Keywords
Cushing’s syndrome - androgens - 11-oxygenated androgens - hyperandrogenemia
Introduction
Cushing’s syndrome (CS) is a rare disease with a prevalence of approximately 0.002%
[1]. It is defined as a state of
cortisol excess with loss of normal hypothalamic-pituitary-adrenal (HPA) axis
feedback loops and disturbed circadian rhythmicity of circulating cortisol levels
[2]. The most common form of CS is
exogenous glucocorticoid excess. Endogenous CS is, in about 80% of cases, caused by
ACTH-dependent CS and about 20% by an ACTH-independent adrenal oversecretion of
cortisol. Of those patients with endogenous CS, 70% are due to excessive secretion
of adrenocorticotropic hormone (ACTH) by pituitary adenomas, also known as
ACTH-dependent CS, and about 10% are due to ectopic ACTH-secreting tumors [3]
[4].
Patients with CS usually present with a broad range of clinical symptoms, some of
which resemble the clinical phenotype of patients with polycystic ovary syndrome
(PCOS) [2]
[5]
[6]
[7]
[8]. As PCOS, with a prevalence of up to
18% in women, is far more common, CS is sometimes initially misdiagnosed or the
final diagnosis delayed, which can have detrimental effects on morbidity and
mortality of the disease [9]
[10].
To optimize the diagnostic work-up in these patients with clinical signs of
hyperandrogenism, pathogenesis in CS must be properly understood. So far,
hyperandrogenism in ACTH-dependent CS has been proposed to be due to several
different causes, such as a hypercortisolemic inhibition of gonadotropins production
at the pituitary level [11], reduced
production of sex hormone binding globulin (SHBG) by the liver resulting in an
increased percentage of bioactive available androgens or an actual ACTH-mediated
elevation of classical adrenal androgen concentrations [11]
[12]
[13]
[14]
[15]. As adrenal androgen synthesis is ACTH-dependent, concentrations of
androgen precursors vary depending on the subtype of CS. ACTH-dependent CS and CS
due to ectopic ACTH production result in elevations of adrenal androgen
concentrations, while levels of androgens in patients with CS due to adrenal
cortisol production are low to suppressed [12]
[16]. The clinical
hyperandrogenic phenotype in patients with CS is, however, not sufficiently
explained by just the elevation in classical androgen concentrations, for example,
in a comparison of hirsute and non-hirsute females with ACTH-dependent CS, no
discernible difference in adrenal androgen concentrations were observed [11]. Just recently, adrenal-specific
11-oxygenated androgens have been shown to play a major role in hyperandrogenemia in
patients with ACTH-dependent CS [17].
While the direct stimulation via ACTH was proposed to be responsible for the
elevation of this subgroup of androgens, further analysis of different subtypes of
CS, however, revealed elevations among all patient subgroups with an inverse
relationship between 11-deoxygenated and 11-oxygenated androgens in adrenal CS [17].
This review aims to further elucidate the significance of androgen elevations in
patients with CS.
Androgen synthesis in women
Androgen synthesis in women
In women, the ovaries, adrenal glands, and peripheral tissues are involved in
androgen synthesis and metabolism ([Fig.
1]) [18]
[19]
[20]. Most androgen precursors are 19 carbon (C19) steroids, such as
dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and
androstenedione (A4), and are synthesized in the adrenal cortex, more specifically
in the zona reticularis (ZR), after stimulation by the pituitary hormone ACTH [21]. Androgen precursors, which do not
exhibit high androgenic potency, are further converted to the highly potent androgen
testosterone (T) and 5α-dihydrotestosterone (DHT), mainly in the ovaries and
peripheral tissues, but to a small extent also in the adrenal gland [21]
[22].
Fig. 1 Androgen synthesis in females. Illustration depicting adrenal
(orange), ovarian (blue) and peripheral (red) androgen synthesis in females.
[rerif]
This implies that DHEA and DHEAS are predominantly of adrenal origin, while A4
production is equally achieved by both ovarian and adrenal steroid synthesis. In the
adrenal gland, C19 steroids DHEA and DHEA-S are converted to A4 via the enzyme
β-hydroxysteroid dehydrogenase type 2 (HSD3B2) [21]
[23].
Ovarian androgen synthesis is regulated mainly by the pituitary production of
luteinizing hormone (LH) and takes place in the ovarian thecal cells. As in the
adrenal cortex, T, A4, and DHEA result from the conversion of cholesterol and
pregnenolone via the cholesterol side-chain cleavage enzyme and 17α-hydroxylase
[24].
