11-Oxygenated androgens in health and disease

Abstract

The adrenal gland is a source of sex steroid precursors, and its activity is particularly relevant during fetal development and adrenarche. Following puberty, the synthesis of androgens by the adrenal gland has been considered of little physiologic importance. Dehydroepiandrosterone (DHEA) and its sulfate, DHEAS, are the major adrenal androgen precursors, but they are biologically inactive. The second most abundant unconjugated androgen produced by the human adrenals is 11(β-hydroxyandrostenedione (11OHA4). 11-Ketotestosterone, a downstream metabolite of 11OHA4 (which is mostly produced in peripheral tissues), and its 5α-reduced product, 11-ketodihydrotestosterone, are bioactive androgens, with potencies equivalent to those of testosterone and dihydrotestosterone. These adrenal-derived androgens all share an oxygen atom on carbon 11, so we have collectively termed them 11-oxyandrogens. Over the past decade, these androgens have emerged as major components of several disorders of androgen excess, such as congenital adrenal hyperplasia, premature adrenarche and polycystic ovary syndrome, as well as in androgen-dependent tumours, such as castration-resistant prostate cancer. Moreover, in contrast to the more extensively studied, traditional androgens, circulating concentrations of 11-oxyandrogens do not demonstrate an age-dependent decline. This Review focuses on the rapidly expanding knowledge regarding the implications of 11-oxyandrogens in human physiology and disease.

The adrenal gland is a recognized source of sex steroid precursors, largely dehydroepiandrosterone (DHEA) and its sulfate, DHEAS. These steroids are particularly relevant during key developmental stages, including during fetal development and adrenarche. The adrenal contribution to the synthesis of androstenedione and testosterone, which are also produced by the gonads, has been primarily recognized in the context of several disorders of androgen excess, including congenital adrenal hyperplasia (CAH)¹, premature adrenarche^2,3, polycystic ovary syndrome (PCOS)⁴ and androgen-producing or androgen-dependent tumours^5,6. The adrenals are also the principal source of a set of 11-oxygenated metabolites of androstenedione and testosterone, 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT). These metabolites are then converted, mostly in peripheral tissues, to 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT)^7,8. Collectively, these 19-carbon (C₁₉) steroids that all have an oxygen atom on carbon 11 (FIG. 1) are known as 11-oxyandrogens. Although the existence of 11-oxyandrogens has been acknowledged for several decades, exploration of their implications in human physiology and disease has been minimal. Rapidly expanding evidence suggests that 11-oxyandrogens are important hormones in human physiology, from adrenarche through to old age, as well as in several disorders of androgen excess. This Review discusses the physiology and pathology of adrenal C₁₉ steroids, emphasizing the previously under-recognized roles of 11-oxyandrogens.

Fig. 1 ∣. Adrenal androgen synthesis.

The adrenal cortex is structured into three concentric layers, and each layer produces specific steroids: mineralocorticoids are synthesized in the outermost layer (zona glomerulosa); glucocorticoids are produced in the middle layer (zona fasciculata); and androgens are produced in the inner layer (zona reticularis). The enzyme 3β-hydroxysteroid dehydrogenase/Δ^5/4-isomerase type 2 (HSD3B2) catalyses oxidation of the hydroxyl group on carbon 3 of steroids to a keto group and, simultaneously, isomerization of the double bond from the B ring (Δ⁵ steroids) to the A ring (Δ⁴ steroids). Androgens share a 19-carbon structure (C₁₉ steroids), whereas mineralocorticoids and glucocorticoids have a 21-carbon structure (C₂₁ steroids). The most abundant C₁₉ steroids produced by the adrenal glands are dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). The adrenal glands also produce a set of unique androgens, synthesized via the adrenal-specific enzyme, cytochrome P450 11β-hydroxylase (CYP11B1). These steroids are called 11-oxyandrogens, and 11-hydroxyandrostenedione is the most abundant. The relative androgenic potency of tested androgens is depicted based on data from Rege et al.⁸⁵ and Storbeck et al.⁸⁰. Testosterone and 11-ketotestosterone are potent androgens. Steroid 5α-reductases (SRD5A) convert testosterone and 11-ketotestosterone to dihydrotestosterone and 11-ketodihydrotestosterone, respectively. Dihydrotestosterone and 11-ketodihydrotestosterone are the most potent androgens. AKR1C3, aldo-keto reductase family 1 member C3 (also known as 17β-hydroxysteroid dehydrogenase type 5); CYB5A, cytochrome b₅ type A; CYP11A1, cytochrome P450 cholesterol side chain cleavage; CYP17A1, cytochrome P450 17A1; OH, hydroxy; StAR, steroidogenic acute regulatory protein; SULT2A1, steroid sulfotransferase type 2A1.

Biosynthesis of adrenal androgens

Adrenal synthesis of androgens.

The adrenal cortex is the origin for three major types of steroids: mineralocorticoids, glucocorticoids and androgens. Adrenal steroid production is highly dynamic and tightly controlled by adrenocorticotropic hormone (ACTH), angiotensin II and other regulators. All adrenal steroids are synthesized de novo and immediately released, with no stored reserves. To ensure a maximum synthetic efficiency in response to regulatory signals, the adrenal cortex is structured into three different layers (also called zones), each with distinct enzymatic machineries (FIG. 1). Most adrenal androgen synthesis occurs in the innermost adrenal cortical zone, the zona reticularis.

Steroidogenesis begins with the transfer of cholesterol, the common precursor for all steroids, from the cytoplasm to the outer mitochondrial membrane, via incompletely understood mechanisms⁹. Tropic stimulating hormones act via second messengers to induce the acute expression and activation of the steroidogenic acute regulatory protein (StAR), which orchestrates the transfer of cholesterol to the inner mitochondrial membrane, where it is then converted to pregnenolone by the cholesterol side chain cleavage enzyme (CYP11A1)^10,11. StAR’s facilitation of the movement of cholesterol to the inner mitochondrial membrane is considered the rate-limiting step in all steroidogenic tissues except the placenta⁹. The 37-kDa human StAR precursor protein¹² is cleaved to a 30-kDa form^13,14 upon entry into the mitochondria. StAR contains a StAR-related lipid transfer domain that forms a β-sheet rich sterol-binding pocket, which can host a single molecule of cholesterol¹⁵. StAR is thought to repeatedly shuttle cholesterol across the mitochondrial membrane, until it is inactivated by proteolytic cleavage. StAR is also expressed in the gonads and in many tissues that do not produce steroids (such as the nervous system or granulocytes), which suggests that it might have other cellular functions¹⁶.

