Concentrations Of Pregnenolone Research Articles

Multiple androgen pathways contribute to the steroid signature of adrenarche

ContextAdrenarche is a normal developmental event in mid-childhood characterized by increasing adrenal androgen secretion. The role of the classic androgen pathway has been well described in adrenarche, but the role of newer active androgens and additional androgen pathways is less clear. ObjectiveTo study the contribution of novel androgens and related steroid biosynthesis pathways to the development of adrenarche, and to identify additional steroid biomarkers of adrenarche. DesignA longitudinal study of children aged 6–8 years at baseline, followed up at ages 8–10 and 14–16 years. A total of 34 children (20 girls) with clinical and/or biochemical signs of adrenarche (cases) and 24 children (11 girls) without these signs (controls) at age 8–10 years were included. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. Main outcome measuresThirty-two steroids compartmentalized in progestagens, gluco- and mineralocorticoid pathways, and four androgen related pathways, including the classic, backdoor, 11-oxy, and 11-oxy backdoor pathways. ResultsThe classic and 11-oxy androgen pathways were more active, and serum concentrations of main androgens in the classic (dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione and androsterone) and 11-oxy (11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketoandrostenedione, and 11-ketotestosterone) pathways were higher in cases at ages 6–8 and 8–10 years. Pregnenolone concentrations at adrenarchal age (8–10 years) and cortisol concentrations at adolescence (14–16 years) were higher in cases. 11β-hydroxyandrosterone and 11-ketoandrosterone tended to be higher in cases with clinical signs compared to cases who had only biochemical evidence of adrenarche, albeit they were detected at low levels. In biomarker analyses, calculated steroid ratios with cortisol, cortisone, or 11-deoxycortisone as dividers were better classifiers for adrenarche than single steroids. Among these ratios, androstenedione/cortisone was the best. ConclusionsThe classic and 11-oxy androgen pathways are active in adrenarche. Children with earlier timing of adrenarche have higher serum cortisol levels at late pubertal age, suggesting that early adrenarche might have long-term effects on adrenal steroidogenesis by increasing the activity of the glucocorticoid pathway. Future studies should employ comprehensive steroid profiling to define novel classifiers and biomarkers for adrenarche and premature adrenarche.

Reduced urine pregnenolone concentration after clinical response in patients with depression: An open-label short-term prospective study

BackgroundIdentifying biological alterations in patients with depression, particularly those that differ between responders and non-responders, is of interest to clinical practice. Biomarker candidates involve neuroactive steroids, including pregnenolone (PREG) and allopregnanolone (ALLO). However, alterations in PREG and ALLO associated with treatment response are understudied. This study's main aim was to evaluate the effects of antidepressant treatment, clinical response, and treatment duration on PREG and ALLO in depression. Materials and MethodsIn a 4-week, open-label trial, participants were allocated randomly to the venlafaxine (n = 27) or mirtazapine (n = 30) group. Urine concentrations of PREG and ALLO were assessed through gas chromatography-mass spectrometry. Participants collected night urine between 10:30 p.m. and 8:00 a.m. Two primary outcomes were analyzed. Firstly, the effect of treatment (mirtazapine or venlafaxine), clinical response (operationalized through the Hamilton Depression Rating Scale), and time (baseline compared to 28 days) on the urine concentrations of PREG or ALLO in depression. Finally, the effect of clinical response and time on the urine concentration of PREG or ALLO, independently of the antidepressant given (mirtazapine or venlafaxine). Linear mixed models were carried out. ResultsThere was no significant difference in PREG and ALLO concentrations between baseline and 28 days in responders and non-responders when investigating the venlafaxine or the mirtazapine group. However, we found a significant reduction of urine PREG concentration after 28 days of treatment in responders who received either venlafaxine or mirtazapine (estimate = −0.56; p = 0.016; 95CI [−1.003; −0.115]; Cohen’s d = −0.61). ConclusionsOur main results indicate that responders in depression show reduced urinary PREG concentrations after 4-weeks of therapy, independently of the antidepressant used. More studies are needed to confirm these findings.

