylated androgens, 1-methyl androgens and nandrolone and its derivatives (figure 6).

 

FIGURE 6. Testosterone and its pharmacological derivatives. Listed are the most common synthetic androgens displaying their structural relationship with testosterone.

Steriodal Androgens

Most oral androgens are hepatotoxic 17a-alkylated andro­gens (methyltestosterone, fluoxymesterone, oxymetholone, oxandrolone, ethylestrenol, stanozolol, danazol, methandrostenolone, norethandrolone) making them unacceptable for ­long-term androgen replacement therapy. The 1-methyl androgen mesterolone is an orally active DHT analog that undergoes neither amplification by 5a reduction nor aromatization but it is free of hepatotoxicity. Mesterolone is not used for long-term androgen replacement due to the need for multiple daily dosing, its poorly defined pharmacology (691) and suboptimal efficacy at standard dose (570, 577). For historical reasons, the other marketed 1-methyl androgen methenolone is used almost exclusively in anemia due to marrow failure (692, 693) although it has no specific pharmacological advantage over testosterone or other androgens.

Nandrolone (19-nor testosterone) is a widely used injectable androgen in the form of aliphatic fatty acid esters in an oil vehicle mainly for treatment of postmenopusal osteoporosis where it is effective at increasing bone density and reducing fracture rate (694, 695). It is also the most popular androgen abused in sports doping and in body building. Nandrolone is a naturally occuring steroid but is not normally secreted in the human bloodstream although it occurs as an intermediate in the aromatization of testosterone to estradiol by the aromatase enzyme (696). This enzyme complex undertakes two successive hydroxylations on the angular C19 methyl group of testosterone followed by a cleavage of the C10-C19 bond to releases formic acid and aromatize the A ring (697). Nandrolone represents a penultimate step of the molecule undergoing aromatization bound to the enzyme complex with the C19 methyl group excised but a still non-aromatized A ring. Paradoxically, despite being an intermediate in the aromatization reaction, nandrolone is virtually not aromatized after parenteral administration in men (698, 699), presumably because it is a very poor substrate for the human aromatase enzyme (700). It is susceptible to amplification by 5a reductase with its 5a reduced metabolites being moderately activated in androgenic potency (701). The minimal aromatizability of nandrolone makes it suitable for treatment of osteoporosis in women in whom estrogen therapy is contraindicated due to hormone sensitive cancers (breast, uterus) or for older women, although virilization limits its acceptability (702).

Synthetic nandrolone derivatives 7a-methyl 19-nortestosterone (MENT) (703) and 7a, 11b-dimethyl 19-nortestosterone (dimethandrolone) (704) are potent, non-hepatotoxic androgens. MENT is being developed as a depot androgen (705) for androgen replacement (706) and male contraception in an androgen-progestin combination regimen (707) while dimethandrolone has potential for male contraception as a single steroid with dual androgen and progestin activity (708). As nandrolone derivatives, these synthetic androgens are less susceptible to amplification by 5a-reduction (700, 709) whereby their 5a-reduced metabolites have reduced AR binding affinity (710). Disparities in reported susceptibility to aromatization vary from minimal using a recombinant human aromatase assay (700)whereas greater aromatization is reported using purified human or equine placental aromatase (709, 711, 712). The inability of MENT to maintain bone density in androgen deficient men (575) may be due to underdosing rather than an intrinsic feature of this synthetic androgen but illustrates the need for thorough dose titration in different tissues for synthetic androgens that may not possess the full characteristic spectrum of testosterone effects.

Nonsteroidal Androgens

The first nonsteroidal androgen was reported in 1998 (582) and the first placebo-controlled randomized clinical trial in 2013 (713). None are yet approved for clinical use but registration studies are underway for enobosarm (714). Based on structural modifications of the nonsteroidal class of the aryl-propionamide antiandrogens (bicalutamide, flutamide), these compounds offer the possibility of orally active, potent androgens. Subsequently, additional classes of non-steroidal androgen based on structures including quinolines, hydantoins, tetracyclic indoles and oxachrysensones have been reported. Lacking the classical steroid ring structure such androgens are likely to be not subject to androgen activation either by 5a reductase or aromatization but, if taken orally, subject to first-pass hepatic metabolism. Such hepatic metabolism can eliminate in vivo bioactivity of analogs with potent in vitro androgenic effects (715) whereas metabolically resistant analogs can produce potent and disproportionate androgenic effects on the liver in transit. Many of the novel non-steroidal androgens demonstrate potent androgenic effects experimentally on muscle, bone and sexual function while minimizing prostate effects in experimental animals but none have yet undergone full clinical evaluation. These selective effects may be attributable to the tissue-selective distribution of 5a-reductase as a local tissue, pre-receptor androgen amplification mechanism (716) or more complex mechanisms involving ligand-induced receptor conformation changes and/or post-receptor co-regulator interaction mechanisms such as define the tissue selectivity and agonist/antagonist specificity of non-steroidal estrogen partial agonists (717). These features suggest that non-steroidal androgens have potential for development into pharmacologic androgen therapy regimens as tissue-selective mixed or partial androgen agonists (“selective androgen receptor modulators”, SARM) (419, 718). Conversely, they are not ideal for androgen replacement therapy where the full spectrum of testosterone effects including aromatization is idealy required, especially for tissues such as the brain (148, 159) and bone (153) where aromatization is a prominent feature of testosterone action. The clinical efficacy and safety of non-steroidal androgens have yet to be reported and none are yet marketed. Whether the hepatotoxicity of antiandrogens (719, 720) will also be a feature of non-steroidal androgens remains to be determined.

Choice of Preparation

The choice of testosterone product for androgen replacement therapy depends on physician experience and patient preference, involving factors such as convenience, availability, familiarity, cost, and tolerance of frequent injections. Preparations of testosterone or its esters are favored over synthetic androgens for all androgen replacement therapy applications by virtue of their long record of safety and efficacy, ease of dose titration and of monitoring of blood levels as well as the possibility that synthetic androgens lack the full spectrum of testosterone effects through pre-receptor tissue activational mechanisms (5a reduction, aromatization). The hepatotoxicity of synthetic 17a-alkylated androgens (340, 341) makes them unsuitable for long-term androgen replacement therapy.

Cross-over studies indicate that patients prefer testosterone formulations that maintain stable blood levels and smoother clinical effects. This is best achieved by testosterone products that form effective depots for sustained release such as long-acting testosterone implants (6 monthly) (657) and injectable testosterone undecanoate (3 monthly) (721, 722) or shorter-acting daily transdermal gels (71). These are an improvement over the previous standard of intramuscular injections of older testosterone esters (enanthate, mixed esters) in an oil vehicle every 2-3 weeks (655, 657, 658) which produce characteristically wide fluctuations in testosterone levels and corresponding roller-coaster symptomatic effects.

There are few well-established formulation or route-dependent differences between various testosterone products once adequate doses are administered. As with estrogen replacement (723, 724), testosterone effects on SHBG are effectively manifestations of hepatic overdose (725) so that oral ingestion of either 17a-alkylated androgens (726) or oral testosterone undecanoate (657) cause prominent lowering of SHBG levels due to prominent first-pass hepatic effects. By contrast long or short acting sustained-action testosterone depot products cause at most minor transient decreases, mirroring blood testosterone levels, or no effects on blood SHBG (590, 639, 657, 660, 721). The more convenient and well tolerated depot testosterone products which maintain steady-state delivery patterns (71, 590, 657, 721, 722) are supplanting the older, short-term (2-3 week) injectable testosterone esters (enanthate, mixed esters) as the mainstays of androgen replacement therapy.

Side Effects of Androgen Therapy

See also Endotext, Endocrinology of Male Reproduction, Androgens and Cardiovascular Disease in Men

Serious adverse effects from androgen replacement therapy using physiological testosterone doses for appropriate indications are rare. This corresponds to the observation that testosterone is the only hormone without a well defined, spontaneously occurring clinical syndrome of hormone excess in men. However, supraphysiological doses of synthetic androgens in pharmacological androgen therapy or the massive doses of androgen abusers as well as unphysiological use of androgens in chidren or women may produce unwanted androgenic side effects. Oral 17aalkylated androgens also risk a wide range of hepatic adverse effects. Virtually all androgenic side effects are rapidly reversible on cessation of treatment apart from inappropriate virilization in children or women in which voice deepening, ­terminal body hair, or stunting of final height may be irreversible.

STEROIDAL EFFECTS

Androgen replacement therapy activates physical and mental activity to enhance mood, behavior, and libido, thereby reversing their impairment during androgen deficiency (727). In otherwise healthy men, however, additional testosterone at doses equivalent to testosterone replacement doses (eg for male contraception or “andropause”) mood or behaviour changes are not evident (354, 728-734) or minimal (669). Even among healthy young men having very high androgen doses there are few mood or behaviour changes (488, 735-738) except for a small minority (~5%) of paid clinical trial vounteers who display a hypomanic reaction, reversible on androgen discontinuation (488). However, such adverse behavioural reactions were not observed in larger studies of testosterone administration to unpaid healthy men (666, 669, 739, 740). The higher prevalence of adverse behavioral effects reported among androgen abusers may be related not only to the massive androgen doses but also to high levels of background psychological disturbance (527), drug habituation (557), and anticipation (741) which predispose to behavioral disturbances reported during this form of drug abuse (727, 742).

Excessive or undesirable androgenic effects may be experienced during androgen therapy due to intrinsic androgenic effects in inappropriate settings (e.g., virilization in women or children). In a few untreated hypogonadal men, mainly in newly diagnosed older men, initiation of androgen treatment with standard doses occasionally produces an unfamiliar and even intolerable increase in libido and erection frequency. If this occurs, more gradual acclimatization to full testosterone dose with counseling of men and their partners may occasionally be helpful but usually adequate advice before starting treatment is sufficient.

Seborrhea and acne are commonly associated with high androgen levels in either the steep rise in endogenous testosterone during puberty or among androgen abusers. In contrast to the predominantly facial distribution of adolescent acne, androgen-induced acne with onset well after puberty is characteristically truncal in distribution and provides a useful clinical clue to androgen abuse (743). Acne is unusual during testosterone replacement therapy being mainly restricted to a few susceptible individuals during establishment of treatment with shorter-acting ­intramuscular testosterone esters, probably related to their generation of transient supraphysiologic testosterone concentrations in the days after injection (570, 655). Acne is rare with depot testosterone products that maintain steady-state physiologic blood testosterone levels. Androgen-induced acne is usually adequately managed with topical measures and/or broad-spectrum antibiotics, if required, with either dose reduction or a switch to steady-state delivery (gel, long-acting injectable) that avoids supraphysiologic peak blood testosterone concentrations. Increased body hair and temporal hair loss or balding may also be seen even with physiologic testosterone replacement in susceptible men.

Modest weight gain (up to 5kg) reflecting anabolic effects on muscle mass is also common. Gynecomastia is a feature of androgen deficiency but may appear during androgen replacement therapy, especially during use of aromatizable androgens such as testosterone that increase circulating estradiol levels at times when androgenic effects are inadequate (e.g., a too low or infrequent dose or unreliable compliance with treatment).

Obstructive sleep apnea causes a mild lowering of blood testosterone concentrations that is rectified by effective continuous positive airway pressure treatment (744). Although testosterone treatment has precipitated obstructive sleep apnea (745) and has potential adverse effects on sleep in older men (746), the prevalence of obstructive sleep apnea precipitated by testosterone treatment remains unclear. The risk is rare in younger androgen deficient men, but is higher among older men with the steeply rising background prevalence of obstructive sleep apnea with age. Hence, screening for obstructive sleep apnea by asking about daytime sleepiness and partner reports of loud and irregular snoring, especially among overweight men with large collar size, is wise for older men starting testosterone treatment but not routinely required for young men with classic androgen deficiency.

THROMBOEMBOLISM

Testosterone treatment of men without pathological hypogonadism were first reported to be associated with an increased risk of venous thromboembolism in a large UK general practice database (747) especially in the first 3-6 months after starting treatment (747-749). These findings were confirmed in another study (750) whereas other studies (751-754) considering only overall or cumulative thromboembolism risk without distinguishing early from later time periods following start of treatment, did not report an increased risk. The mechanism for early coagulopathies might be underlying thrombophilia-hypofibrinolysis due to Factor V Leiden, high lipoprotein (a) or lupus anticoagulant who are at risk of early thromboembolism (748) with recurrence despite adequate anticoagulation (755); however, whether testosterone treatment increases risk remains unclear. Endogenous testosterone is reportedly a risk factor for thromboembolism in a two sample Mendelian randomization study (756) but not confirmed in a 10-year follow-up of 1350 Norwegian men in a population-based study (757). 

HEPATOTOXICITY

Hepatotoxicity is a well-recognized but uncommon side effect of 17a-alkylated (340) whereas the occurrence of liver disorders in patients using non-17a alkylated androgens such as testosterone, nandrolone, and 1-methyl androgens (methenolone, mesterolone) are no more than by chance (341). This is consistent with the evidence of direct toxic effects on liver cells of alkylated but not non-alkylated androgens (758). The risk of 17a alkylated androgen-induced hepatotoxicity is unrelated to the indication for use, although association with certain underlying conditions may be related to intensity of diagnostic surveillance (341). It is possible, but unproven, that the risks are dose-dependent although relatively few cases are reported among women using low dose methyl-testosterone (759, 760) while clinical management of children using the alkylated androgen oxandolone often omits liver function tests. However, even if the risks are dose-dependent, the therapeutic margin is narrow. By contrast, the rates of hepatotoxicity among androgen abusers who typically use supraphysiological, often massive, doses remain difficult to quantify due to underreporting of the extent of illicit usage and dosage but abnormal liver function tests are common in androgen abusers when checked incidentally as part of other health evaluation.

Biochemical hepatotoxicity may involve either a cholestatic or hepatitic pattern and usually abates with cessation of steroid ingestion. Elevation of blood transaminases without gamma-glutamyl transferase may be attributable to rhabdomyolysis rather than to hepatotoxicity if confirmed by increased creatinine kinase (761). Major hepatic abnormalities are related to use of 17-alkylated androgens include peliosis hepatis (blood-filled cysts) (762) and hepatic rupture, adenoma, angiosarcoma (763, 764) and carcinoma; however, these risks do not apply to testosterone or other nonalkylated androgens such as nandrolone or 1-methyl androgens. Prolonged use of 17a-alkylated androgens, if unavoidable, requires regular clinical examination together with biochemical monitoring of hepatic function, the latter not required for non-alkylated androgens. If biochemical abnormalities are detected, treatment with 17a-alkylated androgens should cease and safer androgens may be substituted without concern. Where structural lesions are suspected, radionuclide scan, ultrasonography, or abdominal computed tomography scan should precede hepatic biopsy during which severe bleeding may be provoked in peliosis hepatis. Because equally effective and safer alternatives exist, the hepatotoxic 17a-alkylated androgens should not be used for long-term androgen replacement therapy. By contrast, pharmacological androgen therapy often uses 17a alkylated androgens for historical reasons rather than the non-hepatotoxic alternatives. In these situations, the risk-benefit analysis needs to be judged according to the clinical circumstances.

FORMULATION-RELATED EFFECTS

Complications related to testosterone products may be related to dosage, mode of administration or idiosyncratic reactions to constituents. Intramuscular injections of oil vehicle may cause local pain, bleeding, or bruising and, rarely, coughing fits or fainting due to pulmonary oil microembolization (POME) (765) as a minor variant of accidental self-injection oil embolism (766, 767). In a study of over 3000 consecutive injections by experienced nurses, POME occurred at a rate of ~2% (768) but is often unrecognised or under-reported (769) due to the transient symptoms. There was also no bruising or bleeding reported even among men using anticoagulants and/or antiplatelet drugs (upper confidence limit of risk ~1%) (768). Inadvertent subcutaneous administration of the oil vehicle is highly irritating and may cause pain, inflammation, or even dermal necrosis. Allergy to the vegetable oil vehicle (sesame, castor, arachis) used in testosterone ester injections is very rare, and even patients allergic to peanuts may tolerate arachis (peanut) oil. Self-injection by body-builders of large volumes of sesame or other oils may cause exuberant local injection site reactions (770) or even oil embolism (766). Long-term fibrosis at intramuscular injection sites might be expected but has not been reported. Oral testosterone undecanoate may causes gastrointestinal intolerance due to the castor oil/propylene glycol laurate suspension vehicle. Testosterone implants may be associated with extrusion of implants or bleeding, infection, or scarring at implant sites (594). Parenteral injection of testosterone undecanoate (721) or biodegradable microspheres (640) involves a large injection volume that may cause discomfort. Transdermal patches applied to the trunk cause skin irritation in most men, some with quite severe burn-like lesions (609, 771) with a significant minority (~20%) are unable to continue use . Skin irritation may be reduced in prevalence or ameliorated by concurrent use of topical corticosteroid cream at the application site (610) while transdermal testosterone gels (571) or solution (621) are rarely irritating. Topical testosterone gels can cause virilization via transfer of ­androgens through topical skin-to-skin contact with children (626-631) or sexual partners (623, 624). These problems can be avoided by covering the application site with clothing or washing off excess gel after a short time (772).

Monitoring of Androgen Replacement Therapy

Monitoring of androgen replacement therapy involves, pri­marily, clinical observations to optimize androgen effects including ensuring the continuation of treatment and surveillance for side effects. Once testosterone dosage is well established, androgen replacement therapy requires only very limited, judicious use of biochemical ­testing or hormone assays to verify adequacy of dosage when in doubt or following changes of product or dosage. Testosterone and its esters at conventional doses for replacement therapy are sufficiently safe not to require routine biochemical monitoring of liver, kidney or electrolytes.

Clinical monitoring depends on serial observation of improvement in the key presenting features of androgen deficiency. Androgen-deficient men as a group may report subjective improvement in one or more of a variety of symptoms (some only recognized in retrospect) including energy, well-being, psychosocial drive, initiative, and assertiveness as well as sexual activity (especially libido and ejaculation frequency), increased truncal and facial hair growth and muscular strength and endurance. Individual men will become familiar with their own leading androgen deficiency symptom(s), and these appear in predictable sequence and at consistent blood testosterone thresholds towards the end of any treatment cyce (315, 773). Subjective symptoms of genuine androgen deficiency are alleviated quickly, typically within 3 weeks and reach plateau within 2-3 months (774) whereas persistent symptoms after 3 months may represent placebo responses reflecting the non-specificity of androgen deficiency symptoms and the unusually prominent expectations in the community for testosterone treatment. Objective and sensitive measures of androgen action are highly desirable but not available for most androgen-responsive tissues (775). The main biochemical measures available for monitoring of androgenic effects include hemoglobin and trough reproductive hormone (testosterone, LH, FSH) levels. In androgen deficient men, hemoglobin typically increases by ~10% (or up to 20 g/L) with standard testosterone doses (352, 570, 776). Excessive hemoglobin responses (hematocrit ≥0.54, or ≥0.50 with higher risk of cardio- or ceberebrovascular ischemia) occur as a rare (~1%) idiosyncratic reaction which is more frequent at older age (352) explaining the higher prevalence of polycythemia in older testosterone-treated men (777). Testosterone-induced polycythemia is dose-dependent (352, 778) being related to the supraphysiological peak blood testosterone levels observed with shorter-acting testosterone ester injections (570) or trough blood testosterone during treatment with injectable testosterone (778) although it can occur at high enough androgen doses in older men even with transdermal products (779). Such androgen-induced secondary polycythemia is characteristically negative for JAK2 mutations distinguishing it from primary polycythemia rubra vera (780) and usually resolves with reducing testosterone dose and/or switching to more steady-state testosterone delivery systems (implants, injectable testosterone undecanoate or transdermal gel) (781) and only very rarely is venesection and/or anticoagulation required. Circulating testosterone and gonadotropin levels must be considered in relation to time since last testosterone dose. Trough levels (immediately before next scheduled dose) may be helpful in establishing adequacy of depot testosterone regimens. In the presence of normal testosterone, negative feedback on hypothalamic GnRH and pituitary LH secretion (in men with hyper­gonadotropic hypogonadism), plasma LH levels are elevated in rough proportion to the degree of androgen deficiency. In severe androgen deficiency, virtually castrate LH levels may be present, and, conversely, circulating LH levels provide a sensitive and specific index of tissue testosterone effects (590, 655) especially with more steady-state testosterone delivery by depot-type products. Suppression of LH into the eugonadal range indicates adequate androgen replacement therapy, whereas persistent nonsuppression after the first few months of treatment is an indication of inadequate dose or pattern of testosterone levels. In hypogonadotropic hypogonadism, however, impaired hypothalamic-pituitary function diminishes circulating LH levels regardless of androgen effects, so blood LH levels do not reflect tissue androgenic effects.

Blood testosterone measurements are valuable before treatment for diagnosis and after start of treatment to check adequacy of dosage if in doubt and, during long-term treatment, only to evaluate changes in treatment dosage or product. During depot testosterone treatment which achieves quasi steady-state blood testosterone levels, trough blood testosterone levels taken prior to the next dose may detect patients whose treatment is suboptimal and whose dose and/or treatment interval need modification. Blood testosterone levels are not helpful for monitoring of oral testosterone undecanoate while pharmacological androgen therapy using any synthetic androgens would lower endogenous blood testosterone levels. Serial evaluation of bone density (especially vertebral trabecular bone) by dual photon absorptiometry at 1- to 2-year intervals may be helpful as a time-integrated measure to verify the adequacy of tissue androgen effects (420, 572).

Although chronic androgen deficiency protects against prostate disease (130, 782, 783), prostate size of androgen-deficient men receiving androgen replacement therapy is restored to, but does not exceed, age-appropriate norms (784, 785). Even prolonged (2 years) high doses of exogenous DHT did not significantly increase age-related prostate growth in middle-aged men without known prostate disease (681). Between-subject variability in response to testosterone replacement is partly explained by genetic sensitivity to testosterone, which is inversely related to length of the CAG triplet (polyglutamine) repeat polymorphism in exon 1 of the androgen receptor (235). Furthermore, because neither endogenous blood testosterone nor circulating levels of other androgen predicts subse­quent development of prostate cancer (454), maintaining physiologic testosterone concentrations should ensure no higher rates of prostate disease than eugonadal men of ­similar age (786).

The potential long-term risks for cardiovascular disease of androgen replacement and pharmacologic androgen therapy remain uncertain. Although men have two to three times the prevalence (430) as well as earlier onset and more severe atherosclerotic cardiovascular disease than women, the precise role of blood testosterone and of androgen treatment in this marked gender disparity is still poorly understood (309). Although low blood testosterone concentration is a risk factor for cardiovascular disease and testosterone effects include vasodilation and amelioration of coronary ischemia as well as potentially deleterious effects, it is not possible to predict the net clinical risk-benefit of androgen replacement therapy on cardiovascular disease. Hence, during androgen replacement therapy, it is prudent to aim at maintaining physiologic testosterone concentrations and surveillance of cardiovascular and prostate disease should be comparable with, and no more intensive than, that for eugonadal men of equivalent age (786). The effects of pharmacologic androgen therapy, in which the androgen dose is not necessarily restricted to eugonadal limits, on cardiovascular and prostate disease are still more difficult to predict, and surveillance then depends on the nature, severity, and life expectancy of the underlying ­disease.

Contraindications and Precautions for Androgen Replacement Therapy

Contraindications to androgen replacement therapy are prostate or breast cancer, because these tumors may be androgen responsive, and pregnancy, in which transplacental passage of androgens may disturb fetal sexual differentiation, notably risking virilization of a female fetus.

The Nobel Prize-winning recognition in the 1940’s that prostate cancer was androgen dependent led to castration being ever since the main treatment for advanced prostate cancer for which it prolongs life but is not curative. This approach led to long-held concern about testosterone treatment of men with advanced prostate cancer (286) for fear of relapse, based however, largely on anecdotal observations (787, 788). Recent studies have challenged this belief as intermittent rather than sustained androgen blockade (789), rapid androgen cycling (790), androgen priming (791, 792) or even testosterone administration (793, 794) have all shown promising, albeit counter-intuitive, results. Meta-analyses suggest that neither ambient circulating testosterone concentrations nor testosterone treatment predict future prostate cancer (454, 455, 795). Furthermore, the increasing diagnosis of organ-confined prostate cancer detected by PSA screening among younger men requires different considerations including the continuation of testosterone replacement therapy following curative treatment of the prostate cancer with careful monitoring (796-798). This is consistent with the fact that endogenous circulating androgens (testosterone, dihydrotestosterone) do not predict subsequent prostate cancer (454) and even prolonged (2 year) administration of high doses of exogenous DHT does not accelerate mid-life prostate growth rate in middle-aged men without prostate disease (681) presumably because exogenous DHT does not increase intra-prostatic androgen concentration (683). Hence local, organ-confined prostate cancer following treatment with curative intent may be an exception to the otherwise absolute contraindication to testosterone for men with a diagnosis of prostate cancer.

