Granulosa Cells Of Polycystic Ovary Syndrome Patients Research Articles

MALAT1 expression in granulosa cells in PCOS patients with different phenotypes

Polycystic ovary syndrome (PCOS) is one of the most common reproductive endocrine metabolic disorders. The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) reportedly can regulate the reproductive system. Despite studies, the alteration of MALAT1 expression in granulosa cells (GCs) from PCOS patients was inconsistent. To evaluate MALAT1 expression in GCs in different PCOS subgroups and its association with PCOS phenotypes, we collected GCs from 110 PCOS cases and 71 controls, and examined MALAT1 expression by quantitative PCR. The results showed MALAT1 expression was upregulated in PCOS cases, especially in insulin resistant (IR) PCOS subgroup, obese PCOS subgroup and non-hyperandrogenic (NHA) PCOS subgroup. MALAT1 expression was positively correlated with BMI and several metabolic parameters in controls. Interestingly, MALAT1 expression was notably associated with some critical endocrine indexes for PCOS, including E2, FSH, LH and LH/FSH ratio. In different PCOS subgroups, we found significant positive correlations with LH/FSH ratio in IR-PCOS and PCOS with normal weight, and with serum T and LH level in NHA-PCOS subgroup. Integrated analysis with lncRNA target databases and PCOS-related databases revealed MALAT1 could participate in PCOS by influencing immune response and lipids metabolism in GCs. In conclusion, MALAT1 was differently expressed in GCs in PCOS, especially in IR, obese and NHA PCOS subgroups. MALAT1 was likely involved in metabolism and immune response in GCs in PCOS. However, more studies are necessary to establish this concept.

Metabolomics approach for the etiopathogenesis of polycystic ovarian syndrome

Polycystic ovary syndrome (PCOS) is a complex and multifaceted disorder that has been extensively studied from various angles, including genomics, metabolomics, and other multiomics approaches. This narrative review seeks to provide an updated understanding of PCOS, with a particular focus on metabolomics. PCOS encompasses four primary phenotypes: Classic PCOS with or without polycystic ovary morphology, ovulatory PCOS, and non-hyperandrogenic PCOS. Each phenotype exhibits distinct clinical and biochemical characteristics. Classic PCOS, characterized by hyperinsulinemia, insulin resistance (IR), and metabolic complications, has garnered significant attention in recent studies. In the pursuit of unraveling PCOS’s pathophysiology, researchers have embraced a multiomics approach, exploring the genome, epigenome, transcriptome, exosomes, proteome, and metabolome. The genomic landscape of PCOS includes genes related to androgen synthesis, regulation, insulin receptors, and growth factors, among others. Epigenetic mechanisms have also been investigated, revealing the roles of non-coding RNA, histone acetylation, and deacetylation. Transcriptomic biomarkers and mitochondrial RNAs targets, such as miR-9 and miR-32, have been explored in granulosa cells of PCOS patients. Metabolomics studies have identified various biomarkers and metabolic pathways that differentiate PCOS from healthy individuals. A diverse range of alterations in carbohydrate and lipid metabolism has been observed, shedding light on the underpinnings of PCOS-related metabolic dysfunction. These studies have uncovered numerous biomolecules with potential relevance to PCOS, including kininogen 1, cytoketatin 9, and many others. In addition, the intricate relationships between IR, inflammation, and metabolic factors have been elucidated. Recent research extends beyond high-end research centers, even using urine samples to identify metabolomic markers for PCOS. This approach has led to the discovery of distinct biomarker panels related to metabolic pathways that differentiate PCOS from healthy subjects. In summary, PCOS is a complex and heterogeneous condition, with multiomics studies offering promising avenues for a deeper understanding of its pathophysiology. These studies may ultimately provide valuable insights that can revolutionize the diagnosis and treatment of PCOS, ending a centurylong scientific expedition with a crescendo of discoveries.

Identification of the hub genes in polycystic ovary syndrome based on disease-associated molecule network.

