Vascular imbalance in polycystic ovary syndrome: Insights into endothelial dysfunction.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder among women of reproductive age, characterized by hyperandrogenism, menstrual irregularities, and polycystic ovarian morphology [ 1 ]. The condition affects approximately 8% to 13% of women globally, with significant implications for metabolic health, reproductive function, and psychological well-being [ 2 ]. Although the exact etiology of PCOS remains unknown, it is believed to result from a complex interplay of genetic predisposition, environmental determinants, and lifestyle factors [ 3 – 5 ]. PCOS is associated with metabolic complications such as insulin resistance, obesity, type 2 diabetes, and an increased risk of cardiovascular diseases [ 6 – 8 ]. The widely adopted Rotterdam Criteria for PCOS diagnosis encompass the presence of hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology confirmed by ultrasound [ 9 ]. Management of PCOS involves lifestyle modifications such as diet and exercise, along with pharmacological interventions. Common treatment options include oral contraceptives, insulin sensitizers, and anti-androgen medications. Current research is focused on understanding the long-term effects of PCOS and developing personalized therapeutic approaches [ 10 , 11 ]. Nitric oxide (NO) is a critical signaling molecule involved in numerous biological functions, including vasodilation, neurotransmission, and immune response. NO is implicated in the regulation of ovarian function and the modulation of insulin sensitivity [ 12 ]. In recent decades, considerable focus has been placed on the involvement of NO in regulating key reproductive processes, such as follicular development, hormone production, granulosa cell apoptosis in atretic follicles, and changes in oocyte maturation potential. Additionally, NO is thought to influence the activity of cyclooxygenases (COX-1, COX-2) and prostaglandin synthesis essential for ovulation. The ovaries have the ability to produce NO, which may play a crucial role in ovarian steroidogenesis, ovulation, and corpus luteum regression [ 13 , 14 ]. Originally identified from bovine stomach extracts as the natural ligand for the orphan APJ receptor, a member of the G-protein-coupled receptor family, apelin is expressed in ovarian tissues, such as granulosa and theca cells, and is considered to play a significant role in regulating ovarian function [ 15 , 16 ]. Apelin exhibits both vasodilatory and vasoconstrictive effects, depending on the physiological context, as its interaction with APJ receptors varies between vascular smooth muscle cells (VSMCs) and endothelial cells, while it generally promotes NO-mediated vasodilation [ 17 ], it can induce vasoconstriction in the absence of endothelial influence [ 18 ]. Noradrenaline is a fundamental neurotransmitter and hormone that regulates various physiological processes, particularly within the cardiovascular system. It plays a pivotal role in modulating vascular tone by mediating vasoconstriction. The interplay between noradrenaline and other signaling molecules, such as NO and prostacyclin, is critical for maintaining vascular homeostasis and adapting to physiological demands [ 19 , 20 ]. The regulation of vascular tone by noradrenaline is influenced by the balance between vasoconstrictors and vasodilators [ 20 ]. Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation, playing a crucial role in maintaining vascular homeostasis. Its synthesis occurs primarily in endothelial cells, where it is derived from arachidonic acid through the cyclooxygenase (COX) pathway [ 21 ]. Changes in the morphology of periovarian adipose tissue, which plays a crucial role in ovarian function by secreting bioactive molecules such as adipokines, cytokines, NO, and prostacyclin to support vascular homeostasis and regulate vascular tension, have been observed in conditions such as menopause and PCOS [ 22 ]. Dysregulation of these vasoactive molecules has also been implicated in various reproductive and vascular disorders. For instance, altered NO signaling is associated with abnormal angiogenesis in endometriosis and ovarian hyperstimulation syndrome [ 14 , 23 ]. Elevated apelin levels have been reported in obese and PCOS-related follicular arrest [ 24 ], while increased noradrenaline activity contributes to sympathetic overactivity and disrupted ovarian function in PCOS [ 25 ]. Given the well-established vasodilatory