Analysis of salivary steroid hormones in boys with autism spectrum disorder

BackgroundAutism spectrum disorders (ASD) is a neurodevelopmental disorder with high incidence rate and difficult diagnosis. The purpose of this study was to explore whether salivary cortisol, dehydroepiandrosterone (DHEA) and pregnenolone can be used as biomarkers of ASD children.MethodsThe saliva samples of 55 boys with ASD were collected as the experimental group, and the saliva samples of 24 neurotypical boys were collected as the control group. The Child Behavior Checklist (CBCL), Autism Behavior Checklist (ABC), Social Responsiveness Scale (SRS), Repetitive Behavior Scale (RBS) were used to assess the severity of symptoms in boys with ASD. Cortisol, DHEA and pregnenolone concentrations in saliva were measured using an ABSSCIEX QTRAP® 6500 + LC/MS/MS system. SPSS 23.0 was used for statistical analysis. Comparisons between the two groups which conform to normal distribution were performed by T-test, and those which don’t conform to normal distribution were performed by Mann–Whitney U test. Correlation analysis between two variables was performed using Spearman's correlation analysis. Receiver operating characteristic curve (ROC) analysis was performed to evaluate the discriminatory sensitivity of each hormone between ASD and normal control groups. Logistic regression models were used to analyze whether DHEA and salivary pregnenolone can be used as a biomarker of ASD.ResultsThere were no significant differences in age, and weight between the ASD group and the normal control group. The ABC, SRS, RBS and CBCL scale scores in the ASD group were significantly higher than those in the normal control group. The salivary DHEA and pregnenolone concentrations in the ASD group were significantly higher than those in the normal control group, but there was no significant difference in cortisol. Spearman's correlation analysis showed that only pregnenolone associated with ABC. Logistic regression model analysis suggested that pregnenolone in saliva was an independent predictor of ASD. ROC analysis found that pregnenolone had good discrimination sensitivity between ASD and normal controls.ConclusionGave salivary preoperative a space for utilization as biomarker as number of cases are limited to this high expectation.

The Comprehensive Steroidome in Complete TSPO/PBR Knockout Mice under Basal Conditions.

The 18 kDa translocator protein (TSPO/PBR) is a multifunctional evolutionary highly conserved outer mitochondrial membrane protein. Decades of research has reported an obligatory role of TSPO/PBR in both mitochondrial cholesterol transport and, thus, steroid production. However, the strict dependency of steroidogenesis on TSPO/PBR has remained controversial. The aim of this study was to provide insight into the steroid profile in complete C57BL/6-Tspotm1GuWu(GuwiyangWurra)-knockout male mice (TSPO-KO) under basal conditions. The steroidome in the brain, adrenal glands, testes and plasma was measured by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We found that steroids present in wild-type (WT) mice were also detected in TSPO-KO mice, including pregnenolone (PREG), progestogens, mineralo-glucocorticosteroids and androgens. The concentrations of PREG and most metabolites were similar between genotypes, except a significant decrease in the levels of the 5α-reduced metabolites of progesterone (PROG) in adrenal glands and plasma and of the 5α-reduced metabolites of corticosterone (B) in plasma in TSPO-KO compared to WT animals, suggesting other regulatory functions for the TSPO/PBR. The expression levels of the voltage-dependent anion-selective channel (VDAC-1), CYP11A1 and 5α-reductase were not significantly different between both groups. Thus, the complete deletion of the tspo gene in male mice does not impair de novo steroidogenesis in vivo.

Saliva and plasma steroidome in mare during reproductive stages: A GC-MS/MS study