Precautions and/or careful monitoring of androgen use are required in (1) initiating treatment in older men with newly diagnosed androgen deficiency who may experience unfamiliar and intolerable initial changes in libido; (2) men subject to occupational monitoring by drug testing (including elite athletes) who may be sanctioned or disqualified for drug use; (3) androgen deficient men with residual spermatogenesis who are planning fertility in the near future who may wish to delay or bank sperm prior to starting treatment; (4) women of reproductive age, especially those who use their voice professionally, who may become irreversibly virilized; (5) prepubertal children in whom inappropriate androgen treatment risks precocious ­sexual development, virilization and premature epiphyseal closure with compromised final adult height; (6) patients with bleeding disorders or those undergoing anticoagulation or antiplatelet treatment when parenteral admin­istration may cause severe bruising or bleeding; (7) sex steroid–sensitive epilepsy or migraine; and (8) older and especially obese men with subclinical obstructive sleep apnea.

Some traditional warnings about risks of androgen treatment which appear on older product information appear to be rarely or never observed in modern clinical practice. An example of this is hypercalcemia, originally described during pharmacological androgen therapy for advanced breast cancer with metastases (799) although direct causation was not well established (800), but this not been reported with androgen use for other indications. Similarly, fluid overload from sodium and fluid retention due to cardiac or renal failure or severe hypertension is rare and probably confined to high dose pharmacological androgen therapy (799) whereas controlled clinical trials suggest androgens may improve cardiac function and quality of life (394), rather than having detrimental effects, in men with chronic heart failure.

REFERENCES

1.       Quigley CA, DeBellis A, Marschke KB, El-Awady MK, Wilson EM, French FF (1995) Androgen receptor defects: historical, clinical and molecular perspectives. Endocrine Reviews 16:271-321

2.       Foradori CD, Weiser MJ, Handa RJ (2008) Non-genomic actions of androgens. Frontiers in Neuroendocrinology 29:169-181

3.       Michels G, Hoppe UC (2008) Rapid actions of androgens. Frontiers in Neuroendocrinology 29:182-198

4.       Gonzalez-Montelongo MC, Marin R, Gomez T, Diaz M (2010) Androgens are powerful non-genomic inducers of calcium sensitization in visceral smooth muscle. Steroids 75:533-538

5.       Kochakian CD ed. 1976 Anabolic-Androgenic Steroids. Berlin: Springer-Verlag

6.       Nieschlag E, Behre HM eds. 2012 Testosterone: Action.Deficiency.Substitution. 4th ed. Cambridge: Cambridge University Press

7.       Hall PF 1988 Testicular steroid synthesis: organization and regulation. In: Knobil E, Neill J eds. The Physiology of Reproduction. New York: Raven Press; 975-998

8.       Miller WL, Auchus RJ (2011) The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine Reviews 32:81-151

9.       Neaves WB, Johnson L, Porter JC, Parker CR, Petty CS (1984) Leydig cell numbers, daily sperm production, and serum gonadotropin levels in aging men. Journal of Clinical Endocrinology and Metabolism 55:756-763

10.     Miller WL, Tee MK (2015) The post-translational regulation of 17,20 lyase activity. Molecular and Cellular Endocrinology 408:99-106

11.     Peng HM, Im SC, Pearl NM, Turcu AF, Rege J, Waskell L, Auchus RJ (2016) Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production. Biochemistry 55:4356-4365

12.     Labrie F (2004) Adrenal androgens and intracrinology. Seminars in Reproductive Medicine 22:299-309

13.     Oesterling JE, Epstein JI, Walsh PC (1986) The inability of adrenal androgens to stimulate the adult human prostate: an autopsy evaluation of men with hypogonadotropic hypogonadism and panhypopituitarism. Journal of Urology 136:1030-1034

14.     Young J, Couzinet B, Nahoul K, Brailly S, Chanson P, Baulieu EE, Schaison G (1997) Panhypopituitarism as a model to study the metabolism of dehydroepiandrosterone (DHEA) in humans. Journal of Clinical Endocrinology and Metabolism 82:2578-2585

15.     Arlt W, Justl HG, Callies F, Reincke M, Hubler D, Oettel M, Ernst M, Schulte HM, Allolio B (1998) Oral dehydroepiandrosterone for adrenal androgen replacement: pharmacokinetics and peripheral conversion to androgens and estrogens in young healthy females after dexamethasone suppression. Journal of Clinical Endocrinology and Metabolism 83:1928-1934

16.     Davison SL, Bell R, Donath S, Montalto JG, Davis SR (2005) Androgen levels in adult females: changes with age, menopause, and oophorectomy. Journal of Clinical Endocrinology and Metabolism 90:3847-3853

17.     Gallegos AM, Atshaves BP, Storey SM, Starodub O, Petrescu AD, Huang H, McIntosh AL, Martin GG, Chao H, Kier AB, Schroeder F (2001) Gene structure, intracellular localization, and functional roles of sterol carrier protein-2. Progress in Lipid Research 40:498-563

18.     Miller WL (2016) Disorders in the initial steps of steroid hormone synthesis. Journal of Steroid Biochemistry and Molecular Biology

19.     Midzak A, Zirkin B, Papadopoulos V (2015) Translocator protein: pharmacology and steroidogenesis. Biochemical Society Transactions 43:572-578

20.     Jarow JP, Zirkin BR (2005) The androgen microenvironment of the human testis and hormonal control of spermatogenesis. Annals of the New York Academy of Sciences 1061:208-220

21.     Gray A, Berlin JA, McKinlay JB, Longcope C (1991) An examination of research design effects on the association of testosterone and male aging: results of a meta-analysis. Journal of Clinical Epidemiology 44:671-684

22.     Kaufman JM, Vermeulen A (2005) The decline of androgen levels in elderly men and its clinical and therapeutic implications. Endocrine Reviews 26:833-876

23.     Wu FC, Tajar A, Pye SR, Silman AJ, Finn JD, O'Neill TW, Bartfai G, Casanueva F, Forti G, Giwercman A, Huhtaniemi IT, Kula K, Punab M, Boonen S, Vanderschueren D (2008) Hypothalamic-pituitary-testicular axis disruptions in older men are differentially linked to age and modifiable risk factors: the European Male Aging Study. Journal of Clinical Endocrinology and Metabolism 93:2737-2745

24.     Sartorius G, Spasevska S, Idan A, Turner L, Forbes E, Zamojska A, Allan C, Ly L, Conway A, McLachlan R, Handelsman D (2012) Serum Testosterone, Dihydrotestosterone and Estradiol Concentrations in Older Men Self-Reporting Very Good Health:The Healthy Man Study. Clinical Endocrinology 77:755-763

25.     Handelsman DJ, Staraj S (1985) Testicular size: the effects of aging, malnutrition, and illness. Journal of Andrology 6:144-151

26.     Gray A, Feldman HA, McKinlay JB, Longcope C (1991) Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachussetts Male Aging Study. Journal of Clinical Endocrinology and Metabolism 73:1016-1025

27.     Feldman HA, Longcope C, Derby CA, Johannes CB, Araujo AB, Coviello AD, Bremner WJ, McKinlay JB (2002) Age trends in the level of serum testosterone and other hormones in middle-aged men: longitudinal results from the Massachusetts male aging study. Journal of Clinical Endocrinology and Metabolism 87:589-598

28.     Andersson AM, Jensen TK, Juul A, Petersen JH, Jorgensen T, Skakkebaek NE (2007) Secular decline in male testosterone and sex hormone binding globulin serum levels in Danish population surveys. Journal of Clinical Endocrinology and Metabolism 92:4696-4705

29.     Travison TG, Araujo AB, Hall SA, McKinlay JB (2009) Temporal trends in testosterone levels and treatment in older men. Curr Opin Endocrinol Diabetes Obes 16:211-217

30.     Perheentupa A, Makinen J, Laatikainen T, Vierula M, Skakkebaek NE, Andersson AM, Toppari J (2013) A cohort effect on serum testosterone levels in Finnish men. European Journal of Endocrinology 168:227-233

31.     Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, Lacroix I, Somma-Delpero C, Boudou P (2003) Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clinical Chemistry 49:1381-1395

32.     Sikaris K, McLachlan RI, Kazlauskas R, de Kretser D, Holden CA, Handelsman DJ (2005) Reproductive hormone reference intervals for healthy fertile young men: evaluation of automated platform assays. Journal of Clinical Endocrinology and Metabolism 90:5928-5936.

33.     Vermeulen A, Desylpere JP, Kaufman JM (1989) Influence of antiopioids on luteinizing hormone pulsatility in aging men. Journal of Clinical Endocrinology and Metabolism 68:68-72

34.     Deslypere JP, Kaufman JM, Vermeulen T, Vogelaers D, Vandalem JL, Vermeulen A (1987) Influence of age on pulsatile luteinizing hormone release and responsiveness of the gonadotrophs to sex hormone feedback in men. Journal of Clinical Endocrinology and Metabolism 64:68-73

35.     Veldhuis JD, Urban RJ, Lizarralde G, Johnson ML, Iranmanesh A (1992) Attenuation of luteinizing hormone secretory burst amplitude as a proximate basis for the hypoandrogenism of healthy aging men. Journal of Clinical Endocrinology and Metabolism 75:707-713

36.     Liu PY, Pincus SM, Takahashi PY, Roebuck PD, Iranmanesh A, Keenan DM, Veldhuis JD (2006) Aging attenuates both the regularity and joint synchrony of LH and testosterone secretion in normal men: analyses via a model of graded GnRH receptor blockade. Am J Physiol Endocrinol Metab 290:E34-E41

37.     Mulligan T, Iranmanesh A, Gheorghiu S, Godschalk M, Veldhuis JD (1995) Amplified nocturnal luteinizing hormone (LH) secretory burst frequency with selective attenuation of pulsatile (but not basal) testosterone secretion in healthy aged men: possible Leydig cell desensitization to endogenous LH signaling--a clinical research center study. Journal of Clinical Endocrinology and Metabolism 80:3025-3031

38.     Mulligan T, Iranmanesh A, Kerzner R, Demers LW, Veldhuis JD (1999) Two-week pulsatile gonadotropin releasing hormone infusion unmasks dual (hypothalamic and Leydig cell) defects in the healthy aging male gonadotropic axis. European Journal of Endocrinology 141:257-266

39.     Liu PY, Takahashi PY, Roebuck PD, Iranmanesh A, Veldhuis JD (2005) Aging in healthy men impairs recombinant human luteinizing hormone (LH)-stimulated testosterone secretion monitored under a two-day intravenous pulsatile LH clamp. Journal of Clinical Endocrinology and Metabolism 90:5544-5550

40.     Regadera J, Nistal M, Paniagua R (1985) Testis, epididymis, and spermatic cord in elderly men: correlation of angiographic and histologic studies with systemic arteriosclerosis. Archives of Pathology and Laboratory Medicine 109:663-667

41.     Veldhuis JD, Keenan DM, Iranmanesh A (2005) Mechanisms of ensemble failure of the male gonadal axis in aging. Journal of Endocrinological Investigation 28:8-13

42.     Veldhuis JD, Keenan DM, Liu PY, Iranmanesh A, Takahashi PY, Nehra AX (2009) The aging male hypothalamic-pituitary-gonadal axis: pulsatility and feedback. Molecular and Cellular Endocrinology 299:14-22

43.     Prostate Cancer Trialists' Collaborative Group (2000) Maximum androgen blockade in advanced prostate cancer: an overview of the randomised trials. Lancet 355:1491-1498

44.     Arlt W, Callies F, Koehler I, van Vlijmen JC, Fassnacht M, Strasburger CJ, Seibel MJ, Huebler D, Ernst M, Oettel M, Reincke M, Schulte HM, Allolio B (2001) Dehydroepiandrosterone supplementation in healthy men with an age-related decline of dehydroepiandrosterone secretion. Journal of Clinical Endocrinology and Metabolism 86:4686-4692

45.     Nair KS, Rizza RA, O'Brien P, Dhatariya K, Short KR, Nehra A, Vittone JL, Klee GG, Basu A, Basu R, Cobelli C, Toffolo G, Dalla Man C, Tindall DJ, Melton LJ, 3rd, Smith GE, Khosla S, Jensen MD (2006) DHEA in elderly women and DHEA or testosterone in elderly men. New England Journal of Medicine 355:1647-1659

46.     Baird DT, Horton R, Longcope C, Tait JF (1969) Steroid dynamics under steady-state conditions. Recent Progress in Hormone Research 25:611-664

47.     Gurpide E 1975 Tracer Methods in Hormone Research. New York: Springer

48.     Setchell BP 1978 The Mammalian Testis. London: Paul Elek

49.     Southren AL, Gordon GG, Tochimoto S (1968) Further studies of factors affecting metabolic clearance rate of testosterone in man. Journal of Clinical Endocrinology and Metabolism 28:1105-1112

50.     Santner S, Albertson B, Zhang GY, Zhang GH, Santulli M, Wang C, Demers LM, Shackleton C, Santen RJ (1998) Comparative rates of androgen production and metabolism in Caucasian and Chinese subjects. Journal of Clinical Endocrinology and Metabolism 83:2104-2109

51.     Wang C, Catlin DH, Starcevic B, Leung A, DiStefano E, Lucas G, Hull L, Swerdloff RS (2004) Testosterone metabolic clearance and production rates determined by stable isotope dilution/tandem mass spectrometry in normal men: influence of ethnicity and age. Journal of Clinical Endocrinology and Metabolism 89:2936-2941

52.     Ishimaru T, Edmiston WA, Pages L, Horton R (1978) Splanchnic extraction and conversion of testosterone and dihydrotestosterone in man. Journal of Clinical Endocrinology and Metabolism 46:528-533

53.     Longcope C, Sato K, McKay C, Horton R (1984) Aromatization by splanchnic tissue in men. Journal of Clinical Endocrinology and Metabolism 58:1089-1093

54.     Diver MJ, Imtiaz KE, Ahmad AM, Vora JP, Fraser WD (2003) Diurnal rhythms of serum total, free and bioavailable testosterone and of SHBG in middle-aged men compared with those in young men. Clinical Endocrinology 58:710-717

55.     Bremner WJ, Vitiello MV, Prinz PN (1983) Loss of circadian rhythmicity in blood testosterone levels with aging in normal men. Journal of Clinical Endocrinology and Metabolism 56:1278-1281

56.     Keenan DM, Takahashi PY, Liu PY, Roebuck PD, Nehra AX, Iranmanesh A, Veldhuis JD (2006) An ensemble model of the male gonadal axis: illustrative application in aging men. Endocrinology 147:2817-2828

57.     Petra P, Stanczyk FZ, Namkung PC, Fritz MA, Novy ML (1985) Direct effect of sex-steroid binding protein (SBP) of plasma on the metabolic clearance rate of testosterone in the rhesus macaque. Journal of Steroid Biochemistry and Molecular Biology 22:739-746

58.     Vanbillemont G, Bogaert V, De Bacquer D, Lapauw B, Goemaere S, Toye K, Van Steen K, Taes Y, Kaufman JM (2009) Polymorphisms of the SHBG gene contribute to the interindividual variation of sex steroid hormone blood levels in young, middle-aged and elderly men. Clinical Endocrinology 70:303-310

59.     Ohlsson C, Wallaschofski H, Lunetta KL, Stolk L, Perry JR, Koster A, Petersen AK, Eriksson J, Lehtimaki T, Huhtaniemi IT, Hammond GL, Maggio M, Coviello AD, Ferrucci L, Heier M, Hofman A, Holliday KL, Jansson JO, Kahonen M, Karasik D, Karlsson MK, Kiel DP, Liu Y, Ljunggren O, Lorentzon M, Lyytikainen LP, Meitinger T, Mellstrom D, Melzer D, Miljkovic I, Nauck M, Nilsson M, Penninx B, Pye SR, Vasan RS, Reincke M, Rivadeneira F, Tajar A, Teumer A, Uitterlinden AG, Ulloor J, Viikari J, Volker U, Volzke H, Wichmann HE, Wu TS, Zhuang WV, Ziv E, Wu FC, Raitakari O, Eriksson A, Bidlingmaier M, Harris TB, Murray A, de Jong FH, Murabito JM, Bhasin S, Vandenput L, Haring R (2011) Genetic determinants of serum testosterone concentrations in men. PLoS Genet 7:e1002313

60.     Jin G, Sun J, Kim ST, Feng J, Wang Z, Tao S, Chen Z, Purcell L, Smith S, Isaacs WB, Rittmaster RS, Zheng SL, Condreay LD, Xu J (2012) Genome-wide association study identifies a new locus JMJD1C at 10q21 that may influence serum androgen levels in men. Human Molecular Genetics 21:5222-5228

61.     Xita N, Tsatsoulis A (2010) Genetic variants of sex hormone-binding globulin and their biological consequences. Molecular and Cellular Endocrinology 316:60-65

62.     Goldman AL, Bhasin S, Wu FCW, Krishna M, Matsumoto AM, Jasuja R (2017) A Reappraisal of Testosterone's Binding in Circulation: Physiological and Clinical Implications. Endocr Rev 38:302-324

63.     Dunn JF, Nisula BC, Rodbard D (1981) Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. Journal of Clinical Endocrinology and Metabolism 53:58-68

64.     Fournier T, Medjoubi NN, Porquet D (2000) Alpha-1-acid glycoprotein. Biochimica et Biophysica Acta 1482:157-171

65.     Hammond GL, Wu TS, Simard M (2012) Evolving utility of sex hormone-binding globulin measurements in clinical medicine. Curr Opin Endocrinol Diabetes Obes 19:183-189

66.     Wu TS, Hammond GL (2014) Naturally occurring mutants inform SHBG structure and function. Molecular Endocrinology 28:1026-1038

67.     Luppa PB, Thaler M, Schulte-Frohlinde E, Schreiegg A, Huber U, Metzger J, Fahrner CL, Hackney AC (2006) Unchanged androgen-binding properties of sex hormone-binding globulin in male patients with liver cirrhosis. Clinical Chemistry and Laboratory Medicine 44:967-973

68.     Selva DM, Hammond GL (2006) Human sex hormone-binding globulin is expressed in testicular germ cells and not in sertoli cells. Hormone and Metabolic Research 38:230-235

69.     Diaz L, Queipo G, Carino C, Nisembaum A, Larrea F (1997) Biologically active steroid and thyroid hormones stimulate secretion of sex hormone-binding globulin by human term placenta in culture. Archives of Medical Research 28:29-36

70.     Handelsman DJ, Conway AJ, Boylan LM (1990) Pharmacokinetics and pharmacodynamics of testosterone pellets in man. Journal of Clinical Endocrinology and Metabolism 71:216-222

71.     Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Berman N, Hull L, Swerdloff RS (2004) Long-term testosterone gel (AndroGel) treatment maintains beneficial effects on sexual function and mood, lean and fat mass, and bone mineral density in hypogonadal men. Journal of Clinical Endocrinology and Metabolism 89:2085-2098

72.     Wittert GA, Harrison RW, Buckley MJ, Wlodarczyk J (2016) An open-label, phase 2, single centre, randomized, crossover design bioequivalence study of AndroForte 5 testosterone cream and Testogel 1% testosterone gel in hypogonadal men: study LP101. Andrology 4:41-45

73.     Jaruvongvanich V, Sanguankeo A, Riangwiwat T, Upala S (2017) Testosterone, Sex Hormone-Binding Globulin and Nonalcoholic Fatty Liver Disease: a Systematic Review and Meta-Analysis. Ann Hepatol 16:382-394

74.     Sarkar M, VanWagner LB, Terry JG, Carr JJ, Rinella M, Schreiner PJ, Lewis CE, Terrault N (2019) Sex Hormone-Binding Globulin Levels in Young Men Are Associated With Nonalcoholic Fatty Liver Disease in Midlife. Am J Gastroenterol 114:758-763

75.     Ahrentsen OD, Jensen HK, Johnsen SG (1982) Sex-hormone-binding globulin deficiency. Lancet 2:377

76.     Hogeveen KN, Cousin P, Pugeat M, Dewailly D, Soudan B, Hammond GL (2002) Human sex hormone-binding globulin variants associated with hyperandrogenism and ovarian dysfunction. Journal of Clinical Investigation 109:973-981

77.     Vos MJ, Mijnhout GS, Rondeel JM, Baron W, Groeneveld PH (2014) Sex hormone binding globulin deficiency due to a homozygous missense mutation. Journal of Clinical Endocrinology and Metabolism 99:E1798-1802

78.     Pardridge WM (1987) Plasma protein-mediated transport of steroid and thyroid hormones. American Journal of Physiology 252:E157-164

79.     Mendel CM (1989) The free hormone hypothesis: a physiologically based mathematical model. Endocrine Reviews 10:232-274

80.     Ekins R (1990) Measurement of free hormones in blood. Endocrine Reviews 11:5-46

81.     Rowland M, Tozer TN 2011 Chapter 17: Drug Interactions. In: Clinical Pharmacokinetics and Pharmacodynamics: Concepts and Applications. 4th ed. Baltimore: Wolters Kluwer/Lippincott Willaims & Wilkins; 483-525

82.     Queipo G, Deas M, Arranz C, Carino C, Gonzalez R, Larrea F (1998) Sex hormone-binding globulin stimulates chorionic gonadotrophin secretion from human cytotrophoblasts in culture. Human Reproduction 13:1368-1373

83.     Rosner W, Hryb DJ, Khan MS, Nakhla AM, Romas NA (1999) Sex hormone-binding globulin mediates steroid hormone signal transduction at the plasma membrane. Journal of Steroid Biochemistry and Molecular Biology 69:481-485

84.     Rosner W, Hryb DJ, Khan MS, Nakhla AM, Romas NA (1999) Androgen and estrogen signaling at the cell membrane via G-proteins and cyclic adenosine monophosphate. Steroids 64:100-106

85.     Kahn SM, Li YH, Hryb DJ, Nakhla AM, Romas NA, Cheong J, Rosner W (2008) Sex hormone-binding globulin influences gene expression of LNCaP and MCF-7 cells in response to androgen and estrogen treatment. Advances in Experimental Medicine and Biology 617:557-564

86.     Hamada A, Sissung T, Price DK, Danesi R, Chau CH, Sharifi N, Venzon D, Maeda K, Nagao K, Sparreboom A, Mitsuya H, Dahut WL, Figg WD (2008) Effect of SLCO1B3 haplotype on testosterone transport and clinical outcome in caucasian patients with androgen-independent prostatic cancer. Clinical Cancer Research 14:3312-3318

87.     Hammes A, Andreassen TK, Spoelgen R, Raila J, Hubner N, Schulz H, Metzger J, Schweigert FJ, Luppa PB, Nykjaer A, Willnow TE (2005) Role of endocytosis in cellular uptake of sex steroids. Cell 122:751-762

88.     Adams JS (2005) "Bound" to work: the free hormone hypothesis revisited. Cell 122:647-649

89.     Poole CN, Roberts MD, Dalbo VJ, Sunderland KL, Kerksick CM (2011) Megalin and androgen receptor gene expression in young and old human skeletal muscle before and after three sequential exercise bouts. J Strength Cond Res 25:309-317

90.     Holt SK, Karyadi DM, Kwon EM, Stanford JL, Nelson PS, Ostrander EA (2008) Association of megalin genetic polymorphisms with prostate cancer risk and prognosis. Clinical Cancer Research 14:3823-3831

91.     Handelsman DJ (2017) Free Testosterone: Pumping up the Tires or Ending the Free Ride? Endocr Rev 38:297-301

92.     Hsu B, Cumming RG, Blyth FM, Naganathan V, Waite LM, Le Couteur DG, Seibel MJ, Handelsman DJ (2018) Evaluating Calculated Free Testosterone as a Predictor of Morbidity and Mortality Independent of Testosterone for Cross-sectional and 5-Year Longitudinal Health Outcomes in Older Men: The Concord Health and Ageing in Men Project. J Gerontol A Biol Sci Med Sci 73:729-736

93.     Sodergard R, Backstrom T, Shanbhag V, Carstensen H (1982) Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. Journal of Steroid Biochemistry 16:801-810

94.     Vermeulen A, Verdonck L, Kaufman JM (1999) A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 84:3666-3672

95.     Zakharov MN, Bhasin S, Travison TG, Xue R, Ulloor J, Vasan RS, Carter E, Wu F, Jasuja R (2015) A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin. Mol Cell Endocrinol 399:190-200

96.     Ly LP, Sartorius G, Hull L, Leung A, Swerdloff RS, Wang C, Handelsman DJ (2010) Accuracy of calculated free testosterone formulae in men. Clin Endocrinol (Oxf) 73:382-388

97.     Sartorius G, Ly LP, Sikaris K, McLachlan R, Handelsman DJ (2009) Predictive accuracy and sources of variability in calculated free testosterone estimates. Ann Clin Biochem 46:137-143

98.     Nanjee MN, Wheeler MJ (1985) Plasma free testosterone--is an index sufficient? Ann Clin Biochem 22 ( Pt 4):387-390

99.     Hackbarth JS, Hoyne JB, Grebe SK, Singh RJ (2011) Accuracy of calculated free testosterone differs between equations and depends on gender and SHBG concentration. Steroids 76:48-55

100.   Salameh WA, Redor-Goldman MM, Clarke NJ, Reitz RE, Caulfield MP (2010) Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: total and free testosterone reference ranges. Steroids 75:169-175

101.   Ellis GB, Desjardins C (1982) Male rats secrete luteinizing hormone and testosterone episodically. Endocrinology 110:1618-1627

102.   Coquelin A, Desjardins C (1982) Luteinizing hormone and testosterone secretion in young and old male mice. American Journal of Physiology 243:E257-263

103.   Shackleton C (2010) Clinical steroid mass spectrometry: a 45-year history culminating in HPLC-MS/MS becoming an essential tool for patient diagnosis. Journal of Steroid Biochemistry and Molecular Biology 121:481-490

104.   Rosner W, Auchus RJ, Azziz R, Sluss PM, Raff H (2007) Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. Journal of Clinical Endocrinology and Metabolism 92:405-413

105.   Herold DA, Fitzgerald RL (2003) Immunoassays for testosterone in women: better than a guess? Clinical Chemistry 49:1250-1251

106.   Umstot ES, Baxter JE, Andersen RN (1985) A theoretically sound and practicable equilibrium dialysis method for measuring percentage of free testosterone. Journal of Steroid Biochemistry 22:639-648

107.   Swinkels LM, Ross HA, Benraad TJ (1987) A symmetric dialysis method for the determination of free testosterone in human plasma. Clinica Chimica Acta 165:341-349

108.   Hammond GL, Nisker JA, Jones LA, Siiteri PK (1980) Estimation of the percentage of free steroid in undiluted serum by centrifugal ultrafiltration-dialysis. Journal of Biological Chemistry 255:5023-5026

109.   Vlahos I, MacMahon W, Sgoutas D, Bowers W, Thompson J, Trawick W (1982) An improved ultrafiltration method for determining free testosterone in serum. Clinical Chemistry 28:2286-2291

110.   Vermeulen A, Verdonck L, Kaufman JM (1999) A critical evaluation of simple methods for the estimation of free testosterone in serum. Journal of Clinical Endocrinology and Metabolism 84:3666-3672

111.   Rosner W (2001) An extraordinarily inaccurate assay for free testosterone is still with us. Journal of Clinical Endocrinology and Metabolism 86:2903.