and anti-thrombotic functions of prostacyclin, and its protective role in endothelial homeostasis as observed in pulmonary hypertension [ 26 ], it is plausible that reduced prostacyclin bioavailability may also contribute to endothelial dysfunction in PCOS, which is characterized by impaired endothelium-dependent vasodilation and insulin resistance [ 27 ]. These findings suggest that these molecules not only support physiological processes but also participate in pathophysiological mechanisms relevant to ovarian and metabolic diseases. These four biomarkers were selected due to their complementary roles in vascular tone regulation, including endothelial relaxation (NO and prostacyclin), vasoconstriction (noradrenaline), and angiogenic signaling (apelin), all of which are known to be altered in PCOS. Given that PCOS is characterized by endothelial dysfunction, chronic low-grade inflammation, and insulin resistance, simultaneous evaluation of these markers allows for a more integrative understanding of the pathophysiological disturbances underlying vascular dysregulation in affected individuals. Although the physiological roles of NO, apelin, noradrenaline, and prostacyclin have been individually investigated, no study to date has systematically examined their combined contribution to vascular tone imbalance in PCOS. Furthermore, their collective diagnostic value in distinguishing PCOS patients from healthy individuals remains largely unexplored. This study addresses this gap by providing an integrative evaluation of these markers, thereby offering a more comprehensive approach to understanding endothelial dysfunction in PCOS. We hypothesize that the characteristic hormonal and metabolic alterations in PCOS -including hyperandrogenism, insulin resistance, and chronic inflammation- may disrupt the balance between these vasodilatory and vasoconstrictive factors, contributing to endothelial dysfunction and cardiovascular risk. This study aims to comprehensively explore the relationship between serum NO, apelin, noradrenaline, and prostacyclin levels with vascular tone regulation and dysfunction in women with PCOS and to delineate their potential contributions to the pathophysiological mechanisms underlying PCOS.

Forty-four female patients with PCOS and 44 female healthy controls were included in the study. No significant difference was observed between the PCOS and control groups in terms of marital status (p = 0.243) ( Table 1 ). The mean ages of PCOS patients and control subjects were 29.23 ± 4.2 and 29.5 ± 6.82, respectively. No significant difference was observed between the PCOS and control groups in terms of age (p = 0.822) ( Table 1 ). The mean BMI value of PCOS patients and control subjects were 22.54 ± 2.06 and 21.91 ± 2.01, respectively. No significant difference was observed between the PCOS and control groups in terms of BMI (p = 0.151) ( Table 1 ). n, frequencies; %, percent; PCOS, polycystic ovary syndrome; SD, standard deviation; BMI, body mass index; p < 0.05, there is a statistical difference between the groups. Within the study, no statistically significant differences were observed between the PCOS and control groups in terms of estradiol, TSH, prolactin, β-hCG, DHESO 4 , insulin, HOMA-IR, HbA1c, triglyceride, total cholesterol, and LDL levels (p > 0.05) ( Table 2 ). However, FSH, LH, testosterone, SHBG, glucose, and HDL levels were found to be statistically significant higher in the PCOS group compared to the control group (p < 0.05) ( Table 2 ). PCOS, polycystic ovary syndrome; SD, standard deviation; t, independent t-test; FSH, follicle-stimulating hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone, β-hCG, human chorionic gonadotropin; SHBG, sex hormone-binding globulin; DHESO 4 , dehydroepiandrosterone sulfate; HOMA-IR, homeostasis model assessment for insulin resistance; HbA1c, glycated hemoglobin; LDL, low-density lipoprotein; HDL, high-density lipoprotein; p < 0.05, there is a statistical difference between the groups (Bold values). The mean NO levels of PCOS patients and control subjects were 104.35 ± 44.96 and 83.85 ± 22.65, respectively. NO levels were found to be significantly higher in the PCOS group compared to the control group (p = 0.008) ( Table 3 ). The mean apelin levels of PCOS patients and control subjects were 379.57 ± 40.11 and 190.88 ± 16.44, respectively. Apelin levels were found to be significantly higher in the PCOS group compared to the control group (p = 0.0001) ( Table 3 ). The mean noradrenaline levels of PCOS patients and control subjects were 27.48 ± 5.36 and 24.63 ± 4.59, respectively. Noradrenaline levels were found to be significantly higher in the PCOS group compared to the control group (p = 0.009) ( Table 3 ). The mean prostacyclin levels of PCOS patients and control subjects were 5.85 ± 1.28 and 6.78 ± 1.99, respectively. Prostacyclin levels were found to be significantly lower in the PCOS group compared to the control group (p = 0.011) ( Table 3 ). PCOS, polycystic ovary syndrome; NO, nitric oxide; SD, standard deviation; t, independent t-test; p < 0.05, there is a statistical difference between the groups (Bold values). According to binary logistic regression models, the model established with NO, apelin, noradrenaline, and prostacyclin variables was found to be statistically sufficient for predicting the data groups (χ 2  = 12.931, df = 8, p = 0.114; χ 2  = 8.492, df = 7, p = 0.291; χ 2  = 14.079, df = 8, p = 0.080; χ 2  = 4.76, df = 8, p = 0.783, respectively). It was found that the measurement values of NO, apelin, noradrenaline, and prostacyclin had a statistically significant effect in predicting the difference between the PCOS and control groups (p = 0.012, p = 0.0001, p = 0.019, p = 0.007, respectively) ( Table 4 ). The difference between the study and control groups is 1.017 times more affected by the NO value (OR = 1.017, 95% CI 1.004–1.031). An increase of 1 unit in the NO value reduces the disease risk by 1.017 times ( Table 4 ). The difference between the study and control groups is 1.044 times more affected by the apelin value (OR = 1.044, 95% CI 1.025–1.064). An increase of 1 unit in the apelin value reduces the disease risk by 1.044 times ( Table 4 ). The difference between the study and control groups is 1.115 times more affected by the noradrenaline value (OR = 1.115, 95% CI 1.018–1.221). An increase of 1 unit in the noradrenaline value reduces the disease risk by 1.115 times ( Table 4 ). The difference between the study and control groups is 0.658 times more affected by the prostacyclin value (OR = 0.658, 95% CI 0.486–0.891). An increase of 1 unit in the apelin value reduces the disease risk by 0.658 times ( Table 4 ). NO, nitric oxide; β, parameter estimate; SE, standard error; W, Wald statistic; df, degrees of freedom; Exp (β), odds ratio; 95% CI, confidence interval; p < 0.05, there is a statistical difference (Bold values). When the cut-off value for NO was set at 44.17 µmol/L, it demonstrated 97.7% sensitivity and 93.2% specificity in detecting PCOS (AUC = 0.536, 95% CI = 0.404–0.667, p = 0.046). When the cut-off value for apelin was set at 161.34 ng/L, it demonstrated 98.7% sensitivity and 95.5% specificity in detecting PCOS (AUC = 0.983, 95% CI = 0.935–0.991, p = 0.001). When the cut-off value for noradrenaline was set at 18.19 ng/L, it demonstrated 93.2% sensitivity and 95.5% specificity in detecting PCOS (AUC = 0.659, 95% CI = 0.542–0.775, p = 0.010). When the cut-off value for prostacyclin was set at 4.23 ng/L, it demonstrated 88.6% sensitivity and 95.5% specificity in detecting PCOS (AUC = 0.351, 95% CI = 0.236–0,466, p = 0.016) ( Fig 1 ).

The increased levels of vasodilators such as NO and apelin in PCOS patients contribute to enhanced vascular relaxation, while the elevated noradrenaline levels and reduced prostacyclin levels promote vasoconstriction. The simultaneous rise in apelin, NO, and noradrenaline may create an imbalance between abnormal vessel relaxation and constriction, leading to dysregulation of vascular tone and impaired vascular responses. Additionally, the core features of PCOS, including irregular cycles, polycystic ovary morphology, increased adiposity, insulin resistance, hormonal imbalances, and chronic inflammation, further complicate the production and effects of these molecules. These molecular alterations in PCOS can disrupt proper vascular function and potentially contribute to the development of cardiovascular issues. Further investigation into the interactions between these molecules is essential to gain a deeper understanding of the underlying mechanisms and to develop targeted therapeutic strategies. The novel contribution of this study lies in its integrative evaluation of endothelial markers and the identification of apelin as a highly sensitive and specific biomarker for distinguishing PCOS patients from healthy individuals. This highlights a potential avenue for non-invasive diagnostic strategies in PCOS management.

