Monitoring adrenal insufficiency through salivary steroids: a pilot study.

BACKGROUND: Various glucocorticoid replacement therapies (GRTs) are available for adrenal insufficiency (AI). However, their effectiveness in restoring glucocorticoid rhythm and exposure lacks adequate biochemical markers. We described the diurnal salivary cortisol (SalF) and cortisone (SalE) rhythm among different GRTs and analysed the associations between saliva-derived parameters and life quality questionnaires. METHODS: Control subjects (CSs, n = 28) and AI patients receiving hydrocortisone (HC, n = 9), cortisone acetate (CA, n = 23), and dual-release hydrocortisone once (DRHC-od, n = 10) and twice a day (DRHC-td, n = 6) collected 9 saliva samples from 07:00 to 23:00. Patients compiled Pittsburgh Sleep Quality Index, Hospital Anxiety and Depression Scale, and Addison disease-specific quality-of-life questionnaires. SalE and SalF were measured by liquid chromatography-mass spectrometry. Exposure was monitored using SalE for HC and DRHC and SalF for CA. Area under the curve (AUC) was computed. Different GRTs were compared by Z-scores calculated from saliva-derived parameters. Questionnaire results predictors were evaluated with multiple regression analysis. RESULTS: Compared with controls, all GRTs resulted in glucocorticoid overexposure in the morning. Hydrocortisone, CA, and DRHC-td caused overexposure also in afternoon and evening. Compared with other treatments, CA determined increased Z-score-07:00 (P < .001), DRHC-td determined increased Z-score-AUC07:00→14:00 (P = .007), and DRHC-od induced lower Z-score-AUC14:00→23:00 (P = .015). Z-scores-AUC14:00→16:00 ≥ .619 best predicted questionnaire scores. CONCLUSIONS: None of the GRTs mimics normal glucocorticoid rhythmicity and exposure. SalE, SalF, and Z-score may be useful markers for monitoring and comparing different GRTs. Excess glucocorticoid in early afternoon best associated with depressive symptoms and worse life and sleep quality.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Comparative targeted metabolomic profiles of porcine plasma and serum.

Metabolomics has been used to characterise many biological matrices and obtain detailed pictures of biological systems based on many metabolites. Plasma and serum are two blood-derived biofluids commonly used to assess and monitor the organismal metabolism and obtain information on the physiological and health conditions of an animal. Plasma is the supernatant that is separated from the cellular components after centrifugation of the blood that is first added with an anticoagulant. Serum is obtained after centrifugation of the blood that has been coagulated. The choice of one or the other biofluid for metabolomic analyses is related to specific analytical needs and technical issues, to problems derived by the collection and preparation steps, in particular when specimens are sampled from animals involved in field studies. Thus far, most of the metabolomic studies that compared plasma and serum have been carried out in humans and very little is known on the pigs. In this study, we used a targeted metabolomic platform that can detect about 180 metabolites of five biochemical classes to compare plasma and serum profiles of samples collected from 24 pigs. To also obtain a cross-species comparative metabolomic analysis, information for human plasma and serum derived from the same platform was retrieved from previous studies. Statistical analyses included univariate and multivariate approaches aimed at identifying stable and/or differentially abundant metabolites between the two porcine biofluids. A total of 154 (∼83%) metabolites passed the initial quality control, indicating a good repeatability of the analytical platform in pigs. Discarded metabolites included aspartate and biogenic amines that were already reported to be unstable in human studies. More than 80% of the metabolites had similar profiles in both porcine biofluids (average correlation was 0.75). Concentrations were usually higher in serum than in plasma, in agreement with what was already reported in humans. The univariate analysis identified 44 metabolites that had statistically different concentrations between porcine plasma and serum, of which 28 metabolites were also confirmed by the multivariate analysis. The obtained picture described similarities and differences between these two biofluids in pigs and the related human-pig comparisons. The obtained information can be useful for the choice of one or the other matrix for the implementation of metabolomic studies in this livestock species. The results can also provide useful hints to valuing the pig as animal model, in particular when metabolite-derived physiological states are relevant.