112.   Fritz KS, McKean AJ, Nelson JC, Wilcox RB (2008) Analog-based free testosterone test results linked to total testosterone concentrations, not free testosterone concentrations. Clinical Chemistry 54:512-516

113.   Kapoor P, Luttrell BM, Williams D (1993) The free androgen index is not valid for adult males. Journal of Steroid Biochemistry and Molecular Biology 45:325-326

114.   Mazer NA (2009) A novel spreadsheet method for calculating the free serum concentrations of testosterone, dihydrotestosterone, estradiol, estrone and cortisol: with illustrative examples from male and female populations. Steroids 74:512-519

115.   Ly LP, Handelsman DJ (2005) Empirical estimation of free testosterone from testosterone and sex hormone-binding globulin immunoassays. European Journal of Endocrinology 152:471-478

116.   Ly LP, Sartorius G, Hull L, Leung A, Swerdloff RS, Wang C, Handelsman DJ (2010) Accuracy of calculated free testosterone formulae in men. Clinical Endocrinology 73:382-388

117.   Sartorius G, Ly LP, Sikaris K, McLachlan R, Handelsman DJ (2009) Predictive accuracy and sources of variability in calculated free testosterone estimates. Annals of Clinical Biochemistry 46:137-143

118.   Egleston BL, Chandler DW, Dorgan JF (2010) Validity of estimating non-sex hormone-binding globulin bound testosterone and oestradiol from total hormone measurements in boys and girls. Annals of Clinical Biochemistry 47:233-241

119.   Giton F, Fiet J, Guechot J, Ibrahim F, Bronsard F, Chopin D, Raynaud JP (2006) Serum bioavailable testosterone: assayed or calculated? Clinical Chemistry 52:474-481

120.   Van Eenoo P, Delbeke FT (2006) Metabolism and excretion of anabolic steroids in doping control--new steroids and new insights. Journal of Steroid Biochemistry and Molecular Biology 101:161-178

121.   Kumar N, Crozat A, Li F, Catterall JF, Bardin CW, Sundaram K (1999) 7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens. Journal of Steroid Biochemistry and Molecular Biology 71:213-222

122.   Deslypere JP, Young M, Wilson JD, McPhaul MJ (1992) Testosterone and 5 alpha-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Molecular and Cellular Endocrinology 88:15-22

123.   Zhou ZX, Lane MV, Kemppainen JA, French FS, Wilson EM (1995) Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability. Molecular Endocrinology 9:208-218

124.   McRobb L, Handelsman DJ, Kazlauskas R, Wilkinson S, McLeod MD, Heather AK (2008) Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay. Journal of Steroid Biochemistry and Molecular Biology 110:39-47

125.   Russell DW, Wilson JD (1994) Steroid 5 alpha-reductase: two genes/two enzymes. Annual Review of Biochemistry 63:25-61

126.   Thigpen AE, Davis DL, Milatovich A, Mendonca B, Imperato-McGinley J, Griffin JE, Francke U, Wilson JD, Russell DW (1992) Molecular genetics of steroid 5a-reductase 2 deficiency. Journal of Clinical Investigations 90:799-809

127.   Cai LQ, Fratiani CM, Gautier T, Imperato-McGinley J (1994) Dihydrotestosterone regulation of semen in males pseudohermaphrodites with 5-a reductase deficiency. Journal of Clinical Endocrinology and Metabolism 79:409-414

128.   Sobel V, Schwartz B, Zhu YS, Cordero JJ, Imperato-McGinley J (2006) Bone mineral density in the complete androgen insensitivity and 5alpha-reductase-2 deficiency syndromes. Journal of Clinical Endocrinology and Metabolism 91:3017-3023

129.   Imperato-McGinley J, Peterson RE, Gautier T, Sturla E (1979) Androgens and the evolution of male gender identity among male pseudohemaphrodites with 5-a reductase deficiency. New England Journal of Medicine 300:1233-1237

130.   Imperato-McGinley J, Gautier T, Zirinsky K, Hom T, Palomo O, Stein E, Vaughan ED, Markisz JA, deArellano ER, Kazam E (1992) Prostate visualization studies in males homozygous and heterozygous for 5-a reductase deficiency. Journal of Clinical Endocrinology and Metabolism 75:1022-1026

131.   Imperato-McGinley J, Zhu YS (2002) Androgens and male physiology the syndrome of 5alpha-reductase-2 deficiency. Molecular and Cellular Endocrinology 198:51-59

132.   Steers WD (2001) 5alpha-reductase activity in the prostate. Urology 58:17-24; discussion 24.

133.   Frick J, Aulitzky W (1991) Physiology of the prostate. Infection 19 Suppl 3:S115-118

134.   Gisleskog PO, Hermann D, Hammarlund-Udenaes M, Karlsson MO (1998) A model for the turnover of dihydrotestosterone in the presence of the irreversible 5 alpha-reductase inhibitors GI198745 and finasteride. Clinical Pharmacology and Therapeutics 64:636-647

135.   Miller LR, Partin AW, Chan DW, Bruzek DJ, Dobs AS, Epstein JI, Walsh PC (1998) Influence of radical prostatectomy on serum hormone levels. Journal of Urology 160:449-453

136.   Toorians AW, Kelleher S, Gooren LJ, Jimenez M, Handelsman DJ (2003) Estimating the contribution of the prostate to blood dihydrotestosterone. Journal of Clinical Endocrinology and Metabolism 88:5207-5211

137.   Zhu YS, Katz MD, Imperato-McGinley J (1998) Natural potent androgens: lessons from human genetic models. Baillieres Clinical Endocrinology and Metabolism 12:83-113

138.   Uemura M, Tamura K, Chung S, Honma S, Okuyama A, Nakamura Y, Nakagawa H (2008) Novel 5 alpha-steroid reductase (SRD5A3, type-3) is overexpressed in hormone-refractory prostate cancer. Cancer Sci 99:81-86

139.   Cantagrel V, Lefeber DJ, Ng BG, Guan Z, Silhavy JL, Bielas SL, Lehle L, Hombauer H, Adamowicz M, Swiezewska E, De Brouwer AP, Blumel P, Sykut-Cegielska J, Houliston S, Swistun D, Ali BR, Dobyns WB, Babovic-Vuksanovic D, van Bokhoven H, Wevers RA, Raetz CR, Freeze HH, Morava E, Al-Gazali L, Gleeson JG (2010) SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder. Cell 142:203-217

140.   Thompson IM, Goodman PJ, Tangen CM, Lucia MS, Miller GJ, Ford LG, Lieber MM, Cespedes RD, Atkins JN, Lippman SM, Carlin SM, Ryan A, Szczepanek CM, Crowley JJ, Coltman CA, Jr. (2003) The influence of finasteride on the development of prostate cancer. New England Journal of Medicine 349:215-224

141.   Andriole GL, Bostwick DG, Brawley OW, Gomella LG, Marberger M, Montorsi F, Pettaway CA, Tammela TL, Teloken C, Tindall DJ, Somerville MC, Wilson TH, Fowler IL, Rittmaster RS (2010) Effect of dutasteride on the risk of prostate cancer. New England Journal of Medicine 362:1192-1202

142.   Lucia MS, Epstein JI, Goodman PJ, Darke AK, Reuter VE, Civantos F, Tangen CM, Parnes HL, Lippman SM, La Rosa FG, Kattan MW, Crawford ED, Ford LG, Coltman CA, Jr., Thompson IM (2007) Finasteride and high-grade prostate cancer in the Prostate Cancer Prevention Trial. Journal of the National Cancer Institute 99:1375-1383

143.   Redman MW, Tangen CM, Goodman PJ, Lucia MS, Coltman CA, Jr., Thompson IM (2008) Finasteride does not increase the risk of high-grade prostate cancer: a bias-adjusted modeling approach. Cancer Prev Res (Phila) 1:174-181

144.   Kim J, Amos CI, Logothetis C (2011) 5alpha-Reductase inhibitors for prostate-cancer prevention. New England Journal of Medicine 365:2340

145.   Simpson ER, Zhao Y, Agarwal VR, Michael MD, Bulun SE, Hinshelwood MM, Graham-Lorence S, Sun T, Fisher CR, Qin K, Mendelson CR (1997) Aromatase expression in health and disease. Recent Progress in Hormone Research 52:185-213; discussion 213-184

146.   Bulun SE, Takayama K, Suzuki T, Sasano H, Yilmaz B, Sebastian S (2004) Organization of the human aromatase p450 (CYP19) gene. Seminars in Reproductive Medicine 22:5-9

147.   Naftolin F (1994) Brain aromatization of androgens. Journal of Reproductive Medicine 39:257-261

148.   Roselli CF (2007) Brain aromatase: roles in reproduction and neuroprotection. Journal of Steroid Biochemistry and Molecular Biology 106:143-150

149.   Jones ME, Boon WC, Proietto J, Simpson ER (2006) Of mice and men: the evolving phenotype of aromatase deficiency. Trends Endocrinol Metab 17:55-64

150.   Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O (1993) Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proceedings of the National Academy of Sciences USA 90:11162-11166

151.   Couse JE, Mahato D, Eddy EM, Korach KS (2001) Molecular mechanism of estrogen action in the male: insights from the estrogen receptor null mice. Reproduction, Fertility, and Development 13:211-219

152.   Gennari L, Nuti R, Bilezikian JP (2004) Aromatase activity and bone homeostasis in men. Journal of Clinical Endocrinology and Metabolism 89:5898-5907

153.   Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, Ohlsson C (2004) Androgens and bone. Endocrine Reviews 25:389-425

154.   Vandenput L, Swinnen JV, Boonen S, Van Herck E, Erben RG, Bouillon R, Vanderschueren D (2004) Role of the androgen receptor in skeletal homeostasis: the androgen-resistant testicular feminized male mouse model. Journal of Bone and Mineral Research 19:1462-1470

155.   Chesnut CH, 3rd, Ivey JL, Gruber HE, Matthews M, Nelp WB, Sisom K, Baylink DJ (1983) Stanozolol in postmenopausal osteoporosis: therapeutic efficacy and possible mechanisms of action. Metabolism: Clinical and Experimental 32:571-580

156.   Need AG, Nordin BEC, Chatterton BE (1993) Double-blind placebo-controlled trial of treatment of osteoporosis with the anabolic steroid nandrolone decanoate. Osteoporosis International 3 Suppl 1:S218-222

157.   Venken K, De Gendt K, Boonen S, Ophoff J, Bouillon R, Swinnen JV, Verhoeven G, Vanderschueren D (2006) Relative impact of androgen and estrogen receptor activation in the effects of androgens on trabecular and cortical bone in growing male mice: a study in the androgen receptor knockout mouse model. Journal of Bone and Mineral Research 21:576-585

158.   Callewaert F, Venken K, Ophoff J, De Gendt K, Torcasio A, van Lenthe GH, Van Oosterwyck H, Boonen S, Bouillon R, Verhoeven G, Vanderschueren D (2009) Differential regulation of bone and body composition in male mice with combined inactivation of androgen and estrogen receptor-alpha. FASEB Journal 23:232-240

159.   Zuloaga DG, Puts DA, Jordan CL, Breedlove SM (2008) The role of androgen receptors in the masculinization of brain and behavior: what we've learned from the testicular feminization mutation. Hormones and Behavior 53:613-626

160.   Finkelstein JS, Lee H, Burnett-Bowie SA, Pallais JC, Yu EW, Borges LF, Jones BF, Barry CV, Wulczyn KE, Thomas BJ, Leder BZ (2013) Gonadal steroids and body composition, strength, and sexual function in men. New England Journal of Medicine 369:1011-1022

161.   Sartorius GA, Ly LP, Handelsman DJ (2014) Male sexual function can be maintained without aromatization: randomized placebo-controlled trial of dihydrotestosterone (DHT) in healthy, older men for 24 months. J Sex Med 11:2562-2570

162.   Vanderschueren D, Gaytant J, Boonen S, Venken K (2008) Androgens and bone. Curr Opin Endocrinol Diabetes Obes 15:250-254

163.   Zhou SF (2008) Drugs behave as substrates, inhibitors and inducers of human cytochrome P450 3A4. Current Drug Metabolism 9:310-322

164.   Chouinard S, Yueh MF, Tukey RH, Giton F, Fiet J, Pelletier G, Barbier O, Belanger A (2008) Inactivation by UDP-glucuronosyltransferase enzymes: the end of androgen signaling. Journal of Steroid Biochemistry and Molecular Biology 109:247-253

165.   Jakobsson J, Ekstrom L, Inotsume N, Garle M, Lorentzon M, Ohlsson C, Roh HK, Carlstrom K, Rane A (2006) Large differences in testosterone excretion in Korean and Swedish men are strongly associated with a UDP-glucuronosyl transferase 2B17 polymorphism. Journal of Clinical Endocrinology and Metabolism 91:687-693

166.   Schulze JJ, Lundmark J, Garle M, Skilving I, Ekstrom L, Rane A (2008) Doping test results dependent on genotype of uridine diphospho-glucuronosyl transferase 2B17, the major enzyme for testosterone glucuronidation. Journal of Clinical Endocrinology and Metabolism 93:2500-2506

167.   Johnsen SG, Bennet EP, Jensen VG (1974) Therapeutic effectiveness of oral testosterone. Lancet ii:1473-1475

168.   Frey H, Aakvag A, Saanum D, Falch J (1979) Bioavailability of testosterone in males. European Journal of  Clinical Pharmacology 16:345-349

169.   Parkes AS (1938) Effective absorption of hormones. British Medical Journal:371-373

170.   Lisser H, Escamilla RF, Curtis LE (1942) Testosterone therapy of male eunuchoids. III Sublingual administration of testosterone compounds. Journal of Clinical Endocrinology 2:351-360

171.   Korbonits M, Slawik M, Cullen D, Ross RJ, Stalla G, Schneider H, Reincke M, Bouloux PM, Grossman AB (2004) A comparison of a novel testosterone bioadhesive buccal system, striant, with a testosterone adhesive patch in hypogonadal males. Journal of Clinical Endocrinology and Metabolism 89:2039-2043

172.   Wang C, Eyre DR, Clark R, Kleinberg D, Newman C, Iranmanesh A, Veldhuis J, Dudley RE, Berman N, Davidson T, Barstow TJ, Sinow R, Alexander G, Swerdloff RS (1996) Sublingual testosterone replacement improves muscle mass and strength, decreases bone resorption, and increases bone formation markers in hypogonadal men--a clinical research center study. Journal of Clinical Endocrinology and Metabolism 81:3654-3662

173.   Shackleford DM, Faassen WA, Houwing N, Lass H, Edwards GA, Porter CJ, Charman WN (2003) Contribution of lymphatically transported testosterone undecanoate to the systemic exposure of testosterone after oral administration of two andriol formulations in conscious lymph duct-cannulated dogs. Journal of Pharmacology and Experimental Therapeutics 306:925-933

174.   Foss GL (1939) Clinical administration of androgens. Lancet i:502-504

175.   Bousfield GR, Butnev VY, Gotschall RR, Baker VL, Moore WT (1996) Structural features of mammalian gonadotropins. Molecular and Cellular Endocrinology 125:3-19

176.   Gromoll J, Eiholzer U, Nieschlag E, Simoni M (2000) Male hypogonadism caused by homozygous deletion of exon 10 of the luteinizing hormone (LH) receptor: differential action of human chorionic gonadotropin and LH. Journal of Clinical Endocrinology and Metabolism 85:2281-2286

177.   O'Shaughnessy PJ, Baker P, Sohnius U, Haavisto AM, Charlton HM, Huhtaniemi I (1998) Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology 139:1141-1146

178.   Abreu AP, Dauber A, Macedo DB, Noel SD, Brito VN, Gill JC, Cukier P, Thompson IR, Navarro VM, Gagliardi PC, Rodrigues T, Kochi C, Longui CA, Beckers D, de Zegher F, Montenegro LR, Mendonca BB, Carroll RS, Hirschhorn JN, Latronico AC, Kaiser UB (2013) Central precocious puberty caused by mutations in the imprinted gene MKRN3. N Engl J Med 368:2467-2475

179.   Dauber A, Cunha-Silva M, Macedo DB, Brito VN, Abreu AP, Roberts SA, Montenegro LR, Andrew M, Kirby A, Weirauch MT, Labilloy G, Bessa DS, Carroll RS, Jacobs DC, Chappell PE, Mendonca BB, Haig D, Kaiser UB, Latronico AC (2017) Paternally Inherited DLK1 Deletion Associated With Familial Central Precocious Puberty. J Clin Endocrinol Metab 102:1557-1567

180.   Roberts S, Kaiser UB (2020) GENETICS IN ENDOCRINOLOGY: Genetic etiologies of central precocious puberty and the role of imprinted genes. Eur J Endocrinol

181.   Howard SR (2019) The Genetic Basis of Delayed Puberty. Front Endocrinol (Lausanne) 10:423

182.   Meikle AW, Bishop DT, Stringham JD, West DW (1986) Quantitating genetic and nongenetic factors that determine plasma sex steroid variation in normal male twins. Metabolism 35:1090-1095

183.   Eaves L, Silberg J, Foley D, Bulik C, Maes H, Erkanli A, Angold A, Costello EJ, Worthman C (2004) Genetic and environmental influences on the relative timing of pubertal change. Twin Res 7:471-481

184.   Grotzinger AD, Mann FD, Patterson MW, Herzhoff K, Tackett JL, Tucker-Drob EM, Paige Harden K (2018) Twin models of environmental and genetic influences on pubertal development, salivary testosterone, and estradiol in adolescence. Clin Endocrinol (Oxf) 88:243-250

185.   Silventoinen K, Haukka J, Dunkel L, Tynelius P, Rasmussen F (2008) Genetics of pubertal timing and its associations with relative weight in childhood and adult height: the Swedish Young Male Twins Study. Pediatrics 121:e885-891

186.   Day FR, Perry JR, Ong KK (2015) Genetic Regulation of Puberty Timing in Humans. Neuroendocrinology 102:247-255

187.   Busch AS, Hollis B, Day FR, Sørensen K, Aksglaede L, Perry JRB, Ong KK, Juul A, Hagen CP (2019) Voice break in boys-temporal relations with other pubertal milestones and likely causal effects of BMI. Hum Reprod 34:1514-1522

188.   Sultan C, Gaspari L, Maimoun L, Kalfa N, Paris F (2018) Disorders of puberty. Best Pract Res Clin Obstet Gynaecol 48:62-89

189.   Sisk CL, Foster DL (2004) The neural basis of puberty and adolescence. Nature Neuroscience 7:1040-1047

190.   Abreu AP, Kaiser UB (2016) Pubertal development and regulation. Lancet Diabetes Endocrinol 4:254-264

191.   Veldhuis JD (2003) Neuroendocrine facets of human puberty. Neurobiology of Aging 24 Suppl 1:S93-S119; discussion S121-112

192.   Terasawa E, Fernandez DL (2001) Neurobiological mechanisms of the onset of puberty in primates. Endocrine Reviews 22:111-151

193.   de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E (2003) Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proceedings of the National Academy of Sciences of the United States of America 100:10972-10976

194.   Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O'Rahilly S, Carlton MB, Crowley WF, Jr., Aparicio SA, Colledge WH (2003) The GPR54 gene as a regulator of puberty. New England Journal of Medicine 349:1614-1627

195.   Lomniczi A, Ojeda SR (2016) The Emerging Role of Epigenetics in the Regulation of Female Puberty. Endocrine Development 29:1-16

196.   Wu FC, Borrow SM, Nicol K, Elton R, Hunter WM (1989) Ontogeny of pulsatile gonadotrophin secretion and pituitary responsiveness in male puberty in man: a mixed longitudinal and cross-sectional study. Journal of Endocrinology 123:347-359

197.   Pedersen-White JR, Chorich LP, Bick DP, Sherins RJ, Layman LC (2008) The prevalence of intragenic deletions in patients with idiopathic hypogonadotropic hypogonadism and Kallmann syndrome. Molecular Human Reproduction 14:367-370

198.   Delemarre-van de Waal HA (2005) Secular trend of timing of puberty. Endocrine Development 8:1-14

199.   Ong KK, Ahmed ML, Dunger DB (2006) Lessons from large population studies on timing and tempo of puberty (secular trends and relation to body size): the European trend. Molecular and Cellular Endocrinology 254-255:8-12

200.   Parent AS, Teilmann G, Juul A, Skakkebaek NE, Toppari J, Bourguignon JP (2003) The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration. Endocrine Reviews 24:668-693

201.   Gluckman PD, Hanson MA (2006) Changing times: the evolution of puberty. Molecular and Cellular Endocrinology 254-255:26-31

202.   Slyper AH (2006) The pubertal timing controversy in the USA, and a review of possible causative factors for the advance in timing of onset of puberty. Clinical Endocrinology 65:1-8

203.   Juul A, Teilmann G, Scheike T, Hertel NT, Holm K, Laursen EM, Main KM, Skakkebaek NE (2006) Pubertal development in Danish children: comparison of recent European and US data. International Journal of Andrology 29:247-255; discussion 286-290

204.   Day FR, Elks CE, Murray A, Ong KK, Perry JR (2015) Puberty timing associated with diabetes, cardiovascular disease and also diverse health outcomes in men and women: the UK Biobank study. Sci Rep 5:11208

205.   Day FR, Bulik-Sullivan B, Hinds DA, Finucane HK, Murabito JM, Tung JY, Ong KK, Perry JR (2015) Shared genetic aetiology of puberty timing between sexes and with health-related outcomes. Nat Commun 6:8842

206.   Veldhuis JD, Keenan DM, Pincus SM (2010) Regulation of complex pulsatile and rhythmic neuroendocrine systems: the male gonadal axis as a prototype. Progress in Brain Research 181:79-110

207.   Chin WW, Boime I eds. 1990 Glycoprotein Hormones: Structure, Synthesis and Biologic Function. Norwell: Serono Symposia, USA

208.   Boime I, Ben-Menahem D (1999) Glycoprotein hormone structure-function and analog design. Recent Progress in Hormone Research 54:271-288; discussion 288-279

209.   Ascoli M, Fanelli F, Segaloff DL (2002) The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocrine Reviews 23:141-174

210.   Rosa C, Amr S, Birken S, Wehmann R, Nisula B (1984) Effect of desialylation of human chorionic gonadotropin on its metabolic clearance rate in humans. Journal of Clinical Endocrinology and Metabolism 59:1215-1219

211.   Muyan M, Furuhashi M, Sugahara T, Boime I (1996) The carboxy-terminal region of the beta-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers. Molecular Endocrinology 10:1678-1687

212.   Bouloux PM, Handelsman DJ, Jockenhovel F, Nieschlag E, Rabinovici J, Frasa WL, de Bie JJ, Voortman G, Itskovitz-Eldor J (2001) First human exposure to FSH-CTP in hypogonadotrophic hypogonadal males. Human Reproduction 16:1592-1597.

