Journal of the Endocrine Society

Pregnancy induces or represses hepatic drug metabolism. Whether pregnancy affects hepatic drug transport is unexplored. We previously showed that a cocktail of pregnancy-related hormones (PRHC) induces mRNA expression and activity of sodium/taurocholate cotransporting polypeptide (NTCP), organic anion transporter 2 (OAT2), and organic cation transporter 1 (OCT1, mRNA only) in differentiated HepaRG cells. Here, using HepaRG cells, we identified cortisol as the hormone primarily responsible for this induction and explored the underlying mechanisms. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated knockdown studies in HepaRG cells showed that the glucocorticoid receptor (GR) is the primary mediator of this response. GR knockdown markedly attenuated cortisol-induced NTCP, OAT2, and OCT1 mRNA expression and activity. Cortisol also induced the mRNA expression of regulatory factors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and hepatocyte nuclear factor (HNF) 4 alpha (HNF4). HNF4 knockdown selectively attenuated OAT2 and OCT1 induction, whereas HNF1 knockdown enhanced NTCP induction, attenuated OCT1 induction, and reduced basal organic anion transporting polypeptide 1B1 (OATP1B1) expression. In contrast, knockdown of CAR or PXR did not significantly alter cortisol-mediated transporter regulation. These data identify cortisol as the principal PRH driving regulation of the hepatic OAT2, NTCP, and OCT1 in HepaRG cells and indicate that this response is mediated primarily by GR, with selective downstream contributions from HNF4 and HNF1. These findings provide mechanistic insights into pregnancy-associated changes in hepatic transporter-mediated drug disposition, including when antenatal corticosteroids are administered to pregnant women to prevent respiratory distress syndrome in their prematurely born infants. Significance StatementThe extent and mechanisms by which pregnancy-related hormones regulate hepatic uptake transporters remain poorly defined. This study identifies cortisol as the principal pregnancy-related hormone driving NTCP, OAT2, and OCT1 induction in HepaRG cells and shows that this response is mediated primarily through GR, with transporter-specific contributions from HNF4 and HNF1.

Background: C-terminal binding protein 2 (CtBP2) has been implicated in metabolic regulation, but its association with specific measures of adiposity and lipid profiles in humans remains unclear. This study examined the relationship between circulating CtBP2 levels and key components of metabolic syndrome, focusing on body fat distribution and lipid markers. Methods: Data from 508 participants (259 men, 249 women) from a publicly available dataset were analyzed. Serum CtBP2 concentrations were measured using ELISA. Associations with obesity markers (BMI, waist circumference, waist-to-hip ratio) and lipid profiles (triglycerides, HDL cholesterol) were assessed using Spearman correlation and linear regression, adjusting for age and sex. Results: CtBP2 levels showed weak but statistically significant positive correlations with all measures of adiposity, with the strongest association observed for waist circumference ({rho} = 0.150, p < 0.001), followed by BMI ({rho} = 0.120, p = 0.007) and waist-to-hip ratio ({rho} = 0.098, p = 0.027). No significant correlations were found with triglycerides or HDL cholesterol. In the regression model predicting BMI, age, and sex were significant predictors, while CtBP2 demonstrated a trend toward association ({beta} = 0.080, p = 0.052). Conclusion: Circulating CtBP2 appears to be modestly associated with measures of adiposity, particularly abdominal fat, but not with lipid abnormalities. These findings suggest a potential role for CtBP2 in obesity-related metabolic dysregulation and underscore the need for further mechanistic studies to clarify its clinical relevance.

