Gender-Affirming Hormone Therapy Modifies the CpG Methylation Pattern of the ESR1 Gene Promoter After Six Months of Treatment in Transmen.

BACKGROUND: Brain sexual differentiation is a process that results from the effects of sex steroids on the developing brain. Evidence shows that epigenetics plays a main role in the formation of enduring brain sex differences and that the estrogen receptor α (ESR1) is one of the implicated genes. AIM: To analyze whether the methylation of region III (RIII) of the ESR1 promoter is involved in the biological basis of gender dysphoria. METHODS: We carried out a prospective study of the CpG methylation profile of RIII (-1,188 to -790 bp) of the ESR1 promoter using bisulfite genomic sequencing in a cisgender population (10 men and 10 women) and in a transgender population (10 trans men and 10 trans women), before and after 6 months of gender-affirming hormone treatment. Cisgender and transgender populations were matched by geographical origin, age, and sex. DNAs were treated with bisulfite, amplified, cloned, and sequenced. At least 10 clones per individual from independent polymerase chain reactions were sequenced. The analysis of 671 bisulfite sequences was carried out with the QUMA (QUantification tool for Methylation Analysis) program. OUTCOMES: The main outcome of this study was RIII analysis using bisulfite genomic sequencing. RESULTS: We found sex differences in RIII methylation profiles in cisgender and transgender populations. Cismen showed a higher methylation degree than ciswomen at CpG sites 297, 306, 509, and at the total fragment (P ≤ .003, P ≤ .026, P ≤ .001, P ≤ .006). Transmen showed a lower methylation level than trans women at sites 306, 372, and at the total fragment (P ≤ .0001, P ≤ .018, P ≤ .0107). Before the hormone treatment, transmen showed the lowest methylation level with respect to cisgender and transgender populations, whereas transwomen reached an intermediate methylation level between both the cisgender groups. After the hormone treatment, transmen showed a statistically significant methylation increase, whereas transwomen showed a non-significant methylation decrease. After the hormone treatment, the RIII methylation differences between transmen and transwomen disappeared, and both transgender groups reached an intermediate methylation level between both the cisgender groups. CLINICAL IMPLICATIONS: Clinical implications in the hormonal treatment of trans people. STRENGTHS & LIMITATIONS: Increasing the number of regions analyzed in the ESR1 promoter and increasing the number of tissues analyzed would provide a better understanding of the variation in the methylation pattern. CONCLUSIONS: Our data showed sex differences in RIII methylation patterns in cisgender and transgender populations before the hormone treatment. Furthermore, before the hormone treatment, transwomen and transmen showed a characteristic methylation profile, different from both the cisgender groups. But the hormonal treatment modified RIII methylation in trans populations, which are now more similar to their gender. Therefore, our results suggest that the methylation of RIII could be involved in gender dysphoria. Fernández R, Ramírez K, Gómez-Gil E, et al. Gender-Affirming Hormone Therapy Modifies the CpG Methylation Pattern of the ESR1 Gene Promoter After Six Months of Treatment in Transmen. J Sex Med 2020;17:1795-1806.

Translational evidence of prothrombotic and inflammatory endothelial damage in Cushing syndrome after remission.

OBJECTIVE: Sustained evidence from observational studies indicates that after remission of Cushing syndrome (CS) a cardiovascular risk phenotype persists. Here, we performed a translational study in active CS and CS in remission (RCS) to evaluate the subclinical cardiometabolic burden and to explore the direct pro-inflammatory and prothrombotic potential of their sera on the endothelium in an in vitro translational atherothrombotic cell model. PATIENTS: Cross sectional study. The groups were (n = 9/group): I. RCS; II. Active CS (ACS) and III. Controls (CTR), all matched for age, body mass index, sex, without other hormonal deficits. DESIGN: We evaluated in vivo: cardiometabolic profile; endothelial markers (sVCAM-1, NO); endothelial dysfunction (FMD); intima-media thickness and body composition (DEXA). In vitro endothelial cells (EC) were exposed to sera taken from the different subjects to evaluate inflammatory EC response (tisVCAM) and thrombogenicity of the generated extracellular matrix (ECM): von Willebrand factor (VWF) and platelet reactivity. RESULTS: Three of the 9 RCS subjects were on glucocorticoid replacement therapy (GC-RT). Patients on GC-RT had a shorter period of time in stable remission. In vivo analysis ACS showed typically metabolic features, while cardiometabolic markers reached statistical significance for RCS only for Hs-CRP (P < .01). In vitro:EC exposed to ACS and RCS sera displayed increased tisVCAM-1 (P < .01 for ACS and P < .05 for RCS vs CTR), VWF (P < .01 for ACS and P < .05 for RCS vs CTR) and platelet adhesion on ECM (P < .01 for ACC and P < .05 for RCS vs CTR). No statistically significant differences were observed between GC-RT RSC and RCS without GC-RT. CONCLUSIONS: The sera of premenopausal women with CS in remission, without atherothrombotic disease, contain circulatory endothelial deleterious factors with a direct thrombogenic and pro-inflammatory endothelial effect that could increase cardiovascular risk.

