miRNA-mRNA Network in PBMCs of PCOS Women Identifies Overactivated Stress-Activated Kinases.

BACKGROUND/AIMS: Earlier studies have revealed the miRNAs and mRNAs involved in Polycystic Ovarian Syndrome (PCOS), but little is known about their regulatory networks. METHODS: To address this issue, we applied a comprehensive miRNA, mRNA profiling approach in peripheral blood of PCOS patients. We identified 30 differential miRNAs and 3310 differential transcripts. A robust computational framework was created to integrate matched miRNA and mRNA expression profiles in PCOS using feed-forward loops. RESULTS: The network consisted of differential miRNAs, transcription factors (TFs), and their common predicted target genes. The key network consisted of 14 non-orphan network clusters with 50 TF-gene pairs, 8 TF-TF pairs, 6 miRNA-TF pairs and 36 miRNA- gene pairs which were later dissected into 16 subclusters. Gene ontology annotations revealed that a host of signals (hormone, growth factors -EGF/ PDGF, thrombopoietin, oxidative stress and vitamin/nutrition) regulate MAPK signaling altering angiogenesis, JAK-STAT signaling, apoptosis, inflammatory and immune response and steroidogenesis in PCOS women. CONCLUSION: MAPK signaling is identified as the syndrome´s major dysregulated pathway. Our data imparts a robust foundation to expand the work and pave the way to focus efforts on p38MAPK targeted therapeutic strategies in PCOS.

Derailed peripheral circadian genes in polycystic ovary syndrome patients alters peripheral conversion of androgens synthesis.

STUDY QUESTION: Do circadian genes exhibit an altered profile in peripheral blood mononuclear cells (PBMCs) of polycystic ovary syndrome (PCOS) patients and do they have a potential role in androgen excess? SUMMARY ANSWER: Our findings revealed that an impaired circadian clock could hamper the regulation of peripheral steroid metabolism in PCOS women. WHAT IS KNOWN ALREADY: PCOS patients exhibit features of metabolic syndrome. Circadian rhythm disruption is involved in the development of metabolic diseases and subfertility. An association between shift work and the incidence of PCOS in females was recently reported. STUDY DESIGN, SIZE, DURATION: This is a retrospective case-referent study in which peripheral blood samples were obtained from 101 control and 101 PCOS subjects. PCOS diagnoses were based on Rotterdam Consensus criteria. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study comprised 101 women with PCOS and 101 control volunteers, as well as Swiss albino mice treated with dehydroepiandrosterone (DHEA) to induce PCOS development. Gene expression analyses of circadian and steroidogenesis genes in human PBMC and mice ovaries and blood were executed by quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: We observed aberrant expression of peripheral circadian clock genes in PCOS, with a significant reduction in the core clock genes, circadian locomotor output cycles kaput (CLOCK) (P ≤ 0.00001), brain and muscle ARNT-like 1 (BMAL1) (P ≤ 0.00001) and NPAS2 (P ≤ 0.001), and upregulation of their negative feedback loop genes, CRY1 (P ≤ 0.00003), CRY2 (P ≤ 0.00006), PER1 (P ≤ 0.003), PER2 (P ≤ 0.002), DEC1 (P ≤ 0.0001) and DEC2 (P ≤ 0.00005). Transcript levels of an additional feedback loop regulating BMAL1 showed varied expression, with reduced RORA (P ≤ 0.008) and increased NR1D1 (P ≤ 0.02) in PCOS patients in comparison with the control group. We also demonstrated the expression pattern of clock genes in PBMCs of PCOS women at three different time points. PCOS patients also exhibited increased mRNA levels of steroidogenic enzymes like StAR (P ≤ 0.0005), CYP17A1 (P ≤ 0.005), SRD5A1 (P ≤ 0.00006) and SRD5A2 (P ≤ 0.009). Knockdown of CLOCK/BMAL1 in PBMCs resulted in a significant reduction in estradiol production, by reducing CYP19A1 and a significant increase in dihydrotestosterone production, by upregulating SRD5A1 and SRD5A2 in PBMCs. Our data also showed that CYP17A1 as a direct CLOCK-BMAL1 target in PBMCs. Phenotypic classification of PCOS subgroups showed a higher variation in expression of clock genes and steroidogenesis genes with phenotype A of PCOS. In alignment with the above results, altered expression of ovarian core clock genes (Clock, Bmal1 and Per2) was found in DHEA-treated PCOS mice. The expression of peripheral blood core clock genes in DHEA-induced PCOS mice was less robust and showed a loss of periodicity in comparison with that of control mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We could not evaluate the circadian oscillation of clock genes and clock-controlled genes over a 24-h period in the peripheral blood of control versus PCOS subjects. Additionally, circadian genes in the ovaries of PCOS women could not be evaluated due to limitations in sample availability, hence we employed the androgen excess mouse model of PCOS for ovarian circadian assessment. Clock genes were assessed in the whole ovary of the androgen excess mouse model of PCOS rather than in granulosa cells, which is another limitation of the present work. WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggest that the biological clock is one of the contributing factors in androgen excess in PCOS, owing to its potential role in modulating peripheral androgen metabolism. Considering the increasing prevalence of PCOS and the rising frequency of delayed circadian rhythms and insufficient sleep among women, our study emphasizes the potential in modulating circadian rhythm as an important strategy in PCOS management, and further research on this aspect is highly warranted. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the RGCB-DBT Core Funds and a grant (#BT/PR29996/MED/97/472/2020) from the Department of Biotechnology (DBT), India, to M.L. B.S.J. was supported by a DST/INSPIRE Fellowship/2015/IF150361 and M.B.K. was supported by the Research Fellowship from Council of Scientific & Industrial Research (CSIR) (10.2(5)/2007(ii).E.U.II). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Time-in-range as a target in type 2 diabetes: An urgent need.

