Journal of the Endocrine Society

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.

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.

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.

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.

Importance: Higher coffee intake has been associated with lower risk of type 2 diabetes (T2D), but the underlying biological pathways remain incompletely understood. Objective: To examine associations of coffee intake with insulin sensitivity, adiposity, and T2D risk, and assess whether coffee intake modifies associations between pathway-specific genetic susceptibility and incident T2D. Design, Setting, and Participants: Cross-sectional analyses among 806 participants without T2D in the VITamin D and OmegA-3 TriaL (VITAL) clinical sub-cohort, who underwent repeated dietary assessment, clinical phenotyping, and dual-energy X-ray absorptiometry imaging at baseline and year-2. Prospective analyses among 333,053 UK Biobank participants without T2D at baseline who had dietary and genetic data and were followed for a median of 13.3 years. Exposures: Coffee intake assessed by food frequency questionnaires. In UK Biobank, 12 pathway-specific polygenic scores (pPS) representing distinct T2D pathophysiological mechanisms were evaluated. Main Outcomes and Measures: The primary outcomes, in VITAL, were HbA1c, oral glucose tolerance test-derived measures of glucose response and insulin sensitivity, beta-cell function, and overall, truncal, and visceral adiposity; in UK Biobank, was incident T2D. Results: In VITAL, higher coffee intake was associated with higher insulin sensitivity (standardized beta; per cup/day, 0.046; P = .004) and lower visceral adipose tissue mass (beta -0.047; P = .006), after adjusting for demographic, lifestyle, and clinical factors, including body mass index. In UK Biobank, higher coffee intake was associated with lower T2D incidence (hazard ratio per cup/day, 0.96; 95% CI, 0.95-0.97), lower triglyceride-to-HDL cholesterol ratio (beta: -0.01; P = 2.51 x 10-19), and lower visceral adipose tissue mass (beta: -0.01; P = 4.28 x 10-9). Associations of 3 pPS related to insulin resistance and fat distribution with incident T2D were attenuated among participants consuming higher amount of coffee than among non-consumers (P for interaction < .0043). Conclusions and Relevance: Higher coffee intake was associated with greater insulin sensitivity, lower visceral adiposity, and lower risk of T2D. Together with the attenuation of associations between pathway-specific genetic susceptibility and T2D risk among higher coffee consumers, these findings suggest that insulin resistance and visceral adiposity-related pathways may contribute to the association between coffee intake and T2D risk.

Comprehensive detection of steroids, beyond the limited panels typically analyzed in clinical chemistry laboratories, has become increasingly important given their pivotal roles in diverse biological processes. However, steroid quantification poses several analytical challenges, including differences in ionization efficiency and structural similarities across the entire steroid metabolic network. To address these challenges, we developed a targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay to analyze 21 steroids using reverse-phase chromatography combined with rapid polarity switching. Mass spectrometry (MS) analysis was performed in scheduled multiple reaction monitoring (sMRM) mode. Depending on the steroid and matrix, the validated lower limits of quantitation (LLOQ) ranged from 12.0 pM to 1216 pM in plasma and 41.1 pM to 384 pM in fecal sample homogenates. In adipose tissue, it was from 0.01 pmol/g to 9 pmol/g. Measured steroid concentrations obtained from the commercial control samples (MassTrak Steroid Serum QC Set 1 and the MassCheck Steroid Panel 1 Serum Control) showed close agreement with the reference values. As a proof of concept, the method was successfully applied to 469 plasma samples in several projects, 15 adipose tissue samples, and 332 fecal samples, demonstrating its applicability to large-scale studies. In conclusion, the method enables sensitive, derivatization-free quantification of an expanded steroid panel in plasma and complex biological matrices, including adipose tissue and fecal samples, representing a significant advancement in comprehensive steroid profiling.