Adipose tissue is an important peripheral tissue of androgen metabolism. Insulin is
also a potent regulator through direct stimulation of thecal cell androgen
production, by potentiating the function of LH on the ovaries, stimulating androgen
production, and by suppressing hepatic SHBG production, thus increasing free
circulating T concentrations [25]. In
women with PCOS and simple obesity, insulin was shown to upregulate AKR1C3
expression, promoting peripheral A4 to T conversion [26].
Skin, liver, brain, and breast tissue are further peripheral tissues involved in the
conversion of DHEA to A4 via 3βHSD1 and 2 [27]
[28]. T can then also
further be reduced to DHT via 5α-reductase (5αR), which has the highest androgenic
potential [29].
In the same way as in classical androgen synthesis in the adrenal gland,
11-oxygenated androgen (11oxC19) synthesis starts with the Δ5-pathway,
where pregnenolone is converted to DHEA via cytochrome P450
17α-hydroxylase/17,20-lyase (CYP17A1) [24]
[30]. The following
synthesis steps are illustrated in [Fig.
1]. Of the A4, which is the result of conversion from DHEA via HSD3B2,
some is converted to 11β-hydroxyandrostenedione (11OHA4) via cytochrome P450
11β-hydroxylase (CYP11B1) [31]. 11OHA4 is
the most abundant 11oxC19 steroid in the adrenal cortex [32]. To a lesser degree,
11-ketoandrotenedione (11KA4) and 11-ketotestosterone (11KT) are produced via
CYP11B1 and mainly result from peripheral conversion of adrenal-derived 11OHA4 [32]. More precisely, 11OHA4 can be
converted to 11KA4 by 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2), which is
highly prevalent in the kidney [33].
Peripheral tissues, such as adipose tissue, express high levels of aldo-keto
reductase 1C3 (AKR1C3), which can convert 11KA4 to 11KT [24]
[34]. Adipose and other tissues responsive to glucocorticoids produce an
enzyme called 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), which converts
11KA4 and 11KT to 11OHA4 and 11β-hydroxytestosterone (11OHT), respectively. 11OHT
can be converted to 11KT in mineralocorticoid target tissues in a similar way like
11OHA4 [35]. 11KT can also be converted
back to 11OHT by the enzyme HSD11B1 in peripheral tissues [36]
[37]. Other oxidative enzymes like 17β-hydroxysteroid dehydrogenases can
convert 11KT and 11OHT to 11KA4 and 11OHA4 [34]
[37]. Furthermore, 11KT can
be further reduced to 11-keto-5α-dihydrotestosterone (11KDHT) [34]. 11KT and 11KDHT are highly potent
androgens that activate the androgen receptor with the same affinity as T and
dihydrotestosterone (DHT) [38]
[39].
Regarding 11oxC19 androgen synthesis, it is important to consider its dependency upon
ACTH, which results in circadian patterns of circulating 11oxC19 concentrations,
closely resembling cortisol circadian rhythmicity with highest levels in the early
morning and declining salivary and serum concentrations over the day [40]
[41]. The diagnostic benefit and potential of 11oxC19 androgens originate
from their stable expression throughout the lifespan. In contrast to classical
adrenal androgens DHEA and DHEA-S, which decline in postmenopausal women due to
involution of the ZR [42]
[43]
[44]
[45]
[46]
[47]
[48]
[49], 11oxC19 androgen concentrations do
not decline with age and are therefore of higher prevalence than T and A4
concentrations in postmenopausal women [50]. In humans, concentrations of DHEA and DHEA-S show a rapid decline
right after birth until adrenarche [51].
During adrenarche, between ages 6–10 years, a marked increase in adrenal cortex
androgen synthesis is observed [52].
During this period, the distinct ZR forms exhibit a slight alteration in enzyme
expression compared to the zona fasciculata (ZF). The expression of cytochrome b5,
which facilitates the 17,20-lyase activity of CYP17A1, is upregulated, whereas the
expression of 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2) is downregulated [52]. Before the re-discovery of 11oxC19
androgens and investigation of their clinical relevance, adrenarche was attributed
to increased synthesis of DHEA and DHEA-S [53]. In 2018, however, one study comparing steroid production in girls
during adrenarche compared to premature adrenarche found that 11KT is actually the
dominant bioactive androgen in these patients [38]. In postmenopausal women, involution of the ZR results in the ZF
containing 3β-hydroxysteroid-dehydrogenase (HSD3B2) and the ZR containing cytochrome
B5 type A (CYB5A), to become closely juxtaposed. Consequently, this proximity allows
for the conversion of DHEA to A4 and the subsequent formation of 11oxC19 androgens
[50]. In reproductive-age women, 11KT
and T levels are similar. During postmenopause, the 11KT to T ratio increases,
suggesting 11KT as the primary mediator for processes like adrenarche in prepubertal
children and postmenopausal women due to its comparable androgenic potency to T
[22].
