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.

The influence of ghrelin agonist ipamorelin acetate on the hypothalamic-pituitary-testicular axis in a cichlid fish, Oreochromis mossambicus.

Ghrelin, a peptide found in the brain and gut, is predicted to play a significant role in the control of various physiological systems in fish. The objective of this study was to examine the impact of ipamorelin acetate (IPA), a ghrelin agonist, on the reproductive axis of the tilapia Oreochromis mossambicus. The administration of either 5 or 30 µg of IPA for 21 days led to a significant and dose-dependent rise in food intake concomitant with a significant increase in the numbers of primary spermatocytes, secondary spermatocytes, and early spermatids compared to the control group. There was a significant rise in the number of late spermatids, as well as the areas of the lobule and lumen, in fish treated with 30 µg of IPA, compared to the control group. Moreover, there was no significant difference in the percentage of gonadotropin-releasing hormone (GnRH)-immunoreactive fibres in the hypothalamus and anterior pituitary gland across different groups. However, a significant elevation in the expression of androgen receptor protein was observed in fish treated with 30 µg of IPA. Furthermore, the concentrations of luteinizing hormone (LH) and 11-ketotestosterone (11-KT) in the serum of fish treated with either 5 or 30 µg of IPA were significantly elevated in comparison to the control group. Collectively, these findings suggest that the administration of ghrelin enhances the development of germ cells during the meiosis-I phase and that this effect might be mediated via the stimulation of 11-KT and androgen receptors at the testicular level and LH at the pituitary level in the tilapia.

Human Gonads Do Not Contribute to the Circulating Pool of 11-Oxygenated Androgens.

CONTEXT: Androstenedione (A4) and testosterone (T) are produced by both the adrenal glands and the gonads. The adrenal enzyme 11ß-hydroxylase (CYP11B1) executes the final step in cortisol synthesis; CYP11B1 also uses A4 and T as substrates, generating 11-hydroxyandrostenedione and 11-hydroxytestosterone, respectively. It has been suggested that CYP11B1 is expressed in the gonads, yet the circulating levels of all 11-oxygenated androgens (11-oxyandrogens) are similar in males and females of reproductive ages, despite enormous differences in T. OBJECTIVE: To assess the gonadal contribution to the circulating pool of 11-oxyandrogens. METHODS: We used liquid chromatography-tandem mass spectrometry to measure 13 steroids, including traditional and 11-oxyandrogens in: (I) paired gonadal and peripheral vein blood samples obtained during gonadal venograms from 11 patients (7 women), median age 37 (range 31-51 years); and (II) 17 women, median age 57 (range 41-81 years) before and after bilateral salpingo-oophorectomy (BSO). We also compared CYP11B1, 17α-hydroxylase/17,20-lyase (CYP17A1), and 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA expression in adrenal, ovarian, and testicular tissue. RESULTS: A4, T, estradiol, estrone, progesterone, 17α- and 16α-hydroxyprogesterone were all higher in gonadal veins vs. periphery (p < 0.05 for all), while four 11-oxyandrogens were similar between matched gonadal and peripheral vein samples. Equally, in women who underwent BSO, A4 (median [interquartile range]: 59.7 [47.7-67.6] ng/dL vs. 32.7 [27.4-47.8] ng/dL, p < 0.001) and T (24.1 [16.4-32.3] vs.15.5 [13.7-19.0] ng/dL, p < 0.001) declined, while 11-oxyandrogens remained stable. Gonadal tissue displayed negligible CYP11B1 mRNA. CONCLUSION: Despite producing substantial amounts of A4 and T, human gonads are not relevant sources of 11-oxyandrogens.

Diurnal 11-ketotestosterone and 17-hydroxyprogesterone saliva profiles in paediatric classical congenital adrenal hyperplasia.

OBJECTIVES: The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. METHODS: Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. RESULTS: Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. CONCLUSIONS: 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI.

Diversity of Androgens; Comparison of Their Significance and Characteristics in Vertebrate Species.

Androgen(s) is one of the sex steroids that are involved in many physiological phenomena of vertebrate species. Although androgens were originally identified as male sex hormones, it is well known now that they are also essential in females. As in the case of other steroid hormones, androgen is produced from cholesterol through serial enzymatic reactions. Although testis is a major tissue to produce androgens in all species, androgens are also produced in ovary and adrenal (interrenal tissue). Testosterone is the most common and famous androgen. It represents a major androgen both in males and females of almost vertebrate species. In addition, testosterone is a precursor for producing significant androgens such as11-ketotestosterone, 5α-dihydrotestosterone, 11-ketodihydrotestosterones and 15α-hydroxytestosterone in a species- or sex-dependent manner for their homeostasis. In this article, we will review the significance and characteristics of these androgens, following a description of the history of testosterone discovery and its synthetic pathways.

Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess.

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone (11KT), by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation.

The opioid peptide leucine enkephalin modulates hypothalamic-hypophysial axis in the cichlid fish Oreochromis mossambicus.

In vertebrates, opioid peptides are thought to be involved in the regulation of reproduction; however, the significance of enkephalins in testicular function remains unclear. We examined the influence of δ-opioid receptor agonist leucine enkephalin (L-ENK) on the hypophysial-testicular axis of the cichlid fish Oreochromis mossambicus. Treatment with a low dose of L-ENK (60 µg) caused a significant increase in the numbers of primary and secondary spermatocytes and early and late spermatids, concomitant with intense immunolabelling of testicular androgen receptors, but did not significantly alter serum luteinizing hormone (LH) and 11-ketotestosterone (11-KT) levels compared to those of controls. Nevertheless, treatment with a high dose of L-ENK (200 µg) caused a significant reduction in the numbers of secondary spermatocytes as well as late spermatids associated with marginal immunolabelling of androgen receptors and significantly lower concentrations of serum 11-KT and LH compared to controls. In addition, the serum cortisol level was not affected in low-dose L-ENK-treated fish, but its level was significantly increased in the high-dose L-ENK-treated group. Together, these findings indicate that a low dose of L-ENK stimulates the germ cells at the meiosis stage and promotes further stages of spermatogenesis, whereas a high concentration of L-ENK inhibits spermatogenesis at the advanced stages. This effect appears to be mediated through the suppression of testicular steroidogenesis and the reduction of LH release in the pituitary gland of tilapia. The findings also suggest that elevated L-ENK levels in teleosts may exert their inhibitory influence on the hypophysial-testicular axis via glucocorticoids.

Letrozole induced a polycystic ovary syndrome model in zebrafish by interfering with the hypothalamic-pituitary-gonadal axis.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of childbearing age, with an incidence of 5-10%. This study compared the traits of zebrafish with three diagnostic criteria for human PCOS, and the diagnostic criteria for zebrafish PCOS were proposed: decreased fecundity, elevated testosterone (T) or 11-ketotestosterone (11-KT) levels and increased cortical-alveolar oocyte (CO) ratio, enhancing the zebrafish PCOS model's accuracy. According to the mammalian PCOS classification, the type of zebrafsh PCOS is divided into four phenotypes (A, B, C and D), but the four phenotypes of zebrafish PCOS are not fully covered in the existing studies (A and D). In this study, we successfully induced phenotype B zebrafish PCOS model using the aromatase inhibitor, letrozole (LET). That is, wild-type female zebrafish were exposed to 1000 µg/L LET for 30 days. Reproductive tests showed decreased fecundity in female zebrafish exposed to LET (Control: 132.63, 146.00, 173.00; LET: 29.20, 90.00, 82.71). Hormone analysis showed that female zebrafish exposed to LET had significantly lower 17ß-estradiol/testosterone (E2/T) ratios, indicating elevated T levels. Meanwhile, levels of 11-KT in the ovaries exposed to LET were significantly up-regulated (Control: 0.0076 pg/µg; LET: 0.0138 pg/µg). Pathological sections of the ovary showed fewer CO in the LET-exposed group (Control: 16.27%; LET: 8.38%). In summary, the zebrafish PCOS model summarized and studied in this study provide a reliable and economical tool for the screening of therapeutic drugs, as well as for the etiology research and treatment strategies of PCOS.

Employing 11-Ketotestosterone as a Target Analyte for Adrenosterone (11OXO) Administration in Doping Controls.

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11ß-hydroxy-androsterone and 11ß-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.

Investigating the biosynthesis and metabolism of 11ß-hydroxyandrostenedione.