Effects of sardine-enriched diet on metabolic control, inflammation and gut microbiota in drug-naïve patients with type 2 diabetes: a pilot randomized trial.

BACKGROUND: Nutrition therapy is the cornerstone of treating diabetes mellitus. The inclusion of fish (particularly oily fish) at least two times per week is recommended by current international dietary guidelines for type 2 diabetes. In contrast to a large number of human studies examining the effects of oily fish on different cardiovascular risk factors, little research on this topic is available in patients with type 2 diabetes. The aims of this pilot study were to investigate the effects of a sardine-enriched diet on metabolic control, adiponectin, inflammatory markers, erythrocyte membrane fatty acid (EMFA) composition, and gut microbiota in drug-naïve patients with type 2 diabetes. METHODS: 35 drug-naïve patients with type 2 diabetes were randomized to follow either a type 2 diabetes standard diet (control group: CG), or a standard diet enriched with 100 g of sardines 5 days a week (sardine group: SG) for 6 months. Anthropometric, dietary information, fasting glycated hemoglobin, glucose, insulin, adiponectin, inflammatory markers, EMFA and specific bacterial strains were determined before and after intervention. RESULTS: There were no significant differences in glycemic control between groups at the end of the study. Both groups decreased plasma insulin (SG: -35.3%, P = 0.01, CG: -22.6%, P = 0.02) and homeostasis model of assessment--insulin resistance (HOMA-IR) (SG: -39.2%, P = 0.007, CG: -21.8%, P = 0.04) at 6-months from baseline. However only SG increased adiponectin in plasma compared to baseline level (+40.7%, P = 0.04). The omega-3 index increased 2.6% in the SG compared to 0.6% in the CG (P = 0.001). Both dietary interventions decreased phylum Firmicutes (SG and CG: P = 0.04) and increased E. coli concentrations (SG: P = 0.01, CG: P = 0.03) at the end of the study from baseline, whereas SG decreased Firmicutes/Bacteroidetes ratio (P = 0.04) and increased Bacteroides-Prevotella (P = 0.004) compared to baseline. CONCLUSIONS: Although enriching diet with 100 g of sardines 5 days a week during 6 months to a type 2 diabetes standard diet seems to have neutral effects on glycemic control in drug-naïve patients with type 2 diabetes, this nutritional intervention could have beneficial effects on cardiovascular risk. Furthermore, both dietary interventions decreased HOMA-IR and altered gut microbiota composition of drug-naïve patients with type 2 diabetes. TRIAL REGISTRATION: Trial number and name of the registry: NCT02294526, ClinicalTrials.gov.

Weight loss is a major contributor to improved sexual function after bariatric surgery.

BACKGROUND: The relative contribution of anthropometric, hormonal, and metabolic changes after bariatric surgery (BS) on sexual function (SF) in severely obese subjects is not well established. METHODS: Prospective observational case series study of 39 men undergoing BS. SF was assessed by means of the international index of erectile function (IIEF) before and at 1 year after surgery. At the same time points, anthropometric (body mass index, waist circumference), hormonal (testosterone, sex hormone binding globulin, estradiol, gonadotropins, inhibin B, prolactin, leptin), and metabolic parameters (insulin sensitivity, C-reactive protein, lipid profile, hemoglobin A1c, presence of hypertension or sleep apnea) were assessed. RESULTS: BS was associated with marked weight loss (77.18% excess weight loss), improved IIEF score (baseline: 54.85 ± 16.59, 1 year: 61.21 ± 14.10; p < 0.01), gonadal function (testosterone: baseline 256.36 ± 120.98, 1 year: 508.01 ± 161.90; p < 0.001), and improved metabolic profile. However, on multivariate regression analysis whereas changes in body mass index (beta: -0.677, p = 0.001), and baseline IIEF score (beta: -0.397, p = 0.023), were independent predictors of the changes in the IIEF score at 1 year after surgery, changes in hormonal and metabolic factors were not. Variables in the model accounted for 66% of the postsurgical variation in the IIEF score. Similar results were found when the different IIEF-sexual domains were evaluated, except for intercourse satisfaction for which no independent predictor was identified. CONCLUSIONS: Weight loss's beneficial effects on SF occurring after BS are beyond the parallel improvement in gonadal and metabolic profiles.