Revealing the key genes involved in polycystic ovary syndrome (PCOS) and elucidating its pathogenic mechanism is of extreme importance for the development of targeted clinical therapy for PCOS. Investigating disease by integrating several associated and interacting molecules in biological systems will make it possible to discover new pathogenic genes. In this study, an integrative disease-associated molecule network, combining protein-protein interactions and protein-metabolites interactions (PPMI) network was constructed based on the PCOS-associated genes and metabolites systematically collected. This new PPMI strategy identified several potential PCOS-associated genes, which have unreported in previous publications. Moreover, the systematic analysis of five benchmarks data sets indicated the DERL1 was identified as downregulated in PCOS granulosa cell and has good classification performance between PCOS patients and healthy controls. CCR2 and DVL3 were upregulated in PCOS adipose tissues and have good classification performance. The expression of novel gene FXR2 identified in this study is significantly increased in ovarian granulosa cells of PCOS patients compared with controls via quantitative analysis. Our study uncovers substantial differences in the PCOS-specific tissue and provides a plethora of information on dysregulated genes and metabolites that are linked to PCOS. This knowledgebase could have the potential to benefit the scientific and clinical community. In sum, the identification of novel gene associated with PCOS provides valuable insightsinto the underlying molecular mechanisms of PCOS and could potentially lead to the development of new diagnostic and therapeutic strategies.

Astaxanthin treatment ameliorates ER stress in polycystic ovary syndrome patients: a randomized clinical trial

Astaxanthin (ASX), as a natural carotenoid compound, exists in various types of seafood and microorganisms. It has several possible beneficial therapeutic effects for patients with polycystic ovary syndrome (PCOS). Patients with PCOS also suffer from endoplasmic reticulum (ER) stress. In the present work, it was hypothesized that ER stress could be improved by ASX in PCOS patients. Granulosa cells (GCs) were obtained from 58 PCOS patients. The patients were classified into ASX treatment (receiving 12 mg/day for 60 days) and placebo groups. The expression levels of ER stress pathway genes and proteins were explored using Western blotting and quantitative polymerase chain reaction. To assess oxidative stress markers, follicular fluid (FF) was gained from all patients. The Student’s t test was used to perform statistical analysis. After the intervention, ASX led to a considerable reduction in the expression levels of 78-kDa glucose-regulated protein (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and X-box-binding protein 1 compared to the placebo group, though the reduction in the messenger RNA (mRNA) expression level of activating transcription factor 6 was not statistically significant. However, ASX significantly increased the ATF4 expression level. GRP78 and CHOP protein levels represented a considerable decrease in the treatment group after the intervention. In addition, a statistically significant increase was found in the FF level of total antioxidant capacity in the treatment group. Based on clinical outcomes, no significant differences were found between the groups in terms of the oocyte number, fertilization rate, and fertility rate, but the ASX group had higher rates of high-quality oocytes, high-quality embryo, and oocyte maturity compared to the placebo group. Our findings demonstrated that ER stress in the GCs of PCOS patients could be modulated by ASX by changing the expression of genes and proteins included in the unfolding protein response.Trial registration This study was retrospectively registered on the Iranian Registry of Clinical Trials website (www.irct.ir; IRCT-ID: IRCT20201029049183N, 2020-11-27).

Granulosa cells from immature follicles exhibit restricted glycolysis and reduced energy production: a dominant problem in polycystic ovary syndrome.

We hypothesized that immature oocytes are associated with impaired energy production in surrounding granulosa cells (GCs) in polycystic ovary syndrome (PCOS). Thus, this study investigated mitochondrial function, determined expression of glycolytic regulatory enzymes, and measured ATP levels in GCs of PCOS patients. GCs were isolated from forty-five PCOS patients and 45 control women. Intracellular concentration of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), the rate of glycolysis, total antioxidant capacity (TAC), activities of catalase (CAT) and superoxide dismutase (SOD), and ATP level were measured in GCs. The gene expression and protein levels of glycolytic enzymes (hexokinase, muscular phosphofructokinase, platelet derived phosphofructokinase, and muscular pyruvate kinase) were determined. Association of GC energy level with oocyte maturation was further validated by measuring glycolysis rate and ATP level in GCs isolated from mature and immature follicles from new set of fifteen PCOS patients and 15 controls. PCOS patients showed higher ROS level, decreasedTAC, reduced CAT and SOD activities, and lower Δψm together with reduced expression of key glycolytic enzymes. ATP concentration and biochemical pregnancy were lower in PCOS compared with control group. ATP levels werefound to besignificantly correlated with ROS and Δψm (r = - 0.624 and r = 0.487, respectively). GCs isolated from immature follicles had significantly lower ATP levels and rate of glycolysis compared with the GCs separated from mature follicles in both PCOS patients and control. Declined energy due to the mitochondrial dysfunction and restrained glycolysis in GCs is associated with the immature oocytes and lower biochemical pregnancy in PCOS.