Precision livestock farming using omics approach to acquire precise and real-time data can help farmers in individual animal management and decision making. Since steroid hormones play a key role for the regulation of reproductive functions, reproduction management could be improved by characterizing the steroidome during reproductive stages. Moreover, saliva collection is a non-invasive, painless, inexpensive and easy sampling method. Thus, this prospective study proposes a steroidomic analysis in mare saliva during reproductive stages, that could help to identify potential biomarkers to accurately detect their reproductive stage in a welfare friendly production system, for real-time decision making at the individual animal level. Correlation between saliva and plasma steroidome was also investigated. Saliva and blood samples from 6 mares were collected in anestrus, in the follicular phase 3 days, 2 days and 1 day before ovulation and the day when ovulation was detected, in the luteal phase 6 days after ovulation and in gestation 18 days after ovulation and insemination. Steroidome analysis was performed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). We characterized 25 and 36 steroids in saliva and plasma respectively. Pregnenolone concentrations in saliva during gestation were significantly higher than during anestrus or follicular phase and tended to be higher than during luteal phase. Most of the 5α-reduced metabolites of progesterone showed higher salivary concentrations during the luteal phase 6 days post-ovulation and during gestation 18 days post-ovulation compared to anestrus and follicular phase. These steroids could be potential salivary biomarkers of the reproductive stage of the mare. Further studies with a greater number of animals are in progress to confirm the reliability of these potential candidate biomarkers and to develop field-friendly assays.

Bovine In Vitro Oocyte Maturation and Embryo Production Used as a Model for Testing Endocrine Disrupting Chemicals Eliciting Female Reproductive Toxicity With Diethylstilbestrol as a Showcase Compound.

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10–9 M, 10–7 M, 10–5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10–9 M or 10–7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10–5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e., DES), albeit at high concentrations. In principle, this method provides a valuable tool to assess the oocyte maturation process and early embryo development that can be used for reproductive toxicity screening and possibly EDC identification. Further studies should include EDCs with different mechanisms of action and additional endpoints to further demonstrate the applicability of the bovine oocyte model for chemical risk assessment purposes and EDC identification.

Development and validation of an LC-MS/MS assay for the quantification of allopregnanolone and its progesterone-derived isomers, precursors, and cortisol/cortisone in pregnancy.

Neuroactive steroids are potent neuromodulators that play a critical role in both maternal and fetal health during pregnancy. These stress-responsive compounds are reportedly low in women with perinatal depression and may be associated with poor pregnancy outcomes in animal models. Chronic stress is a risk factor for adverse birth outcomes. Simultaneous quantification of neuroactive steroids, in combination with stress hormones cortisol/cortisone, provides an opportunity to investigate the synergistic relationship of these analytes within the convenience of one assay. A simple, reliable, and sensitive method for quantifying these endogenous compounds is necessary for further research with the potential to advance clinical diagnostic tools during pregnancy. Analytes were extracted from serum with a simple protein precipitation using methanol and then separated and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After online extraction, analytes were separated using an Agilent Poroschell 120, 50 × 4.6mm, 2.7μm particle size, EC-C18 analytical column. The reliable quantification range was from 0.78 to 1000ng/mL. QC sample inter- and intraday trueness was between 90 and 110% while inter- and intraday imprecision was less than 10%. Extracted samples were stable up to 7days at 4°C and extraction recovery was above 95%. Serum samples from 54 women in pregnancy were analyzed using this method. Here, we provide a validated, fast, and specific assay with sufficient sensitivity that allows for simultaneous quantification of blood serum concentrations of allopregnanolone (3α-hydroxy-5α-pregnan-20-one), pregnanolone (3α-hydroxy-5β-pregnan-20-one), epipregnanolone (3β-hydroxy-5β-pregnan-20-one), pregnenolone, progesterone, cortisol, and cortisone in pregnancy for clinical study samples and clinical diagnostics.

Abstract 1250: ODM-208, a novel, small-molecule CYP11A1 inhibitor, demonstrates strong inhibition of steroid biosynthesis and antitumor activity in castration-resistant prostate cancer (CRPC) model

