Steroids are critical for numerous physiological processes; disruption in their metabolism is associated with numerous endocrine disorders. Steroid quantification is essential to improve the understanding and diagnosis of these pathologies. Historically, urinary steroid profiling has been performed using low-throughput gas chromatography mass spectrometry (GC-MS), providing holistic coverage of steroid classes with low cross-reactivity. Here, we translate our previous GC-MS urinary steroid profile to a liquid chromatography tandem-MS (LC-MS/MS) platform, offering a validated, comprehensive overview of steroid metabolism with comparatively low sample preparation times and increased throughput. Urinary steroids were enzymatically hydrolysed and extracted via C18 solid-phase extraction. Quantification was conducted using a triple quadrupole mass spectrometer. Chromatographic separation of 27 analytes was completed in 16 min using a C18-T3 column. Due to chromatographic co-elution of tetrahydrocortisol and 5α-tetrahydrocortisol, a second injection was required on a BEH-C18 column for their separation. Lower limit of quantification (LLOQ) ranged from 2 to 20 ng/mL, witha ccuracy (bias) ranging from -18.7% to 19.9%, and precision (percentage coefficient of variation [%CV]) ranging from 4.0% to 18.6%. Matrix effects remained within the ideal range <±15% for all steroids. Recovery ranged from 76% to 103%, and intra- and inter-assay imprecision (CV) ranged from 0.8% to 14.9%. In 40 healthy volunteers, most analytes were detected above the LLOQ in over 95% of samples, although tetrahydro-aldosterone (85%), 5-pregenediol (68%), and pregnanetriol-one (59%) demonstrated lower quantification rates. Diurnal and sex-based variations were observed, with excretion levels significantly higher during daytime and in males. This robust, high-throughput LC-MS/MS method facilitates the simultaneous quantification of multiple steroid classes, enhancing its utility for clinical and research applications in endocrine science.

Abstract Background Steroid sulfatase (STS) cleaves sulfate groups from steroid hormones. In humans, STS deficiency is associated with X-linked ichthyosis (a dermatological disorder), neurodevelopmental/mood conditions, and cardiac arrhythmias. Until recently, no single-gene ‘knockout’ mammalian model existed to investigate these associations; previous work in such a model has been limited to skin phenotypes. Methods We generated a novel C57BL/6J mouse model with a deletion in critical exon 2 of Sts . We then examined gene expression and enzyme activity in liver and brain samples of homozygous mice, and assessed the breeding performance and health of male and female deletion-carriers. Subsequently, we compared performance across a range of behavioural paradigms in wildtype and homozygous male and female mice: elevated plus maze, open field, rotarod, spontaneous alternation, and acoustic startle/prepulse inhibition. We also investigated serum steroid hormone levels by liquid chromatography-mass spectrometry and measured heart weights and two morphological indices (bodyweight/tibia length) post mortem . Results Homozygous mice almost completely lacked STS expression/activity. Genetically-altered mice exhibited grossly-normal breeding performance, health, and endocrinology. Homozygous mice were more active, and had higher normalised heart weights, than wildtype mice. We also found significant genotype x sex interactions on bodyweight, and on two behavioural measures (potentially reflecting lower anxiety in homozygous males and heightened anxiety in homozygous females). Conclusions The ‘ Sts -deletion’ mouse represents an experimentally-tractable model in which to identify and characterise phenotypes associated with STS deficiency. The mechanistic basis of the genotype-phenotype associations described here requires further investigation, and whether such associations translate to humans remains to be tested.

Abstract Androgen excess drives metabolic and reproductive complications in polycystic ovary syndrome (PCOS), affecting 10-15% of women globally. Aldo-keto reductase 1C3 (AKR1C3) converts inactive precursors from both the classic and the recently identified 11-oxygenated androgen pathways, generating testosterone and 11-ketotestosterone, respectively, which exert comparable androgen receptor activation. Both circulate in similar concentrations in premenopausal women while 11-ketotestosterone is predominant after menopause and in PCOS. Here, we show that adipocytes are a major site of AKR1C3 and androgen receptor expression, with increased expression in women and individuals with obesity. Using human female adipose tissue explants, we find a much higher activation of 11-oxygenated over classic androgens, observing a decrease in 11-oxygenated but not classic androgen activation by AKR1C3 inhibition. Correspondingly, we demonstrate that AKR1C3 inhibitor treatment in premenopausal women selectively disrupts the activation of 11-oxygenated androgens. Pharmacological targeting of AKR1C3 provides a novel strategy to alleviate systemic and intra-adipose 11-oxygenated androgen excess. One Sentence Summary Inhibition of the androgen-activating enzyme AKR1C3 results in a major decrease in 11-oxygenated but not classic androgens in women.

