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.

Autoimmune regulator (AIRE): Takes a hypoxia-inducing factor 1A (HIF1A) route to regulate FOXP3 expression in PCOS.

PROBLEM: Autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy (APECED) pathology due to autoimmune regulator (AIRE) gene mutations leads to loss of central tolerance triggering immune attack, a factor causing infertility. One of the targets of autoimmune attack is ovary and its repercussion results in polycystic ovarian syndrome (PCOS). Although reduced Tregs have been reported in PCOS, a lacunae exists on the status of AIRE gene expression and its role in treg insufficiency via HIF1A-FOXP3 axis in PCOS. METHOD OF STUDY: This is a case-control cohort study recruiting 40 normal and 40 PCOS volunteers for peripheral blood sample collection and PCOS diagnoses were based on Rotterdam Consensus criteria. AIRE and HIF1A expression status was analysed by qRT PCR and western blot. FACS analyses was conducted on AIRE silenced peripheral blood mononuclear cells (PBMCs) after Treg induction. RESULTS: Our results indicate a reduced AIRE (fold change log2 (RQ) = -2.6, P < .01) and increased HIF1A (fold change log2 (RQ) = 3.6, P < .02) in PBMCs of PCOS subjects compared to age-matched controls. Western blot of AIRE and HIF1A corroborates with qRT PCR data. Our CHIP data demonstrate AIRE mediated HIF1A promoter regulation. Silencing of AIRE in PBMCs contributes to the upregulation of HIF1A transcripts by two-fold (P < .0015) and downregulation in FOXP3 expression by three-fold (P < .0017). FACS analyses revealed that silencing of AIRE reduces Tcell to Treg conversion. CONCLUSIONS: Our consolidated results derive a new connection among AIRE-HIF1A-FOXP3 with AIRE reduction enabling increased HIF1A resulting in reduced FOXP3 in PBMCs of PCOS patients leading to Treg insufficiency.

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.

Oestrogen, progesterone and stem cells: the discordant trio in endometriosis?

Oestrogen-progesterone signalling is highly versatile and critical for the maintenance of healthy endometrium in humans. The genomic and nongenomic signalling cascades initiated by these hormones in differentiated cells of endometrium have been the primary focus of research since 1920s. However, last decade of research has shown a significant role of stem cells in the maintenance of a healthy endometrium and the modulatory effects of hormones on these cells. Endometriosis, the growth of endometrium outside the uterus, is very common in infertile patients and the elusiveness in understanding of disease pathology causes hindrance in selection of treatment approaches to enhance fertility. In endometriosis, the stem cells are dysfunctional as it can confer progesterone resistance to their progenies resulting in disharmony of hormonal orchestration of endometrial homeostasis. The bidirectional communication between stem cell signalling pathways and oestrogen-progesterone signalling is found to be disrupted in endometriosis though it is not clear which precedes the other. In this paper, we review the intricate connection between hormones, stem cells and the cross-talks in their signalling cascades in normal endometrium and discuss how this is deregulated in endometriosis. Re-examination of the oestrogen-progesterone dependency of endometrium with a focus on stem cells is imperative to delineate infertility associated with endometriosis and thereby aid in designing better treatment modalities.

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.

Expression of protocadherin 11Yb (PCDH11Yb) in seminal germ cells is correlated with fertility status in men.

Protocadherin 11 Y-linked (PCDH11Y), a member of the cadherin superfamily, is predominantly expressed in the central nervous system, is encoded by the Yp11.2 locus and exists in three isoforms: 11Ya, 11Yb and 11Yc. PCDH11Y is upregulated by retinoic acid signalling and is essential for spermatogonial differentiation and initiation of meiosis. PCDH11Y mediates Wnt signalling, which plays a crucial role in the differentiation of various cell types. PCDH11Y has been implicated in neuronal cell differentiation and proliferation, but its association with spermatogenesis has not yet been addressed. Hence, in order to address the possible role of PCDH11Y in relation to spermatogenesis, the expression analysis of PCDH11Y in the seminal germ cells of fertile and infertile males were carried out employing RT-PCR, western blotting and immunofluorescence analysis. In the present study, PCDH11Yb, but not PCDH11Ya or PCDH11Yc, was expressed in germ cells isolated from the semen of all 13 men with proven fertility. However, in several subjects from various infertility classes, there was complete absence or a significant reduction in the expression of PCDH11Yb. PCDH11Y exhibited prominent localisation on the head and midpiece region of spermatozoa from fertile men, whereas spermatozoa from infertile subjects had either weak or abnormal localisation patterns for PCDH11Y. In addition, downregulation of canonical Wnt signalling was correlated with defective expression of PCDH11Y in spermatozoa of infertile men, as evidenced by downregulation of the Wnt targets C-Myc and C-Jun. In conclusion, expression levels of PCDH11Yb in germ cells in the semen were correlated with the fertility status of men.

Aberrant expression of TAR DNA binding protein-43 is associated with spermatogenic disorders in men.

Loss of function of TAR DNA-binding protein (TDP-43) has been implicated in neurodegenerative disorders in both humans and animal models. TDP-43 has also been shown to be cis-acting transcriptional repressor of the acrosome vesicle (Acrv) gene in mice. In the present study, we investigated the expression of the TDP-43 transcript (TARDBP) and protein in germ cells from 11 fertile and 98 subfertile men to verify its potential association with poor seminograms. The expression profile of TDP-43 was characterised in immature germ cells and spermatozoa from semen from fertile and subfertile men using reverse transcription-polymerase chain reaction, western blotting and immunofluorescence. Although germ cells from subfertile men tested negative for TARDBP, the full-length message of the same was detected in fertile men. TDP-43 was detected in spermatozoa from fertile men using western blot analysis and immunofluorescence. The expression of this protein was negligible in spermatozoa from men with primary spermatogenic dysfunction. We conclude that a deficiency in the TDP-43 expression is associated with defective spermatogenesis and male infertility. We propose that TDP-43 could be used as a marker of male factor infertility.

Aberrant Expression of Dynein light chain 1 (DYNLT1) is Associated with Human Male Factor Infertility.

DYNLT1 is a member of a gene family identified within the t-complex of the mouse, which has been linked with male germ cell development and function in the mouse and the fly. Though defects in the expression of this gene are associated with male sterility in both these models, there has been no study examining its association with spermatogenic defects in human males. In this study, we evaluated the levels of DYNLT1 and its expression product in the germ cells of fertile human males and males suffering from spermatogenic defects. We screened fertile (n = 14), asthenozoospermic (n = 15), oligozoospermic (n = 20) and teratozoospermic (n = 23) males using PCR and Western blot analysis. Semiquantitative PCR indicated either undetectable or significantly lower levels of expression of DYNLT1 in the germ cells from several patients from across the three infertility syndrome groups, when compared with that of fertile controls. DYNLT1 was localized on head, mid-piece, and tail segments of spermatozoa from fertile males. Spermatozoa from infertile males presented either a total absence of DYNLT1 or its absence in the tail region. Majority of the infertile individuals showed negligible levels of localization of DYNLT1 on the spermatozoa. Overexpression of DYNLT1 in GC1-spg cell line resulted in the up-regulation of several cytoskeletal proteins and molecular chaperones involved in cell cycle regulation. Defective expression of DYNLT1 was associated with male factor infertility syndromes in our study population. Proteome level changes in GC1-spg cells overexpressing DYNLT1 were suggestive of its possible function in germ cell development. We have discussed the implications of these observations in the light of the known functions of DYNLT1, which included protein trafficking, membrane vesiculation, cell cycle regulation, and stem cell differentiation.

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.