IntroductionPrenatal dibutyl phthalate (DBP) exposure is associated with increased risks of adverse fetal outcomes as well as metabolic and cardiovascular diseases in the offspring in a fetal sex-specific manner. However, mechanisms underlying these prenatal DBP exposure-associated adverse fetal/offspring outcomes are unclear. We hypothesize that environmentally relevant low-dose prenatal DBP exposure dysregulates fetal-placental vascular function and lipid metabolism in a fetal sex-specific manner, thereby impairing placental efficiency and programming adverse offspring metabolic outcomes. MethodsFemale CD-1 adult mice (8-10wks) were orally dosed with vehicle or an environmentally relevant low-dose DBP (0.1 g/kg/day) daily from 30 days pre-pregnancy through gestational day (GD) 18.5. Fetal-placental vascular hemodynamics of these dams were examined using high-frequency ultrasound at multiple timepoints. The effect of prenatal environmentally relevant low-dose DBP exposure on placental efficiency, spatial transcriptomic profiles, lipid homeostasis, and placental vascular endothelial cells function in male and female fetuses were evaluated at gestational day (GD) 18.5. ResultsThe prenatal low-dose DBP exposure dysregulated the fetal-placental vascular hemodynamic indices from mid-to late gestation. DBP exposure impairs placental efficiency in male, but not female placenta at GD18.5. Further, female placentas exhibited fetal labyrinth vasculature-specific transcriptomic adaptations that preserves placental efficiency and endothelial function. In contrast, male placentas exhibited minimum transcriptomic adaptation, together with compromised placental efficiency and endothelial function associated with lipotoxic lipid profile. ConclusionsIn conclusion, prenatal low-dose DBP exposure dysregulates placental vascular function and lipid homeostasis in a fetal sex-specific manner, with male fetuses being more susceptible to DBP exposure.

Aberrant changes in circulating cell-free mitochondrial DNA (ccf-mtDNA) across gestation are associated with adverse pregnancy outcomes. Given the inflammatory properties of ccf-mtDNA via pattern recognition receptors such as Toll-like receptor 9 (TLR9), we hypothesized that extracellular mtDNA induces placental inflammation via TLR9 signaling and that this response differs by fetal sex. Pregnant Sprague-Dawley rats were treated intravenously with purified mtDNA (300 g/kg), nuclear DNA (nDNA), saline, and/or the TLR9 antagonist ODN2088 across five studies. Placental responses were evaluated 4 h (Studies 1-3) and 24 h (Study 4) post-treatment; pregnancy and neonatal outcomes were assessed at delivery (Study 5). Exposure to mtDNA, but not nDNA, increased placental il1{beta}, tnf, and il10 mRNA (p < 0.05), establishing response specificity. mtDNA-induced placental inflammation was fetal sex-dependent: mtDNA increased il6 and il1{beta} mRNA in male placentas (p [&le;] 0.0004) but not female placentas, whereas ifn{gamma} was selectively induced in female placentas (p = 0.0004). TLR9 and MyD88 abundance increased in female but not male placentas, and TLR9 antagonism modified selected inflammatory responses with sex-specific patterns. The 4 h inflammatory transcriptional signature resolved by 24 h, whereas mtDNA exposure was associated with a sex-specific shift in antioxidant enzyme expression persisting to 24 h. Despite no effects on gestational length or neonatal biometrics, mtDNA exposure was associated with a higher estimated stillbirth count per litter (IRR = 4.23, 95% CI [0.89, 20.1], p = 0.069). These findings establish extracellular mtDNA as an acute, sex-differentiated placental inflammatory stimulus with partial TLR9 dependence and a potential impact on fetal viability. New & NoteworthyThis study demonstrates that acute exposure to extracellular mtDNA induces placental inflammatory responses in vivo. This response is specific to mtDNA, fetal-sex dependent, and partially mediated by TLR9, with male and female placentas engaging distinct inflammatory signals within hours of exposure. The biological effects extend beyond the initial inflammatory window, with mtDNA exposure producing lasting, sex-specific changes in antioxidant enzyme expression. mtDNA-exposed dams had higher expected stillbirth counts, suggesting extracellular mtDNA may affect fetal viability.

Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD) and associated dyslipidemia is a growing health issue that gives rise to cardiovascular risk. Men are more prone to development of MASLD than women. Understanding mechanisms underlying sex differences in MASLD may lead to improved prevention and treatment approaches. Cholesteryl ester transfer protein (CETP) is a lipid transfer protein that shuttles triglycerides and cholesteryl esters between blood lipoproteins and tissues. In this study investigate the impact of hepatic CETP expression on MASLD. Hepatic CETP expression (L-HuCETP) was achieved by injecting liver-targeted CETP-expressing adeno-associated virus into C57BL/6J mice. In females, L-HuCETP improved glucose tolerance, consistent with our prior clamp results in global human CETP transgenic mice. Whereas in males, L-HuCETP worsened glucose metabolism and impaired insulin signaling. Correspondingly, L-HuCETP expression reduced the expression of gluconeogenic pathway genes in females but upregulated these genes in males. In males, L-HuCETP mice exhibited increased hepatic lipid droplet accumulation, lipogenesis proteins and these changes were not observed in females. L-HuCETP expression resulted in sex-specific hepatic responses, with increased expression of inflammation and fibrosis related genes in male, but decreased expression of these genes in females. Mechanistic studies indicate that L-HuCETP had sex specific effects on transcription factors ChREBP and HNF4, which are important for glucose and lipid metabolism. Our studies suggest that sex-specific roles of L-HuCETP with regard to liver metabolic adaptation and MASLD risk in obesity, highlighting CETP-mediated pathways as potential targets for sex-specific precision medicine approaches to improve MASLD.

Background Besides hyperglycemia, type 2 diabetes (T2D) is characterized by dyslipidemia, which is typically assessed using traditional clinical lipid measurements. However, molecular plasma lipids beyond these traditional markers can provide additional information about an individuals health status. For molecular lipids to be used effectively, certain characteristics, such as their temporal variability, need to be determined. Methods We analyzed the plasma lipidome for three consecutive time points, each three months apart, of 51 individuals with T2D using targeted liquid chromatography coupled to mass spectrometry (LC-MS). 513 lipid species across 25 (sub)classes were quantified by this approach and the temporal variability were calculated. Moreover, to identify sex differences in the plasma lipidome, we analyzed 914 samples of a cross-sectional T2D cohort with the same approach. Results Neutral lipids and phosphatidylserine had the highest temporal variability which was independent of their platform-specific variability. In contrast, glycosphingolipids were found to be relatively stable over time in individuals with T2D. Acyl-chain analysis revealed generally similar variability in the acyl-chain groups but indicated a higher temporal variability in medium-length acyl-chains. Lipid-sex association analysis showed markedly higher sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines in women and higher acylcarnitines in men. Overall, approximately one-third of measured lipids showed significant sex differences independent of age, BMI, diabetes duration, glycemic control, and medication use. Conclusions Our findings provide insights into temporal variability of molecular lipids. This variability should be considered when assessing novel lipid biomarkers. Likewise, sex differences in these lipids need to be considered in precision medicine for diabetes management.

BackgroundPrenatal cannabis use is becoming increasingly more commonplace. However, cannabis exposure is linked to adverse pregnancy outcomes, including gestational hypertension, preeclampsia, and preterm birth. The aim of this study was to determine the morphological and molecular effects of prenatal cannabinoid exposure on the placenta. MethodsPregnant Sprague-Dawley rats were exposed daily to vaporized THC (100 mg/mL) starting at gestational day (GD)5 until GD19 when dams were sacrificed and fetuses and placentas collected. Fetuses were genotyped for genetic sex and transcriptomic analysis was performed on male and female THC-exposed and control placentas. ResultsOn GD19, both the fetuses and placentas from the THC group were significantly larger than the control. When separated by sex, both male and female THC fetuses were significantly larger; however, only male THC placentas were significantly larger than male control placentas with no significant difference in placental weight between female control and THC placentas. RNA-sequencing revealed enriched biological processes related to nutrient transport and lipid catabolism, protein-lipid complex formation, and lipoprotein particle remodeling and organization. Further transcriptomic analysis determined that the differentially expressed genes and enriched biological processes related to lipid metabolism were preferentially enriched in the female THC placentas compared to the male, suggesting a sex-specific effect. DiscussionCollectively, these data present sex-specific effects of prenatal cannabinoid exposure on placental growth and global gene expression. These data also suggest that sex influences gene expression of genes related to lipid metabolism in the THC-exposed placentas.