Long-Term Follow-Up of Single-Fiber Multiple Low-Intensity Energy Laser Ablation Technique of Benign Thyroid Nodules.

AIM: The short-term and long-term efficacy of different thermal percutaneous ablation techniques remains a topical issue. Our group implemented percutaneous laser ablation (LA), a moving-shot technique to increase efficiency and reduce costs and variability of LA by applying multiple lower-intensity energy illuminations (MLIEI) covering the nodular volume (V) through changes in position of a single laser fiber within the thyroid nodule. The aim of the present study was to evaluate the efficacy of the single-fiber LA-MLIEI during a 5-year follow-up and to identify possible predictors of the final outcome. METHODS: Prospective study: Thirty outpatients (23 women and seven men) with benign symptomatic thyroid nodules were assigned to single-fiber LA-MLIEI, between 2012 and 2015. A single LA session was performed under real-time ultrasound (US) guidance using a 1,064-nm continuous-wave laser at 3 W. A 400-µm optical fiber was inserted through a 21-gauge needle, and 3-10 illuminations were performed per nodule, administering between 400 and 850 J/illumination. The total administered energy was calculated on the initial V of the nodule and the estimated ablation area. US evaluation was performed after LA-MLIEI at 1 week and 1, 3, 6, and 12 months and after that annually up to 5 years. Clinical symptoms, laboratory thyroid function during follow-up, and acute and chronic complications of treatment were registered. RESULTS: On follow-up, 67% (n: 20) were responders to single-fiber LA-MLIEI, while 33% (n: 10) were non-responders. The responder group initiated V reduction (ΔV) at 1 month, with remission of symptoms, and presented a 50% ΔV at 3 months of treatment; the maximum response was achieved at 24 months and remained stable until the end of the study. The non-responder group presented a ΔV of less than 50% at 12 months; though a tendency to >50% ΔV was observed at 24-36 months, there was subsequent regrowth, and 40% of this group required surgery. ΔV was positively correlated with the total administered energy/V (J/V) and inversely with nodule V. No severe adverse effects were observed. Thyroid function remained normal in all patients. Remission of symptoms occurred rapidly after 1 month. CONCLUSIONS: LA with multiple fractional discharges employing a single fiber in a unique session is a safe and inexpensive technique that allows rapid reduction of thyroid nodules, with a stable response up to 5 years, similarly to what has been reported with the conventional LA. Total nodule volume appears as a predictive factor of the reduction.

Cardiovascular Risk Associated With Gender Affirming Hormone Therapy in Transgender Population.

Transgender men and women represent about 0.6 -1.1%% of the general population. Gender affirming hormone therapy (GAHT) helps ameliorate gender dysphoria and promote well-being. However, these treatments' cardiovascular (CV) effects are difficult to evaluate due to the limited number of extensive longitudinal studies focused on CV outcomes in this population. Furthermore, these studies are mainly observational and difficult to interpret due to a variety of hormone regimens and observation periods, together with possible bias by confounding factors (comorbidities, estrogen types, smoking, alcohol abuse, HIV infection). In addition, the introduction of GAHT at increasingly earlier ages, even before the full development of the secondary sexual characteristics, could lead to long-term changes in CV risk compared to current data. This review examines the impact of GAHT in the transgender population on CV outcomes and surrogate markers of CV health. Furthermore, we review available data on changes in DNA methylation or RNA transcription induced by GAHT that may translate into changes in metabolic parameters that could increase CV risk.

Endogenous cortisol excess confers a unique lipid signature and metabolic network.

Chronic cortisol excess induces several alterations on protein, lipid and carbohydrate metabolism resembling those found in the metabolic syndrome. However, patients exposed to prolonged high levels of cortisol in Cushing syndrome (CS) present exceeding cardiometabolic alterations not reflected by conventional biomarkers. Using 3 ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) platforms, we aimed to characterise the serum metabolome of 25 patients with active endogenous CS and 25 control subjects matched by propensity score (sex, BMI, diabetes mellitus type 2 (T2D), high blood pressure (HBP) and dyslipidaemia) to search for potential disease-specific biomarkers and pathways associated to the clinical comorbidities. A total of 93 metabolites were significantly altered in patients with CS. Increased levels of sulfur amino acids (AA), triacylglycerols, glycerophospholipids, ceramides and cholesteryl esters were observed. Contrarily, concentrations of essential and non-essential AA, polyunsaturated fatty acids, conjugated bile acids and second messenger glycerolipids were decreased. Twenty-four-hour urinary free cortisol (24h-UFC) independently determined the concentration of 21 lipids and 4 AA. A metabolic signature composed by 10 AA and 10 lipid metabolites presented an AUC-ROC of 95% for the classification of CS patients. Through differential network analysis, 152 aberrant associations between metabolites involved in the Lands cycle and Kennedy pathway were identified. Our data indicates that chronic hypercortisolemia confers a unique lipidomic signature and several alterations in numerous AA even when compared to patients with similar metabolic comorbidities providing novel insights of the increased cardiometabolic burden of CS. KEY MESSAGES: • Cortisol excess induces metabolic alterations beyond conventional biomarkers. • The hypercortisolism extent determines the concentration of 21 lipids and 5 aa. • Cortisol excess confers a unique metabolic signature of 20 metabolites. • Kennedy and Lands cycle are profoundly disturbed by cortisol excess.