Notch Signaling Induced by Endoplasmic Reticulum Stress Regulates Cumulus-Oocyte Complex Expansion in Polycystic Ovary Syndrome.

Endoplasmic reticulum (ER) stress activated in granulosa cells contributes to the pathophysiology of polycystic ovary syndrome (PCOS). In addition, recent studies have demonstrated that Notch signaling plays multiple roles in the ovary via cell-to-cell interactions. We hypothesized that ER stress activated in granulosa cells of antral follicles in PCOS induces Notch signaling in these cells, and that activated Notch signaling induces aberrant cumulus-oocyte complex (COC) expansion. Expression of Notch2 and Notch-target transcription factors was increased in granulosa cells of PCOS patients and model mice. ER stress increased expression of Notch2 and Notch-target transcription factors in cultured human granulosa-lutein cells (GLCs). Inhibition of Notch signaling abrogated ER stress-induced expression of genes associated with COC expansion in cultured human GLCs, as well as ER stress-enhanced expansion of cumulus cells in cultured murine COCs. Furthermore, inhibition of Notch signaling reduced the areas of COCs in PCOS model mice with activated ER stress in the ovary, indicating that Notch signaling regulates COC expansion in vivo. Our findings suggest that Notch2 signaling is activated in granulosa cells in PCOS and regulates COC expansion. It remains to be elucidated whether aberrant COC expansion induced by the ER stress-Notch pathway is associated with ovulatory dysfunction in PCOS patients.

Mitochondrial and glucose metabolic dysfunctions in granulosa cells induce impaired oocytes of polycystic ovary syndrome through Sirtuin 3

Mitochondrial function and glucose metabolism play important roles in bidirectional signaling between granulosa cells (GCs) and oocytes. However, the factors associated with mitochondrial function and glucose metabolism of GCs in polycystic ovary syndrome (PCOS) are poorly understood, and their potential downstream effects on oocyte quality are still unknown. The aim of this study was to investigate whether there are alterations in mitochondrial-related functions and glucose metabolism in ovarian GCs of women with PCOS and the role of Sirtuin 3 (SIRT3) in this process. Here, we demonstrated that women with PCOS undergoing in vitro fertilization and embryo transfer had significantly lower rates of metaphase II oocytes, two-pronuclear fertilization, cleavage, and day 3 good-quality embryos. Germinal vesicle- and metaphase I-stage oocytes from women with PCOS exhibited increased mitochondrial reactive oxygen species (ROS), decreased mitochondrial membrane potential, and downregulation of glucose-6-phosphate dehydrogenase. GCs from women with PCOS presented significant alterations in mitochondrial morphology, amount, and localization, decreased membrane potential, reduced adenosine triphosphate (ATP) synthesis, increased mitochondrial ROS and oxidative stress, and insufficient oxidative phosphorylation (OXPHOS) together with decreased glycolysis. SIRT3 expression was significantly decreased in GCs of PCOS patients, and knockdown of SIRT3 in KGN cells could mimic the alterations in mitochondrial functions and glucose metabolism in PCOS GCs. SIRT3 knockdown changed the acetylation status of NDUFS1, which might induce altered mitochondrial OXPHOS, the generation of mitochondrial ROS, and eventually defects in the cellular insulin signaling pathway. These findings suggest that SIRT3 deficiency in GCs of PCOS patients may contribute to mitochondrial dysfunction, elevated oxidative stress, and defects in glucose metabolism, which potentially induce impaired oocytes in PCOS.

Effect of lncRNA MALAT1 on the Granulosa Cell Proliferation and Pregnancy Outcome in Patients With PCOS.