Purpose: Gonadotropin releasing hormone (GnRH) agonists are clinically used to delay pubertal progression by suppressing the hypothalamic-pituitary-gonadal (HPG) axis. While GnRH agonists have long been used clinically, the developmental characterization of HPG axis suppression during puberty remains incompletely understood. Thus, we examined the effects of GnRH receptor agonism in juvenile rats. Hypothesis: Sustained GnRH receptor agonism will result in lower gonadal mass, blunt peripheral pubertal landmarks, and alter hormonal signaling dynamics within the HPG axis. Methods: Animals received a single injection of extended-release leuprolide acetate depot (LA) or vehicle control on postnatal day (PND) 23. Animals were assessed for body mass and peripheral markers of puberty. On PND 44, animals were euthanized and tissues were evaluated to assess additional markers of pubertal maturation, pituitary gene transcript levels, and hormone concentrations in serum and gonads. Results: In females, LA treatment resulted in a smaller gonad size, increased body mass, and less vaginal openings. In males, LA treatment resulted in smaller gonads but did not significantly alter body mass or preputial separation. In the pituitary, LA-treated rats had lower Gnrhr, Fshb, and Lhb transcript levels regardless of sex, while females exhibited higher Cga and Nr5a1. Serum FSH and ACTH were lower in LA-treated animals, and treated females also had lower progestins and androstenedione, and higher LH. Conclusions: LA treatment reduced aspects of pubertal maturation and HPG axis output, with sex specific outcomes. These findings highlight the need for integrated, multi-level approaches to understand how altered GnRH signaling impacts pubertal and long-term physiology.

Alternaria mycotoxins represent an emerging concern due to their frequent occurrence in food and feed. However, available toxicological data remain limited. Under the current EU regulatory framework, and in line with the EFSA/ECHA/JRC guidance for the identification of endocrine disruptors (EDs), assessment of endocrine activity relies on standardized assays performed according to OECD Test Guidelines (TGs) for the estrogen-, androgen- and steroidogenesis- (EAS) modalities. Within the framework of the European Partnership for the Assessment of Risks from Chemicals (PARC), standardized in vitro methods of regulatory relevance were performed for six chemically characterized Alternaria toxins, aiming to address current regulatory gaps on EAS-mediated activity. Alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altertoxin-I (ATX-I), tentoxin (TEN) and altenuene (ALT) were assessed over a broad concentration range, from 0.001 up to 60 M, depending on cytotoxicity and solubility profile of each compound. Our findings indicate estrogenic activity for AOH (PC50: 3.9 - 4.6 {micro}M) and AME (PC50: 5.2 - 8.5 {micro}M) in the estrogen receptor transactivation assay (OECD TG 455), as well as an anti-estrogenic activity for ATX-I (IC30: 0.27 - 0.37 {micro}M). Minimal positive responses were observed at high concentrations for AOH (from the dose of 3 {micro}M) and for AME (from the dose of 10 {micro}M) in the agonistic part of the androgen receptor transactivation assay (OECD TG 458), which may also reflect glucocorticoid receptor activation. No effects on estradiol or testosterone production were observed for any of the tested Alternaria compounds in the steroidogenesis assay (OECD TG 456).