Besides their relevance in premature adrenarche, 11oxC19 androgens have been
described as potential diagnostic and therapeutic monitoring tools in other
endocrine disorders, such as congenital adrenal hyperplasia (CAH). Owing to their
ACTH dependence and adrenal specificity, 11oxC19 androgens have been proposed as
biomarkers of androgen excess in CAH due to 21-hydroxylase deficiency (21OHD). In
patients with 21OHD, concentrations of 11oxC19 androgens are usually up to fourfold
higher than in healthy controls [33]
[41]. Especially in male patients with
21OHD, 11KT has been found to better resemble disease control compared to T. Levels
of 11KT and 11OHT inversely correlate with T and luteinizing hormone concentrations,
suggesting suppression of the hypothalamic-pituitary-gonadal axis and testicular T
synthesis in poorly controlled male patients with 21OHD [33]
[54]. 11OHT and 11KT concentrations were also significantly higher in
patients with testicular adrenal rest tumors (TART) – which results from poor
disease control – and also in female patients with menstrual disturbances compared
to those patients without [54]. In
patients with PCOS and clinical signs of hyperandrogenism, the origin of androgen
excess is diverse. While traditionally, the ovaries have been identified as the
primary source of heightened androgens in PCOS, emerging clinical and biochemical
evidence suggests a nuanced perspective [55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]. The 11oxC19
androgens, particularly 11OHA4 and 11KA4, have garnered attention as significant
contributors to the androgen pool in this disorder [11]
[63]
[64]. This, as along with
increased DHEA and DHEA-S concentrations in some patients with PCOS, points towards
adrenal involvement [60]
[61]. Besides elevations in 11oxC19
androgens, studies could also demonstrate correlations to factors, including body
mass index, insulin levels, and homeostatic model assessment-insulin resistance, as
important evaluation parameters for metabolic risk in these women [11]. Moreover, the adrenal
hyper-reactivity to ACTH stimulation also implies a distinct adrenal role in certain
PCOS cases, shedding light on the complexity of androgen regulation in this syndrome
[62]
[65].
Androgens in adrenocorticotropic hormone-dependent Cushing’s syndrome
Androgens in adrenocorticotropic hormone-dependent Cushing’s syndrome
Signs of hyperandrogenism, such as menstrual disturbances, acne and hirsutism are
common in female patients with ACTH-dependent CS. Baseline data from the European
Registry on CS (ERCUSYN) on 481 CS patients collected from 36 centres in 23
countries revealed presence of skin alterations in 78% of patients with
ACTH-dependent CS, hirsutism and menstrual irregularities in 63% and hair loss in
34% of patients with ACTH-dependent CS [7]. Another study on 45 premenopausal women with ACTH-dependent CS
discovered an even higher rate of 80% of menstrual irregularities at the time of
disease diagnosis, with amenorrhea being the most prevalent type of menstrual
dysregulation [66]. Despite the presence
of clinical signs of hyperandrogenism, the cause of hyperandrogenism in these
patients was, for a long time, not sufficiently understood, but is most likely of
multifactorial origin ([Fig. 2]). One
plausible rationale for the extent of hyperandrogenism in these individuals has been
postulated to involve heightened concentrations of classical androgens coupled with
diminished levels of SHBG. This interplay leads to elevated concentrations of
unbound androgens, facilitating efficient binding to androgen receptors. Levels of
classical androgens DHEA-S, A4, and T have, however, only rarely been found to be
elevated [11]. A study evaluating
androgen concentrations in a subset of 36 women with ACTH-dependent CS, mostly due
to ACTH-dependent CS (30/36) revealed elevations in DHEA-S in 8/30 patients, A4
elevations in 18/30 and T elevations in 17/30 cases, with normalization of androgen
levels after pituitary surgery. In patients with ectopic ACTH secretion, A4
elevations were higher compared to those with ACTH-dependent CS [12]. Another study on 47 patients with
ACTH-dependent CS and six patients with ectopic CS also presented elevated levels of
androgens and their metabolites compared to a healthy control cohort [15]. In other studies, DHEA-S, A4 and T
have been shown to be in the normal reference range mostly [13]
[67] or DHEA-S elevations, at least in some patients (11/37) with
ACTH-dependent CS, while the majority (70% of patients) however also presented with
DHEA-S levels within the reference range [68]. Another retrospective study on 222 patient samples tested for CS
with analysis of a panel of 15 plasma steroid hormones via mass spectrometry,
identified higher concentrations of adrenal androgens A4, DHEA, and DHEA-S in
ACTH-dependent CS compared to adrenal CS. DHEA-S levels were shown to be higher in
patients with ACTH-dependent CS compared to healthy patients without
hypercortisolism [14]. We have moreover
learned from a study on pediatric patients with ACTH-dependent CS that those
patients with excessive clinical symptoms of virilization actually go in hand with
elevations of classical androgens A4, DHEA-S, and T and also decreased SHBG levels
in a direct comparison to those patients with ACTH-dependent CS without clinical
signs of virilization [69].