Background: Recent primary aldosteronism (PA) guidelines proposed probability-based stratifications, and use of aldosterone suppression testing, to predict lateralizing PA subtype. This guideline framework was based on very low-quality evidence. Methods: The discriminatory capacity of guideline-endorsed probability frameworks for PA subtyping were evaluated in this retrospective study of 319 PA patients, from two large tertiary centers in Bangkok, Thailand, who underwent subtyping assessments regardless of probability status. PA subtypes were determined by adrenal venous sampling (AVS) and/or post-adrenalectomy outcomes using PASO criteria. The main objectives were to evaluate the accuracy of predicting PA subtype using: 1) guideline-endorsed classification to high, intermediate, and low probabilities of lateralization; and 2) the seated saline suppression test (SST). Results: The majority of PA patients were characterized as having intermediate probability for lateralizing PA (75%); however, lateralizing PA was ultimately confirmed in 61-78% of all patients, regardless of guideline-based probability classification. The vast majority of SST results were positive using guideline-derived criteria, regardless of probability stratification or ultimate subtype: 89.3% of patients with lateralizing PA and 80.6% of those with bilateral PA had a positive SST. Among patients with intermediate probability of lateralizing PA, where guidelines specifically endorse the value of SST, the SST had a sensitivity of 89.4% and specificity of 22.0% for detecting lateralizing PA, with 78.0% false-positive and 10.6% false-negative rates. Consistently, post-SST aldosterone concentrations exhibited near-complete overlap between those with and without lateralizing PA. Conclusion: Guideline-endorsed probability frameworks, and the use of SST, lacked discriminatory capacity to predict PA subtype.

Polyendocrine metabolic ovarian syndrome (PMOS), previously called polycystic ovary syndrome (PCOS) - the most common reproductive endocrinopathy in women of reproductive age, is frequently associated with chronic low-grade inflammation and immune dysregulation. Beyond hyperandrogenism and ovulatory dysfunction, women with PMOS exhibit reduced regulatory T cell (Treg) levels and impaired STAT5 phosphorylation. This study investigates the molecular basis of the defective STAT5 signalling in PMOS. No significant difference in plasma IL2 levels is observed in PMOS women versus normal subjects. Analysis of 102 PMOS patients and 102 controls reveals significantly decreased JAK2 expression alongside increased expression and activity of the phosphatases PTP1B (Protein Tyrosine Phosphatase 1B), TCPTP (T cell Protein Tyrosine Phosphatase), and DUSP4 (Dual Specificity Protein Phosphatase), in leukocytes of PMOS women. In isolated Tregs, only PTP1B and DUSP4 were significantly upregulated. DUSP4 expression positively correlates with serum testosterone and luteinizing hormone levels, linking hormonal imbalance with immune defects. Functional experiments show that silencing PTP1B and DUSP4 enhances IL2-induced Treg generation. Our collective findings identify phosphatase-mediated inhibition of STAT5 signalling as a key mechanism underlying Treg deficiency in PMOS and highlight PTP1B and DUSP4 as potential therapeutic targets to restore immune tolerance and improve reproductive outcomes.

ObjectiveThe aryl hydrocarbon receptor (AhR) pathway primarily mediates pollutant responses by activating xenobiotic metabolism enzymes like cytochrome P450 1A1 and 1A2 (CYP1A). Although AhR has also been implicated in systemic metabolic dysfunction and is inducible in pancreatic islets, its role in islet physiology remains unclear. MethodsWe analyzed a publicly available bulk human islet transcriptomic dataset to identify pathways associated with CYP1A1 expression. We also assessed islet responses to the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and glucolipotoxicity (GLT) in vitro using two mouse models: a global Cyp1a1/1a2 double knockout (CypKO) model, which disrupts canonical AhR-CYP1A signaling in whole islets, and a {beta}-cell-specific Ahr knockout ({beta}AhrKO) model, which abolishes AhR signaling selectively in {beta}-cells. We then examined the role of {beta}-cell Ahr in early adaptation to high-fat diet (HFD) feeding in vivo. ResultsXenobiotic and nutrient metabolism pathways were enriched in donors with high CYP1A1 expression. Global Cyp1a1/1a2 deletion increased susceptibility of female mouse islets to TCDD-induced impairments in insulin secretion but had minimal effects on GLT responses in either sex. In contrast, {beta}-cell Ahr deletion did not affect islet responses to TCDD, but exacerbated GLT-induced islet dysfunction in male islets and increased baseline insulin secretion in both vehicle- and GLT-exposed female islets in vitro. Lastly, {beta}-cell Ahr deletion prevented adaptive HFD-induced hyperinsulinemia in both sexes in vivo. ConclusionIslet AhR signaling shapes responses to chemical and nutrient stressors in a context- and sex-dependent manner. While the canonical AhR-CYP1A axis supports female islet resilience to TCDD, {beta}-cell AhR signaling more broadly regulates nutrient stress responses in both sexes.