Once StAR delivers cholesterol within the mitochondrion, CYP11A1 converts cholesterol to pregnenolone via three serial reactions: 20-hydroxylation, 22-hydroxylation and scission of the C20–C22 bond^9,17-19. Both StAR and CYP11A1 are expressed in all adrenal cortical zones, and zone-specific repertoires of downstream enzymes determine the compartmentalized steroidogenic flux within the adrenal cortex. ACTH is the primary regulator of CYP11A1 expression²⁰ and the synthesis of C₁₉ steroids in the zona reticularis²¹.

The conversion of pregnenolone to DHEA (the initial C₁₉ steroid) occurs in two steps, which are both mediated by cytochrome P450 17A1 (CYP17A1; also known as 17α-hydroxylase or 17,20-lyase)²². CYP17A1 first hydroxylates pregnenolone in position 17, and subsequently breaks the C17–C20 bond of 17-hydroxypregnenolone (FIG. 1). From a single gene, a 2.1-kb human CYP17A1 mRNA is translated into a 57-kDa protein common to the adrenals and gonads^23,24. A type II P450 enzyme, CYP17A1, is located in the endoplasmic reticulum, where it receives electrons from NADPH via a flavoprotein called P450 oxidoreductase (POR)²⁵. Human CYP17A1 catalyses the 17α-hydroxylation of both pregnenolone and progesterone with similar efficiencies; conversely, for the 17,20-lyase reaction, CYP17A1 yields approximately 50-fold more DHEA from 17-hydroxypregnenolone than androstenedione does from 17-hydroxyprogesterone (17OHP4)^26,27. The 17,20-lyase reaction is further enhanced by the cofactor cytochrome b₅ type A (CYB5A) in both the Δ⁴ and Δ⁵ pathways^28-31 (FIG. 1). The mechanisms by which CYB5A modulates the 17,20-lyase reaction are incompletely understood, but CYB5A proportionately increases the coupling of NADPH consumption to product formation in vitro^29,32.

CYP17A1 is essential for the synthesis of both cortisol and C₁₉ steroids and is abundantly expressed in the zonae fasciculata and reticularis. ACTH is the primary regulator of CYP17A1 expression in both zones^33,34. Growth factors, including insulin-like growth factor 1 (IGF1) and IGF2, also enhance adrenal CYP17A1 expression, whereas transforming growth factor-β inhibits CYP17A1 expression^35-37. During adrenarche, the thin zona reticularis starts to expand, CYB5A expression increases and the augmented 17,20-lyase activity of CYP17A1 promotes the synthesis of C₁₉ steroids in this innermost cortical zone^38-40 (FIG. 2).

Fig. 2 ∣. Synthesis, circulation and metabolism of 11-oxyandrogens.

19-carbon (C₁₉) steroids are synthesized in the adrenal cortex, including androgen precursors, predominantly dehydroepiandrosterone (DHEA), its sulfate (DHEAS), androstenedione and 11β-hydroxyandrostenedione (11OHA4), and smaller quantities of bioactive androgens, such as testosterone and 11β-hydroxytestosterone (11OHT).The kidney expresses 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2), which converts the adrenal-derived 11OHA4 and 11OHT into 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT), respectively. Other target tissues, such as the adipose tissue, prostate, hair follicles and genital skin, are able to convert the adrenal androgen precursors androstenedione, 11OHA4 and 11KA4 into bioactive androgens via 17β-hydroxysteroid dehydrogenase (HSD17B) enzymes and steroid 5α-reductases (SRD5A). The bioactive androgens bind the androgen receptors, located in the cytoplasm, and the hormone-receptor complex translocates to the nucleus, where it activates several androgen-responsive genes. 11KDHT, 11-ketodihydrotestosterone; 11OHDHT, 11β-hydroxydihydrotestosterone; DHT, dihydrotestosterone; OH, hydroxy.

The nascent DHEA can be either diverted towards downstream androgens or sulfonated to DHEAS. The sulfotransferase SULT2A1 catalyses the sulfonation of the 3β-hydroxyl group of DHEA using the co-substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS)⁴¹. During fetal development, SULT2A1 utilizes DHEA and other Δ⁵ steroids (including pregnenolone, 17-hydroxypregnenolone and androst-5-ene-3β,17β-diol (Adiol)) as substrates. The DHEAS generated in the fetal adrenal gland is utilized in the fetal liver and placenta as the substrate for oestriol synthesis⁴². The expression of SULT2A1 and CYB5A increases in the adrenal gland during adrenarche^39,43. As the adrenal gland begins to produce C₁₉ steroids, SULT2A1 preferentially utilizes DHEA as a substrate. As DHEA is a precursor for androstenedione and testosterone, the conjugation of DHEA acts as a kind of trap, preventing excessive adrenal androgen production. Defects in DHEA sulfonation allow abundant production of DHEA, which is subsequently diverted to the active androgen testosterone, after it is first converted to androstenedione, by 3β-hydroxysteroid dehydrogenase/Δ^5/4-isomerase type 2 (HSD3B2) in the adrenal glands or HSD3B1 in peripheral tissues⁴⁴. Although defects in SULT2A1 itself have not been described, defects in the enzyme that synthesizes PAPS, the mandatory sulfate donor, compromise DHEA sulfonation^44-46. In humans, PAPS synthase (PAPSS) has a ubiquitously expressed isoform, PAPSS1, and the PAPSS2 isoform is expressed in cartilage, the adrenal glands and the liver, with the latter two organs being the major sites of DHEA sulfonation⁴⁶. Deficiency of PAPSS2 prevents DHEA sulfonation and leads to adrenal androgen excess, as reported in a girl with premature pubarche, advanced bone age, acne, hirsutism and secondary amenorrhoea⁴⁴.