Fig. 2 Multifactorial origin of hyperandrogenism in ACTH-dependent and
ACTH-independent Cushing’s syndrome. Hyperandrogenemia in ACTH-dependent
Cushing’s syndrome seems to result from multifactorial origin due to
hypercortisolemic inhibition of the production of gonadotropins LH and FSH
at the pituitary level and inhibition of the release of
gonadotropin-releasing hormone synthesis at hypothalamic level (left); due
to reduced direct stimulation of adrenal androgen production and decreased
SHBG synthesis, which results in increased percentage of bioactive available
androgens (middle) and direct stimulation of 11oxC19 androgen synthesis due
to increased ACTH concentrations (right). Hyperandrogenemia in
ACTH-independent Cushing’s syndrome is most likely the result of the direct
conversion of approximately 10% of cortisol to 11oxC19 androgens. ACTH,
adrenocorticotropic hormone; LH, luteinizing hormone; FSH,
follicle-stimulating hormone; 11oxC19, 11-oxygenated androgen. [rerif]
Despite some reports of elevations of classical androgens in patients with
ACTH-dependent CS, they do not sufficiently explain the pronounced clinical symptoms
as described above. Another contributing factor to menstrual disturbances in
patients with ACTH-dependent CS may be attributed to diminished gonadotropin
concentrations. Analysis of 45 female patients with ACTH-dependent CS with menstrual
irregularities in 80% of these patients revealed positive correlations of menstrual
cycle disturbances and cortisol levels as well as inverse correlations with E2
concentrations, while gonadotropin levels were, however, still in the normal range.
Therefore, Lado-Abeal et al. suggested at least menstrual irregularities are the
results of a hypercortisolemic inhibition of hypothalamic gonadotropin production,
rather than high androgen concentrations [66]. White et al. confirmed in 22 patients with CS that gonadotropin
levels were only subnormal in patients with the highest elevation in cortisol levels
[70]. Instead of increased androgen
concentrations, inverse correlations of urinary free cortisol and SHBG, estradiol,
and T were also observed in a second study on 13 women with CS, while again, serum
E2 levels were similar to those in patients in the early follicular phase of the
menstrual cycle and only low in seven of the 13 women with CS [67].
More recent data from our lab revealed the relevance of 11oxC19 elevations for
hyperandrogenism in patients with ACTH-dependent CS. We measured elevated levels of
11oxC19 androgens 11OHA4 and 11KT in saliva day profiles of 23 female patients with
ACTH-dependent CS compared to healthy controls, while A4, T, and DHEA-S levels were
comparable to those of controls and gonadotropin concentrations also in the normal
range [17]. We could also demonstrate
normalization of salivary 11oxC19 androgen concentrations after successful
transsphenoidal surgery in 13 of the patients mentioned above as well as in those
medically controlled with Osilodrostat or metyrapone treatment [17]. ACTH-dependence was also confirmed by
a positive correlation between the decrease of 11oxC19 steroids in saliva and their
urinary metabolites and ACTH concentrations. We, therefore, concluded that ACTH
stimulation in ACTH-dependent CS leads to direct adrenal stimulation of 11oxC19
synthesis and that elevated 11oxC19 androgen concentrations are the most relevant
factor for hyperandrogenemia in this patient cohort, which could also be supported
by correlations of cortisol and cortisone levels and 11OHA4 concentrations as well
as cortisol/cortisone and ACTH concentrations. However, disturbances of the HPA axis
were not observed in our study cohort; moreover, as 11oxC19 estrogens seem not to be
aromatizable in vivo, symptoms of hyperandrogenemia in ACTH-dependent CS seem to
rather result from the direct androgenic effect of 11oxC19 steroids on skin, hair,
and ovaries, than due to disturbance in the HPG axis [17]
[34].