The "rediscovery" 11ß-hydroxyandrostenedione (11OHA4) placed the spotlight on this unique adrenal-derived hormone with researchers and clinicians once again focusing on the steroid's presence in endocrine pathology. Little was known about the steroid other than its chemical characterisation and that a mitochondrial cytochrome P450 enzyme catalysed the 11ß-hydroxylation of 11OHA4. The fact that neither the biosynthesis nor metabolism of 11OHA4 had been fully characterised presented an ideal opportunity to investigate the metabolic pathways. In addition, methodologies and analytical tools have improved vastly since 11OHA4 was first identified in the 1950s. Cell models, recombinant DNA technology and steroid quantification using liquid chromatography mass spectrometry have greatly facilitated investigations in the field of steroidogenesis. Evident from the structure is that 11OHA4 can be metabolised by hydroxysteroid dehydrogenases and reductases acting on the C4/C5 double bond and on functional moieties at specific carbons on the cyclopentane-perhydro-phenanthrene backbone of the steroid. In this chapter, the biosynthesis and metabolism of 11OHA4 is followed using two strategies that complement each another; (i) human cell models either transiently transfected with recombinant DNA or expressing endogenous steroidogenic enzymes and (ii) steroid identification and quantification using high resolution mass spectrometry. These methodologies have proven invaluable in the determination of 11OHA4's metabolic route. Both strategies are presented with the focus on the accurate identification and quantification of steroids using UHPLC-MS/MS and UPC2-MS/MS. The protocols described in this chapter lay a sound foundation which can aid the researcher and be adapted and implement in future studies.

Reproductive toxicity induced by lead exposure: Effects on gametogenesis and sex steroid signaling in teleost fish.

Lead (Pb) is an emerging contaminant widely distributed in aquatic environments, which has serious effects on human and animal health. In this study, we determined whether Pb exposure affects gametogenesis, sex steroids, estrogen (ERα and ERß), and androgen (AR) receptors. Adult specimens of Astyanax bimaculatus were exposed in duplicate to 15, 50, and 100 µg/L of lead acetate, whereas the control group was not exposed. After 28 days of exposure, fish were euthanized and samples of the gonads, liver, and blood were collected for analysis. The results indicated a reduction in the gonadosomatic index as well as the diameters of the vitellogenic follicles and seminiferous tubules in the exposed groups. Morphometry of gametogenesis revealed inhibition of the secondary oocyte growth and a reduction in the number of spermatozoa in the 50 and 100 µg/L Pb-treated groups. In females, plasma 17ß-estradiol (E2) increased following 15 and 50 µg/L Pb treatment, whereas males exhibited an increase in E2 and 11-ketotestosterone following treatment with 15 and 100 µg/L Pb, respectively. Vitellogenin was significantly reduced in females exposed to 100 µg/L Pb, but metallothionein levels were unchanged. ERα, ERß, and AR were immunolocalized in the somatic and germ cells, with increased ovarian expression of ERα and Erß in the 100 µg/L Pb-treated group, but no significant difference in AR among the groups. In males, only ERα increased in the 100 µg/L Pb-treated group. These results indicate that Pb exposure impairs gametogenesis, disrupts estrogen receptor signaling, and affects the expression of major reproductive biomarkers in A. bimaculatus.

The serum steroid signature of PCOS hints at the involvement of novel pathways for excess androgen biosynthesis.

CONTEXT: Polycystic ovary syndrome (PCOS) is defined by androgen excess and ovarian dysfunction in the absence of a specific physiological diagnosis. The best clinical marker of androgen excess is hirsutism, while the best biochemical parameter is still a matter of debate. Current consensus guidelines recommend, among other hormones, serum free testosterone as an important serum parameter to measure androgen excess. Recently, however, novel active androgens and androgen metabolic pathways have been discovered. OBJECTIVE: To assess the contribution of novel androgens and related steroid biosynthetic pathways to the serum steroid pool in PCOS women in comparison to healthy controls. DESIGN: This is a case control study, wherein PCOS was diagnosed according to the AE-PCOS 2009 criteria. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. SETTING: Yeditepe University and associated clinics in Istanbul, Turkey, together with Bern University Hospital Inselspital, Bern, Switzerland. PARTICIPANTS: 42 PCOS women and 42 matched, healthy control women. MAIN OUTCOME MEASURES: Assessment of 34 steroids compartmentalized in four androgen related pathways: the classic androgen pathway, the backdoor pathway, the C11-oxy backdoor pathway, and the C11-oxy (11ß-hydroxyandrostenedione) pathway. RESULTS: Metabolites of all four pathways were identified in healthy and PCOS women. Highest concentrations were found for progesterone in controls and androstenedione in PCOS. Lowest levels were found for 11-ketotestosterone in controls compared to PCOS, and for 20α-hydroxyprogesterone in PCOS compared to controls. PCOS also had higher serum testosterone levels compared to the controls. PCOS women had overall higher levels of steroid metabolites of all four androgen pathways compared to healthy controls. CONCLUSIONS: Novel alternative pathways contribute to the androgen production in healthy and PCOS women. Hyperandrogenism in PCOS is characterized by an overall increase of serum androgens in the classic, backdoor and C11-oxy pathways. While monogenetic disorders of steroid biosynthesis can be recognized by a specific pattern in the steroid profile, no diagnostic pattern or classifier was found in the serum for PCOS.

