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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.

Background/Objective: Ectopic Cushing syndrome can be challenging to diagnose when its presentation is atypical. Herein, we highlight features of ectopic adrenocorticotropic hormone (ACTH) syndrome in a patient with worsening hypertension, hypokalemia, ACTH-dependent hypercortisolism, and disproportionate elevation in serum androstenedione levels. Case Report: A 59-year-old woman presented with rapidly progressing hypertension, severe hypokalemia, confusion, and weakness. Her medical history included well-controlled hypertension receiving amlodipine 5 mg/day, which worsened 3 months prior to admission requiring losartan and spironolactone therapy, with twice daily potassium supplementation. Physical examination was notable for bruising, muscle wasting, thin extremities, facial fullness, and abdominal adiposity despite body mass index 17 kg/m2. Laboratory evaluation showed potassium 2.6 mEq/L (3.5-5.3), morning cortisol >50 mcg/dL (8-25), 24-hour urine cortisol 8369 mcg/day (

The transcriptomic Point of Departure (tPOD) is increasingly used in ecotoxicology to derive quantitative endpoints from RNA sequencing studies. Utilizing transcriptomic data in zebrafish embryos as a New Approach Methodology (NAM) is beneficial due to its acknowledgment as an alternative to animal testing under EU Directive 2010/63/EU. Transcriptomic profiles are available in zebrafish for various modes of action (MoA). The limited literature available suggest that tPOD values from Fish Embryo Toxicity (FET) tests align with, but are generally lower than, No Observed Effect Concentrations (NOEC) from long-term chronic fish toxicity tests. In studies with the androgenic hormone androstenedione in a Fish Sexual Development Test (FSDT), a significant shift in the sex ratio towards males was noted at all test concentrations, making it impossible to determine a NOEC (NOEC <4.34 μg/L). To avoid additional animal testing in a repetition of the FSDT and adhere to the 3Rs principle (replacement, reduction, and refinement), a modified zebrafish FET (zFET) was conducted aiming to determine a regulatory acceptable effect threshold. This involved lower concentration ranges (20 to 6105 ng/L), overlapping with the masculinization-observed concentrations in the FSDT. The tPOD analysis in zFET showed consistent results with previous FSDT findings, observing strong expression changes in androgen-dependent genes at higher concentrations but not at lower ones, demonstrating a concentration-response relationship. The tPOD values for androstenedione were determined as 24 ng/L (10th percentile), 60 ng/L (20th gene), and 69 ng/L (1st peak). The 10th percentile tPOD value in zFET was 200 times lower than the lowest concentration in the FSDT. Comparing the tPOD values to literature suggests their potential to inform on the NOEC range in FSDT tests., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The study was financed by Bayer AG. The study was conducted and analyzed under the quality assurance system good laboratory practice (FSDT) or according to good scientific practice (tPOD) at Fraunhofer IME. Bayer AG had no influence on the conduction or evaluation of the study., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Porcine adrenocorticotrophic hormone (ACTH) has been considered valid for the ACTH stimulation test (ACTHST) in humans and dogs; however, its safety and efficacy for use in cats are unknown. Also, the equivalence between 5 µg/kg and 125 µg/cat dose of synthetic corticotropin (1-24 ACTH - cosyntropin/tetracosactide) is assumed for ACTHST in cats. This study evaluated the safety and effectiveness of different porcine recombinant ACTH doses for the ACTHST in healthy cats and its equivalence with tetracosactide. The study was divided into two arms. The first evaluated safety and equivalence of intravenous 1 µg/kg, 5 µg/kg, or 125 µg/cat porcine ACTH in seven healthy cats for the ACTHST evaluating basal and post-ACTH androstenedione, aldosterone, cortisol, and progesterone concentrations. In the second arm, the equivalence of the 125 µg/cat porcine ACTH dose was evaluated compared to results obtained using 125 µg/cat of tetracosactide in ten healthy cats regarding cortisol responses. In all tests, several cat-friendly strategies were adopted, and the ACTHST protocol involved basal and 60-minute post-ACTH blood sampling and intravenous ACTH injection. No adverse reactions were documented, and no tested cat showed any complications during the study. No porcine ACTH tested dose significantly increased androstenedione secretion. In contrast, all tested doses were able to increase progesterone concentration significantly (P < 0.05), and Δ-progesterone in response to 5 µg/kg or 125 µg/cat was considered equivalent (P > 0.99). The 125 µg/cat dose promoted greater responses for both cortisol and aldosterone, characterized by Δ-cortisol (P = 0.009) and Δ-aldosterone (P = 0.004). Despite equivalent Δ-cortisol results in response to 5 µg/kg or 125 µg/cat (P = 0.18); post-ACTH results of cortisol in response to 5 µg/kg only approximate statistical significance when compared with basal (P = 0.07). Porcine ACTH and tetracosactide significantly increased post-ACTH cortisol concentration (P < 0.0001) while the Δ-cortisol was slightly greater in response to the porcine ACTH (P = 0.006). These results suggest porcine ACTH could be an alternative source of corticotropin for the ACTHST in cats; however, maximum corticoadrenal stimulation seemed more reliable in response to a 125 µg/cat regarding cortisol and aldosterone., Competing Interests: Declaration of competing interest While preparing this work, the authors used the Grammar Check Service from Grammarly, Inc. (USA) to warrant correct spelling and grammar construction and for clarity and conciseness. After using this service, the authors reviewed and edited the content as needed and took full responsibility for the publication's content., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Androstenedione (ASD) is a biomarker used in the diagnostic workup of hyperandrogenism, congenital adrenal hyperplasia, premature adrenarche, and polycystic ovary syndrome (PCOS). The Elecsys ASD competitive electrochemiluminescence immunoassay (Roche Diagnostics, Indianapolis, IN) is a new assay recently available in the US. Objective: This study evaluated the analytical and clinical performance of the Elecsys ASD assay. Design & Methods: We evaluated the linearity/analytical measuring range (AMR), precision, and accuracy of the Elecsys ASD assay on the cobas e601 analyzer. ASD was measured in serum/plasma in the Elecsys ASD, Immulite (Siemens Medical Solutions USA, Inc. Malvern, PA), and LC-MS/MS assays. Reference intervals (RI) were evaluated across genders, menopausal status, and in children. Statistical analysis was performed using EP evaluator and R program. Results: The Elecsys ASD assay had a linear response across the AMR. The intra- and inter-assay coefficients of variation at various concentrations were ≤4.5%. The Elecsys ASD assay had a mean difference of −0.04 ng/mL (−1.7%) with the LC-MS/MS assay, whereas the Immulite assay had a mean difference of 1.17 ng/mL (66%) and −1.22 ng/mL (−38%) compared to the LC-MS/MS and Elecsys ASD assays, respectively. The Roche recommended RIs for healthy men (0.280–1.52 ng/mL) and postmenopausal women (0.187–1.07 ng/mL) were successfully verified. The RIs for children were adopted from published data. For pre-menopausal women, a RI of