Follicle arrest is one of the main characteristics of polycystic ovary syndrome (PCOS), the most common endocrinological disorder in reproductive-aged women. Increasing evidence proves that high anti-Mullerian hormone (AMH) levels may play an important role in follicular development. Long noncoding RNA (lncRNA) with a length of more than 200 nt is widely involved in the directional differentiation, growth, and development of cells, whereas whether lncRNA is involved in AMH’s role in follicular development is unknown. In this study, we analyzed lncRNA expression in ovarian granulosa cells (GCs) collected from women with and without PCOS via high-throughput sequencing. The results showed that a total of 79 noncoding transcripts were differently expressed in GCs of PCOS patients, including upregulated lncRNA MALAT1. The upregulation of MALAT1 was further confirmed by RT-qPCR in GCs from a larger cohort of PCOS patients. Furthermore, knockdown MALAT1 can promote the proliferation of KGN cell in vitro. These data suggested a role for MALAT1 in the development of PCOS. Meanwhile, MALAT1 and phosphorylated SMAD 1/5 (Ser463/465) protein were upregulated in KGN cells after exogenous AMH stimulation, which identified AMH perhaps as a regulator for the expression of MALAT1. We also found that MALAT1 can predict clinical pregnancy outcome to a certain extent by ROC curve analysis (area: 0.771, p = 0.007, 95% CI: 0.617–0.925, sensitivity: 57.1%, specificity: 91.7%). Thus, our findings revealed a role of lncRNA MALAT1 in inhibiting granulosa cell proliferation and may be correlated with pregnancy outcome in PCOS.

Effects of Androgen Excess-Related Metabolic Disturbances on Granulosa Cell Function and Follicular Development.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease in women of reproductive age. Ovarian dysfunction including abnormal steroid hormone synthesis and follicular arrest play a vital role in PCOS pathogenesis. Hyperandrogenemia is one of the important characteristics of PCOS. However, the mechanism of regulation and interaction between hyperandrogenism and ovulation abnormalities are not clear. To investigate androgen-related metabolic state in granulosa cells of PCOS patients, we identified the transcriptome characteristics of PCOS granulosa cells by RNA-seq. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) revealed that genes enriched in lipid metabolism pathway, fatty acid biosynthetic process and ovarian steroidogenesis pathway were abnormally expressed in PCOS granulosa cells in comparison with that in control. There are close interactions among these three pathways as identified by analysis of the protein-protein interaction (PPI) network of DEGs. Furthermore, in vitro mouse follicle culture system was established to explore the effect of high androgen and its related metabolic dysfunction on follicular growth and ovulation. RT-qPCR results showed that follicles cultured with dehydroepiandrosterone (DHEA) exhibited decreased expression levels of cumulus expansion-related genes (Has2, Ptx3, Tnfaip6 and Adamts1) and oocyte maturation-related genes (Gdf9 and Bmp15), which may be caused by impaired steroid hormone synthesis and lipid metabolism, thus inhibited follicular development and ovulation. Furthermore, the inhibition effect of DHEA on follicle development and ovulation was ameliorated by flutamide, an androgen receptor (AR) antagonist, suggesting the involvement of AR signaling. In summary, our study offers new insights into understanding the role of androgen excess induced granulosa cell metabolic disorder in ovarian dysfunction of PCOS patients.

Melatonin alleviates insulin resistance through the PI3K/AKT signaling pathway in ovary granulosa cells of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine gynecological disorder. Insulin resistance (IR) is a major cause of PCOS. Melatonin, a critical endogenous hormone, has beneficial effects on the female reproductive system. This study aims to investigate the molecular effect of melatonin on IR in human ovarian granulosa cells (GCs). Hormone levels of the subjects were determined through clinical examination. The expression levels of insulin receptor substrate (IRS)-1 and glucose transporter (GLUT4) in GCs from PCOS patients and a human granulosa cell line (SVOG) were examined using qRT-PCR and western blot. The IR cell model was established by inducing SVOG cells with palmitic acid (PA). IR was detected in GCs of PCOS patients and SVOG by measuring glucose content and glucose uptake. Cell viability and apoptosis levels were detected by CCK-8 assay and flow cytometry. PI3K/Akt pathway expression in SVOG was assessed by western blot. PCOS patients had higher levels of luteinizing hormone (LH), testosterone, and LH/follicle-stimulating hormone. PA decreased cell viability, promoted apoptosis, and reduced glucose uptake in SVOG cells. IRS-1 and GLUT4 mRNA and protein expression was downregulated, and glucose uptake capacity was reduced in PCOS GCs and SVOG cells. Melatonin significantly upregulated IRS-1 and GLUT4 expression, downregulated p-IRS-1 (Ser307), and improved glucose uptake in PCOS patients' GCs and SVOG cells. PA decreased PI3K and Akt phosphorylation, whereas melatonin increased p-PI3K and p-Akt levels. Melatonin can reduce IR in GCs and PA-induced SVOG cells via the PI3K/Akt signaling pathway, providing more evidence for treating polycystic ovary syndrome.