In addition to DHEA, SULT2A1 can also efficiently sulfonate pregnenolone, 17-hydroxypregenolone and Adiol; ACTH acutely stimulates the formation of pregnenolone sulfate and 17-hydroxypregenolone sulfate⁴⁷. Although the intra-adrenal expression of SULT2A1 is much higher in the zona reticularis than in the zona fasciculata⁴⁸, ACTH acutely stimulates the production of pregnenolone sulfate and 17-hydroxypregenolone sulfate within the zona reticularis and in the inner zona fasciculata. By contrast, the synthesis of the androgen precursors DHEAS and androstenediol sulfate occurs exclusively in the zona reticularis⁴⁹. Owing to its long half-life (7–10h)^50,51, which reduces its diurnal fluctuations⁵², measuring the levels of DHEAS has been used to assess the chronic integrity of the hypothalamic–pituitary–adrenal axis⁵³, such as in excluding secondary adrenal insufficiency^54,55 and screening for ACTH-independent hypercortisolism^56,57, which are both associated with low levels of DHEAS. The use of DHEAS as a measure of integrated ACTH synthesis is limited in certain scenarios, for example in pubertal children who could be undergoing ACTH-independent adrenarche^58,59 or in patients with adrenal cortical carcinomas, which can co-produce cortisol and DHEAS⁵.

Another key enzyme for the synthesis of all bioactive steroids, including androgens, is HSD3B2. HSD3B2 catalyses oxidation of the hydroxyl group on carbon 3 of the steroids to a keto group and, simultaneously, isomerization of the double bond from the B ring (Δ⁵ steroids) to the A ring (Δ⁴ steroids)^60-62 (FIG. 1). Pregnenolone, 17-hydroxypregnenolone, DHEA and Adiol are all substrates for HSD3B2 (REFS^63,64), which are irreversibly converted to progesterone, 17OHP4, androstenedione and testosterone, respectively. Of the two human isoforms⁶⁵, HSD3B2 is the major isoform expressed in the adrenals and gonads^66,67. Within the adrenal gland, HSD3B2 expression is abundant in the zonae glomerulosa and fasciculata, but negligible in the zona reticularis⁴⁸. In contrast to the zonae glomerulosa and fasciculata, the zona reticularis expresses considerably more SULT2A1 than HSD3B2, favouring the synthesis of DHEAS rather than androstenedione and testosterone. The efficient synthesis of androgens from pregnenolone requires co-expression of CYB5A (to produce DHEA) and HSD3B2 (to convert DHEA to androstenedione); this co-expression is present in the testicular Leydig cells and the ovarian theca cells. Conversely, within the adrenal gland, the expression of HSD3B2 and CYB5A is segregated: HSD3B2 within the zonae glomerulosa and fasciculata, and CYB5A in the zona reticularis. It is believed that the synthesis of androstenedione and downstream Δ⁴ C₁₉ steroids occurs at the interface between the zona fasciculata and the zona reticularis, where cells expressing HSD3B2 and CYB5A, respectively, are in close contact⁶⁸. The areas of HSD3B2 and CYB5A proximity have been reported to be largest in people aged 13–20 years⁶⁸.

Androstenedione is converted to testosterone by 17β-hydroxysteroid dehydrogenase (HSD17B) enzymes. In the testis, 17βHSD type 3 (17βHSD3) catalyses this reaction⁶⁹. The adrenal gland expresses a much less efficient enzyme in the aldo-keto reductase (AKR) family, AKR1C3 (also known as HSD17B type 5)⁷⁰, which is also found in non-steroidogenic tissues^71-73. An increase in AKR1C3 transcription contributes to hyperandrogenism in PCOS⁷⁴.

The canonical adrenal pathways to aldosterone, cortisol and DHEAS omit the 11-oxyandrogen pathway. Androstenedione and testosterone are both substrates for the adrenal enzyme cytochrome P450 11β-hydroxylase (CYP11B1), yielding 11OHA4 and 11OHT, respectively. CYP11B1, which also catalyses the last step in cortisol synthesis under the regulation of ACTH⁷⁵, is abundantly expressed in the zonae fasciculata and reticularis of the adrenal gland⁴⁸; however, in humans, CYP11B1 expression in the gonads is negligable⁷⁶. Little or no 11OHA4 derives from cortisol, via CYP17A1 (REF.⁷⁷). Primarily in the periphery and, to a lesser extent within the adrenal glands, 11OHA4 and 11OHT can be oxidized to 11KA4 and 11KT, respectively, by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2). In addition, 11KT derives from the reduction of 11KA4, via AKR1C3, which is analogous to androstenedione conversion to testosterone in the periphery⁴⁸.

Activation of adrenal androgens in peripheral tissues.

As noted in a previous section, the majority of C₁₉ steroids produced by the adrenal gland consist of androgen precursors that can be activated in target tissues, such as the prostate or skin (FIG. 2). DHEA in the circulation can be converted to androstenedione by peripheral HSD3B1; subsequently, androstenedione is further metabolized to testosterone by AKR1C3 (REF.⁷⁸). The abundant amounts of 11OHA4 produced by the adrenal glands can be oxidized to 11KA4 in the periphery, predominately by HSD11B2 in the kidney. 11KA4 is then converted to 11KT by AKR1C3 in other peripheral tissues expressing this enzyme, including adipose tissue⁷⁹. The catalytic efficiency of AKR1C3 is in fact eightfold higher when it utilizes 11KA4 rather than androstenedione as a substrate⁷⁹.

