Circulating adrenal and gonadal steroid hormones heterogeneity in active young males and the contribution of 11-oxy androgens.

The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency.

Resumption of ovulation in anovulatory women with PCOS and obesity is associated with reduction of 11ß-hydroxyandrostenedione concentrations.

STUDY QUESTION: Is resumption of ovulation after a 6-month lifestyle intervention in women with PCOS and obesity associated with differential changes in endocrine and metabolic parameters (weight, insulin resistance, anti-Müllerian hormone (AMH), and androgens) compared to women with PCOS who remained anovulatory? SUMMARY ANSWER: Resumption of ovulation after a 6-month lifestyle intervention in women with PCOS and obesity is associated with changes in serum 11ß-hydroxyandrostenedione (11OHA4) concentrations. WHAT IS KNOWN ALREADY: Lifestyle interventions have been shown to reduce clinical and biochemical hyperandrogenism in women with PCOS. Weight loss of 5-10% may reverse anovulatory status, thereby increasing natural conception rates. However, the mechanisms underlying why some women with PCOS remain anovulatory and others resume ovulation after weight loss are unclear. Reproductive characteristics at baseline and a greater degree of change in endocrine and metabolic features with lifestyle intervention may be crucial for ovulatory response. STUDY DESIGN, SIZE, DURATION: We used data and samples originating from an earlier randomized controlled trial (RCT), which examined the efficacy of a 6-month lifestyle intervention prior to infertility treatment compared to prompt infertility treatment on live birth rate in women with obesity. A total of 577 women with obesity (BMI > 29 kg/m2) were randomized between 2009 and 2012. Anovulatory women with PCOS who were allocated to the intervention arm of the original RCT (n = 95) were included in the current analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: We defined women as having resumed ovulation (RO+) based on the following criteria: spontaneous pregnancy; or assignment to expectant management; or IUI in natural cycles as the treatment strategy after lifestyle intervention. Steroid hormones were measured using liquid chromatography tandem mass spectrometry. Generalized estimating equations with adjustment for baseline measures and interaction between group and time was used to examine differences in changes of endocrine and metabolic parameters between RO+ (n = 34) and persistently anovulatory women (RO-, n = 61) at 3 and 6 months after intervention. MAIN RESULTS AND THE ROLE OF CHANCE: At baseline, the mean ± SD age was 27.5 ± 3.6 years in the RO+ group and 27.9 ± 4.1 years in the RO- group (P = 0.65), and the mean ± SD weights were 101.2 ± 9.5 kg and 105.0 ± 14.6 kg, respectively (P = 0.13). Baseline AMH concentrations showed significant differences between RO+ and RO- women (median and interquartile range [IQR] 4.7 [3.2; 8.3] versus 7.2 [5.3; 10.8] ng/ml, respectively). Baseline androgen concentrations did not differ between the two groups. During and after lifestyle intervention, both groups showed weight loss; changes in 11OHA4 were significantly different between the RO+ and RO groups (P-value for interaction = 0.03). There was a similar trend for SHBG (interaction P-value = 0.07), and DHEA-S (interaction P-value = 0.06), with the most pronounced differences observed in the first 3 months. Other parameters, such as AMH and FAI, decreased over time but with no difference between the groups. LIMITATIONS, REASONS FOR CAUTION: No high-resolution transvaginal ultrasonography was used to confirm ovulatory status at the end of the lifestyle program. The small sample size may limit the robustness of the results. WIDER IMPLICATIONS OF THE FINDINGS: Reduction of androgen concentrations during and after lifestyle intervention is associated with recovery of ovulatory cycles. If our results are confirmed in other studies, androgen concentrations could be monitored during lifestyle intervention to provide individualized recommendations on the timing of resumption of ovulation in anovulatory women with PCOS and obesity. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by a grant from ZonMw, the Dutch Organization for Health Research and Development (50-50110-96-518). The Department of Obstetrics and Gynecology of the UMCG received an unrestricted educational grant from Ferring Pharmaceuticals BV, The Netherlands. A.H. reports consultancy for the development and implementation of a lifestyle App MyFertiCoach developed by Ferring Pharmaceutical Company. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: The LIFEstyle RCT was registered at the Dutch trial registry (NTR 1530).

