A single-cell atlas of the aging mouse ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Female humans experience a sharp decline in fertility around 35 years of age, which corresponds to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging mouse ovary to identify early drivers of ovarian decline. To fill this gap we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress-response, immunogenic and fibrotic signaling pathway inductions with aging. This report provides critical insights into mechanisms responsible for ovarian aging phenotypes. The data can be explored interactively via a Shiny-based web application.

17α-estradiol Alleviates High-Fat Diet-Induced Inflammatory and Metabolic Dysfunction in Skeletal Muscle of Male and Female Mice.

Skeletal muscle has a central role in maintaining metabolic homeostasis. 17α-estradiol (17α-E2), a naturally-occurring non-feminizing diastereomer of 17ß-estradiol that demonstrates efficacy for improving metabolic outcomes in male, but not female, mice. Despite several lines of evidence showing that 17α-E2 treatment improves metabolic parameters in middle-aged obese and old male mice through effects in brain, liver, and white adipose tissue little is known about how 17α-E2 alters skeletal muscle metabolism, and what role this may play in mitigating metabolic declines. Therefore, this study aimed to determine if 17α-E2 treatment improves metabolic outcomes in skeletal muscle from obese male and female mice following chronic high fat diet (HFD) administration. We hypothesized that male, but not female, mice, would benefit from 17α-E2 treatment during HFD. To test this hypothesis, we used a multi-omics approach to determine changes in lipotoxic lipid intermediates, metabolites, and proteins related to metabolic homeostasis. In male mice, we show that 17α-E2 alleviates HFD-induced metabolic detriments of skeletal muscle by reducing the accumulation of diacylglycerol (DAGs) and ceramides, inflammatory cytokine levels, and reduced the abundance of most of the proteins related to lipolysis and beta-oxidation. In contrast to males, 17α-E2 treatment in female mice had little effect on the DAGs and ceramides content, muscle inflammatory cytokine levels, or changes to the relative abundance of proteins involved in beta-oxidation. These data support to the growing evidence that 17α-E2 treatment could be beneficial for overall metabolic health in male mammals.

Metabolic benefits of 17α-estradiol in liver are partially mediated by ERß in male mice.

Metabolic dysfunction underlies several chronic diseases. Dietary interventions can reverse metabolic declines and slow aging but remaining compliant is difficult. 17α-estradiol (17α-E2) treatment improves metabolic parameters and slows aging in male mice without inducing significant feminization. We recently reported that estrogen receptor α is required for the majority of 17α-E2-mediated benefits in male mice, but that 17α-E2 also attenuates fibrogenesis in liver, which is regulated by estrogen receptor ß (ERß)-expressing hepatic stellate cells (HSC). The current studies sought to determine if 17α-E2-mediated benefits on systemic and hepatic metabolism are ERß-dependent. We found that 17α-E2 treatment reversed obesity and related systemic metabolic sequela in both male and female mice, but this was partially blocked in female, but not male, ERßKO mice. ERß ablation in male mice attenuated 17α-E2-mediated benefits on hepatic stearoyl-coenyzme A desaturase 1 (SCD1) and transforming growth factor ß1 (TGF-ß1) production, which play critical roles in HSC activation and liver fibrosis. We also found that 17α-E2 treatment suppresses SCD1 production in cultured hepatocytes and hepatic stellate cells, indicating that 17α-E2 directly signals in both cell-types to suppress drivers of steatosis and fibrosis. We conclude that ERß partially controls 17α-E2-mediated benefits on systemic metabolic regulation in female, but not male, mice, and that 17α-E2 likely signals through ERß in HSCs to attenuate pro-fibrotic mechanisms.

A single-cell atlas of the aging murine ovary.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling, and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Around 35 years old, women experience a sharp decline in fertility, corresponding to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging ovary to identify early drivers of ovarian decline. To fill this gap, we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress response, immunogenic, and fibrotic signaling pathway inductions with aging. This report raises provides critical insights into mechanisms responsible for ovarian aging phenotypes.