Androstenedione (AED) is a naturally occurring androgen that is released into the environment through human and animal excretion. Several studies have shown that AED can interfere with the normal growth, development, and reproduction of organisms. However, studies examining the thyroid system, an important part of endocrine system of fish, are still lacking. This study examines thyroid hormone levels including T3 and T4, and transcriptional expression levels of genes associated with the hypothalamic-pituitary-thyroid axis (HPT axis) in adult zebrafish. Fish were exposed to a solvent control and three other concentrations of AED (0.2, 2.3, and 23.7 μg/L) for 60 days. Results indicated that AED significantly increased T4 levels but inhibited transcription of some HPT axis-related genes (nis, tpo, tg, dio1, dio2, sult1 st5, pax8, and nkx2.1) in female zebrafish. The adverse effects of AED on the thyroid system in males were very low, only affecting down-regulation of the tshb gene and up-regulation of the thrb gene. The overall results demonstrated that AED interferes with the thyroid endocrine system in fish.

Background: Several in vitro and in vivo studies have demonstrated the effects of phthalates on androgen synthesis, and the adverse outcomes of phthalate exposure on male reproductive function have been reported. However, the direct relationship among these three factors remains unknown. Objective: To explore the potential roles of steroids involved in androgen synthesis in the association between phthalate exposure and semen quality. Methods: Eighteen phthalate metabolites (mPAEs) and nine steroids were analyzed in semen samples of 403 male participants aged 18–54 years from a hospital in Shenzhen, China. The associations across phthalate metabolites, steroids, and eleven semen quality parameters were evaluated by multivariate linear regression and logistical regression models. The potential contributions of steroids to the associations between phthalate metabolites and semen quality outcomes were explored by mediation effect analysis. Results: In this cross-sectional study, mono-n-butyl phthalate (MnBP) was inversely associated with nine continuous semen quality parameters in a dose-dependent manner (all p for trend

A new potent Pseudomonas aeruginosa isolate capable for biotransformation of corn oil phytosterol (PS) to 4-androstene-3, 17-dione (AD), testosterone (T) and boldenone (BOL) was identified by phenotypic analysis and 16S rRNA gene sequencing. Sequential statistical strategy was used to optimize the biotransformation process mainly concerning BOL using Factorial design and response surface methodology (RSM). The production of BOL in single step microbial biotransformation from corn oil phytosterols by P. aeruginosa was not previously reported. Results showed that the pH concentration of the medium, (NH4)2SO4 and KH2PO4 were the most significant factors affecting BOL production. By analyzing the statistical model of three-dimensional surface plot, BOL production increased from 36.8% to 42.4% after the first step of optimization, and the overall biotransformation increased to 51.9%. After applying the second step of the sequential statistical strategy BOL production increased to 53.6%, and the overall biotransformation increased to 91.9% using the following optimized medium composition (g/l distilled water) (NH4)2SO4, 2; KH2PO4, 4; Na2HPO4. 1; MgSO4·7H2O, 0.3; NaCl, 0.1; CaCl2·2H2O, 0.1; FeSO4·7H2O, 0.001; ammonium acetate 0.001; Tween 80, 0.05%; corn oil 0.5%; 8-hydroxyquinoline 0.016; pH 8; 200 rpm agitation speed and incubation time 36 h at 30 °C. Validation experiments proved the adequacy and accuracy of model, and the results showed the predicted value agreed well with the experimental values.