The LUCA device: a multi-modal platform combining diffuse optics and ultrasound imaging for thyroid cancer screening.

We present the LUCA device, a multi-modal platform combining eight-wavelength near infrared time resolved spectroscopy, sixteen-channel diffuse correlation spectroscopy and a clinical ultrasound in a single device. By simultaneously measuring the tissue hemodynamics and performing ultrasound imaging, this platform aims to tackle the low specificity and sensitivity of the current thyroid cancer diagnosis techniques, improving the screening of thyroid nodules. Here, we show a detailed description of the device, components and modules. Furthermore, we show the device tests performed through well established protocols for phantom validation, and the performance assessment for in vivo. The characterization tests demonstrate that LUCA device is capable of performing high quality measurements, with a precision in determining in vivo tissue optical and dynamic properties of better than 3%, and a reproducibility of better than 10% after ultrasound-guided probe repositioning, even with low photon count-rates, making it suitable for a wide variety of clinical applications.

Ultrasound measures of abdominal fat layers correlate with metabolic syndrome features in patients with obesity.

OBJECTIVE: Abdominal fat ultrasound (US) is a simple clinical tool that may allow measures of fat depots not visible using common dual-energy X-ray absorptiometry (DEXA) or computerized tomography (CT) imaging. The aim of this study was to validate the technique, give measures of superficial and profound subcutaneous, preperitoneal, omental and perirenal (retroperitoneal) fat and correlate them with MS markers. METHODS: Sequential US measures of these five abdominal fat layers were done at 397 adults. Blood pressure (BP), body mass index (BMI), waist, body fat %, HOMA-IR index (homeostatic model assessment of insulin resistance), lipid profile and leptin were recorded. Metabolic syndrome (MS) was defined according to Cholesterol education programme adult treatment panel III (ATPIII) criteria. RESULTS: Subcutaneous and omental fat were increased among people with obesity, whereas preperitoneal and perirenal fat did not show any difference according to BMI or waist. Women showed thicker subcutaneous fat (both superficial and profound), whereas men had bigger omental fat. Both postmenopausal and diabetic patients had changes in omental fat only, whereas patients with fatty liver showed thicker preperitoneal and perirenal fat, as well. MS patients showed both thicker perirenal and omental fat. A cut-off of 54 mm in male (M)/34 mm in female (F) of omental fat and 22.5 mm (M)/12.5 mm (F) of perirenal fat could be predictive of later MS onset. CONCLUSIONS: US is a valid method to measure all different abdominal fat depots. Omental and perirenal fat measures may classify patients at risk for MS. Preperitoneal fat depot may also correlate with fatty liver disease.

Effects of sex steroids on cardiovascular risk profile in transgender men under gender affirming hormone therapy.

PURPOSE: Gender affirming hormone therapy (HT) in transgender men both improves and impairs several surrogate cardiovascular risk markers. However, few prospective works with long follow-up and control group are available. In this context, this work aimed to assess the changes in the metabolic and cardiovascular risk pattern after 12 months of HT in transgender men. Furthermore, we aimed to investigate early effects on target tissues that may reflect an initial vascular damage. METHODS: Prospective observational study, including 20 transgender men, attended in the Gender Identity Unit (UIG) of the Hospital Clinic from July 2013 to November 2015. Anthropometric and body composition by dual-energy X-ray absorptiometry (DXA), hormonal, metabolic and coagulation parameters, endothelial dysfunction by flow-mediated dilation (FMD) and intima-media thickness (IMT) by carotid ultrasound, were assessed at baseline, at 6 and 12 months of HT. RESULTS: We observed an impairment of lipid profile, and increase of homocysteine and leucocytes count, as well as changes in body composition with increased total lean mass together with decreased total fat mass. In addition, higher mean-maximum common IMT was observed after 12 months of HT. CONCLUSION: Our work shows changes in metabolic and inflammatory parameters after HT after short-medium follow-up, which could increase cardiovascular risk in this setting, together with initial evidence of vascular changes.

