Circular RNAs: Emerging Modulators in the Pathophysiology of Polycystic Ovary Syndrome and their Clinical Implications.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine/metabolic disorder in women of reproductive age. PCOS is characterized by hyperandrogenism, polycystic ovary morphology, and ovulatory dysfunction/anovulation. It involves multiple effects in patients, including granulosa/theca cell hyperplasia, menstrual disturbances, infertility, acne, obesity, insulin resistance, and cardiovascular disorders. Biochemical analyses and the results of RNA sequencing studies in recent years have shown a type of non-coding RNAs as a splicing product known as circular RNAs (circRNAs). Several biological functions have been identified in relation to circRNAs, including a role in miRNA sponge, protein sequestration, increased parental gene expression, and translation leading to polypeptides. These circular molecules are more plentiful and specialized than other types of RNAs. For this reason, they are referred to as potential biomarkers in different diseases. Evidence suggests that circRNAs may have regulatory potentials through different signaling pathways, such as the miRNA network. Probably most experts in the field of obstetricians are not aware of circRNAs as a useful biomarker. Therefore, this review focused on the researches that have been done on the involvement of circRNAs in PCOS and summarized recent supportive evidence, and evaluated the circRNA association and mechanisms involved in PCOS.

The Association Between Increment of Interleukin-1 and Interleukin-6 in Women with Polycystic Ovary Syndrome and Body Mass Index.

Background: There is an association between inflammatory factors and polycystic ovary syndrome (PCOS) and most of women with PCOS experience the symptoms of hirsutism. The purpose of this study was to evaluate the role of obesity in PCOS occurrence, which is linked with inflammation and hirsutism. Methods: This study was designed as a case-control research. It was performed on 102 women with PCOS and 102 healthy women as controls who were age-matched. Serum concentrations of testosterone, estradiol (E2), IL-1, IL-6, high-sensitivity c-reactive protein (hs-CRP), and aromatase activity were measured in blood samples. Statistical tests including unpaired t-tests, Mann-Whitney U test, Kruskal-Wallis, Spearman's correlation, and Chi-square tests were used for data analysis. Statistical significance was set at p<0.05. Results: A significant difference was found between hs-CRP, IL-1, and IL-6 in PCOS patients and healthy individuals (p<0.001). Aromatase activity was markedly lower in PCOS cases. The serum level of IL-1 (p=0.392) and IL-6 (p=0.764) was not different between overweight and normal weight women. In both studied groups (case and control), hirsutism frequency was markedly higher in individuals with BMI ≥25 kg/m 2 (p<0.05). Inflammatory factors significantly affected the PCOS group (p<0.05). However, logistic regression showed that hs-CRP increment is more effective on increasing the risk of PCOS (OR: 6.324, p<0.001). Conclusion: In this study, hs-CRP, IL-1, and IL-6 levels increased in all PCOS women. Although the incidence of hirsutism in PCOS is associated with obesity, in PCOS pathogenesis, only IL-1 and IL-6 were independent of BMI.

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

PURPOSE: 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. METHODS: 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. RESULTS: PCOS patients showed higher ROS level, decreased TAC, 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 were found to be significantly 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. CONCLUSION: Declined energy due to the mitochondrial dysfunction and restrained glycolysis in GCs is associated with the immature oocytes and lower biochemical pregnancy in PCOS.

The Association of Mitochondrial Translocator Protein and Voltage-Dependent Anion Channel-1 in Granulosa Cells with Estradiol Levels and Presence of Immature Follicles in Polycystic Ovary Syndrome.

