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Background: Children with classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency require treatment with glucocorticoids, usually at supraphysiologic doses, to address cortisol insufficiency and reduce excess adrenal androgens. However, such treatment confers a predisposition to glucocorticoid-related complications. In 2-week phase 2 trials, patients with CAH who received crinecerfont, a new oral corticotropin-releasing factor type 1 receptor antagonist, had decreases in androstenedione levels. Methods: In this phase 3, multinational, randomized trial, we assigned pediatric participants with CAH, in a 2:1 ratio, to receive crinecerfont or placebo for 28 weeks. A stable glucocorticoid dose was maintained for 4 weeks, and the dose was then adjusted to a target of 8.0 to 10.0 mg per square meter of body-surface area per day (hydrocortisone dose equivalents), provided that the androstenedione level was controlled (≤120% of the baseline level or within the reference range). The primary efficacy end point was the change in the androstenedione level from baseline to week 4. A key secondary end point was the percent change in the glucocorticoid dose from baseline to week 28 while androstenedione control was maintained. Results: A total of 103 participants underwent randomization, of whom 69 were assigned to crinecerfont and 34 to placebo; 100 (97%) remained in the trial at 28 weeks. At baseline, the mean glucocorticoid dose was 16.4 mg per square meter per day, and the mean androstenedione level was 431 ng per deciliter (15.0 nmol/liter). At week 4, androstenedione was substantially reduced in the crinecerfont group (-197 ng per deciliter [-6.9 nmol/liter]) but increased in the placebo group (71 ng per deciliter [2.5 nmol/liter]) (least-squares mean difference [LSMD], -268 ng per deciliter [-9.3 nmol/liter]; P<0.001); the observed mean androstenedione value, obtained before the morning glucocorticoid dose, was 208 ng per deciliter (7.3 nmol/liter) in the crinecerfont group, as compared with 545 ng per deciliter (19.0 nmol/liter) in the placebo group. At week 28, the mean glucocorticoid dose had decreased (while androstenedione control was maintained) by 18.0% with crinecerfont but increased by 5.6% with placebo (LSMD, -23.5 percentage points; P<0.001). Headache, pyrexia, and vomiting were the most common adverse events. Conclusions: In this phase 3 trial, crinecerfont was superior to placebo in reducing elevated androstenedione levels in pediatric participants with CAH and was also associated with a decrease in the glucocorticoid dose from supraphysiologic to physiologic levels while androstenedione control was maintained. (Funded by Neurocrine Biosciences; CAHtalyst Pediatric ClinicalTrials.gov number, NCT04806451.). [ABSTRACT FROM AUTHOR]

Per‐ and poly‐fluoroalkyl substances (PFAS), which are long‐lasting environmental contaminants that are released into the environment during the e‐waste disassembly process, pose a threat to human health. Human milk is a complex and dynamic mixture of endogenous and exogenous substances, including steroid hormones and PFAS. Therefore, in this study, we aimed to investigate the association between PFAS and steroid hormones in human milk from women living close to an e‐waste disassembly area. In 2021, we collected milk samples from 150 mothers within 4 weeks of delivery and analyzed them via liquid chromatography‐tandem mass spectrometry to determine the levels of 21 perfluorinated compounds and five steroid hormones (estrone, estriol, testosterone, progesterone, and androstenedione [A‐dione]). We also performed multiple linear regression analysis to clarify the association between maternal PFAS exposure and steroid hormone concentrations. Our results indicated that PFOA and PFOS were positively associated with estrone (β, 0.23; 95% CI, 0.08–0.39) and A‐dione (β, 0.186; 95% CI, 0.016–0.357) concentrations in human milk, respectively. Further, the average estimated daily intake of PFOA and PFOS were 36.5 ng/kg bw/day (range, 0.52–291.7 ng/kg bw/day) and 5.21 ng/kg bw/day (range, 0.26–32.3 ng/kg bw/day), respectively. Of concern, the PFAS intake of breastfeeding infants in the study area was higher than the recommended threshold. These findings suggested that prenatal exposure to PFAS from the e‐waste disassembly process can influence steroid hormones levels in human milk. Increased efforts to mitigate mother and infant exposure to environmental pollutants are also required. Plain Language Summary: We recruited 150 mothers who provided human milk samples within 4 weeks of delivery. Using liquid chromatography‐tandem mass spectrometry, we measured concentrations of 21 perfluorinated compounds and five steroid hormones (estrone, estriol, testosterone, progesterone, and androstenedione [A‐dione]) in the human milk samples. Multiple linear regression analysis was performed to examine the association between maternal PFAS exposure and steroid hormone concentrations. Our findings revealed significant positive associations between specific PFAS compounds (PFOA and PFOS) and certain steroid hormone concentrations in human milk. Notably, PFOA showed positive associations with estrone concentrations, PFOS was associated with A‐dione levels. These results suggest that prenatal exposure to PFAS from e‐waste disassembly areas can impact the levels of steroid hormones in human milk. Key Points: Per‐ and poly‐fluoroalkyl substances and steroid hormones concentrations in human milk measured in an e‐waste disassembly areaPFOA was positively associated with DHEA and estrone concentrations in human milkPFOS was positively associated with A‐dione in human milk [ABSTRACT FROM AUTHOR]