C11-hydroxy and C11-oxo C19 and C21 Steroids: Pre-Receptor Regulation and Interaction with Androgen and Progesterone Steroid Receptors.

C11-oxy C19 and C11-oxy C21 steroids have been identified as novel steroids but their function remains unclear. This study aimed to investigate the pre-receptor regulation of C11-oxy steroids by 11ß-hydroxysteroid dehydrogenase (11ßHSD) interconversion and potential agonist and antagonist activity associated with the androgen (AR) and progesterone receptors (PRA and PRB). Steroid conversions were investigated in transiently transfected HEK293 cells expressing 11ßHSD1 and 11ßHSD2, while CV1 cells were utilised for agonist and antagonist assays. The conversion of C11-hydroxy steroids to C11-oxo steroids by 11ßHSD2 occurred more readily than the reverse reaction catalysed by 11ßHSD1, while the interconversion of C11-oxy C19 steroids was more efficient than C11-oxy C21 steroids. Furthermore, 11-ketodihydrotestosterone (11KDHT), 11-ketotestosterone (11KT) and 11ß-hydroxydihydrotestosterone (11OHDHT) were AR agonists, while only progestogens, 11ß-hydroxyprogesterone (11ßOHP4), 11ß-hydroxydihydroprogesterone (11ßOHDHP4), 11α-hydroxyprogesterone (11αOHP4), 11α-hydroxydihydroprogesterone (11αOHDHP4), 11-ketoprogesterone (11KP4), 5α-pregnan-17α-diol-3,11,20-trione (11KPdione) and 21-deoxycortisone (21dE) exhibited antagonist activity. C11-hydroxy C21 steroids, 11ßOHP4, 11ßOHDHP4 and 11αOHP4 exhibited PRA and PRB agonistic activity, while only C11-oxo steroids, 11KP4 and 11-ketoandrostanediol (11K3αdiol) demonstrated PRB agonism. While no steroids antagonised the PRA, 11OHA4, 11ß-hydroxytestosterone (11OHT), 11KT and 11KDHT exhibited PRB antagonism. The regulatory role of 11ßHSD isozymes impacting receptor activation is clear-C11-oxo androgens exhibit AR agonist activity; only C11-hydroxy progestogens exhibit PRA and PRB agonist activity. Regulation by the downstream metabolites of active C11-oxy steroids at the receptor level is apparent-C11-hydroxy and C11-oxo metabolites antagonize the AR and PRB, progestogens the former, androgens the latter. The findings highlight the intricate interplay between receptors and active as well as "inactive" C11-oxy steroids, suggesting novel regulatory tiers.

Analysis of 52 C19 and C21 steroids by UPC2-MS/MS: Characterising the C11-oxy steroid metabolome in serum.