213.   Joshi L, Murata Y, Wondisford FE, Szkudlinski MW, Desai R, Weintraub BD (1995) Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance. Endocrinology 136:3839-3848

214.   Fares F, Ganem S, Hajouj T, Agai E (2007) Development of a long-acting erythropoietin by fusing the carboxyl-terminal peptide of human chorionic gonadotropin beta-subunit to the coding sequence of human erythropoietin. Endocrinology 148:5081-5087

215.   Saunders PT (2003) Germ cell-somatic cell interactions during spermatogenesis. Reproduction Supplement 61:91-101

216.   Rudolfsson SH, Wikstrom P, Jonsson A, Collin O, Bergh A (2004) Hormonal regulation and functional role of vascular endothelial growth factor a in the rat testis. Biology of Reproduction 70:340-347

217.   Schnorr JA, Bray MJ, Veldhuis JD (2001) Aromatization mediates testosterone's short-term feedback restraint of 24-hour endogenously driven and acute exogenous gonadotropin-releasing hormone-stimulated luteinizing hormone and follicle-stimulating hormone secretion in young men. Journal of Clinical Endocrinology and Metabolism 86:2600-2606

218.   Pitteloud N, Dwyer AA, DeCruz S, Lee H, Boepple PA, Crowley WF, Jr., Hayes FJ (2008) Inhibition of luteinizing hormone secretion by testosterone in men requires aromatization for its pituitary but not its hypothalamic effects: evidence from the tandem study of normal and gonadotropin-releasing hormone-deficient men. Journal of Clinical Endocrinology and Metabolism 93:784-791

219.   Wilson JD (2001) The role of 5alpha-reduction in steroid hormone physiology. Reproduction, Fertility, and Development 13:673-678

220.   Simpson ER (2004) Aromatase: biologic relevance of tissue-specific expression. Seminars in Reproductive Medicine 22:11-23

221.   Cooke PS, Nanjappa MK, Ko C, Prins GS, Hess RA (2017) Estrogens in Male Physiology. Physiol Rev 97:995-1043

222.   Naftolin F, Ryan KJ, Petro Z (1971) Aromatization of androstenedione by the diencephalon. J Clin Endocrinol Metab 33:368-370

223.   Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS (1994) Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331:1056-1061

224.   Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O (1998) Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci U S A 95:15677-15682

225.   Finkelstein JS, Lee H, Burnett-Bowie SA, Pallais JC, Yu EW, Borges LF, Jones BF, Barry CV, Wulczyn KE, Thomas BJ, Leder BZ (2013) Gonadal steroids and body composition, strength, and sexual function in men. N Engl J Med 369:1011-1022

226.   Vanderschueren D, Laurent MR, Claessens F, Gielen E, Lagerquist MK, Vandenput L, Borjesson AE, Ohlsson C (2014) Sex steroid actions in male bone. Endocr Rev 35:906-960

227.   Russell N, Grossmann M (2019) MECHANISMS IN ENDOCRINOLOGY: Estradiol as a male hormone. Eur J Endocrinol 181:R23-R43

228.   Gronemeyer H, Gustafsson JA, Laudet V (2004) Principles for modulation of the nuclear receptor superfamily. Nature Reviews Drug Discovery 3:950-964

229.   Shi Y (2007) Orphan nuclear receptors in drug discovery. Drug Discov Today 12:440-445

230.   Adachi M, Takayanagi R, Tomura A, Imasaki K, Kato S, Goto K, Yanase T, Ikuyama S, Nawata H (2000) Androgen-insensitivity syndrome as a possible coactivator disease. New England Journal of Medicine 343:856-862

231.   McEwan IJ, Lavery D, Fischer K, Watt K (2007) Natural disordered sequences in the amino terminal domain of nuclear receptors: lessons from the androgen and glucocorticoid receptors. Nucl Recept Signal 5:e001

232.   Rajender S, Singh L, Thangaraj K (2007) Phenotypic heterogeneity of mutations in androgen receptor gene. Asian Journal of Andrology 9:147-179

233.   Zitzmann M, Nieschlag E (2003) The CAG repeat polymorphism within the androgen receptor gene and maleness. International Journal of Andrology 26:76-83.

234.   Simanainen U, Brogley M, Gao YR, Jimenez M, Harwood DT, Handelsman DJ, Robins DM (2011) Length of the human androgen receptor glutamine tract determines androgen sensitivity in vivo. Molecular and Cellular Endocrinology 342:81-86

235.   Zitzmann M, Depenbusch M, Gromoll J, Nieschlag E (2003) Prostate volume and growth in testosterone-substituted hypogonadal men are dependent on the CAG repeat polymorphism of the androgen receptor gene: a longitudinal pharmacogenetic study. Journal of Clinical Endocrinology and Metabolism 88:2049-2054

236.   Zitzmann M, Nieschlag E (2007) Androgen receptor gene CAG repeat length and body mass index modulate the safety of long-term intramuscular testosterone undecanoate therapy in hypogonadal men. Journal of Clinical Endocrinology and Metabolism 92:3844-3853

237.   Zeegers MP, Kiemeney LA, Nieder AM, Ostrer H (2004) How strong is the association between CAG and GGN repeat length polymorphisms in the androgen receptor gene and prostate cancer risk? Cancer Epidemiology, Biomarkers and Prevention 13:1765-1771

238.   Davis-Dao CA, Tuazon ED, Sokol RZ, Cortessis VK (2007) Male infertility and variation in CAG repeat length in the androgen receptor gene: a meta-analysis. Journal of Clinical Endocrinology and Metabolism 92:4319-4326

239.   Ioannidis JP, Ntzani EE, Trikalinos TA, Contopoulos-Ioannidis DG (2001) Replication validity of genetic association studies. Nature Genetics 29:306-309

240.   Palazzolo I, Gliozzi A, Rusmini P, Sau D, Crippa V, Simonini F, Onesto E, Bolzoni E, Poletti A (2008) The role of the polyglutamine tract in androgen receptor. Journal of Steroid Biochemistry and Molecular Biology 108:245-253

241.   Thomas PS, Jr., Fraley GS, Damian V, Woodke LB, Zapata F, Sopher BL, Plymate SR, La Spada AR (2006) Loss of endogenous androgen receptor protein accelerates motor neuron degeneration and accentuates androgen insensitivity in a mouse model of X-linked spinal and bulbar muscular atrophy. Human Molecular Genetics 15:2225-2238

242.   Atsuta N, Watanabe H, Ito M, Banno H, Suzuki K, Katsuno M, Tanaka F, Tamakoshi A, Sobue G (2006) Natural history of spinal and bulbar muscular atrophy (SBMA): a study of 223 Japanese patients. Brain 129:1446-1455

243.   Adachi H, Waza M, Katsuno M, Tanaka F, Doyu M, Sobue G (2007) Pathogenesis and molecular targeted therapy of spinal and bulbar muscular atrophy. Neuropathology and Applied Neurobiology 33:135-151

244.   Ross CA, Poirier MA (2005) Opinion: What is the role of protein aggregation in neurodegeneration? Nature Reviews Molecular Cell Biology 6:891-898

245.   Palazzolo I, Stack C, Kong L, Musaro A, Adachi H, Katsuno M, Sobue G, Taylor JP, Sumner CJ, Fischbeck KH, Pennuto M (2009) Overexpression of IGF-1 in muscle attenuates disease in a mouse model of spinal and bulbar muscular atrophy. Neuron 63:316-328

246.   Rinaldi C, Bott LC, Chen KL, Harmison GG, Katsuno M, Sobue G, Pennuto M, Fischbeck KH (2012) IGF-1 administration ameliorates disease manifestations in a mouse model of spinal and bulbar muscular atrophy. Molecular Medicine

247.   Katsuno M, Banno H, Suzuki K, Takeuchi Y, Kawashima M, Yabe I, Sasaki H, Aoki M, Morita M, Nakano I, Kanai K, Ito S, Ishikawa K, Mizusawa H, Yamamoto T, Tsuji S, Hasegawa K, Shimohata T, Nishizawa M, Miyajima H, Kanda F, Watanabe Y, Nakashima K, Tsujino A, Yamashita T, Uchino M, Fujimoto Y, Tanaka F, Sobue G (2010) Efficacy and safety of leuprorelin in patients with spinal and bulbar muscular atrophy (JASMITT study): a multicentre, randomised, double-blind, placebo-controlled trial. Lancet Neurol 9:875-884

248.   Banno H, Katsuno M, Suzuki K, Tanaka F, Sobue G (2012) Pathogenesis and molecular targeted therapy of spinal and bulbar muscular atrophy (SBMA). Cell and Tissue Research 349:313-320

249.   Grunseich C, Fischbeck KH (2015) Spinal and Bulbar Muscular Atrophy. Neurologic Clinics 33:847-854

250.   Prescott J, Coetzee GA (2006) Molecular chaperones throughout the life cycle of the androgen receptor. Cancer Letters 231:12-19

251.   Heinlein CA, Chang C (2002) Androgen receptor (AR) coregulators: an overview. Endocrine Reviews 23:175-200

252.   Smith CL, O'Malley BW (2004) Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocrine Reviews 25:45-71

253.   Gottlieb B, Beitel LK, Nadarajah A, Paliouras M, Trifiro M (2012) The androgen receptor gene mutations database: 2012 update. Human Mutation 33:887-894

254.   Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, Chen Y, Mohammad TA, Chen Y, Fedor HL, Lotan TL, Zheng Q, De Marzo AM, Isaacs JT, Isaacs WB, Nadal R, Paller CJ, Denmeade SR, Carducci MA, Eisenberger MA, Luo J (2014) AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. New England Journal of Medicine 371:1028-1038

255.   McCrea E, Sissung TM, Price DK, Chau CH, Figg WD (2016) Androgen receptor variation affects prostate cancer progression and drug resistance. Pharmacological Research 114:152-162

256.   Sarpel U, Palmer SK, Dolgin SE (2005) The incidence of complete androgen insensitivity in girls with inguinal hernias and assessment of screening by vaginal length measurement. Journal of Pediatric Surgery 40:133-136; discussion 136-137

257.   Hiort O, Holterhus PM, Horter T, Schulze W, Kremke B, Bals-Pratsch M, Sinnecker GH, Kruse K (2000) Significance of mutations in the androgen receptor gene in males with idiopathic infertility. Journal of Clinical Endocrinology and Metabolism 85:2810-2815

258.   Belgorosky A, Rivarola MA (1985) Sex hormone binding globulin response to testosterone. An androgen sensitivity test. Acta Endocrinologica 109:130-138

259.   Sinnecker GH, Hiort O, Nitsche EM, Holterhus PM, Kruse K (1997) Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene. German Collaborative Intersex Study Group. European Journal of Pediatrics 156:7-14

260.   Cheikhelard A, Morel Y, Thibaud E, Lortat-Jacob S, Jaubert F, Polak M, Nihoul-Fekete C (2008) Long-term followup and comparison between genotype and phenotype in 29 cases of complete androgen insensitivity syndrome. Journal of Urology 180:1496-1501

261.   Marcus R, Leary D, Schneider DL, Shane E, Favus M, Quigley CA (2000) The contribution of testosterone to skeletal development and maintenance: lessons from the androgen insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism 85:1032-1037

262.   Danilovic DL, Correa PH, Costa EM, Melo KF, Mendonca BB, Arnhold IJ (2007) Height and bone mineral density in androgen insensitivity syndrome with mutations in the androgen receptor gene. Osteoporosis International 18:369-374

263.   Han TS, Goswami D, Trikudanathan S, Creighton SM, Conway GS (2008) Comparison of bone mineral density and body proportions between women with complete androgen insensitivity syndrome and women with gonadal dysgenesis. European Journal of Endocrinology 159:179-185

264.   Wisniewski AB, Migeon CJ, Meyer-Bahlburg HF, Gearhart JP, Berkovitz GD, Brown TR, Money J (2000) Complete androgen insensitivity syndrome: long-term medical, surgical, and psychosexual outcome. Journal of Clinical Endocrinology and Metabolism 85:2664-2669

265.   Hines M, Ahmed SF, Hughes IA (2003) Psychological outcomes and gender-related development in complete androgen insensitivity syndrome. Archives of Sexual Behavior 32:93-101

266.   Minto CL, Liao KL, Conway GS, Creighton SM (2003) Sexual function in women with complete androgen insensitivity syndrome. Fertility and Sterility 80:157-164

267.   Brinkmann L, Schuetzmann K, Richter-Appelt H (2007) Gender assignment and medical history of individuals with different forms of intersexuality: evaluation of medical records and the patients' perspective. J Sex Med 4:964-980

268.   Hughes IA, Werner R, Bunch T, Hiort O (2012) Androgen insensitivity syndrome. Seminars in Reproductive Medicine 30:432-442

269.   Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, Shimura N, Tait AD, Hughes IA (2000) Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism 85:658-665

270.   Deeb A, Mason C, Lee YS, Hughes IA (2005) Correlation between genotype, phenotype and sex of rearing in 111 patients with partial androgen insensitivity syndrome. Clinical Endocrinology 63:56-62

271.   Migeon CJ, Wisniewski AB, Gearhart JP, Meyer-Bahlburg HF, Rock JA, Brown TR, Casella SJ, Maret A, Ngai KM, Money J, Berkovitz GD (2002) Ambiguous genitalia with perineoscrotal hypospadias in 46,XY individuals: long-term medical, surgical, and psychosexual outcome. Pediatrics 110:e31

272.   Gottlieb B, Beitel LK, Wu JH, Trifiro M (2004) The androgen receptor gene mutations database (ARDB): 2004 update. Human Mutation 23:527-533

273.   Rodien P, Mebarki F, Mowszowicz I, Chaussain JL, Young J, Morel Y, Schaison G (1996) Different phenotypes in a family with androgen insensitivity caused by the same M780I point mutation in the androgen receptor gene. Journal of Clinical Endocrinology and Metabolism 81:2994-2998

274.   Boehmer AL, Brinkmann O, Bruggenwirth H, van Assendelft C, Otten BJ, Verleun-Mooijman MC, Niermeijer MF, Brunner HG, Rouwe CW, Waelkens JJ, Oostdijk W, Kleijer WJ, van der Kwast TH, de Vroede MA, Drop SL (2001) Genotype versus phenotype in families with androgen insensitivity syndrome. Journal of Clinical Endocrinology and Metabolism 86:4151-4160

275.   Kohler B, Lumbroso S, Leger J, Audran F, Grau ES, Kurtz F, Pinto G, Salerno M, Semitcheva T, Czernichow P, Sultan C (2005) Androgen insensitivity syndrome: somatic mosaicism of the androgen receptor in seven families and consequences for sex assignment and genetic counseling. Journal of Clinical Endocrinology and Metabolism 90:106-111

276.   Boehmer AL, Brinkmann AO, Nijman RM, Verleun-Mooijman MC, de Ruiter P, Niermeijer MF, Drop SL (2001) Phenotypic variation in a family with partial androgen insensitivity syndrome explained by differences in 5alpha dihydrotestosterone availability. Journal of Clinical Endocrinology and Metabolism 86:1240-1246

277.   MacLean HE, Favaloro JM, Warne GL, Zajac JD (2006) Double-strand DNA break repair with replication slippage on two strands: a novel mechanism of deletion formation. Human Mutation 27:483-489

278.   Rogowski W (2006) Genetic screening by DNA technology: a systematic review of health economic evidence. International Journal of Technology Assessment in Health Care 22:327-337

279.   Hiort O, Sinnecker GH, Holterhus PM, Nitsche EM, Kruse K (1998) Inherited and de novo androgen receptor gene mutations: investigation of single-case families. Journal of Pediatrics 132:939-943

280.   Yeh SH, Chiu CM, Chen CL, Lu SF, Hsu HC, Chen DS, Chen PJ (2007) Somatic mutations at the trinucleotide repeats of androgen receptor gene in male hepatocellular carcinoma. International Journal of Cancer 120:1610-1617

281.   Hiort O, Naber SP, Lehners A, Muletta-Feurer S, Sinnecker GH, Zollner A, Komminoth P (1996) The role of androgen receptor gene mutations in male breast carcinoma. Journal of Clinical Endocrinology and Metabolism 81:3404-3407

282.   Paul R, Breul J (2000) Antiandrogen withdrawal syndrome associated with prostate cancer therapies: incidence and clinical significance. Drug Safety 23:381-390

283.   Suzuki H, Okihara K, Miyake H, Fujisawa M, Miyoshi S, Matsumoto T, Fujii M, Takihana Y, Usui T, Matsuda T, Ozono S, Kumon H, Ichikawa T, Miki T (2008) Alternative nonsteroidal antiandrogen therapy for advanced prostate cancer that relapsed after initial maximum androgen blockade. Journal of Urology 180:921-927

284.   Hara T, Miyazaki J, Araki H, Yamaoka M, Kanzaki N, Kusaka M, Miyamoto M (2003) Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. Cancer Research 63:149-153

285.   Miyamoto H, Rahman MM, Chang C (2004) Molecular basis for the antiandrogen withdrawal syndrome. Journal of Cellular Biochemistry 91:3-12

286.   Huggins C, Hodges CV (1941) Studies on prostatic cancer. I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. Cancer Research 1:293-297

287.   Labrie F, Cusan L, Gomez JL, Martel C, Berube R, Belanger P, Belanger A, Vandenput L, Mellstrom D, Ohlsson C (2008) Comparable amounts of sex steroids are made outside the gonads in men and women: Strong lesson for hormone therapy of prostate and breast cancer. Journal of Steroid Biochemistry and Molecular Biology

288.   Attard G, Reid AH, Yap TA, Raynaud F, Dowsett M, Settatree S, Barrett M, Parker C, Martins V, Folkerd E, Clark J, Cooper CS, Kaye SB, Dearnaley D, Lee G, de Bono JS (2008) Phase I clinical trial of a selective inhibitor of CYP17, abiraterone acetate, confirms that castration-resistant prostate cancer commonly remains hormone driven. Journal of Clinical Oncology 26:4563-4571

289.   Anonymous (2000) Maximum androgen blockade in advanced prostate cancer: an overview of the randomised trials. Prostate Cancer Trialists' Collaborative Group. Lancet 355:1491-1498

290.   Pezaro CJ, Mukherji D, De Bono JS (2012) Abiraterone acetate: redefining hormone treatment for advanced prostate cancer. Drug Discov Today 17:221-226

291.   Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, de Wit R, Mulders P, Chi KN, Shore ND, Armstrong AJ, Flaig TW, Flechon A, Mainwaring P, Fleming M, Hainsworth JD, Hirmand M, Selby B, Seely L, de Bono JS, Investigators A (2012) Increased survival with enzalutamide in prostate cancer after chemotherapy. New England Journal of Medicine 367:1187-1197

292.   Meikle AW (2004) The interrelationships between thyroid dysfunction and hypogonadism in men and boys. Thyroid 14 Suppl 1:S17-25

293.   Payne KL, Loidl NM, Lim CF, Topliss DJ, Stockigt JR, Barlow JW (1997) Modulation of T3-induced sex hormone-binding globulin secretion by human hepatoblastoma cells. European Journal of Endocrinology 137:415-420

294.   Isojarvi J (2008) Disorders of reproduction in patients with epilepsy: antiepileptic drug related mechanisms. Seizure 17:111-119

295.   Wheeler MJ, Toone BK, ADannatt, Fenwick PB, Brown S (1991) Metabolic clearance rate of testosterone in male epileptic patients on anti-convulsant therapy. Journal of Endocrinology 129:465-468

296.   Death AK, McGrath KC, Handelsman DJ (2005) Valproate is an anti-androgen and anti-progestin. Steroids 70:946-953

297.   Green JRB, Goble HL, Edwards CRW, Dawson AM (1977) Reversible insensitivity to androgens in men with untreated gluten enteropathy. Lancet i:280-282

298.   Farthing MJR, Rees LH, Edwards CRW, Dawson AM (1983) Male gonadal dysfunction in coeliac disease: 2. Sex hormones. Gut 24:127-135

299.   Frydman M, Kauschansky A, Bonne-Tamir B, Nassar F, Homburg R (1991) Assessment of the hypothalamic-pituitary-testicular function in male patients with Wilson's disease. Journal of Andrology 12:180-184

300.   Herrick AL, McColl KE, Wallace AM, Moore MR, Goldberg A (1990) Elevation of hormone-binding globulins in acute intermittent porphyria. Clinica Chimica Acta 187:141-148

301.   Iturriaga H, Lioi X, Valladares L (1999) Sex hormone-binding globulin in non-cirrhotic alcoholic patients during early withdrawal and after longer abstinence. Alcohol and Alcoholism 34:903-909

302.   Handelsman DJ, Strasser S, McDonald JA, Conway AJ, McCaughan GW (1995) Hypothalamic-pituitary testicular function in end-stage non-alcoholic liver disease before and after liver transplantation. Clinical Endocrinology 43:331-337

303.   Hamilton JB, Mestler GE (1969) Mortality and survival: comparison of eunuchs with intact men and women in a mentally retarded population. Journal of Gerontology 24:395-411

304.   Nieschlag E, Nieschlag S, Behre HM (1993) Lifespan and testosterone. Nature 366:215

305.   Jenkins JS (1998) The voice of the castrato. Lancet 351:1877-1880

306.   Eyben FE, Graugaard C, Vaeth M (2005) All-cause mortality and mortality of myocardial infarction for 989 legally castrated men. European Journal of Epidemiology 20:863-869

307.   Min KJ, Lee CK, Park HN (2012) The lifespan of Korean eunuchs. Current Biology 22:R792-793

308.   Bojesen A, Juul S, Birkebaek N, Gravholt CH (2004) Increased mortality in Klinefelter syndrome. Journal of Clinical Endocrinology and Metabolism 89:3830-3834

309.   Liu PY, Death AK, Handelsman DJ (2003) Androgens and cardiovascular disease. Endocrine Reviews 24:313-340

310.   Liu PY, Handelsman DJ 2004 Androgen therapy in non-gonadal disease. In: Nieschlag E, Behre HM eds. Testosterone: Action, Deficiency and Substitution. 3rd ed. Berlin: Springer-Verlag; 445-495

311.   Zuraw BL (2008) Clinical practice. Hereditary angioedema. New England Journal of Medicine 359:1027-1036

312.   Longhurst H, Cicardi M (2012) Hereditary angio-oedema. Lancet 379:474-481

313.   Handelsman DJ (2007) Update in andrology. Journal of Clinical Endocrinology and Metabolism 92:4505-4511

314.   Bojesen A, Juul S, Gravholt CH (2003) Prenatal and postnatal prevalence of Klinefelter syndrome: a national registry study. Journal of Clinical Endocrinology and Metabolism 88:622-626

315.   Kelleher S, Conway AJ, Handelsman DJ (2004) Blood testosterone threshold for androgen deficiency symptoms. Journal of Clinical Endocrinology and Metabolism 89:3813-3817

316.   Schaison G, Young J, Pholsena M, Nahoul K, Couzinet B (1993) Failure of combined follicle-stimulating hormone-testosterone administration to initiate and/or maintain spermatogenesis in men with hypogonadotropic hypogonadism. Journal of Clinical Endocrinology and Metabolism 77:1545-1549

317.   Pitteloud N, Hayes FJ, Dwyer A, Boepple PA, Lee H, Crowley WF, Jr. (2002) Predictors of outcome of long-term GnRH therapy in men with idiopathic hypogonadotropic hypogonadism. Journal of Clinical Endocrinology and Metabolism 87:4128-4136

318.   Wang C, Tso SC, Todd D (1989) Hypogonadotropic hypogonadism in severe beta-thalassemia: effect of chelation and pulsatile gonadotropin-releasing hormone therapy. Journal of Clinical Endocrinology and Metabolism 68:511-516

319.   Liu PY, Baker HW, Jayadev V, Zacharin M, Conway AJ, Handelsman DJ (2009) Induction of spermatogenesis and fertility during gonadotropin treatment of gonadotropin-deficient infertile men: predictors of fertility outcome. Journal of Clinical Endocrinology and Metabolism 94:801-808

320.   Dwyer AA, Raivio T, Pitteloud N (2015) Gonadotrophin replacement for induction of fertility in hypogonadal men. Best Practice and Research Clinical Endocrinology and Metabolism 29:91-103

321.   Handelsman DJ, Goebel C, Idan A, Jimenez M, Trout G, Kazlauskas R (2009) Effects of recombinant human LH and hCG on serum and urine LH and androgens in men. Clinical Endocrinology 71:417-428

322.   Barrio R, de Luis D, Alonso M, Lamas A, Moreno JC (1999) Induction of puberty with human chorionic gonadotropin and follicle-stimulating hormone in adolescent males with hypogonadotropic hypogonadism. Fertility and Sterility 71:244-248

323.   Bouvattier C, Maione L, Bouligand J, Dode C, Guiochon-Mantel A, Young J (2011) Neonatal gonadotropin therapy in male congenital hypogonadotropic hypogonadism. Nat Rev Endocrinol 8:172-182

324.   Howard S, Dunkel L (2016) Sex Steroid and Gonadotropin Treatment in Male Delayed Puberty. Endocrine Development 29:185-197