In this study, we synthesized androsta-4,14-diene-3,16-dione, 12β-hydroxyandrosta-4,14-diene-3,16-dione, and other 3,16-androstenedione derivatives from commercially available dehydroepiandrosterone as a starting material in 9-13 steps with high yields. The bioactivity of the obtained compounds was evaluated. Compounds 14a and 23a were shown to have high antitumor activity against acute lymphoblastic leukemia cell lines Nalm-6 and BALL-1, respectively. Network pharmacology analysis showed that the anti-leukemia activity of compounds 14a and 23a might be related to the JAK2, ABL1 protein, and PI3K/Akt signaling pathways. The molecular docking of compounds 14a and 23a identified possible active sites, with the lowest docking scores for PTGS2 and MAPK14, respectively. In addition, the absorption, distribution, metabolism, and excretion prediction results revealed the drug-likeness of the two compounds. Therefore, compounds 14a and 23a should be considered anti-leukemia candidates in future studies., 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 © 2023 Elsevier Inc. All rights reserved.)

An increasing number of microplastics have been detected in aquatic environments, causing various damage to organisms. The size of microplastics affects the toxicity once they enter the organisms. Meanwhile, there is an increasing variety of Endocrine-disrupting chemicals (EDCs) present in aquatic environments. Androstenedione (AED) is a typical EDC. In this study, we used polystyrene microspheres of 80 nm (NPs) and 8 μm (MPs) as materials to simulate environmental contaminants in the aquatic environment with AED. We used female mosquitofish (Gambusia affinis) as the research object to investigate the effects of microplastics on fish in waters containing AED. We compared different sizes of particles accumulation in some tissues of fish and variation of enzyme activities (SOD, LDH, CAT), and the content of MDA in the gut. MPs, NPs, and AED combined exposure test investigated mRNA profiles of immune-related genes (IL-1β, IL-6, IL-8, IL-10) and hormone receptor genes (ARα, ARβ, ERα, ERβ) in the liver of fish. Our results indicated that MPs emerged in various tissues (gill, gut, and liver) of mosquitofish. Besides, NPs and MPs caused enteric abnormal enzyme activity after 48 h of exposure, which was particularly pronounced in the MPs-AED group. MPs induced significant upregulation of inflammatory factors and gonadal factor genes after 96 h of exposure, which was more pronounced when co-exposed with AED. In conclusion, NPs and MPs caused mechanisms of immune damage and inflammatory response. MPs were found to be more likely to cause adverse reactions than NPs, and these responses were enhanced by the combined effects of AED. This study demonstrated that AED can exacerbate the negative effects of MPs and NPs on mosquitofish. It provided an important basis for the effective assessment of MPs and NPs on bioaccumulation and biochemical status of mosquitofish. Additionally, it serves as a foundation to investigate the interactive effects of microplastics and EDCs in living organisms., 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 © 2023. Published by Elsevier Ltd.)

Maternal effects are thought to be essential tools for females to modulate offspring development. The selective deposition of avian maternal hormones could therefore allow females to strategically adjust the phenotype of their offspring to the environmental situation encountered. However, at the time of egg formation, several contaminants are also transferred to the egg, including per- and polyfluoroalkyl substances (PFAS) which are ubiquitous organic contaminants with endocrine disrupting properties. It is, however, unknown if they can disrupt maternal hormone deposition. In this study we explored relationships between female PFAS burden and maternal deposition in the eggs of steroids (dihydrotestosterone, androstenedione and testosterone), glucocorticoids (corticosterone) and thyroid hormones (triiodothyronine and thyroxine) in a population of the Arctic-breeding black-legged kittiwake (Rissa tridactyla). Egg yolk hormone levels were unrelated to female hormone plasma levels. Second-laid eggs had significantly lower concentrations of androstenedione than first-laid eggs. Triiodothyronine yolk levels were decreasing with increasing egg mass but increasing with increasing females' body condition. Testosterone was the only transferred yolk hormone correlated to maternal PFAS burden: specifically, we found a positive correlation between testosterone in yolks and circulating maternal perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDcA) and perfluoroundecanoic acid (PFUnA) in first-laid eggs. This correlative study provides a first insight into the potential of some long-chain perfluoroalkyl acids to disrupt maternal hormones deposition in eggs and raises the question about the consequences of increased testosterone deposition on the developing embryo., 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 © 2023 Elsevier B.V. All rights reserved.)