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. [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]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Abstract Per‐ and poly‐fluoroalkyl substances (PFAS), which are long‐lasting environmental contaminants that are released into the environment during the e‐waste disassembly process, pose a threat to human health. Human milk is a complex and dynamic mixture of endogenous and exogenous substances, including steroid hormones and PFAS. Therefore, in this study, we aimed to investigate the association between PFAS and steroid hormones in human milk from women living close to an e‐waste disassembly area. In 2021, we collected milk samples from 150 mothers within 4 weeks of delivery and analyzed them via liquid chromatography‐tandem mass spectrometry to determine the levels of 21 perfluorinated compounds and five steroid hormones (estrone, estriol, testosterone, progesterone, and androstenedione [A‐dione]). We also performed multiple linear regression analysis to clarify the association between maternal PFAS exposure and steroid hormone concentrations. Our results indicated that PFOA and PFOS were positively associated with estrone (β, 0.23; 95% CI, 0.08–0.39) and A‐dione (β, 0.186; 95% CI, 0.016–0.357) concentrations in human milk, respectively. Further, the average estimated daily intake of PFOA and PFOS were 36.5 ng/kg bw/day (range, 0.52–291.7 ng/kg bw/day) and 5.21 ng/kg bw/day (range, 0.26–32.3 ng/kg bw/day), respectively. Of concern, the PFAS intake of breastfeeding infants in the study area was higher than the recommended threshold. These findings suggested that prenatal exposure to PFAS from the e‐waste disassembly process can influence steroid hormones levels in human milk. Increased efforts to mitigate mother and infant exposure to environmental pollutants are also required.

Background: Primary angle closure glaucoma (PACG) is the leading cause of irreversible blindness in Asia, and no reliable, effective diagnostic, and predictive biomarkers are used in clinical routines. A growing body of evidence shows metabolic alterations in patients with glaucoma. We aimed to develop and validate potential metabolite biomarkers to diagnose and predict the visual field progression of PACG. Methods: Here, we used a five-phase (discovery phase, validation phase 1, validation phase 2, supplementary phase, and cohort phase) multicenter (EENT hospital, Shanghai Xuhui Central Hospital), cross-sectional, prospective cohort study designed to perform widely targeted metabolomics and chemiluminescence immunoassay to determine candidate biomarkers. Five machine learning (random forest, support vector machine, lasso, K-nearest neighbor, and GaussianNaive Bayes [NB]) approaches were used to identify an optimal algorithm. The discrimination ability was evaluated using the area under the receiver operating characteristic curve (AUC). Calibration was assessed by Hosmer-Lemeshow tests and calibration plots. Results: Studied serum samples were collected from 616 participants, and 1464 metabolites were identified. Machine learning algorithm determines that androstenedione exhibited excellent discrimination and acceptable calibration in discriminating PACG across the discovery phase (discovery set 1, AUCs=1.0 [95% CI, 1.00-1.00]; discovery set 2, AUCs = 0.85 [95% CI, 0.80-0.90]) and validation phases (internal validation, AUCs = 0.86 [95% CI, 0.81-0.91]; external validation, AUCs = 0.87 [95% CI, 0.80-0.95]). Androstenedione also exhibited a higher AUC (0.92-0.98) to discriminate the severity of PACG. In the supplemental phase, serum androstenedione levels were consistent with those in aqueous humor (r=0.82, p=0.038) and significantly (p=0.021) decreased after treatment. Further, cohort phase demonstrates that higher baseline androstenedione levels (hazard ratio = 2.71 [95% CI: 1.199-6.104], p=0.017) were associated with faster visual field progression. Conclusions: Our study identifies serum androstenedione as a potential biomarker for diagnosing PACG and indicating visual field progression. [ABSTRACT FROM AUTHOR]