Steroid 5α-reductases (SRD5A) are known for converting testosterone to the more potent androgen dihydrotestosterone (DHT), but SRD5A can also utilize androstenedione and all of the 11-oxygenated derivatives of androstenedione and testosterone as substrates⁸⁰. Two distinct isoenzymes exist in humans: type 1 (SRD5A1) is expressed in multiple tissues, including the liver and skin, whereas type 2 (SRD5A2) is found in male reproductive tissues. SRD5A2 preferentially utilizes testosterone, whereas SRD5A1 catalyses reduction of androstenedione more efficiently than testosterone⁸¹. The DHT homologue, 11-ketodihydrotestosterone (11KDHT), can be generated either from 11β-hydroxydihydrotestosterone, via 11βHSD2, or from 11-keto-5α-androstanedione, via AKR1C3 (REF.⁸⁰).

Androgenic potency

Although DHEA and DHEAS are the staple adrenal androgens, their bioactivity is negligible (FIG. 1). Similarly, androstenedione and its 11-oxygenated metabolites have little biological activity^82,83. By contrast, testosterone and 11KT are potent agonists of the androgen receptor (AR, encoded by NR3C4). Using cell models engineered to express the human AR and a luciferase reporter, we found that the maximum androgenic activity of 11KT approached that of testosterone, whereas that of 11OHT was more modest^7,84. The induction of expression of the AR target gene was also similar for both testosterone and 11KT (REF.⁸⁵). Storbeck and colleagues obtained similar results in a COS-1 cell system expressing human AR; in addition, they demonstrated that 11KDHT is equipotent to DHT⁸⁰ (FIG. 1). Moreover, using two androgen-dependent prostate cancer cell lines, the same group showed that 11KT and 11KDHT bind to the human AR with similar affinities to testosterone and DHT, respectively⁸⁶. In the same study, all four steroids activated the expression of AR-regulated genes and promoted cell growth⁸⁶.

Circulation patterns

During human fetal development, the adrenal glands are a major source of DHEA and DHEAS, which are utilized by the placenta for oestrogen synthesis⁴². At birth, the adrenal glands are similar in size to the kidneys, largely due to the presence of the fetal zone, which involutes soon after birth⁴². Levels of DHEA and DHEAS also decline rapidly after birth, and their concentrations remain negligible until adrenarche⁸⁷. Adrenarche is an endocrine phenomenon characterized by a rise in the synthesis of DHEA and DHEAS in parallel with the developmental expansion of the adrenal zona reticularis^88-90. This process begins sometime in childhood and progresses gradually, prior to and independent of puberty⁹¹. The hormonal changes of adrenarche precede its clinical manifestations, including the appearance of axillary and/or pubic hair growth (pubarche), adult body odour and mild acne, and they typically initiate around age 6–8 years in both boys and girls^91-95. Although a rise in the circulating concentrations of the major adrenal C₁₉ steroids, DHEA and DHEAS, has been long associated with adrenarche, we demonstrated in a study published in 2018 that levels of the potent androgens testosterone and 11KT also rise during adrenarche, with circulating concentrations of 11KT exceeding those of testosterone in girls with normal or premature adrenarche⁸⁵. The phenotypic manifestations associated with adrenarche could be attributed to the action of androgens directly released from the adrenal gland and/or to the peripheral conversion of DHEA to testosterone^96-100 and of 11OHA4 to 11KT (REFS^80,101) in other organs or in target tissues, such as hair follicles or genital skin.

Following adrenarche, the adrenal production of DHEA and DHEAS continues to rise gradually and peaks in the mid-20s (FIG. 3). Subsequently, levels of DHEA and DHEAS decline with ageing, such that by age 80 years, DHEA and DHEAS concentrations are only about 20% of their mid-20s peak levels¹⁰². Throughout life, DHEAS concentrations are generally higher in boys and men than in age-matched girls and women; however, the cause of this sexual dimorphism is poorly understood. Beginning with puberty, both the gonads and the adrenals contribute to the production of androstenedione and testosterone^103,104, either directly or indirectly by providing androgen precursors. The contribution of the adrenal gland to the circulating levels of testosterone throughout reproductive ages is similar to that of the ovaries in women¹⁰⁵, whereas in men the overwhelming proportion of testosterone originates from the testes. Like DHEA and DHEAS, androstenedione and testosterone decline gradually with ageing in both sexes^106-112. In contrast to these traditional androgens, we have found that the circulating concentrations of 11-oxyandrogens remain fairly stable, or even show a modest up-trend in some postmenopausal women¹¹³ (FIG. 3).

Fig. 3 ∣. Distribution of key C₁₉ steroids across the human female lifespan.

Levels of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), the major adrenal androgen precursors, begin to increase around age 6 years, peak around the mid-20s and, subsequently, decline gradually. In women during reproductive years, androstenedione and testosterone are produced in similar amounts by both the adrenal glands and the ovaries, and their concentrations decline gradually with ageing. By contrast, levels of 11β-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) remain elevated in postmenopausal women. Data obtained from Rege et al.⁸⁵ and Nanba et al.¹¹³, and expressed in medians. Total n = 283 girls and women (aged 4–5 years, n = 22; aged 6–8 years, n = 38; aged 9–10 years, n = 23; aged 20–40 years, n = 100; aged 60–80 years, n = 100).

Ageing is accompanied by an involution of the zona reticularis, which explains the concurrent decline in levels of DHEA and DHEAS^114,115. Nonetheless, we have observed that the sharp zonal separation of the expression of HSD3B2 and CYB5A that is characteristic of adrenal glands in young people becomes less distinct in women aged >60 years¹¹³. Such morphological changes (that is, the sharp zonal separation becoming less distinct in women aged >60 years) would facilitate the conversion of DHEA to androstenedione and, subsequently, to downstream androgens, including 11-oxyandrogens.