Background: A rapid and accurate measurement approach for 17α-hydroxyprogesterone (17-OHP) and related steroids in amount/volume-limited clinic samples is of importance for precise newborn diagnosis of congenital adrenal hyperplasia (CAH) and its subtypes in clinic., Methods: Sixteen steroids (17-OHP, androstenedione, cortisol, tetrahydro-11-deoxycortisol, pregnenolone, progesterone, 11-deoxycorticosterone, corticosterone, 21-deoxycortisol, 11-deoxycortisol, dehydroepiandrosterone, testosterone, aldosterone, 17α-hydroxypregnenolone, dihydrotestosterone and 18-hydroxycorticosterone) were included in the panel of high-throughput microbore ultra-performance liquid chromatography-tandem mass spectrometry. Samples were collected from 126 normal subjects and 65 patients including different subtypes of CAH., Results: The method was validated with satisfactory analytical performance in linearity, repeatability, recovery and limit of detection. Reference intervals for 16 steroids were established by quantifying the level of steroids detected in normal infants. The applicability of the method was tested by differentiating steroid metabolic characteristics between normal infants and infants with CAH, as well as between infants with different CAH subtypes. The relevance of 17-OHP, 21-deoxycortisol, and 17-OHP/11-deoxycortisol for 21-hydroxylase deficiency screening was demonstrated. The level of 11-deoxycorticosterone, 11-deoxycortisol, progesterone and androstenedione can be used for the diagnosis of different rare subtypes of CAH., Conclusion: This study provides a strategy for highly efficient steroid analysis of amount/volume-limited clinic samples and holds great potential for clinical diagnosis of CAH., 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 © 2023 Elsevier B.V. All rights reserved.)

Background: Neighborhood walkability (NW) has been linked to increased physical activity, which in turn is associated with lower concentrations of sex hormones and higher concentration of SHBG in women. However, no study has directly examined the association of NW with female sex hormone levels., Objective: We conducted a cross-sectional study to evaluate the association between NW and circulating levels of sex hormones and SHBG in pre- and post-menopausal women., Methods: We included 797 premenopausal and 618 postmenopausal women from the New York University Women's Health Study (NYUWHS) who were healthy controls in previous nested case-control studies in which sex hormones (androstenedione, testosterone, DHEAS, estradiol and estrone) and SHBG had been measured in serum at enrollment. Baseline residential addresses were geo-coded and the Built Environment and Health Neighborhood Walkability Index (BEH-NWI) was calculated. Generalized Estimating Equations were used to assess the association between BEH-NWI and sex hormone and SHBG concentrations adjusting for individual- and neighborhood-level factors., Results: In premenopausal women, a one standard deviation (SD) increment in BEH-NWI was associated with a 3.5% (95% CI 0.9%-6.1%) lower DHEAS concentration. In postmenopausal women, a one SD increment in BEH-NWI was related to an 8.5% (95% CI 5.4%-11.5%) lower level of DHEAS, a 3.7% (95% CI 0.5%-6.8%) lower level of testosterone, a 1.8% (95% CI 0.5%-3.0%) lower level of estrone, and a 4.2% (95% CI 2.7%-5.7%) higher level of SHBG. However, the associations with respect to DHEAS and estrone became apparent only after adjusting for neighborhood-level variables. Sensitivity analyses using fixed effects meta-analysis and inverse probability weighting accounting for potential selection bias yielded similar results., Conclusion: Our findings suggest that NW is associated with lower concentrations of androgens and estrone, and increased SHBG, in postmenopausal women, and lower levels of DHEAS in premenopausal women., 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 © 2022 Elsevier Inc. All rights reserved.)

Background: Maternal hormonal risk factors for autism spectrum disorder (ASD) in offspring could intersect genetic and environmental risk factors., Objectives: This analysis explored ASD risk in association with maternal testosterone, androstenedione, and dehydroepiandrosterone (DHEA) measured in first, second, and third trimesters of pregnancy., Methods: MARBLES 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., Results: Non-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., Conclusion: No significant associations were found between maternal androgen concentrations and risk of ASD or Non-TD in the child., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: RJ Schmidt receives research funding from the Simons Foundation, was a paid consultant for a research study at Drexel University, and has received honoraria from NIH for grant reviews. A-M Iosif has received honoraria for reviewing activities from Elsevier, NIH and Department of Defense. L Granillo and NW Snyder declare no competing financial interests.

Background: The aim of the study was to produce age and sex specific pediatric reference intervals (RIs) on a fully automated chemiluminescent immunoassay (CLIA) system., Material and Method: A total of 1586 patients' remnant sera were included in the study and free testosterone (FT), 17-OH progesterone (17OHP), androstenodione (A4) and insulin-like growth factor-1 (IGF-1) parameters were measured on MAGLUMI 2000 (Shenzhen New Industries Biomedical Engineering Co., Ltd. (Snibe), Shenzhen, China) CLIA analyser. After appropriate age and gender partitioning, specific intervals were calculated according to Clinical Laboratory Standart Institute's (CLSI) C28-A3 protocol., Results: All analytes showed sex and age dependent concentrations requiring several subgroups with specific reference intervals. 17OHP and A4 were found high with birth, declined thereafter: 17OHP by the end of 12 months and A4 by 6 months. So this period was also partitioned for these two hormones. All showed gradual increases by the end of 18 years. 17OHP, A4 and IGF-1 of girls were higher than boys around puberty as the result of earlier sexual development and maturation. FT values of boys and girls didn't differ from each other upto 10 years of age but boys had significantly higher values than girls afterwards. IGF-1 values gradually increase in both sexes upto the ages of 13, girls with significantly higher values than boys. In 13-18 years no significant gender difference was found., Conclusions: We present method specific pediatric RIs, which are comparable with medical literature, necessary for interpretation of patient results., 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 © 2022 Elsevier Inc. All rights reserved.)