Metabolic benefits of 17α-estradiol in liver are partially mediated by ERß in male mice.

Metabolic dysfunction underlies several chronic diseases. Dietary interventions can reverse metabolic declines and slow aging but remaining compliant is difficult. 17α-estradiol (17α-E2) treatment improves metabolic parameters and slows aging in male mice without inducing significant feminization. We recently reported that estrogen receptor α is required for the majority of 17α-E2-mediated benefits in male mice, but that 17α-E2 also attenuates fibrogenesis in liver, which is regulated by estrogen receptor ß (ERß)-expressing hepatic stellate cells (HSC). The current studies sought to determine if 17α-E2-mediated benefits on systemic and hepatic metabolism are ERß-dependent. We found that 17α-E2 treatment reversed obesity and related systemic metabolic sequela in both male and female mice, but this was partially blocked in female, but not male, ERßKO mice. ERß ablation in male mice attenuated 17α-E2-mediated benefits on hepatic stearoyl-coenyzme A desaturase 1 (SCD1) and transforming growth factor ß1 (TGF-ß1) production, which play critical roles in HSC activation and liver fibrosis. We also found that 17α-E2 treatment suppresses SCD1 production in cultured hepatocytes and hepatic stellate cells, indicating that 17α-E2 directly signals in both cell-types to suppress drivers of steatosis and fibrosis. We conclude that ERß partially controls 17α-E2-mediated benefits on systemic metabolic regulation in female, but not male, mice, and that 17α-E2 likely signals through ERß in HSCs to attenuate pro-fibrotic mechanisms.

Increase in PPARγ inhibitory phosphorylation by Fetuin-A through the activation of Ras-MEK-ERK pathway causes insulin resistance.

Obesity induced insulin resistance is primarily regulated by the inhibitory phosphorylation of peroxisome proliferator-activated receptor γ at serine 273 (PPARγS273) which has been shown to be regulated by MEK and ERK. An upstream regulatory molecule of this pathway could be a therapeutic option. Here we analyzed the involvement of Fetuin-A (FetA), a key hepato-adipokine implicated in insulin resistance, as an upstream regulator molecule for the regulation of PPARγ inhibitory phosphorylation. Mice fed with standard diet (SD), high fat diet (HFD) and HFD with FetA knockdown (HFD-FetAKD) were used to examine the role of FetA on PPARγS273 phosphorylation in adipocytes. The mechanism of regulation and its effect on skeletal muscle were studied using primary adipocytes, 3T3-L1 (preadipocyte) and C2C12 (myotube) cell lines. Increased FetA in HFD mice strongly correlated with augmentation of PPARγS273 phosphorylation in inflamed adipocytes while knockdown of FetA suppressed it. This effect of FetA was mediated through the activation of Ras which in turn activated MEK and ERK. On addressing how FetA could stimulate activation of Ras, we found that FetA triggered TNFα in inflamed adipocytes which induced Ras activation. The ensuing sharp fall in adiponectin level attenuated AMPK activation in skeletal muscle cells affecting mitochondrial ATP production. Our data reveal the essential role of FetA induced activation of Ras in regulating PPARγ inhibitory phosphorylation through Ras-MEK-ERK pathway which downregulates adiponectin disrupting skeletal muscle mitochondrial bioenergetics. Thus, FetA mediated PPARγ inactivation has adverse consequences upon adipocyte-myocyte crosstalk leading to disruption of energy homeostasis and loss of insulin sensitivity.

Profile of Auto-antibodies (Disease Related and Other) in Children with Type 1 Diabetes.