The clinical and biochemical significance of 11-oxygenated androgens in human health and disease.

For many decades, the prevailing paradigm in endocrinology was that testosterone and 5α-dihydrotestosterone are the only potent androgens in the context of human physiology. The more recent identification of adrenal derived 11-oxygenated androgens and particularly 11-ketotestosterone have challenged these established norms, prompting a revaluation of the androgen pool, particularly in women. Since being recognized as bone fide androgens in humans, numerous studies have focused their attention on understanding the role of 11-oxygenated androgens in human health and disease and have implicated them as role players in conditions such as castration resistant prostate cancer, congenital adrenal hyperplasia, polycystic ovary syndrome, Cushing's syndrome, and premature adrenarche. This review therefore provides an overview of our current knowledge on the biosynthesis and activity of 11-oxygenated androgens with a focus on their role in disease states. We also highlight important analytical considerations for measuring this unique class of steroid hormone.

Adrenal gland involvement in 11-ketotestosterone production analyzed using LC-MS/MS.

Introduction: 11-ketotestosterone (11KT), which is derived by the bioconversion of testosterone via 11ß-hydroxytestosterone (11OHT), is a potent agonist of the human androgen receptor. The adrenal gland is considered an important organ in 11KT production because CYP11B1, which catalyzes testosterone to 11OHT, is expressed in the adrenal glands. The present study aimed to demonstrate adrenal gland involvement in 11KT production in prepubertal children, a topic which has not yet been addressed by any previous studies. Methods: Three, retrospective, observational studies were performed. Study 1 enrolled patients aged 8 months to 7 years with severe Kawasaki disease (KD) who were treated with mPSL pulse. Studies 2 and 3 included patients who had received a corticotropin-releasing hormone (CRH) stimulation test and adrenocorticotropic hormone (ACTH) stimulation test, respectively. Samples were collected before and after treatment or drug administration, and serum 11KT, 11OHT, and other 11-oxygenated androgens were measured by LC-MS/MS. Steroid hormone values before and after medication were analyzed using the Wilcoxon signed rank test. Results: Studies 1, 2, and 3 included twenty patients with severe KD, eight patients with a CRH stimulation test, and eight patients with an ACTH stimulation test, respectively. Study 1 demonstrated that the median (IQR) 11KT level was significantly higher before, than after, mPSL pulse (0.39 (0.28-0.47) nmol/L versus 0.064 (0.012-0.075) nmol/L; P < 0.001). Studies 2 and 3 indicated no significant difference in the median 11KT value before and after the CRH or ACTH stimulation test while the 11OHT value was significantly higher after the test. Conclusion: In conclusion, the mediation of 11KT production by ACTH demonstrated the importance of the adrenal glands in the synthesis of this androgen in prepubertal children.

Congenital adrenal hyperplasia: New biomarkers and adult treatments.

Congenital adrenal hyperplasia (CAH) is a genetic disease caused by an enzyme deficiency interrupting adrenal steroidogenesis. It most frequently involves 21-hydroxylase, which induces adrenal insufficiency with hyperandrogenism. Restoring hormonal balance is difficult with glucocorticoids, which are the gold-standard treatment. Strict normalization of conventional biomarkers (17-hydroxyprogesterone and delta-4 androstenedione) is often obtained at the cost of iatrogenic hypercortisolism. Optimizing the management of these patients first involves using more specific biomarkers of adrenal steroidogenesis in difficult situations, and secondly using therapeutics targeting the induced hypothalamic-pituitary-adrenal axis disorder. 11-oxygenated androgens are candidates for biochemical monitoring of Congenital adrenal hyperplasia (CAH), in particular 11-ketotestosterone. Numerous new therapeutic agents are currently being explored, the prime goal being to reduce glucocorticoid exposure, as no strategy can fully replace it at present. They can be divided into 3 categories. The first includes "more physiological" hydrocortisone administration (modified-release hydrocortisone and continuous subcutaneous infusion of hydrocortisone hemisuccinate); the second includes corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) receptor antagonists and anti-ACTH antibodies; and the third includes steroidogenesis inhibitors. Finally, experiments on gene and cell therapies suggest the possibility of lasting remission or even cure in the future.