Background: Repeated metabolic-bariatric surgery (MBS, r-BS) represents 10-25% of all MBS procedures and is commonly performed for recurrent weight gain after initial weight loss. How weight loss followed by regain reshapes adipose tissue biology remains unclear. We hypothesized that women undergoing r-BS exhibit a distinct adipose tissue signature compared with those undergoing primary bariatric surgery (p-BS). Methods: We analyzed subcutaneous and visceral adipose tissues (SAT, VAT, respectively) from women undergoing either p-BS, or r-BS with documented >15% weight loss after prior MBS. Tissues were assessed histologically, molecularly, and functionally (activation of human microglia cells (HMC3) by SAT secretome). Results: Consistent with other cohorts, women undergoing r-BS (n=21) trended to be older (47.2 vs. 40.5 y, p=0.06) than those undergoing p-BS (n=35), with a lower BMI (42.3 vs. 45.6 kg/m2, respectively, p=0.103), and a trend for improved cardiometabolic risk parameters such as fasting insulin, CRP and HDL-c. Adipose tissue histological features (adipocyte size, fibrosis, macrophage and crown-like structure abundance) were similar, while adipose mast cells were slightly (though insignificantly) more prevalent in r-BS. A single-nucleus RNA-seq-based deconvolution algorithm applied to bulk RNA-seq confirmed the absence of a major shift in adipose tissue cell-type composition. Yet, it uncovered a unique SAT transcriptome, with activation of inflammatory pathways in r-BS. Consistently, SAT explants from r-BS secreted higher protein concentrations of NFkB-regulated cytokines IL6 and IL8. Biological impact of the more inflammatory secretome was demonstrated by its increased ability to activate human microglia cells. Conclusions: Prior BS with significant weight loss-regain in women is associated with an inflammatory SAT transcriptome and secretome, possibly reflecting altered adipose-brain endocrine communication.

Context: Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder involving complex interactions among endocrine, metabolic, inflammatory, and thyroid pathways. However, the systems-level organization of these interactions remains poorly understood. Objective: To reconstruct the endocrine-metabolic biomarker network in women with PCOS and identify bridge biomarkers integrating distinct physiological domains. Design: Retrospective cross-sectional study. Setting: Single tertiary referral center. Participants: A total of 1,286 women diagnosed with PCOS according to the revised Rotterdam criteria. Methods: Twenty-nine routinely measured laboratory biomarkers representing endocrine, metabolic, hematological/inflammatory, and thyroid domains were analyzed. Sparse Gaussian graphical models were estimated using Graphical LASSO with Extended Bayesian Information Criterion model selection. Network topology, node centrality, bridge centrality, bootstrap resampling, and predefined sensitivity analyses were performed. Results: The reconstructed network comprised 29 biomarkers connected by 73 conditional dependency edges (network density, 0.18), demonstrating a modular but highly integrated endocrine-metabolic architecture. Conventional centrality analysis primarily identified biomarkers organizing local physiological modules, whereas bridge-centrality analysis revealed biomarkers coordinating communication between biological domains. Sex hormone-binding globulin exhibited the highest bridge strength, followed by fasting insulin, triglycerides, and high-density lipoprotein cholesterol. Additional reproducible bridge biomarkers included free thyroxine, white blood cell count, 2-hour plasma glucose, absolute neutrophil count, androstenedione, and anti-thyroglobulin antibodies. The leading bridge biomarkers remained stable across bootstrap resampling, complete-case reconstruction, and alternative network specifications. Conclusions: PCOS is characterized by an integrated endocrine-metabolic network organized around a limited number of reproducible bridge biomarkers linking multiple physiological systems. Network analysis provides complementary systems-level information beyond conventional biomarker evaluation and may facilitate future biological phenotyping and precision medicine approaches in PCOS.