SPIOMET4HEALTH-efficacy, tolerability and safety of lifestyle intervention plus a fixed dose combination of spironolactone, pioglitazone and metformin (SPIOMET) for adolescent girls and young women with polycystic ovary syndrome: study protocol for a multicentre, randomised, double-blind, placebo-controlled, four-arm, parallel-group, phase II clinical trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most prevalent, chronic endocrine-metabolic disorder of adolescents and young women (AYAs), affecting 5-10% of AYAs worldwide. There is no approved pharmacological therapy for PCOS. Standard off-label treatment with oral contraceptives (OCs) reverts neither the underlying pathophysiology nor the associated co-morbidities. Pilot studies have generated new insights into the pathogenesis of PCOS, leading to the development of a new treatment consisting of a fixed, low-dose combination of two so-called insulin sensitisers [pioglitazone (PIO), metformin (MET)] and one mixed anti-androgen and anti-mineralocorticoid also acting as an activator of brown adipose tissue [spironolactone (SPI)], within a single tablet (SPIOMET). The present trial will evaluate the efficacy, tolerability and safety of SPIOMET, on top of lifestyle measures, for the treatment of PCOS in AYAs. METHODS: In this multicentre, randomised, double-blind, placebo-controlled, four-arm, parallel-group, phase II clinical trial, AYAs with PCOS will be recruited from 7 clinical centres across Europe. Intention is to randomise a total of 364 eligible patients into four arms (1:1:1:1): Placebo, PIO, SPI + PIO (SPIO) and SPI + PIO + MET (SPIOMET). Active treatment over 12 months will consist of lifestyle guidance plus the ingestion of one tablet daily (at dinner time); post-treatment follow-up will span 6 months. Primary endpoint is on- and post-treatment ovulation rate. Secondary endpoints are clinical features (hirsutism, menstrual regularity); endocrine-metabolic variables (androgens, lipids, insulin, inflammatory markers); epigenetic markers; imaging data (carotid intima-media thickness, body composition, abdominal fat partitioning, hepatic fat); safety profile; adherence, tolerability and acceptability of the medication; and quality of life in the study participants. Superiority (in this order) of SPIOMET, SPIO and PIO will be tested over placebo, and if present, subsequently the superiority of SPIOMET versus PIO, and if still present, finally versus SPIO. DISCUSSION: The present study will be the first to evaluate-in a randomised, double-blind, placebo-controlled way-the efficacy, tolerability and safety of SPIOMET treatment for early PCOS, on top of a lifestyle intervention. TRIAL REGISTRATION: EudraCT 2021-003177-58. Registered on 22 December 2021. https://www.clinicaltrialsregister.eu/ctr-search/search?query=%092021-003177-58 .

Efficacy of very low-calorie ketogenic diet with the Pronokal® method in obese women with polycystic ovary syndrome: a 16-week randomized controlled trial.

