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Offspring phenotypes can be affected by maternal testosterone and androstenedione (A4), which are considered a tool of mothers to adjust offspring to a fluctuating environment. Yet testosterone and A4 are very rapidly metabolized by developing avian embryos, suggesting that either the maternal testosterone and A4 have potent organizational effects on the embryos extremely early before being metabolized or it is the metabolites that evoke phenotypic variation in the offspring. One of the metabolites, etiocholanolone, increases substantially during early embryonic development and is a likely candidate for mediating maternal effects as it can promote erythropoiesis. To investigate and compare the effects of testosterone and A4 with the possible effects of etiocholanolone during prenatal embryonic development, we increased their levels in black-headed gull eggs (Larus ridibundus), and used sham-injected eggs as controls. This species usually has 3-egg clutches in which maternal androgen levels increase with the egg-laying sequence. We analysed embryonic heart rate, peri-hatching biometric traits, the ratio of white to red blood cells (W/R ratio) and bursa development. We found that testosterone and A4 treatment increased embryonic heart rate irrespective of egg-laying sequence and decreased bill length and W/R ratio, whereas etiocholanolone did not mimic these effects. Instead, etiocholanolone treatment decreased tarsus length and brain mass. Our finding that etiocholanolone does not mimic the effects induced by testosterone and A4 suggests that the embryonic metabolism of maternal testosterone and A4 can potentially diversify the function of these maternal androgens., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The World Anti-Doping Agency (WADA) has introduced an Athlete Biological Passport (ABP) with a steroidal module, which is intended for the monitoring of longitudinal profiles of an athlete's steroid variables in urine to identify endogenous anabolic androgenic steroids that are administered exogenously. It has been in use since 2014. The prevalence of UGT2B17 gene deletion with relatively low levels of testosterone (T) glucuronide in urine is high in the Asian region. There are cases in which urinary T is below the detection limit in specific urine samples, for example, diluted urine, urine collected from females, or urine collected from UGT2B17 deletion individuals. Additional steroid markers T, 4-androstenedione (A4), and the T/A4 ratio in serum were newly added to the ABP steroidal module by WADA in 2023 to compensate for the urine steroid profile. In this study, populations of blood steroid markers in Asians (n = 510) were investigated and classified according to UGT2B17 polymorphism to confirm the effectiveness of blood steroid markers in monitoring Asian athletes. No significant difference in the T/A4 ratio was observed between the genotypes. Furthermore, an administration study of T enanthate in females (n = 10) who were classified according to UGT2B17 genotypes was performed. The concentration of T and the T/A4 ratio were found to be significantly increased in all post-administration samples until 15 days after administration (p < 0.01). The overall results supported the high effectiveness of subject-based monitoring for serum T and T/A4 ratio for recently identified shortcomings in the detection of T abuse in Asians., (© 2023 John Wiley & Sons Ltd.)