P–710 The possible relation between kisspeptin and apoptosis on granulosa cells of PCOS patients

Abstract Study question Does kisspeptin administration affect the apoptotic factors of granulosa cells of Polycystic Ovary Syndrome (PCOS) patients? Summary answer Kisspeptin administration significantly increased apoptosis related factors. What is known already Kisspeptin plays a critical role in central gonadotropin secretion by acting on GnRH neurons in the hypothalamus. Kisspeptin receptors are also expressed on the ovaries. Kisspeptin–54 has recently been known as a prominent agent in oocyte maturation. Although there are many studies on the possible roles of kisspeptin administration in vivo on hypothalamic gonadotropin secretion and ovarian function and the possible roles of these effects in the pathogenesis of PCOS, there is not enough information about the effect of in vitro application of kisspeptin on ovarian function and conditions that lead to the development of PCOS. Study design, size, duration This basic research study was an in vitro experimental approach involving the use of granulosa cell from a PCOS and normal cases between July to December in 2020. 32 women were included in both control and PCOS groups. Participants/materials, setting, methods Women with (n = 16) and without (n = 16) PCOS were included in the study. Granulosa cells, follicular fluids and blood samples were collected on oocyte pick-up day. Cells were isolated from follicular fluids. Kisspeptin–54 levels were determined by ELISA in serum and follicle fluids. Also, FSH and LH levels were determined in serum. Kisspeptin–54 administrated in vitro to the half of the samples. Fluorescence spectrophotometer and laser scanning confocal microscope were used to evaluate calcium concentrations Main results and the role of chance The serum kisspeptin level was found significantly higher in PCOS patients compared to the control group (p < 0.05). Also, the level of kisspeptin in follicular fluid was statistically significantly higher in PCOS patients compared to the control group (p < 0.05). The mean serum FSH level of PCOS patients was found to be significantly lower than the control group (p < 0.05). Furthermore, the mean serum LH level of PCOS patients was significantly higher than control groups (p < 0.05). The intracellular calcium concentration in granulosa cells obtained from PCOS patients was significantly lower than the control group (p < 0.05). Limitations, reasons for caution The KISS1, KISS1R, BCL–2, CASPASE–3, FSHR, LHCGR, STAR, ESR1, ESR2, INHBA gene expression levels of apoptotic process is still under analyses. The protein expression analyses of these genes may strength the results. Wider implications of the findings: Using kisspeptin antagonists may be supposed as an additional treatment for patients with PCOS. Trial registration number OMU KAEK 2019 /724

Circadian Clock Genes REV-ERBs Inhibits Granulosa Cells Apoptosis by Regulating Mitochondrial Biogenesis and Autophagy in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is an endocrinopathy with complex pathophysiology that is a common cause of anovulatory infertility in women. Although the disruption of circadian rhythms is indicated in PCOS, the role of the clock in the etiology of these pathologies has yet to be appreciated. The nuclear receptors REV-ERBα and REV-ERBβ are core modulators of the circadian clock and participate in the regulation of a diverse set of biological functions. However, in PCOS, the expression of REV-ERBs and their effects remain unclear. Here, we demonstrate that the levels of REV-ERBα and REV-ERBβ expression were lower in the granulosa cells of PCOS patients than in control subjects. In vitro, we found that the overexpression of REV-ERBα and REV-ERBβ, and their agonist SR9009, promoted the expression of mitochondrial biosynthesis genes PGC-1α, NRF1, and TFAM and inhibited autophagy in KGN cells. Our results also indicate that REV-ERBα and REV-ERBβ can inhibit apoptosis in granulosa cells and promote proliferation. Importantly, the REV-ERB agonist SR9009 ameliorates abnormal follicular development by promoting mitochondrial biosynthesis and inhibiting autophagy in a mouse PCOS model. This allows us to speculate that SR9009 has potential as a therapeutic agent for the treatment of PCOS.