BACKGROUND:Chronic kidney disease (CKD) is associated with increased incidence of sarcopenia and muscle weakness in aging patients. The glucocorticoid-activating enzyme11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) within skeletal muscle in these patients has been implicated in driving this process. We hypothesized that skeletal muscle 11β-HSD1 is elevated in CKD and aging, where it would represent a promising therapeutic target. METHODS:We measured 11β-HSD1 activity in quadriceps biopsies from 17 CKD patients (eGFR <30 mL/min) and 14 age-matched controls (mean age 71 ± 5 years). Muscle strength, mass, and gait speed were assessed in relation to tissue 11β-HSD1 activity. Primary human myotubes were treated with patient-derived sera to evaluate regulatory drivers. Murine models of CKD (adenine diet) and aging (21 months) in wild-type (WT) and 11β-HSD1 knockout (11βKO) animals were examined to model therapeutic targeting. RESULTS:Muscle 11β-HSD1 activity was not elevated with CKD, but increased with age (r = 0.559, P = .001), predicting lower grip strength (P = .008). Markers of inflammation, including C-reactive protein and IL-6, strongly predicted 11β-HSD1 activity. Pro-inflammatory mediators such as TNFα directly upregulated 11β-HSD1 activity in myotubes, whilst sera from CKD donors did not. Whilst mice with 11βKO deletion were not protected from CKD-driven muscle atrophy, 11βKO animals showed marked protection against age-related muscle loss. CONCLUSIONS:Skeletal muscle 11β-HSD1 activity is not induced by CKD itself, but increases significantly over aging, where it is a predictor of muscle weakness. Its targeted deletion preserves muscle mass in aged mice, supporting its efficacy in preventing sarcopenia in an aging CKD population.

OBJECTIVES:The strong female sex bias in Sjögren's disease (SjD) remains poorly understood. We evaluated hormonal risk factors and steroidal hormones in a well-characterized cohort. METHODS:Newly presenting patients investigated for SjD at a multidisciplinary clinic completed a hormonal risk factor questionnaire. One hundred eighty-four females with a diagnosis of SjD or non-SjD Sicca were included. Steroids were extracted from 88 consecutively recruited SjD and 59 Sicca serum samples and analysed via liquid chromatography tandem mass spectrometry. Sex steroid, glucocorticoid and mineralocorticoid data were compared with those from 165 healthy controls. RESULTS:A history of hirsutism was negatively associated with SjD even when corrected for smoking status, ethnicity, age and symptom duration (OR 0.14, 95% CI 0.04, 0.57; P = 0.006), whereas hysterectomy and ovary removal were positively associated, whether up to time of recruitment (hysterectomy OR 3.29, 95% CI 1.19, 9.11; P = 0.02) or to symptom onset (hysterectomy OR 5.8, 95% 1.52, 22.1; P = 0.01). Participants reporting hirsutism had higher androstenedione levels (2.51 vs 1.80 nmol/l; P = 0.03) and in pre-menopause, androstenedione and testosterone were higher in non-SjD sicca than both SjD and healthy controls. Within the whole cohort, androstenedione levels were inversely correlated with minor salivary gland focus score (r = -0.44; P = 0.03). Lower glucocorticoids were seen in both SjD and Sicca compared with healthy controls and negatively correlated with symptoms. CONCLUSION:Participants with SjD were less likely to report hirsutism and had lower androstenedione and testosterone pre-menopause when compared with non-SjD sicca. The role of androgens in modulating salivary gland inflammation should be further investigated.

Steroid sulfatase (STS) cleaves sulphate groups from steroid hormones. In humans, STS deficiency is associated with X-linked ichthyosis, an increased predisposition to neurodevelopmental and mood conditions (including Attention Deficit Hyperactivity Disorder, autism, depression and anxiety), and cardiac arrhythmia risk. Until recently, no single-gene 'knockout' mammalian model existed; previous work in such a model is limited to skin phenotypes. We generated a novel C57BL/6J mouse model with a deletion in exon 2 of Sts. We examined gene expression and enzyme activity in liver and brain samples of homozygous mice, and assessed the breeding performance and health of male and female deletion-carriers. Subsequently, we compared performance across a range of behavioural paradigms in wildtype and homozygous male and female mice: elevated plus maze, open field, rotarod, spontaneous alternation, and acoustic startle/prepulse inhibition. We also investigated serum steroid hormone levels by liquid chromatography-mass spectrometry and measured heart weights and two morphological indices (bodyweight/tibia length) post mortem. Homozygous mice almost completely lacked STS expression/activity. Genetically-altered mice exhibited grossly-normal breeding performance, health, and endocrinology. Homozygous mice were more active and had higher normalised heart weights than wildtype mice. We also found significant genotype × sex interactions on bodyweight and on two behavioural measures (potentially reflecting lower anxiety in homozygous males and heightened anxiety in homozygous females). The 'Sts-deletion' mouse represents an experimentally-tractable model in which to identify and characterise phenotypes associated with STS deficiency. The mechanistic basis of the associations described here requires further investigation, and whether these translate to humans remains to be tested.