Capillary dried blood spot (DBS) analysis coupled with multi-analyte steroid liquid chromatography mass spectrometry (LCMS) is attractive for field studies, home-based self-sampling as well as clinical trials by eliminating costly and laborious sample processing involving venipuncture and frozen storage/shipping while providing multiple steroid measurements from a single small sample. We investigated steroid measurements in DBS samples stored for four years at room temperature prior to analysis compared with the original venipuncture serum samples. Healthy women (n=12) provided paired DBS and blood samples over two weeks run-in before seven days treatment with daily transdermal T gel (12.5 mg) and after the end of treatment on days 0, 1, 2, 4, 7 and 14. Compliance with treatment and sampling was high and no adverse effects were reported. Testosterone (T), androstenedione (A 4 ), 17 hydroxyprogesterone (17OHP) and progesterone (P 4 ) were measured in extracted DBS samples as whole blood concentrations with and without adjustment for hematocrit. Using the same LCMS methods, DBS T and A 4 measurements had high correlation with minimal bias from prior serum measurements with DBS T displaying the same pattern as serum, with or without hematocrit adjustment. However, serial whole blood measurements of T without hematocrit adjustment provided the best fitting model compared with serum, urine, or hematocrit-adjusted whole blood T measurements. These finding facilitate and simplify DBS methodology for wider field and home-based self-sampling studies of reproductive steroids indicating the need for hematocrit adjustment may be superfluous., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Background: Dried blood spots have recently been approved by the World Anti-Doping Agency as an alternative biological matrix for testing of doping substances. However, their use is limited to the detection of non-threshold compounds without a Minimum Reporting Level due to the numerous issues related to quantitative analyses and the limitation on testing capabilities of a haemolysed matrix., Aim: In this study androstenedione, testosterone and IGF-1 were longitudinally monitored in four different blood matrices to evaluate the potential of liquid capillary blood as an alternative matrix for quantitative determination in doping control analysis., Methodology: The analytical protocols developed to pretreat 20 μL of the blood matrices selected were based: i) for testosterone and androstenedione, on supported liquid extraction for liquid blood matrices, and on ultrasonication in the presence of methanol for dried blood matrices; ii) for IGF-1, proteins precipitation followed by evaporation of the supernatant was used to pretreat both liquid and dried blood matrices. The detection for all the target analytes was performed using liquid chromatography coupled to mass spectrometry. The analytical workflows, once optimized, were fully validated according to the requirements of World Anti-Doping Agency and ISO 17025 standard and used for the analysis of venous (serum) and capillary (liquid plasma and dried whole blood collected using either volumetric or non-volumetric devices) blood samples collected from 7 healthy subjects., Results: The validation results showed satisfactory performance as related to specificity, sensitivity, matrix effects, linearity, accuracy, and precision in all the blood matrices evaluated despite the limited volume of sample used. The analysis of the different blood matrices collected from the subjects showed non-significant differences between the levels of testosterone and androstenedione measured in dried (fixed volume collected) and liquid matrices. An acceptable underestimation (lower than 15 %) was observed in capillary plasma compared to venous serum. The testosterone/androstenedione ratio was similar in all the blood matrices considered (bias lower than 5 %), indicating this parameter was not affected by either the blood matrix or collection device selected. For IGF-1, the levels measured in liquid blood matrices differed significantly (bias higher than 20 %) from those measured in dried whole blood matrices, suggesting haemolyzed blood might represent a challenge for the determination of macromolecules, mainly due to the complexity of the whole blood matrix in comparison to plasma/serum., Novelty: The outcomes of our study suggest that liquid capillary blood might open new avenues to blood microsampling in doping control field. It represents an efficient alternative to overcome the issues related to venous blood and dried blood spot sampling. Furthermore, it also allows greater frequency of blood sampling, with minor discomfort and without needing a phlebotomist, for analyses that can only be performed in blood samples, with an increased probability to detect and report Adverse Analytical Finding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Testosterone biosynthesis from its precursor androstenedione is thought to be exclusively catalysed by the 17β-hydroxysteroid dehydrogenases-HSD17B3 in testes, and AKR1C3 in the ovary, adrenal and peripheral tissues. Here we show for the first time that the glucocorticoid activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) can also catalyse the 17β-reduction of androstenedione to testosterone, using a combination of in vitro enzyme kinetic assays, mathematical modelling, and molecular docking analysis. Furthermore, we show that co-expression of HSD11B1 and AKR1C3 increases testosterone production several-fold compared to the rate observed with AKR1C3 only, and that HSD11B1 is likely to contribute significantly to testosterone production in peripheral tissues., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification., Methods: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued., Results: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively., Conclusions: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Androgens are synthesized from cholesterol through sequential conversions by enzymes in the adrenal glands and gonads. Serum levels of androgens change during the different phases of life and regulate important developmental and maturational processes. Androgen excess or deficiency can therefore present at various ages in various ways., Content: The diagnostic approach for atypical genitalia, premature pubarche, delayed pubertal onset or progression, and hirsutism or virilization, including measurement of androgens (testosterone, androstenedione, 17-OHprogesterone, dehydroepiandrosterone, and dihydrotestosterone) is discussed in the current review. Androgens can be measured in serum, saliva, urine, or dried blood spots. Techniques to measure androgens, including immunoassays and LC-MS, have their own advantages and pitfalls. In addition, pre- and postanalytical issues are important when measuring androgens., Summary: During clinical interpretation of androgen measurements, it is important to take preanalytical circumstances, such as time of blood withdrawal, into account. As immunoassays have major drawbacks, especially in samples from women and neonates, concentrations measured using these assays should be interpreted with care. Reference intervals can only be used in relation to the measurement technique and the standardization of the assay. In the near future, new androgens will probably be added to the current repertoire to further improve the diagnosis and follow-up of androgen excess or deficiency., (© Association for Diagnostics & Laboratory Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17β-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17β-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17β-OH (14α and 14β) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14β) with an average IC 50 of 13.3 μM. In LNCaP cells, 14β was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC 50 of 12.0 μM vs. 59.6 μM, respectively). At low concentrations (0.25-0.5 μM), 14α and 14β were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Raised brain levels of testosterone (Tes), as well as single nucleotide polymorphisms of P-glycoprotein (P-gp) that cause impaired transport function, are associated with increased risk of suicide. Here, we examined whether Tes and its precursors and metabolites are substrates of P-gp, using several in vitro methods. In ATPase assay, increased ATP consumption was observed as the concentrations of Tes, dihydroepiandrosterone (Dhea), androstenedione (Ado), and dihydrotestosterone (Dht), but not androstenediol (Adol), were increased, suggesting that these four androgens are transported by P-gp. Furthermore, Tes and Ado, though not Dhea or Dht, increased the intracellular accumulation of Rhodamine 123 (Rho123), a typical substrate of P-gp, in a P-gp-overexpressing cell line, suggesting that they inhibit Rho123 efflux and thus are substrates or inhibitors of P-gp. A membrane permeability study using P-gp-overexpressing cells in Transwell inserts indicated that the permeability coefficients of both Ado and Tes in the basal-to-apical direction (excretion) are significantly higher than those in the apical-to-basal direction. Moreover, transport of both Ado and Tes was significantly suppressed by verapamil, a typical P-gp inhibitor. These results indicate that Tes and Ado are endogenous substrates of P-gp. These findings provide a physiological basis for understanding previously reported associations of P-gp dysfunction and raised brain levels of Tes with suicidal behavior, and may open up new possibilities for treating patients at risk of suicide., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The role of androgens in shaping "masculine" traits in males is a core focus in behavioral endocrinology, but relatively little is known about an androgenic role in female aggression and social dominance. In mammalian models of female dominance, including the ring-tailed lemur (Lemur catta), links to androgens in adulthood are variable. We studied the development of ring-tailed lemurs to address the behavioral basis and ontogenetic mechanisms of female dominance. We measured behavior and serum androgen concentrations in 24 lemurs (8 males, 16 females) from infancy to early adulthood, and assessed their 'prenatal' androgen milieu using serum samples obtained from their mothers during gestation. Because logistical constraints limited the frequency of infant blood sampling, we accounted for asynchrony between behavioral and postnatal hormone measurements via imputation procedures. Imputation was unnecessary for prenatal hormone measurements. The typical sex difference in androgen concentrations in young lemurs was consistent with adult conspecifics and most other mammals; however, we found no significant sex differences in rough-and-tumble play. Female (but not male) aggression increased beginning at approximately 15 months, coincident with female puberty. In our analyses relating sexually differentiated behavior to androgens, we found no relationship with activational hormones, but several significant relationships with organizational hormones. Notably, associations of prenatal androstenedione and testosterone with behavior were differentiated, both by offspring sex and by type of behavior within offspring sexes. We discuss the importance of considering (1) missing data in behavioral endocrinology research, and (2) organizational androgens other than testosterone in studies of female dominance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prostate cancer is the most common malignancy among men in the Western world. Treatment of this patient population, e.g. by (chemical) castration, is primarily focused on depletion of tumor-stimulating androgens, with testosterone being the major androgenic hormone. After initial therapy, prostate cancer may progress to metastatic castration-resistant prostate cancer. Anti-hormonal drugs abiraterone acetate and enzalutamide are commonly used to treat patients with this disease as both drugs reduce tumor growth and increase time to tumor progression. To evaluate the pharmacodynamic effects of anti-hormonal drugs in this patient population, we developed an LC-MS/MS method for the quantification of testosterone, dihydrotestosterone, androstenedione, cortisol and prednisone in human plasma. The validated assay ranges from 10-10,000 pg/mL for testosterone and androstenedione, 100-10,000 pg/mL for dihydrotestosterone, 50-5000 pg/mL for cortisol and 500-50,000 pg/mL for prednisone. Intra-assay and inter-assay variabilities were within ±15% of the nominal concentrations for quality control (QC) samples at low, medium and high concentrations and within ±20% at the lower limit of quantification (LLOQ), respectively. The applicability of the method was demonstrated in plasma from patients with metastatic castrated-resistant prostate cancer using either abiraterone acetate or enzalutamide., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Androgens modulate maternal ovarian activity, embryo implantation and correct placental development. The objective of this study was to establish reference values for testosterone (T), androstenedione (A 4 ) and dehydroepiandrosterone (DHEA) concentrations in pregnant mares. A total of 30 healthy Spanish Purebred mares with an age range of 9.33 ± 3.31 years, were studied during the 11 months of gestation. T, A 4 and DHEA concentrations were determined using EIA validated specifically for equines. T increased in the 2nd and 3rd month (P < 0.05), showing a plateau between the 4th and 6th month, decreased from the 7th to the 9th month (P < 0.05) and increased in the 10th month (P < 0.05), reaching the maximum value in the last month of pregnancy (P < 0.05). A 4 increased in the 2nd month (P < 0.05), reaching the maximum value in the 3rd month (P < 0.05), decreased in the 4th month, increasing in the 5th and 6th month (P < 0.05). From the 7th month the average values decreased until reaching the minimum at the end of gestation. DHEA progressively increased from the 1st to the 5th month, at which time the maximum mean value was reached (P < 0.05), after a decrease in the 6th and 7th month occurred (P < 0.05), reaching the minimum value in the last month of gestation. T, A 4 and DHEA were positive and significantly correlated (P < 0.05). The androgens analyzed in this study can be used as predictive markers of pregnancy in the mare., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dioxins have been suspected to be potential substances causing endocrine disruptions in humans. We are conducting the research in one of three dioxin exposure areas (hotspots) in Vietnam. We previously reported that the salivary dehydroepiandrosterone (DHEA) level decreased in 3-year-old Vietnamese children and that it was significantly inversely correlated with polychlorinated dibenzodioxin/dibenzofuran levels in their mother's breast milk. In this study, we investigated the influence of exposure to dioxin on steroid hormone biosynthesis in the same children when they reached 5 years of age, focusing on androgens. Thirty-five and 50 mother-child pairs from dioxin hotspot and non-sprayed areas, respectively, participated in this study. Maternal breast milk was donated at 4 to 16 weeks postpartum in 2008 to measure dioxin levels by gas chromatography/high-resolution mass spectrometry. Serum was collected from 5-year-old children in 2013. Seven steroid hormones were measured by liquid chromatography/mass spectrometry. Most dioxin congeners in breast milk were 2- to 10-fold higher in the hotspot than in the non-sprayed area. DHEA and testosterone (T) were significantly lower in the hotspot and showed negative correlations with most dioxin congeners. Similar results were observed for the activities of cytochrome P450-17, 20 lyase (CYP17 lyase), and 17β-hydroxysteroid dehydrogenase (HSD). Conversely, the elevated androstenedione (A-dione) level and 3β-HSD activity in children from the hotspot were positively correlated with dioxin levels. Moreover, a positive correlation was shown between T and 17β-HSD. It is possible that dioxin inhibits 17β-HSD activity, leading to a decrease in the T level. Multiple regression analysis indicated that dioxin had a strong association with the DHEA, A-dione, and T levels. In conclusion, the present study suggests that dioxin is associated with low levels of DHEA and T and inhibition of the activity of steroidogenic enzymes such as CYP17 lyase and 17β-HSD in 5-year-old children., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Recently, measurements of steroids like testosterone, androstenedione, cortisol and cortisone in saliva are more and more applied in diagnostics and scientific studies. This is mainly due to the simple and non-invasive collection of saliva. We aimed to evaluate the optimal way to collect saliva for steroid hormone measurement., Methods: We investigated in twenty volunteers whether there is a difference between steroid hormone concentrations in unstimulated and stimulated saliva collected while chewing, using cotton and synthetic Salivettes®, citric acid or chewing gum. Furthermore, total unstimulated saliva was compared to parotid gland saliva. Testosterone, androstenedione, cortisol and cortisone were measured using Liquid-Chromatography Tandem Mass Spectrometry (LC-MS/MS)., Results: Salivary testosterone, androstenedione and cortisol concentrations were unaffected by stimulation upon mouth and tongue movements, cortisone levels were on average 16% lower. Concentrations of all hormones were lower in parotid gland saliva compared to total unstimulated saliva (on average 51%, 26%, 66% and 49% lower, for testosterone, androstenedione, cortisol and cortisone, respectively). Concentrations of testosterone as well as androstenedione were lower when using synthetic Salivettes® (58% and 41%, respectively) and were higher when using cotton Salivettes® (217% and 46%, respectively). Cortisol levels in saliva were unaffected by using Salivettes®. However, cortisol and testosterone levels were higher in with chewing gum stimulated saliva (16% and 55%, respectively). Cortisone concentrations were lower in all types of stimulations (on average 25%-35%)., Conclusion: The way saliva is collected should be considered when analysing and interpreting salivary hormone concentrations. We advocate unstimulated saliva collection in simple polypropylene tubes for all steroid measurements., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A large body of research links testosterone and cortisol to male-male competition. Yet, little work has explored acute steroid hormone responses to coalitional, physical competition during middle childhood. Here, we investigate testosterone, dehydroepiandrosterone (DHEA), androstenedione, and cortisol release among ethnically Chinese boys in Hong Kong (N = 102), aged 8-11 years, during a soccer match (n = 84) and an intrasquad soccer scrimmage (n = 81), with 63 participants competing in both treatments. The soccer match and intrasquad soccer scrimmage represented out-group and in-group treatments, respectively. Results revealed that testosterone showed no measurable change. DHEA increased during both treatments in the majority of participants and the degree of change had no relation to independent variables (e.g., performance, age, treatment, outcome) or covariate measures (Body Mass Index, Pubertal Development Scale). Most boys experienced androstenedione increases during match play, but no significant differences during the intrasquad soccer scrimmage competitions. The magnitude of change differed significantly between treatments and was positively associated with age. These latter findings suggest boys' androstenedione responses may be sensitive to competitor type (i.e., unknown competitors vs. peers). For most subjects, cortisol significantly increased during match play, decreased during the intrasquad soccer scrimmage, and differed significantly between treatments, suggesting each treatment promoted a different psychological state among competitors. Cortisol/DHEA molar ratio decreased during the intrasquad scrimmage, suggestive of a more relaxed mental state. These data shed new light on potential proximate mechanisms associated with coalitional competition among prepubescent boys, with relevance to adrenarche and life history theory.