Background: Granulosa cells (GCs) play key roles in oocyte maturation by providing required estradiol (E2). Since the presence of immature oocytes has been reported in cases with polycystic ovary syndrome (PCOS), in this study, the levels of mitochondrial membrane transporter proteins involved in E2 synthesis were determined. E2 concentration and parameters of oxidative status were also measured in follicular fluids of PCOS women. Methods: Forty-three women with PCOS and 43 healthy women who were candidates for IVF procedure due to their husbands' infertility were enrolled in this case-control study. The gene expression and protein levels of mitochondrial translocator protein (TSPO) and voltage-dependent anion channel 1 (VDAC1) were determined in GCs using RT-qPCR and immunocytochemistry assay, respectively. E2 level was measured with electrochemiluminescence, whereas total cholesterol, total antioxidant capacity (TAC), total oxidant status (TOS), and malondialdehyde (MDA) were determined using colorimetric methods in follicular fluids. Data were analyzed using unpaired t-test or Mann-Whitney U test, and Spearman's correlation coefficient. Results: VDAC1 and TSPO were significantly lower in mRNA (p<0.05) and protein levels (p<0.001) of PCOS patients. PCOS patients had lower cholesterol, estradiol, and TAC levels, and higher TOS and MDA contents. E2 level had direct correlation with VDAC1, TSPO, and TAC while it was negatively correlated with TOS, oxidative stress index (OSI), and MDA (p<0.001). Higher E2 levels were associated with higher numbers of high-quality oocytes and conceived embryos (p<0.001). Conclusion: Decreased E2 levels and increased oxidative stress in the follicular fluid may be the cause of immature oocytes in PCOS cases.

Association of Prx4, Total Oxidant Status, and Inflammatory Factors with Insulin Resistance in Polycystic Ovary Syndrome.

INTRODUCTION: Chronic inflammation and oxidative stress conditions have been reported in women with polycystic ovary syndrome (PCOS). Peroxiredoxin 4 (Prx4) is a related antioxidant in insulin synthesis. We hypothesized that insulin resistance in these women is associated with total oxidant status (TOS) and inflammatory factors. MATERIALS AND METHODS: Two hundred three people including 104 PCOS patients and 99 healthy women, who were matched for age and body mass index (BMI), entered the study. Waist circumference of the participants was measured; serum glucose, lipid profile, insulin, Prx4, TOS, hs-CRP, and TNF-α were also evaluated. RESULTS: The Prx4 level was significantly lower in PCOS than in the control group. In addition, marked increase was observed in the concentration of TOS, hs-CRP, and TNF-α in PCOS, compared to the healthy women. There was a positive correlation of TOS with hs-CRP, TNF-α, and HOMA-IR. The risk of PCOS for subjects with high hs-CRP was 60 times greater than those who had low serum hs-CRP concentration; after performing multiple logistic regression analyses with the backward method, TNF-α was considered as an effective biomarker to predict PCOS ß = 49.087 (all p < 0.05). CONCLUSION: This study identified increased oxidative stress and inflammation in PCOS; this may be due to decrease in the antioxidants, such as Prx4.

Association of glutamate cystein ligase (GCL) activity Peroxiredoxin 4 (prxR4) and apelin levels in women with preeclampsia.

INTRODUCTION: Preeclampsia is a common disease of pregnancy that is characterized by symptoms such as high blood pressure and proteinuria. Peroxiredoxin 4 (Prx4), is a protein with antioxidant properties which is produced in placenta and protects it from antioxidant stress and recurrent miscarriage. For regeneration of Peroxiredoxin 4 need to glutathione (GSH) and Glutamate-cysteine ligase (GCL) enzyme controls the pathway of glutathione regeneration. Apelin is a paired internal ligand with a G protein coupled receptor and is associated with angiotensin receptor (AT1) as a blood pressure regulator. This study was designed to evaluate GCL enzyme activity and Peroxiredoxin 4, glutathione and apelin levels in serum of women with preeclampsia. MATERIAL AND METHODS: Thirty pregnant women with preeclampsia and 30 healthy pregnant women were enrolled in this study. All participants were diagnosed by clinical examination and confirmation by Obstetrician-Gynecologist. The GCL enzyme activity and concentration of Prx4 and apelin in serum samples were measured using ready-to-use non-competitive ELISA methods while glutathione level was determined using Ellman's reagent. RESULTS: The GCL enzyme activity and Prx4 level were significantly lower in preeclampsia compared with control group (p < 0.05). In addition, marked reductions were observed in the concentrations of glutathione and apelin in preeclampsia compared to the healthy pregnant women (p < 0.05). CONCLUSION: This study identified the role of the GCL and Prx4 system in preeclampsia disorder and may be one of the ways to prevent and reduce the risks of preeclampsia in high-risk women using diet control and stress reduction.

Correlation of thioredoxin reductase (TrxR) and nitric oxide synthase (NOS) activities with serum trace elements in preeclampsia.