Objective: The aim of this study isto assess the efficacy of a very low-calorie ketogenic diet (VLCKD) method vs a Mediterranean low-calorie diet (LCD) in obese polycystic ovary syndrome (PCOS) women of a reproductive age. Design: Randomized controlled open-label trial was performed in this study. The treatment period was 16 weeks; VLCKD for 8 weeks then LCD for 8 weeks, according to the Pronokal® method (experimental group; n = 15) vs Mediterranean LCD for 16 weeks (control group; n = 15). Ovulation monitoring was carried out at baseline and after 16 weeks, while a clinical exam, bioelectrical impedance analysis (BIA), anthropometry, and biochemical analyses were performed at baseline, at week 8, and at week 16. Results: BMI decreased significantly in both groups and to a major extent in the experimental group (-13.7% vs -5.1%, P = 0.0003). Significant differences between the experimental and the control groups were also observed in the reduction of waist circumference (-11.4% vs -2.9%), BIA-measured body fat (-24.0% vs -8.1%), and free testosterone (-30.4% vs -12.6%) after 16 weeks (P = 0.0008, P = 0.0176, and P = 0.0009, respectively). Homeostatic model assessment for insulin resistance significantly decreased only in the experimental group (P = 0.0238) but without significant differences with respect to the control group (-23% vs -13.2%, P > 0.05). At baseline, 38.5% of participants in the experimental group and 14.3% of participants in the control group had ovulation, which increased to 84.6% (P = 0.031) and 35.7% (P > 0.05) at the end of the study, respectively. Conclusion: In obese PCOS patients, 16 weeks of VLCKD protocol with the Pronokal® method was more effective than Mediterranean LCD in reducing total and visceral fat, and in ameliorating hyperandrogenism and ovulatory dysfunction. Significance statements: To the best of our knowledge, this is the first randomized controlled trial on the use of the VLCKD method in obese PCOS. It demonstrates the superiority of VLCKD with respect to Mediterranean LCD in reducing BMI with an almost selective reduction of fat mass and a unique effect of VLCKD in reducing visceral adiposity, insulin resistance, and in increasing SHBG with a consequent reduction of free testosterone. Interestingly, this study also demonstrates the superiority of the VLCKD protocol in improving ovulation, whose occurrence increased by 46.1% in the group treated by the VLCKD method against a rise of 21.4% in the group treated by Mediterranean LCD. This study extends the therapeutic approach possibilities in obese PCOS women.

Mild autonomous cortisol secretion in adrenal incidentalomas and risk of fragility fractures: a large cross-sectional study.

OBJECTIVE: Mild autonomous cortisol secretion (MACS) has been associated with a higher prevalence of osteoporosis, although most data rely on single-center studies with limited sample size. We aimed to assess the prevalence of fragility fractures and contributing factors in a large cohort of patients with adrenal incidentalomas. DESIGN AND METHODS: Medical records of 1023 patients with adrenal incidentalomas from 1990 to 2019 were reviewed, and 735 patients were selected. Clinically obtained electronic radiological images closest to first endocrine evaluation, such as lateral views of spine X-rays or CT thoraco-abdominal scans, were reviewed to screen for asymptomatic morphometric vertebral fractures. Clinical fragility fractures, hormonal, and dual-energy x-ray absorptiometry (DXA) indices were also recorded. RESULTS: Four hundred seventy-four patients had nonfunctioning (NF) adrenal incidentalomas, 238 had MACS and 23 adrenal Cushing's syndrome (AC). Prevalence of fragility fractures was different (P = .018) between groups, respectively, 24.1% (NF), 34.0% (MACS), and 30.4% (AC), with significant difference between NF and MACS (P = .012). When analyzed separately by sex and menopausal status, this difference remained significant in postmenopausal women (P = .011), with a fracture prevalence of 22.2% (NF) and 34.6% (MACS). Fracture prevalence was similar in males. Women with MACS aged ≥65 years reported a 48.8% prevalence of fractures, as compared with 29.5% in NF (P < .01). In postmenopausal women, fragility fractures were associated with age (odds ratio [OR] 1.1, P < .001), smoking (OR 1.8, P = .048), and 1 mg-dexamethasone suppression test (DST) cortisol (OR 3.1, P = .029), while in men, only age was associated with fragility fractures. CONCLUSIONS: A considerable fracture burden was shown in postmenopausal women with adrenal incidentalomas and MACS, with clinical implications for the evaluation and management of bone metabolism.

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel.

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.

Determinants of sexual function in men living with HIV younger than 50 years old: Focus on organic, relational, and psychological issues.