Time-in-range emerged as a valuable blood glucose metric, 'beyond HbA1c' for a deeper insight into glycemic control in people with diabetes. It denotes the proportion of time that a person's glucose level remains within the desired target range (usually 70-180 mg/dL or 3.9-10.0 mmol/L). Though clinical targets in the current recommendations for type 1 and type 2 diabetes are close enough, their clinical profiles and prevalences are quite different. Type 2 diabetes is the commonest form of diabetes. Many clinical trials have challenged the usefulness of HbA1c as a glycemic target for Type 2 diabetes mellitus. On account of the higher prevalence and complications of type 2 diabetes, more outcomes-based studies are needed to associate time-in-range with its ongoing risk. These studies strongly support the dependability of time-in-range to identify patients with elevated risk in type 2 diabetes. We discuss the utility of time-in-range, a new metric of continuous glucose monitoring as an outcome measure to correlate with type 2 diabetes risks and complications and to analyze the effectiveness of type 2 diabetes management. This approach may support the use of time-in-range as a metric for long-term health outcomes in the type 2 diabetes population.

Evolution of Insulin Delivery Devices: From Syringes, Pens, and Pumps to DIY Artificial Pancreas.

The year 2021 will mark 100 years since the discovery of insulin. Insulin, the first medication to be discovered for diabetes, is still the safest and most potent glucose-lowering therapy. The major challenge of insulin despite its efficacy has been the occurrence of hypoglycemia, which has resulted in sub-optimal dosages being prescribed in the vast majority of patients. Popular devices used for insulin administration are syringes, pens, and pumps. An artificial pancreas (AP) with a closed-loop delivery system with > 95% time in range is believed to soon become a reality. The development of closed-loop delivery systems has gained momentum with recent advances in continuous glucose monitoring (CGM) and computer algorithms. This review discusses the evolution of syringes, disposable, durable pens and connected pens, needles, tethered and patch insulin pumps, bionic pancreas, alternate controller-enabled infusion (ACE) pumps, and do-it-yourself artificial pancreas systems (DIY-APS).

Impaired Arginine Metabolism Coupled to a Defective Redox Conduit Contributes to Low Plasma Nitric Oxide in Polycystic Ovary Syndrome.