Of the unconjugated C₁₉ steroids produced in the adrenal glands, 11OHA4 is the second most abundant (with DHEA being the first) throughout most life stages (FIG. 3), and its circulating concentrations far exceed those of androstenedione, both under baseline conditions and after stimulation with cosyntropin (a synthetic form of ACTH). The predominance of 11OHA4 over androstenedione has been demonstrated both in cultured human adrenal cells¹¹⁶ and in serum obtained from human adrenal veins^7,8. Notably, 11OHA4 is the most abundant unconjugated C₁₉ steroid in postmenopausal women, with concentrations over twofold higher than those of DHEA¹¹³ (FIG. 3 and TABLE 1). The adrenal gland also produces smaller amounts of 11OHT, 11KA4 and 11KT, and ACTH enhances the synthesis of all four 11-oxyandrogens^7,8. Peripheral concentrations of 11KT exceed those of testosterone before puberty in both sexes⁸⁵. In women of reproductive age, circulating levels of 11KT and testosterone are fairly similar, whereas after menopause, the ratio of 11KT to testosterone increases again¹¹³ (FIG. 3 and TABLE 1). Considering that the androgenic potency of 11KT is similar to that of testosterone, its quantitative dominance in prepubertal children and postmenopausal women suggests that 11KT is the primary mediator of many processes previously attributed to testosterone, including adrenarche. The physiological functions of 11-oxyandrogens throughout adulthood and ageing remain to be determined. As gonadal function also undergoes an age-dependent decline in both sexes, albeit more abruptly in women than in men, the persistent production of bioactive adrenal androgens might have residual benefits on bone, muscle, the brain or other tissues and functions.

Table 1 ∣.

Concentrations of major C₁₉ steroids in healthy individuals and those with androgen excess

Study details	Steroid level (ng/dl)								Ref.
	DHEAS	DHEA	Androstenedione	Testosterone	11OHA4	11KA4	11OHT	11KT
100 premenopausal women (aged 20–40 years) vs 100 postmenopausal women (aged 60–80 years)	116,268 (71,112–206,477) vs 38,747 (22,258–53,873); P < 0.0001	232 (155–403) vs 79 (52–134); P < 0.0001	99 (76–140) vs 34 (23–51); P < 0.0001	30 (22–40) vs 19 (14–31); P < 0.0001	172 (118–261) vs 196 (141–289); P = 0.06	37 (26–55) vs 34 (23–46); P = 0.05	14 (9–23) vs 20 (13–28); P = 0.0002	26 (19–38) vs 28 (22–37); P = 0.3	110
37 girls with premature adrenarche vs 54 control girls (all aged 4–7 years)	38,227 (23,035–57,073) vs 9,321 (4,333–17,637); P < 0.001	242 (162–429) vs 52.3 (32.8–89.5); P < 0.001	17.0 (11.4–22.8) vs 13.1 (9.2–22.4); P = 0.238	5.2 (4.6–5.8) vs 4.6 (3.7–5.2); P < 0.001	45.7 (29.3–67.5) vs 26.1 (16.7–38.9); P < 0.001	15.0 (12.4–22.1) vs 10.8 (8.2–13.8); P < 0.001	6.2 (4.9–7.4) vs 3.8 (2.6–6.1); P < 0.001	18.1 (12.3–22.9) vs 11.2 (8.4–16.5); P < 0.001	84
38 patients with 21OHD (19 females) vs 38 control individuals (19 females); all aged 3–59 years	18,744 (7,847–64,308) vs 139,784 (58,409–186,697); P < 0.0001	29 (16–85) vs 175 (118–318); P < 0.0001	155 (72–390) vs 42 (22–63); P < 0.0001	80 (38–162) vs 26 (12–309); P = 0.09	351 (188–792) vs 118 (70–154); P < 0.0001	96 (58–143) vs 31 (20–42); P < 0.0001	59 (21–104) vs 15 (9–21); P < 0.0001	171 (105–366) vs 50 (29–78); P < 0.0001	8
114 women with PCOS vs 49 control women; all aged 18–40 years	300,111 (203,506–451,661) vs 222,804 (125,535–351,365); P < 0.001	406 (300–524) vs 205 (121–340); P < 0.001	768 (484–1,009) vs 169 (103–264); P < 0.001	20 (14–29) vs 9 (6–14); P < 0.001	958 (508–1,444) vs 205 (148–372); P < 0.001	402 (255–565) vs 81 (60–114); P < 0.001	12 (9–15) vs 6 (3–9); P < 0.01	73 (54–118) vs 45 (36–54); P < 0.01	194

Data are expressed as medians (interquartile ranges) and are extracted from four publications^8,83,109,193. 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; 11OHA4, 11β-hydroxyandrostenedione; 11OHT, 11β-hydroxytestosterone; 21OHD, 21-hydraxylase deficiency; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; PCOS, polycystic ovary syndrome.

In teleost fish, 11KT is produced in the gonads as a male-specific androgen^117-121; however, in human beings and other primates, 11-oxyandrogens are of adrenal origin¹²². Patients with adrenal insufficiency produce negligible amounts of 11-oxyandrogens^8,123,124, and 11-oxyandrogens circulate at similar concentrations in healthy men and women, including during the reproductive lifespan, when males have a high testicular testosterone output^8,76,122. An important clinical implication for disorders of androgen excess is that circulating levels of 11OHA4 and 11OHT primarily reflect adrenal androgen output, with rare exceptions. One of our studies provided a comprehensive analysis of 11-oxyandrogens across 18 animal species and demonstrated that, among non-primate animals, only pigs and guinea pigs synthesize 11-oxyandrogens and therefore might serve as suitable animal models for future investigations¹²².

Adrenal androgens in human disorders

Congenital adrenal hyperplasia.

A group of autosomal recessive defects in genes encoding enzymes required for cortisol synthesis cause CAH. These genetic defects can lead to complete or partial enzymatic insufficiencies, resulting in the wide spectrum of clinical severity seen in this disorder. In the most severe forms of CAH, also called classic CAH, cortisol and/or aldosterone synthesis are inadequate, and patients require lifelong glucocorticoid replacement therapy. Milder, or non-classic, CAH features normal cortisol synthesis; however, this normal cortisol synthesis is achieved at the expense of elevated levels of androgens. In all CAH forms, however, the hypothalamic–pituitary axis is overactivated, in an attempt to overcome the enzymatic blockade (that is, the lack of enzymes in the cortisol and/or aldosterone synthesis pathways) and preserve cortisol synthesis. Stimulated by the ensuing increase in levels of ACTH that are occurring as a result of overactivation of the hypothalamic–pituitary axis, the steroidogenic stream favours the accessible pathways (that is, pathways with normally functioning enzymes), leading to excessive amounts of steroids that are usually only present at low levels.