BACKGROUND: Sexual dysfunctions, particularly erectile dysfunction, are common in men living with HIV, whose organic and psychological components remain to be clarified. The aim of the study is to investigate the impact of risk factors of sexual dysfunctions, including organic, relational, and psychological determinants of erectile function, in men living with HIV younger than 50 years old. METHODS: A cross-sectional, observational study was conducted in men living with HIV < 50 years. The questionnaire International Index of Erectile Function-15 was used to assess the prevalence and degree of erectile dysfunction. The structured interview of erectile dysfunction was used to explore the organic (Scale 1), relational (Scale 2), and psychological (Scale 3) components of erectile dysfunction. Total testosterone, estradiol, and dihydrotestosterone were measured by liquid chromatography-tandem-mass spectrometry; free testosterone was calculated by the Vermeulen equation. RESULTS: A total of 313 consecutive men living with HIV were prospectively enrolled (median age 47.0 years; median HIV-infection duration 16.2 years). 187 patients (59.7%) had erectile dysfunction, with a higher prevalence of non-heterosexual (138 out of 187, 73.8%) than heterosexual patients (p = 0.003). Patients with erectile dysfunction showed a worse score of structured interview of erectile dysfunction scale 3 compared to patients without erectile dysfunction (p = 0.025); the International Index of Erectile Function-15 was inversely related to structured interview of erectile dysfunction scale 3 (p = 0.042). No difference was found for sex steroids (total testosterone, estradiol, free testosterone, and dihydrotestosterone) between men living with HIV with and without erectile dysfunction. In the multivariate analysis sexual orientation, and lack of stable relationships were major determinants for erectile dysfunction. Only 35 of 187 patients with erectile dysfunction (18.7%) reported the use of erectile dysfunction medications. CONCLUSIONS: Within the multidimensional network of erectile dysfunction in men living with HIV, the psychological component is predominant, highlighting the contribution of peculiar factors related to HIV distress (e.g., fear of virus transmission, stigma) rather than gonadal status and other classical risk factors. In contrast to the high prevalence, only a few patients reported the use of erectile dysfunction medications suggesting a general under-management of such issues.

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.

Multi-omics gut microbiome signatures in obese women: role of diet and uncontrolled eating behavior.

BACKGROUND: Obesity and related co-morbidities represent a major health challenge nowadays, with a rapidly increasing incidence worldwide. The gut microbiome has recently emerged as a key modifier of human health that can affect the development and progression of obesity, largely due to its involvement in the regulation of food intake and metabolism. However, there are still few studies that have in-depth explored the functionality of the human gut microbiome in obesity and even fewer that have examined its relationship to eating behaviors. METHODS: In an attempt to advance our knowledge of the gut-microbiome-brain axis in the obese phenotype, we thoroughly characterized the gut microbiome signatures of obesity in a well-phenotyped Italian female cohort from the NeuroFAST and MyNewGut EU FP7 projects. Fecal samples were collected from 63 overweight/obese and 37 normal-weight women and analyzed via a multi-omics approach combining 16S rRNA amplicon sequencing, metagenomics, metatranscriptomics, and lipidomics. Associations with anthropometric, clinical, biochemical, and nutritional data were then sought, with particular attention to cognitive and behavioral domains of eating. RESULTS: We identified four compositional clusters of the gut microbiome in our cohort that, although not distinctly associated with weight status, correlated differently with eating habits and behaviors. These clusters also differed in functional features, i.e., transcriptional activity and fecal metabolites. In particular, obese women with uncontrolled eating behavior were mostly characterized by low-diversity microbial steady states, with few and poorly interconnected species (e.g., Ruminococcus torques and Bifidobacterium spp.), which exhibited low transcriptional activity, especially of genes involved in secondary bile acid biosynthesis and neuroendocrine signaling (i.e., production of neurotransmitters, indoles and ligands for cannabinoid receptors). Consistently, high amounts of primary bile acids as well as sterols were found in their feces. CONCLUSIONS: By finding peculiar gut microbiome profiles associated with eating patterns, we laid the foundation for elucidating gut-brain axis communication in the obese phenotype. Subject to confirmation of the hypotheses herein generated, our work could help guide the design of microbiome-based precision interventions, aimed at rewiring microbial networks to support a healthy diet-microbiome-gut-brain axis, thus counteracting obesity and related complications.

Overall Sexual Function in Dysmetabolic Obese Men with Low Testosterone Levels Treated with Clomiphene Citrate.