Objectives: Levels of steroid hormones such as androgens and cortisol exhibit circadian variation, and their fluctuations are related to the sleep-wake cycle. Currently, the functional role of different stages of sleep in steroid hormone secretion remains unclear. The present study aims to explore the effect of slow-wave sleep (SWS) suppression on morning levels of cortisol and androgens., Methods: Twelve healthy male volunteers participated in two experimental sessions: a session with selective SWS suppression during night sleep and a session with regular night sleep (control). SWS suppression was achieved by stimulation using an acoustic tone. Salivary samples were collected in the morning immediately after awakening and again 40 min later. The samples were analysed by liquid chromatography-tandem mass spectrometry for testosterone, androstenedione (Ad), dehydroepiandrosterone (DHEA), 17α-hydroxyprogesterone (17-OHP), and cortisol., Results: SWS suppression reduced overall SWS duration by 54.2% without significant changes in total sleep time and sleep efficiency. In the session with selective SWS suppression, the average level of morning testosterone was lower than in the control session (p = 0.017). Likewise, 17-OHP was lower in the SWS suppression condition (p = 0.011) whereas the ratio of DHEA/Ad was higher (p = 0.025). There were no significant differences between sessions in cortisol, Ad, or DHEA concentrations., Conclusions: The effect of selective SWS suppression on morning levels of testosterone and 17-OHP points to the importance of SWS for the synthesis and secretion of androgens. These results suggest that chronic sleep problems, which lead to reduced SWS, increase the risk for the development of androgen deficiency in the long term., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cytochrome P450 aromatase (AROM) catalyzes the biosynthesis of estrogen from androgen. Previously crystal structures of human AROM in complex with the substrate androstenedione, and inhibitors exemestane, as well as the newly designed steroidal compounds, have been reported. Here we report the first crystal structure of testosterone complex of human placental AROM. Testosterone binds at the androgen-specific heme distal pocket. The polar and hydrophobic interactions with the surrounding residues resemble the interactions observed for other ligands. The heme proximal region comprises the intermolecular interface in AROM, and also the putative interaction surface of its redox partner cytochrome P450 reductase. Unreported previously, the proximal region is characterized by a large surface cavity, unlike most known P450's. Using five best X-ray data sets from androstenedione and testosterone complexes of AROM, we now unequivocally show the presence of an unexplained ligand electron density inside the proximal cavity. The density is interpreted as ordered five ethylene glycol units of polyethylene glycols used as a solvent for steroids and also in crystallization. Interestingly, polyethylene glycol exhibits weak inhibition of AROM enzyme activity in a time dependent manner. Besides its critical role in the redox partner coupling and electron transfer process, the proximal cavity possibly serves as the interaction site for other molecules that may have regulatory effects on AROM activity. In addition, the new data also reveal a previously unidentified water channel linking the active site to the lipid interface. The channel could be the predicted passage for water molecules involved in catalysis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Salivary androgen testing represents a valuable source of biological information. However, the proper measurement of such low levels is challenging for direct immunoassays, lacking adequate accuracy. In the last few years, many conflicting findings reporting low correlation with the serum counterparts have hampered the clinical application of salivary androgen testing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it possible to overcome previous analytical limits, providing new insights in endocrinology practice., Methods: Salivary testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17OHprogesterone (17OHP) were extracted from 500µL of saliva, separated in 9.5 min LC-gradient and detected by positive electrospray ionization - multiple reaction monitoring. The diurnal variation of salivary and serum androgens was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects., Results: The assay allowed the quantitation of T, A, DHEA and 17OHP down to 3.40, 6.81, 271.0 and 23.7 pmol/L, respectively, with accuracy between 83.0 and 106.1% for all analytes. A parallel diurnal rhythm in saliva and serum was observed for all androgens, with values decreasing from the morning to the evening time points. Salivary androgen levels revealed a high linear correlation with serum counterparts in both sexes (T: R>0.85; A: R>0.90; DHEA: R>0.73 and 17OHP: R>0.89; p<0.0001 for all)., Conclusions: Our LC-MS/MS method allowed a sensitive evaluation of androgen salivary levels and represents an optimal technique to explore the relevance of a comprehensive androgen profile as measured in saliva for the study of androgen secretion modulation and activity in physiologic and pathologic states.