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 Quality of life (QoL) has been inconsistently reported in children and young people (CYP) with congenital adrenal hyperplasia (CAH). Objective Assess QoL in CYP with CAH in the UK alongside biometric and androgen profiles. Design To define the evidence base for health care delivery, we conducted a cross-sectional study in CYP with CAH in the UK. Questionnaire results were compared with normative data and between groups, and modelled for association with sex, height, weight, body mass index, or steroid biomarkers of CAH control. Setting Tertiary care in 14 UK centers. Patients Results from 104 patients, 55% female, mean age 12.7 years (SD 3.0), paired responses from parents. Interventions Strengths and Difficulties questionnaire (SDQ) and pediatric QoL questionnaire. Main Outcome Measure Total QoL scores as assessed by SDQ and a pediatric QoL questionnaire in comparison to normative data. Results Total scores were worse in parents than normative data, but similar in patients. Patient QoL was rated better in social functioning but worse in emotional, school, and peer domains by patients, and worse in total scores and domains of peer problems, and psychosocial, emotional, and school functioning by parents. Parents consistently scored QoL of their children lower than their child. Larger height-SD score and lower weight-SD score were associated with better QoL. Girls with lower steroid biomarkers had worse SDQ scores. Conclusions In CYP with CAH, reduced height, increased weight, and hormonal biomarkers consistent with overtreatment were associated with worse QoL; addressing these problems should be prioritized in clinical management. Clinical Trials Registration Number: SCH/15/088 [ABSTRACT FROM AUTHOR]

17-β-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-β-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-β-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-β-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-β-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-β-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-β-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-β-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-β-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-β-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care.

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.

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.)

17-β-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-β-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-β-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-β-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-β-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-β-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-β-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-β-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-β-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-β-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care. [Display omitted] • E2 increased in healthy asbestos-exposed subjects and MPM patients. • DHEA-S-androstenedione-E2 signature score increased in asbestos-exposed subjects. • The signature differentiates subjects exposed to asbestos from those not exposed. • DHEA-S-androstenedione-E2 as biomarkers of MPM risk and early diagnosis of MPM. [ABSTRACT FROM AUTHOR]

Androstenedione (ADD), an important precursor for steroid hormone drug synthesis, can be obtained through the transformation of phytosterols by members of the Mycobacteria genus. In this study, multidirectional pathway engineering of Mycobacterium neoaurum LY-2 was applied to improve ADD production from phytosterols. Initially, the nonessential gene KasB encoding a cell wall β-ketoacyl acyl carrier protein synthase in Mycobacterium neoaurum LY-2 was knocked out, and the production of ADD was increased by 21.0%. Then, the key enzymes involved in ADD synthetic metabolism were systematically explored. These assays revealed that propionyl coenzyme A carboxylase (PCC) and Hsd4A played important roles in the synthesis of ADD. In addition, the heterologous expression of Vitreoscilla hemoglobin (VHb), a protein with oxygen-binding capacity, was successfully realized in Mycobacterium neoaurum LY-2, and the ADD concentration reached 4.42 g/L. Finally, the recombinant strain LY-2- ΔKasB-hsd4A-pcc-vgb with high ADD production was constructed by combining the above strategies. Through optimization of conversion conditions, the recombinant strain LY-2-Δ KasB - hsd4A - pcc-vgb produced an ADD concentration of 11.12 g/L when adding 20 g/L phytosterols. [Display omitted] • Mycobacterium neoaurum LY-2 efficiently catalyzes phytosterols to produce ADD. • Deletion of KasB enhanced the productivity of M. neoaurum LY-2. • PCC, Hsd4A, and VHb were co-expressed in the recombinant strain LY-2-Δ KasB. • The recombinant strain LY-2-Δ KasB - hsd4A - pcc-vgb produced 11.12 g/L of ADD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• A unified total synthetic route to 18- and 19-trideuteromethyl steroids is described. • The method can also be used to prepare 18,19-hexadeuteromethyl steroids. • Compounds are useful for preparing calibration curves for mass spec analysis of these steroid hormones. • Compounds are useful for distinguishing between these endogenous and exogenous steroids under various conditions. A unified total synthesis route has been used to prepare 18- and 19-trideuterated testosterone, androstenedione and progesterone. The 18-trideuterated steroid synthetic method starts with the synthesis of 2-(methyl- d 3)-1,3-cyclopentanedione from CD 3 I and 1,3-cyclopentanedione and is subsequently converted into the Hajos-Parrish ketone for synthesis of these trideuterated steroids. The 19-trideuterated steroid synthesis proceeds through non-deuterated Hajos-Parrish ketone with incorporation of the 19-methyl- d 3 group from CD 3 I at a later stage of the same synthetic route. Utilization of CD 3 I at both the initial and later stages of the synthesis provides a route to 18,19-hexadeuterated steroids. The deuterated steroids are useful for studies of steroid biosynthesis and metabolism. [ABSTRACT FROM AUTHOR]