BACKGROUND: Preeclampsia is a dangerous disease of pregnancy with symptoms such as high blood pressure and proteinuria. The nitric oxide synthase (NOS) enzyme produces nitric oxide (NO) as a mediator of vasodilation and need to calcium and zinc ions for activity. The thioredoxin reductase (TrxR) as an enzyme containing selenium has been neutralized the damaging effects of oxidants. In this study, our aim was to evaluate the activity of eNOS and TrxR enzymes and the amounts of calcium, zinc, and selenium elements in serum of women with preeclampsia. MATERIAL AND METHODS: Thirty preeclampsia and 30 healthy pregnant women were enrolled in the study after clinical examination and confirmation by Obstetrician-Gynecologist. Venous blood samples were collected and the activity of NOS, TrxR enzymes, and the concentration of zinc, calcium, and selenium elements were measured in serum. RESULTS: The results of NOS and TrxR activities showed significant decreases in preeclampsia compared with control group (p < .05). In addition, concentrations of zinc, calcium, and selenium were markedly lower in preeclampsia compared to the healthy control group (p < .05). CONCLUSION: This study identified the role of the NOS and TrxR activities in preeclampsia disorder and may be one of the ways to prevent and reduce the risks of preeclampsia in high-risk women using diet control and stress reduction.

Evaluation of methylenetetrahydrofolate reductase (MTHFR) activity and the levels of homocysteine and malondialdehyde (MDA) in the serum of women with preeclampsia.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) has a key role in converting homocysteine to methionine so its deficiency might cause homocysteine accumulation and arterial destruction. The aim of this study was to evaluate the activity of methylenetetrahydrofolate reductase (MTHFR), homocysteine, and MDA levels in preeclampsia patients. MATERIAL AND METHODS: In this case-control study, 30 preeclampsia patients and 30 healthy pregnant women as control group were selected and matched for maternal age, gestational age, and BMI. Preeclampsia was confirmed by examining hypertension and urinary proteinuria. Serum homocysteine and MDA level as well as MTHFR activity were measured manually. RESULTS: Homocysteine and MDA levels were significantly different between women with preeclampsia and normal pregnancies (p-value <0.001). Also, the results indicate that MTHFR activity in the two groups was statistically different (p-value <0.001). CONCLUSION: It can be concluded that the regulation of homocysteine and its metabolic enzymes plays a significant role in controlling blood pressure and consequently in the pathophysiology of Preeclampsia and it can be a goal for prevention and treatments.

Preliminary study showing no association between G238A (rs361525) tumor necrosis factor-α (TNF-α) gene polymorphism and its serum level, hormonal and biochemical aspects of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the main cause of female infertility. Interactions among genetic, biochemical, and immunological factors can affect the pathogenesis of PCOS. As a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) plays an important role in this regard. The present study aimed to evaluate the association of the rs361525 gene single-nucleotide polymorphism (SNP) and TNF-α serum levels with the hormonal and biochemical characteristics of PCOS in Iranian individuals. METHODS: The SNP rs361525 in the TNF-α gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a total of 111 PCOS patients and 105 healthy females. Serum levels of TNF-α, lipid and hormone profiles, and biochemical factors were measured using enzyme-linked immunosorbent assay (ELISA) and calorimetric methods, as appropriate. RESULTS: The TNF-α serum level was higher in women with PCOS compared with the control group (p <  0.0001), and it was significantly correlated with the homeostasis model assessment (HOMA) factor (r = 0.138, p <  0.05). No significant differences were found in the genotype and allelic frequencies between the two groups (p >  0.05). Higher levels and significant differences were found for the HOMA factor, luteinizing hormone/follicle-stimulating hormone (LH/FSH), testosterone, and body mass index (BMI) in the PCOS group compared with the control group (p <  0.0001). High LH/FSH ratios (odds ratio [OR] = 1.98, 95% confidence interval [CI] = 1.20-3.28, p <  0.01), and high HOMA factor (OR = 5.04, 95% CI = 2.82-9.01, p <  0.001) were significantly associated with an increased risk of PCOS. CONCLUSIONS: Despite the lack of significant difference between rs361525 polymorphism of the TNF-α gene and PCOS, the serum level of TNF-α was increased in PCOS patients and positively correlated with the HOMA factor. Elevation of the LH/FSH ratio and HOMA for insulin resistance (HOMA-IR) increased the risk of PCOS. Therefore, TNF-α could indirectly contribute to PCOS progression.