BACKGROUND: Type 1 diabetes is associated with several disease-related and other organ-specific autoimmune disorders. Data related to various auto-antibodies in Type 1 diabetes in India is limited. MATERIALS AND METHODS: In this cross sectional study, 92 subjects with T1DM (33 males, 59 females) were evaluated for T1DM related antibodies (autoantibodies to glutamic acid decarboxylase (anti-GAD), autoantibodies to protein tyrosine phosphatise (anti-IA2), anti-islet cell antibody (ICA), insulin autoantibody (IAA), anti-Zinc Transporter(ZnT8) and other organ specific auto antibodies like anti-thyroid peroxidase (anti-TPO), anti-thyroglobulin (TgAb), IgA anti-tissue transglutaminase (IgA anti-tTG), anti-21-hydroxylase, and anti-ovarian antibody (in females). RESULTS: Anti-GAD, IA-2, islet cell antibody, insulin autoantibodies (IAA), ZnT8 antibody were present in 79.3%, 32.6%, 61.9%, 63%, and 20.65% subjects, respectively. Only 2.2% patients with Type 1 diabetes were antibody negative. At least one antibody was found in 97.8% and at least two antibodies in 67.3%. The presence of anti-TPO, anti-thyroglobulin, IgA anti-tissue transglutaminase, anti 21-hydroxylase were found in 51%, 25%, 22.8%, and 2.1%, respectively. Anti-ovarian antibody was absent in all females of our study population. The duration of diabetes positively correlated with the number of T1DM specific antibody and also with GAD antibody positivity. Anti TPO positivity correlated with the age of onset of T1DM, but not with the duration of disease or presence of other T1DM specific autoantibody. CONCLUSIONS: T1DM is associated with a high prevalence of autoantibodies and antibody negative T1DM is rare. The association with other organ specific antibody (especially thyroid and adrenal glands) and celiac disease is also substantial, which reinforces the importance of regular thyroid and celiac disease screening in T1DM subjects. The duration of diabetes positively correlated with number of T1DM specific antibodies.

Trimester-Specific Reference Intervals for Thyroid Function Parameters in Indian Pregnant Women during Final Phase of Transition to Iodine Sufficiency.

BACKGROUND: Interpretation of thyroid function tests during pregnancy depends on gestational age, method, and population-specific reference intervals. Therefore, there is a worldwide trend to establish trimester-specific levels for different populations. The aim of this study was to establish a trimester-specific reference range for thyroid function parameters during pregnancy in Indian women. MATERIALS AND METHODS: Thyroid function tests (TSH, FT4, TT4, TT3) of 80, 76, and 73 women at 1st, 2nd, and 3rd trimester, respectively, and 168 nonpregnant women were analyzed after exclusion of low UIC(<150 µg/L) and anti-TPO positivity(>35 IU/ml). Urinary iodine excretion (UIC) was assessed in all. The 2.5th and 97.5th percentile values were used to determine the reference ranges for thyrotropin (TSH), free thyroxine (FT4), total thyroxine (TT4), and total triiodothyronine (TT3) for each trimester of pregnancy. RESULTS: The reference range for TSH for first trimester was 0.19-4.34 µIU/ml, for second trimester 0.46-4.57 µIU/ml, and for third trimester 0.61-4.62 µIU/ml. The reference range during three trimesters for FT4 (ng/dl) was 0.88-1.32, 0.89-1.60, and 0.87-1.54, for total T4 (µg/dl) was 5.9-12.9, 7.4-15.2, and 7.9-14.9. In nonpregnant women, FT4 was 0.83-1.34, total T4 was 5.3-11.8, and TSH was 0.79-4.29. The mean UIC in nonpregnant women was 176 ± 15.7 µg/L suggesting iodine-sufficiency in the cohort. CONCLUSION: The trimester-specific TSH range in pregnant women in this study is not significantly different from nonpregnant reference range in the final phase of transition to iodine sufficiency in India.

Neck Circumference to Height Ratio is a Reliable Predictor of Liver Stiffness and Nonalcoholic Fatty Liver Disease in Prediabetes.