BACKGROUND: Though oxidative stress is associated with Polycystic Ovary Syndrome (PCOS), the status of nitric oxide is still unclear. Nitric Oxide (NO) plays pivotal roles in many physiological functions which are compromised in PCOS. Our recent study reveals lowered T-regulatory cells (Tregs) in PCOS, and Treg generation is known to be regulated by NO levels. However concrete evidences are lacking on mechanisms modulating NO levels under PCOS. METHODS: This is a retrospective case-control cohort study, comprised of PCOS women (N=29) and normal menstruating women as controls (N=20). We analysed NOx (nitrite+nitrate) and hydrogen peroxide (H2O2) concentrations, transcript levels of endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and arginine modulators, hydrogen peroxide regulators in the cohort. RESULTS: PCOS women showed reduced plasma NOx(nitrate+nitrite) and H2O2 compared to controls. We report reduction in transcript levels of iNOS/NOS2 and eNOS/NOS3 in PCOS peripheral blood. The transcripts involved in arginine bioavailability: Argininosuccinate lyase (ASL), Solute Carrier Family1, member 7 (SLC7A1) and Arginase 1 (ARG1) and Asymmetric Dimethyl Arginine (ADMA) metabolism: Protein arginine methyltransferase 1 (PRMT1) and Dimethylarginine dimethylaminohydrolase 2 (DDAH2) also showed differential expression. H2O2 concentration in PCOS women was also found to be reduced. The reduction can be attributed to increase in catalase levels as a consequence of the body's effort to alleviate the oxidative burden in the system. CONCLUSION: Our study advocates that PCOS women have lowered NO due to reduced iNOS/eNOS expression, low H2O2, high ADMA synthesis and reduced arginine bioavailability. An in-depth analysis of redox biology of PCOS to open up potential therapeutic strategies is highly recommended.

Reduced Tregs in peripheral blood of PCOS patients - a consequence of aberrant Il2 signaling.

CONTEXT: The immunesupressive action of CD4(+)CD25(+) CD127(-/low) T regulatory cells (Tregs) is vital for an efficient reproductive function. However no data exists on their number or functionality in polycystic ovary syndrome (PCOS). OBJECTIVE: The study aimed to analyze the frequency of circulating Tregs and key factors modulating them in women with PCOS. DESIGN, SETTING, AND PARTICIPANTS: This is a retrospective, case-control cohort study conducted in women with PCOS recruited from Samad IVF hospitals and Women and Children Hospital, Thiruvananthapuram, India. Women with PCOS (N = 20) were diagnosed according to Rotterdam Consensus and normal menstruating women were taken as controls (N = 2331). MAIN OUTCOME MEASURES: We analyzed the proportion of CD4(+)CD25(+) CD127(-/low) Tregs in women with PCOS by fluorescent activated cell sorting. RESULTS: The study discovered that the women with PCOS have reduced numbers of Tregs (2.626 ± 0.62) compared with controls (4.253 ± 0.87) (t = 6.963, P < .0001, mean difference = -1.627; 95% confidence interval = -2.099--1.155). We documented a decrease in the follicular phase Treg expansion in women with PCOS. Our results revealed a reduced STAT5A (fold change [FC] = 7.642, P < .0004)/STAT5B (FC = 3.824, P < .0001), FOXP3 (FC = 4.1343, P = .0004)/CTLA4 (FC = 2.569, P = .0001) and elevated AKT (FC = 7.39, P = .05)/PIK3 (FC = 5.326, P = .0002) expression in women with PCOS. Recombinant interleukin 2 (rIL2) treatment failed to improve FOXP3/CTLA4 levels but caused a reduction of AKT/PIK3 arm, possibly due to an elevated PTEN in women with PCOS. CONCLUSION: The study suggests that women with PCOS have reduced Tregs due to an inherent hyporesponsiveness to IL2, which is unable to activate STAT5B and reduce FOXP3 expression. IL2-based therapeutic strategies can ameliorate complications in PCOS by suppressing the AKT/PIK3 arm.