Ameliorating Effect of Ginseng on Epididymo-Orchitis Inducing Alterations in Sperm Quality and Spermatogenic Cells Apoptosis following Infection by Uropathogenic Escherichia coli in Rats.

OBJECTIVE: Epididymo-orchitis (EO) potentially results in reduced fertility in up to 60% of affected patients. The anti-inflammatory effects of Korean red ginseng (KRG) and its ability to act as an immunoenhancer in parallel with the beneficial effects of this ancient herbal medicine on the reproductive systems of animals and humans led us to evaluate its protective effects against acute EO. MATERIALS AND METHODS: This animal experimental study was conducted in the Department of Anatomical Sciences, Faculty of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran during 2013-2015. We divided 50 Wistar rats into five following groups (n=10 per group): i. Control-intact animals, ii. Vehicle-phosphate buffered saline (PBS) injection into the vas deferens, iii. KRG-an intraperitoneal (IP) injection of KRG, iv. EO-an injection of uropathogenic Escherichia coli (UPEC) strain M39 into the vas defer- ens, and v. EO/ KRG-injections of both UPEC strain M39 and KRG. The treatment lasted seven days. We then evaluated sperm parameters, number of germ cell layers, Johnson's criteria, germ cell apoptosis, body weight and relative sex organs weight. RESULTS: Acute EO increased the relative weight of prostate and seminal vesicles (P≤0.05). It also reduced sperm quality such as total motility, sperm concentration (P≤0.01), and the percentage of normal sperm (P≤0.001). Moreover, acute EO decreased Miller's (P≤0.05) and Johnsen's scores and increased apoptotic indexes of spermatogenic cells (P≤0.001). KRG treatment decreased prostate weight gain (P≤0.05) and improved the percentage of sperm with normal morphology, total motility (P≤0.01), and progressive motility (P≤0.05). The apoptotic indexes of spermatogenic cells reduced (P≤0.001), whereas both Johnsen's (P≤0.01) and Miller's criteria increased in the KRG-treated EO testis (P≤0.05). CONCLUSION: Consequently, KRG ameliorated the devastating effects of EO on the sperm retrieved from either epididymis or testicle in rats.

Association between Hypoadiponectinemia and Low Serum Concentrations of Calcium and Vitamin D in Women with Polycystic Ovary Syndrome.

Objective. To investigate the possible association of calcium and vitamin D deficiency with hypoadiponectinemia in women with PCOS. Subjects and Methods. In this case-control study, 103 PCOS cases and 103 controls included. The concentrations of calcium, 25-OH-vitamin D (25OHD), adiponectin, insulin, glucose, total cholesterol, HDL-cholesterol, triglyceride (TG), and androgens were measured in fasting blood samples. Results. Adiponectin (8.4 ± 2.7 ng/mL versus 13.6 ± 5 ng/mL in control group, P : 0.00), calcium (2 ± 0.1 mmol/L versus 2.55 ± 0.17 mmol/L in controls, P : 0.00), and 25-OH-Vit D (30 ± 2.99 nmol/L versus 43.7 ± 5.2 nmol/L in control group, P : 0.00) levels were decreased in women with PCOS. Subjects with PCOS had higher concentrations of TG (1.4 ± 0.77 mmol/L versus 1.18 ± 0.75 mmol/L in control group, P : 0.019) and dehydroepiandrosterone sulfate (DHEA-S) (10.7 ± 11 mmol/L versus 9.7 ± 10.4, P : 0.02 in control group). There were significant correlations between adiponectin concentrations with calcium (r : 0.78, P : 0.00) and 25OHD levels (r : 0.82, P : 0.00). The association of hypoadiponectinemia and PCOS was not significant considering 25OHD as a confounding factor. Conclusion. The present findings indicate that the association of hypoadiponectinemia with PCOS is dependent on vitamin D. A possible beneficiary effect of vitamin D on the metabolic parameters in PCOS may be suggested.

Serum magnesium concentrations in polycystic ovary syndrome and its association with insulin resistance.