BACKGROUND AND OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) and dysglycemia are public health challenges. There is urgent need for anthropometric surrogates for NAFLD screening. This study evaluated role of neck circumference (NC) and neck-height ratio (NHtR) as predictors of liver stiffness measure (LSM) in individuals with prediabetes (IPD). METHODS: In a cross-sectional study, 188 IPD from 1130 screened individuals underwent anthropometry, ultrasonography, Fibroscan® for LSM, dyslipidemia, insulin resistance (IR), and fetuin-A assessment. RESULTS: Hypertension, hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C), metabolic syndrome (MetS), NAFLD, and significant liver stiffness (SLS) (LSM >8.5kPa) were observed in 53.7%, 31.4%, 71.3%, 73.9%, 24.5%, and 11.2% prediabetes individuals, respectively. Prediabetes with NAFLD had significantly higher body mass index (BMI), NC, NHtR, glycated hemoglobin, triglycerides, fatty liver index (FLI), and LSM. Prediabetes in highest NHtR quartile had significantly higher BMI, hypertension, MetS, fasting glucose, glycated hemoglobin, homeostatic model assessment-IR, NAFLD, LSM, SLS, and lower HDL-C. Stepwise forward linear regression revealed that NHtR, FLI, and LDL-C were best predictors of LSM, at baseline (Model-1), after adjusting for age and sex (Model-2), and adjusting model-2 plus systolic and diastolic blood pressure (Model-3). NHtR and NC (in females) and NHtR and BMI (in males) had largest area under the curves for predicting LSM, NAFLD, and MetS. NHtR ≥21.54 cm/m (sensitivity: 90%; specificity: 52.5%; females) and ≥21.62 cm/m (sensitivity: 80%; specificity: 49.4%; males) was best predictor of SLS. INTERPRETATION AND CONCLUSION: NHtR is a reliable tool for community screening of NAFLD and liver stiffness in prediabetes.

Thyroid Status in Patients with Type 2 Diabetes Attending a Tertiary Care Hospital in Eastern India.

OBJECTIVE: Type 2 diabetes mellitus and thyroid dysfunction (TD) are two major public health endocrine problem, but the prevalence of TD and iodine status in patients with T2 DM in India is less studied. The study objective was to explore the prevalence of TD and to evaluate iodine health in type 2 diabetes patients attending a tertiary care center in Eastern India. METHODS: Consecutive 100 patients with diabetes attending outpatient department were evaluated clinically and biochemically (thyrotropin [TSH], free thyroxine, anti-TPO antibody, and urinary iodine). We excluded pregnant women or patients taking drugs that can alter thyroid function. Subclinical hypothyroid and overt hypothyroidism were diagnosed as per standard definitions. RESULTS: Out of 100 patients were analyzed, 51 (51%) were male. Mean (±standard deviation) age was 45.4 ± 11.2 years, body mass index 24.1 ± 4.28 kg/m2, and duration of diabetes 7.76 ± 5.77 years. The prevalence of subclinical hypothyroidism and overt hypothyroidism was 23/100 (23%) and 3/100 (3%), respectively. Thyroid autoantibody was positive in 13 (13.1%) patients. All patients were iodine sufficient. A trend toward increased neuropathy (r = 0.45) and nephropathy (r = -0.29) was associated with rising TSH. CONCLUSION: Almost one in four people living with diabetes are suffering from TD. Thus, routine screening should be implemented. Salt iodination program is a huge success in this part of the country.

Vitamin D supplementation reduces thyroid peroxidase antibody levels in patients with autoimmune thyroid disease: An open-labeled randomized controlled trial.