Abstract Several new treatments have been developed to target the androgen signaling axis in CRPC over the last decade. Despite the new treatment options, the challenge for managing CRPC is the limited duration of clinical benefit from treatments due to primary and acquired resistance. Altered steroid biosynthesis and adaptive signaling by endogenous steroids especially in AR mutated tumors have been suggested to be one of the resistance mechanisms. Inhibition of CYP11A1 enzyme (cytochrome P450scc) that catalyzes the first step in the steroidogenic pathway and leads to suppression of the synthesis of all steroid hormones and their precursors is expected to be a target with therapeutic potential in CRPC patients. Here we show, using multiple in vitro and in vivo models, selectivity and anti-tumor activity of a potent, orally bioavailable, inhibitor of CYP11A1 enzyme, ODM-208. The potency and specificity of ODM-208 was tested by using targeted screening assay and human recombinant CYP11A1 enzyme. The inhibition potential of ODM-208 in vitro was studied in H295R cells. The selectivity of ODM-208 to wide panel of different target classes including GPCRs, nuclear receptors, ion channels and enzymes was evaluated. Inhibition of steroidogenesis in vivo was studied in intact male dogs after repeated 4-week dosing of ODM-208, with and without corticosteroid replacement therapy. Steroid hormone concentrations in plasma and cell culture media were analyzed by LC-MS/MS. In vivo antitumor effects of ODM-208 were assessed in murine subcutaneous CRPC VCaP xenograft model. Strong reduction of androgen and other steroid hormones in H295R cells was shown with ODM-208, whereas with abiraterone clear increases in concentrations of pregnenolone, progesterone and corticosterone were observed. ODM-208 showed high selectivity to CYP11A1 and no significant off-target binding was detected in the broad panel. ODM-208 exposure-dependent decreases in plasma testosterone and cortisol concentrations were detected. ODM-208 inhibited substantially tumor growth in murine CRPC VCaP xenograft model. A phase 1/2 study of ODM-208 administered concomitantly with corticosteroid replacement and androgen deprivation therapy in metastatic CRPC subjects is ongoing (NCT03436485). In subjects with ODM-208 treatment all studied serum steroid hormones were decreased to under lower limit of quantification in the LC-MS/MS assays. These preclinical and clinical data suggest that ODM-208 has potential to provide clinical benefit to patients with treatment-resistant CRPC bearing altered steroid synthesis and/or mutated AR. Citation Format: Mari Karimaa, Henna Kettunen, Meri Ramela, Suvi Mansikka-Savolainen, Marcin Chrusciel, Outi Simola, Päivi Taavitsainen, Gerd Wohlfahrt, Petteri Rummakko, Annamari Vuorela, Karim Fizazi, Riikka Oksala. ODM-208, a novel, small-molecule CYP11A1 inhibitor, demonstrates strong inhibition of steroid biosynthesis and antitumor activity in castration-resistant prostate cancer (CRPC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1250.

Endogenous Progestogens and Colorectal Cancer Risk among Postmenopausal Women.

The role of progestogens in colorectal cancer development is poorly characterized. To address this, our group developed a highly sensitive assay to measure concentrations of seven markers of endogenous progestogen metabolism among postmenopausal women. The markers were measured in baseline serum collected from postmenopausal women in a case-cohort study within the breast and bone follow-up to the fracture intervention trial (B∼FIT). We followed women not using exogenous hormones at baseline (1992-1993) for up to 12 years: 187 women with incident colorectal cancer diagnosed during follow-up and a subcohort of 495 women selected on strata of age and clinical center. We used adjusted Cox regression models with robust variance to estimate risk for colorectal cancer [hazard ratios (HR), 95% confidence intervals (CI)]. High concentrations of pregnenolone and progesterone were not associated with colorectal cancer [quintile(Q)5 versus Q1: pregnenolone HR, 0.71, 95% CI, 0.40-1.25; progesterone HR, 1.25; 95% CI, 0.71-2.22]. A trend of increasing risk was suggested, but statistically imprecise across quintiles of 17-hydroxypregnenolone (Q2 to Q5 HRs, 0.75-1.44; P trend, 0.06). We used sensitive and reliable assays to measure multiple circulating markers of progestogen metabolism. Progestogens were generally unassociated with colorectal cancer risk in postmenopausal women. Our findings are consistent with most prior research on circulating endogenous sex hormones, which taken together suggest that sex hormones may not be major drivers of colorectal carcinogenesis in postmenopausal women.

Reduced concentrations of vaginal metabolites involved in steroid hormone biosynthesis are associated with increased vulvar vestibular pain and vaginal muscle tenderness in provoked vestibulodynia: An exploratory metabolomics study.