Thyroid cancer (TC) is substantially more common in women than in men, pointing to a possible role of sex steroid hormones. We investigated the association between circulating sex steroid hormones, sex hormone binding globulin (SHBG) and the risk of differentiated TC in men and women within the European Prospective Investigation into Cancer and nutrition (EPIC) cohort. During follow-up, we identified 333 first primary incident cases of differentiated TC (152 in pre/peri-menopausal women, 111 in post-menopausal women, and 70 in men) and 706 cancer-free controls. Women taking exogenous hormones at blood donation were excluded. Plasma concentrations of testosterone, androstenedione, dehydroepiandrosterone, estradiol, estrone and progesterone (in pre-menopausal women only) were performed using liquid chromatography/mass spectrometry method. SHBG concentrations were measured by immunoassay. Odds ratios (ORs) were estimated using conditional logistic regression models adjusted for possible confounders. No significant associations were observed in men and postmenopausal women, while a borderline significant increase in differentiated TC risk was observed with increasing testosterone (adjusted OR T3 vs T1: 1.68, 95% CI: 0.96-2.92, p trend  = .06) and androstenedione concentrations in pre/perimenopausal women (adjusted OR T3 vs T1: 1.78, 95% CI: 0.96-3.30, p trend  = .06, respectively). A borderline decrease in risk was observed for the highest progesterone/estradiol ratio (adjusted OR T3 vs T1: 0.54, 95% CI: 0.28-1.05, p trend  = .07). Overall, our results do not support a major role of circulating sex steroids in the etiology of differentiated TC in post-menopausal women and men but may suggest an involvement of altered sex steroid production in pre-menopausal women., (© 2024 The World Health Organization. The World Health Organization retains copyright and all other rights in the manuscript of this article as submitted for publication.)

Background: Androgen could impact cervical remodelling during pregnancy, and a higher level is associated with adverse pregnancy outcomes. A population-based gestation age-specific reference interval (RI) of total testosterone (TT), androstenedione (A4), and 17-hydroxyprogesterone (17-OHP) can help to diagnose maternal hyperandrogenism., Methods: We enrolled 600 healthy Chinese women to obtain longitudinal serum samples across gestation. The serum androgen profile was measured by liquid chromatography-tandem mass spectrometry. The equations for medians of TT, A4, and 17-OHP were generated by MedCal, and the variances adjusted for 2-level modeling were generated by MLwiN, a system for the specification and analysis of a range of multilevel models., Results: A4 and TT levels increased across the gestation, and they closely correlated with each other (R = 0.90, P=<0.001), whereas 17-OHP level decreased from 5th gestational week to 16th gestational week and then increased afterward towards the end of pregnancy. Women diagnosed with preeclampsia (PE) were found to have a significantly higher level of A4, TT, and 17-OHP when compared with non-PE cases with p ≤0.01, whereas mothers carrying male versus female fetuses have comparable levels of A4, TT, and 17-OHP., Conclusion: The study highlights a methodology for constructing gestational age-specific TT, A4, and 17-OHP levels to provide a better interpretation of results in a cohort of healthy Chinese women. The observation in PE supports previous findings, and the higher levels of TT, A4, and 17-OHP were observed before the onset of PE., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

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. [ABSTRACT FROM AUTHOR]