BACKGROUND: Sexual disorders are the most common clinical manifestations of hypogonadism. Functional hypogonadism is the most frequent form, and clomiphene citrate (CC) has been recently introduced as a possible off-label therapeutic option for these patients. OBJECTIVES: This study aimed to evaluate the effects of CC on the overall sexual function in dysmetabolic obese men with low testosterone (T) levels. METHODS: This was a sub-study of a randomized, double-blind, cross-over, placebo-controlled trial that included twenty-four obese or overweight subjects with impaired glucose tolerance or type 2 diabetes and confirmed low total T (≤10.4 nmol/l) levels. Subjects were treated with CC or placebo (Plac) for 12 weeks, with an interval wash-out period of 6 weeks between treatments. All subjects were on metformin 2gr/day and a low-calorie diet. The between-treatment difference in the overall sexual function was assessed by IIEF-15 and a qADAM questionnaire. RESULTS: IIEF-15 and qADAM questionnaire data were available for 18 individuals. In unadjusted analyses, CC was associated with lower IIEF-15 total, erectile function, and intercourse satisfaction domain scores than Plac. After adjustments for multiple variables, CC was associated with a higher IIEF-15 sexual desire domain score (+0.9 ± 0.8; p<.001) despite a lower qADAM score (-2.1 ± 0.9; p=.008) with respect to Plac. No differences were found for the other domains between groups. DISCUSSION: The clinical significance of the absolute changes in IIEF-15 and qADAM scores during CC versus Plac is limited. However, CC has a reliable effect on sexual desire and is also as safe as Plac. According to the sample size, duration of follow-up, and inclusion criteria defined for the main study, further studies are therefore needed to assess the long-term efficacy of CC. CONCLUSION: Compared to Plac, CC was found to be associated with a neutral effect on overall sexual function.

Primary, secondary and compensated male biochemical hypogonadism in people living with HIV (PLWH): relevance of sex hormone-binding globulin (SHBG) measurement and comparison between liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemiluminescent immunoassay for sex steroids assay.

BACKGROUND: Data about classification of hypogonadism and estrogen deficiency in male people living with HIV (PLWH) are scanty. AIM: To investigate the prevalence and characterization of biochemical hypogonadism and relative estrogen deficiency in male PLWH aged < 50 comparing liquid chromatography-tandem mass spectrometry (LC-MS/MS) with chemiluminescent immunoassay (CI), and combining gonadotropin, sex hormone-binding globulin (SHBG) and serum estradiol (E2) measurements. METHODS: Prospective, cross-sectional, observational study. Serum total testosterone (TT), E2, gonadotropins, SHBG were measured by CI. TT and E2 were also assessed by LC-MS/MS. Free testosterone (cFT) was calculated by Vermeulen equation. RESULTS: A total of 316 PLWH (45.3 ± 5.3 years) were enrolled. TT and cFT by LC-MS/MS were lower compared to CI (p < 0.0001). The prevalence of biochemical hypogonadism was higher with LC-MS/MS than CI, both for TT (5.1% vs 3.2%, p < 0.0001) or cFT (9.5% vs 7%, p < 0.0001). The prevalence of hypogonadism (overt + compensated) was 17.1% for cFT using LC-MS/MS. Secondary form of hypogonadism was more prevalent than primary. The prevalence of relative estrogen deficiency was of 30.0% among hypogonadal patients and 15.5% among eugonadal. CONCLUSIONS: The prevalence of male hypogonadism results underestimated by CI compared to LC-MS/MS in PLWH, both for TT and cFT. SHBG and gonadotropins are essential for detecting T deficiency.

Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

Biomediators in Polycystic Ovary Syndrome and Cardiovascular Risk.

Polycystic ovary syndrome (PCOS) is extremely heterogeneous in terms of clinical manifestations. The variability of the syndrome's phenotype is derived from the genetic and molecular heterogeneity, with a great deal of environmental factors that may have long-term health consequences, such as metabolic and cardiovascular (CV) diseases. There is no doubt that women with PCOS suffer from metabolic complications more than their age-matched counterparts in the general population and at an earlier age. Obesity, low steroid hormone-binding globulin (SHBG), hyperandrogenemia, insulin resistance, and compensatory hyperinsulinemia are biomediators and early predictors of metabolic complications in PCOS. Doubts remain about the real risk of CV diseases in PCOS and the molecular mechanisms at the basis of CV complications. Based on that assumption, this review will present the available evidence on the potential implications of some biomediators, in particular, hyperandrogenism, estrogen-progesterone imbalance, insulin resistance, and low SHBG, in the processes leading to CV disease in PCOS, with the final aim to propose a more accurate CV risk assessment.