325.   Belanger A, Candas B, Dupont A, Cusan L, Diamond P, Gomez JL, Labrie F (1994) Changes in serum concentrations of conjugated and unconjugated steroids in 40- to 80-year-old men. Journal of Clinical Endocrinology and Metabolism 79:1086-1090

326.   Orwoll E, Lambert LC, Marshall LM, Phipps K, Blank J, Barrett-Connor E, Cauley J, Ensrud K, Cummings S (2006) Testosterone and estradiol among older men. Journal of Clinical Endocrinology and Metabolism 91:1336-1344

327.   Travison TG, Araujo AB, Kupelian V, O'Donnell AB, McKinlay JB (2007) The relative contributions of aging, health, and lifestyle factors to serum testosterone decline in men. Journal of Clinical Endocrinology and Metabolism 92:549-555

328.   Andersson AM, Jorgensen N, Frydelund-Larsen L, Rajpert-De Meyts E, Skakkebaek NE (2004) Impaired Leydig cell function in infertile men: a study of 357 idiopathic infertile men and 318 proven fertile controls. Journal of Clinical Endocrinology and Metabolism 89:3161-3167

329.   Howell SJ, Radford JA, Adams JE, Shalet SM (2000) The impact of mild Leydig cell dysfunction following cytotoxic chemotherapy on bone mineral density (BMD) and body composition. Clinical Endocrinology 52:609-616

330.   Howell SJ, Radford JA, Smets EM, Shalet SM (2000) Fatigue, sexual function and mood following treatment for haematological malignancy: the impact of mild Leydig cell dysfunction. British Journal of Cancer 82:789-793

331.   Gerl A, Muhlbayer D, Hansmann G, Mraz W, Hiddemann W (2001) The impact of chemotherapy on Leydig cell function in long term survivors of germ cell tumors. Cancer 91:1297-1303

332.   Somali M, Mpatakoias V, Avramides A, Sakellari I, Kaloyannidis P, Smias C, Anagnostopoulos A, Kourtis A, Rousso D, Panidis D, Vagenakis A (2005) Function of the hypothalamic-pituitary-gonadal axis in long-term survivors of hematopoietic stem cell transplantation for hematological diseases. Gynecological Endocrinology 21:18-26

333.   Howell SJ, Radford JA, Adams JE, Smets EM, Warburton R, Shalet SM (2001) Randomized placebo-controlled trial of testosterone replacement in men with mild Leydig cell insufficiency following cytotoxic chemotherapy. Clinical Endocrinology 55:315-324

334.   Merza Z, Blumsohn A, Mah PM, Meads DM, McKenna SP, Wylie K, Eastell R, Wu F, Ross RJ (2006) Double-blind placebo-controlled study of testosterone patch therapy on bone turnover in men with borderline hypogonadism. International Journal of Andrology 29:381-391

335.   Nieschlag E (2010) Male hormonal contraception. Handb Exp Pharmacol:197-223

336.   Handelsman DJ 2011 Androgen therapy in non-gonadal disease. In: Nieschlag E, Behre HM eds. Testosterone: Action, Deficiency and Substitution. 4th ed. Cambridge: Cambridge University Press; 372-407

337.   Teruel JL, Aguilera A, Marcen R, Antolin JN, Otero GG, Ortuno J (1996) Androgen therapy for anaemia of chronic renal failure. Scandanavian Journal of Urology and Nephrology 30:403-408

338.   Gascon A, Belvis JJ, Berisa F, Iglesias E, Estopinan V, Teruel JL (1999) Nandrolone decanoate is a good alternative for the treatment of anemia in elderly male patients on hemodialysis. Geriatric Nephrology and Urology 9:67-72

339.   Navarro JF, Mora C, Macia M, Garcia J (2002) Randomized prospective comparison between erythropoietin and androgens in CAPD patients. Kidney International 61:1537-1544.

340.   Ishak KG, Zimmerman HJ (1987) Hepatotoxic effects of the anabolic-androgenic steroids. Seminars in Liver Disease 7:230-236

341.   Velazquez I, Alter BP (2004) Androgens and liver tumors: Fanconi's anemia and non-Fanconi's conditions. American Journal of Hematology 77:257-267

342.   Sloane DE, Lee CW, Sheffer AL (2007) Hereditary angioedema: Safety of long-term stanozolol therapy. Journal of Allergy and Clinical Immunology 120:654-658

343.   Banerji A, Sloane DE, Sheffer AL (2008) Hereditary angioedema: a current state-of-the-art review, V: attenuated androgens for the treatment of hereditary angioedema. Annals of Allergy, Asthma, and Immunology 100:S19-22

344.   Bork K, Bygum A, Hardt J (2008) Benefits and risks of danazol in hereditary angioedema: a long-term survey of 118 patients. Annals of Allergy, Asthma, and Immunology 100:153-161

345.   Sabharwal G, Craig T (2015) Recombinant human C1 esterase inhibitor for the treatment of hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE). Expert Rev Clin Immunol 11:319-327

346.   Bhasin S, Storer TW, Berman N, Callegari C, Clevenger B, Phillips J, Bunnell TJ, Tricker R, Shirazi A, Casaburi R (1996) The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. New England Journal of Medicine 335:1-7

347.   Elashoff JD, Jacknow AD, Shain SG, Braunstein GD (1991) Effects of anabolic-androgenic steroids on muscular strength. Annals of Internal Medicine 115:387-393

348.   Storer TW, Magliano L, Woodhouse L, Lee ML, Dzekov C, Dzekov J, Casaburi R, Bhasin S (2003) Testosterone dose-dependently increases maximal voluntary strength and leg power, but does not affect fatigability or specific tension. Journal of Clinical Endocrinology and Metabolism 88:1478-1485

349.   Storer TW, Woodhouse L, Magliano L, Singh AB, Dzekov C, Dzekov J, Bhasin S (2008) Changes in muscle mass, muscle strength, and power but not physical function are related to testosterone dose in healthy older men. Journal of the American Geriatrics Society

350.   Bhasin S, Woodhouse L, Casaburi R, Singh AB, Mac RP, Lee M, Yarasheski KE, Sinha-Hikim I, Dzekov C, Dzekov J, Magliano L, Storer TW (2005) Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle. Journal of Clinical Endocrinology and Metabolism 90:678-688

351.   Coviello AD, Lakshman K, Mazer NA, Bhasin S (2006) Differences in the apparent metabolic clearance rate of testosterone in young and older men with gonadotropin suppression receiving graded doses of testosterone. Journal of Clinical Endocrinology and Metabolism 91:4669-4675

352.   Coviello AD, Kaplan B, Lakshman KM, Chen T, Singh AB, Bhasin S (2008) Effects of graded doses of testosterone on erythropoiesis in healthy young and older men. Journal of Clinical Endocrinology and Metabolism 93:914-919

353.   Singh AB, Hsia S, Alaupovic P, Sinha-Hikim I, Woodhouse L, Buchanan TA, Shen R, Bross R, Berman N, Bhasin S (2002) The effects of varying doses of T on insulin sensitivity, plasma lipids, apolipoproteins, and C-reactive protein in healthy young men. Journal of Clinical Endocrinology and Metabolism 87:136-143

354.   Gray PB, Singh AB, Woodhouse LJ, Storer TW, Casaburi R, Dzekov J, Dzekov C, Sinha-Hikim I, Bhasin S (2005) Dose-dependent effects of testosterone on sexual function, mood and visuospatial cognition in older men. Journal of Clinical Endocrinology and Metabolism 90:3838-3846

355.   Basaria S, Coviello AD, Travison TG, Storer TW, Farwell WR, Jette AM, Eder R, Tennstedt S, Ulloor J, Zhang A, Choong K, Lakshman KM, Mazer NA, Miciek R, Krasnoff J, Elmi A, Knapp PE, Brooks B, Appleman E, Aggarwal S, Bhasin G, Hede-Brierley L, Bhatia A, Collins L, LeBrasseur N, Fiore LD, Bhasin S (2010) Adverse events associated with testosterone administration. The New England Journal of Medicine 363:109-122

356.   Kotler DP, Tierney AR, Wang J, Pierson RN (1989) Magnitude of body-cell-mass depletion and the timing of death from wasting in AIDS. American Journal of Clinical Nutrition 50:444-447

357.   Moyle GJ, Schoelles K, Fahrbach K, Frame D, James K, Scheye R, Cure-Bolt N (2004) Efficacy of selected treatments of HIV wasting: a systematic review and meta-analysis. Journal of Acquired Immune Deficiency Syndromes 37 Suppl 5:S262-276

358.   Johns K, Beddall MJ, Corrin RC (2005) Anabolic steroids for the treatment of weight loss in HIV-infected individuals. Cochrane Database Syst Rev:CD005483

359.   Bolding G, Sherr L, Maguire M, Elford J (1999) HIV risk behaviours among gay men who use anabolic steroids. Addiction 94:1829-1835

360.   Lambert CP, Sullivan DH, Freeling SA, Lindquist DM, Evans WJ (2002) Effects of testosterone replacement and/or resistance exercise on the composition of megestrol acetate stimulated weight gain in elderly men: a randomized controlled trial. Journal of Clinical Endocrinology and Metabolism 87:2100-2106

361.   Mulligan K, Zackin R, Von Roenn JH, Chesney MA, Egorin MJ, Sattler FR, Benson CA, Liu T, Umbleja T, Shriver S, Auchus RJ, Schambelan M (2007) Testosterone supplementation of megestrol therapy does not enhance lean tissue accrual in men with human immunodeficiency virus-associated weight loss: a randomized, double-blind, placebo-controlled, multicenter trial. Journal of Clinical Endocrinology and Metabolism 92:563-570

362.   Vuong C, Van Uum SH, O'Dell LE, Lutfy K, Friedman TC (2010) The effects of opioids and opioid analogs on animal and human endocrine systems. Endocrine Reviews 31:98-132

363.   Bawor M, Bami H, Dennis BB, Plater C, Worster A, Varenbut M, Daiter J, Marsh DC, Steiner M, Anglin R, Coote M, Pare G, Thabane L, Samaan Z (2015) Testosterone suppression in opioid users: a systematic review and meta-analysis. Drug and Alcohol Dependence 149:1-9

364.   O'Rourke TK, Jr., Wosnitzer MS (2016) Opioid-Induced Androgen Deficiency (OPIAD): Diagnosis, Management, and Literature Review. Current Urology Reports 17:76

365.   Daniell HW, Lentz R, Mazer NA (2006) Open-label pilot study of testosterone patch therapy in men with opioid-induced androgen deficiency. J Pain 7:200-210

366.   Basaria S, Travison TG, Alford D, Knapp PE, Teeter K, Cahalan C, Eder R, Lakshman K, Bachman E, Mensing G, Martel MO, Le D, Stroh H, Bhasin S, Wasan AD, Edwards RR (2015) Effects of testosterone replacement in men with opioid-induced androgen deficiency: a randomized controlled trial. Pain 156:280-288

367.   Huang G, Travison TG, Edwards RR, Basaria S (2016) Effects of Testosterone Replacement on Pain Catastrophizing and Sleep Quality in Men with Opioid-Induced Androgen Deficiency. Pain Med

368.   Wierman ME, Basson R, Davis SR, Khosla S, Miller KK, Rosner W, Santoro N (2006) Androgen therapy in women: an Endocrine Society Clinical Practice guideline. Journal of Clinical Endocrinology and Metabolism 91:3697-3710

369.   Arlt W, Callies F, van Vlijmen JC, Koehler I, Reincke M, Bidlingmaier M, Huebler D, Oettel M, Ernst M, Schulte HM, Allolio B (1999) Dehydroepiandrosterone replacement in women with adrenal insufficiency New England Journal of Medicine 341:1013-1020

370.   Gurnell EM, Hunt PJ, Curran SE, Conway CL, Pullenayegum EM, Huppert FA, Compston JE, Herbert J, Chatterjee VK (2008) Long-term DHEA replacement in primary adrenal insufficiency: a randomized, controlled trial. Journal of Clinical Endocrinology and Metabolism 93:400-409

371.   Johannsson G, Burman P, Wiren L, Engstrom BE, Nilsson AG, Ottosson M, Jonsson B, Bengtsson BA, Karlsson FA (2002) Low dose dehydroepiandrosterone affects behavior in hypopituitary androgen-deficient women: a placebo-controlled trial. Journal of Clinical Endocrinology and Metabolism 87:2046-2052

372.   Lovas K, Gebre-Medhin G, Trovik TS, Fougner KJ, Uhlving S, Nedrebo BG, Myking OL, Kampe O, Husebye ES (2003) Replacement of dehydroepiandrosterone in adrenal failure: no benefit for subjective health status and sexuality in a 9-month, randomized, parallel group clinical trial. Journal of Clinical Endocrinology and Metabolism 88:1112-1118

373.   Miller KK, Biller BM, Beauregard C, Lipman JG, Jones J, Schoenfeld D, Sherman JC, Swearingen B, Loeffler J, Klibanski A (2006) Effects of testosterone replacement in androgen-deficient women with hypopituitarism: a randomized, double-blind, placebo-controlled study. Journal of Clinical Endocrinology and Metabolism 91:1683-1690

374.   Shifren JL, Braunstein GD, Simon JA, Casson PR, Buster JE, Redmond GP, Burki RE, Ginsburg ES, Rosen RC, Leiblum SR, Caramelli KE, Mazer NA (2000) Transdermal testosterone treatment in women with impaired sexual function after oophorectomy. New England Journal of Medicine 343:682-688

375.   Davis S, Papalia MA, Norman RJ, O'Neill S, Redelman M, Williamson M, Stuckey BG, Wlodarczyk J, Gard'ner K, Humberstone A (2008) Safety and efficacy of a testosterone metered-dose transdermal spray for treating decreased sexual satisfaction in premenopausal women: a randomized trial. Annals of Internal Medicine 148:569-577

376.   Somboonporn W, Davis S, Seif MW, Bell R (2005) Testosterone for peri- and postmenopausal women. Cochrane Database Syst Rev:CD004509

377.   Greenblatt RB, Barfield WE, Garner JF, Calk GL, Harrod JP (1950) Evaluation of an estrogen, androgen, estrogen-androgen combination, and a placebo in the treatment of the menopause. Journal of Clinical Endocrinology and Metabolism 10:1547-1558

378.   Sherwin BB, Gelfand MM (1987) The role of androgens in the maintenance of sexual functioning in oophorectomized women. Psychosomatic Medicine 49:397-409

379.   Urman B, Pride SM, Yuen BH (1991) Elevated serum testosterone, hirsutism, and virilism associated with combined androgen-estrogen hormone replacement therapy. Obstetrics and Gynecology 77:1124-1131

380.   Gerritsma EJ, Brocaar MP, Hakkesteegt MM, Birkenhager JC (1994) Virilization of the voice in post-menopausal women due to the anabolic steroid nandrolone decanoate (Decadurabolin). The effects of medication for one year. Clin Otolaryngol Allied Sci 19:79-84

381.   Baker J (1999) A report on alterations to the speaking and singing voices of four women following hormonal therapy with virilizing agents. Journal of Voice 13:496-507

382.   Davis SR, McCloud P, Strauss BJG, Burger H (1995) Testosterone enhances estradiol's effects on postmenopausal bone density and sexuality. Maturitas 21:227-236

383.   Elraiyah T, Sonbol MB, Wang Z, Khairalseed T, Asi N, Undavalli C, Nabhan M, Firwana B, Altayar O, Prokop L, Montori VM, Murad MH (2014) Clinical review: The benefits and harms of systemic testosterone therapy in postmenopausal women with normal adrenal function: a systematic review and meta-analysis. Journal of Clinical Endocrinology and Metabolism 99:3543-3550

384.   Davis SR, Wahlin-Jacobsen S (2015) Testosterone in women--the clinical significance. Lancet Diabetes Endocrinol 3:980-992

385.   Reed BG, Bou Nemer L, Carr BR (2016) Has testosterone passed the test in premenopausal women with low libido? A systematic review. Int J Womens Health 8:599-607

386.   Miller KK, Grieco KA, Klibanski A (2005) Testosterone administration in women with anorexia nervosa. Journal of Clinical Endocrinology and Metabolism 90:1428-1433

387.   Choi HH, Gray PB, Storer TW, Calof OM, Woodhouse L, Singh AB, Padero C, Mac RP, Sinha-Hikim I, Shen R, Dzekov J, Dzekov C, Kushnir MM, Rockwood AL, Meikle AW, Lee ML, Hays RD, Bhasin S (2005) Effects of testosterone replacement in human immunodeficiency virus-infected women with weight loss. Journal of Clinical Endocrinology and Metabolism 90:1531-1541

388.   Gordon C, Wallace DJ, Shinada S, Kalunian KC, Forbess L, Braunstein GD, Weisman MH (2008) Testosterone patches in the management of patients with mild/moderate systemic lupus erythematosus. Rheumatology 47:334-338

389.   Ferreira IM, Verreschi IT, Nery LE, Goldstein RS, Zamel N, Brooks D, Jardim JR (1998) The influence of 6 months of oral anabolic steroids on body mass and respiratory muscles in undernourished COPD patients. Chest 114:19-28.

390.   Casaburi R, Bhasin S, Cosentino L, Porszasz J, Somfay A, Lewis MI, Fournier M, Storer TW (2004) Effects of testosterone and resistance training in men with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 170:870-878

391.   Svartberg J, Aasebo U, Hjalmarsen A, Sundsfjord J, Jorde R (2004) Testosterone treatment improves body composition and sexual function in men with COPD, in a 6-month randomized controlled trial. Respiratory Medicine 98:906-913

392.   Weisberg J, Wanger J, Olson J, Streit B, Fogarty C, Martin T, Casaburi R (2002) Megestrol acetate stimulates weight gain and ventilation in underweight COPD patients. Chest 121:1070-1078

393.   Jankowska EA, Biel B, Majda J, Szklarska A, Lopuszanska M, Medras M, Anker SD, Banasiak W, Poole-Wilson PA, Ponikowski P (2006) Anabolic deficiency in men with chronic heart failure: prevalence and detrimental impact on survival. Circulation 114:1829-1837

394.   Malkin CJ, Pugh PJ, West JN, van Beek EJ, Jones TH, Channer KS (2006) Testosterone therapy in men with moderate severity heart failure: a double-blind randomized placebo controlled trial. European Heart Journal 27:57-64

395.   Handelsman DJ, Dong Q 1992 Ontogenic regression: a model of stress and reproduction. In: Sheppard K, Boublik JH, Funder JW eds. Stress and Reproduction. New York: Raven Press; 333-345

396.   Reid IR, Wattie DJ, Evans MC, Stapleton JP (1996) Testosterone therapy in glucocorticoid-treated men. Archives of Internal Medicine 156:1173-1177

397.   Crawford BA, Liu PY, Kean M, Bleasel J, Handelsman DJ (2003) Randomised, placebo-controlled trial of androgen effects on bone and muscle in men requiring long-term systemic glucocorticoid therapy. Journal of Clinical Endocrinology and Metabolism 88:3167-3176

398.   Jeschke MG, Finnerty CC, Suman OE, Kulp G, Mlcak RP, Herndon DN (2007) The effect of oxandrolone on the endocrinologic, inflammatory, and hypermetabolic responses during the acute phase postburn. Annals of Surgery 246:351-360; discussion 360-352

399.   Bulger EM, Jurkovich GJ, Farver CL, Klotz P, Maier RV (2004) Oxandrolone does not improve outcome of ventilator dependent surgical patients. Annals of Surgery 240:472-478; discussion 478-480

400.   Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR (2001) Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. Journal of Clinical Endocrinology and Metabolism 86:724-731.

401.   Travison TG, Shackelton R, Araujo AB, Hall SA, Williams RE, Clark RV, O'Donnell AB, McKinlay JB (2008) The natural history of symptomatic androgen deficiency in men: onset, progression, and spontaneous remission. Journal of the American Geriatrics Society 56:831-839

402.   Deslypere JP, Vermeulen A (1981) Aging and tissue androgens. Journal of Clinical Endocrinology and Metabolism 53:430-434

403.   Deslypere JP, Vermeulen A (1985) Influence of age on steroid concentration in skin and striated muscle in women and in cardiac muscle and lung tissue in men. Journal of Clinical Endocrinology and Metabolism 60:648-653

404.   Mohr BA, Guay AT, O'Donnell AB, McKinlay JB (2005) Normal, bound and nonbound testosterone levels in normally ageing men: results from the Massachusetts Male Ageing Study. Clinical Endocrinology 62:64-73

405.   Corona G, Monami M, Rastrelli G, Aversa A, Tishova Y, Saad F, Lenzi A, Forti G, Mannucci E, Maggi M (2011) Testosterone and metabolic syndrome: a meta-analysis study. J Sex Med 8:272-283

406.   Brand JS, van der Tweel I, Grobbee DE, Emmelot-Vonk MH, van der Schouw YT (2011) Testosterone, sex hormone-binding globulin and the metabolic syndrome: a systematic review and meta-analysis of observational studies. International Journal of Epidemiology 40:189-207

407.   Ruige JB, Mahmoud AM, De Bacquer D, Kaufman JM (2011) Endogenous testosterone and cardiovascular disease in healthy men: a meta-analysis. Heart 97:870-875

408.   Araujo AB, Dixon JM, Suarez EA, Murad MH, Guey LT, Wittert GA (2011) Clinical review: Endogenous testosterone and mortality in men: a systematic review and meta-analysis. Journal of Clinical Endocrinology and Metabolism 96:3007-3019

409.   Toma M, McAlister FA, Coglianese EE, Vidi V, Vasaiwala S, Bakal JA, Armstrong PW, Ezekowitz JA (2012) Testosterone supplementation in heart failure: a meta-analysis. Circ Heart Fail 5:315-321

410.   Borst SE, Shuster JJ, Zou B, Ye F, Jia H, Wokhlu A, Yarrow JF (2014) Cardiovascular risks and elevation of serum DHT vary by route of testosterone administration: a systematic review and meta-analysis. BMC Med 12:211

411.   Onasanya O, Iyer G, Lucas E, Lin D, Singh S, Alexander GC (2016) Association between exogenous testosterone and cardiovascular events: an overview of systematic reviews. Lancet Diabetes Endocrinol

412.   Alexander GC, Iyer G, Lucas E, Lin D, Singh S (2016) Cardiovascular Risks of Exogenous Testosterone Use Among Men: A Systematic Review and Meta-Analysis. American Journal of Medicine

413.   Shores MM, Smith NL, Forsberg CW, Anawalt BD, Matsumoto AM (2012) Testosterone treatment and mortality in men with low testosterone levels. Journal of Clinical Endocrinology and Metabolism 97:2050-2058

414.   Wu FC (2012) Caveat emptor: does testosterone treatment reduce mortality in men? Journal of Clinical Endocrinology and Metabolism 97:1884-1886

415.   Gruenewald DA, Matsumoto AM (2003) Testosterone supplementation therapy for older men: potential benefits and risks. Journal of the American Geriatrics Society 51:101-115

416.   Ly LP, Jimenez M, Zhuang TN, Celermajer DS, Conway AJ, Handelsman DJ (2001) A double-blind, placebo-controlled, randomized clinical trial of transdermal dihydrotestosterone gel on muscular strength, mobility, and quality of life in older men with partial androgen deficiency. Journal of Clinical Endocrinology and Metabolism 86:4078-4088.