BACKGROUND AND AIMS: Although Vitamin D deficiency has been linked to autoimmune thyroid disorders (AITD), the impact of Vitamin D supplementation on thyroid autoimmunity is not known. This study aimed to evaluate the impact of Vitamin D supplementation on thyroid autoimmunity (thyroid peroxidase antibody [TPO-Ab] titers) in patients with newly diagnosed AITD in a randomized controlled trial. MATERIALS AND METHODS: One hundred two patients with newly diagnosed AITD (TPO-Ab > 34 kIU/L and/or sonographic evidence of thyroiditis) patients were randomized into Group-1 (intervention group) and Group-2 (control group). Group-1 received cholecalciferol 60,000 IU weekly and calcium 500 mg/day for 8 weeks; Group-2 received calcium 500 mg/day for 8 weeks. Responders were defined as ≥25% fall in TPO-Ab titers. Individuals with at least 3-month follow-up were analyzed. Trial is registered at ctri.nic.in (CTRI/2015/04/005713). RESULTS: Data from 100 AITD patients (68 with thyroid stimulating hormone [TSH] ≤10 mIU/L, 32 with TSH > 10 mIU/L), 93% having Vitamin D insufficiency, were analyzed. TPO-Ab titers were highest among patients in the lowest 25-hydroxyvitamin D quartile (P = 0.084). At 3 months follow-up, there was significant fall in TPO-Ab in Group-1 (-46.73%) as compared to Group-2 (-16.6%) (P = 0.028). Sixty-eight percentage patients in Group-1 were responders compared to 44% in Group-2 (P = 0.015). Kaplan-Meier analysis revealed significantly higher response rate in Group-1 (P = 0.012). Significantly greater reduction in TPO-Ab titers was observed in AITD with TSH ≤ 10 mIU/L compared to TSH > 10 mIU/L. Cox regression revealed Group-1 followed by TPO-Ab and free tetraiodothyronine levels to be a good predictor of response to therapy (P = 0.042, 0.069, and 0.074, respectively). CONCLUSION: Vitamin D supplementation in AITD may have a beneficial effect on autoimmunity as evidence by significant reductions in TPO-Ab titers.

Increased Soluble TNF Receptor-1 and Glutathione Peroxidase may Predict Carotid Intima Media Thickness in Females with Cushing Syndrome.

OBJECTIVE: Cardiovascular events are the most common cause of mortality in Cushing syndrome (CS). This study aimed to evaluate the impact of novel factors on atherosclerosis in endogenous CS. METHODS: A total of 22 female patients with CS and 33 normal female controls underwent evaluation of fibrinogen, high-sensitivity C-reactive protein (hsCRP), inflammatory cytokines (interleukin [IL]-6, IL-1ß, soluble tumor necrosis factor receptor [sTNFR]-1, and sTNFR2), glutathione peroxidase (GPx; measure of oxidative stress), carotid intima media thickness (CIMT; a measure of atherosclerosis), and percent change in flow-mediated vasodilation (%FMV) of the brachial artery, a measure of endothelial dysfunction. Stepwise multiple linear regressions were done after adjusting for variables in models 1 through 3 to evaluate their role in predicting CIMT and %FMV. Model 1 consisted of age and body mass index (BMI). Model 2 consisted of model 1 plus blood pressure (BP), fasting blood glucose (FBG), and 2-hour postglucose blood glucose (2hPGBG). Model 3 consisted of model 2 plus triglycerides and low- and high-density lipoprotein. RESULTS: Females with CS had significantly higher BMI, BP, FBG, 2hPGBG, total cholesterol, triglycerides, fibrinogen, IL-6, IL-1ß, sTNFR1, and GPx. CIMT and %FMV were significantly higher and lower, respectively, in CS patients. Regression analyses revealed sTNFR1 to be a consistent predictor of CIMT after adjusting for model 1 (ß = 0.656; P = .004), model 2 (ß = 0.571; P = .047), and model 3 (ß = 0.683; P =.026). GPx was a predictor of CIMT after adjusting for model 1 (ß = 0.565; P = .033) and model 3 (ß = 0.756; P= .038). CONCLUSION: This study highlights increased CIMT and endothelial dysfunction in CS, associated with an altered inflammatory milieu. sTNFR1 and GPx may predict CIMT in females with CS.

Vitamin-D supplementation in prediabetes reduced progression to type 2 diabetes and was associated with decreased insulin resistance and systemic inflammation: an open label randomized prospective study from Eastern India.

