The Journal of Steroid Biochemistry and Molecular Biology

Humans and other vertebrates contain two estrogen receptors (ERs), ER-alpha and ER-beta, which mediate the physiological actions of three estrogens: estrone (E1), estradiol (E2) and estriol (E3). Of these three estrogens, in vivo, E2 is the strongest transcriptional activator of ER-alpha and ER-beta, E1 is next most active, followed by E3. We studied transcriptional activation of human ER-alpha and ER-beta by E2, E1 and E3 in African green monkey kidney (COS-7) cells, which we compared with studies of estrogen stimulation of ER transcription in human em-bryonic kidney (HEK-293) cells. To our surprise, in COS-7 cells, E3 had the lowest half-maximal response (EC50) for human ER-alpha and ER-beta than either E2, which was second most active estrogen, or E1. In contrast, for human ER-alpha and ER-beta transfected into HEK-293 cells, E2 was the most active estrogen, followed by E1 and E3. Similar results were found in COS-7 cells and HEK-293 cells transfected with elephant shark ER-alpha and ER-beta. Thus, under some conditions, E3 is a more active estrogen than either E2 or E1. This suggests that E3 may be a novel physiological ligand for the ER in some mammalian cells.

The steroidal module of the Athlete Biological Passport (ABP) targets the use of exogenous androgenous anabolic steroids (EAAS) in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive OCP cycles (8 weeks) in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and/or epitestosterone (E) were below the limit of quantification of 1 ng/mL in 62% of the samples. Biomarkers variability ranged between 31% and 41%, with a significantly lesser variability for ratios (with the exception of T/E (41%)): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5-androstane-3,17{beta}-diol/5{beta}-androstane-3,17{beta}-diol (p < 0.001). In serum, variability for testosterone (T; 24%), androstenedione (A4; 23%), dihydrotestosterone (DHT; 19%) and T/A4 (16%) was significantly lower than urinary biomarkers (p < 0.001). Urinary A/Etio increased by > 18% after the first two weeks (p < 0.05) following blood loss. In contrast, T (0.98 nmol/L during the first week), and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p<0.05), respectively in the following weeks. Our results outline steroidal variations during the OCP cycle highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear detrimental impact on the subsequent interpretation of steroidal variations for the ABP. With a greater analytical sensitivity and lesser variability for steroids in serum vs. urine in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.

Deficiency of Cytochrome P450 Steroid 21-hydroxylase (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction along with functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in the Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of 21OH deficiency.

Understanding the tissue-specific distribution and metabolism of sex steroids is critical for elucidating their physiological roles and pathological alterations. However, existing in vivo approaches often require surgical castration to suppress endogenous hormones, disrupting systemic endocrine balance and limiting physiological relevance. Here, we present a novel mass spectrometry method combining systemic administration of deuterium-labeled sex steroids with high-sensitivity gas chromatography-tandem mass spectrometry (GC-MS/MS) to simultaneously quantify exogenous, endogenous, and metabolized steroid species in serum and multiple tissues of non-castrated mice. This approach enables temporally resolved tracking of steroid uptake and biotransformation without perturbing endogenous hormonal status. We demonstrate the methods high sensitivity, specificity, and capacity to reveal tissue-specific steroid metabolism previously inaccessible in physiological models. Our technique offers a versatile platform for studying steroid dynamics in vivo and has broad applications in endocrinology, neurobiology, and pharmacology. TeaserDeuterium tracing illuminates hidden steroid pathways in living animals.