Androgens are endogenous hormones that play a crucial role in the paracrine and intracrine hormone system to perform and maintain vital physiological functions. Altered levels of androgens are implicated in many diseases such as sexual dysregulation, breast cancer, prostate cancer, and heart diseases etc. In this manuscript we describe a liquid chromatography-mass spectrometry (LC-MS) method using multiple reaction monitoring (MRM) for quantitatively measuring specific androgens such as dehydroepiandrosterone, testosterone, androsterone sulphate, androstenedione, and dihydrotestosterone in serum and urine samples. Serum acquired from nine different subjects (three pre-menopausal women, three postmenopausal women, and three healthy males) were used to evaluate the developed methods. In the sample preparation methods for serum either protein precipitation or liquid-liquid extraction (LLE) was used while the analysis of urinary androgens used LLE. The extracted androgens were quantitatively measured using LC-MRM-MS to which known amounts of stable isotope labeled standards were added. This manuscript also presents a LC-MRM-MS method mode for the analysis of oxime derivatized androgens potentially to enhance the sensitivity of the assay if required, from urine and venous-drawn serum samples., (Published by Elsevier Inc.)

Background: Newborn screening for congenital adrenal hyperplasia (CAH) using 17-hydroxyprogesterone dissociation-enhanced, lanthanide fluorescence immunoassay (DELFIA) generates a large number of false-positive results. The present study aimed to improve the sensitivity of the CAH neonatal screening by including second-tier steroid profiling in dried blood spots (DBS) using liquid chromatography tandem mass spectrometry (LC-MS/MS)., Methods: We developed and validated a LC-MS/MS method for simultaneous determination of six steroids in DBS, including androstenedione, testosterone, 17-hydroxyprogesterone, 11-deoxycortisol, 21-deoxycortisol, and cortisol. Two 5-mm blood spots were eluted by internal standard working solution. We analyzed 1170 DBS samples from neonates to determine gestational age-specific reference intervals. In order to test the specificity of the second-tier method, we analyzed 707 cards with a positive screening by DELFIA., Results: Values of intra- and inter-day precision coefficients of variance and accuracy were 2.0%-13.3% and 85.8%-114.5%, respectively. Recovery ranged from 85.0% to 106.9%. The lower limit of quantification was 0.5 ng/mL for 21-deoxycortisol, 0.25 ng/mL for 17-hydroxyprogesterone and cortisol, and 0.1 ng/mL for testosterone, androstenedione, and 11-deoxycortisol. In addition, the linearity range was 0.25-50 ng/mL (R 2  > 0.99). According to the 17-hydroxyprogesterone levels and ratios of (androstenedione + 17-hydroxyprogesterone)/cortisol in the 707 positive screening samples, 77 neonates should receive recall visit. The number of false-positive results reduced by 89.1%. Totally, 18 newborns were diagnosed with 21-hydroxylase deficiency, one with P450 oxidoreductase deficiency and one with 11β-hydroxylase deficiency. With two-tier screening, the positive predictive value increased to 26.0%., Conclusions: The second-tier steroid profiling by LC-MS/MS reduced the false-positive rate and improved the positive predictive value of CAH screening. We suggest applying this steroid profiling assay as a second-tier test for CAH screening in China., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A development of robust and rapid method with simple sample preparation for the analysis of steroids of C 18 -, C 19 -, C 21 - families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C 18 - steroids (estrone, estradiol), nineteen C 19 - steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C 21 - steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Although they are involved in the progression of PCa, the use of sex steroid hormones in urinary exosomes as biomarkers for PCa remains obscure. Here, the potential use of sex steroid hormones in urinary exosomes as biomarkers was investigated for the prediction of early-stage PCa to assist in clinical diagnosis., Methods: Two hundred and eighty-six participants were randomly recruited, 231 patients with PCa and 55 healthy controls. According to their Gleason scores (GSs), the patients with PCa were divided into two groups, mild PCa (GS6) (n = 116) and severe (≥GS7) group (n = 115). The concentrations of 8 sex steroid hormones in urinary exosomes were quantitated using liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS)., Results: The results showed that the levels of 7 out of 8 sex steroids including dehydroepiandrosterone (DHEA), dehydroepiandrosteronesulfate (DHEAS), androstenedione (A4), testosterone (T), progesterone (P), dihydrotestosterone (DHT), and estrone (E1), but not estradiol (E2) in urinary exosomes, were not only distinguished the PCa patients from healthy controls, can also differentiate between patients with mild and severe PCa. Of the 8 selected urinary exosomal biomarkers, DHEA, DHEAS, T, and DHT were finally screened further to build the regression model, and the detection method of the 4 biomarkers-combined achieved an area under the ROC curve (AUC) of 0.854 and predictive accuracy of 78.2%., Conclusion: Our data showed the use of exosomal sex steroids in urine could be as biomarkers for predicting PCa for the first time. This finding would supply a novel insight for PCa diagnosis., (Copyright © 2022. Published by Elsevier B.V.)