Objective: To identify the androgen ratio that best predicts insulin resistance and metabolic syndrome among women with polycystic ovary syndrome (PCOS)., Methods: Data for 180 women with PCOS and 180 healthy controls were extracted from two previous studies in Iran (conducted during 2008-2010 and 2011-2013). The diagnosis of PCOS was based on the Rotterdam criteria. The serum concentration of different androgens was measured. Receiver operating characteristic curve analysis was used to assess the ability of various androgen ratios to predict insulin resistance and metabolic syndrome., Results: Among women with PCOS, the testosterone-to-androstenedione ratio was the best predictor of insulin resistance (sensitivity 0.83, specificity 0.42) and metabolic syndrome (sensitivity 0.85, specificity 0.70). Among healthy controls, the ratio of free androgen index to testosterone was the best predictor of insulin resistance (sensitivity 0.84, specificity 0.33) and metabolic syndrome (sensitivity 0.91, specificity 0.17)., Conclusion: The prediction of insulin resistance and metabolic syndrome among women with PCOS was best accomplished with the testosterone-to-androstenedione ratio., (© 2017 International Federation of Gynecology and Obstetrics.)

An increasing number of men are being diagnosed with hypogonadism. While many benefit from testosterone supplementation therapy, others who do not meet the criteria for hormone supplementation have turned to dietary adjuncts as a way or gaining improvements in libido, energy, and physical performance. These oral adjunct medications include controlled substances such as androstenedione, androstenediol as well as other "over-the-counter" options like DHEA (dehydroepiandrosterone) and herbal remedies like Tribulus terrestris This review will focus on the use of these adjunct medications in isolation, or in combination with testosterone supplementation therapy as well as the biochemical nature of the supplements, the results of scientific trials as well as the side effects that limit their use. At the end of this review, physicians will have an improved understanding of the popular testosterone adjuncts being used currently as well as the availability of these substances and how they are used., (© The Author(s) 2015.)

Background: The measurement of androgens in many laboratories is often limited to testosterone. To more accurately determine the androgen status in both sexes, the measurement of other androgens such as dihydrotestosterone, the more potent metabolite of testosterone, and androstenendione and dehydroepiandrosterone, the most abundant circulating androgens in women would be informative. We report a combined liquid chromatography tandem mass spectrometry method for the measurement of these androgens., Methods: Internal standards in methanol (10 µL) were added to 100 µL serum followed by the addition of zinc sulphate (100 µL). After mixing, 100 µL of acetonitrile was added and was further mixed. The samples were centrifuged and the steroids extracted using an automated online solid phase extraction on a C18 cartridge by a Waters Acquity with online sample manager coupled to a TQS mass spectrometer., Results: Separation of the androgens was achieved by liquid chromatography. The run time was 6.5 min per sample. The lower limit of quantitation was 0.1 nmol/L for testosterone, androstenedione and dihydrotestosterone and 1 nmol/L for dehydroepiandrosterone. The coefficient of variation of the assay in serum for testosterone was <6%, androstenedione <8% and dihydrotestosterone and dehydroepiandrosterone <10%., Discussion: We have developed a rapid assay for the liquid chromatography tandem mass spectrometry measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone in a routine clinical laboratory. The assay requires a small volume of serum, and all analytes are measured simultaneously. The assay is rapid and simple to execute offering the potential for routine clinical application., (© The Author(s) 2016.)

Background: Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC-MS/MS methods is available. We compared eight routine unpublished LC-MS/MS methods for the simultaneous measurement of testosterone and androstenedione., Methods: Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC-MS/MS methods. We performed Passing-Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated., Results: The concentrations ranged from 0.05-1.26 nmol/L, 6.15-24.44 nmol/L and 0.15-4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7-16.0%, 0.9-5.2% and 1.2-9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90-1.25, 0.87-1.24 and 0.94-1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods., Conclusion: Although most routine LC-MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved., (Copyright © 2016. Published by Elsevier B.V.)

Chronic hepatitis C (HCV) infection is a serious problem all over the world and has a special importance in Egypt, where the prevalence of infection is 14.7% of population. In males, HCV is associated with sexual dysfunction and changes in the semen parameters. This study aimed at estimation of a panel of the most important related hormones in the serum of patients and illustration of their correlation to the routine laboratory investigations. The four studied hormones showed alteration in the patients in comparison with the controls. While androstenedione, prolactin and testosterone were significantly increased in patients, dehydroepiandrosterone sulphate was decreased. These changes in the hormones were not related to the liver functions, pathological grade or even viral load. We hypothesised a model of how HCV can induce these hormonal changes and recommended to add these hormones to the follow-up panel of male patients with HCV., (© 2015 Blackwell Verlag GmbH.)

Avian egg yolks contain various amounts of maternally derived androgens that can modify offspring phenotype and adjust their development to the post-hatching environment. Seemingly adaptive variation in yolk androgen levels with respect to breeding density conditions or male attractiveness has been found in numerous studies. One important consideration that has been overlooked in previous research is the likely non-linear nature of hormone effects. To examine possible complex dose-response effects of maternal androgens on chick development, we experimentally administered three different androgen doses of the naturally occurring mixture of yolk testosterone and androstenedione to spotless starling eggs (Sturnus unicolor). We found that yolk androgens induce a non-linear dose-response pattern in several traits. Androgens had a stimulatory effect on hatchling body mass and nestling skeletal growth, but maximum values were found at intermediate doses, whereas our highest dose resulted in a decrease. However, the opposite U-shaped effect was found on nestling body mass. We also detected linear negative and positive effects on embryonic development period and nestling gape width, respectively. Our results suggest differential tissue responsiveness to yolk androgens, which may result in compromises in maternal allocation to produce adapted phenotypes. Because of the non-linear dose-response pattern, future investigations should carefully consider a wide range of concentrations, as the balance of costs and benefits may strongly differ depending on concentration., (© 2015. Published by The Company of Biologists Ltd.)