The C11-oxy androgens have been implicated in the progression of many diseases and endocrine-linked disorders, such as polycystic ovarian syndrome (PCOS), congenital adrenal hyperplasia, specifically 21-hydroxylase deficiency (21OHD), castration resistant prostate cancer (CRPC), as well as premature adrenarche. While the C11-oxy C19 steroids have been firmly established in the steroid arena, the C11-oxy C21 steroids are now also of significance. The current study reports on a high-throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) method for the separation and quantification of 52 steroids in peripheral serum, which include the C11-oxy C19 and C11-oxy C21 steroids. Fifteen deuterium-labelled steroids were included for absolute quantification, which incorporates steroid extraction efficiency, together with one steroid and four non-steroidal compounds serving as quality controls (QC). The 15 min run-time per sample (16 min injection-to-injection time with an 8-step gradient) quantifies 68 analytes in a 2 µL injection volume. A single chromatographic step simultaneously identifies steroids in the mineralocorticoid, glucocorticoid and androgen pathways in adrenal steroidogenesis, together with steroid metabolites produced in the periphery, presenting an analytical method for the application of screening in vivo clinical samples. This study highlights cross-talk between the C11-oxy steroids, and describes the optimisation of multiple reaction monitoring required to measure steroids accurately. The limit of detection for the steroid metabolites ranged from 0.002 to 20 ng/mL and the limit of quantification from 0.02 to 100 ng/mL. The calibration range for the steroids ranged from 0.002 to 1000 ng/mL and for the QC compounds from 0.075 to 750 ng/mL. The method is fully validated in terms of accuracy (%RSD, <13%), precision (including inter-day variability across a three-day period) (%RSD, <16%), recovery (average 102.42%), matrix effect (ranging from -15.25 to 14.25%) and process efficiency (average 101.79%). The dilution protocol for the steroids, internal standards and QC compounds were validated, while the ion ratios of the steroid metabolites (%RSD, <16%) and QC compounds were monitored and the accuracy bias values (%RSD, <9%) were within acceptable limits. The method was subsequently used to quantify steroid levels in a cohort of healthy women. C11-oxy steroid metabolites produced as intermediates in steroidogenic pathways, together with end-products included in the method can potentially characterise the 11ß-hydroxyandrostenedione-, C21- and C11-oxy backdoor pathways in vivo. The identification of these C11-oxy C19 and C11-oxy C21 intermediates would allow insight into active pathways, while steroid metabolism could be traced in patients and reference ranges established in both normal and abnormal conditions. Furthermore, conditions currently undefined in terms of the C11-oxy steroids would benefit from the analysis provided by this method, while the C11-oxy steroids could be further explored in PCOS, 21OHD, CRPC and adrenarche.

Development of a total serum testosterone, androstenedione, 17-hydroxyprogesterone, 11ß-hydroxyandrostenedione and 11-ketotestosterone LC-MS/MS assay and its application to evaluate pre-analytical sample stability.

Background Classically, serum testosterone (T) and androstenedione (A4) have been the mainstay for the biochemical assessment of hyperandrogenism. However, recent evidence suggests 11ß-hydroxyandrostenedione (11OHA4) and 11-ketotestosterone (11KT) may also be important. Here, we describe the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitation of total serum T, A4, 17-hydroxyprogesterone (17OHP), 11OHA4 and 11KT. In addition, we applied the method to assess pre-analytical stability. Methods An isotopically labelled internal standard was added to samples prior to supported liquid extraction (SLE). Extracts were analysed using LC-MS/MS to detect T/A4/17OHP/11OHA4 and 11KT along with their corresponding internal standards. Samples (n = 7) were collected from healthy volunteers (n = 14) and left incubated at 20 °C for up to 72 h. Tubes were retrieved at select time points, centrifuged, separated and frozen prior to analysis. Results The total run time was 4 min. For all analytes, intra- and inter-assay imprecision did not exceed 7.9% and 5.3%, respectively; matrix effects were negligible and mean recoveries ranged from 95.3 to 111.6%. The limits of quantitation (LOQs) were 0.25 nmol/L for T, A4 and 11OHA4, 0.50 nmol/L for 17OHP, and 0.24 nmol/L for 11KT. No significant change was observed in pre-centrifugation A4 or female T concentrations over 72 h. Significant increases (p < 0.01) in concentrations of 11KT, 17OHP, 11OHA4 and male T were observed after 2, 8, 12 and 24 h, respectively. Conclusions We developed a robust LC-MS/MS assay for the quantitation of total serum T/A4/17OHP/11OHA4 and 11KT. Applying the method to determine pre-analytical stability suggests samples requiring 11KT need separating from the cells within 2 h.

The role of adrenal derived androgens in castration resistant prostate cancer.