Objective Endometriosis is a chronic, hormone-dependent condition affecting an estimated 190 million women worldwide. Our understanding of hormonal contributions to endometriosis pathophysiology is incomplete, hindering the identification of diagnostic biomarkers and novel therapeutic targets. Although the role of estrogens is well established, research on androgens in endometriosis is limited and the contribution of adrenal-derived 11-oxygenated androgens remains largely unknown. Methods We performed steroid androgen profiling to measure androgen concentrations in serum from healthy controls and women with laparoscopically confirmed endometriosis. We found that women with endometriosis had a distinct hormone signature characterized by systemic differences in adrenal androgen concentrations and 11-ketotestosterone excess. Using metabolomic data, we generated statistical models that showed robust discrimination between healthy controls and women with endometriosis (AUC = 0.99; positive predictive power = 96.84%, negative predictive power = 92.86%) consistent with an endometriosis-specific signature. Data were partitioned into train and validation groups to assess diagnostic potential and a refined model identified >95% of endometriosis patients in a blinded sample set. Conclusions Collectively, these data reframe endometriosis as an androgen-dependent disorder and highlight 11-oxygenated androgens as potential diagnostic biomarkers and future therapeutic targets.

Introduction: Oxidized low density lipoproteins (OxLDL) present in atherosclerotic plaques. Epidemiological studies demonstrate that IgM antibody against malondialdehyde LDL (MDA-LDL), the major subtype of OxLDL, is inversely associated with severity of coronary artery disease (CAD), heart attack and death. In human, circulating marginal zone B cells (MZB) were recently identified as producers of these atheroprotective IgM to MDA-LDL. The frequency of circulating MZB cells correlates with CAD severity. We further discover that MZB cells from subjects with high CAD burden produce less atheroprotective IgM to MDA-LDL. This study explores phenotypic changes in MZB cells in relation to CAD severity and investigates underlying mechanisms Hypothesis: The chemokine receptor CCR6 regulates the production of atheroprotective IgM against MDA-LDL by MZB cells and is downregulated in subjects with severe CAD. Methods: Humanized mice were adoptively transferred with sort-purified human MZB cells from low and high CAD severity subjects. Single cell multi-omics (CITE-Seq) was performed on B cells from both low and high CAD groups. In-vitro migration study, imaging-flow cytometry, as well as CRISPR-mediated gene knock down were utilized to further investigate mechanisms. Results: Humanized mice receiving MZB cells from low-CAD subjects (n = 7) produced higher levels of atheroprotective IgM than those receiving cells from high-CAD subjects (n = 6). Single-cell analysis of B cells from low (n = 30) and high (n = 30) CAD subjects revealed higher CCR6 expression in MZB cells from the low-CAD group. In vitro activation of MZB cells with MDA antigen led to downregulation of both CD24 and CCR6. Imaging flow cytometry and proximity ligation assays suggested co-localization of CD24 and CCR6. Blocking CD24 with a monoclonal antibody reduced surface CCR6 expression and impaired CCL20-induced migration. Finally, CCR6-knockdown MZB cells (n = 4) exhibited reduced splenic migration and IgM production in humanized mice compared to wild-type controls (n = 6). Conclusion: MZB cells from individuals with severe CAD exhibit reduced CCR6 expression and impaired atheroprotective IgM production. CCR6 is critical for splenic migration of human MZB cells and subsequent IgM production.

OBJECTIVES:Vamorolone, a dissociated steroidal compound with reduced side effects, offers a promising alternative to traditional glucocorticoids for inflammatory diseases. Unlike conventional glucocorticoids, vamorolone lacks the hydroxyl or ketone groups required for metabolism by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a key enzyme that modulates glucocorticoid activity. This study investigates vamorolone's resistance to 11β-HSD1 metabolism and assesses its therapeutic efficacy in the murine tumour necros factor-alpha-overexpressing (TNFtg) model of polyarthritis. METHODS:11β-HSD1 metabolism and action were examined in Hs68 and primary leucocyte culture. Vamorolone 20 mg/kg/day, prednisolone (standard of care) or vehicle were administered by gavage to TNFtg or TNFtg 11β-HSD1 knock-out (TNFtg11BKOKO) animals. Body weight and disease severity were scored daily, and markers of inflammation, joint destruction and side effects assessed at day 56 of age. RESULTS:Vamorolone was entirely resistant to 11β-HSD1 metabolism in vitro. Vamorolone demonstrated comparable anti-inflammatory actions in TNFtg mice, with a comparable reduction in joint inflammation, serum interleukin-6 (IL-6) and synovitis relative to prednisolone. However, vamorolone-treated mice did not experience typical glucocorticoid side effects, including adrenal atrophy, body weight reduction, muscle wasting or inhibition of anabolic bone metabolism. These benefits persisted in 11β-HSD1 knockout mice, indicating that the efficacy of vamorolone is largely independent of 11β-HSD1 metabolism. CONCLUSION:The findings suggest that at the effective anti-inflammatory dose examined in this study, vamorolone possesses a reduced profile of deleterious systemic effects relative to prednisolone. Whilst highlighting its potential for broader clinical application in inflammatory conditions, it remains unclear whether these side effects would remain mild at markedly higher doses.