417.   Kunelius P, Lukkarinen O, Hannuksela ML, Itkonen O, Tapanainen JS (2002) The effects of transdermal dihydrotestosterone in the aging male: a prospective, randomized, double blind study. Journal of Clinical Endocrinology and Metabolism 87:1467-1472

418.   Liu PY, Wishart SM, Handelsman DJ (2002) A double-blind, placebo-controlled, randomized clinical trial of recombinant human chorionic gonadotropin on muscle strength and physical function and activity in older men with partial age-related androgen deficiency. Journal of Clinical Endocrinology and Metabolism 87:3125-3135

419.   Bhasin S, Calof OM, Storer TW, Lee ML, Mazer NA, Jasuja R, Montori VM, Gao W, Dalton JT (2006) Drug insight: Testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging. Nat Clin Pract Endocrinol Metab 2:146-159

420.   Isidori AM, Giannetta E, Greco EA, Gianfrilli D, Bonifacio V, Isidori A, Lenzi A, Fabbri A (2005) Effects of testosterone on body composition, bone metabolism and serum lipid profile in middle-aged men: a meta-analysis. Clinical Endocrinology 63:280-293

421.   Tracz MJ, Sideras K, Bolona ER, Haddad RM, Kennedy CC, Uraga MV, Caples SM, Erwin PJ, Montori VM (2006) Testosterone use in men and its effects on bone health. A systematic review and meta-analysis of randomized placebo-controlled trials. Journal of Clinical Endocrinology and Metabolism 91:2011-2016

422.   Ottenbacher KJ, Ottenbacher ME, Ottenbacher AJ, Acha AA, Ostir GV (2006) Androgen treatment and muscle strength in elderly men: A meta-analysis. Journal of the American Geriatrics Society 54:1666-1673

423.   Isidori AM, Giannetta E, Gianfrilli D, Greco EA, Bonifacio V, Aversa A, Isidori A, Fabbri A, Lenzi A (2005) Effects of testosterone on sexual function in men: results of a meta-analysis. Clinical Endocrinology 63:381-394

424.   Bolona ER, Uraga MV, Haddad RM, Tracz MJ, Sideras K, Kennedy CC, Caples SM, Erwin PJ, Montori VM (2007) Testosterone use in men with sexual dysfunction: a systematic review and meta-analysis of randomized placebo-controlled trials. Mayo Clinic Proceedings 82:20-28

425.   Calof OM, Singh AB, Lee ML, Kenny AM, Urban RJ, Tenover JL, Bhasin S (2005) Adverse events associated with testosterone replacement in middle-aged and older men: a meta-analysis of randomized, placebo-controlled trials. Journals of Gerontology Series A, Biological Sciences and Medical Sciences 60:1451-1457

426.   Liverman CT, Blazer DG eds. 2004 Testosterone and Aging: Clinical Research Directions. Washington, DC: Institute of Medicine: The National Academies Press

427.   Snyder PJ, Bhasin S, Cunningham GR, Matsumoto AM, Stephens-Shields AJ, Cauley JA, Gill TM, Barrett-Connor E, Swerdloff RS, Wang C, Ensrud KE, Lewis CE, Farrar JT, Cella D, Rosen RC, Pahor M, Crandall JP, Molitch ME, Cifelli D, Dougar D, Fluharty L, Resnick SM, Storer TW, Anton S, Basaria S, Diem SJ, Hou X, Mohler ER, 3rd, Parsons JK, Wenger NK, Zeldow B, Landis JR, Ellenberg SS, Testosterone Trials I (2016) Effects of Testosterone Treatment in Older Men. New England Journal of Medicine 374:611-624

428.   Orwoll ES (2016) Establishing a Framework--Does Testosterone Supplementation Help Older Men? New England Journal of Medicine 374:682-683

429.   Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J (2002) Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial. Jama 288:321-333

430.   Kalin MF, Zumoff B (1990) Sex hormones and coronary disease: a review of the clinical studies. Steroids 55:330-352

431.   Zhang Y, Ouyang P, Post WS, Dalal D, Vaidya D, Blasco-Colmenares E, Soliman EZ, Tomaselli GF, Guallar E (2011) Sex-steroid hormones and electrocardiographic QT-interval duration: findings from the third National Health and Nutrition Examination Survey and the Multi-Ethnic Study of Atherosclerosis. American Journal of Epidemiology 174:403-411

432.   Carnes CA, Dech SJ (2002) Effects of dihydrotestosterone on cardiac inward rectifier K(+) current. International Journal of Andrology 25:210-214

433.   Liu XK, Katchman A, Whitfield BH, Wan G, Janowski EM, Woosley RL, Ebert SN (2003) In vivo androgen treatment shortens the QT interval and increases the densities of inward and delayed rectifier potassium currents in orchiectomized male rabbits. Cardiovascular Research 57:28-36

434.   Fulop L, Banyasz T, Szabo G, Toth IB, Biro T, Lorincz I, Balogh A, Peto K, Miko I, Nanasi PP (2006) Effects of sex hormones on ECG parameters and expression of cardiac ion channels in dogs. Acta Physiol (Oxf) 188:163-171

435.   Ridley JM, Shuba YM, James AF, Hancox JC (2008) Modulation by testosterone of an endogenous hERG potassium channel current. Journal of Physiology and Pharmacology 59:395-407

436.   Wu ZY, Chen K, Haendler B, McDonald TV, Bian JS (2008) Stimulation of N-terminal truncated isoform of androgen receptor stabilizes human ether-a-go-go-related gene-encoded potassium channel protein via activation of extracellular signal regulated kinase 1/2. Endocrinology 149:5061-5069

437.   Charbit B, Christin-Maitre S, Demolis JL, Soustre E, Young J, Funck-Brentano C (2009) Effects of testosterone on ventricular repolarization in hypogonadic men. American Journal of Cardiology 103:887-890

438.   Pecori Giraldi F, Toja PM, Filippini B, Michailidis J, Scacchi M, Stramba Badiale M, Cavagnini F (2010) Increased prevalence of prolonged QT interval in males with primary or secondary hypogonadism: a pilot study. International Journal of Andrology 33:e132-138

439.   van Noord C, Rodenburg EM, Stricker BH (2011) Invited commentary: sex-steroid hormones and QT-interval duration. American Journal of Epidemiology 174:412-415

440.   Sieveking DP, Lim P, Chow RW, Dunn LL, Bao S, McGrath KC, Heather AK, Handelsman DJ, Celermajer DS, Ng MK (2010) A sex-specific role for androgens in angiogenesis. Journal of Experimental Medicine 207:345-352

441.   Lecce L, Lam YT, Lindsay LA, Yuen SC, Simpson PJ, Handelsman DJ, Ng MK (2014) Aging impairs VEGF-mediated, androgen-dependent regulation of angiogenesis. Molecular Endocrinology 28:1487-1501

442.   Wu FC, von Eckardstein A (2003) Androgens and coronary artery disease. Endocrine Reviews 24:183-217

443.   Khaw KT, Dowsett M, Folkerd E, Bingham S, Wareham N, Luben R, Welch A, Day N (2007) Endogenous testosterone and mortality due to all causes, cardiovascular disease, and cancer in men: European prospective investigation into cancer in Norfolk (EPIC-Norfolk) Prospective Population Study. Circulation 116:2694-2701

444.   Laughlin GA, Barrett-Connor E, Bergstrom J (2008) Low serum testosterone and mortality in older men. Journal of Clinical Endocrinology and Metabolism 93:68-75

445.   Smith GD, Ben-Shlomo Y, Beswick A, Yarnell J, Lightman S, Elwood P (2005) Cortisol, testosterone, and coronary heart disease: prospective evidence from the Caerphilly study. Circulation 112:332-340

446.   Araujo AB, Kupelian V, Page ST, Handelsman DJ, Bremner WJ, McKinlay JB (2007) Sex steroids and all-cause and cause-specific mortality in men. Archives of Internal Medicine 167:1252-1260

447.   Maggio M, Lauretani F, Ceda GP, Bandinelli S, Ling SM, Metter EJ, Artoni A, Carassale L, Cazzato A, Ceresini G, Guralnik JM, Basaria S, Valenti G, Ferrucci L (2007) Relationship between low levels of anabolic hormones and 6-year mortality in older men: the aging in the Chianti Area (InCHIANTI) study. Archives of Internal Medicine 167:2249-2254

448.   Xu L, Freeman G, Cowling BJ, Schooling CM (2013) Testosterone therapy and cardiovascular events among men: a systematic review and meta-analysis of placebo-controlled randomized trials. BMC Med 11:108

449.   Handelsman DJ (2011) An old emperor finds new clothing: rejuvenation in our time. Asian Journal of Andrology 13:125-129

450.   Haring R, Teumer A, Volker U, Dorr M, Nauck M, Biffar R, Volzke H, Baumeister SE, Wallaschofski H (2013) Mendelian randomization suggests non-causal associations of testosterone with cardiometabolic risk factors and mortality. Andrology 1:17-23

451.   Zhao J, Jiang C, Lam TH, Liu B, Cheng KK, Xu L, Au Yeung SL, Zhang W, Leung GM, Schooling CM (2014) Genetically predicted testosterone and cardiovascular risk factors in men: a Mendelian randomization analysis in the Guangzhou Biobank Cohort Study. International Journal of Epidemiology 43:140-148

452.   Gong J, Zubair N (2015) Commentary: Mendelian randomization, testosterone, and cardiovascular disease. International Journal of Epidemiology 44:621-622

453.   Swerdlow AJ, Higgins CD, Schoemaker MJ, Wright AF, Jacobs PA (2005) Mortality in patients with Klinefelter syndrome in Britain: a cohort study. Journal of Clinical Endocrinology and Metabolism 90:6516-6522

454.   Roddam AW, Allen NE, Appleby P, Key TJ (2008) Endogenous sex hormones and prostate cancer: a collaborative analysis of 18 prospective studies. Journal of the National Cancer Institute 100:170-183

455.   Boyle P, Koechlin A, Bota M, d'Onofrio A, Zaridze DG, Perrin P, Fitzpatrick J, Burnett AL, Boniol M (2016) Endogenous and exogenous testosterone and the risk of prostate cancer and increased prostate specific antigen (PSA): a meta-analysis. BJU International

456.   Millar AC, Lau AN, Tomlinson G, Kraguljac A, Simel DL, Detsky AS, Lipscombe LL (2016) Predicting low testosterone in aging men: a systematic review. Cmaj 188:E321-330

457.   Huo S, Scialli AR, McGarvey S, Hill E, Tugertimur B, Hogenmiller A, Hirsch AI, Fugh-Berman A (2016) Treatment of Men for "Low Testosterone": A Systematic Review. PLoS One 11:e0162480

458.   Conway AJ, Handelsman DJ, Lording DW, Stuckey B, Zajac JD (2000) Use, misuse and abuse of androgens: The Endocrine Society of Australia consensus guidelines for androgen prescribing. Medical Journal of Australia 172:220-224

459.   Petak SM, Nankin HR, Spark RF, Swerdloff RS, Rodriguez-Rigau LJ, American Association of Clinical E (2002) American Association of Clinical Endocrinologists Medical Guidelines for clinical practice for the evaluation and treatment of hypogonadism in adult male patients--2002 update. Endocr Pract 8:440-456

460.   Nieschlag E, Swerdloff R, Behre HM, Gooren LJ, Kaufman JM, Legros JJ, Lunenfeld B, Morley JE, Schulman C, Wang C, Weidner W, Wu FC (2005) Investigation, treatment and monitoring of late-onset hypogonadism in males: ISA, ISSAM, and EAU recommendations. International Journal of Andrology 28:125-127

461.   Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS, Montori VM (2006) Testosterone therapy in adult men with androgen deficiency syndromes: an endocrine society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 91:1995-2010

462.   Handelsman DJ (2012) Pharmacoepidemiology of testosterone prescribing in Australia, 1992-2010. Medical Journal of Australia 196:642-645

463.   Gan EH, Pattman S, S HSP, Quinton R (2013) A UK epidemic of testosterone prescribing, 2001-2010. Clinical Endocrinology 79:564-570

464.   Nigro N, Christ-Crain M (2012) Testosterone treatment in the aging male: myth or reality? Swiss Medical Weekly 142:w13539

465.   Bhasin S, Singh AB, Mac RP, Carter B, Lee MI, Cunningham GR (2003) Managing the risks of prostate disease during testosterone replacement therapy in older men: recommendations for a standardized monitoring plan. Journal of Andrology 24:299-311

466.   Tan RS, Salazar JA (2004) Risks of testosterone replacement therapy in ageing men. Expert Opin Drug Saf 3:599-606

467.   Baillargeon J, Urban RJ, Ottenbacher KJ, Pierson KS, Goodwin JS (2013) Trends in androgen prescribing in the United States, 2001 to 2011. JAMA Intern Med 173:1465-1466

468.   Handelsman DJ (2013) Global trends in testosterone prescribing, 2000-2011: expanding the spectrum of prescription drug misuse. Medical Journal of Australia 199:548-551

469.   Yeap BB, Grossmann M, McLachlan RI, Handelsman DJ, Wittert GA, Conway AJ, Stuckey BG, Lording DW, Allan CA, Zajac JD, Burger HG (2016) Endocrine Society of Australia position statement on male hypogonadism (part 1): assessment and indications for testosterone therapy. Medical Journal of Australia 205:173-178

470.   Yeap BB, Grossmann M, McLachlan RI, Handelsman DJ, Wittert GA, Conway AJ, Stuckey BG, Lording DW, Allan CA, Zajac JD, Burger HG (2016) Endocrine Society of Australia position statement on male hypogonadism (part 2): treatment and therapeutic considerations. Medical Journal of Australia 205:228-231

471.   Vandekerckhove P, Lilford R, Vail A, Hughes E (2000) Androgens versus placebo or no treatment for idiopathic oligo/asthenospermia. Cochrane Database Syst Rev:CD000150

472.   Gabrielsen JS, Najari BB, Alukal JP, Eisenberg ML (2016) Trends in Testosterone Prescription and Public Health Concerns. Urologic Clinics of North America 43:261-271

473.   Handelsman DJ (2004) Trends and regional differences in testosterone prescribing in Australia, 1991-2001. Medical Journal of Australia 181:419-422

474.   Layton JB, Li D, Meier CR, Sharpless J, Sturmer T, Jick SS, Brookhart MA (2014) Testosterone Lab Testing and Initiation in the United Kingdom and the United States, 2000-2011. Journal of Clinical Endocrinology and Metabolism:jc20133570

475.   Rao PK, Boulet SL, Mehta A, Hotaling J, Eisenberg ML, Honig SC, Warner L, Kissin DM, Nangia AK, Ross LS (2016) Trends in Testosterone Replacement Therapy Use Among Reproductive-Age US Men, 2003-2013. Journal of Urology

476.   Jasuja GK, Bhasin S, Reisman JI, Berlowitz DR, Rose AJ (2015) Ascertainment of Testosterone Prescribing Practices in the VA. Medical Care 53:746-752

477.   Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS, Montori VM (2010) Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 95:2536-2559

478.   Wang C, Nieschlag E, Swerdloff R, Behre HM, Hellstrom WJ, Gooren LJ, Kaufman JM, Legros JJ, Lunenfeld B, Morales A, Morley JE, Schulman C, Thompson IM, Weidner W, Wu FC (2009) Investigation, treatment, and monitoring of late-onset hypogonadism in males: ISA, ISSAM, EAU, EAA, and ASA recommendations. Journal of Andrology 30:1-9

479.   Handelsman DJ 2010 Androgen Physiology, Pharmacology and Abuse. In: DeGroot LJ, Jameson JL eds. Endocrinology. 6th ed. Philadelphia: Elsevier Saunders; 2469-2498

480.   Herlihy AS, Halliday JL, Cock ML, McLachlan RI (2011) The prevalence and diagnosis rates of Klinefelter syndrome: an Australian comparison. Medical Journal of Australia 194:24-28

481.   Mulligan T, Frick MF, Zuraw QC, Stemhagen A, McWhirter C (2006) Prevalence of hypogonadism in males aged at least 45 years: the HIM study. International Journal of Clinical Practice 60:762-769

482.   Araujo AB, O'Donnell AB, Brambilla DJ, Simpson WB, Longcope C, Matsumoto AM, McKinlay JB (2004) Prevalence and incidence of androgen deficiency in middle-aged and older men: estimates from the Massachusetts Male Aging Study. Journal of Clinical Endocrinology and Metabolism 89:5920-5926

483.   Haring R, Ittermann T, Volzke H, Krebs A, Zygmunt M, Felix SB, Grabe HJ, Nauck M, Wallaschofski H (2010) Prevalence, incidence and risk factors of testosterone deficiency in a population-based cohort of men: results from the study of health in Pomerania. Aging Male 13:247-257

484.   Wu FC, Tajar A, Beynon JM, Pye SR, Silman AJ, Finn JD, O'Neill TW, Bartfai G, Casanueva FF, Forti G, Giwercman A, Han TS, Kula K, Lean ME, Pendleton N, Punab M, Boonen S, Vanderschueren D, Labrie F, Huhtaniemi IT (2010) Identification of late-onset hypogonadism in middle-aged and elderly men. New England Journal of Medicine 363:123-135

485.   Hoberman JM, Yesalis CE (1995) The history of synthetic testosterone. Scientific American 272:76-81

486.   Franke WW, Berendonk B (1997) Hormonal doping and androgenization of athletes: a secret program of the German Democratic Republic government. Clinical Chemistry 43:1262-1279

487.   Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X, Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim I, Shen R, Storer TW (2001) Testosterone dose-response relationships in healthy young men. Am J Physiol Endocrinol Metab 281:E1172-1181

488.   Pope HG, Jr., Kouri EM, Hudson JI (2000) Effects of supraphysiologic doses of testosterone on mood and aggression in normal men: a randomized controlled trial. Archives of General Psychiatry 57:133-140; discussion 155-136

489.   Kanayama G, Hudson JI, Pope HG, Jr. (2008) Long-term psychiatric and medical consequences of anabolic-androgenic steroid abuse: A looming public health concern? Drug and Alcohol Dependence 98:1-12

490.   Sagoe D, Molde H, Andreassen CS, Torsheim T, Pallesen S (2014) The global epidemiology of anabolic-androgenic steroid use: a meta-analysis and meta-regression analysis. Annals of Epidemiology 24:383-398

491.   Buckley WE, Yesalis CE, Freidl KE, Anderson WA, Streit AL, Wright JE (1988) Estimated prevalence of anabolic steroid use among male high school students. Journal of the American Medical Association 260:3441-3445

492.   Nilsson S (1995) Androgenic anabolic steroid use among male adolescents in Falkenberg. Europen Journal of Clinical Pharmacology 48:9-11

493.   Handelsman DJ, Gupta L (1997) Prevalence and risk factors for anabolic-androgenic steroid abuse in Australian secondary school students. International Journal of Andrology 20:159-164

494.   Lambert MI, Titlestad SD, Schwellnus MP (1998) Prevalence of androgenic-anabolic steroid use in adolescents in two regions of South Africa. South African Medical Journal 88:876-880

495.   Ferenchick GS (1996) Validity of self-report in identifying anabolic steroid use among weightlifters. Journal of General Internal Medicine 11:554-556

496.   Pope HG, Kouri EM, Powell KF, Campbell C, Katz DL (1996) Anabolic-androgenic steroid use among 133 prisoners. Comprehensive Psychiatry 37:322-327

497.   Isacsson G, Garle M, Ljung EB, Asgard U, Bergmen U (1998) Anabolic steroids and violent crime - an epidemiological study at a jail in Stockholm, Sweden. Comprehensive Psychiatry 39:203-205

498.   Catlin DH, Ahrens BD, Kucherova Y (2002) Detection of norbolethone, an anabolic steroid never marketed, in athletes' urine. Rapid Communication in Mass Spectrometry 16:1273-1275

499.   Death AK, McGrath KC, Kazlauskas R, Handelsman DJ (2004) Tetrahydrogestrinone is a potent androgen and progestin. Journal of Clinical Endocrinology and Metabolism 89:2498-2500

500.   Catlin DH, Sekera MH, Ahrens BD, Starcevic B, Chang YC, Hatton CK (2004) Tetrahydrogestrinone: discovery, synthesis, and detection. Rapid Communication in Mass Spectrometry 18:1245-1249

501.   Sekera MH, Ahrens BD, Chang YC, Starcevic B, Georgakopoulos C, Catlin DH (2005) Another designer steroid: discovery, synthesis, and detection of 'madol' in urine. Rapid Communication in Mass Spectrometry 19:781-784

502.   Handelsman DJ, Heather A (2008) Androgen abuse in sports. Asian Journal of Andrology 10:403-415

503.   vandenBerg P, Neumark-Sztainer D, Cafri G, Wall M (2007) Steroid use among adolescents: longitudinal findings from Project EAT. Pediatrics 119:476-486

504.   McCabe SE, Brower KJ, West BT, Nelson TF, Wechsler H (2007) Trends in non-medical use of anabolic steroids by U.S. college students: results from four national surveys. Drug and Alcohol Dependence 90:243-251

505.   Holma PK (1977) Effects of an anabolic steroid (metandienone) on spermatogenesis. Contraception 15:151-162

506.   Knuth UA, Maniera H, Nieschlag E (1989) Anabolic steroids and semen parameters in bodybuilders. Fertility and Sterility 52:1041-1047

507.   Turek PJ, Williams RH, Gilbaugh JH, Lipshultz LI (1995) The reversibility of anabolic steroid-induced azoospermia. Journal of Urology 153:1628-1630

508.   Sorensen M, Ingerslev HJ (1995) Azoospermia in two bodybuilders taking anabolic steroids. Ugeskrift for Laeger 157:1044-1045

509.   Gazvani MR, Buckett W, Luckas MJ, Aird IA, Hipkin LJ, Lewis-Jones DI (1997) Conservative management of azoospermia following steroid abuse. Human Reproduction 12:1706-1708

510.   Reyes RJ, Zicchi S, Hamed H, Chaudary MA, Fentiman IS (1995) Surgical correction of gynaecomastia in bodybuilders. British Journal of Clinical Practice 49:177-179

511.   Friedl KE (1990) Reappraisal of health risks associated with use of high doses of oral and injectable androgenic steroids. NIDA Research Monograph 102:142-177

512.   Sklarek HM, Mantovani RP, Erens E, Heisler D, Niederman MS, Fein AM (1984) AIDS in a bodybuilder using anabolic steroids [letter]. New England Journal of Medicine 311:1701

513.   Nemechek PM (1991) Anabolic steroid users--another potential risk group for HIV infection [letter]. New England Journal of Medicine 325:357

514.   Henrion R, Mandelbrot L, Delfieu D (1992) HIV contamination after injections of anabolic steroids (letter). Presse Medicale 21:218

515.   Rich JD, Dickinson BP, Merriman NA, Flanigan TP (1998) Hepatitis C virus infection related to anabolic-androgenic steroid injection in a recreational weight lifter [letter]. American Journal of Gastroenterology 93:1598

516.   Aitken C, Delalande C, Stanton K (2002) Pumping iron, risking infection? Exposure to hepatitis C, hepatitis B and HIV among anabolic-androgenic steroid injectors in Victoria, Australia. Drug and Alcohol Dependence 65:303-308

517.   Rich JD, Dickinson BP, Feller A, Pugatch D, Mylonakis E (1999) The infectious complications of anabolic-androgenic steroid injection. International Journal of Sports Medicine 20:563-566

518.   Khankhanian NK, Hammers YA (1992) Exuberant local tissue reaction to intramuscular injection of nandrolone decanoate (Deca-Durabolin) - a steroid compound in sesame seed oil base - mimicking soft tissue malignant tumors: a case report and review of the literature. Military Medicine 157:670-674

519.   Evans NA (1997) Local complications of self administered anabolic steroid injections. British Journal of Sports Medicine 31:349-350

520.   Freeman BJ, Rooker GD (1995) Spontaneous rupture of the anterior cruciate ligament after anabolic steroids. British Journal of Sports Medicine 29:274-275

521.   Daniels JM, van Westerloo DJ, de Hon OM, Frissen PH (2006) [Rhabdomyolysis in a bodybuilder using steroids]. Nederlands Tijdschrift Voor Geneeskunde 150:1077-1080

522.   Lepori M, Perren A, Gallino A (2002) The popliteal-artery entrapment syndrome in a patient using anabolic steroids. New England Journal of Medicine 346:1254-1255

523.   Sahraian MA, Mottamedi M, Azimi AR, Moghimi B (2004) Androgen-induced cerebral venous sinus thrombosis in a young body builder: case report. BMC Neurol 4:22

524.   Liljeqvist S, Hellden A, Bergman U, Soderberg M (2008) Pulmonary embolism associated with the use of anabolic steroids. Eur J Intern Med 19:214-215

525.   Alaraj AM, Chamoun RB, Dahdaleh NS, Haddad GF, Comair YG (2005) Spontaneous subdural haematoma in anabolic steroids dependent weight lifters: reports of two cases and review of literature. Acta Neurochirurgica 147:85-87; discussion 87-88

526.   Petersson A, Garle M, Granath F, Thiblin I (2007) Convulsions in users of anabolic androgenic steroids: possible explanations. Journal of Clinical Psychopharmacology 27:723-725

527.   Pope HG, Katz DL (1988) Affective and psychotic symptoms associated with anabolic steroid use. American Journal of Psychiatry 145:487-490

528.   Bahrke MS, Yesalis CE, Wright JE (1996) Psychological and behavioural effects of endogenous testosterone levels and anabolic-androgenic steroids among male. An update. Sports Medicine 22:367-390

529.   Rockhold RW (1993) Cardiovascular toxicity of anabolic steroids. Annual Review of Pharmacology and Toxicology 33:497-520

530.   Melchert RB, Welder AA (1995) Cardiovascular effects of androgenic-anabolic steroids. Medicine and Science in Sports and Exercise 27:1252-1262

531.   Sullivan ML, Martinez CM, Gennis P, Gallagher EJ (1998) The cardiac toxicity of anabolic steroids. Progress in Cardiovascular Diseases 41:1-15

532.   Dhar R, Stout CW, Link MS, Homoud MK, Weinstock J, Estes NA, 3rd (2005) Cardiovascular toxicities of performance-enhancing substances in sports. Mayo Clinic Proceedings 80:1307-1315

533.   Furlanello F, Serdoz LV, Cappato R, De Ambroggi L (2007) Illicit drugs and cardiac arrhythmias in athletes. Eur J Cardiovasc Prev Rehabil 14:487-494