Pregnant women are frequently exposed to various endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), causing harm to both the developing placenta and fetus. BPA can promote placental dysfunction by altering key cellular processes such as differentiation, invasion, and migration in trophoblast cells. These cellular processes are also tightly managed by the ubiquitin proteasomal system via maintenance of the ubiquitinated protein pool. However, the BPA-mediated dysregulation of this ubiquitin proteasomal homeostasis is poorly understood. Therefore, we identified 19 deubiquitinases (DUBs) and a dynamic ubiquitinome profile of extravillous trophoblast cells (HTR8/SVneo), which reduced trophoblast cell migration post-BPA exposure. Further investigation using an integrated substrate-ligase-deubiquitinase network shows that BPA binding to PPAR-alpha or indirect regulation of its E3 Ligase MuRF1 and DUB USP5 via BPA resulted in hyper-ubiquitination of PPAR-alpha, triggering its nuclear localization. In the nucleus, the ubiquitinated PPAR-alpha can deregulate its migration-associated target gene expression, causing a reduction in the migration of HTR8/SVneo cells. This physiological alteration of extravillous trophoblast cells (EVTs) through BPA can disrupt placental homeostasis. Hence, we assumed that BPA-induced cellular alteration in EVTs can promote placental defects, which might contribute to adverse pregnancy outcomes.

BackgroundDietary sodium restriction is a common nutritional and physiological challenge that activates electrolyte-conserving endocrine pathways, but its impact on systemic lipid metabolism remains incompletely defined. We examined whether short-term dietary sodium deprivation alters the circulating lipidome and identifies lipid signatures of metabolic adaptation. MethodsMale Sprague-Dawley rats were maintained on sodium-sufficient (NaS) or sodium-deprived (NaD) diets for 7 days (n=3 per group). Serum lipids were profiled by untargeted LC-MS/MS in positive and negative ion modes. Lipidomic differences were evaluated using class-level and species-level analyses, principal component analysis, volcano plots, heatmaps, and pathway-oriented interpretation. ResultsNaD rats exhibited a distinct serum lipidomic profile compared with NaS controls, indicating global remodeling of circulating lipid composition. Sodium deprivation produced class-specific and species-resolved changes, including selective depletion of subsets of neutral lipid species, prominent wax ester remodeling, increased phosphatidylcholine and lysophosphatidylcholine abundance, and altered acylcarnitine profiles. These signatures are consistent with coordinated changes in lipid storage, membrane phospholipid turnover, and mitochondrial fatty-acid handling. ConclusionsDietary sodium deprivation induces coordinated serum lipidome remodeling in rats, supporting the concept that nutritional electrolyte status can influence systemic lipid metabolism. These exploratory findings identify sodium deprivation as a metabolic stressor linked to neutral lipid mobilization, phospholipid remodeling, and altered mitochondrial substrate handling, and provide a foundation for future mechanistic studies.