Systematic study of the effect of ultrasound gel on the performances of time-domain diffuse optics and diffuse correlation spectroscopy.

Recently, multimodal imaging has gained an increasing interest in medical applications thanks to the inherent combination of strengths of the different techniques. For example, diffuse optics is used to probe both the composition and the microstructure of highly diffusive media down to a depth of few centimeters, but its spatial resolution is intrinsically low. On the other hand, ultrasound imaging exhibits the higher spatial resolution of morphological imaging, but without providing solid constitutional information. Thus, the combination of diffuse optical imaging and ultrasound may improve the effectiveness of medical examinations, e.g. for screening or diagnosis of tumors. However, the presence of an ultrasound coupling gel between probe and tissue can impair diffuse optical measurements like diffuse optical spectroscopy and diffuse correlation spectroscopy, since it may provide a direct path for photons between source and detector. A systematic study on the effect of different ultrasound coupling fluids was performed on tissue-mimicking phantoms, confirming that a water-clear gel can produce detrimental effects on optical measurements when recovering absorption/reduced scattering coefficients from time-domain spectroscopy acquisitions as well as particle Brownian diffusion coefficient from diffuse correlation spectroscopy ones. On the other hand, we show the suitability for optical measurements of other types of diffusive fluids, also compatible with ultrasound imaging.

Molecular basis of Gender Dysphoria: androgen and estrogen receptor interaction.

BACKGROUND: Polymorphisms in sex steroid receptors have been associated with transsexualism. However, published replication studies have yielded inconsistent findings, possibly because of a limited sample size and/or the heterogeneity of the transsexual population with respect to the onset of dysphoria and sexual orientation. We assessed the role of androgen receptor (AR), estrogen receptors alpha (ERα) and beta (ERß), and aromatase (CYP19A1) in two large and homogeneous transsexual male-to-female (MtF) and female-to-male (FtM) populations. METHODS: The association of each polymorphism with transsexualism was studied with a twofold subject-control analysis: in a homogeneous population of 549 early onset androphilic MtF transsexuals versus 728 male controls, and 425 gynephilic FtMs versus 599 female controls. Associations and interactions were investigated using binary logistic regression. RESULTS: Our data show that specific allele and genotype combinations of ERß, ERα and AR are implicated in the genetic basis of transsexualism, and that MtF gender development requires AR, which must be accompanied by ERß. An inverse allele interaction between ERß and AR is characteristic of the MtF population: when either of these polymorphisms is short, the other is long. ERß and ERα are also associated with transsexualism in the FtM population although there was no interaction between the polymorphisms. Our data show that ERß plays a key role in the typical brain differentiation of humans. CONCLUSION: ERß plays a key role in human gender differentiation in males and females.

Broadband (550-1350 nm) diffuse optical characterization of thyroid chromophores.

Thyroid plays an important role in the endocrine system of the human body. Its characterization by diffuse optics can open new path ways in the non-invasive diagnosis of thyroid pathologies. Yet, the absorption spectra of tyrosine and thyroglobulin-key tissue constituents specific to the thyroid organ-in the visible to near infrared range are not fully available. Here, we present the optical characterization of tyrosine (powder), thyroglobulin (granular form) and iodine (aqueous solution) using a time domain broadband diffuse optical spectrometer in the 550-1350 nm range. Various systematic errors caused by physics of photo migration and sample inherent properties were effectively suppressed by means of advanced time domain diffuse optical methods. A brief comparison with various other known tissue constituents is presented, which reveals key spectral regions for the quantification of the thyroid absorbers in an in vivo scenario.

Effects of sex steroids on the pattern of methylation and expression of the promoter region of estrogen and androgen receptors in people with gender dysphoria under cross-sex hormone treatment.

Cross-sex hormone therapy (CHT) is critical for phenotypical and physiological transition in adults with gender dysphoria (GD). However, the impact of the CHT onto the molecular level/epigenetic regulation has not been comprehensively addressed. We postulate that CHT in GD could drive changes at the androgen receptor (AR), estrogen receptor alpha (ESR1) and estrogen receptor beta (ESR2), affecting their DNA methylation pattern and mRNA expression that may influence in the phenotypical changes associated to CHT. We carried out a prospective observational study on individuals with a diagnosis of GD. 18 subjects (no previous CHT): 12 female to male (FtoM) and 6 male to female (MtoF). An Epityper Mass array TM method was used to study the DNA methylation and Real-time PCR quantitative reverse transcription PCR (qRT-PCR) was used to quantify the gene expression. The analysis of AR, ESR1 and ESR2 receptor was performed at baseline, 6 and 12 months after CHT. No differences in DNA methylation of ESR were found in MtoF, while DNA methylation was increased in FtoM at 6 and 12 months of CHT. The AR showed a significant increase of methylation in MtoF group after 12 months of estrogenic treatment. Regarding the expression analysis, AR expression was significantly decreased in FtoM upon CHT treatment. AR, ESR1 and ESR2 methylation were correlated with anthropometric, metabolic and hormonal parameters in FtoM and MtoF. Our results support that CHT is associated to epigenetic changes that might affect the response to treatment with sex steroids.