OBJECTIVE: It has been revealed that low serum magnesium (Mg) is often associated with insulin resistance (IR), cardiovascular problems, diabetes mellitus, and hypertension. Patients with polycystic ovary syndrome (PCOS) are known to have a high incidence of insulin resistance. This study was designed to determine whether women with PCOS exhibit serum magnesium deficiency and its potential association with IR. SUBJECTS AND METHODS: In this cross-sectional study, 103 cases with PCOS and 103 normal women who were matched for their age and body mass index (BMI) were included. Blood samples were collected after an overnight fast and concentrations of calcium, magnesium, testosterone, dehydroepianderosterone sulfate, insulin, fasting plasma glucose (FPG), triglyceride, total cholesterol, and HDL-cholesterol were measured. RESULTS: The risk of PCOS for subjects with Mg deficiency was 19 times greater than those who had normal serum Mg concentrations (p ≤ 0.0001). No correlation was found between Mg and insulin sensitivity or secretion, FPG, dyslipidemias, and also androgen concentrations. After adjustment for calcium concentration the role of magnesium to predict PCOS attenuated and became non-significant (ß:-1.9, p: 0.7). CONCLUSION: The present study provides the first evidence showing that magnesium deficiency is not associated with IR in PCOS. According the evidences of this study, serum calcium concentration is more potent predictor of PCOS than serum Mg and only calcium, not Mg, is related to insulin resistance in PCOS.

Multiple correlations between cord blood leptin concentration and indices of neonatal growth.

BACKGROUND AND AIMS: The discovery of a role for leptin in controlling fetal and neonatal growth suggests a fetal origin of some adult chronic diseases and has stimulated research into the mechanisms of action of leptin early in life. The aim of this study was to determine umbilical cord blood leptin levels and to evaluate their association with newborn growth indices. METHODS: Two hundred healthy newborns (89 males, 110 females, and one of undetermined gender; gestational ages ranging from 34-43 weeks) and their healthy mothers were enrolled in this study conducted at Moovsavi Hospital in Zanjan, Iran. The body size index of each newborn was determined in terms of birth weight, birth length, head circumference, body mass index (BMI) and ponderal index. Umbilical cord blood leptin levels were measured by ELISA. RESULTS: Umbilical cord leptin concentration was found to positively correlate with birth weight (r=0.322; p<0.0001), neonatal BMI (r=0.247; p<0.0001), ponderal index (r=0.206; p=0.04), and gestational age (r=0.221; p=0.002). There was no significant correlation between cord leptin and birth length or umbilical glucose concentration. Umbilical cord leptin concentrations (15.20+/-12.3 vs. 12.08+/-11.7; p=0.01) were significantly greater in female as compared to male newborns, respectively. Linear regression analysis indicated that umbilical cord leptin levels correlated with birth weight, umbilical triglyceride concentration, neonatal gender, and method of delivery. CONCLUSIONS: Our findings confirm the association of leptin concentrations with weight gain in fetal and newborn infants.

Decreased adiponectin levels in polycystic ovary syndrome, independent of body mass index.

BACKGROUND: Insulin resistance has been shown to have an association with polycystic ovary syndrome (PCOS). This study was designed to evaluate the potential role of adiponectin, which is linked with insulin resistance, in the etiology of PCOS and its relationship to obesity. METHODS: This case-control study consisted of 103 newly diagnosed PCOS cases and 73 female controls seen at a referral university hospital in Zanjan, Iran. Serum adiponectin, insulin, plasma fasting glucose, and lipid levels were measured. The homeostasis model assessment index was used to determine the level of insulin resistance. Women were classified as follows: Group I (normal nonlean women); group II (normal lean women); group III (nonlean women with PCOS); and group IV (lean women with PCOS) RESULTS: Adiponectin levels were decreased in women with PCOS (8.4 +/- 2.7 ng/mL vs. 13.6 +/- 5 ng/mL in the control group, P < 0.001). There was no significant difference between the adiponectin concentrations of women in group III and that in group IV (8.1 +/- 2.8 ng/mL vs. 9.2 +/- 2.6 ng/mL, respectively, P = 0.1). Adiponectin levels were significantly lower in group I compared with group II. A weak but significant negative correlation was found between adiponectin and insulin levels in all the subjects. Multiple regression analyses showed that the presence of PCOS was the only significant determinant of serum adiponectin levels. CONCLUSIONS: Adiponectin levels were reduced in all the women with PCOS. There seemed to be an interaction between adiponectin and PCOS pathogenesis that was independent of body mass index.