Vitamin-D supplementation in vitamin-D insufficient/deficient prediabetes individuals is associated with significantly lower progression to diabetes (6/55 vs. 13/49; p=0.04) and higher reversal to normoglycemia (23/55 vs. 10/49; p=0.02), associated with decreased insulin resistance and systemic inflammation (TNFα and IL6). Baseline vitamin-D and 2h blood glucose independently predicted progression to diabetes.

Urinary albumin : creatinine ratio predicts prediabetes progression to diabetes and reversal to normoglycemia: role of associated insulin resistance, inflammatory cytokines and low vitamin D.

BACKGROUND: The relationship between albumin : creatinine ratio (ACR), insulin resistance (IR), cytokines, dyslipidemia, and 25-hydroxy vitamin D (25-OHD) in individuals with prediabetes (IPD) was investigated to evaluate their role in predicting future risk of progression to diabetes. METHODS: The aforementioned parameters were evaluated in 147 IPD with persistent impaired fasting glucose and/or impaired glucose tolerance over two oral glucose tolerance tests, who were then followed up at 3-monthly intervals for progression to diabetes or reversal to normoglycemia. RESULTS: Data were analyzed for 137 IPD with at least 1-year follow-up. Forty-three IPD reversed to normoglycemia (Group I), 69 continued with prediabetes (Group II), and 25 progressed to diabetes (Group III) over a mean follow-up period of 28.36 ± 8.19 months. Baseline fasting blood glucose levels (BGLs), 2-h post-glucose BGLs, and ACR were lowest in Group I and highest in Group III. Of the 137 IPD, 54.75% (n = 75) had microalbuminuria. The IPD in the lowest ACR quartile had the highest reversal to normoglycemia. Cox regression revealed that baseline IL-6 was predictive of progression to diabetes (P = 0.03) and ACR was an independent predictor of reversal to normoglycemia (P = 0.007). Kaplan-Meier analysis showed higher reversal to normoglycemia in IPD without microalbuminuria (P < 0.001). CONCLUSION: An increased ACR is associated with higher creatinine, IR, and cytokine levels and lower 25-OHD levels in IPD. Microalbuminuria is associated with decreased reversal to normoglycemia and increased progression to diabetes. Low 25-OHD may be associated with increased progression to diabetes, perhaps via modulation of the ACR.

Tumor necrosis factor alpha -238G/A (rs 361525) gene polymorphism predicts progression to type-2 diabetes in an Eastern Indian population with prediabetes.

Prediabetes (IPD; n=122) and normoglycemic individuals (n=100) underwent assessment of polymorphisms of TNFα (-238, -308) and IL6 (-174). After 27.25±5.64 months, 16 IPD had reverted to normoglycemia and 18 progressed to diabetes. TNFα -238AA/GA genotypes were significantly more common in IPD, had higher TNFα, higher progression to diabetes and lower reversal.

Urinary level of CA19-9 as a tumor marker in urothelial carcinoma of the bladder.

PURPOSE: To diagnose the urothelial carcinoma of the bladder by measuring CA19-9 level in the urine. MATERIALS AND METHODS: This study was conducted on 47 patients with histopathologically confirmed urothelial cancer and 50 control subjects. The urinary level of CA19-9 was measured in both groups by enzyme-linked immunosorbent assay after concentration of urine with Bio-Gel dry beads. Urine cytology was also done in both controls and patients. RESULTS: The mean urinary level of CA19-9 was 194.59 ± 110.56 u/mL in patients and 11.67 ± 8.42 u/mL in controls (P = .0001). The mean urinary level of CA19-9 in patients with low-grade and high-grade bladder cancer was 206.56 ± 114.56 u/mL and 174.80 ± 94.06 u/mL, respectively (P = .56). Urine cytology by Papanicolaou stain was mostly negative. CONCLUSION: It can be concluded that CA19-9 may be a useful non-invasive test to diagnose the urothelial carcinoma of the bladder.