Zinc alpha-2 glycoprotein is overproduced in Cushing's syndrome.

INTRODUCTION: Cushing syndrome (CS), an endogenous hypercortisolemic condition with increased cardiometabolic morbidity, leads to development of abdominal obesity, insulin resistance, diabetes and proatherogenic dyslipidemia. Zinc alpha-2 glycoprotein (ZAG) is a recently characterized lipolytic adipokine implicated in regulation of adipose tissue metabolism and fat distribution. In vitro and animal studies suggest that glucocorticoids interact with ZAG secretion and action. To assess the relationship between ZAG and glucocorticoids in a human model of hypercortisolism, circulating ZAG levels were tested in patients with CS and its counterpart controls. METHODS: An observational, cross-sectional study on 39 women, 13 with active CS and 26 controls matched by age and body mass index. Plasma ZAG levels (µg/ml) were measured by ELISA and correlated with hypercortisolism, metabolic, and phenotypic parameters. RESULTS: Plasma ZAG levels were significantly higher in patients with CS compared to controls (64.3±16.6 vs. 44.0±16.1, p=0.002). In a univariate analysis, ZAG levels positively correlated to 24-h urinary free cortisol (p=0.001), body mass index (p=0.02), non-esterified fatty acids (p=0.05), glucose (p=0.003), LDL-C (p=0.028), and type 2 diabetes mellitus (p=0.016), and were inversely related to total adiponectin levels (p=0.035). In a multivariate analysis, after adjusting for CS, ZAG levels only correlated with body mass index (p=0.012), type 2 diabetes mellitus (p=0.004), and glucose (p<0.001). CONCLUSION: This study provides initial evidence that plasma ZAG levels are higher in patients with CS as compared to controls. The close relationship of ZAG with metabolic and phenotypic changes in CS suggests that ZAG may play a significant role in adipose tissue changes in hypercortisolism.

Circulatory Immune Cells in Cushing Syndrome: Bystanders or Active Contributors to Atherometabolic Injury? A Study of Adhesion and Activation of Cell Surface Markers.

Glucocorticoids (GC) induce cardiometabolic risk while atherosclerosis is a chronic inflammation involving immunity. GC are immune suppressors, and the adrenocorticotrophic hormone (ACTH) has immune modulator activities. Both may act in atherothrombotic inflammation involving immune cells (IMNC). Aim. To investigate adhesion and activation surface cell markers (CDs) of peripheral IMNC in endogenous Cushing syndrome (CS) and the immune modulator role of ACTH. Material and Methods. 16 ACTH-dependent CS (ACTH-D), 10 ACTH-independent (ACTH-ID) CS, and 16 healthy controls (C) were included. Leukocytes (Leuc), monocytes (MN), lymphocytes (Lym), and neutrophils (N) were analyzed by flow cytometry for atherosclerosis previously associated with CDs. Results. Leuc, N, and MN correlated with CS (p < 0.05), WC (p < 0.001), WHR (p = 0.003), BMI (p < 0.001), and hs-CRP (p < 0.001). CD14++CD16+ (p = 0.047); CD14+CD16++ (p = 0.053) MN; CD15+ (p = 0.027); CD15+CD16+ (p = 0.008) N; and NK-Lym (p = 0.019) were higher in CS. CD14+CD16++ MN were higher in ACTH-ID (8.9 ± 3.5%) versus ACTH-D CS (4.2 ± 1.9%) versus C (4.9 ± 2.3%). NK-Lym correlated with c-LDL (r = 0.433, p = 0.039) and CD15+ N with hs-CRP (r = 0.446, p = 0.037). In multivariate analysis, Leuc, N, and MN depended on BMI (p = 0.021), WC (p = 0.002), and WHR (p = 0.014), while CD15+ and CD15+CD16+ N on hypercortisolism and CS (p = 0.035). Conclusion. In CS, IMNC present changes in activation and adhesion CDs implicated in atherothrombotic inflammation. ACTH-IDCS presents a particular IMNC phenotype, possibly due to the absence of the immune modulator effect of ACTH.