Castration resistant prostate cancer (CRPC) remains androgen dependant despite castrate levels of circulating testosterone following androgen deprivation therapy, the first line of treatment for advanced metstatic prostate cancer. CRPC is characterized by alterations in the expression levels of steroidgenic enzymes that enable the tumour to derive potent androgens from circulating adrenal androgen precursors. Intratumoral androgen biosynthesis leads to the localized production of both canonical androgens such as 5α-dihydrotestosterone (DHT) as well as less well characterized 11-oxygenated androgens, which until recently have been overlooked in the context of CRPC. In this review we discuss the contribution of both canonical and 11-oxygenated androgen precursors to the intratumoral androgen pool in CRPC. We present evidence that CRPC remains androgen dependent and discuss the alterations in steroidogenic enzyme expression and how these affect the various pathways to intratumoral androgen biosynthesis. Finally we summarize the current treatment strategies for targeting adrenal derived androgen biosynthesis.

The 11ß-hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-oxy C19 steroids with 11ßHSD2 favouring 11-ketotestosterone, 11-ketoandrostenedione and 11-ketoprogesterone biosynthesis.

The 11ß-hydroxysteroid dehydrogenase (11ßHSD) types 1 and 2 are primarily associated with glucocorticoid inactivation and reactivation. Several adrenal C11-oxy C19 and C11-oxy C21 steroids, which have been identified in prostate cancer, 21-hydroxylase deficiency and polycystic ovary syndrome, are substrates for these isozymes. This study describes the kinetic parameters of 11ßHSD1 and 11ßHSD2 towards the C11-keto and C11-hydroxy derivatives of the C19 and C21 steroids. The apparent Km and Vmax values indicate the more prominent 11ßHSD2 activity towards 11ß-hydroxy androstenedione, 11ß-hydroxytestosterone and 11ß-hydroxyprogesterone in contrast to the 11ßHSD1 reduction of the C11-keto steroids, as was demonstrated in the LNCaP cell model in the production of 11-ketotestosterone and 11-ketodihydrotestosterone. Data highlighted the role of 11ßHSD2 and cytochrome P450 17A1 in the contribution of C11-oxy C21 steroids to the C11-oxy C19 steroid pool in the C11-oxy backdoor pathway. In addition, 11ßHSD2 activity, catalysing 11-ketotestosterone biosynthesis, was shown to be key in the production of prostate specific antigen and in the progression of prostate cancer to castration resistant prostate cancer. The study at hand thus provides evidence that 11ßHSD isozymes play key roles in pathophysiological states, more so than was previously put forward.

Canonical and Noncanonical Androgen Metabolism and Activity.

Androgens are critical drivers of prostate cancer. In this chapter we first discuss the canonical pathways of androgen metabolism and their alterations in prostate cancer progression, including the classical, backdoor and 5α-dione pathways, the role of pre-receptor DHT metabolism, and recent findings on oncogenic splicing of steroidogenic enzymes. Next, we discuss the activity and metabolism of non-canonical 11-oxygenated androgens that can activate wild-type AR and are less susceptible to glucuronidation and inactivation than the canonical androgens, thereby serving as an under-recognized reservoir of active ligands. We then discuss an emerging literature on the potential non-canonical role of androgen metabolizing enzymes in driving prostate cancer. We conclude by discussing the potential implications of these findings for prostate cancer progression, particularly in context of new agents such as abiraterone and enzalutamide, which target the AR-axis for prostate cancer therapy, including mechanisms of response and resistance and implications of these findings for future therapy.

The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase.

Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol, forming 11ß-hydro-xyandrostenedione. A cortisol-inducible operon (desABCD) was previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes a protein annotated as a member of the major facilitator family, predicted to function as a cortisol transporter. The desA and desB genes are annotated as N-terminal and C-terminal transketolases, respectively. We hypothesized that the DesAB forms a complex and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C. scindens ATCC 35704 in pETDuet for overexpression in Escherichia coli The purified recombinant DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This was achieved by coupling DesAB-dependent formation of 11ß-hydroxyandrostenedione with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17ß-HSDH from the filamentous fungus, Cochliobolus lunatus The pH optimum for the coupled assay was 7.0 and kinetic constants using cortisol as substrate were Km of 4.96 ± 0.57 µM and kcat of 0.87 ± 0.076 min-1 Substrate-specificity studies revealed that rDesAB recognized substrates regardless of 11ß-hydroxylation, but had an absolute requirement for 17,21-dihydroxy 20-ketosteroids.