Most cases of CAH are due to 21-hydroxylase (CYP21A2) deficiency. Classic 21-hydroxylase deficiency (21OHD) occurs in ~1:16,000 neonates¹²⁵; non-classic 21OHD is much more common, with a prevalence of roughly 1:1,000 white people and an even higher prevalence in selected ethnicities, such as Ashkenazi Jewish people, Hispanic people and people with Mediterranean origins¹²⁶.

In 21OHD (both forms), the synthesis of mineralocorticoids and glucocorticoids is obstructed, which leads to accumulation of 17OHP4, the main substrate of CYP21A2. As a result of stimulation by ACTH, 17OHP4 is diverted towards formation of adrenal androgens and androgen precursors¹²⁷. Although 17-hydroxypregnenolone is the preferred substrate of the human 17,20-lyase¹²⁸, the supraphysiologic concentrations of 17OHP4 in 21OHD allow some conversion of 17OHP4 to androstenedione, which can be further metabolized to testosterone by AKR1C3. Subsequently, CYP11B1 catalyses the reactions from androstenedione and testosterone to 11OHA4 and 11OHT, respectively, which are further metabolized to 11KA4 and 11KT, predominantly in the periphery.

Serum levels of 17OHP4, androstenedione and, in women, testosterone, are normally used to assess disease control and to guide treatment in patients with 21OHD; however, they are unreliable biomarkers^129,130. The marked elevations in levels of 17OHP4 in the serum of patients with 21OHD fluctuate widely with time after glucocorticoid exposure, and only normalize when supraphysiological glucocorticoid doses are used, which can lead to adverse effects¹³¹. Additionally, 17OHP4, androstenedione and testosterone are also synthesized by the gonads, further confounding their clinical utility after puberty. In contrast to 17OHP4, levels of the major adrenal C₁₉ steroids, DHEA and DHEAS, are paradoxically low in patients with classic 21OHD, even when disease control is suboptimal^8,132, making them unreliable biomarkers of disease control.

Owing to their adrenal origin, 11-oxyandrogens have been proposed as biomarkers of androgen excess in 21OHD. In an early study using immunoassays, it was found that the ratio of androstenedione to 11OHA4 was lower in women with non-classic 21OHD and higher in women with chronic anovulation than in control women¹³³. Another study found that levels of 11OHA4 were higher at baseline in women with untreated non-classic 21OHD than in unaffected control women matched for age and BMI, whereas the levels were similar after cosyntropin stimulation¹³⁴. Using mass spectrometry, we have shown that not only levels of 11OHA4 but also levels of 11KA4, 11OHT and 11KT are threefold to fourfold higher in patients with classic 21OHD of both sexes during usual glucocorticoid treatment than in control individuals matched for age and sex⁸ (TABLE 1). A subsequent study measured the daily urinary excretion of androgen metabolites in 99 children with classic 21OHD who were receiving treatment and showed that the 11-oxyandrogen metabolite 11β-hydroxyandrosterone was the most abundant urinary androgen metabolite and the only one that was higher than in healthy children¹³⁵.

In girls, women and prepubertal boys with classic 21OHD, levels of 11KT correlate directly with those of testosterone. However, it is notable that circulating concentrations of 11KT are twice as high as those of testosterone in these patients⁸. Conversely, levels of both 11KT and 11OHT are inversely correlated with levels of testosterone and luteinizing hormone in post-pubertal male individuals with 21OHD (REFS^8,136). These findings suggest that 11KT is a better biomarker of disease control than testosterone in men, with evidence that the high concentrations of 11KT in male patients with 21OHD that is poorly controlled suppress the hypothalamic–pituitary–gonadal axis and subsequently testicular testosterone synthesis. Additionally, this inverse correlation between levels of 11KT and testosterone in sexually mature men with 21OHD demonstrates that testicular testosterone is not a source of 11KT, but rather that 11KT derives predominately from adrenal precursors.

To further explore the clinical utility of 11-oxyandrogens, we studied the association between a set of 23 steroids and clinical findings suggestive of poor 21OHD control, such as adrenal volume, testicular adrenal rest tumours (TART), bone age, menstrual irregularities and hirsutism¹³⁶. In a cross-sectional study of 114 patients with classic 21OHD, we found that, along with 21-deoxycortisl, the four 11-oxyandrogens correlated best with adrenal volume and levels of ACTH¹³⁶. In addition, levels of all four 11-oxyandrogens, and in particular 11OHA4, were threefold to sevenfold higher in male patients with TART than in those without TART of similar ages. Furthermore, as previously reported¹³⁷, levels of testosterone were similar between patients who had 21OHD with or without TART. Levels of 11OHT and 11KT were also higher in women with 21OHD who had menstrual disturbances (such as irregular menses or amenorrhoea) and hirsutism than in those without these additional symptoms. These initial studies suggest that 11-oxyandrogens are excellent biomarkers of 21OHD control, and prospective longitudinal studies are needed to determine the dynamics of these 11-oxyandrogens in individuals with 21OHD, in particular in response to therapy.

Premature adrenarche.