In conclusion, the clinical signs of hyperandrogenism in ACTH-dependent CS appear to
be multifactorial, stemming from a combination of dysregulations involving cortisol
levels, hypothalamic-gonadotropin inhibition, and altered classical and 11oxC19
androgen concentrations, however, the major relevance of 11oxC19 androgens has so
far been neglected as 11oxC19 concentrations have not been routinely measured in
patients with ACTH-dependent CS.
Androgens in adrenocorticotropic hormone-independent Cushing’s syndrome
Androgens in adrenocorticotropic hormone-independent Cushing’s syndrome
Patients with CS due to adrenal origin of cortisol excess present with similar
clinical signs of hyperandrogenism [67],
although, to a lower degree in comparison to ACTH-dependent subtypes of CS [71]. In contrast, in all studies comparing
concentrations of androgens between the different subtypes of CS, patients with
adrenal disease had the lowest concentrations of androgens [14]. Further studies evaluating androgen
concentrations in comparison to a healthy control cohort demonstrated significantly
lower levels of classical androgens DHEA, DHEA-S, and A4 in both patients with
adrenal adenomas and primary bilateral macronodular adrenocortical hyperplasia
(PBMAH) [15]
[72]. Further studies analyzing the
recovery of DHEA-S secretion after achieving remission of CS due to unilateral
adrenalectomy in patients with CS due to adrenocortical adenoma revealed persisting
suppression of DHEA-S levels for a further two years [68].
Urinary steroid metabolome analysis by gas chromatography-mass spectrometry in a
cohort of 168 patients with CS, including 44 patients with ACTH-dependent CS, 18
with adrenal adenoma, and 13 patients with PBMAH as well as 93 healthy controls,
revealed a striking increase in excretion rates of all adrenal androgen metabolites
in patients with ACTH-dependent CS with 1.5 times higher levels of 11-deoxygenated
androgens and 2.7 higher 11oxC19 androgen concentrations compared to the levels in
the control group [73]. This resulted in
an increased ratio between the 11oxC19 androgens and the 11-deoxygenated adrenal
androgen levels. In the same way, 11oxC19 androgen concentrations were elevated 1.6
times in patients with CS due to unilateral cortisol-producing adrenal adenoma, most
likely due to the high expression of CYP11B1 in these adrenal adenomas [74]. In those patients however, levels of
classical androgens were rather low as expected due to the lack of ACTH-stimulation,
resulting in an inverse relation and an even higher ratio of the 11oxC19 androgens
and the 11-deoxygenated adrenal androgens. In patients with PBMAH, 11oxC19 androgen
concentrations were even further elevated up to 2.3 times those of the control
cohort, while classical androgen metabolites were downregulated 0.5 times in
comparison to healthy controls, however not as much as in those patients with
unilateral adrenal CS [73]. The elevation
of 11oxC19 androgens in patients with CS is further exacerbated by the direct
conversion of approximately 10% of cortisol to these androgens [75]. As we have demonstrated that these
11oxC19 androgens predominate in all forms of CS, it is most likely that they are
also responsible for the clinical signs of hyperandrogenism, such as menstrual
disorders, acne, and hirsutism in all subtypes of CS. To distinguish between the two
major subtypes of ACTH-dependent and ACTH-independent CS, estradiol and SHBG
concentrations, as well as the ratio of 11oxC19 to classical androgen
concentrations, should therefore be considered ([Fig. 2]).
Conclusion
In conclusion, a nuanced understanding of androgen dysregulation in CS reveals the
multifactorial nature of hyperandrogenism, involving cortisol levels, gonadotropin
inhibition, and alterations in both classical and 11oxC19 androgen concentrations.
While classical androgens alone do not fully explain the pronounced clinical
symptoms, the prominence of 11oxC19 androgens, particularly 11OHA4 and 11KT, so far
overlooked in routine assessments, add a crucial dimension to the understanding of
hyperandrogenism. These highly potent adrenal-specific androgens demonstrate stable
expression throughout a womanʼs lifespan, distinguishing them as valuable diagnostic
biomarkers. Their predominant role in all CS subtypes, irrespective of the
underlying cause, underscores their pivotal contribution to hyperandrogenic
symptoms. The distinct androgen profile provided by 11oxC19 androgens not only aids
in subtype differentiation but also offers insights into the multifaceted nature of
androgen dysregulation in CS. Recognizing the diagnostic potential of 11oxC19
androgens promises to refine diagnostic approaches, enhancing the precision and
effectiveness of clinical management strategies for patients with CS.