534.   Larkin GL (1991) Carcinoma of the prostate. New England Journal of Medicine 324:1892

535.   Roberts JT, Essenhigh DM (1986) Adenocarcinoma of prostate in 40 year old body-builder. Lancet 2:742

536.   Nakata S, Hasumi M, Sato J, Ogawa A, Yamanaka H (1997) Prostate cancer associated with long-term intake of patent medicine containing methyltestosterone: a case report. Hinyokika Kiyo - Acta Urologica Japonica 43:791-793

537.   Hartgens F, Cheriex EC, Kuipers H (2003) Prospective echocardiographic assessment of androgenic-anabolic steroids effects on cardiac structure and function in strength athletes. International Journal of Sports Medicine 24:344-351

538.   Chung T, Kelleher S, Liu PY, Conway AJ, Kritharides L, Handelsman DJ (2007) Effects of testosterone and nandrolone on cardiac function: a randomized, placebo-controlled study. Clinical Endocrinology 66:235-245

539.   Jin B, Turner L, Walters WAW, Handelsman DJ (1996) Androgen or estrogen effects on the human prostate. Journal of Clinical Endocrinology and Metabolism 81:4290-4295

540.   Palatini P, Giada F, Garavelli G, Sinisi F, Mario L, Michieletto M, Baldo-Enzi G (1996) Cardiovascular effects of anabolic steroids in weight-trained subjects. Journal of Clinical Pharmacology and New Drugs 36:1132-1140

541.   Krieg A, Scharhag J, Albers T, Kindermann W, Urhausen A (2007) Cardiac tissue Doppler in steroid users. International Journal of Sports Medicine 28:638-643

542.   D'Andrea A, Caso P, Salerno G, Scarafile R, De Corato G, Mita C, Di Salvo G, Severino S, Cuomo S, Liccardo B, Esposito N, Calabro R (2007) Left ventricular early myocardial dysfunction after chronic misuse of anabolic androgenic steroids: a Doppler myocardial and strain imaging analysis. British Journal of Sports Medicine 41:149-155

543.   Lane HA, Grace F, Smith JC, Morris K, Cockcroft J, Scanlon MF, Davies JS (2006) Impaired vasoreactivity in bodybuilders using androgenic anabolic steroids. European Journal of Clinical Investigation 36:483-488

544.   Nottin S, Nguyen LD, Terbah M, Obert P (2006) Cardiovascular effects of androgenic anabolic steroids in male bodybuilders determined by tissue Doppler imaging. American Journal of Cardiology 97:912-915

545.   Urhausen A, Albers T, Kindermann W (2004) Are the cardiac effects of anabolic steroid abuse in strength athletes reversible? Heart 90:496-501

546.   Climstein M, O'Shea P, Adams KJ, DeBeliso M (2003) The effects of anabolic-androgenic steroids upon resting and peak exercise left ventricular heart wall motion kinetics in male strength and power athletes. Journal of Science and Medicine in Sport 6:387-397

547.   Grace F, Sculthorpe N, Baker J, Davies B (2003) Blood pressure and rate pressure product response in males using high-dose anabolic androgenic steroids (AAS). Journal of Science and Medicine in Sport 6:307-312

548.   Karila TA, Karjalainen JE, Mantysaari MJ, Viitasalo MT, Seppala TA (2003) Anabolic androgenic steroids produce dose-dependant increase in left ventricular mass in power atheletes, and this effect is potentiated by concomitant use of growth hormone. International Journal of Sports Medicine 24:337-343

549.   Sader MA, Griffiths KA, McCredie RJ, Handelsman DJ, Celermajer DS (2001) Androgenic anabolic steroids and arterial structure and function in male bodybuilders. Journal of the American College of Cardiology 37:224-230

550.   Di Bello V, Giorgi D, Bianchi M, Bertini A, Caputo MT, Valenti G, Furioso O, Alessandri L, Paterni M, Giusti C (1999) Effects of anabolic-androgenic steroids on weight-lifters' myocardium: an ultrasonic videodensitometric study. Medicine and Science in Sports and Exercise 31:514-521

551.   Dickerman RD, Schaller F, Zachariah NY, McConathy WJ (1997) Left ventricular size and function in elite bodybuilders using anabolic steroids. Clinical Journal of Sports Medicine 7:90-93

552.   Di Bello V, Pedrinelli R, Giorgi D, Bertini A, Talarico L, Caputo MT, Massimiliano B, Dell'Omo G, Paterni M, Giusti C (1997) Ultrasonic videodensitometric analysis of two different models of left ventricular hypertrophy. Athlete's heart and hypertension. Hypertension 29:937-944

553.   Thiblin I, Petersson A (2005) Pharmacoepidemiology of anabolic androgenic steroids: a review. Fundamental and Clinical Pharmacology 19:27-44

554.   Sarna S, Sahi T, Koskenvuo M, Kaprio J (1993) Increased life expectancy of world class male athletes. Medicine and Science in Sports and Exercise 25:237-244

555.   Sarna S, Kaprio J, Kujala UM, Koskenvuo M (1997) Health status of former elite athletes. The Finnish experience. Aging 9:35-41

556.   Parssinen M, Kujala U, Vartiainen E, Sarna S, Seppala T (2000) Increased premature mortality of competitive powerlifters suspected to have used anabolic agents. International Journal of Sports Medicine 21:225-227

557.   Kashkin KB, Kleber HD (1989) Hooked on hormones? An anabolic steroid addiction hypothesis. Journal of the American Medical Association 262:3166-3170

558.   Fingerhood MI, Sullivan JT, Testa M, Jasinski DR (1997) Abuse liability of testosterone. Journal of Psychopharmacology 11:59-63

559.   Gazvani MR, Buckett W, Luckas MJ, Aird IA, Hipkin LJ, Lewis-Jones DI (1997) Conservative management of azoospermia following steroid abuse. Hum Reprod 12:1706-1708

560.   Gill GV (1998) Anabolic steroid induced hypogonadism treated with human chorionic gonadotropin. Postgrad Med J 74:45-46

561.   Boyadjiev NP, Georgieva KN, Massaldjieva RI, Gueorguiev SI (2000) Reversible hypogonadism and azoospermia as a result of anabolic-androgenic steroid use in a bodybuilder with personality disorder. A case report. J Sports Med Phys Fitness 40:271-274

562.   Torres-Calleja J, Gonzalez-Unzaga M, DeCelis-Carrillo R, Calzada-Sanchez L, Pedron N (2001) Effect of androgenic anabolic steroids on sperm quality and serum hormone levels in adult male bodybuilders. Life Sci 68:1769-1774

563.   Shankara-Narayana N, Yu C, Savkovic S, Desai R, Fennell C, Turner L, Jayadev V, Conway AJ, Kockx M, Ridley L, Kritharides L, Handelsman DJ (2020) Rate and Extent of Recovery from Reproductive and Cardiac Dysfunction Due to Androgen Abuse in Men. J Clin Endocrinol Metab 105

564.   Menon DK (2003) Successful treatment of anabolic steroid-induced azoospermia with human chorionic gonadotropin and human menopausal gonadotropin. Fertility and Sterility 79 Suppl 3:1659-1661

565.   Drakeley A, Gazvani R, Lewis-Jones I (2004) Duration of azoospermia following anabolic steroids. Fertility and Sterility 81:226

566.   Nejat RJ, Rashid HH, Bagiella E, Katz AE, Benson MC (2000) A prospective analysis of time to normalization of serum testosterone after withdrawal of androgen deprivation therapy. Journal of Urology 164:1891-1894

567.   Kaku H, Saika T, Tsushima T, Ebara S, Senoh T, Yamato T, Nasu Y, Kumon H (2006) Time course of serum testosterone and luteinizing hormone levels after cessation of long-term luteinizing hormone-releasing hormone agonist treatment in patients with prostate cancer. Prostate 66:439-444

568.   Goldberg L, MacKinnon DP, Elliot DL, Moe EL, Clarke G, Cheong J (2000) The adolescents training and learning to avoid steroids program: preventing drug use and promoting health behaviors. Archives of Pediatrics and Adolescent Medicine 154:332-338

569.   Behre HM, Kliesch S, Leifke E, Link TM, Nieschlag E (1997) Long-term effect of testosterone therapy on bone mineral density in hypogonadal men. Journal of Clinical Endocrinology and Metabolism

 82:2386-2390

570.   Jockenhovel F, Vogel E, Reinhardt W, Reinwein D (1997) Effects of various modes of androgen substitution therapy on erythropoiesis. European Journal of Medical Research 2:293-298

571.   Wang C, Swedloff RS, Iranmanesh A, Dobs A, Snyder PJ, Cunningham G, Matsumoto AM, Weber T, Berman N (2000) Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. Testosterone Gel Study Group. Journal of Clinical Endocrinology and Metabolism 85:2839-2853

572.   Aminorroaya A, Kelleher S, Conway AJ, Ly LP, Handelsman DJ (2005) Adequacy of androgen replacement influences bone density response to testosterone in androgen-deficient men. European Journal of Endocrinology 152:881-886

573.   Wong FH, Pun KK, Wang C (1993) Loss of bone mass in patients with Klinefelter's syndrome despite sufficient testosterone replacement. Osteoporosis International 3:3-7

574.   Ishizaka K, Suzuki M, Kageyama Y, Kihara K, Yoshida K (2002) Bone mineral density in hypogonadal men remains low after long-term testosterone replacement. Asian Journal of Andrology 4:117-121

575.   Anderson RA, Wallace AM, Sattar N, Kumar N, Sundaram K (2003) Evidence for tissue selectivity of the synthetic androgen 7 alpha-methyl-19-nortestosterone in hypogonadal men. Journal of Clinical Endocrinology and Metabolism 88:2784-2793

576.   Zacharin MR, Pua J, Kanumakala S (2003) Bone mineral density outcomes following long-term treatment with subcutaneous testosterone pellet implants in male hypogonadism. Clinical Endocrinology 58:691-695

577.   Schubert M, Bullmann C, Minnemann T, Reiners C, Krone W, Jockenhovel F (2003) Osteoporosis in male hypogonadism: responses to androgen substitution differ among men with primary and secondary hypogonadism. Hormone Research 60:21-28

578.   Finkelstein JS, Klibanski A, Neer RM (1996) A longitudinal evaluation of bone mineral density in adult men with histories of delayed puberty. Journal of Clinical Endocrinology and Metabolism 81:1152-1155

579.   Finkelstein JS, Neer RM, Biller BM, Crawford JD, Klibanski A (1992) Osteopenia in men with a history of delayed puberty. New England Journal of Medicine 326:600-604

580.   Huhtaniemi IT, Pye SR, Limer KL, Thomson W, O'Neill TW, Platt H, Payne D, John SL, Jiang M, Boonen S, Borghs H, Vanderschueren D, Adams JE, Ward KA, Bartfai G, Casanueva F, Finn JD, Forti G, Giwercman A, Han TS, Kula K, Lean ME, Pendleton N, Punab M, Silman AJ, Wu FC (2009) Increased estrogen rather than decreased androgen action is associated with longer androgen receptor CAG repeats. Journal of Clinical Endocrinology and Metabolism 94:277-284

581.   Kicman AT (2008) Pharmacology of anabolic steroids. British Journal of Pharmacology 154:502-521

582.   Dalton JT, Mukherjee A, Zhu Z, Kirkovsky L, Miller DD (1998) Discovery of nonsteroidal androgens. Biochemical and Biophysical Research Communications 244:1-4

583.   Thevis M, Schanzer W (2008) Mass spectrometry of selective androgen receptor modulators. Journal of Mass Spectrometry 43:865-876

584.   Lubahn D, Joseph DR, Sullivan PM, Williard HF, French FS, Wilson EM (1988) Cloning of the human androgen receptor complementary DNA and localisation to the X-chromosome. Science 240:327-330

585.   Chang CS, Kokontis J, Liao ST (1988) Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science 240:324-326

586.   Trapman J, Klaassen P, Kuiper GG, van der Korput JA, Faber PW, van Rooij HC, Geurts van Kessel A, Voorhorst MM, Mulder E, Brinkmann AO (1988) Cloning, structure and expression of a cDNA encoding the human androgen receptor. Biochemical and Biophysical Research Communications 153:241-248

587.   Wilson JD (1980) The use and misuse of androgens. Metabolism: Clinical and Experimental 29:1278-1295

588.   Negro-Vilar A (1999) Selective androgen receptor modulators (SARMs): a novel approach to androgen therapy for the new millennium. Journal of Clinical Endocrinology and Metabolism 84:3459-3462

589.   Bhasin S, Jasuja R (2009) Selective androgen receptor modulators as function promoting therapies. Current Opinion in Clinical Nutrition and Metabolic Care 12:232-240

590.   Handelsman DJ, Conway AJ, Boylan LM (1990) Pharmacokinetics and pharmacodynamics of testosterone pellets in man. Journal of Clinical Endocrinology and Metabolism 71:216-222

591.   Deansley R, Parkes AS (1938) Further experiments on the administration of hormones by the subcutaneous implantation of tablets. Lancet ii:606-608

592.   Kelleher S, Howe C, Conway AJ, Handelsman DJ (2004) Testosterone release rate and duration of action of testosterone pellet implants. Clinical Endocrinology 60:420-428

593.   Vierhapper H, Nowotny P, Waldhausl W (2003) Reduced production rates of testosterone and dihydrotestosterone in healthy men treated with rosiglitazone. Metabolism: Clinical and Experimental 52:230-232

594.   Handelsman DJ, Mackey MA, Howe C, Turner L, Conway AJ (1997) Analysis of testosterone implants for androgen replacement therapy. Clinical Endocrinology 47:311-316

595.   Kelleher S, Turner L, Howe C, Conway AJ, Handelsman DJ (1999) Extrusion of testosterone pellets: a randomized controlled clinical study. Clinical Endocrinology 51:469-471

596.   Kelleher S, Conway AJ, Handelsman DJ (2002) A randomised controlled clinical trial of antibiotic impregnation of testosterone pellet implants to reduce extrusion rate. European Journal of Endocrinology 146:513-518

597.   Kelleher S, Conway AJ, Handelsman DJ (2001) Influence of implantation site and track geometry on the extrusion rate and pharmacology of testosterone implants. Clinical Endocrinology 55:531-536

598.   Cavender RK, Fairall M (2009) Subcutaneous testosterone pellet implant (Testopel) therapy for men with testosterone deficiency syndrome: a single-site retrospective safety analysis. J Sex Med 6:3177-3192

599.   Pastuszak AW, Mittakanti H, Liu JS, Gomez L, Lipshultz LI, Khera M (2012) Pharmacokinetic evaluation and dosing of subcutaneous testosterone pellets. Journal of Andrology 33:927-937

600.   McCullough AR, Khera M, Goldstein I, Hellstrom WJ, Morgentaler A, Levine LA (2012) A multi-institutional observational study of testosterone levels after testosterone pellet (Testopel((R))) insertion. J Sex Med 9:594-601

601.   Bals-Pratsch M, Knuth UA, Yoon YD, Nieschlag E (1986) Transdermal testosterone substitution therapy for male hypogonadism. Lancet 2:943-946

602.   Findlay JC, Place VA, Snyder PJ (1987) Transdermal delivery of testosterone. Journal of Clinical Endocrinology and Metabolism 64:266-268

603.   Bals-Pratsch M, Langer K, Place VA, Nieschlag E (1988) Substitution therapy of hypogonadal men with transdermal testosterone over one year. Acta Endocrinologica 118:7-13

604.   Jordan WP, Jr., Atkinson LE, Lai C (1998) Comparison of the skin irritation potential of two testosterone transdermal systems: an investigational system and a marketed product. Clinical Therapeutics 20:80-87

605.   Jordan WP, Jr. (1997) Allergy and topical irritation associated with transdermal testosterone administration: a comparison of scrotal and nonscrotal transdermal systems. American Journal of Contact Dermatitis 8:108-113

606.   Meikle AW, Mazer NA, Moellmer JF, Stringham JD, Tolman KG, Sanders SW, Odell WD (1992) Enhanced transdermal delivery of testosterone across nonscrotal skin produces physiological concentrations of testosterone and its metabolites in hypogonadal men. Journal of Clinical Endocrinology and Metabolism 74:623-628

607.   Arver S, Dobs AS, Meikle AW, Caramelli KE, Rajaram L, Sanders SW, Mazer NA (1997) Long-term efficacy and safety of a permeation-enhanced testosterone transdermal system in hypogonadal men. Clinical Endocrinology 47:727-737

608.   Shomaker TS, Zhang J, Ashburn MA (2001) A pilot study assessing the impact of heat on the transdermal delivery of testosterone. Journal of Clinical Pharmacology 41:677-682

609.   Bennett NJ (1998) A burn-like lesion caused by a testosterone transdermal system. Burns 24:478-480

610.   Wilson DE, Kaidbey K, Boike SC, Jorkasky DK (1998) Use of topical corticosteroid pretreatment to reduce the incidence and severity of skin reactions associated with testosterone transdermal therapy. Clinical Therapeutics 20:299-306

611.   Guerin JF, Rollet J (1988) Inhibition of spermatogenesis in men using various combinations of oral progestagens and percutaneous or oral androgens. International Journal of Andrology 11:187-199

612.   Fiet J, Morville R, Chemana D, Villette JM, Gourmel B, Brerault JL, Dreux C (1982) Percutaneous absorption of 5a-dihydrotestosterone in man. I Plasma androgen and gonadotrophin levels in normal adult men after percutaneous administration of 5a-dihydrotestosterone. International Journal of Andrology 5:586-594

613.   Chemana D, Morville R, Fiet J, Villette JM, Tabuteau F, Brerault JL, Passa P (1982) Percutaneous absorption of 5a-dihydrotestosterone in man. II Percutaneous administration of 5a-dihydrotestosterone in hypogonadal men with idiopathic haemochromatosis; clinical, metabolic and hormonal effectiveness. International Journal of Andrology 5:595-606

614.   Wang C, Iranmanesh A, Berman N, McDonald V, Steiner B, Ziel F, Faulkner SM, Dudley RE, Veldhuis JD, Swerdloff RS (1998) Comparative pharmacokinetics of three doses of percutaneous dihydrotestosterone gel in healthy elderly men--a clinical research center study. Journal of Clinical Endocrinology and Metabolism 83:2749-2757

615.   Swerdloff RS, Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Longstreth J, Berman N (2000) Long-term pharmacokinetics of transdermal testosterone gel in hypogonadal men. Journal of Clinical Endocrinology and Metabolism 85:4500-4510

616.   Rolf C, Kemper S, Lemmnitz G, Eickenberg U, Nieschlag E (2002) Pharmacokinetics of a new transdermal testosterone gel in gonadotrophin-suppressed normal men. European Journal of Endocrinology 146:673-679

617.   Steidle C, Schwartz S, Jacoby K, Sebree T, Smith T, Bachand R (2003) AA2500 testosterone gel normalizes androgen levels in aging males with improvements in body composition and sexual function. Journal of Clinical Endocrinology and Metabolism 88:2673-2681

618.   McNicholas TA, Dean JD, Mulder H, Carnegie C, Jones NA (2003) A novel testosterone gel formulation normalizes androgen levels in hypogonadal men, with improvements in body composition and sexual function. BJU International 91:69-74

619.   Kuhnert B, Byrne M, Simoni M, Kopcke W, Gerss J, Lemmnitz G, Nieschlag E (2005) Testosterone substitution with a new transdermal, hydroalcoholic gel applied to scrotal or non-scrotal skin: a multicentre trial. European Journal of Endocrinology 153:317-326

620.   Wang C, Ilani N, Arver S, McLachlan RI, Soulis T, Watkinson A (2011) Efficacy and safety of the 2% formulation of testosterone topical solution applied to the axillae in androgen-deficient men. Clinical Endocrinology 75:836-843

621.   Muram D, Melby T, Alles Kingshill E (2012) Skin reactions in a phase 3 study of a testosterone topical solution applied to the axilla in hypogonadal men. Current Medical Research and Opinion 28:761-766

622.   Iyer R, Mok SF, Savkovic S, Turner L, Fraser G, Desai R, Jayadev V, Conway AJ, Handelsman DJ (2017) Pharmacokinetics of testosterone cream applied to scrotal skin. Andrology 5:725-731

623.   Delanoe D, Fougeyrollas B, Meyer L, Thonneau P (1984) Androgenisation of female partners of men on medroxyprogesterone acetate/percutaneous testosterone contraception. Lancet i:276

624.   Moore N, Paux G, Noblet C, Andrejak M (1988) Spouse-related drug side-effects. Lancet 1:468

625.   de Ronde W (2009) Hyperandrogenism after transfer of topical testosterone gel: case report and review of published and unpublished studies. Human Reproduction 24:425-428

626.   Yu YM, Punyasavatsu N, Elder D, D'Ercole AJ (1999) Sexual development in a two-year-old boy induced by topical exposure to testosterone. Pediatrics 104:e23

627.   Bhowmick SK, Ricke T, Rettig KR (2007) Sexual precocity in a 16-month-old boy induced by indirect topical exposure to testosterone. Clinical Pediatrics 46:540-543

628.   Brachet C, Vermeulen J, Heinrichs C (2005) Children's virilization and the use of a testosterone gel by their fathers. European Journal of Pediatrics 164:646-647

629.   Svoren BM, Wolfsdorf JI (2005) Sexual development in a 21 month-old boy caused by testosterone contamination of a topical hydrocortisone cream. Journal of Pediatric Endocrinology and Metabolism 18:507-510

630.   Kunz GJ, Klein KO, Clemons RD, Gottschalk ME, Jones KL (2004) Virilization of young children after topical androgen use by their parents. Pediatrics 114:282-284

631.   Franklin SL, Geffner ME (2003) Precocious puberty secondary to topical testosterone exposure. Journal of Pediatric Endocrinology and Metabolism 16:107-110

632.   Stahlman J, Britto M, Fitzpatrick S, McWhirter C, Testino SA, Brennan JJ, Zumbrunnen TL (2012) Serum testosterone levels in non-dosed females after secondary exposure to 1.62% testosterone gel: effects of clothing barrier on testosterone absorption. Current Medical Research and Opinion 28:291-301

633.   Mazer N, Fisher D, Fischer J, Cosgrove M, Bell D, Eilers B (2005) Transfer of transdermally applied testosterone to clothing: a comparison of a testosterone patch versus a testosterone gel. J Sex Med 2:227-234

634.   Stahlman J, Britto M, Fitzpatrick S, McWhirter C, Testino SA, Brennan JJ, Zumbrunnen TL (2012) Effect of application site, clothing barrier, and application site washing on testosterone transfer with a 1.62% testosterone gel. Current Medical Research and Opinion 28:281-290

635.   Rolf C, Kemper S, Lemmnitz G, Eickenberg U, Nieschlag E (2002) Pharmacokinetics of a new transdermal testosterone gel in gonadotrophin-suppressed normal men. Eur J Endocrinol 146:673-679

636.   de Ronde W, Vogel S, Bui HN, Heijboer AC (2011) Reduction in 24-hour plasma testosterone levels in subjects who showered 15 or 30 minutes after application of testosterone gel. Pharmacotherapy 31:248-252

637.   Rolf C, Knie U, Lemmnitz G, Nieschlag E (2002) Interpersonal testosterone transfer after topical application of a newly developed testosterone gel preparation. Clin Endocrinol (Oxf) 56:637-641

638.   Burris AS, Ewing LL, Sherins RJ (1988) Initial trial of slow-release testosterone microspheres in hypogonadal men. Fertility and Sterility 50:493-497

639.   Bhasin S, Swerdloff RS, Steiner B, Peterson MA, Meridores T, Galmirini M, Pandian MR, Goldberg R, Berman N (1992) A biodegradable testosterone microcapsule formulation provides uniform eugonadal levels of testosterone for 10-11 weeks in hypogonadal men. Journal of Clinical Endocrinology and Metabolism 74:75-83

640.   Amory JK, Anawalt BD, Blaskovich PD, Gilchriest J, Nuwayser ES, Matsumoto AM (2002) Testosterone release from a subcutaneous, biodegradable microcapsule formulation (Viatrel) in hypogonadal men. Journal of Andrology 23:84-91

641.   Page ST, Bremner WJ, Clark RV, Bush MA, Zhi H, Caricofe RB, Smith PM, Amory JK (2008) Nanomilled oral testosterone plus dutasteride effectively normalizes serum testosterone in normal men with induced hypogonadism. Journal of Andrology 29:222-227

642.   Amory JK, Bremner WJ (2005) Oral testosterone in oil plus dutasteride in men: a pharmacokinetic study. Journal of Clinical Endocrinology and Metabolism 90:2610-2617

643.   Amory JK, Page ST, Bremner WJ (2006) Oral testosterone in oil: pharmacokinetic effects of 5alpha reduction by finasteride or dutasteride and food intake in men. Journal of Andrology 27:72-78

644.   Johnsen SG, Kampmann JP, Bennet EP, Jorgensen F (1976) Enzyme induction by oral testosterone. Clinical Pharmacology and Therapeutics 20:233-237