Premature adrenarche is defined as the precocious appearance of pubic and/or axillary hair before age 8 years in girls and 9 years in boys^138-142. Unlike precocious puberty, premature adrenarche does not manifest with the development of secondary sexual characteristics, such as breast development and testicular growth¹⁴³. Along with elevated age-adjusted DHEA and DHEAS synthesis, children with premature adrenarche might have modestly advanced bone age and accelerated linear growth^144,145. Steroid profiles of infants with fine genital hair showed a mild elevation in levels of DHEAS compared with healthy pre-adrenarchal children, thus indicating that pubic hair in infancy might represent an early-onset variant of premature adrenarche¹⁴⁶. Premature adrenarche is more common in girls than boys, with a ratio of 9:1 (REF.¹⁴⁷), and it has been reported to occur in 9.5% of African-American girls and 1.5% of white girls¹⁴⁸. Although premature adrenarche had long been considered a benign condition, studies from the past few years have indicated that dysregulation of adrenarche predisposes to increased cardiovascular risk and increased insulin resistance later in life, including in adolescence and adulthood^140,142,149. Many studies have attempted to determine the link between the early androgen excess seen in premature adrenarche and factors related to the metabolic syndrome^150-153. A subgroup of girls with premature adrenarche have an increased susceptibility for developing PCOS in adult life^154-158. Moreover, in some cases, early or exaggerated adrenarche is an early manifestation of PCOS in girls who progress rapidly from premature adrenarche to puberty to oligomenorrhoea and hyperandrogenism, and these patients tend to have marked insulin resistance and/or obesity^153,159-162.

Although the molecular causes of premature adrenarche remain unclear, there is supporting evidence for early expansion of the zona reticularis¹⁶³, elevated 17,20-lyase activity¹⁶⁴, and increased IGF1 (REF.¹⁶⁵) and anti-Müllerian hormone¹⁶⁶ production being involved. Apparent cortisone reductase deficiency due to decreased 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) activity causes adrenal hyperandrogenaemia and premature pubarche, which results in symptoms similar to those seen in children with premature adrenarche^167,168. Other genetic causes of premature adrenarche are apparent DHEA sulfotransferase deficiency due to PAPSS2 deficiency⁴⁴, glucocorticoid resistance¹⁶⁹ and increased androgen sensitivity owing to a CAG repeat polymorphism in the gene that encodes the androgen receptor¹⁷⁰.

Although high serum concentrations of DHEA, DHEAS and androstenedione have been historically associated with normal and premature adrenarche^{2,3,93,95,140,171,172}, these steroids exhibit weak androgen activity and are thereby unlikely to directly cause the clinical manifestations observed in these conditions. It should be noted, however, that DHEA, DHEAS and androstenedione act as a reservoir of precursors for production of potent androgens, including testosterone and DHT, in the periphery or target tissues^173-175. Testosterone has been used as the conventional marker for androgen excess in prepubertal children, and several studies have demonstrated elevations in serum concentrations of testosterone and its urinary metabolites that are related to premature adrenarche^{2,3,85,93,144,171,176}. Our group has highlighted the 11-oxyandrogens as major contributors to the androgen excess of premature adrenarche⁸⁵. We found that premature adrenarche is characterized by marked alterations in the circulating steroid profile, including increases in levels of adrenal-derived steroid sulfates, testosterone and all of the 11-oxyandrogens, earlier than in control children. Notably, circulating concentrations of 11KT were higher than levels of testosterone in prepubertal girls and more so in girls with premature adrenarche (3.5-fold) than in age-matched reference girls (2.4-fold) (TABLE 1). This observation supports the theory that the 11-oxyandrogens, especially 11KT, contribute to the precocious androgenic phenotype of premature adrenarche.

Polycystic ovary syndrome.

PCOS is the most common ovarian disorder, affecting 5–15% of reproductive-aged women^177-179. PCOS and non-classic 21OHD share several phenotypic features, including reproductive abnormalities and hyperandrogenism^177,180, which makes their clinical distinction difficult. In contrast to the monogenic aetiology of 21OHD, PCOS is a mixed, heterogenic condition with poorly understood pathogenesis. The diagnosis of PCOS is based on a set of criteria, of which ovarian dysfunction and clinical and/or biochemical evidence of hyperandrogenism are included in all expert guidelines^181,182. Patients with PCOS have an increased risk of metabolic abnormalities, such as obesity, insulin resistance, type 2 diabetes mellitus, cardiovascular disease and dyslipidaemia^183-185. These risks are also observed in patients with 21OHD, but are as a consequence of therapies featuring supraphysiologic levels of glucocorticoid¹⁸⁶.

Clinical and/or biochemical evidence of androgen excess is common¹⁷⁷ but not universal¹⁸² in women with PCOS. The ovaries have been long considered the primary source of androgen excess in women with PCOS^187,188. The androgen excess in women with PCOS is enhanced with gonadotropin-releasing hormone agonists^189,190, and testosterone and androstenedione respond less to chronic dexamethasone suppression than in healthy women¹⁹¹, suggesting that the androgens predominantly originate in the ovary. Although testosterone and androstenedione are produced by both the ovaries and the adrenal glands, androstenedione has been suggested to be superior to testosterone as a marker of hyperandrogenism and metabolic risk in women with PCOS^192-194. Notably, a subgroup of women with PCOS often have elevated circulating concentrations of DHEA and DHEAS^4,195, and, in contrast to testosterone and androstenedione, DHEA concentrations are markedly suppressed with long-term night time dexamethasone use in these patients¹⁹¹. These two findings suggest that the adrenal glands have a role as contributors towards androgen excess in a subset of patients with PCOS. Moreover, along with cortisol, the response of adrenal androgens to stimulation with cosyntropin is exaggerated in patients with PCOS^196,197, suggesting that hyper-reactivity of the adrenal zona reticularis to ACTH might be implicated in the aetiology of hyperandrogenism for certain patients with PCOS.

Another indication for an adrenal contribution to the pool of androgens in PCOS is the elevation of circulating levels of 11OHA4 in many of these women, which was initially demonstrated by studies using immunoassays in the early 1990s (REFS^133,198). In a more recent study, levels of 11OHA4, 11KA4, 11OHT and 11KT, as well as their urinary metabolite 11β-hydroxyandrosterone, were all higher in 114 women with PCOS than in 49 healthy women¹⁹⁹ (TABLE 1). In addition, this study found that serum concentrations of 11OHA4 and 11KA4, but not the urinary 11-oxygenated testosterone metabolites, correlated weakly with BMI, insulin levels and HOMA-IR.