Provoked vestibulodynia (PVD) is a chronic vulvar pain disorder characterized by hypersensitivity and severe pain with pressure localized to the vulvar vestibule. Knowledge regarding pathophysiological mechanisms contributing to the etiology and production of symptoms in PVD remains incomplete but is considered multifactorial. Using a cross-sectional observational study design, data from untargeted metabolomic profiling of vaginal fluid and plasma in women with PVD and healthy women was combined with pain testing and brain imaging in women with PVD to test the hypotheses that women with PVD compared to healthy women show differences in vaginal and plasma metabolites involved in steroid hormone biosynthesis. Steroid hormone metabolites showing group differences were correlated with vulvar vestibular pain and vaginal muscle tenderness and functional connectivity of brain regions involved in pain processing in women with PVD to provide insight into the functional mechanisms linked to the identified alterations. Sensitivity analyses were also performed to determine the impact of hormonal contraceptive use on the study findings. Women with PVD compared to healthy controls had significant reductions primarily in vaginal fluid concentrations of androgenic, pregnenolone and progestin metabolites involved in steroidogenesis, suggesting localized rather than systemic effects in vagina and vulvar vestibule. The observed reductions in androgenic metabolite levels showed large effect size associations with increased vulvar vestibular pain and vulvar muscle tenderness and decreases in androgenic and progestin metabolites were associated with decreased connectivity strength in primary sensorimotor cortices. Women with PVD showed symptom-associated reductions in vaginal fluid concentrations of metabolites involved in the biosynthesis of steroid hormones previously shown to affect the integrity of vulvar and vaginal tissue and nociceptive processing. Deficiency of certain steroids may be an important mechanism contributing to the pathophysiology of symptoms in PVD may provide potential diagnostic markers that could lead to new targets for therapeutic intervention.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB−. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3β-hydroxysteroid dehydrogenase (3β-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3β-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− had an opposite effect on 3β-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11β1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB− extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Association of Circulating Progesterone With Breast Cancer Risk Among Postmenopausal Women

The role of endogenous progesterone in the development of breast cancer remains largely unexplored to date, primarily owing to assay sensitivity limitations and low progesterone concentrations in postmenopausal women. Recently identified progesterone metabolites may provide insights as experimental data suggest that 5α-dihydroprogesterone (5αP) concentrations reflect cancer-promoting properties and 3α-dihydroprogesterone (3αHP) concentrations reflect cancer-inhibiting properties. To evaluate the association between circulating progesterone and progesterone metabolite levels and breast cancer risk. Using a sensitive liquid chromatography-tandem mass spectrometry assay, prediagnostic serum levels of progesterone and progesterone metabolites were quantified in a case-cohort study nested within the Breast and Bone Follow-up to the Fracture Intervention Trial (n = 15 595). Participation was limited to women not receiving exogenous hormone therapy at the time of blood sampling (1992-1993). Incident breast cancer cases (n = 405) were diagnosed during 12 follow-up years and a subcohort of 495 postmenopausal women were randomly selected within 10-year age and clinical center strata. Progesterone assays were completed in July 2017; subsequent data analyses were conducted between July 15, 2017, and December 20, 2018. Circulating concentrations of pregnenolone, progesterone, and their major metabolites. Development of breast cancer, with hazard ratios (HRs) and 95% CIs was estimated using Cox proportional hazards regression adjusted for key confounders, including estradiol. Evaluation of hormone ratios and effect modification were planned a priori. The present study included 405 incident breast cancer cases and a subcohort of 495 postmenopausal women; the mean (SD) age at the time of the blood draw was 67.2 (6.2) years. Progesterone concentrations were a mean (SD) of 4.6 (1.7) ng/dL. Women with higher circulating progesterone levels were at an increased risk for breast cancer per SD increase in progesterone levels (HR, 1.16; 95% CI, 1.00-1.35; P = .048). The association with progesterone was linear in a 5-knot spline and stronger for invasive breast cancers (n = 267) (HR, 1.24; 95% CI, 1.07-1.43; P = .004). Among women in the lowest quintile (Q1) of circulating estradiol (<6.30 pg/mL) elevated progesterone concentrations were associated with reduced breast cancer risk per SD increase in progesterone levels (HR, 0.38; 95% CI, 0.15-0.95; P = .04) and increased risk among women in higher quintiles of estradiol (Q2-Q5; ≥6.30 pg/mL) (HR, 1.18; 95% CI, 1.04-1.35; P = .01; P = .04 for interaction). In this case-cohort study of postmenopausal women, elevated circulating progesterone levels were associated with a 16% increase in the risk of breast cancer. Additional research should be undertaken to assess how postmenopausal breast cancer risk is associated with both endogenous progesterone and progesterone metabolites and their interactions with estradiol.