IntroductionIt is now widely recognized that environmental exposures can predispose unexposed descendants to disease across multiple generations without inducing genetic mutations. Among the numerous unknowns that multigenerational effects still hold, identifying the most probable windows of susceptibility for multigenerational environmental disease predisposition remains a crucial challenge in preventing such effects. We have proposed that multigenerational environmental effects can be mediated by perturbations in chromatin organization that originate from environmental exposures causing alterations in gamete elements necessary for establishing chromatin organization immediately after fertilization. Based on this hypothesis, it is likely that the period preceding conception serves as a relevant window of susceptibility for multigenerational effects, and that such susceptibility may vary between female and male preconception exposures due to the distinct characteristics of oocytes and sperm. Here, we test this framework using nicotine--a well-established endocrine- and metabolism-disrupting chemical with documented multigenerational effects--and assess whether windows of nicotine cessation prior to conception that span the last stages of gamete maturation mitigate these effects. MethodsWe conducted two asynchronous studies to determine the direct and offspring effects of female preconception exposure (FPE) and male preconception exposure (MPE) to nicotine and nicotine cessation. We exposed C57BL/6J female (FPE) or male (MPE) mice to deionized water (control), continuous nicotine (300 {micro}g/mL), or one of two nicotine cessation windows whose durations did or did not encompass one full round of gamete maturation. Following exposure, we mated exposed mice with unexposed mice of the same age to produce their offspring. We measured the water and food consumption and body weight of exposed mice to determine the efficacy and direct effect of the assayed exposures. We also measured body weight, fasting body weight, fasting glucose, gonadal white adipose tissue and liver weights, and plasma concentrations of twelve metabolic hormones in the offspring of exposed mice to determine the offspring effect of nicotine exposure and its mitigation upon nicotine cessation. We determined the significance of comparisons between nicotine and control groups using the Monte Carlo-Wilcoxon testing framework that we have previously developed. ResultsPreconception nicotine exposure elicited sexually dimorphic metabolic effects in the offspring of exposed mice that differed between FPE and MPE studies. Nicotine cessation mitigated F1 metabolic perturbations only after maternal--not paternal--preconception exposure, and only when the cessation window encompassed one full round of oocyte maturation. ConclusionsThese findings support the hypothesis that preconception exposures perturb offspring metabolism through sex-specific gamete mechanisms and highlight that the efficacy of cessation strategies depends on the parental sex exposed.

Endometriosis is a steroid-dependent gynecologic disease characterized by progesterone (P4) resistance, subfertility/infertility, and pelvic pain; however, the molecular mechanisms underlying impaired P4 responsiveness in endometriosis tissue are not fully understood. RE-1 silencing transcription factor (REST), a transcriptional regulator implicated in steroid hormone signaling, has emerged as a potential mediator of P4 responsiveness. Here, we investigated the role of REST in endometriosis using human tissues and a uterine-specific Rest conditional knockout mouse model. Immunohistochemical analysis of eutopic endometrium and ectopic lesions from patients with endometriosis revealed significantly reduced nuclear REST expression compared with control endometrium, suggesting loss of functional REST in disease. To assess the physiological consequences of REST deficiency, uterine-specific Rest knockout (Rest d/d) mice were generated. Rest d/d females exhibited progressive subfertility and hyper-estrogenic uterine tissue characteristics that displayed a blunted responsiveness to P4 treatment. Loss of Rest selectively altered expression of P4-responsive genes associated with endometriosis pathology, despite preserved P4 receptor expression. Following induction of experimental endometriosis, female mice that developed endometriotic-like lesions using Rest-deficient donor tissue developed significantly larger lesions that were less responsive to P4 treatment compared to lesions induced using control tissue. Mechanical sensitivity was modestly increased in mice receiving Rest-deficient tissue, whereas vaginal hyperalgesia was unaffected. These findings identify loss of nuclear REST as a feature of endometriosis and support a role of REST in subfertility, lesion progression, and blunted response to P4. REST may represent a novel molecular contributor to altered P4 responsiveness and a potential therapeutic target in endometriosis. Significance StatementEndometriosis is a common disease in women characterized by altered steroid hormone signaling, infertility, and pelvic pain. RE-1 silencing transcription factor (REST) is a candidate regulator of steroid hormone signaling in gynecologic disease but a role in endometriosis pathophysiology remains unexplored. To fill this knowledge gap, our study utilizes human endometrial and endometriotic tissues coupled with a conditional knockout mouse model for uterine Rest deficiency. We show that REST is significantly reduced in eutopic and ectopic endometrial tissue from women with endometriosis and that deletion from mouse uterine tissue recapitulates clinical characteristics in women with endometriosis including progesterone resistance, sub-fertility and pelvic pain. These findings will further guide future research to understand impaired steroid signaling in the pathophysiology of endometriosis.