Androstenedione (ADSD) is one of the widely detected androgens in diverse aquatic environments. However, there were few reports on the molecular mechanism of Chlorella vulgaris exposure to ADSD. In our previous research, we have investigated the genes associated with chlorophyll metabolism in Chlorella vulgaris response to ADSD. In this study, we focus on continuously up-regulated genes to explore the mechanism underlying Chlorella vulgaris resistance to ADSD toxicity. Chlorella vulgaris was exposed to ADSD with five concentration gradients. The continuously up-regulated genes were enriched by Series Test of Cluster (STC) analysis and verified by qRT-PCR. Microalgae Super Oxidase Dimutase (SOD) and Microalgae Malonic dialdehyde (MDA), two indicators of oxidative stress, were determined by ELISA after exposure to ADSD. The results showed that ADSD can stimulate the production of extracellular polymeric substances (EPS) and lead to enlargement in the cell body of Chlorella vulgaris. In addition, steroid biosynthesis and oxidoreductase activity processes were consistently up-regulated upon exposure to ADSD. In conclusion, our study highlighted the crucial role of phenotypic modification, hormone synthesis, and redox mechanisms in protecting Chlorella vulgaris cells from the harmful effects of ADSD contamination. [Display omitted] • ADSD exposure can cause EPS increase and cell body enlargement. • Alterations phenotypic traits are crucial for chlorella withstand ADSD toxic. • Steroid biosynthesis was up-regulated after ADSD exposure. • Oxidoreductase activity was up-regulated after ADSD exposure. [ABSTRACT FROM AUTHOR]

• Influence of ethanol consumption and food intake on serum concentrations of endogenous steroids. • Ethanol consumption increased testosterone/androstenedione ratio in blood. • Meal-induced ACTH and cortisol peaks coincided with pulsatile androstenedione peaks. • Female testosterone levels are more susceptible to perturbations by ethanol or ACTH. Steroid biosynthesis and biotransformation are based on a cascade of enzymatic processes being highly sensitive to various external influences. Amongst those, ethanol was shown to affect testosterone metabolism. For doping analyses, athlete steroid profiles comprise seven urinary steroid metabolites, of which relevant ratios are significantly increased following ethanol consumption. This effect is presumably based on the lack of hepatic NAD+-coenzyme as a consequence of ethanol oxidation. Only recently, testosterone (T) and androstenedione (A4) blood profiles have been introduced as additional approach for doping control. However, a potential influence of ethanol intake on testosterone biosynthesis and thus on blood steroid profiles has not been investigated so far. Therefore, steroid concentrations from 10 males and 10 females receiving an ethanol infusion up to a breath alcohol concentration of 0.5 mg/L which was hold as a plateau for two hours were conducted. Blood samples were drawn every 15 min for steroid quantification. An ethanol-dependent T/A4 increase up to 385 % resulting from A4 suppression was observed in 14 volunteers. In addition, we observed sporadic A4 increases coinciding with cortisol and ACTH pulses pointing to a meal-induced adrenal stimulation. While testosterone levels in males showed diurnal variation solely, testosterone levels in some females were found to be susceptible to ethanol- and ACTH-dependent perturbations, which is thought to be due to its predominant adrenal synthesis in females. In conclusion, the results of the present study emphasize the importance of blood sampling at a sufficient time interval from food and ethanol intake. This is of interest if T and A4 are used for diagnostics in doping control. [ABSTRACT FROM AUTHOR]

C19 steroids and C22 steroids are vital intermediates for the synthesis of steroid drugs. Compared with C19 steroids, C22 steroids are more suitable for synthesizing progesterone and adrenocortical hormones, albeit less developed. 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one(9-OHBA), due to its substituents at positions C-9 and C-22, is a beneficial and innovative steroid derivative for synthesizing corticosteroids. We focused on the C22 pathway in Mycobacterium fortuitum ATCC 35855, aiming to develop a productive strain that produces 9-OHBA. We used a mutant strain, MFΔ kstD , that knocked out kstds from Mycobacterium fortuitum ATCC 35855 named MFKD in this study as the original strain. Hsd4A and FadA5 are key enzymes in controlling the C19 metabolic pathway of steroids in Mycobacterium fortuitum ATCC 35855. After knocking out hsd4A , MFKDΔ hsd4A accumulated 81.47% 9-OHBA compared with 4.13% 9-OHBA in the strain MFKD. The double mutant MFKDΔ hsd4A Δ fadA5 further improved the selectivity of 9-OHBA to 95.13%, and 9α-hydroxy-4-androstenedione (9-OHAD) decreased to 0.90% from 4.19%. In the end, we obtained 6.81 g/L 9-OHBA from 10 g/L phytosterols with a molar yield of 80.33%, which showed the best performance compared with formerly reported strains.