Development of mass spectrometry-based methods for the detection of 11-ketotestosterone and 11-ketodihydrotestosterone.

The anabolic properties of 11-hydroxyandrostenedione (OHA4) and its physiologically active metabolites 11-ketotestosterone (KT) and 11-ketodihydrotestosterone (KDHT) have been discussed in several recent publications. Especially KT has become readily available via internet-based providers. No doping control methods for the detection of KT or KDHT exist, neither on the initial testing procedure level nor as confirmatory assay. Probing for the misuse of adrenosterone, the prohormone of OHA4, has already been addressed, and the suggested marker for its misuse was mainly the urinary concentration of 11-hydroxyandrosterone (OHA). In addition, for confirmation purposes, the carbon isotope ratios (CIR) were taken into consideration. The urinary concentration of OHA is highly variable, and the endogenous dilution after exogenous administration may therefore be considerable; hence, described approaches resulted in short detection times. In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry-based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration.

Androgen and prolactin manipulation do not induce changes in immunocompetence measures in a fish with male parental care.

Prolactin and 11-ketotestosterone (11-KT) are important reproductive hormones in fishes, which may also influence immunocompetence. The immunocompetence handicap hypothesis states that higher androgen concentrations that support secondary sex traits are traded off against a decrease in immune system function. To test the relationships between these hormones and immunocompetence, we experimentally manipulated 11-ketotestosterone and prolactin in the freshwater fish, bluegill (Lepomis macrochirus) during parental care using implants that contained either 11-KT, prolactin, or an inert control. We vaccinated individuals to stimulate the acquired immune response, then measured immunocompetence as the number of granulocytes, lymphocytes and monocytes, and the expression of interleukin 8 in each sample. We did not observe any significant differences in the immune measures among the hormone treatments. Our results indicate that in bluegill, there is no trade-off between androgens or prolactin and immunocompetence.

Ultra-performance convergence chromatography tandem mass spectrometry analysis of adrenal and gonadal steroid hormones in southern white rhinoceros(Ceratotherium simum simum) faeces and serum.

Steroid hormone analysis is routinely undertaken in the assessment of stress response and reproductive function in the management of both captive and free-ranging wildlife species. Faecal samples have become the preferred sample type for analysis as collection is non-invasive and easily assessable. These investigations are generally aimed at aiding successful translocations, enhanced survival outcomes in captivity and improvement of reproductive rate. Immunoassays are the most common approach in the analysis of hormones, particularly in the case of the southern white rhinoceros (Ceratotherium simum simum). Non-specificity, attributed to structural similarity of steroid metabolites impedes accurate evaluations which can be eliminated by chromatographic techniques which are more specific, selective and provide comprehensive analyses. This study developed and validated three methods using ultra-performance convergence chromatography tandem mass spectrometry for the assessment of classical androgens, progestogens and adrenal steroids, as well as the C11-oxy androgens and C11-oxy progestogens in serum and faeces from white rhinoceros. The limit of detection and quantification were determined for each steroid, parameters such as accuracy (<19.8 % RSD) and precision (<20.2 % RSD) were established with recovery, matrix effect, and process efficiency within acceptable limits. Subsequent analysis of serum and faecal samples from five white rhinoceros identified novel steroids for the first time in this species. In addition to the classical adrenal steroids, the following C11-oxy steroids were detected in faecal samples: 11α-hydroxydihydroprogesterone (168 ng/g), 11α-hydroxyprogesterone (125.9 ng/g), 11ß-hydroxyprogesterone (210.2 ng/g) and 11-ketoandrostenedione (3.3-19.6 ng/g) with 11-deoxycortisol being the major glucocorticoid (24.2-67.3 ng/g) together with 21-deoxycortisone (40.7 ng/g) and deoxycorticosterone (7.6-14.6 ng/g). In serum samples 11ß-hydroxyandrostenedione (0.35-2.34 ng/mL) and 11ß-hydroxytestosterone (0.18-1.62 ng/mL) were the predominant androgens with cortisol (5.8-20.5 ng/mL), the predominant glucocorticoid, while corticosterone, 18-hydroxycorticosterone and aldosterone were also detected. These methods can be applied independently to assess either androgens, progestogens, or adrenal steroid panels or in combination to assess the cohort of gonadal and adrenal steroids in faeces and/or serum, in southern white rhinoceros as well as other wildlife species. Analysis would enable the accurate assessment of reproductive health and stress responses while also distinguishing between stress and distress thus contributing to the conservation of wildlife species.