Endocrine-disrupting chemicals (EDCs) may impact the development of Prostate Cancer (PCa) by altering the steroid metabolism. Although their exact mechanism of action in controlling tumor growth is not known, EDCs may inhibit steroidogenic enzymes such as Cytochrome P450 c17 (CYP17A1) or aromatase (CYP19A1) involved in the production of Androgens or Estrogens. High levels of circulating androgens are linked to PCa in men and Polycystic Ovary Syndrome (PCOS) in women. Essential Oils or their metabolites (EOs) like lavender oil and tea tree oil have been reported to act as potential EDCs and contribute towards sex steroid imbalance in case of prepubertal gynecomastia in boys and premature thelarche in girls due to the regular exposure to lavender-based fragrances among Hispanic population. We screened a range of EO components to determine their effects on CYP17A1 and CYP19A1 Computational docking was performed to predict the binding of EOs with CYP17A1 and CYP19A1 and functional assays were done using the radiolabeled substrates or Liquid Chromatography high-resolution Mass Spectrometry and cell viability assays were carried out in LNCaP cells. Many of the tested compounds bind close to the active site of CYP17A1, and (+)-Cedrol had the best binding with CYP17A1 and CYP19A1. Eucalyptol, Dihydro-{beta}-Ionone & (-)--pinene showed 20% to 40% inhibition of dehydroepiandrosterone production; and some compounds also effected CYP19A1. Extensive use of these EOs in various beauty and hygiene products is common, but only a limited knowledge about their potential detrimental side effects exists. Our results suggest that prolonged exposure to some of these essential oils may result in steroid imbalances. On the other hand, due to their effect on lowering androgen output, ability to bind at the active site of steroidogenic cytochrome P450s, these compounds may provide design ideas for the novel compounds against hyperandrogenic disorders such as PCa and PCOS.

1Sex steroid hormones are not exclusively localised in the circulation and can be found in numerous extragonadal tissues, in concentrations unrelated to the circulating fraction. Existing methodology to measure intramuscular steroid hormone concentrations includes both immune-based assays and liquid chromatography-mass spectrometry (LC-MS), the gold standard for hormone measurements. To date, no LC-MS based methods validation has been published on the measurement of intramuscular sex steroid hormones, despite clear biological relevance. Here, we describe the development and validation of a simple, high-throughput LC-MS Orbitrap method for the measurement of 10 intramuscular sex steroid hormones, including pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, epitestosterone, dihydrotestosterone, oestrone, oestradiol, and oestriol. In brief, isotope labelled standards were added to 5-6 milligrams of lyophilised muscle tissue, homogenised and extracted with ethyl acetate. The extracts were dried down and sequentially derivatised with 1-methylimidazole-2-sulfonyl chloride and hydroxylamine hydrochloride to target both the phenolic hydroxyl groups and ketone groups. The limit of detection was 1.0 {+/-} 1.0 pg/mg (range 0.36 - 3.26 pg/mg), with a R2 > 0.99 for all analytes. Matrix effects were 90-110% for all analytes except for dihydrotestosterone (143.6%), and precision was <10 CV% for all analytes in the presence of a muscle matrix. Our method allows for 20-40 samples to be prepared in [~]4 h, with a sample data acquisition time of 13 minutes. Moreover, our method provides the opportunity for specific analysis of steroid hormone concentrations in skeletal muscle, allowing target tissue specificity instead of relying on proxy measures from the circulation.

ObjectivesGlucocorticoids, mediated by the activation of the HPA axis, affect metabolic responses, insulin resistance, lipolysis and body fat distribution. Dehydroepiandrosterone-sulfate (DHEA-S) is a hormone produced by the adrenal glands and a precursor of sex hormones. The balance and interaction between cortisol and DHEA-S can significantly affect body composition. This study aimed to investigate the relationship between cortisol and DHEA-S levels, cortisol/DHEA-S ratio, and body composition in Korean men and women. MethodsIn total, 802 adults participated in this study between January 2018 and March 2023. Socio-demographic data and lifestyle factors were assessed using questionnaires. Body composition, clinical blood pressure, and metabolic variables, including cortisol and DHEA-S levels, were assessed. Cortisol and DHEA-S scores were analyzed in relation to height, body weight(BW), body mass index(BMI) and waist circumference(WC) according to age and sex. ResultsParticipants had a mean age of 52.6{+/-}11.7 years. Cortisol levels adjusted for age and gender were negatively correlated with BW, WC and BMI. This result was more significant in women than in men. DHEA-S levels were positively correlated with height, BW and WC after adjusting for age. The cortisol/DHEA-S ratio was associated with lower height and BW after adjusting for gender. Logistic regression for cortisol, DHEA-S and the cortisol/DHEA-S ratio in the prediction of central obesity was significant for men after adjusting for age and BMI. ConclusionsElevated cortisol concentrations are associated with lower adiposity. DHEA-S levels were positively correlated with height and body mass. The prediction of central obesity was associated with cortisol and the cortisol/DHEA-S ratio in men and negatively associated with DHEA-S.