Castration-resistant prostate cancer.

Prostate cancer is the second most common cancer in men globally. Prostate cancer growth is promoted by androgens, which in men are produced predominantly in the testes. Medical or surgical androgen deprivation is usually implemented when there is evidence of metastatic disease that requires systemic therapy²⁰⁰. Progression of disease despite androgen deprivation is referred to as castration-resistant prostate cancer (CRPC). Although circulating levels of testosterone fall dramatically after androgen deprivation therapy, intratumoural DHT synthesis continues^201,202, probably from precursors produced by the adrenal glands. Suppression of adrenal-derived androgen synthesis with abiraterone acetate, a potent P450 17A1 inhibitor, and inhibition of androgen receptor signalling with enzalutamide improves survival in men with CRPC^203-205, which supports the idea that CRPC is androgen dependent. Molecular alterations that promote both intratumoural androgen synthesis and androgen signalling promote cancer cell growth. For instance, upregulation of androgen receptors and of enzymes involved in androgen synthesis (such as AKR1C3, HSD3B1, HSD17B3 and SRD5A1) have been reported in CRPC tissue samples^206-210. CRPC cells from patients exhibit an isoenzyme shift from SRD5A2 to SRD5A1 (REF.²¹⁰). SRD5A1 preferentially utilizes androstenedione rather than testosterone as a substrate, which is funnelled to DHT via 5α-androstanedione²¹¹. Consequently, inhibiting both SRD5A1 and SRD5A2 is important to prevent cancer growth in patients with CRPC.

11OHA4 from the adrenal glands, which circulates in higher concentrations than androstenedione, is an additional substrate for potent androgen synthesis within CRPC cells from patients. The LNCaP androgen-dependent prostate cancer cell line metabolizes 11OHA4 to the potent androgens 11KT and 11KDHT, via the enzymes HSD11B2, AKR1C3 and SRD5A1 (REFS^79,80). The dynamic interplay between key androgenic enzymes within prostate cancer tissue seems to favour the formation of active androgens, which are essential for tumour growth. Using in vitro human prostate cancer cell models, it was shown that the kinetics of the two HSD11B isoenzymes favour the formation of 11-keto androgens via HSD11B2 (REF.²¹²), which is expressed in prostate cancer cells^213,214. Owing to the increased expression of SRD5A1 and AKR1C3, along with downregulation of HSD17B2 (REF.²¹⁵), the enzymatic milieu of prostate cancer cells favours the synthesis of the potent androgens DHT and 11KDHT. Furthermore, 11-oxyandrogens have been identified in tumour tissue from three patients with CRPC; of these, 11OHA4 was the most abundant^216,217. In the same patients, the serum concentrations of 11KT and 11KDHT were considerably higher than those of testosterone and DHT, respectively.

Additional intratumoural metabolism that could contribute to cancer progression includes reduced inactivation of bioactive androgens via conjugation, such as the sulfonation or glucuronidation reactions carried out by sulfotransferase and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes, respectively. Using in vitro androgen-dependent prostate cancer cell-line models, inactivation of 11KT and 11KDHT was found to occur considerably more slowly than that of testosterone and DHT⁸⁶. Similarly, in contrast to testosterone, which conjugates to testosterone–glucuronide within 48 h in LNCaP cells, this glucuronidation was inefficient for 11KT and 11KDHT (REFS^216,217). Consequently, intracellular availability of 11KT and 11KDHT is prolonged, allowing these AR agonists to activate the expression of endogenous AR-regulated genes and contribute to cancer progression⁸⁶.

Conclusions

The high circulating levels of DHEAS throughout most of adulthood and the weak capacity of the zona reticularis to produce testosterone have focused most studies of adrenal androgens on the Δ⁵ pathway and their subsequent conversion to androstenedione and testosterone. Although the ability of the human adrenal to produce 11OHA4 has been recognized for several decades mainly as a curiosity, an explosion of observations made over the past 5 years has illuminated the profound contributions of 11-oxyandrogens to human health and disease. With the advent of clinical assays for 11-oxyandrogens, studies are underway to define the utility of their measurements in the management of disorders such as 21OHD and in the prognosis of CRPC. Additional studies to define the kinetics of 11-oxyandrogen disposition and responses to treatment will provide further insight regarding the adrenal contributions to androgen-derived implications.

Key points.

• Dehydroepiandrosterone (DHEA) and its sulfate, DHEAS, are the most abundant adrenal androgen precursors, but they are biologically inactive as androgens.

• The adrenal gland is the primary source of a set of 11-oxygenated 19-carbon steroids, also termed 11-oxyandrogens, which have several roles in human physiology and disease.

• 11-Ketotestosterone is a bioactive 11-oxyandrogen, with a potency similar to that of testosterone, and its concentrations exceed those of testosterone in prepubertal children and in postmenopausal women.

• Concentrations of 11-oxyandrogens are elevated in several disorders of androgen excess, including premature adrenarche, congenital adrenal hyperplasia and polycystic ovary syndrome, and they can contribute to the progression of castration-resistant prostate cancer.

Adrenarche.

An early stage in sexual maturation specific to humans and other primates that is caused by androgen synthesis from the adrenal glands and is associated with the development of pubic hair, body odour, skin oiliness and acne.

Steroidogenesis.

The process of steroid synthesis.

Tropic.

Related to hormones that stimulate the growth and activity of target glands.

Second messengers.

Small intracellular molecules that mediate the effects of first messengers, that is, neurotransmitters and hormones.

Pubarche.

The first appearance of pubic hair at puberty.

Sulfonation.

The transfer of a sulfonate group (SO₃⁻¹) from the universal sulfonate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to an appropriate acceptor molecule.

Glucuronidation.

A process of steroid metabolism that consists of transfer of the glucuronic acid component of uridine diphosphate glucuronic acid to a substrate by any of several types of UDP-glucuronosyltransferase.