Case report: 11-ketotestosterone may potentiate advanced bone age as seen in some cases of Wiedemann-Steiner Syndrome.

Context: Wiedemann-Steiner Syndrome (WSS) is a genetic disorder associated with an array of clinical phenotypes, including advanced bone age and short stature. 11-ketotestosterone (11KT) is a member of the group known as 11-oxygenated C19 androgens that are implicated in premature adrenarche. Case description: Case 1: The patient is a 3 year and 11-month-old female diagnosed with WSS due to deletion of KMT2A detected on CGH microarray. At two years and 11 months, imaging revealed an advanced bone age. We obtained an 11KT level on this patient. 11KT in case 1 was elevated at 26.3 ng/dL, while the normal reference range is 7.3-10.9 ng/dL and the reference interval for premature adrenarche is 12.3-22.9 ng/dL, The repeat 11KT at follow up (chronological age 4 years and 6 months) was still elevated at 33.8 ng/dL Case 2: A second child with WSS and a 5kb intragenic KMT2A deletion was evaluated at 11 months of age; his 11KT was 4.5 ng/dL. Conclusions: The elevated 11KT may indicate maturational changes related to increasing adrenal gland androgenic activation and may explain the advanced bone age seen in some patients with WSS. To our knowledge, this is the first case report that describes 11KT as a bioactive androgen potentially causing bone age advancement in WSS. Lack of elevation of 11KT in the second child who is an infant suggests increasing androgenic precursors and metabolites related to premature adrenarche may need to be longitudinally followed.

Steroid implants for the induction of vitellogenesis in feminized European silver eels (Anguilla anguilla L.).

Assisted propagation of the European eel will lead to a closed production cycle supplying the aquaculture industry with juvenile glass eels. Females require long-term weekly treatment with pituitary extract (PE), which is stressful and causes abnormalities in oogenesis. We tested the effects of 17α-methyltestosterone (17 MT), as potent androgen activating the androgen receptor, and 17ß-estradiol (E2), as an inducer of vitellogenesis, to shorten the duration of PE treatment.Four groups of feminized eels were subjected to a simulated migration and subsequent injection with implants containing 17 MT (17 MT-group), E2 (E2-group) or 17 MT plus E2 (17 MT + E2-group) to test for synergistic effects, or without any steroids as controls (C-group). The effects of a 2-months treatment were investigated by determining the eye index (EI), hepatosomatic and gonadosomatic index (HSI and GSI, respectively), plasma steroid concentrations by liquid chromatography mass spectrometry (LCMS), gonadal histology, expression of androgen receptors a and b (ara, arb); estrogen receptor 1 (esr1); FSH receptor (fshr); vitellogenin receptor (vtgr) and aromatase (cyp19), and the required number of weekly PE injections to fully mature. For many parameters, both the 17 MT and E2 groups showed an increase vs. controls, with the 17 MT + E2 group showing a synergistic effect, as seen for EI, GSI (3.4 for 17 MT and for E2, 6.6 for 17 MT + E2), oocyte diameter and ara, arb and esr1 expression. Concentrations of almost all focal steroids decreased with simulated migration and steroid treatment. Only eels of the 17 MT-group showed increased expression of cyp19 and of fshr, while fshr expression increased 44-fold in the 17 MT + E2 group, highlighting that co-implantation is most effective in raising fshr mRNA levels. Specific for eels of the E2 groups were vitellogenesis-associated changes such as an increase of HSI, plasma E2, and presence of yolk in the oocytes. Steroid treatments reduced the duration of PE treatment, again synergistically for co-implantation. In conclusion, E2 is necessary to start vitellogenesis, but 17 MT has specific effects on cyp19 and fshr expression. The combination is necessary for synergistic effects and as such, steroid implants could be applied in assisted reproduction protocols for European eel to improve oocyte quality leading to the production of more vital larvae.