Circulating free androgens are important indicators for a variety of diseases, so accurate determination of these hormones in serum is of great clinical significance. However, there are still many challenges for the accurate quantification of free androgens using mass methods because of their very low levels and complex interferences in serum, as well as the high serum protein binding rate and high nonspecific binding (NSB) rate. Here, an HPLC-MS/MS method coupled with magnetic solid phase extraction (MSPE) and in-situ derivatization was developed to quantify two free androgens-free testosterone (FT) and free androstenedione (FA 4 )-in human serum simultaneously and accurately. Ultrafiltration was used to obtain free androgens in serum. To minimize the NSB rate and obtain accurate results, the ultrafiltration membrane was doubly modified with surfactant followed by a silane-coupling agent. Multiple pre-saturation with the tested samples was also used. With these strategies, the ultrafiltration recoveries were up to 95.4% for FT and 94.0% for FA 4 , so the NSB was negligible. After that, the extremely low levels of free androgens in ultrafiltrates were extracted and enriched using MSPE with core-shell structured ferroferric oxide coated with graphene oxide. Hydroxylamine hydrochloride was used to derivatize the analytes and the reaction took place on the surface of the adsorbent. All the extraction and derivatization conditions were optimized. Under such conditions, the assays were linear for FT within the range of 2-100 pg mL -1 and for FA 4 within the range of 5-500 pg mL -1 . The intra- and inter-run CV was less than 12.3% and 10.9% for FT and less than 7.2% and 8.3% for FA 4 , respectively. For the intra- and inter-run accuracies, the relative error of the mean was 9.9% and 8.4% for FT, and 11.5% and 7.3% for FA 4 , respectively. The total extraction recoveries with MSPE in-situ derivatization were 93.2% for FT, 93.8% for FA 4 and 95.7% for the internal standard. The method was validated and was used to quantify the trace level of these two free androgens in serum samples from female patients suspected of having polycystic ovary syndrome (PCOS) accurately. It is expected to improve the diagnosis accuracy of PCOS when combined with other clinical indicators., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Although DHEA sulfate (DS) is the most abundant steroid in the circulation, breast fluid contains an approximately 80-fold greater concentration than serum. Transport of DS into cells requires organic anion transporting polypeptides (OATPs), which are specific for cell type, cell location, and substrate, but may have a broader specificity for housekeeping functions. Specific classes, which may be modified by soluble factors including neutral steroids, have been identified in the breast. After transport, DS may be cleaved to DHEA by ubiquitous sulfatases, which may be modified by the cell milieu, or DHEA may enter by diffusion. Synthesis from cholesterol does not occur because CYP17B12 and cytochrome b5 are lacking in breast tissues. Case-control studies reveal a positive association of serum DS with risk of breast cancer. The association is even greater with DHEA, particularly in postmenopausal women with HR + invasive tumors. Metabolites of DHEA, androstenedione and testosterone, are associated with breast cancer but DHEA is likely to have an independent role as well. Mechanisms by which DHEA may promote breast cancer relate to its effect in increasing circulating IGF-I, by inhibiting the suppressive effect of glucocorticoids, and by promoting retention of pre-adipocytes with aromatase activity. In addition, DHEA may interact with the G-protein coupled receptor GPER for stimulation of miR-21 and subsequent activation of the MAPK pathway. DHEA also has antitumor properties that relate to stimulation of immunity, suppression of inflammation, and elevation of adipose tissue adiponectin synthesis. The net effect may depend on the which factors predominate., (Copyright © 2022. Published by Elsevier Inc.)