Plasma steroid hormone concentrations and their relationships in Suffolk ewes during gestation and parturition.

In this study, we measured plasma concentrations of progesterone, pregnenolone, estradiol, estrone, estrone sulfate, and cortisol and analyzed the correlations between these hormones during gestation in 13 Suffolk ewes, the main breed in Japan. Progesterone increased during gestation and decreased a few days before parturition; however, this pattern was different in samples with high progesterone concentrations (P4 spike samples). This P4 spike was associated with a high pregnenolone concentration. Apart from the P4 spike, the progesterone change was similar to that in other sheep breeds. Pregnenolone increased during gestation and decreased after parturition. A significant correlation between progesterone and pregnenolone was observed a few days before parturition. Estrone sulfate and estradiol concentrations increased during gestation, but estrone did not. They increased shortly before parturition, and then decreased immediately after parturition. At parturition, the correlation between estrone and estrone sulfate was significantly stronger. Moreover, a strong correlation between estrone sulfate and estradiol was observed after parturition. Cortisol did not change during gestation and increased shortly before parturition. The results showed steroid hormone dynamics in normal pregnant Suffolk ewes, which were mostly in line with those of other sheep breeds. It should be noted that high progesterone concentrations altered the typical patterns.

Conformational selection dominates binding of steroids to human cytochrome P450 17A1

Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent Kd and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.

Sex‐dependent changes in neuroactive steroid concentrations in the rat brain following acute swim stress

Sex differences in hypothalamic‐pituitary‐adrenal (HPA) axis activity are well established in rodents. In addition to glucocorticoids, stress also stimulates the secretion of progesterone and deoxycorticosterone (DOC) from the adrenal gland. Neuroactive steroid metabolites of these precursors can modulate HPA axis function; however, it is not known whether levels of these steroids differ between male and females following stress. In the present study, we aimed to establish whether neuroactive steroid concentrations in the brain display sex‐ and/or region‐specific differences under basal conditions and following exposure to acute stress. Brains were collected from male and female rats killed under nonstress conditions or following exposure to forced swimming. Liquid chromatography‐mass spectrometry was used to quantify eight steroids: corticosterone, DOC, dihydrodeoxycorticosterone (DHDOC), pregnenolone, progesterone, dihydroprogesterone (DHP), allopregnanolone and testosterone in plasma, and in five brain regions (frontal cortex, hypothalamus, hippocampus, amygdala and brainstem). Corticosterone, DOC and progesterone concentrations were significantly greater in the plasma and brain of both sexes following stress; however, the responses in plasma were greater in females compared to males. This sex difference was also observed in the majority of brain regions for DOC and progesterone but not for corticosterone. Despite observing no stress‐induced changes in circulating concentrations of pregnenolone, DHDOC or DHP, concentrations were significantly greater in the brain and this effect was more pronounced in females than males. Basal plasma and brain concentrations of allopregnanolone were significantly higher in females; moreover, stress had a greater impact on central allopregnanolone concentrations in females. Stress had no effect on circulating or brain concentrations of testosterone in males. These data indicate the existence of sex and regional differences in the generation of neuroactive steroids in the brain following acute stress, especially for the 5α‐reduced steroids, and further suggest a sex‐specific expression of steroidogenic enzymes in the brain. Thus, differential neurosteroidogenesis may contribute to sex differences in HPA axis responses to stress.