The adrenal and gonadal androgens, testosterone, androstenedione and dehydroepiandrosterone (DHEA) play an important role in sexual development as well as in other processes. We developed a method for simultaneous quantitative analysis of serum and plasma testosterone, androstenedione and DHEA levels using Isotope-Dilution Liquid-Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS). Samples underwent liquid-liquid extraction and were analyzed on an Acquity 2D-UPLC-System and a Xevo TQ-S tandem mass spectrometer (Waters). The intra-assay and inter-assay coefficients of variation were <4.0%, <6.3% and <7.0% and <6.0%, <8.1% and <7.7% for testosterone, androstenedione and DHEA, respectively. Inter-assay CVs at the lower limit were 10.6%, 16.9% and 9.0% for testosterone (0.10nmol/L), androstenedione (0.10nmol/L) and DHEA (1.0nmol/L), respectively. Recoveries of spiked analytes were 93-107%. The present testosterone method compared well (y=1.00x-0.04; r=0.998) to a published ID-LC-MS/MS method for testosterone in our lab. The latter method being concordant with a published reference method (Bui et al., 2013). The present method compared well to a published ID-LC-MS/MS method (Kushnir et al., 2010) (y=1.06x-0.06; r=0.996 for testosterone; y=1.04x-0.04; r=0.995 for androstenedione and y=1.03x+0.01; r=0.991 for DHEA). In conclusion, we developed a sensitive and accurate ID-LC-MS/MS method to simultaneously measure serum testosterone, androstenedione and DHEA in serum and plasma., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Context Several challenges still exist to adopt the anti-müllerian hormone (AMH) as a marker of polycystic ovary morphology, as included in the recently updated international guideline. Although different evaluations of age- and assay-specific reference ranges have been published in the past few years, these studies have mainly been conducted in normo-ovulatory or infertile women. Objective To develop an age-specific percentile distribution of AMH in patients with polycystic ovary syndrome (PCOS) measured by 3 different assays. Design Retrospective cross-sectional study. Patients A total of 2725 women aged 20 to 40 years with PCOS diagnosis were included. Interventions Serum AMH measurement by the Gen II (Beckman Coulter), the picoAMH (Ansh Labs), and the Elecsys (Roche) assays. Main outcome measures Age-specific percentile curves for all the assays and correlations between AMH, clinical, hormonal, and ultrasound characteristics. Results Age-related nomograms for the 5th, 10th, 25th, 50th, 75th, 90th, and 95th percentiles of AMH were calculated using the Lambda-Mu-Sigma method for all the assays. AMH levels were significantly different between PCOS phenotypes. AMH levels were positively correlated to LH, LH/FSH ratio, testosterone, androstenedione, free androgen index, mean follicular number, and mean ovarian volume. Conclusion To our knowledge, this is the first study reporting age-specific percentile nomograms of serum AMH levels measured by the Gen II, the picoAMH, and the Elecsys assays in a large population of women with PCOS. These findings may help to interpret AMH levels in patients with PCOS and facilitate the use of AMH as a diagnostic tool across age ranges. [ABSTRACT FROM AUTHOR]

Background: Few studies are available on the relationship of androstenedione with inflammation and obesity and the effect of androstenedione and inflammation on the association between testosterone and obesity. This study intended to examine the mediation effect of inflammatory markers on the association of testosterone with obesity and the moderation effect of androstenedione on the association of testosterone with inflammation and obesity in Chinese rural men. Materials and methods: This cross‐sectional research enrolled 2536 male rural inhabitants from the Henan Rural Cohort study. The serum concentrations of testosterone and androstenedione were determined by liquid chromatography–tandem mass spectrometry. Linear and logistic regression were used to examine the relationships between testosterone, inflammatory markers, and obesity. Mediation and moderation analyses were carried out to evaluate the potential effects of inflammatory markers on the relationship between testosterone and obesity, as well as androstenedione on the relationships of testosterone with inflammation and obesity. Results: After adjusting for confounding factors, the results showed that testosterone and androstenedione were negatively related to obesity, and inflammatory markers were positively associated with obesity. Besides, testosterone and androstenedione were negatively associated with inflammatory markers. Mediation analysis showed that white blood cell, neutrophil, monocyte, and high‐sensitivity C‐reactive protein had mediating effects on the association between testosterone and obesity. The most vital mediator was high‐sensitivity C‐reactive protein, and its proportion of the effect was 11.02% (defined by waist circumference), 11.15% (defined by waist‐to‐hip ratio), 12.92% (defined by waist‐to‐height ratio), and full mediating effect (defined by body mass index). Moreover, androstenedione played negative moderation effects on the associations of testosterone with inflammation and obesity. Conclusion: Inflammatory markers and androstenedione were first found to have modifying effects on the association of testosterone with obesity. Higher levels of testosterone and androstenedione could reduce the inflammation level and risk of obesity, indicating their potential roles in the prevention and treatment of chronic diseases. [ABSTRACT FROM AUTHOR]

Urine marking, aggression, and other behavioral concerns are common reasons for cat owners to seek veterinary care. Empiric treatment for lower urinary tract disease or primary behavior disorders are commonly pursued, especially in those cases with normal routine laboratory evaluations. Herein, we report the clinicopathologic findings in eight sexually altered cats that were diagnosed with androgen-secreting adrenocortical tumors. Nearly all cats (n = 7) initially were evaluated for inappropriate urination and pungent urine, with additional behavioral concerns including aggression (n = 3) and excess vocalization (n = 4) commonly reported. Penile barbs (n = 5) were identified in all five male cats, and an enlarged clitoris was observed in one female cat. Testing of serum androgen concentrations revealed abnormally high androstenedione (n = 1) or testosterone (n = 7) concentrations. In the five cases with available adrenal tissue, histopathologic evaluation identified either an adrenocortical adenoma (n = 3) or adrenocortical carcinoma (n = 2). Hormonal abnormalities resolved and clinical signs improved in the four cats that underwent surgical adrenalectomy, with each of these cats surviving >1 year. However, clinical signs were minimally impacted with medical treatments, including one cat in which trilostane treatment failed to improve clinical signs or testosterone concentrations. This collection of cases underscores the importance of a detailed physical examination as well as the consideration of endocrine disturbances in cats undergoing evaluation for inappropriate urination or aggression. Furthermore, this report adds to the growing body of evidence that sex-hormone secreting adrenal tumors in cats may be an under-recognized syndrome.

BackgroundMaternal hormonal risk factors for autism spectrum disorder (ASD) in offspring could intersect genetic and environmental risk factors.ObjectivesThis analysis explored ASD risk in association with maternal testosterone, androstenedione, and dehydroepiandrosterone (DHEA) measured in first, second, and third trimesters of pregnancy.MethodsMARBLES is a prospective pregnancy cohort study based at the MIND Institute in Northern California that enrolls mothers who have at least one child previously diagnosed with ASD and are expecting, or planning to have another child. At 36 months the younger sibling is clinically classified as having ASD, or as non-typically developing (Non-TD), or typically developing (TD). Maternal androgens during pregnancy were measured in serum samples from 196 mothers. Multivariable logistic regression models estimated risk of ASD and Non-TD in offspring compared to TD, in relation to the log-transformed maternal androgen concentrations, at each trimester.ResultsNon-significant associations were observed, and borderline significant associations were only observed in some stratified unadjusted models. Second trimester maternal testosterone was non-significantly associated with ASD in female offspring, although not after adjustment, aRR 1.54 (95% CI 0.71, 3.33), and second trimester maternal DHEA was non-significantly associated with non-TD in male offspring, again not after adjustment, aRR 0.50 (95% CI 0.21, 1.21). Secondary analysis suggested that third trimester androgen concentrations in mothers with male offspring had significant or near significant associations with their child's Social Responsiveness Scale score.ConclusionNo significant associations were found between maternal androgen concentrations and risk of ASD or Non-TD in the child.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences. [ABSTRACT FROM AUTHOR]