This study was conducted between August 2022 and November 2022 at the Department of Obstetrics and Gynecology Faculty of Medicine Malatya Turgut Özal University, Malatya, Turkey. The patient and control groups in the study were composed of volunteers who applied to Faculty of Medicine Malatya Turgut Özal University. At the beginning of the study, the individuals’ medical histories were taken. Demographic and anthropometric data, including marital status, age, weight, height, and body mass index (BMI), were recorded and ultrasound evaluation (Samsung RS85 Prestige, Gangwon-do, Republic of Korea) was performed. Forty-four individuals aged 18–40 years diagnosed with PCOS according to Rotterdam Criteria were included in the study [ 28 ]. According to the 2003 Rotterdam Criteria, a diagnosis of PCOS is confirmed when at least two of the following conditions are present: (1) oligomenorrhea or amenorrhea, (2) clinical or biochemical signs of hyperandrogenemia, and (3) polycystic ovarian morphology detected through ultrasonography [ 29 ]. Individuals who were pregnant or breastfeeding, or those with irregular menstrual cycles, androgen excess conditions (such as Cushing’s syndrome), gestational diabetes mellitus, arterial disease, hypertension, congestive heart failure, chronic liver or kidney failure, type 1 or type 2 diabetes, and hyperlipidemia were excluded from the study. Forty-four individuals of the same ethnic origin, aged between 18 and 40 years, with similar demographic characteristics, who met the exclusion criteria, had no diseases, had regular menstrual cycles, and had normal ovarian morphology on ultrasonography, were included in the control group. To minimize hormonal variability, blood samples from cycling control participants were collected during the early follicular phase (days 2–5) of the menstrual cycle. Written informed consent was obtained from all volunteers in the study. The study was conducted according to the Declaration of Helsinki. The study was approved by the Ethics Committee of Malatya Turgut Özal University, Turkey (Number: 2022/35, Date: July 28, 2022). The collected data were accessed between 01.12.2022 and 05.01.2023 for research purposes. Early in the morning, after an overnight fast, blood samples were collected by a single specialist phlebotomist, from the brachial veins of all volunteers into two gel separator tubes (serum collection) and one K 2 EDTA tube. After the collection process, all tubes were properly transported to the biochemistry laboratory for routine analysis. Glycated hemoglobin (HbA1c) measurements in K 2 EDTA tubes were performed using an automatic glycohemoglobin analyzer (Arkray, Adams A1c HA-8180V, Japan). The serum collection tubes were left for 20–30 minutes to allow clotting. Once coagulation was complete, the serum collection tubes were centrifuged at 1800 g for 10 minutes. First serum collection tube was used for biochemical analysis (Glucose, triglyceride, total cholesterol, low-density lipoprotein [LDL], and high-density lipoprotein [HDL]) and hormone analysis (Follicle-stimulating hormone [FSH], luteinizing hormone [LH], estradiol [E2], thyroid-stimulating hormone [TSH], prolactin, human chorionic gonadotropin [β-hCG], testosterone, sex hormone-binding globulin [SHBG], dehydroepiandrosterone sulfate [DHESO 4 ], and insulin). The analyses were conducted using a biochemistry analyzer (Abbott Architect c16000, Illinois, USA) and a hormone analyzer (Roche Diagnostics Cobas E601, Tokyo, Japan), respectively. Serum samples in the second serum collection tube (obtained at the end of centrifugation) were transferred to 1.5 mL micro-volume tubes. These serum samples were placed in a −80°C deep freezer for NO, apelin, noradrenaline, and prostacyclin analyses and stored there until the analyses were conducted. On the day of analysis, serum NO, apelin, noradrenaline, and prostacyclin (Bioassay Technology Laboratory, Cat. No: E1510Hu, E2014Hu, EA0069Hu, and EA0019Hu, China respectively) levels were measured using human-specific enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s instructions. The data collected for the study were analyzed using SPSS (Statistical Program for Social Sciences) version 25. The normality of the data was assessed using the Kolmogorov-Smirnov test. Descriptive data are presented as mean, standard deviation, frequency, and percentage values. The significance level (p) for comparison tests was set at 0.05. Since the data followed a normal distribution (p > 0.05), parametric test methods were used for further analysis. Comparisons between independent two groups were made using the t-test, as the normality assumption was met. To determine the cutoff point for a measurement value, ROC analysis was performed and indices were calculated. Binary logistic regression models were established in which the groups were the dependent variables and the NO, apelin, noradrenaline, and prostacyclin values were the independent variables. The Hosmer-Lemeshow statistic was used to test the model’s goodness of fit in the binary logistic regression analysis. The sample size for this study was calculated using the G*Power 3.1 program. According to the results, a minimum of 84 participants (42 per group) was required, with an effect size of 0.73, a margin of error of 0.05, a confidence level of 95%, and a statistical power of 0.95 [ 30 ].

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