Vitamin D deficiency presents a significant public health concern, especially in regions where reference intervals from Western populations may not apply due to differences in sun exposure and ethnicity. This study aimed to establish population-specific reference intervals for serum 25-hydroxyvitamin D [25(OH)D] and to determine a deficiency cutoff for healthy adults in Bangladesh. In a cross-sectional design, we assessed serum 25(OH)D and intact parathyroid hormone (iPTH) levels in 125 coastal fishermen (Group 1) and 371 urban residents (Group 2), comprising healthy adults aged 18 years or older. Group 1 served as a reference to establish baseline vitamin D levels, while Group 2 data aided in determining the deficiency cutoff. Measurements were conducted using chemiluminescent immunoassay, and reference intervals were calculated according to Clinical and Laboratory Standards Institute (CLSI) Guidelines C28-A3. The deficiency cutoff was identified at the deflection point of iPTH levels. Results indicate a reference interval for serum 25(OH)D of 15.88-45.27 ng/ml among coastal fishermen. Among urban residents, mean serum 25(OH)D was 21.53 {+/-} 15.98 ng/ml, with iPTH levels showing significant increases below 12.16 ng/ml (95% CI: 11.04-13.28), establishing this as the deficiency cutoff. Urban residents exhibited significantly lower vitamin D levels than coastal fishermen (21.53 ng/ml vs. 27.36 ng/ml, p < 0.001). Limitations include potential selection bias due to convenience sampling and the use of chemiluminescent immunoassay instead of the gold-standard LC-MS/MS assay. This study provides the first population-specific reference intervals for serum 25(OH)D in Bangladesh, accounting for unique sun exposure patterns and ethnic factors, and sets a deficiency threshold at 12.16 ng/ml. These findings are critical for guiding targeted interventions against vitamin D deficiency in this region.

LH and FSH are two important hormones in the regulation of granulosa cells. Their effects are mediated mainly by cAMP/PKA signaling, bit the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated as well. We studied the involvement of the ERK cascade in LH and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production, downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased-expression of the steroidogenic acute regulatory (StAR) protein, a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is downregulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis, but also activates downregulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents, may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysone receptors (EcRs) and at least one membrane GPCR receptor (DopEcR) and displays numerous pharmacological effects in mammals. However, its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ER{beta} receptor. The goal of our study was to better understand 20E mechanism of action. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors. 17{beta}-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and indeed this mechanism may explain the close similarity between angiotensin-(1-7) and 20E effects. Our findings open a lot of possible therapeutic developments by stimulating the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

Adipogenesis involves adipocyte differentiation and synthesis and storage of fats. PPAR-{gamma} is the master regulator of adipogenesis and regulates genes for adipocyte differentiation, lipogenesis, adipocyte survival and adipokine secretion. Central carbon metabolism (CCM) comprises of three key pathways, glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. CCM utilizes carbon sources to provide energy and building blocks for lipogenesis. Although several targets of PPAR-{gamma} have been identified in the CCM pathways, the exact process of how PPAR-{gamma} modulates adipogenesis via CCM remains elusive. In this study, we used real time-qPCR and metabolic arrays for CCM to understand the effect of PPAR activation by PPAR-{gamma} agonist 15d-PGJ2 on CCM and its role in adipogenesis. Our data show that PPAR-{delta} is likely the target of 15d-PGJ2 and the key modulator of adipogenesis in human SGBS adipocytes at least under current experimental conditions. Further studies are warranted to fully understand the regulation of CCM in adipocytes.