Context: Salivary androgens represent non-invasive biomarkers of puberty that may have utility in clinical and population studies. Objective: To understand normal age-related variation in salivary sex steroids and demonstrate their correlation to pubertal development in young adolescents. Design, setting and participants: School-based cohort study of 1495 adolescents at two time points for collecting saliva samples approximately 2 years apart. Outcome measures: The saliva samples were analyzed for five androgens (testostero ne, androstenedione (A4), 17-hydroxyprogesterone, 11-ketotestosterone and 11β-hydroxyandrostenedione) using liquid chromatography-mass spectrometry; in addition, salivary dehydroepiandrosterone (DHEA) and oestradiol (OE2) were analysed by ELISA. The pubertal staging was self-reported using the Pubertal Development Scale (PDS). Results: In 1236 saliva samples from 903 boys aged between 11 and 16 years, salivary androgens except DHEA exhibited an increasing trend with an advancing age (ANOVA, P < 0.001), with salivary testosterone and A4 concentration showing the strongest correlation (r = 0.55, P < 0.001 and r = 0.48, P < 0.001, respectively). In a subgroup analysis of 155 and 63 saliva samples in boys and girls, respectively, morning salivary testosterone concentrations showed the highest correlation with composite PDS scores and voice-breaking category from PDS self-report in boys (r = 0.75, r = 0.67, respectively). In girls, salivary DHEA and OE2 had negligible correlations with age or composite PDS scores. Conclusion: In boys aged 11–16 years, an increase in salivary testosterone and A4 is associated with self-reported pubertal progress and represents valid non-invasive biomarkers of puberty in boys.

Background: Androgen deprivation therapy (ADT) is a mainstay of prostate cancer in both adjuvant and palliative settings. Since androgens are crucial for functional status and psychological functions, we evaluated whether blood testosterone, androstenedione, or DHEA concentrations were associated with functional status and psychological alterations in patients with localised (PCa) or metastatic prostate cancer (mPCa) receiving ADT with analogues of luteinising hormone-releasing hormone (LHRH). Methods: The five Fried criteria were considered to identify frailty syndrome. In addition, complementary evaluations were carried out to measure other variables of interest. Sleep quality was assessed using the Athens Insomnia Scale, cognitive functions were assessed using the Mini-Mental State Examination, and symptoms of depression were measured using the Yesavage Geriatric Depression Scale. Logistic regression analysis was performed to determine if the androgens level could be related to frailty syndrome, sleep impairment, depressive symptoms, and cognitive functions. Results: The results of the multivariate analyses show that high concentrations of androstenedione were significantly associated with frailty syndrome in both groups (p = 0.018; odds ratio = 4.66, 95% confidence interval [1.30–16.6]). There were significant relationships between frailty syndrome and the systemic concentration of androstenedione (p = 0.01), but not the concentration of testosterone (p = 0.60) or DHEA (p = 0.42). In addition, the results of the non-parametric tests show significant results between a decreased gait speed in the two groups (metastatic and localised) and the concentration of androstenedione (p = 0.015). High androstenedione levels were associated with a slow walking speed in the mCaP group (p = 0.016), while high testosterone levels were associated with a better walking speed in the localised CaP group (p = 0.03). For the concentration of androstenedione in plasma, the area under the curve was 0.72, with a 95% CI of 0.55–0.88 with acceptable values, and with a cut-off point of 4.51 pg/mL, a sensitivity of 82.9%, and specificity of 53.8%. No relationships between the concentration of androgens in plasma and sleep quality, cognitive functions, or symptoms of depression suggest that the changes were specific to frailty syndrome. Conclusions: Further research into the role of androstenedione should be evaluated in follow-up studies in order to recommend its use as a suitable biomarker of frailty syndrome in prostate cancer patients. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study is to analyze correlation between vitamin D level and body mass index (BMI) in polycystic ovary syndrome (PCOS) women. Material and methods: The study group consisted of 311 patients with PCOS. Patients were categorized according to four phenotypes. All of the women participating in the study had their blood tested in the appropriate phase of the menstrual cycle and after proper preparation for the tests. The ultrasound examination and anthropometric measurements were performed. Results: Vitamin D concentration was assessed in all study subgroups. The majority of patients had vitamin D deficiency or insufficient level. Variables included in the study, such as level of vitamin D, low density lipoprotein (LDL), sex hormone binding globulin (SHBG), testosterone, androstenedione, Anti-Müllerian Hormone (AMH) and BMI were correlated. A negative correlation was observed with the the level of SHBG, vitamin D and AMH. Subsequently, positive correlations were shown with testosterone, LDL and free testosterone level. An analysis of the correlation between BMI and vitamin D concentration showed that in phenotype I of PCOS this correlation was statistically significant and in the remaining PCOS phenotypes the correlation was close to statistical significance. Conclusions: Most PCOS patients have a deficiency or insufficient level of vitamin D. Women with PCOS have shown a significant negative correlation between BMI and SHBG serum level and between BMI and AMH level. A positive correlation exists between BMI and total and free testosterone and LDL. There is a negative correlation between BMI and vitamin D level in PCOS patients and in phenotype I this correlation was statistically significant. [ABSTRACT FROM AUTHOR]

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17β-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17β-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17β-OH (14α and 14β) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14β) with an average IC50 of 13.3 μM. In LNCaP cells, 14β was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 μM vs. 59.6 μM, respectively). At low concentrations (0.25-0.5 μM), 14α and 14β were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

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Context Ovarian and adrenal steroidogenesis underlie endocrine-metabolic dysfunction in polycystic ovary syndrome (PCOS). Adipocytes express aldo-keto reductase 1C3 and type 1 11β-hydroxysteroid dehydrogenase, which modulate peripheral androgen and cortisol production. Objectives To compare serum adrenal steroids, including 11-oxygenated androgens (11-oxyandrogens), cortisol, and cortisone between normal-weight women with PCOS and body mass index- and age-matched ovulatory women with normal-androgenic profiles (controls), and assess whether adrenal steroids associate with abdominal adipose deposition. Design Prospective, cross-sectional, cohort study. Setting Academic medical center. Patients Twenty normal-weight women with PCOS and 20 body mass index-/age-matched controls. Intervention(s) Blood sampling, IV glucose tolerance testing, and total-body dual-energy x-ray absorptiometry. Main Outcome Measure(s) Clinical characteristics, hormonal concentrations, and body fat distribution. Results Women with PCOS had higher serum total/free testosterone (T) and androstenedione (A4) levels and a greater android/gynoid fat mass than controls (androgens P <.001; android/gynoid fat mass ratio, P =.026). Serum total/free T and A4 levels correlated positively with android/gynoid fat mass ratio in all women combined (P <.025, all values). Serum 11ß-hydroxyA4, 11-ketoA4, 11ß-hydroxyT, 11-ketoT, cortisol, and cortisone levels were comparable between female types and unrelated to body fat distribution. Serum 11-oxyandrogens correlated negatively with % total body fat, but lost significance adjusting for cortisol. Serum cortisol levels, however, correlated inversely with android fat mass (P =.021), with a trend toward reduced serum cortisol to cortisone ratio in women with PCOS vs controls (P =.075), suggesting diminished 11β-hydroxysteroid dehydrogenase activity. Conclusion Reduced cortisol may protect against preferential abdominal fat mass in normal-weight PCOS women with normal serum 11-oxyandrogens. [ABSTRACT FROM AUTHOR]