Cortisol, progesterone, 17α[sbnd]OH[sbnd]progesterone, and pregnenolone in foals born from mare's hormone-treated for experimentally induced ascending placentitis

This study aimed to evaluate steroid hormones in foals born from mares treated for ascending placentitis with different combinations of trimethoprim-sulfamethoxazole (TMS), flunixin meglumine (FM), long-acting altrenogest (ALT) and estradiol cypionate (ECP) for ten consecutive days, starting two days after experimental induction of placentitis with Streptococcus zooepidemicus. Fourty-six pregnant mares and respective foals were assigned as healthy group (Control, n = 8) or treated groups as follows: TMS+FM (n = 8), TMS+FM+ALT (n = 8), TMS+FM+ALT+ECP (n = 6), TMS+FM+ECP (n = 6) and no treatment (NO TREAT n = 10). At delivery, foals were classified as high-risk or low-risk based on clinical and hematologic findings, and survival rates were recorded during the first week of life for comparisons across groups. Cortisol, progesterone, 17αOHprogesterone, and pregnenolone concentrations were determined via immunoassays in 31 of the 46 foals immediately after foaling (0 h), at 12, 24, 48 h, and seven days post-partum (168h). At birth, serum cortisol concentrations were higher in Control and TMS+FM+ECP foals than in remaining groups (p < 0.05). Foals in TMS+FM+ALT and TMS+FM groups had higher 17αOHprogesterone concentrations at 24 h and 48 h, respectively (p < 0.05). Pregnenolone concentrations were higher in TMS+FM than TMS+FM+ALT+ECP foals at 7 days (p < 0.05). High-risk and non-surviving foals had decreased concentrations of cortisol at parturition, but increased concentrations of progesterone from 0 h to 48 h. Pregnenolone and 17αOHprogesterone concentrations were increased and pregnenolone after 12 h in high-risk and non-surviving foals (p < 0.05). In conclusion, adding ECP to the treatment of experimentally-induced placentitis appears to improve foal viability and endocrine response. Cortisol and progestogen profiles were abnormal in high-risk and non-surviving foals, and those treated with ALT or TMS+FM only.

Regulation of steroid hormones in the placenta and serum of women with preeclampsia.

Preeclampsia (PE) is a pregnancy‑specific hypertensive syndrome that results in substantial maternal and fetal morbidity and mortality. The exact cause of PE has not been completely elucidate, although abnormal formation of the placenta has been considered. The placenta connects the developing fetus to the uterine wall, producing a large quantity of steroid hormones to maintain pregnancy. Although steroid hormones, particularly progesterone (P4) and estrogen (E2), in the serum of women with PE have been studied, steroidogenesis in the placenta has not well been established. The present study compared the concentrations of steroid hormones, including pregnenolone (PG), P4, dehydroepiandrosterone (DHEA), testosterone (T) and E2, in the serum and placenta of women with PE. PG, P4, DHEA and E2 concentrations tended to be decreased in PE serum and placentas, and the results were statistically significant for P4 and E2 in the serum. Quantification of genes associated with steroidogenesis in the placenta was performed, and the expression of the P4‑ and E2‑synthesizing enzymes testosterone 17‑β‑dehydrogenase 3 and 3 β‑hydroxysteroid dehydrogenase/δ5 4‑isomerase type 1 was reduced. Notably, aromatase, an enzyme required for the production of E2, was upregulated in the PE placenta, suggesting that steroidogenic enzymes may be dynamically regulated and may affect the symptoms of PE. In conclusion, the results of the present study suggested that the levels of steroid hormones, including P4 and E2, in the serum and placenta of women with PE are downregulated, which may be mediated by the regulation of steroidogenic enzyme expression in the PE placenta.

Steroid hormones and persistent organic pollutants in plasma from North-eastern Atlantic pilot whales

Persistent organic pollutants (POPs) are known to have endocrine disruptive effects, interfering with endogenous steroid hormones. The present study examined nine steroid hormones and their relationships with the concentrations of selected POPs in pilot whales (Globicephala melas) from the Faroe Islands, NE Atlantic. The different steroids were detected in 15 to all of the 26 individuals. High concentrations of progesterone (83.3–211.7pmol/g) and pregnenolone (PRE; 4.68–5.69pmol/g) were found in three adult females indicating that they were pregnant or ovulating. High androgen concentrations in two of the males reflected that one was adult and that one (possibly) had reached puberty. In males a significant positive and strong correlation between body length and testosterone (TS) levels was identified. Furthermore, positive and significant correlations were found between 4-OH-CB107/4’-OH-CB108 and 17β-estradiol in males. In adult females significant positive correlations were identified between PRE and CB149 and t-nonachlor, between estrone and CB138, -149, -187 and p,p’-DDE, between androstenedione and CB187, and between TS and CB-99 and -153. Although relationships between the POPs and the steroid hormones reported herein are not evidence of cause-effect relationships, the positive correlations between steroids and POPs, particularly in females, suggest that POPs may have some endocrine disrupting effects on the steroid homeostasis in this species.