Effect of cross-sex hormone treatment on cardiovascular risk factors in transsexual individuals. Experience in a specialized unit in Catalonia.

BACKGROUND AND AIMS: Since the onset of cross hormone therapy (CHT) in transsexual individuals, there has been concern about possible chronic side effects. Our objective was to assess baseline differences in lipid profile in individuals with gender identity disorder in relation to prior CHT, and changes in the lipid profile and other cardiovascular (CV) risk factors after 24 months of treatment. METHODS: Retrospective longitudinal study including all individuals assisted for the first time in the Gender Identity Unit of Catalonia from 2006 to 2010. Socio-demographical, anthropometric and laboratory data were collected. RESULTS: We evaluated 247 transsexuals, 150 male to female (MtF: 60.7%) and 97 female to male (FtM; 39.3%). At baseline, FtM transsexuals were younger and had started prior CHT less often than MtF (13.4% vs. 64.7%; p<0.001). During follow up, in MtF weight and BMI increased significantly, as well as systolic and diastolic blood pressure, though these latter remained within normal range. No significant differences in lipid profile were observed. FtM transsexuals also presented an increase in weight and BMI, without differences in blood pressure. A general worsening in lipid profile was observed in this group, with increased total cholesterol (166.0 ± 35.1 vs. 175.6 ± 38.2mg/dL; p=0.001), triglycerides (70.6 ± 30.7 vs. 102.3 ± 68.5 mg/dL; p<0.001) and LDL cholesterol (103.8 ± 28.7 vs. 112.8 ± 30.3 mg/dL; p=.013) and decreased HDL cholesterol (52.2 ± 12.2 vs. 45.4 ± 13.8 mg/dL; p=0.001), even though final levels were all within normal range. CONCLUSION: There is no detectable increase in CV risk factors in MtF transsexuals who were treated with currently prescribed estrogenic compounds, while a slight worsening in lipid profile takes place in the FtM group, though within normal limits.

New splice site acceptor mutation in AIRE gene in autoimmune polyendocrine syndrome type 1.

Autoimmune polyglandular syndrome type 1 (APS-1, OMIM 240300) is a rare autosomal recessive disorder, characterized by the presence of at least two of three major diseases: hypoparathyroidism, Addison's disease, and chronic mucocutaneous candidiasis. We aim to identify the molecular defects and investigate the clinical and mutational characteristics in an index case and other members of a consanguineous family. We identified a novel homozygous mutation in the splice site acceptor (SSA) of intron 5 (c.653-1G>A) in two siblings with different clinical outcomes of APS-1. Coding DNA sequencing revealed that this AIRE mutation potentially compromised the recognition of the constitutive SSA of intron 5, splicing upstream onto a nearby cryptic SSA in intron 5. Surprisingly, the use of an alternative SSA entails the uncovering of a cryptic donor splice site in exon 5. This new transcript generates a truncated protein (p.A214fs67X) containing the first 213 amino acids and followed by 68 aberrant amino acids. The mutation affects the proper splicing, not only at the acceptor but also at the donor splice site, highlighting the complexity of recognizing suitable splicing sites and the importance of sequencing the intron-exon junctions for a more precise molecular diagnosis and correct genetic counseling. As both siblings were carrying the same mutation but exhibited a different APS-1 onset, and one of the brothers was not clinically diagnosed, our finding highlights the possibility to suspect mutations in the AIRE gene in cases of childhood chronic candidiasis and/or hypoparathyroidism otherwise unexplained, especially when the phenotype is associated with other autoimmune diseases.

Altered clock gene expression in obese visceral adipose tissue is associated with metabolic syndrome.

Clock gene expression was associated with different components of metabolic syndrome (MS) in human adipose tissue. However, no study has been done to compare the expression of clock genes in visceral adipose tissue (VAT) from lean and obese subjects and its clinical implications. Therefore, we studied in lean and obese women the endogenous 24 h expression of clock genes in isolated adipocytes and its association with MS components. VAT was obtained from lean (BMI 21-25 kg/m2; n = 21) and morbidly obese women (BMI >40 kg/m2; n = 28). The 24 h pattern of clock genes was analyzed every 6 hours using RT-PCR. Correlation of clinical data was studied by Spearman analysis. The 24 h pattern of clock genes showed that obesity alters the expression of CLOCK, BMAL1, PER1, CRY2 and REV-ERB ALPHA in adipocytes with changes found in CRY2 and REV-ERB ALPHA throughout the 24 h period. The same results were confirmed in VAT and stromal cells (SC) showing an upregulation of CRY2 and REV-ERB ALPHA from obese women. A positive correlation was observed for REV-ERB ALPHA gene expression with BMI and waist circumference in the obese population. Expression of ROR ALPHA was correlated with HDL levels and CLOCK with LDL. Obese subjects with MS exhibited positive correlation in the PER2 gene with LDL cholesterol, whereas REV-ERB ALPHA was correlated with waist circumference. We identified CRY2 and REV-ERB ALPHA as the clock genes upregulated in obesity during the 24 h period and that REV-ERB ALPHA is an important gene associated with MS.