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

The article is devoted to the review of scientific publications on gynecological and dermatological aspects of polycystic ovary syndrome (PCOS) in different age periods of women’s life. Analysis of domestic and foreign publications presents that the prevalence of PCOS depends on the age of women and the state of their reproductive function, and is accounted 17% in women 21 to 30 years old and significantly decreased with age. More than half of all cases of endocrine infertility (50-75%) and about 20-22% of the causes of infertile marriage in general are associated with PCOS. The phenotypic heterogeneity of women with PCOS affects the pregnancy outcomes in different ways, which increases the risk of its pathological course, early pregnancy loss, the development of gestational diabetes mellitus, hypertensive disorders, the birth of small and large for gestational age babies, etc. “The golden standard” for hyperandrogenism diagnosis in PCOS patients is the determination of the index of free testosterone and androstenedione. Other indicators are important for differential diagnosis and definition of syndrome phenotypes. Different PCOS phenotypes are characterized by different ratios of the contribution of sources of excess androgen synthesis. Depending on the cause, hyperandrogenism in PCOS patients is accompanied by various metabolic risks. Usually, an excess of androgens in women is clinically manifested by hirsutism, seborrheic dermatitis, acne, acanthosis nigricans, and androgenetic alopecia. The presence of dermatological manifestations of hyperandrogenism has different diagnostic value in puberty, women of reproductive age and in menopause. Acne can be the first sign of pubertal maturation. Additional examinations should be planned in extremely severe cases, which are accompanied by signs of androgen excess, or do not respond to treatment. In women of reproductive age and in perimenopause, the need to assess androgenic status is extremely important. Hair loss according to the female type is associated with manifestations of metabolic syndrome, and it is an independent risk factor for the development of diabetes, atherosclerosis and mortality from cardiovascular diseases. Therefore it is recommended to use the modified Rotterdam criteria for PCOS diagnosis. Such criteria include clinical or biochemical hyperandrogenism, signs of oligoanovulation, polycystosis (morphology of the ovaries according to ultrasound diagnostics), if other causes of relevant disorders are excluded. At the same time, any two of the specified criteria have diagnostic value, which makes possible to establish not only the diagnosis, but also clinical variant (phenotype) of PCOS, the diagnosis of which is the basis for choosing individual treatment for this contingent of women. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C–H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/ M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs. [ABSTRACT FROM AUTHOR]

Simple Summary: 17β-Hydroxysteroid dehydrogenase type1 (HSD17B1) is responsible for converting estrone, a weak estrogen, into the more potent estradiol in the ovaries of vertebrates ranging from teleosts to mammals. We evaluated its expression in the placentae of various eutherian species and demonstrated that it was only expressed in the human placenta. The ubiquitous transcription factor SP1-binding site was conserved in the promoter region of the HSD17B1/Hsd17b1 gene across the species. This region conferred the promoter activities both in human and ovine HSD17B1. However, in the ovine placenta, cytosine residues of this SP1 site in the HSD17B1 promoter region were completely methylated. Human HSD17B1 had high activity for testosterone production, similar to its homologs in other species. On the basis of this fact, the significance of placental HSD17B1 expression is discussed. During mammalian gestation, large amounts of progesterone are produced by the placenta and circulate for the maintenance of pregnancy. In contrast, primary plasma estrogens are different between species. To account for this difference, we compared the expression of ovarian and placental steroidogenic genes in various mammalian species (mouse, guinea pig, porcine, ovine, bovine, and human). Consistent with the ability to synthesize progesterone, CYP11A1/Cyp11a1, and bi-functional HSD3B/Hsd3b genes were expressed in all species. CYP17A1/Cyp17a1 was expressed in the placenta of all species, excluding humans. CYP19A/Cyp19a1 was expressed in all placental estrogen-producing species, whereas estradiol-producing HSD17B1 was only strongly expressed in the human placenta. The promoter region of HSD17B1 in various species possesses a well-conserved SP1 site that was activated in human placental cell line JEG-3 cells. However, DNA methylation analyses in the ovine placenta showed that the SP1-site in the promoter region of HSD17B1 was completely methylated. These results indicate that epigenetic regulation of HSD17B1 expression is important for species-specific placental sex steroid production. Because human HSD17B1 showed strong activity for the conversion of androstenedione into testosterone, similar to HSD17B1/Hsd17b1 in other species, we also discuss the biological significance of human placental HSD17B1 based on the symptoms of aromatase-deficient patients. [ABSTRACT FROM AUTHOR]

The role of estrogens and progesterone in the development and progression of endometrial cancer is well-established, but there are very little data about the role of androgens. There are five different androgens produced in women: dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T) and dihydrotestosterone (DHT). The most potent hormones are T and DHT, the latter being mainly produced from T in peripheral tissues, including endometrium. Although they are considered to exert antiproliferative effects in many settings and the expression of their receptors is more often associated with a good prognosis in EC, it is still unknown in which specific settings androgens have carcinogenic or protective effects in EC. [ABSTRACT FROM AUTHOR]

Background: Children with attention-deficit/hyperactivity disorder (ADHD) might have similar problems as in autism spectrum disorder (ASD) and show impairment in social behaviour. Also, there is a relationship between social relationship skills and ToM (theory of mind) skills of children with ADHD. Besides, ASD is associated with prenatal exposure to high levels of androgens, and oxytocin plays a role in the modulation of emotions, coping with stress, and social behaviour like ASD. In this study, the relationship between autistic traits and serum oxytocin, testosterone, and androstenedione levels in prepubertal male drug naive children with ADHD has been investigated. Method: Eighty-three prepubertal children, who were diagnosed with ADHD between the ages of 6-10 years old, are included in the study. For the study, intelligence levels were evaluated by using WISC-4, and autistic traits were measured by using both social responsiveness scale and theory of mind tests. In addition, serum oxytocin, testosterone, and androstenedione levels were measured by using ELISA. Results: It has been found that serum testosterone levels of patients with lower autistic traits are significantly lower than those with moderate and severe autistic traits, while the serum oxytocin levels are significantly higher. Also, patients with severe autistic traits have had significantly higher serum androstenedione levels than those with lower and moderate autistic traits. Conclusion: This study suggests that patients who have higher autistic traits have elevated testosterone and androstenedione levels and lower serum oxytocin levels. Further studies are needed to clarify this relationship. [ABSTRACT FROM AUTHOR]

Urine marking, aggression, and other behavioral concerns are common reasons for cat owners to seek veterinary care. Empiric treatment for lower urinary tract disease or primary behavior disorders are commonly pursued, especially in those cases with normal routine laboratory evaluations. Herein, we report the clinicopathologic findings in eight sexually altered cats that were diagnosed with androgen-secreting adrenocortical tumors. Nearly all cats (n = 7) initially were evaluated for inappropriate urination and pungent urine, with additional behavioral concerns including aggression (n = 3) and excess vocalization (n = 4) commonly reported. Penile barbs (n = 5) were identified in all five male cats, and an enlarged clitoris was observed in one female cat. Testing of serum androgen concentrations revealed abnormally high androstenedione (n = 1) or testosterone (n = 7) concentrations. In the five cases with available adrenal tissue, histopathologic evaluation identified either an adrenocortical adenoma (n = 3) or adrenocortical carcinoma (n = 2). Hormonal abnormalities resolved and clinical signs improved in the four cats that underwent surgical adrenalectomy, with each of these cats surviving >1 year. However, clinical signs were minimally impacted with medical treatments, including one cat in which trilostane treatment failed to improve clinical signs or testosterone concentrations. This collection of cases underscores the importance of a detailed physical examination as well as the consideration of endocrine disturbances in cats undergoing evaluation for inappropriate urination or aggression. Furthermore, this report adds to the growing body of evidence that sex-hormone secreting adrenal tumors in cats may be an under-recognized syndrome.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences.