There is a lack of consensus on the optimal screening strategy for insulin resistance (IR), particularly in lean women with polycystic ovary syndrome (PCOS). Therefore, we conducted a cross-sectional study in 80 women with PCOS (28 lean/52 obese) and 80 age- and body mass index (BMI)-matched controls. Using a 5-point 75-g oral glucose tolerance test (OGTT) (0, 30, 60, 90, 120 min), we examined glucose and insulin excursions, IR, insulin sensitivity, beta-cell function (ßF), and the effect of androgens on IR. Lean and obese women with PCOS had similar glucose but higher insulin (except fasting in lean women) and insulin AUC as compared to their respective controls (p < 0.05). Lean women with PCOS were equally insulin-resistant but more hyperinsulinemic than the obese controls (p < 0.05). Although ßF ([1st phase: 481.71 ± 263.53 vs. 430.56 ± 232.37], [2nd phase: 815.16 ± 447.12 vs. 752.66 ± 428.95]) was comparable in lean and obese women with PCOS, lean women had better insulin sensitivity (112.78 ± 66.26 vs. 75.49 ± 55.6) (p < 0.05). Dehydroepiandrosterone sulfate (DHEAS) and androstenedione decreased with increasing BMI in lean women, and this correlated with deteriorating insulin sensitivity and exaggerated hyperinsulinemia. In obese women with PCOS, sex hormone-binding globulin (SHBG) correlated negatively with BMI and hyperinsulinemia, and positively with insulin sensitivity. This data suggests that estimating only fasting insulin may miss IR in lean women with PCOS; hence, additional time points in OGTT will add value to screening for IR. DHEAS and androstenedione may have a beneficial effect on insulin sensitivity and may be used to screen IR in lean women, while SHBG can be used as a predictive marker for IR in obese women with PCOS., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Background: Primary angle closure glaucoma (PACG) is the leading cause of irreversible blindness in Asia, and no reliable, effective diagnostic, and predictive biomarkers are used in clinical routines. A growing body of evidence shows metabolic alterations in patients with glaucoma. We aimed to develop and validate potential metabolite biomarkers to diagnose and predict the visual field progression of PACG., Methods: Here, we used a five-phase (discovery phase, validation phase 1, validation phase 2, supplementary phase, and cohort phase) multicenter (EENT hospital, Shanghai Xuhui Central Hospital), cross-sectional, prospective cohort study designed to perform widely targeted metabolomics and chemiluminescence immunoassay to determine candidate biomarkers. Five machine learning (random forest, support vector machine, lasso, K-nearest neighbor, and GaussianNaive Bayes [NB]) approaches were used to identify an optimal algorithm. The discrimination ability was evaluated using the area under the receiver operating characteristic curve (AUC). Calibration was assessed by Hosmer-Lemeshow tests and calibration plots., Results: Studied serum samples were collected from 616 participants, and 1464 metabolites were identified. Machine learning algorithm determines that androstenedione exhibited excellent discrimination and acceptable calibration in discriminating PACG across the discovery phase (discovery set 1, AUCs=1.0 [95% CI, 1.00-1.00]; discovery set 2, AUCs = 0.85 [95% CI, 0.80-0.90]) and validation phases (internal validation, AUCs = 0.86 [95% CI, 0.81-0.91]; external validation, AUCs = 0.87 [95% CI, 0.80-0.95]). Androstenedione also exhibited a higher AUC (0.92-0.98) to discriminate the severity of PACG. In the supplemental phase, serum androstenedione levels were consistent with those in aqueous humor (r=0.82, p=0.038) and significantly (p=0.021) decreased after treatment. Further, cohort phase demonstrates that higher baseline androstenedione levels (hazard ratio = 2.71 [95% CI: 1.199-6.104], p=0.017) were associated with faster visual field progression., Conclusions: Our study identifies serum androstenedione as a potential biomarker for diagnosing PACG and indicating visual field progression., Funding: This work was supported by Youth Medical Talents - Clinical Laboratory Practitioner Program (2022-65), the National Natural Science Foundation of China (82302582), Shanghai Municipal Health Commission Project (20224Y0317), and Higher Education Industry-Academic-Research Innovation Fund of China (2023JQ006)., Competing Interests: SL, JR, ZJ, YQ, MS, YL, JW, YS, XS, SG, WC No competing interests declared, (© 2023, Li, Ren, Jiang et al.)