Zinc alpha-2 glycoprotein is overproduced in Cushing's syndrome / Aumento de la glucoproteína zinc alfa-2 en el síndrome de Cushing

Introduction: Cushing syndrome (CS), an endogenous hypercortisolemic condition with increased cardiometabolic morbidity, leads to development of abdominal obesity, insulin resistance, diabetes and proatherogenic dyslipidemia. Zinc alpha-2 glycoprotein (ZAG) is a recently characterized lipolytic adipokine implicated in regulation of adipose tissue metabolism and fat distribution. In vitro and animal studies suggest that glucocorticoids interact with ZAG secretion and action. To assess the relationship between ZAG and glucocorticoids in a human model of hypercortisolism, circulating ZAG levels were tested in patients with CS and its counterpart controls. Methods: An observational, cross-sectional study on 39 women, 13 with active CS and 26 controls matched by age and body mass index. Plasma ZAG levels (μg/ml) were measured by ELISA and correlated with hypercortisolism, metabolic, and phenotypic parameters. Results: Plasma ZAG levels were significantly higher in patients with CS compared to controls (64.3±16.6 vs. 44.0±16.1, p=0.002). In a univariate analysis, ZAG levels positively correlated to 24-h urinary free cortisol (p=0.001), body mass index (p=0.02), non-esterified fatty acids (p=0.05), glucose (p=0.003), LDL-C (p=0.028), and type 2 diabetes mellitus (p=0.016), and were inversely related to total adiponectin levels (p=0.035). In a multivariate analysis, after adjusting for CS, ZAG levels only correlated with body mass index (p=0.012), type 2 diabetes mellitus (p=0.004), and glucose (p<0.001). Conclusion: This study provides initial evidence that plasma ZAG levels are higher in patients with CS as compared to controls. The close relationship of ZAG with metabolic and phenotypic changes in CS suggests that ZAG may play a significant role in adipose tissue changes in hypercortisolism (AU)

Introducción: El síndrome de Cushing (SC) es un estado de hipercortisolismo endógeno en el que se observa un incremento del riesgo cardiovascular asociado al desarrollo de obesidad abdominal, insulinorresistencia, diabetes y dislipidemia aterogénica. La zinc alfa-2 glucoproteína (ZAG) es una adipocina lipolítica recientemente caracterizada que está implicada en la regulación del metabolismo del tejido adiposo y la distribución de la grasa. Estudios in vitro y en animales indican que los glucocorticoides interaccionan con la secreción y acción de ZAG. Para evaluar la relación entre ZAG y los glucocorticoides en un modelo humano de hipercortisolismo, se analizaron los niveles circulantes de ZAG en pacientes con SC y sus correspondientes controles. Métodos: Estudio observacional en 39 mujeres, 13 con SC activo y 26 controles pareadas por edad e índice de masa corporal. Los niveles plasmáticos de ZAG (μg/ml) se determinaron mediante ELISA y se correlacionaron con los parámetros de hipercortisolismo, metabólicos y fenotípicos. Resultados: Las concentraciones plasmáticas de ZAG fueron significativamente más elevadas en los pacientes con SC (64,3±16,6 vs. 44±16,1; p=0,002). En el análisis univariante los niveles de ZAG se correlacionaron positivamente con cortisol libre urinario (p=0,001), índice de masa corporal (p=0,02), ácidos grasos no esterificados (p=0,05), glucosa (p=0,003), c-LDL (p=0,028) y diabetes mellitus (p=0,016) e inversamente con adiponectina total (p=0,035). En el análisis multivariante, después de ajustar por el SC, los niveles de ZAG solo se correlacionaron con el índice de masa corporal (p=0,012), la diabetes mellitus tipo 2 (p=0,004) y la glucosa (p<0,001). Conclusión: Nuestro estudio proporciona la primera evidencia de las concentraciones plasmáticas de ZAG en el SC. Los pacientes con SC presentan concentraciones más elevadas de ZAG que los controles. La estrecha relación de ZAG con las alteraciones metabólicas y fenotípicas del SC indica que ZAG podría desempeñar un papel importante en las alteraciones del tejido adiposo en el hipercortisolismo (AU)