Context: Prader-Willi syndrome is a genetic neurodevelopmental disorder characterized by hyperphagia and early-onset obesity from hypothalamic dysfunction with endocrinopathies and learning disability. Management is challenging with strict control of the food environment needed. While newer glucagon-like peptide-1 receptor agonists, such as semaglutide, have efficacy in non-PWS obesity, there have been limited case reports in PWS. Objective/Design/Setting: Retrospective records review of 12 adults with PWS and overweight/obesity treated with semaglutide at a UK academic hospital centre specialist clinic. Patients: mean +/- SD age 28.3 +/- 10.1 years, 83% female, BMI 46.6 +/- 8.2kg/m2, 75% type 2 diabetes mellitus. Intervention: Median follow-up 17.2 months (range 8.7-36.1) with median semaglutide dose 2.4mg once weekly (1.0-2.4). Results: Although there was no significant weight loss on semaglutide, there was stabilisation of the weight gain prior to treatment over previous 12.4 months (7.6-23.0) (post -3.1 +/- 9.9% vs. pre +5.7 +/- 5.6%: d -0.72, P=0.037). There was a significant decrease in hyperphagia on semaglutide from hyperphagia questionnaire for clinical trials (n=11, -7.3 +/- 6.1 (max 36), d -1.19, P=0.003), having been stable before treatment. HbA1c improved in those with elevated baseline levels (n=6, -4.2 +/- 4.9%, d -0.74, P=0.13). Mild gastrointestinal side effects were seen in 25% but did not lead to discontinuation. Conclusions: In adults with PWS, semaglutide produced weight maintenance, reduced hyperphagia, and improved glycaemic control, with good tolerability. Larger placebo-controlled trials are needed to confirm these findings in adults and adolescents with PWS, especially in those without T2DM, where efficacy may be greater.

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.

ObjectivesLow serum testosterone (T) in men with obesity suggesting T deficiency may be misinterpreted by confounding changes in serum SHBG, Ts circulating carrier protein. Measuring or calculating "free" testosterone (FT) concentrations to define a low T is problematic as cFT is not a valid analytical variable lacking certified standard, quality control or reference range. We developed a novel metric, Scaled Testosterone (ST), comparing standardized serum T (LCMS) and SHBG without invoking hypothetical serum T fractions. MethodsSerum T and SHBG in men (n=10,027) pooled from three population-based studies in Australia were expressed as standardized (Z) scores (ZT, ZSHBG) and their difference ST = ZT-ZSHBG. ST was evaluated in a clinical trial of 51 men with severe obesity undergoing 1 year of diet-induced weight loss. ResultsZT and ZSHBG displayed linear correlation (r=0.44, 10-11) with ST approximating zero (-0.33 {+/-}2.14 SD). In non-obese men with low serum T suggestive of organic hypogonadism displayed very low ST indicating ST can evaluate whether a low serum T is proportionate to a concomitant serum SHBG. In men with obesity, low pre-treatment serum T and SHBG both increased during diet-induced weight loss with no change in serum LH while ST which remained within standard limits at each time. ConclusionsThe low serum T in men with obesity may better be considered as the pseudo-hypogonadism of obesity comprising low serum T with proportionately low serum SHBG in the presence of normal serum LH {+/-} FSH serving as a tissue androgen sensor.