BACKGROUNDAdrenal insufficiency is primarily treated with replacement of cortisol, which is the predominant circulating glucocorticoid. Human adrenals also secrete corticosterone and emerging evidence suggests this may be a safer glucocorticoid replacement therapy. However, little is known about corticosterone in humans, particularly related to its metabolism. METHODSTo investigate the secretion and metabolism of corticosterone in comparison with cortisol, we: 1) investigated the diurnal rhythm of circulating cortisol/ corticosterone in 7 healthy volunteers; 2) quantified A-ring reduction of both hormones in human hepatic cytosol and 3) measured glucocorticoid metabolites in vivo in 24 healthy men; 4) determined the pharmacokinetics of corticosterone via intravenous infusion of 2,2,4,6,6,17,21,21-[2H]8-corticosterone; 5) assessed the response of corticosterone and cortisol to 1mcg ACTH in 279 healthy volunteers. RESULTSThe natural diurnal rhythm of corticosterone closely mirrored that of cortisol, and accounted for [~]3% of total circulating glucocorticoid concentrations. Daily corticosterone production, as measured through urinary steroid profiling, was approximately 10-fold lower than cortisol, and corticosterone demonstrated substantially greater metabolism by both 5- and 5{beta}-reductase than cortisol. In keeping with greater metabolism, the half-life of corticosterone was 28.5 {+/-} 3.3 minutes. Finally, corticosterone demonstrated a greater relative rise in response to ACTH than cortisol, particularly in men, revealing sex-specific differences. CONCLUSIONSCorticosterone is a dynamic glucocorticoid with faster metabolism and greater response to stimulation than cortisol in humans. These data raise the possibility of distinct roles for these two glucocorticoids and highlight important pharmacokinetic differences with implications for the therapeutic potential of corticosterone replacement in humans.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

Infertility, defined as the inability to achieve pregnancy after 12 months of unprotected intercourse, is a major reproductive health concern. In females, hyperandrogenism often contributes to polycystic ovarian syndrome (PCOS), a leading cause of infertility. Although clomiphene and letrozole are widely used as ovulation inducers, their individual efficacy is limited, and the potential benefit of combined therapy remains unclear. This study investigated the effects of clomiphene-letrozole co-administration on gonadotrophic hormones, inflammatory cytokines, antioxidant status, and ovarian histomorphology in a rat model of hyperandrogenism. Thirty female Wistar rats were randomised into five groups (n=6). Group A received saline, while groups B-E were administered testosterone enanthate (10 mg/kg, subcutaneous injection) for 35 days to induce PCOS. Group B served as PCOS control, while groups C, D, and E were additionally treated with clomiphene (100 {micro}g/kg), letrozole (5 mg/kg), or their combination, respectively, for 10 days from day 36. Hormonal assays, cytokine profiling, antioxidant measurements, and ovarian histology were performed. Results showed that the co-administration significantly reduced body and ovary weights, lowered glucose levels, and improved oestradiol and follicle-stimulating hormone profiles compared with PCOS controls. Combination therapy also enhanced antioxidant capacity, reduced lipid peroxidation, and modulated inflammatory cytokines by lowering IL-1{beta} and TNF- while elevating IL-10. Histological evaluation revealed cystic follicles with basement membrane thickening in the PCOS control, consistent with ovarian hyperstimulation; and a reversal with clomiphene and or letrozole treatment. In conclusion, clomiphene-letrozole co-administration demonstrated superior benefits over monotherapy in modulating endocrine, oxidative, and inflammatory parameters, suggesting a potential therapeutic advantage in ovulation induction for PCOS-related infertility.

IntroductionPolycystic ovary syndrome (PCOS) is a common disorder that is not fully understood. Multiple hormonal and metabolic factors impact on disease pathophysiology resulting in various phenotypic characteristics among the PCOS population. Luteinizing hormone beta subunit (LHB) (protein ID P01229) is mapped on (chr19p13.3) and consists of three exons. Luteinizing hormone (LH) has a central role in stimulation ovarian steroidogenesis, in particular androgen production, and the promotion of ovulation. ObjectivesTo determine if genetic variations of LHB are associated with PCOS among Sudanese families. MethodsA prospective laboratory based cross-sectional study to examine genetic mutations in LHB that associate with PCOS in families (cases; n=35 families, 90 females and controls; n=11 families, 30 females) in Khartoum State, Sudan. Quantitative enzyme linked immuno-sorbent assay (ELISA) and polymerase chain reaction (PCR) with Sanger sequencing were used to analyze biochemical parameters and detect polymorphisms. Protein structure and function bioinformatics analysis was conducted using standard software. ResultsPCOS cases had significantly different biochemical parameters from the controls (LH: p<0.001; testosterone: p<0.001; fasting glucose: p=0.02; insulin: p=0.01; triglycerides: p=0.03; total cholesterol: p<0.001; high density lipoprotein (HDL): p=0.012;low density lipoprotein (LDL): p<0.001). There were no differences in follicle stimulating hormone (FSH) (p=0.984) or prolactin (p=0.068). Sanger sequencing revealed 5 single nucleotide polymorphisms (rs5030775, A18T; rs746167425, R22K; rs1800447, W28R; rs35270001, H30R; and rs34349826, I35T) located on (exon 2) of LHB gene that were statistically correlated with serum LH, Testosterone and insulin levels among PCOS families. ConclusionThis is the first molecular family-based study in Sudan exploring the genetics of the LHB gene in women manifesting PCOS. These novel mutations give further information about the role of genetic inheritance and may explain some of the altered ovarian function and responses in women with PCOS.