Androgens androstadienedione (ADD) and androstenedione (AED) are predominant steroid hormones in surface water, and can disrupt the endocrine system in fish. However, little is known about the transgenerational effects of ADD and AED in fish. In the present study, F0 generation was exposed to ADD and AED from 21 to 144 days post-fertilization (dpf) at nominal concentrations of 5 (L), 50 (M) and 500 (H) ng L -1 , and F1 generation was domesticated in clear water for 144 dpf. The sex ratio, histology and transcription in F0 and F1 generations were examined. In the F0 generation, ADD and AED tended to be estrogenic in zebrafish, resulting in female biased zebrafish populations. In the F1 generation, ADD at the H level caused 63.5% females, while AED at the H level resulted in 78.7% males. In brain, ADD and AED had similar effects on circadian rhythm in the F0 and F1 generations. In the F1 eleutheroembryos, transcriptomic analysis indicated that neuromast hair cell related biological processes (BPs) were overlapped in the ADD and AED groups. Taken together, ADD and AED at environmentally relevant concentrations had transgenerational effects on sex differentiation and transcription in zebrafish., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Developing rapid and reliable method for simultaneous hormones quantitation is of great significant because of important roles of hormones in metabolism. However, current methods are faced with problems of low throughput or complicated operation procedure to remove matrices from serum samples in routine clinical diagnosis. In the present work, a multilayer PS-MS method was developed for rapid and simple detection of hormones. In the strategy, multilayer filter paper acted as the Liquid Chromatography in LC-MS/MS for separation of hormones and biological matrices. Qualitative and quantitative analysis of three hormones, testosterone (T), androsterone (ADT) and androstenedione (4-AD) were realized through MS/MS spectra. The method exhibited linearity in the range of 0.02-2 μg/L and the results of recovery and repeatability were satisfactory for standard samples and spiked serum. The time-cost of a whole detection process was less than 3 min. The established multilayer PS-MS realized rapid, simple and reliable quantitative analysis of various hormones and provided broad prospect for clinical analysis of small molecules in different biological samples. Moreover, it provides a novel MS approach with high through-put and free HPLC, meeting the requirements of point-of-care testing (POCT)., (Copyright © 2021. Published by Elsevier B.V.)

Background and Aims: We aimed to investigate the associations of testosterone and androstenedione with coronary heart disease, and the interaction effect of testosterone or androstenedione and age on coronary heart disease., Methods and Results: A total of 6178 participants were included in this study. Serum testosterone and androstenedione were detected by liquid chromatography-tandem mass spectrometry. Logistic regression and restricted cubic splines were used to assess the independent effects of testosterone and androstenedione on coronary heart disease. Interactive plots were employed to examine the interaction effects of testosterone or androstenedione with age on coronary heart disease. After adjusting for multiple variables, serum testosterone and androstenedione levels were negatively associated with coronary heart disease in males (tertile 3 vs tertile 1, odd ratio (OR) = 0.56, 95% confidence interval (CI) (0.33, 0.96), and OR = 0.40, 95% CI (0.22, 0.74)). Per 1 unit increase in ln-testosterone and ln-androstenedione was associated with a 24% (OR = 0.76, 95% CI (0.63, 0.91)) and 30% (OR = 0.69, 95% CI (0.55, 0.86)) lower risk of coronary heart disease, respectively. Additionally, the positive association of age with coronary heart disease was attenuated by increasing concentrations of ln-testosterone and ln-androstenedione concentration in males., Conclusions: The results indicated that serum testosterone and androstenedione were negatively associated with coronary heart disease risk in Chinese rural males. To some extent, this study supports the application of hormone therapy in males with coronary heart disease, which can contribute to reducing the burden of coronary heart disease and related cardiovascular disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

The evidence that androgens regulate the folliculogenesis supports the hypothesis that intrafollicular testosterone (T), androstenedione (A 4 ) and dehydroepiandrosterone (DHEA) could be modified along follicular growth. The objective of this study was to evaluate the changes and related relationships between systemic and intrafollicular T, A 4 and DHEA in post-deviation and impending ovulation follicles. Sixty ovaries were taken after the slaughter of 30 clinically healthy mares. In according to the sizes, the follicles were classified in 3 different categories, as small (20-30 mm), medium (31-40 mm) and large (≥ 41 mm), and the follicular fluid (FF) samplings were extracted from each single follicle. Intrafollicular concentrations of T, A 4 , and DHEA were significantly higher than systemic ones. Intrafollicular and systemic T and A 4 concentrations were strongly and positively correlated, and DHEA negatively. A 4 was the predominant androgen in FF. T and A 4 were positively and DHEA negatively correlated with the follicular diameter. T and A 4 significantly increased in large and medium than small follicle sizes. DHEA was significantly higher in small than medium and large follicle size. The increase of intrafollicular androgens suggests the presence of androgenic environment based in the biotransformation of DHEA in A 4 and later in T in the follicles, and the progressive oestradiol-17β (E 2 ) production with the advance of follicular growth. The evidence of significant correlations between systemic and intrafollicular androgens considerably helps to understanding more deeply the role of these steroids in the physiology of follicular development in the mare, adding a new segment of scientific literature., (Copyright © 2021. Published by Elsevier Inc.)

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of DHT by LC-MS/MS. Derivatization of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of DHT, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies., 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. Published by Elsevier B.V.)