Context Polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS), is a biologically heterogeneous disorder, yet previous clustering studies have reported inconsistent phenotype structures. Whether these discrepancies reflect methodological variability or genuine multidimensional disease biology remains unknown. Objective To determine whether independently derived endocrine, metabolic, inflammatory, and thyroid phenotypes represent the same underlying biological structure or capture distinct dimensions of PMOS heterogeneity. Design Cross-sectional observational study using a cross-space phenotyping framework. Setting Tertiary referral outpatient endocrinology and gynecology clinic. Participants A total of 1,286 women were diagnosed with PCOS according to the Rotterdam criteria. Methods Four predefined biological spaces (endocrine, metabolic, inflammatory, and thyroid) were analyzed independently. Within each space, standardized preprocessing, dimensionality reduction, and unsupervised clustering were performed. Cluster robustness was evaluated using bootstrap resampling, while agreement between independently derived phenotypes was quantified using the adjusted Rand index (ARI). Biological relevance was assessed using independent non-circular validation with variables excluded from phenotype derivation. Sensitivity analyses compared complete-case and imputed datasets. Results All four biological spaces produced highly stable clustering solutions (bootstrap ARI: endocrine 0.915, metabolic 0.964, inflammatory 0.930, thyroid 0.990). Despite this robustness, agreement between independently derived phenotypes remained consistently low. The highest concordance was observed between metabolic and inflammatory phenotypes (ARI = 0.208), followed by endocrine and metabolic phenotypes (ARI = 0.159), whereas agreement involving thyroid phenotypes was close to zero. Independent non-circular validation confirmed that all identified phenotypes represented biologically coherent patient subgroups beyond the variables used for clustering. Sensitivity analyses demonstrated high agreement between complete-case and imputed solutions, supporting the robustness of the findings. Conclusions Stable biological phenotypes exist within individual physiological domains of PMOS but do not converge into a single overarching biological phenotype. These findings support a multidimensional model of PMOS heterogeneity in which endocrine, metabolic, inflammatory, and thyroid systems describe complementary rather than interchangeable aspects of disease biology. Cross-space phenotyping provides a general framework for investigating biological heterogeneity in complex disorders and may facilitate future precision medicine approaches.

Background The placenta serves a critical role in nutrient uptake and waste elimination for the developing fetus. The placenta is also responsible for the uptake and/or exchange of xenobiotics, including medications, between the maternal and fetal bloodstreams. An estimated 40-80% of women take medications or drugs during pregnancy for a variety of conditions. Very little is understood about fetal drug and nutrient exposure during pregnancy and how it may change over the course of fetal development. Objective This study aimed to characterize the abundance of transport proteins in placental tissue, which are important in modulating fetal nutrient and drug exposure, over the duration of pregnancy. Mass spectrometry-based global proteomic analysis revealed trends in the expression of thousands of proteins throughout gestation. Focusing on the membrane-associated proteome enabled an increased emphasis on the solute carrier and ATP-binding cassette families of transporter proteins that are critical for nutrient and xenobiotic transport across the maternal-fetal barrier. Study Design Using data-independent acquisition proteomics, relative abundance of proteins in placental tissue samples was profiled across all three trimesters of pregnancy (Trimester 1 = 16, Trimester 2 = 9, and Term = 9). Membrane fractions were generated to enrich membrane-associated proteins for proteomic analysis. Placental samples were grouped into randomized batches for membrane fraction generation and mass spectrometry analysis. Proteomic search results from each batch were imported into the R programming environment from Skyline, concatenated, and normalized as one data set for downstream analysis. Results A total of 6,331 proteins were detected across all samples with 4,210 proteins identified in every sample. Pathway analysis revealed that as gestational age increases, membrane-associated proteins involved in more complex metabolic pathways increase in relative abundance while those involved in extracellular remodeling events and simple organic ion transport tended to decrease. A total of 139 solute carrier and ATP-binding cassette transport proteins were identified in all samples, and 80 were identified in every sample. In general, membrane-associated proteins, including solute carrier and ATP-binding cassette transport proteins, were significantly enriched in placental tissue collected during early gestation compared to term placental tissue. Conclusion This study presents a comprehensive profiling of membrane-associated proteomic changes during gestation and identifies significant gestational age associated abundance changes at the protein level in several transport protein families. The application of data-independent acquisition global proteomic techniques enabled in-depth analysis of thousands of proteomic changes across pregnancy in a single experiment. These data provide critical information to support future studies into the understanding of fetal exposure to xenobiotics and nutrients circulating in the maternal bloodstream.