RETRACTED ARTICLE: Downregulating lncRNA NEAT1 induces proliferation and represses apoptosis of ovarian granulosa cells in polycystic ovary syndrome via microRNA-381/IGF1 axis

ObjectiveResearchers have revealed the combined functions of long noncoding RNAs (lncRNAs) and microRNA (miRNAs) in polycystic ovary syndrome (PCOS). This study aimed to understand the role of nuclear-enriched abundant transcript 1 (NEAT1) and miR-381 involving insulin-like growth factor 1 (IGF1) in PCOS.MethodsPCOS rat model was established by dehydroepiandrosterone induction. NEAT1, miR-381 and IGF1 expression in ovarian granulosa cells of PCOS patients and ovarian tissues of PCOS rats were tested. Bioinformatics website and dual luciferase reporter gene assay were utilized to verify the relationship between NEAT1 and miR-381 and that between miR-381 and IGF1. Levels of sex hormone, pathological changes and ovarian granulosa cell apoptosis in ovarian tissues of PCOS rats were detected. Ovarian granulosa cell proliferation and apoptosis were analyzed in vitro.ResultsNEAT1 and IGF1 expression increased while miR-381 expression decreased in the ovarian granulosa cells of patients with PCOS and the ovarian tissues of PCOS rats. In in vivo experiments, interference with NEAT1 improved the levels of sex hormones, alleviated pathological changes and suppressed ovarian granulosa cell apoptosis in the ovarian tissues of PCOS rats. In in vitro cell experiments, interference with NEAT1 suppressed apoptosis and enhanced cell proliferation of ovarian granulosa cells. NEAT1 interference-mediated effect would be reversed by up-regulating miR-381. NEAT1 acted as a ceRNA to adsorb miR-381 to target IGF1. Overexpression of IGF1 reversed the inhibitory effect of miR-381 on ovarian granulosa cell apoptosis.ConclusionInterference with NEAT1 increases miR-381 and reduces IGF1 levels, effectively improving the levels of sex hormones and reducing the pathological damage of ovarian tissue in rats with PCOS.

The Release of Peripheral Immune Inflammatory Cytokines Promote an Inflammatory Cascade in PCOS Patients via Altering the Follicular Microenvironment.

BackgroundHormones and immune imbalance are critical factors in polycystic ovary syndrome (PCOS). The alternation of immune microenvironment of oocytes may play a significant role in infertility of PCOS patients.ObjectiveThis study explores the role of follicular fluid microenvironment change in inflammatory pathways activation of granulosa cells (GCs) in PCOS women infertility.MethodsWe enrolled 27 PCOS patients and 30 controls aged 22 to 38 years who underwent IVF and collected their luteinized granulosa cells (LGCs). Meanwhile, a granulosa-like tumor cell line (KGN) as a cell-model assisted this study. Key inflammatory markers in human ovarian GCs and follicular fluid were detected by RT-qPCR, Western blotting, or ELISA. The KGN cells were treated with follicle supernatant mixed with normal medium to simulate the microenvironment of GCs in PCOS patients, and the inflammation indicators were observed. The assembly of NLRP3 inflammasomes was detected by immunofluorescence techniques. Dihydroethidium assay and EdU proliferation assay were used to detect ROS and cell proliferation by flow cytometry.ResultsCompared with normal controls (n = 19), IL-1β (P = 0.0005) and IL-18 (P = 0.021) in the follicular fluid of PCOS patients (n = 20) were significantly increased. The NF-κB pathway was activated, and NLRP3 inflammasome was formatted in ovarian GCs of PCOS patients. We also found that inflammation of KGN cells was activated with LPS irritation or stimulated by follicular fluid from PCOS patients. Finally, we found that intracellular inflammation process damaged mitochondrial structure and function, which induced oxidative stress, affected cellular metabolism, and impaired cell proliferation.ConclusionInflammatory microenvironment alteration in the follicular fluid of PCOS patients leads to activated inflammatory pathway in GCs, serving as a crucial factor that causes adverse symptoms in patients. This study provides a novel mechanism in the inflammatory process of PCOS.