Introduction: Hyperandrogenemia is the defining feature of polycystic ovary syndrome (PCOS). Increasingly androgens are being advocated to be measured through liquid chromatography-mass spectrometry (LC-MS/MS). The role of LC-MS/MS over immunoassay in diagnosis of PCOS has been debated over a long time. We analyzed the role of androgens as measured by LC-MS/MS in diagnosing women with PCOS., Materials and Methods: We performed a prospective case-control study involving 59 women with PCOS compared with 30 age- and BMI-matched controls., Results: In PCOS phenotypes A-C (hyperandrogenic by definition), 19/47 (40%) had normal testosterone (T) levels but 14/19 (75%) had either elevated androstenedione (A4) or dehydroepiandrosterone. A4 had the highest area under curve (0.89) for diagnosing PCOS followed by T (0.81). Even in the PCOS-D phenotype (sonologic polycystic ovaries + oligomenorrhoea), A4 was significantly higher as compared to controls though still in normal range., Conclusion: A4 had a role in diagnosing hyperandrogenism in women with PCOS. Further studies clarifying the role of androgen profiles in diagnosing PCOS and its cost-effectiveness may be required in the future., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Subramaniam et al.)

Background: Endogenous steroid hormones have significant effects on inflammatory and immune processes, but the immunological activities of steroidogenesis precursors remain largely unexplored., Methods: We conducted a systematic approach to examine the association between steroid hormones profile and immune traits in a cohort of 534 healthy volunteers. Serum concentrations of steroid hormones and their precursors (cortisol, progesterone, testosterone, androstenedione, 11-deoxycortisol and 17-OH progesterone) were determined by liquid chromatography-tandem mass spectrometry. Immune traits were evaluated by quantifying cellular composition of the circulating immune system and ex vivo cytokine responses elicited by major human pathogens and microbial ligands. An independent cohort of 321 individuals was used for validation, followed by in vitro validation experiments., Findings: We observed a positive association between 11-deoxycortisol and lymphoid cellular subsets numbers and function (especially IL-17 response). The association with lymphoid cellularity was validated in an independent validation cohort. In vitro experiments showed that, as compared to androstenedione and 17-OH progesterone, 11-deoxycortisol promoted T cell proliferation and Candida-induced Th17 polarization at physiologically relevant concentrations. Functionally, 11-deoxycortisol-treated T cells displayed a more activated phenotype (PD-L1 high CD25 high CD62L low CD127 low ) in response to CD3/CD28 co-stimulation, and downregulated expression of T-bet nuclear transcription factor., Interpretation: Our findings suggest a positive association between 11-deoxycortisol and T-cell function under physiological conditions. Further investigation is needed to explore the potential mechanisms and clinical implications., Funding: Found in acknowledgements., Competing Interests: Declaration of interests The authors declare that there is no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The synthesis of a 14 C-labeled C-18 functionalized steroid (as referred as EM-6798) that will serve as a probe for the research of novel antiandrogens has been accomplished. This radioactive steroid was obtained in nine steps by coupling racemic N-cyclohexyl-1-(3'-hydroxy[U- 14 C]phenyl)propylamine with protected 18-bromomethyl-3,17-androstenedione. Incorporation of the radiolabel on the C-18 side chain was achieved using commercially available 3-bromo[U- 14 C]phenol. Alkylation of N-cyclohexyl-1-(3'-hydroxy[U- 14 C]phenyl)propylamine with 3-ethylenedioxy-18-bromomethyl-3,17-androstenedione furnished after reduction and deprotection, [phenyl-U- 14 C]EM-6798 in a 20% overall yield from 3-bromo[U- 14 C]phenol at a specific activity of 156 μCi/mg with 97.9% radiochemical purity as determined by HPLC., (© 2023 John Wiley & Sons, Ltd.)