Pregnancy leads to many adaptations in the maternal body, most of which are reversible. However, reproductive experience can also result in permanent effects. Here, we investigated how pregnancy influences the somatotrophic system and the lasting effects of reproductive experience on the maternal organism. Reproductive experience induced a pronounced increase in lean body mass and longitudinal growth in both wild-type and growth hormone (GH)-deficient mice compared with age-matched virgins. Body growth was primarily observed during the first pregnancy, whereas a second gestation was mostly associated with increased adiposity. Data from a cohort of women with isolated GH deficiency (IGHD) caused by a loss-of-function mutation in the GHRHR gene revealed that nulliparous women were 7 cm shorter than those with one or more pregnancies. Increased GH secretion was observed in pregnant wild-type mice but not in pregnant GHRHR-deficient mice. Pregnancy-induced body growth is preserved despite disruption of GH-, ghrelin-, and estrogen-related signaling pathways. In conclusion, reproductive experience induces permanent changes in the maternal organism, promoting body growth in models that allow this response. Pregnancy-induced body growth appears to be independent of GH action. These findings underscore the need for further studies to investigate the long-lasting consequences of reproductive experience in females.

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.

Polycystic ovary syndrome (PCOS) is linked to adverse pregnancy outcomes and increased cardiometabolic risk in offspring, yet the placental mechanisms underlying these risks remain poorly understood. Metformin is prescribed during PCOS pregnancies despite limited mechanistic justification. Using multi-modal molecular analyses of placentas from healthy controls and women with PCOS randomized to placebo or metformin (PregMet trial), restricted to uncomplicated pregnancies, we characterized direct PCOS associated placental alterations independent of confounding complications. PCOS placentas showed transcriptional downregulation across multiple cell types and shifts in cell type proportions. Specifically, syncytiotrophoblasts exhibited reduced expression activity of growth hormone receptor signaling and glycosaminoglycan biosynthesis. Endothelial cells displayed diminished receptor tyrosine kinase pathway activity, including VEGFC, despite increased cell proportion and hypervascularity. Intercellular communication networks were globally suppressed, including reductions in PDGF signaling from Hofbauer cells to fibroblasts. Notably, metformin did not reverse most PCOS-associated molecular alterations and induced transcriptional changes correlated to birth weight and childhood BMI. These findings indicate that PCOS-associated placental features are driven by cell type specific dysregulation of growth factor, angiogenic signaling pathways that are largely unresponsive to metformin. This underscores the need to develop mechanism based, placenta targeted therapeutic alternatives for future pregnancy management.

Adrenal chromaffin cells are key effectors of the sympathoadrenal response and play a central role in the organisms adaptation to environmental and physiological challenges. While cholinergic and pituitary adenylate cyclase-activating polypeptide (PACAP)-dependent mechanisms have long been recognized as major regulators of catecholamine secretion, increasing evidence indicates that connexin-mediated gap junctional communication provides an additional and highly dynamic level of control. Whether early-life experience modifies the adult capacity of chromaffin-cell networks to undergo stress-induced connexin remodeling remains unclear. Here, we examined adrenal medullary connexin expression in adult rats exposed to neonatal maternal separation (MS; 3 h daily, postnatal days 2-15) and later challenged with an 8-day unpredictable mild stress (UMS) protocol. Under basal adult conditions, MS did not produce an overt change in adrenal medullary Cx36 or Cx43 immunoreactivity relative to animal-facility-reared controls. In contrast, UMS increased connexin immunoreactivity in the adrenal medulla, and this response was amplified in animals with a history of MS. MS+UMS animals also displayed enhanced corticosterone responses to acute restraint stress. These findings suggest that neonatal MS does not impose a constitutively altered adult chromaffin-cell phenotype, but instead primes the future stress responsiveness of adrenal medullary connexin remodeling. We propose that chromaffin-cell gap junctions represent a substrate sensitive to stress history, through which developmental experience may influence sympathoadrenal and endocrine adaptation in adulthood.