Methylome and transcriptome profiling revealed epigenetic silencing of LPCAT1 and PCYT1A associated with lipidome alterations in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine diseases of fertile women and a major cause of infertility. The regulatory effects of DNA methylation on gene transcription and downstream lipid metabolism have not been explored in PCOS. In this study, MBD-seq and RNA-seq were performed on ovarian granulosa cells of PCOS patients and controls, and methylation specific PCR and quantitative polymerase chain reaction were used to validate the results. Then lipidomic profiling was conducted on serum of PCOS patients and controls using UPLC-MS. We identified 73 genes with differently methylated promoters and 830 differently expressed genes. The promoter regions of LPCAT1 and PCYT1A were hypermethylated, accompanied by downregulation of their messenger RNA expression, which may be involved in the regulation of PCOS through downstream glycerophospholipid metabolism and phosphatidylcholine synthesis. The lipid profiling results showed significant changes in 21 lipids, which demonstrated the disturbance in glycerophospholipid metabolism and glycerolipid metabolism pathways. Furthermore, the metabolites-genes interaction network was constructed to illustrate the association of aberrant methylome and transcriptome with lipidome alterations in glycerolipid and glycerophospholipid metabolism pathways. Our study suggested that the methylation silencing of LPCAT1 and PCYT1A may promote glycerophospholipids metabolism dysregulation, which provided a novel genetic and lipometabolic basis for the pathogenesis of PCOS.

Induction of aryl hydrocarbon receptor in granulosa cells by endoplasmic reticulum stress contributes to pathology of polycystic ovary syndrome.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously showed that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

The role of serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome.

ObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome (PCOS).MethodsSelected serum inflammatory cytokines were analyzed in the particle cells, which were interfered by berberine, from 78 infertile women who were to be treated with In Vitro Fertilization (IVF) /Intracytoplasmic Sperm Injection-Embryo Transfer (icsi-et). Among them, 49 patients had PCOS infertility, and 29 were non-PCOS patients whose infertility resulted from fallopian tube and male factors. The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells. The correlations between the serum inflammatory cytokine expression levels and the corresponding clinical hormones were analyzed. The changes in the expression (mRNA and protein) levels of the serum inflammatory cytokines were studied by real-time quantitative PCR and protein printing. Fluorescence microscope and flow cytometry were used to detect the glucose uptake capacity of ovarian granulosa cells in PCOS patients under the action of insulin after berberine.ResultsIn the PCOS group, IL-17a (P = 0.001), IL-1Ra (P<0.0001), and IL-6 (P = 0.035) were significantly higher than those in the non-PCOS group. In the non-PCOS group, AMH level was negatively correlated with inflammatory cytokines IL-17a (r = -0.819;P = 0.004), IL-1a (r = -0.716;P = 0.0.02), IL-1b (r = -0.678;P = 0.031), IL-2 (r = -0.765;P = 0.01), and IL-8 (r = -0.705;P = 0.023). However, in the PCOS group, AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines. Berberine significantly reduced the expression level of mTOR mRNA (P = 0.001), and increased the expression level of IRS-1 mRNA (P = 0.009) in the PCOS granule cells.ConclusionIn this study, we find that the elevated levels of serum inflammatory factors IL-17a, IL-1Ra, and IL-6 cause women to be in a subclinical inflammatory state for a long time. Abnormal changes in inflammatory factors alter their original negative correlations with AMH levels, thereby weakening the metabolism of glycolipids, promoting insulin resistance, destroying the normal ovulation and fertilization system of women, leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation. Berberine can improve the sensitivity of insulin by regulating the signal pathway of insulin receptor substrate-1 (IRS-1) and mammalian target of rapamycin (mTOR) in PCOS patients and achieve a therapeutic effect of treating PCOS.

MiRNA-155 regulates cumulus cells function, oocyte maturation, and blastocyst formation.