Aberrant androgen metabolism is a characteristic feature of polycystic ovary syndrome (PCOS). Various androgens as well as their precursors and metabolites can accumulate in the blood of PCOS patients. Although these steroids include neuroactive steroids, such as allopregnanolone and androstenedione (Δ4A), it remains unknown whether altered blood steroid levels contribute to the high risk of mood disorders in PCOS. In this study, we measured blood levels of 11 steroids in 25 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay, and assessed the psychological status of these patients using the Hospital Anxiety and Depression Scale (HADS) questionnaire. We also examined age and the degree of metabolic abnormalities of each patient. Steroid values of the patients were compared to our previous data from 31 eumenorrheic women. As a result, 20 patients exhibited aberrant blood levels of one or more of the 11 tested steroids. In most cases, Δ4A and allopregnanolone levels were within or close to the reference ranges. Levels of four steroids were negatively correlated with patients' age, while no correlation was observed between steroid values and metabolic conditions. Seven patients showed high HADS scores. HADS scores were correlated with blood Δ4A levels even after stratifying by body mass indexes, but not with the levels of other steroids or clinical data. These results indicate that the high frequency of anxiety and depression in PCOS patients cannot be ascribed to altered blood levels of a specific steroid, although there may be a weak association between circulating Δ4A levels and psychological conditions of the patients., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The accurate measurement of androstenedione in human serum and plasma is required for steroid profiling to assure the appropriate diagnosis and differential diagnosis of hyperandrogenism. In this work, we introduce an isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedure for the quantification of androstenedione in human serum and plasma. The performance of the procedure enables its use in the evaluation and standardization of routine assays and for the evaluation of patient samples to ensure the traceability of individual patient results. As the primary standard, a certified reference material from NMIA (National Measurement Institute, Australia) was used. Additionally, a quantitative nuclear magnetic resonance (qNMR) method was developed for the value assignment of the primary reference material, which ensures the direct traceability to SI units, as well as the independence from the availability of reference materials. 13 C 3 -labeled androstenedione was used as the internal standard. The introduced method allows the measurement of androstenedione in the range of 0.05-12 ng/mL, and the assay imprecision was found to be <2% between 5 and 12 ng/mL, 3.5% at 1.5 ng/mL, and 5.2% at 0.05 ng/mL, with an accuracy of 95-105% for the serum and 91-103% for the plasma matrix. The transferability to a second laboratory was validated by method comparison based on 112 patient samples. The comparison of the results obtained from the presented method and an LC-MS/MS routine assay, using 150 native patient samples, showed a good correlation with a bias of the routine method of ≤4.0%., Competing Interests: 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., (© 2020 The Author(s).)

AMH (Anti-Müllerian Hormone) is involved in the regulation of follicle growth initiation and inhibits FSH-induced aromatase expression and estrogen production in granulosa cells. However, the function of AMH in steroidogenesis by theca cells remains unclear. The aim of this study is to investigate the role of AMH as a regulator of the basal and stimulated steroid production by pig granulosa cells (pGCs) and theca cells (pTCs). PGCs and pTCs were incubated with hormones AMH, LH (luteinizing hormone), FSH (follicle stimulating hormone), individually or in combination. The expression of CYP19A1, HSD3B1, CYP11A1, LHCGR, and CYP17A1 mRNA were evaluated by quantitative reverse transcriptase PCR. In pGCs, 10 ng/mL AMH significantly decreased the FSH-stimulated effect on FSHR and CYP19A1 expression and estradiol production. In pTCs, LH treatment significantly increased the expression of HSD3B1, CYP11A1, LHCGR, and androstenedione or progesterone production (P < 0.05). Additionally, 10 ng/mL AMH also significantly decreased the LH-stimulated effects on the expression of HSD3B1, CYP11A1, CYP17A1, LHCGR and androstenedione production. Transfection with siAMHR2-I abolished the suppressive effects of AMH on LH-induced HSD3B1 expression and androstenedione production. Taken together, these results demonstrate that AMH is involved in FSH induced estradiol production in pGCs and LH induced androstenedione production in pTCs by regulating the steroidogenesis pathway., (Copyright © 2019 Elsevier Inc. 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.)

Endogenous chemicals specific to human metabolism have been suggested to be good candidates for markers of population size in wastewater-based epidemiology (WBE). So far, creatinine is the only endogenous chemical to be assessed against the criteria of in-sewer stability. This study thus aimed to evaluate the fate of three other endogenous compounds, 5-hydroxy indole acetic acid (5-HIAA), cortisol and androstenedione, under different sewer conditions using laboratory-scale sewer reactors. The results showed that while all compounds were stable in wastewater only (i.e. without biofilm), cortisol and androstenedione degraded quickly in sewers with the presence of sewer biofilms. The degradation followed first-order kinetics similar to that of creatinine. In contrast, 5-HIAA was relatively stable in sewer reactors. This study also recognised the impact of wastewater pH on the detectability of 5-HIAA using a LC-MS/MS direct injection method. In samples acidified to pH 2, the method did not allow routine detection/quantification of 5-HIAA whereas in non-acidified samples the method was sufficiently sensitive for routine quantification of 5-HIAA. The stability of 5-HIAA in sewers and the possibility to measure it using a simple and rapid analytical method corroborate that 5-HIAA may be a suitable biomarker for estimation of population size in WBE., (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.)