11ß-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: a putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

11ß-hydroxyandrostenedione, the product of androstenedione metabolism in the adrenal, is metabolized in LNCaP cells by 5α-reductase yielding 11ß-hydroxy-5α-androstanedione.

11ß-Hydroxyandrostenedione (11OHA4), which is unique to the adrenal, was first isolated from human adrenal tissue in the fifties. It was later shown in the sixties that 11ß-hydroxytestosterone (11OHT) was also produced by the human adrenal. Attention has shifted back to these adrenal androgens once more, as improved analytical techniques have enabled more accurate detection of steroid hormones. In this paper, we investigated the origin of these metabolites as well as their subsequent metabolism and examined a possible physiological role for 11OHA4 in prostate cancer cells. In H295R cells treated with forskolin and trilostane, etomidate, a reported cytochrome P450 11ß-hydroxylase (CYP11B1) inhibitor, blocked the production of corticosterone, cortisol, 11OHA4 and 11OHT. The metabolism of androstenedione and testosterone by CYP11B1 and aldosterone synthase (CYP11B2) was assayed. Androstenedione was converted by CYP11B1, while the conversion by CYP11B2 was negligible. Both enzymes readily converted testosterone. The metabolism of these 11ß-hydroxylated metabolites by 11ß-hydroxysteroid dehydrogenase (11ßHSD) types 1 and 2 was subsequently investigated. 11ßHSD2 catalyzed the conversion of both 11OHA4 and 11OHT to their respective keto-steroids, while 11ßHSD1 catalyzed the conversion of 11-ketoandrostenedione and 11-ketotestosterone to their respective hydroxy-steroids in Chinese hamster ovary cells. Investigating a functional role, steroid 5α-reductase types 1 and 2 converted 11OHA4 to 11ß-hydroxy-5α-androstanedione (11OH-5α-dione), identified by accurate mass detection. UPLC-MS/MS analyses of 11OHA4 metabolism in LNCaP androgen-dependent prostate cancer cells, identified the 5α-reduced metabolite as well as 11-ketoandrostenedione and 11-ketotestosterone, with the latter indicating conversion by 17ß-hydroxysteroid dehydrogenase. Downstream metabolism by 11ßHSD2 and by 5α-reductase may therefore indicate a physiological role for 11OHA4 and/or 11OH-5α-dione in normal and prostate cancer cells.

Steroidogenic acute regulatory protein transcript abundance in the eel, Anguilla australis: changes during the induced reproductive cycle and effects of follicle-stimulating hormone during previtellogenesis.

Steroidogenic acute regulatory protein (StAR) mRNA levels in the eel ovary were assayed by quantitative PCR and related to plasma steroid levels throughout oogenesis in order to shed light on the previously considered 'aberrant' prematurational increase in plasma levels of estradiol-17ß (E2). Total ovarian StAR transcript abundance mirrored circulating levels of E2, but not of 11-ketotestosterone (11KT). The study was complemented by evaluation of in vitro effects of follicle-stimulating hormone (FSH) on ovarian StAR transcript abundance and on short-term ('acute') radiolabelled pregnenolone-supported steroid metabolism by ovarian fragments to understand how the production of steroids during previtellogenic oocyte growth is regulated. We observed a significant effect of FSH on StAR mRNA levels within 24h of incubation, but these were no longer evident by 4 days of culture. Unexpectedly, FSH had no effect on substrate-supported steroidogenesis, as comparable yields of steroid products were detected using semi-quantitative HPLC and scintillation counting. We conclude that the eel ovarian follicle can respond to FSH from a very early stage of development (early oil droplet stage) by increasing StAR mRNA levels, but that there is no evidence for acute effects of FSH on bioactive steroid production downstream of cytochrome P450 side-chain cleavage. Furthermore, the prematurational increase in StAR mRNA in vivo is in keeping with general teleost models and is likely to be a 'normal' response to reaching advanced stages of development.