Human hair is a common contaminant in GMP-controlled manufacturing environments, and its identification is important for contamination source investigation and corrective action. Because human hair can originate from multiple body sites, it is often necessary to determine not only the species of origin but also the anatomical source of the hair. Conventional forensic approaches distinguish scalp hair from body hair by microscopic examination of cuticle patterns, medullary structure, cross-sectional morphology, and pigment distribution. However, these methods depend on examiner expertise, are difficult to apply to damaged specimens, and provide limited quantitative information. In this study, we developed a proteomics-based approach for distinguishing scalp hair from pubic hair using identical sample preparation and analytical workflows. Comparative proteomic analysis identified keratin-associated proteins KAP 4-3 and KAP 9-6 as enriched in scalp hair, whereas cuticular keratins Ha7 and Ha8 were strongly enriched in pubic hair. Amino acid composition analysis further revealed that scalp hair-enriched proteins were highly cysteine-rich, consistent with sulfur-rich cross-linking matrix proteins, whereas pubic hair-enriched proteins exhibited characteristics of structural keratin filaments. These results demonstrate that proteomic signatures can provide a quantitative and objective means of determining the anatomical origin of human hair and may contribute to contamination source tracing in GMP manufacturing and forensic investigations.

The insulin-like growth factor (IGF)/IGF-binding protein (IGFBP) axis has been implicated in diabetes mellitus and the associated burden of cardiovascular complications. Higher circulating levels of IGFBP-1 and IGFBP-2 have been established as markers of protection from incident type 2 diabetes, yet their associations with cardiovascular disease remain unclear. Utilising the UK Biobank (UKB) resource to integrate disease outcomes, plasma proteomics and MRI data, we examined associations of IGFBP-1 and IGFBP-2 with incident diabetes and cardiovascular disease. Approximately 50,000 UKB participants with plasma proteomic measurements for IGFBP-1 and IGFBP-2 were included. Multivariate Cox regression models revealed that participants in the highest quartiles of IGFBP-1 and IGFBP-2 had a substantially lower risk of incident diabetes (hazard ratio (HR) = 0.31 and 0.32 respectively), but, paradoxically, had increased risks of incident macrovascular disease, all-cause and cardiovascular-related mortality (HR = 1.81 and 2.39). Both proteins were negatively associated with HbA1c levels, triglyceride/HDL ratio and abdominal adiposity, yet positively associated with NT-proBNP, troponin I, cardiac chamber size and aortic dimensions. In summary, negative associations of IGFBP-1 and IGFBP-2 with incident diabetes mellitus did not translate to a reduced cardiovascular risk, suggesting potentially complex actions of IGFBP-1 and IGFBP-2 in the pathophysiology of cardiometabolic disease.

Background Unsupervised machine learning has become a cornerstone of computational phenotyping across clinical medicine, genomics, imaging, and multi-omics research. However, phenotype discovery relies on a sequence of analytical decisions - including missing-data handling, preprocessing, dimensionality reduction, clustering methodology, and stochastic initialization - that are rarely evaluated collectively. Although clustering stability has been extensively investigated, the robustness of complete analytical workflows remains largely unexplored. Results We developed an Analytical Perturbation Framework that systematically quantifies the robustness of phenotype discovery by perturbing complete unsupervised learning workflows rather than individual clustering algorithms. Using a real-world cohort of 1,286 women with polycystic ovary syndrome (PCOS), we generated 116 valid analytical pipelines comprising alternative preprocessing strategies, missing-data handling methods, dimensionality reduction approaches, clustering algorithms, and random initializations. Agreement between independently generated phenotype solutions was consistently low (median Adjusted Rand Index = 0.079), indicating substantial sensitivity of phenotype discovery to routine analytical decisions. Variance decomposition identified preprocessing as the largest contributor to phenotype instability (22.8%), followed by clustering methodology (14.6%), whereas stochastic initialization explained only 3.1% of the observed variability. At the patient level, most individuals exhibited reproducible phenotype assignments (median Patient Robustness Score = 0.719), although a substantial subgroup showed markedly lower assignment stability. Feature perturbation analyses identified follicle-stimulating hormone, anti-thyroglobulin antibodies, anti-thyroid peroxidase antibodies, total testosterone, luteinizing hormone, and androstenedione as the strongest contributors to computational robustness, rather than biological importance. Finally, phenotype solutions demonstrating greater computational robustness also exhibited greater biological coherence during independent validation.