Background: There is no consensus regarding markers of optimal treatment or timing between glucocorticoid intake and assessment of hormone levels in the follow-up of female 21-hydroxylase deficient patients. Objective: To examine visit-to-visit repeatability in levels of adrenal hormones in adult female patients, to identify predictors of repeatability in hormone levels and to examine concordance between levels of different adrenal hormones. Method: All patients with confirmed 21-hydroxylase deficiency treated with glucocorticoids, were included. The two most recent blood samples collected on a stable dose of glucocorticoid replacement were compared. Complete concordance was defined as all measured adrenal hormones either within, below or above normal range evaluated in a single-day measurement. Results: Sixty-two patients, median age of 35 (range 18–74) years were included. All hormone levels showed moderate to excellent repeatability with an intraclass correlation coefficient between 0.80 and 0.99. Repeatability of hormone levels was not affected by the use of long-acting glucocorticoids or time of day for blood sample collection. The median difference in time between the two sample collections was 1.5 (range 0–7.5) h. Complete concordance between 17-hydroxyprogesterone, androstenedione, and testosterone was found in 21% of cases. Conclusion: During everyday, clinical practice hormone levels in adult female patients with 21-hydroxylase deficiency showed a moderate to excellent repeatability, despite considerable variation in time of day for blood sample collection. We found no major predictors of hormone level variation. Future studies are needed to address the relationship between the timing of glucocorticoid intake vs adrenal hormone levels and clinical outcome in both adults and children.

Hyperandrogenism (HA) remains an actual problem that raises a large number of questions, in particular, in cases of discrepancy between the clinical signs of HA and biochemical parameters. This review includes an analysis of recent studies of androgen synthesis and metabolism in healthy women of reproductive age; management of pathologies accompanied by HA; as well as contemporary approaches to the principles of prescribing and evaluating the results of the main available markers of biochemical HA. There are сoncepts of clinical and biochemical HA. Biochemical HA is an increase in the levels of various circulating androgens. The concept of clinical HA includes hirsutism as assessed by the modified Ferriman–Gallwey scale. The most common causes of HA include polycystic ovary syndrome, non-classical forms of congenital adrenal hyperplasia, other endocrine disorders. It is necessary to carry out a differential diagnosis of these conditions with more rare causes as a primarily tumor pathology. The choice and evaluation of laboratory markers of biochemical HA should be made taking into account clinical data such as puberty history, anovulation, polycystic ovarian morphotype, obesity; presence and degree of hirsutism and/or other androgen-associated dermatopathies, the characteristics of the course of clinical HA (sudden onset, rapid progression), presence of other endocrinopathies, liver and kidney dysfunction; diabetes mellitus. The objectives of the laboratory assessment of HA are determination of HA severity; determination of the source of excess androgen synthesis (ovaries, adrenal glands); assessment of the free fraction of circulating active androgens; in some cases, assessment of the formation of active androgens in the skin. The following indicators are used to solve the tasks in laboratory diagnostics: total testosterone, free testosterone, free androgen index, androstenedione, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, dihydrotestosterone. A comprehensive assessment of these indicators allows not only assessing the presence of HA and its severity, but also the nature of its leading and secondary components, which determine the presence and prognosis of metabolic risks and allow individualizing therapeutic approaches.

Objective: Patients with congenital adrenal hyperplasia (CAH) in developing countries have limited access to appropriate laboratory facilities for diagnosis and follow‐up. The aim of this study is to evaluate steroid measurement in hair as a diagnostic tool to identify and monitor CAH in these patients. Design: A method was developed to measure steroids in hair, the stability of steroids in hair was assessed, and the concentration range in healthy volunteers was determined. Hair samples of patients, before and after starting therapy, were transported at ambient temperature to The Netherlands for analysis. Patients: Twenty‐two Indonesian CAH patients and 84 healthy volunteers participated. Measurements: Cortisol, 17‐hydroxyprogesterone (17OHP), androstenedione, and testosterone in hair were measured by liquid chromatography with tandem mass spectrometry. Results: Steroids in hair could be measured and remained stable (<4.9% deviation) for at least 3 weeks at 4°C and 30°C. In each of the untreated patients, hair concentrations of 17OHP (9.43–1135 pmol/g), androstenedione (36.1–432 pmol/g), and testosterone (2.85–69.2 pmol/g) were all above the upper limit of the corresponding range in healthy volunteers; 5.5 pmol/g, 13 pmol/g, and 1.8 pmol/g, respectively. After starting glucocorticoid treatment, the steroid concentrations in the hair of CAH patients decreased significantly for androstenedione (73%) and testosterone (59%) after 6 months. Conclusions: CAH could be confirmed in Indonesian patients based on the concentration of 17OHP, androstenedione, and testosterone in hair, and a treatment effect was observed. These findings open up opportunities to diagnose and/or monitor CAH in developing countries with a simple noninvasive technique. [ABSTRACT FROM AUTHOR]

The study aimed to familiarise primary care physicians and specialists with the minimum hormonal diagnostic tests necessary to assay isolated micropenis in healthy children without any phenotypic abnormality. Children aged 6–15 years (mean 11.6 ± 1.68) were assessed from May 2010 to September 2021 (N = 247). Multiple regression analysis showed correlations between stretched penile length (SPL) and hormonal assays as follows: follicle‐stimulating hormone (FSH): r = 0.097, p = 0.035; luteinizing hormone (LH): r = 0.139, p = 0.012, thyroid‐stimulating hormone (TSH): r = −0.001, p = 0.321; testosterone (T): r = 0.118, p = 0.004; dihydrotestosterone (DHT): r = 0.002, p = 0.243; androstenedione (Δ4And): r = −0.004, p = 0.502; insulin‐like growth factor I (IGF‐I): r = −0.003, p = 0.062; and IFG‐binding protein 3 (IGF‐BP3): r = 0.052, p = 0.051. The most hormonal disorder was testosterone deficiency. TSH, Δ4And, and DHT were normal in all boys. SPL was significantly correlated with FSH, LH, and T, but there was no significant correlation between SPL and TSH, DHT, Δ4And, IGF‐I, and IGF‐BP3. Whenever the isolated micropenis is seen without other anomalies, it is sufficient to assay testosterone, FSH, and LH in the first step. [ABSTRACT FROM AUTHOR]

The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first "oxidative" stage, phytosterol (5–10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains ("reductive" mode). The conditions favorable for "oxidative" and "reductive" stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the "reductive" mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes. [ABSTRACT FROM AUTHOR]