Estrogen and estrogen metabolites are commonly measured in human plasma and serum, but there exist almost no reports of estrogen measured in human stool. This methodological limitation in turn limits our understanding of the relationship between systemic and intestinal estrogen. We thus developed a highly sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for measuring free and conjugated forms of 15 estrogens and estrogen metabolites in human stool and plasma. We first investigated human stool and plasma estrogen in healthy control males; follicular and luteal phase premenopausal females; and postmenopausal females. Most estrogens were present in the plasma and stool of all groups, and plasma estrogen levels correlated with stool estrogen levels. In stool, estrogens were higher in premenopausal females, with estrogen levels rising across the menstrual cycle. We further combined these measures with shotgun metagenomic sequencing of the stool microbiomes. The level of estrogen deconjugation enzyme gene copy number (beta-glucuronidase + arylsulfatase) was higher in premenopausal females; while the gene copy numbers of beta-glucuronidase + arylsulfatase, but not beta-glucuronidase alone, correlated with reactivated stool estrogen in all groups. Moreover, deconjugation enzyme gene copy number correlated with plasma total estrogen in males and with individual plasma estrogen metabolites in all groups. These results support the hypothesis that gut microbial beta-glucuronidase and arylsulfatase control the reactivation of gut estrogen while modulating systemic levels through the uptake and recirculation of reactivated estrogen.

ContextThe body has evolved homeostatic mechanisms to maintain free levels of Ca+2 and 1,25-dihydroxyvitamin D [1,25(OH)2D] within narrow physiological ranges. Clinical guidelines emphasize important contributions of PTH in maintaining this homeostasis. ObjectiveTo investigate mechanisms of homeostatic regulation of vitamin D (VitD) metabolism and to apply mechanistic insights to improve clinical assessment of VitD status. DesignCrossover clinical trial studying participants before and after VitD3-supplementation. SettingCommunity. Participants11 otherwise healthy individuals with VitD-deficiency (25-hydroxyvitamin D [25(OH)D] [&le;]20 ng/mL). InterventionsVitD3-supplements (50,000 IU once or twice a week depending on BMI, for 4-6 weeks) were administered to achieve 25(OH)D[&ge;]30 ng/mL. ResultsVitD3-supplementation significantly increased mean 25(OH)D by 2.7-fold and 24,25-dihydroxyvitamin D [24,25(OH)2D] by 4.3-fold. In contrast, mean levels of PTH, FGF23, and 1,25(OH)2D did not change. Mathematical modeling suggested that 24-hydroxylase activity was maximal for 25(OH)D[&ge;]50 ng/mL and achieved a minimum ([~]90% suppression) with 25(OH)D<10-20 ng/mL. The 1,25(OH)2D/24,25(OH)2D ratio better predicted modeled 24-hydroxylase activity (h) ({rho}=-0.85; p=0.001) compared to total plasma 25(OH)D ({rho}=0.51; p=0.01) and the 24,25(OH)2D/25(OH)D ratio ({rho}=0.37; p=0.3). ConclusionsSuppression of 24-hydroxylase provides a first line of defense against symptomatic VitD-deficiency by decreasing metabolic clearance of 1,25(OH)2D. The 1,25(OH)2D/24,25(OH)2D ratio provides a useful index of VitD status since it incorporates 24,25(OH)2D levels and therefore, provides insight into 24-hydroxylase activity. When VitD availability is limited, this suppresses 24-hydroxylase activity - thereby decreasing the level of 24,25(OH)2D and increasing the 1,25(OH)2D/24,25(OH)2D ratio. Thus, an increased 1,25(OH)2D/24,25(OH)2D ratio signifies triggering of homeostatic regulation, which occurs at early stages of VitD-deficiency.