Adrenarche, the pre-pubertal rise in adrenal androgens, particularly dehydroepiandrosterone (DHEA) and its sulfated form DHEAS, is a critical driver of middle childhood cognitive and social development in modern humans. Compared to other apes, modern human adrenarche is more prolonged, with higher DHEAS levels. Whether this uniquely prolonged human adrenarche is a derived trait of Homo sapiens or has deeper hominin roots remains unresolved. Here, we examine the Neanderthal genetic variation in five key DHEAS biosynthesis genes (HSD3B2, CYP17A1, POR, CYB5A, SULT2A1). We also examine archaic introgression in these genes by comparing high-coverage Neanderthal genomes with globally diverse modern human sequences from the 1000 Genomes Project. We identify 29 Neanderthal-derived single nucleotide variants across these genes. Key steroidogenic genes in the biosynthesis pathway show no evidence of introgression, consistent with selection on pleiotropic regulators of steroidogenesis. In contrast, accessory genes carried introgressed Neanderthal haplotypes at moderate frequencies in non-African human populations, indicating Neanderthal variants are compatible with the human DHEAS synthesis pathway. All Neanderthal-specific variants were in non-coding regions, with three variants associated with reduced enzyme efficiency or DHEAS production in adults. Additionally, for all 29 positions, the modern human major allele is ancestral, and there is no evidence for a suite of novel adrenarche-extending variants. We conclude that the genetic foundation for extended adrenarche is shared between Homo sapiens and Neanderthals, and may have deeper hominin roots. Any phenotypic variation in adrenarche between modern humans and Neanderthals is more likely attributable to differential gene expression than to divergence in protein-coding sequences.

This study enrolled 152 hypertensive patients with an ARR > 3.7 to assess the relationship between the traditional hypokalemia cutoff (3.5 mmol/L) and primary aldosteronism (PA) screening, and to establish a new cutoff. Under the traditional cutoff, only 35.7% of PA patients presented with hypokalemia. ROC curve analysis identified a new cutoff of 4.22 mmol/L, which increased sensitivity from 35.7% to 77.5%, with a specificity of 91.1% and an AUC of 0.897. The findings indicate that the traditional cutoff is insufficiently sensitive, while the new cutoff markedly improves screening sensitivity and facilitates early detection of PA.

Context. Teprotumumab (TEP) is an emerging treatment for thyroid eye disease (TED), but real-world evidence outside the United States remains limited, and detailed changes in orbital components have not been fully clarified. Objective. To evaluate the effectiveness of TEP based on clinical manifestations and magnetic resonance imaging (MRI) findings, and compare it with that of intravenous glucocorticoid (IVGC) therapy. Methods: The TEP cohort included all 18 patients who started TEP therapy at Kyoto University Hospital by July 31, 2025. A historical IVGC cohort included 20 patients matched to the TEP cohort. Results: During 24 weeks of TEP therapy, proptosis measured using a Hertel exophthalmometer improved from 22 (20-22) to 19 (16-21) mm (p = 0.025), and clinical activity score decreased from 4 (3-5) to 1 (0-1) point (p < 0.001). Among 15 patients with diplopia, a reduction of at least 1 point in Gorman score was observed in 9 patients (60.0%). Thyroid-stimulating antibody titers decreased from 1,180% (349-4,710) to 282% (132-504) (p = 0.013). MRI-based comparisons with the IVGC cohort showed that TEP reduced both extraocular muscle and orbital fat areas, whereas IVGC reduced extraocular muscle area but conversely increased orbital fat area. Inflamed extraocular muscles identified on MRI were enlarged at baseline and showed marked shrinkage after both therapies. Conclusion: TEP showed robust effectiveness in Japanese real-world patients with TED. MRI-based analyses revealed distinct effects of TEP and IVGC on orbital fat and identified inflamed extraocular muscles as treatment-responsive components.