Gross pathology, physiological and toxicological responses in relation to metals and persistent organic pollutants (POPs) burden in tilapia species from Ogun River, Nigeria

We have investigated gross pathology, physiological (steroid precursors) and toxicological responses (oxidative stress and phase II biotransformation) in relation to tissue contaminant burden in Tilapia species along the entire length (320 km) of Ogun River, Nigeria. The Ogun River is the longest and largest river in Southwestern Nigeria located along heavily industrialized cities and receives complex mixtures of effluents. A total of 1074 tilapias were collected from three sampling points (Abeokuta, Isheri and Ikorodu) and from an upstream control point (Igboho) and evaluated for gross pathological changes, hepatic transcript levels for oxidative stress and phase II biotransformation responses. Trace metal concentrations and POPs in muscle samples were analyzed using ICP-MS and GC-MS respectively. Evaluation of gross pathological changes showed a 50-, 33-, 17 and 0% prevalence of hepatic tumors at the Ikorodu, Abeokuta, Isheri and Igboho sites, respectively. Plasma concentrations of cholesterol and pregnenolone showed apparent significant decreases at downstream sites of the control point in both male and female fish (except for pregnenolone levels of male fish at Ikorodu). Inversely, gst, ugt-1, ZuCu-sod and sod significantly increased in fish collected from downstream sites, compared with the control site and these increases paralleled the significant increase in trace metal and POPs concentrations at these sites. PCA revealed a site related association between measured toxicological responses and contaminant burden, indicating a potential cause-and-effect relationship. Thus, the possible adaptation of Ogun River Tilapia species to contaminants may have significant consequences on cellular, physiological and biochemical processes regulating metabolism, growth, development and reproduction, and also have serious human health consequences, since the Ogun River is used for fisheries and domestic water supply for surrounding neighborhoods.

Differential effects of the 18-kDa translocator protein (TSPO) ligand etifoxine on steroidogenesis in rat brain, plasma and steroidogenic glands: Pharmacodynamic studies

Etifoxine is indicated in humans for treating anxiety. In rodents, besides its anxiolytic-like properties, it has recently shown neuroprotective and neuroregenerative activities. It acts by enhancing GABAA receptor function and by stimulating acute steroid biosynthesis via the activation of the 18-kDa translocator protein. However, the regulatory action of etifoxine on steroid production is not well characterized. In this work, we performed dose-response, acute and chronic time-course experiments on the effects of intraperitoneal injections of etifoxine on steroid levels in adult male rat brain and plasma analyzed by gas chromatography-mass spectrometry. Concentrations of pregnenolone, progesterone and its 5α-reduced metabolites were significantly increased in both tissues in response to 25 and 50mg/kg of etifoxine, as compared with vehicle controls, and reached maximal values at 0.5–1h post-injection. Daily injections of etifoxine (50mg/kg, 15days) kept them increased at day 15. Comparisons between steroidogenic tissues revealed that 1h after 50mg/kg of etifoxine treatment, levels of pregnenolone, progesterone and corticosterone were highest in adrenal glands and markedly increased together with their reduced metabolites. They were also increased by etifoxine in brain and plasma, but not in testis except for corticosterone and its metabolites. In contrast, testosterone level was significantly decreased in testis while with its 5α-reduced metabolites, it was unchanged in brain. Results demonstrate that the modulation of steroid concentrations by etifoxine is dependent on the type of steroid and on the steroidogenic organ. They further suggest that adrenal steroids upregulated by etifoxine make an important contribution to the steroids present in brain. This work provides a precise and complete view of steroids regulated by etifoxine that could be useful in therapeutic research.