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males.

BACKGROUND: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. METHODS: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. FINDINGS: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). INTERPRETATION: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. FUNDING: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo.

Inverse correlation between plasma 2-arachidonoylglycerol levels and subjective severity of depression.

OBJECTIVE: Endocannabinoids have been implicated in the pathophysiology of Major Depressive Disorder (MDD) and might represent potential targets for therapeutic intervention. Objectives of the study were: (1) to measure plasma levels of endocannabinoids in a group of antidepressant-free depressed outpatients; (2) to explore their relationship with the severity of depressive symptoms as subjectively perceived by the patients; and (3) to investigate the effect of the selective serotonin reuptake inhibitor escitalopram on endocannabinoid levels. METHODS: We measured plasma levels of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anadamide), in 12 drug-free outpatients diagnosed with MDD and in 12 matched healthy controls. In the patient group, endocannabinoids plasma levels were assessed at baseline and after 2 months of treatment with escitalopram. RESULTS: Baseline plasma levels of the two endocannabinoids did not differ between depressed patients and healthy controls. However, there was a significant inverse correlation between 2-arachidonoylglycerol levels and the severity of subjectively perceived depressive symptoms. Treatment with escitalopram did not change endocannabinoid levels in depressed patients, although it caused the expected improvement of depressive symptoms. CONCLUSIONS: Our results suggest that 2-arachidonylglycerol, the most abundant endocannabinoid in the central nervous system, might act to mitigate depressive symptoms, and raise the interesting possibility that 2-arachidonylglycerol and anandamide are differentially regulated in patients affected by MDD. Also, our data suggest but do not prove that the endocannabinoid system is not regulated by serotonergic transmission, at least in depressed patients.

Steroid reference intervals in women: influence of menopause, age and metabolism.

OBJECTIVE: To investigate the impact of age, obesity and metabolic parameters on 13 circulating steroids in reproductive and menopausal age. To define reference intervals (RIs). DESIGN: Cross-sectional. METHODS: Three hundred and twenty five drug-free, healthy and eumenorrheic women were selected from the general population. Independent relationships of LC-MS/MS-determined steroid levels with age, BMI and metabolic parameters were estimated. Reference sub-cohorts were defined for calculating upper and lower limits in reproductive age, menstrual phases and menopause, and these were compared with limits in dysmetabolic sub-cohorts. RESULTS: Lower androgens, pro-androgens and estrogens, but higher cortisol and metabolites were found in menopausal compared to reproductive age women. Androgens and precursors decreased during reproductive age (P < 0.001-P = 0.002) but not after menopause. 17OH-progesterone decreased with BMI (P = 0.006) and glucocorticoids with waist circumference (P < 0.001P = 0.002) in reproductive age, but increased with triglycerides (P=0.011P=0.038) after menopause. Inverse associations of dihydrotestosterone with BMI (P=0.004) and HDL-cholesterol (P=0.010), estrone with total cholesterol (P=0.033) and estradiol with triglycerides (P=0.011) were found in reproductive age. After menopause, estrone increased with waist circumference (P<0.001) and decreased with insulin resistance (P=0.012). Ovarian steroid RIs were estimated in menstrual phases and menopause. Age- and reproductive status-specific RIs were generated for androgens, precursors and corticosteroids. Lower limits for reproductive age cortisol (P=0.020) and menopausal 11-deoxycortisol (P=0.003) in dysmetabolic sub-cohorts were reduced and increased, respectively, compared to reference limits. CONCLUSIONS: Obesity and dysmetabolism differently influence circulating steroids in reproductive and menopausal status. Age, menstrual and menopausal status-specific RIs were provided by LC-MS/MS for a broad steroid panel.