645.   Johnsen S (1978) Long-term oral testosterone and liver function. Lancet 1:50

646.   Daggett PR, Wheeler MJ, Nabarro JD (1978) Oral testosterone, a reappraisal. Hormone Research 9:121-129

647.   Lee A, Rubinow K, Clark RV, Caricofe RB, Bush MA, Zhi H, Roth MY, Page ST, Bremner WJ, Amory JK (2012) Pharmacokinetics of modified slow-release oral testosterone over 9 days in normal men with experimental hypogonadism. Journal of Andrology 33:420-426

648.   Amory JK, Bush MA, Zhi H, Caricofe RB, Matsumoto AM, Swerdloff RS, Wang C, Clark RV (2011) Oral testosterone with and without concomitant inhibition of 5alpha-reductase by dutasteride in hypogonadal men for 28 days. Journal of Urology 185:626-632

649.   Salehian B, Wang C, Alexander G, Davidson T, McDonald V, Berman N, Dudley RE, Ziel F, Swerdloff RS (1995) Pharmacokinetics, bioefficacy, and safety of sublingual testosterone cyclodextrin in hypogonadal men: comparison to testosterone enanthate--a clinical research center study. Journal of Clinical Endocrinology and Metabolism 80:3567-3575

650.   Dobs AS, Hoover DR, Chen MC, Allen R (1998) Pharmacokinetic characteristics, efficacy, and safety of buccal testosterone in hypogonadal males: a pilot study. Journal of Clinical Endocrinology and Metabolism 83:33-39

651.   Mazer N, Bell D, Wu J, Fischer J, Cosgrove M, Eilers B (2005) Comparison of the steady-state pharmacokinetics, metabolism, and variability of a transdermal testosterone patch versus a transdermal testosterone gel in hypogonadal men. J Sex Med 2:213-226

652.   Swerdloff RS, Wang C (1998) Dihydrotestosterone: a rationale for its use as a non-aromatizable androgen replacement therapeutic agent. Baillieres Clinical Endocrinology and Metabolism 12:501-506

653.   Junkman K (1957) Long-acting steroids in reproduction. Recent Progress in Hormone Research 13:380-419

654.   Minto C, Howe C, Wishart S, Conway AJ, Handelsman DJ (1997) Pharmacokinetics and pharmacodynamics of nandrolone esters in oil vehicle: effects of ester, injection site and volume. Journal of Pharmacology and Experimental Therapeutics 281:93-102

655.   Snyder PJ, Lawrence DA (1980) Treatment of male hypogonadism with testosterone enanthate. Journal of Clinical Endocrinology and Metabolism 51:1335-1339

656.   Cantrill JA, Dewis P, Large DM, Newman M, Anderson DC (1984) Which testosterone replacement therapy? Clinical Endocrinology 24:97-107

657.   Conway AJ, Boylan LM, Howe C, Ross G, Handelsman DJ (1988) A randomised clinical trial of testosterone replacement therapy in hypogonadal men. International Journal of Andrology 11:247-264

658.   Behre HM, Wang C, Handelsman DJ, Nieschlag E 2004 Pharmacology of testosterone preparations. In: Nieschlag E, Behre HM eds. Testosterone: Action Deficiency Substitution. 3rd ed. Cambridge: Cambridge University Press; 405-444

659.   Weinbauer GF, Marshall GR, Nieschlag E (1986) New injectable testosterone ester maintains serum testosterone of castrated monkeys in the normal range for four months. Acta Endocrinol 113:128-132

660.   Behre HM, Nieschlag E (1992) Testosterone buciclate (20 Aet-1) in hypogonadal men: pharmacokinetics and pharmacodynamics of the new long-acting androgen ester. Journal of Clinical Endocrinology and Metabolism 75:1204-1210

661.   Behre HM, Baus S, Kliesch S, Keck C, Simoni M, Nieschlag E (1995) Potential of  testosterone buciclate for male contraception: endocrine differences between responders and nonresponders. Journal of Clinical Endocrinology and Metabolism 80:2394-2403

662.   Zhang GY, Gu YQ, Wang XH, Cui YG, Bremner WJ (1998) A pharmacokinetic study of injectable testosterone undecanoate in hypogonadal men. Journal of Andrology 19:761-768

663.   Nieschlag E, Buchter D, Von Eckardstein S, Abshagen K, Simoni M, Behre HM (1999) Repeated intramuscular injections of testosterone undecanoate for substitution therapy in hypogonadal men. Clinical Endocrinology 51:757-763.

664.   von Eckardstein S, Nieschlag E (2002) Treatment of male hypogonadism with testosterone undecanoate injected at extended intervals of 12 weeks: a phase II study. Journal of Andrology 23:419-425

665.   Schubert M, Minnemann T, Hubler D, Rouskova D, Christoph A, Oettel M, Ernst M, Mellinger U, Krone W, Jockenhovel F (2004) Intramuscular testosterone undecanoate: pharmacokinetic aspects of a novel testosterone formulation during long-term treatment of men with hypogonadism. Journal of Clinical Endocrinology and Metabolism 89:5429-5434

666.   Gu YQ, Wang XH, Xu D, Peng L, Cheng LF, Huang MK, Huang ZJ, Zhang GY (2003) A multicenter contraceptive efficacy study of injectable testosterone undecanoate in healthy Chinese men. Journal of Clinical Endocrinology and Metabolism 88:562-568

667.   Gu YQ, Tong JS, Ma DZ, Wang XH, Yuan D, Tang WH, Bremner WJ (2004) Male hormonal contraception: effects of injections of testosterone undecanoate and depot medroxyprogesterone acetate at eight-week intervals in chinese men. Journal of Clinical Endocrinology and Metabolism 89:2254-2262

668.   Qoubaitary A, Meriggiola C, Ng CM, Lumbreras L, Cerpolini S, Pelusi G, Christensen PD, Hull L, Swerdloff RS, Wang C (2006) Pharmacokinetics of testosterone undecanoate injected alone or in combination with norethisterone enanthate in healthy men. Journal of Andrology 27:853-867

669.   Mommers E, Kersemaekers WM, Elliesen J, Kepers M, Apter D, Behre HM, Beynon J, Bouloux PM, Costantino A, Gerbershagen HP, Gronlund L, Heger-Mahn D, Huhtaniemi I, Koldewijn EL, Lange C, Lindenberg S, Meriggiola MC, Meuleman E, Mulders PF, Nieschlag E, Perheentupa A, Solomon A, Vaisala L, Wu FC, Zitzmann M (2008) Male hormonal contraception: a double-blind, placebo-controlled study. Journal of Clinical Endocrinology and Metabolism 93:2572-2580

670.   Jockenhovel F, Minnemann T, Schubert M, Freude S, Hubler D, Schumann C, Christoph A, Ernst M (2009) Comparison of long-acting testosterone undecanoate formulation versus testosterone enanthate on sexual function and mood in hypogonadal men. European Journal of Endocrinology 160:815-819

671.   Singh GK, Turner L, Desai R, Jimenez M, Handelsman DJ (2014) Pharmacokinetic-pharmacodynamic study of subcutaneous injection of depot nandrolone decanoate using dried blood spots sampling coupled with ultrapressure liquid chromatography tandem mass spectrometry assays. J Clin Endocrinol Metab 99:2592-2598

672.   Kaminetsky JC, McCullough A, Hwang K, Jaffe JS, Wang C, Swerdloff RS (2019) A 52-Week Study of Dose Adjusted Subcutaneous Testosterone Enanthate in Oil Self-Administered via Disposable Auto-Injector. J Urol 201:587-594

673.   Olson J, Schrager SM, Clark LF, Dunlap SL, Belzer M (2014) Subcutaneous Testosterone: An Effective Delivery Mechanism for Masculinizing Young Transgender Men. LGBT Health 1:165-167

674.   Spratt DI, Stewart, II, Savage C, Craig W, Spack NP, Chandler DW, Spratt LV, Eimicke T, Olshan JS (2017) Subcutaneous Injection of Testosterone Is an Effective and Preferred Alternative to Intramuscular Injection: Demonstration in Female-to-Male Transgender Patients. J Clin Endocrinol Metab 102:2349-2355

675.   Wilson DM, Kiang TKL, Ensom MHH (2018) Pharmacokinetics, safety, and patient acceptability of subcutaneous versus intramuscular testosterone injection for gender-affirming therapy: A pilot study. Am J Health Syst Pharm 75:351-358

676.   Turner L, Ly LP, Desai R, Singh GKS, Handelsman TD, Savkovic S, Fennell C, Jayadev V, Conway A, Handelsman DJ (2019) Pharmacokinetics and Acceptability of Subcutaneous Injection of Testosterone Undecanoate. J Endocr Soc 3:1531-1540

677.   Kohn FM, Schill WB (2003) A new oral testosterone undecanoate formulation. World Journal of Urology 21:311-315

678.   Bagchus WM, Hust R, Maris F, Schnabel PG, Houwing NS (2003) Important effect of food on the bioavailability of oral testosterone undecanoate. Pharmacotherapy 23:319-325

679.   Schnabel PG, Bagchus W, Lass H, Thomsen T, Geurts TB (2007) The effect of food composition on serum testosterone levels after oral administration of Andriol Testocaps. Clinical Endocrinology 66:579-585

680.   Roth MY, Dudley RE, Hull L, Leung A, Christenson P, Wang C, Swerdloff R, Amory JK (2011) Steady-state pharmacokinetics of oral testosterone undecanoate with concomitant inhibition of 5alpha-reductase by finasteride. International Journal of Andrology 34:541-547

681.   Idan A, Griffiths KA, Harwood DT, Seibel MJ, Turner L, Conway AJ, Handelsman DJ (2010) Long-term effects of dihydrotestosterone treatment on prostate growth in healthy, middle-aged men without prostate disease: a randomized, placebo-controlled trial. Annals of Internal Medicine 153:621-632

682.   Gooren LJ (1994) A ten-year safety study of the oral androgen testosterone undecanoate. Journal of Andrology 15:212-215.

683.   Page ST, Lin DW, Mostaghel EA, Marck BT, Wright JL, Wu J, Amory JK, Nelson PS, Matsumoto AM (2011) Dihydrotestosterone administration does not increase intraprostatic androgen concentrations or alter prostate androgen action in healthy men: a randomized-controlled trial. Journal of Clinical Endocrinology and Metabolism 96:430-437

684.   Tauber U, Schroder K, Dusterberg B, Matthes H (1986) Absolute bioavailability of testosterone after oral administration of testosterone-undecanoate and testosterone. European Journal of Drug Metabolism and Pharmacokinetics 11:145-149

685.   Ahmed SF, Tucker P, Mayo A, Wallace AM, Hughes IA (2004) Randomized, crossover comparison study of the short-term effect of oral testosterone undecanoate and intramuscular testosterone depot on linear growth and serum bone alkaline phosphatase. Journal of Pediatric Endocrinology and Metabolism 17:941-950

686.   Swerdloff RS, Wang C, White WB, Kaminetsky J, Gittelman MC, Longstreth JA, Dudley RE, Danoff TM (2020) A New Oral Testosterone Undecanoate Formulation Restores Testosterone to Normal Concentrations in Hypogonadal Men. J Clin Endocrinol Metab 105

687.   Nieschlag E, Mauss J, Coert A, Kicovic P (1975) Plasma androgen levels in men after oral administration of testosterone or testosterone undecanoate. Acta Endocrinol (Copenh) 79:366-374

688.   Butler GE, Sellar RE, Walker RF, Hendry M, Kelnar CJH, Wu FCW (1992) Oral testosterone undecanoate in the management of delayed puberty in boys: pharmacokinetics and effects on sexual maturation and growth. Journal of Clinical Endocrinology and Metabolism 75:37-44

689.   Brown DC, Butler GE, Kelnar CJ, Wu FC (1995) A double blind, placebo controlled study of the effects of low dose testosterone undecanoate on the growth of small for age, prepubertal boys. Archives of Disease in Childhood 73:131-135

690.   Albanese A, Kewley GD, Long A, Pearl KN, Robins DG, Stanhope R (1994) Oral treatment for constitutional delay of growth and puberty in boys: a randomised trial of an anabolic steroid or testosterone undecanoate. Archives of Disease in Childhood 71:315-317

691.   Luisi M, Franchi E (1980) Double-blind group comparative study of testosterone undecanoate and mesterolone in hypogonadal male patients. Journal of Endocrinological Investigations 3:305-308

692.   (1979) Androgen therapy of aplastic anaemia--a prospective study of 352 cases. Scandinavian Journal of Haematology 22:343-356

693.   Shimoda K, Shide K, Kamezaki K, Okamura T, Harada N, Kinukawa N, Ohyashiki K, Niho Y, Mizoguchi H, Omine M, Ozawa K, Haradaa M (2007) The effect of anabolic steroids on anemia in myelofibrosis with myeloid metaplasia: retrospective analysis of 39 patients in Japan. International Journal of Hematology 85:338-343

694.   Gennari C, AgnusDei D, Gonnelli S, Nardi P (1989) Effects of nandrolone decanoate therapy on bone mass and calcium metabolism in women with established post-menopausal osteoporosis: a double-blind placebo-controlled study. Maturitas 11:187-197

695.   Frisoli A, Jr., Chaves PH, Pinheiro MM, Szejnfeld VL (2005) The effect of nandrolone decanoate on bone mineral density, muscle mass, and hemoglobin levels in elderly women with osteoporosis: a double-blind, randomized, placebo-controlled clinical trial. Journals of Gerontology Series A, Biological Sciences and Medical Sciences 60:648-653

696.   Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinshelwood MM, Graham-Lorence S, Amarneh B, Ito Y, Fisher CR, Michael MD, et al. (1994) Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews 15:342-355

697.   Hong Y, Yu B, Sherman M, Yuan YC, Zhou D, Chen S (2007) Molecular basis for the aromatization reaction and exemestane-mediated irreversible inhibition of human aromatase. Molecular Endocrinology 21:401-414

698.   Hobbs CJ, Jones RE, Plymate SR (1996) Nandrolone, a 19-nortestosterone, enhances insulin-independent glucose uptake in normal men. Journal of Clinical Endocrinology and Metabolism 81:1582-1585

699.   Behre HM, Kliesch S, Lemcke B, von Eckardstein S, Nieschlag E (2001) Suppression of spermatogenesis to azoospermia by combined administration of GnRH antagonist and 19-nortestosterone cannot be maintained by this non-aromatizable androgen alone. Human Reproduction 16:2570-2577

700.   Attardi BJ, Pham TC, Radler LC, Burgenson J, Hild SA, Reel JR (2008) Dimethandrolone (7alpha,11beta-dimethyl-19-nortestosterone) and 11beta-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase. Journal of Steroid Biochemistry and Molecular Biology 110:214-222

701.   Lemus AE, Enriquez J, Garcia GA, Grillasca I, Perez-Palacios G (1997) 5alpha-reduction of norethisterone enhances its binding affinity for androgen receptors but diminishes its androgenic potency. Journal of Steroid Biochemistry and Molecular Biology 60:121-129

702.   Geusens P (1995) Nandrolone decanoate: pharmacological properties and therapeutic use in osteoporosis. Clinical Rheumatology 14:32-39.

703.   Sundaram K, Kumar N (2000) 7alpha-methyl-19-nortestosterone (MENT): the optimal androgen for male contraception and replacement therapy. International Journal of Andrology 23 Suppl 2:13-15

704.   Cook CE, Kepler JA (2005) 7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties. Bioorganic and Medicinal Chemistry Letters 15:1213-1216

705.   Suvisaari J, Sundaram K, Noe G, Kumar N, Aguillaume C, Tsong YY, Lahteenmaki P, Bardin CW (1997) Pharmacokinetics and pharmacodynamics of 7a-methyl-19-nortestosterone after intramuscular administration in healthy men. Human Reproduction 12:967-973

706.   Sundaram K, Kumar N, Bardin CW (1994) 7 alpha-Methyl-19-nortestosterone: an ideal androgen for replacement therapy. Recent Progress in Hormone Research 49:373-376

707.   Sundaram K, Kumar N, Bardin CW (1993) 7a-Methyl-nortestosterone (MENT): the optimal androgen for male contraception. Annals of Medicine 25:199-205

708.   Attardi BJ, Hild SA, Reel JR (2006) Dimethandrolone undecanoate: a new potent orally active androgen with progestational activity. Endocrinology 147:3016-3026

709.   Sundaram K, Kumar N, Monder C, Bardin CW (1995) Different patterns of metabolism determine the relative anabolic activity of 19-norandrogens. Journal of Steroid Biochemistry and Molecular Biology 53:253-257

710.   Toth M, Zakar T (1982) Relative binding affinities of testosterone, 19-nortestosterone and their 5 alpha-reduced derivatives to the androgen receptor and to other androgen-binding proteins: a suggested role of 5 alpha-reductive steroid metabolism in the dissociation of "myotropic" and "androgenic" activities of 19-nortestosterone. Journal of Steroid Biochemistry 17:653-660

711.   LaMorte A, Kumar N, Bardin CW, Sundaram K (1994) Aromatization of 7 alpha-methyl-19-nortestosterone by human placental microsomes in vitro. Journal of Steroid Biochemistry and Molecular Biology 48:297-304

712.   Moslemi S, Dintinger T, Dehennin L, Silberzahn P, Gaillard JL (1993) Different in vitro metabolism of 7 alpha-methyl-19-nortestosterone by human and equine aromatases. European Journal of Biochemistry 214:569-576

713.   Dobs AS, Boccia RV, Croot CC, Gabrail NY, Dalton JT, Hancock ML, Johnston MA, Steiner MS (2013) Effects of enobosarm on muscle wasting and physical function in patients with cancer: a double-blind, randomised controlled phase 2 trial. Lancet Oncology 14:335-345

714.   Crawford J, Prado CM, Johnston MA, Gralla RJ, Taylor RP, Hancock ML, Dalton JT (2016) Study Design and Rationale for the Phase 3 Clinical Development Program of Enobosarm, a Selective Androgen Receptor Modulator, for the Prevention and Treatment of Muscle Wasting in Cancer Patients (POWER Trials). Current Oncology Reports 18:37

715.   Yin D, Xu H, He Y, Kirkovsky LI, Miller DD, Dalton JT (2002) Pharmacology, pharmacokinetics, and metabolism of  acetothiolutamide, a novel nonsteroidal agonist for the  androgen receptor. Journal of Pharmacology and Experimental Therapeutics 304:1323-1333.

716.   Gao W, Dalton JT (2007) Ockham's razor and selective androgen receptor modulators (SARMs): are we overlooking the role of 5alpha-reductase? Mol Interv 7:10-13

717.   Sengupta S, Jordan VC (2008) Selective estrogen modulators as an anticancer tool: mechanisms of efficiency and resistance. Advances in Experimental Medicine and Biology 630:206-219

718.   Gao W, Dalton JT (2007) Expanding the therapeutic use of androgens via selective androgen receptor modulators (SARMs). Drug Discov Today 12:241-248

719.   Thole Z, Manso G, Salgueiro E, Revuelta P, Hidalgo A (2004) Hepatotoxicity induced by antiandrogens: a review of the literature. Urologia Internationalis 73:289-295

720.   Manso G, Thole Z, Salgueiro E, Revuelta P, Hidalgo A (2006) Spontaneous reporting of hepatotoxicity associated with antiandrogens: data from the Spanish pharmacovigilance system. Pharmacoepidemiol Drug Saf 15:253-259

721.   Minnemann T, Schubert M, Hubler D, Gouni-Berthold I, Freude S, Schumann C, Oettel M, Ernst M, Mellinger U, Sommer F, Krone W, Jockenhovel F (2007) A four-year efficacy and safety study of the long-acting parenteral testosterone undecanoate. Aging Male 10:155-158

722.   Saad F, Kamischke A, Yassin A, Zitzmann M, Schubert M, Jockenhel F, Behre HM, Gooren L, Nieschlag E (2007) More than eight years' hands-on experience with the novel long-acting parenteral testosterone undecanoate. Asian Journal of Andrology 9:291-297

723.   Stege R, Frohlander N, Carlstrom K, Pousette A, von Schoultz B (1987) Steroid-sensitive proteins, growth hormone and somatomedin C in prostatic cancer: effects of parenteral and oral estrogen therapy. Prostate 10:333-338

724.   Serin IS, Ozcelik B, Basbug M, Aygen E, Kula M, Erez R (2001) Long-term effects of continuous oral and transdermal estrogen replacement therapy on sex hormone binding globulin and free testosterone levels. European Journal of Obstetrics, Gynecology, and Reproductive Biology 99:222-225

725.   von Schoultz B, Carlstrom K (1989) On the regulation of sex-hormone-binding globulin. A challenge of an old dogma and outlines of an alternative mechanism. Journal of Steroid Biochemistry and Molecular Biology 32:327-334

726.   Small M, Beastall GH, Semple CG, Cowan RA, Forbes CD (1984) Alterations of hormone levels in normal males given the anabolic steroid stanozolol. Clinical Endocrinology 21:49-55

727.   Christiansen K 2004 Behavioural correlates of testosterone. In: Nieschlag E, Behre HM eds. Testosterone: Action Deficiency Substitution. 3rd ed. Cambridge: Cambridge University Press; 125-172

728.   Anderson RA, Bancroft J, Wu FCW (1992) The effects of exogenous testosterone on sexuality and mood of normal men. Journal of Clinical Endocrinology and Metabolism 75:1503-1507

729.   Buena F, Peterson MA, Swerdloff RS, Pandian MR, Steiner BS, Galmarini M, Lutchmansingh P, Bhasin S (1993) Sexual function does not change when serum testosterone levels are pharmacologically varied within the normal male range. Fertility and Sterility 59:1118-1123

730.   Bagatell CJ, Heiman JR, Matsumoto AM, Rivier JE, Bremner WJ (1994) Metabolic and behavioral effects of high-dose, exogenous testosterone in healthy men. Journal of Clinical Endocrinology and Metabolism 79:561-567

731.   Tricker R, Casaburi R, Storer TW, Clevenger B, Berman N, Shirazi A, Bhasin S (1996) The effects of supraphysiological doses of testosterone on angry behavior in healthy eugonadal men--a clinical research center study. Journal of Clinical Endocrinology and Metabolism 81:3754-3758

732.   O'Connor DB, Archer J, Hair WM, Wu FC (2002) Exogenous testosterone, aggression, and mood in eugonadal and hypogonadal men. Physiology and Behavior 75:557-566

733.   O'Connor DB, Archer J, Wu FC (2004) Effects of testosterone on mood, aggression, and sexual behavior in young men: a double-blind, placebo-controlled, cross-over study. Journal of Clinical Endocrinology and Metabolism 89:2837-2845

734.   Meriggiola MC, Cerpolini S, Bremner WJ, Mbizvo MT, Vogelsong KM, Martorana G, Pelusi G (2006) Acceptability of an injectable male contraceptive regimen of norethisterone enanthate and testosterone undecanoate for men. Human Reproduction 21:2033-2040

735.   Su TP, Pagliaro M, Schmidt PJ, Pickar D, Wolkowitz O, Rubinow DR (1993) Neuropsychiatric effects of anabolic steroids in male normal volunteers. Journal of the American Medical Association 269:2760-2764

736.   Yates WR, Perry PJ, MacIndoe J, Holman T, Ellingrod V (1999) Psychosexual effects of three doses of testosterone cycling in normal men. Biological Psychiatry 45:254-260

737.   Daly RC, Su TP, Schmidt PJ, Pagliaro M, Pickar D, Rubinow DR (2003) Neuroendocrine and behavioral effects of high-dose anabolic steroid administration in male normal volunteers. Psychoneuroendocrinology 28:317-331

738.   Schmidt PJ, Berlin KL, Danaceau MA, Neeren A, Haq NA, Roca CA, Rubinow DR (2004) The effects of pharmacologically induced hypogonadism on mood in healthy men. Archives of General Psychiatry 61:997-1004

739.   WHO Task Force on Methods for the Regulation of Male Fertility (1996) Contraceptive efficacy of testosterone-induced azoospermia and oligozoospermia in normal men. Fertility and Sterility 65:821-829

740.   WHO Task Force on Methods for the Regulation of Male Fertility (1990) Contraceptive efficacy of testosterone-induced azoospermia in normal men. Lancet 336:955-959

741.   Bjorkvist K, Nygren T, Bjorklund AC, Bjorkvist SE (1994) Testosterone intake and aggressiveness: real effect or anticipation? Aggressive Behavior 20:17-26

742.   Archer J (1991) The influence of testosterone on human aggression. British Journal of Psychiatry 82:1-28

743.   Melnik B, Jansen T, Grabbe S (2007) Abuse of anabolic-androgenic steroids and bodybuilding acne: an underestimated health problem. J Dtsch Dermatol Ges 5:110-117

744.   Grunstein RR, Handelsman DJ, Lawrence SJ, Blackwell C, Caterson ID, Sullivan CE (1989) Hypothalamic dysfunction in sleep apnea: reversal by nasal continuous positive airways pressure. Journal of Clinical Endocrinology and Metabolism 68:352-358

745.   Sandblom RE, Matsumoto AM, Scoene RB, Lee KA, Giblin EC, Bremner WJ, Pierson DJ (1983) Obstructive sleep apnea induced by testosterone administration. New England Journal of Medicine 308:508-510