Numerous oocytes are retrieved during in vitro fertilization from patients with polycystic ovary syndrome (PCOS). The poor quality of these oocytes leads to lower fertilization and decreases in cleavage and implantation. MiR-155 is one of the microRNA (miRNA) that is increased in serum and granulosa cells of PCOS patients. In this study, we investigate the effects of miR-155 expression and its target genes on oocyte maturation and embryo development. We used the calcium phosphate protocol to transfect vectors that contained miR-155 or miR-off 155 and alone eGFP into cumulus oophorus complex (COCs) of B6D2F1 female mice for in vitro maturation. Cumulus expansion, nuclear, and cytoplasmic maturation, as well as cleavage rates were determined in groups transfected and compared with the control groups. Quantitative real-time polymerase chain reaction was performed to analyze expression levels of miR-155 and the target genes in the cumulus cells, oocytes, and blastocysts. MiR-155 overexpression in COCs suppressed cumulus expansion, oocyte maturation, and inhibition of endogenous miR-155 by miR-off 155 improved cumulus expansion and oocyte maturation by downregulation and expression increase of the Smad2 and Bcl2 genes. On the other hand, overexpression and downregulation of miR-155 in the COCs led to increase and decrease in cleavage rates by changes in expressions of the Mecp2, Jarid2, and Notch1 genes, respectively (P<0.05). These results suggested that miR-155 overexpression in granulosa cells of PCOS patients can negatively affect nuclear and cytoplasmic maturation, but this miRNA expression has a positive impact on embryo development.

HMGB1-induced aberrant autophagy contributes to insulin resistance in granulosa cells in PCOS.

Insulin resistance (IR) disrupts ovarian functions in polycystic ovary syndrome (PCOS). The contributing factors remains elusive. High mobility group box 1 (HMGB1), a damage-associated molecular pattern molecule, has been shown to be related to IR and autophagy, respectively, in peripheral tissues. Here, we investigated whether increased HMGB1 contributes to IR in granulosa cells of PCOS patients via induction of aberrant autophagy. Results showed that HMGB1 abundance in the follicular fluid was significantly increased with enhanced autophagy in granulosa cells in PCOS patients with IR. HMGB1 exacerbated autophagy in granulosa cells as evinced by increased LC3B II/I ratio and ATG7 as well as decreased p62, the markers for autophagy. Concurrently, HMGB1 impaired insulin sensitivities by attenuating the abundance of insulin receptor substrate-1, Akt phosphorylation, GLUT4 translocation, and glucose uptake in granulosa cells, which were reversed by blocking autophagy pathways with siRNA-mediated knockdown of ATG7 or with chloroquine and bafilomycin A1, the lysosome inhibitors. In conclusion, our results indicate that increased HMGB1 contributes to IR development in granulosa cells of PCOS patients, which is associated with exacerbation of autophagy by HMGB1. Control of HMGB1 production may be benefical for the improvement of insulin sensitivity in granulosa cells in PCOS.

Melatonin ameliorates excessive PINK1/Parkin-mediated mitophagy by enhancing SIRT1 expression in granulosa cells of PCOS.

Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against mitochondrial injury in the granulosa cells of PCOS remain unclear. In this study, decreased mitochondrial membrane potential and mtDNA content, increased number of autophagosomes were found in the granulosa cells of PCOS patients and the dihydrotestosterone (DHT)-treated KGN cells, with decreased protein level of the autophagy substrate p62 and increased levels of the cellular autophagy markers Beclin 1 and LC3B-II, while the protein levels of PTEN-induced kinase-1 (PINK1) and Parkin were increased and the level of sirtuin 1 (SIRT1) was decreased. DHT-induced PCOS-like mice also showed enhanced mitophagy and decreased SIRT1 mRNA expression. Melatonin treatment significantly increased the protein level of SIRT1 and decreased the levels of PINK1/Parkin, whereas it ameliorated the mitochondrial dysfunction and PCOS phenotype in vitro and in vivo. However, when the KGN cells were treated with SIRT1 siRNA to knock down SIRT1 expression, melatonin treatment failed to repress the excessive mitophagy. In conclusion, melatonin protects against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression to inhibit excessive PINK1/Parkin-mediated mitophagy.