Health status is related to testosterone, estrone and body fat: moving to functional hypogonadism in adult men with HIV.

OBJECTIVE: Hypogonadism is common in HIV-infected men. The relationship between health status, sex steroids and body composition is poorly known in HIV. The aim was to investigate the association between health status (comorbidities/frailty), body composition, and gonadal function in young-to-middle-aged HIV-infected men. DESIGN: Prospective, cross-sectional, observational study. METHODS: HIV-infected men aged <50 years and ongoing Highly Active Antiretroviral Therapy were enrolled. Serum total testosterone (TT), estradiol (E2), estrone (E1) were measured by liquid chromatography-tandem mass spectrometry, LH and FSH by immunoassay. Free testosterone (cFT) was calculated by Vermeulen equation. Body composition was assessed by dual-energy X-ray absorptiometry and abdominal CT scan. Multimorbidity (MM) and frailty were defined as ≥3 comorbidities and by a 37-item index, respectively. RESULTS: A total of 316 HIV-infected men aged 45.3 ± 5.3 years were enrolled. Body fat parameters were inversely related to cFT and TT, and directly related to E1 and E2/testosterone (TS) ratio. Patients with MM had lower cFT (P < 0.0001) and TT (P = 0.036), and higher E1 (P < 0.0001) and E2/TS ratio (P = 0.002). Frailty was inversely related to cFT (R2 = 0.057, P < 0.0001) and TT (R2 = 0.013, P = 0.043), and directly related to E1 (R2 = 0.171, P < 0.0001), E2 (R2 = 0.041, P = 0.004) and E2/TS ratio (R2 = 0.104, P < 0.0001). CONCLUSIONS: Lower TT and cFT, higher E1, E2/TS ratio and visceral fat were independently associated to poor health status and frailty, being possible hallmarks of unhealthy conditions in adult HIV-infected men. Overall, MM, frailty and body fat mass are strictly associated to each other and to sex steroids, concurring together to functional male hypogonadism in HIV.

Body mass index rather than the phenotype impacts precocious ultrasound cardiovascular risk markers in polycystic ovary syndrome.

OBJECTIVE: Research into cardiovascular disease (CV) prevention has demonstrated a variety of ultrasound (US) markers predicting risk in the general population but which have been scarcely used for polycystic ovary syndrome (PCOS). Obesity is a major factor contributing to CV disease in the general population, and it is highly prevalent in PCOS. However, it is still unclear how much risk is attributable to hyperandrogenism. This study evaluates the most promising US CV risk markers in PCOS and compares them between different PCOS phenotypes and BMI values. DESIGN: Women fulfilling the Rotterdam criteria for PCOS were recruited from our outpatient clinic for this cross-sectional study. METHODS: Participants (n = 102) aged 38.9 ± 7.4 years were stratified into the four PCOS phenotypes and the three BMI classes (normal-weight, overweight, obese). They were assessed for clinical and biochemical parameters together with the following US markers: coronary intima-media thickness (cIMT), flow-mediated vascular dilation (FMD), nitroglycerine-induced dilation (NTG), and epicardial fat thickness (EFT). RESULTS: There was no statistical difference among the four phenotypes in terms of cIMT, FMD, NTG or EFT, however all the US parameters except NTG showed significant differences among the three BMI classes. Adjusting for confounding factors in multiple regression analyses, EFT retained the greatest direct correlation with BMI and cIMT remained directly correlated but to a lesser degree. CONCLUSIONS: This study showed that obesity rather than the hyperandrogenic phenotype negatively impacts precocious US CV risk markers in PCOS. In addition, EFT showed the strongest association with BMI, highlighting its potential for estimating CV risk in PCOS.

Age-related changes in human Leydig cell status.

STUDY QUESTION: What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER: Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY: There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION: This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies were obtained from 15 heart beating organ donors (age range: 19-85 years) and 24 patients (age range: 19-45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing age was negatively associated with Leydig cell number (R = -0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= -0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= -0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. LIMITATIONS, REASONS FOR CAUTION: In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.