Humans and other vertebrates contain two estrogen receptors (ERs), ER and ER{beta}. Among cartilaginous fish (sharks, rays, skates), which are chondrichthyans that evolved about 425 million years ago, only activation by steroids of ER{beta} orthologs has been characterized. To remedy this gap in understanding estrogen signaling in chondrichthyans, we studied estrogen activation of orthologs of human ER and ER{beta} from elephant shark (Callorhynchus milii). Unexpectedly, we found that C. milii contained three estrogen-responsive ER genes: ER1 (596 amino acids), ER2 (600 amino acids), and ER3 (599 amino acids) with strong sequence similarity to each other. We also found an estrogen-unresponsive gene, ER4 (561 amino acids), with a 39 amino acid deletion in the DNA-binding domain. An estrogen-responsive ER{beta} ortholog (580 amino acids) also was present in C. milii. The three active C. milii ERs are of similar length to human ER (595 amino acids); however, C. milii ER{beta} is longer than human ER{beta} (530 amino acids). We studied transcriptional activation of ER and ER{beta} by estradiol (E2), the main reproductive estrogen in humans. We also studied estrone (E1), the main postmenopausal estrogen, and estriol (E3), which is synthesized during pregnancy. We determined the half-maximal response (EC50) and fold-activation to E2, E1, and E3 of C. milii ER1, ER2, ER3, and ER{beta}. Among these estrogens, E2 had the lowest EC50 for all four ERs. Fold-activation by E2 and E3 was similar for ER1, ER2, ER3, and ER{beta}. Overall, estrogen activation of C. milii ER and ER{beta} was similar to that for human ER and ER{beta}, indicating substantial conservation of the vertebrate ER during the 425 million years since the divergence of cartilaginous fish and humans from a common ancestor.

BackgroundBicalutamide is a potential anti-androgen for transgender individuals with feminizing embodiment goals, but use has been limited because of hepatotoxicity in cisgender men with prostate cancer. This study compared transaminase changes in transfeminine adolescents and young adults (AYA) using low-dose bicalutamide with individuals using other methods of androgen blockade. MethodsA retrospective analysis was conducted using electronic health record data for patients starting gender affirming hormone therapy with at least 10 months of follow-up data between 2015 and 2023. Linear mixed models compared change in ALT and AST from baseline and maximum ALT and AST values in bicalutamide and comparison groups. Secondary outcomes included % individuals with ALT and AST elevation more than 1, 2, or 3 times the upper limit of normal (ULN) (Fishers exact test), standardized mean estradiol dose by group (t test), and Tanner staging of breast tissue by group (Fishers exact test). ResultsEighty-four transfeminine AYA (median age 18) taking bicalutamide were compared to 69 transfeminine AYA (median age 19) taking GnRH agonists, spironolactone or no agent in addition to estradiol. In linear mixed models adjusted for baseline age, BMI, baseline ALT or AST, and alcohol use, there was no difference in delta or maximum ALT or AST in bicalutamide and comparison groups. No individuals had an AST or ALT level > 3x ULN. Estradiol doses and Tanner stages were similar between groups in a subgroup analysis of individuals receiving pediatric care. ConclusionBicalutamide was not associated with significant change in transaminases as compared with other anti-androgen regimens over one year. Bicalutamide appears to be a safe anti-androgen for transfeminine individuals at low dose with close monitoring and deserves further study.

Androgen excess in women is associated with the development of PCOS and its abnormalities. The Hypothalamus Pituitary Ovarian axis signaling is altered with excessed androgens, leading to anovulation and infertility. Previous studies in the lab have shown that AR signaling in the pituitary alters gonadotrophin release. Hence, the present pioneering study was an approach to determine the transcriptomic changes responsible to the phenotype seen with DHT excess. RNA seq data showed that 583 genes were differentially regulated by DHT in pituitary, of which 344 were upregulated and 239 downregulated. Pathways involved for these genes included endoplasmic reticulum, Golgi apparatus, calcium signaling and vesicles. Meanwhile, Transcriptional factor analysis showed that majority of the genes changed had Androgen responsive elements.