Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age, with an estimated global prevalence ranging from 4% to 20%, depending on the diagnostic criteria and population studied.¹ It is a heterogeneous disorder characterized by chronic anovulation, hyperandrogenism, and polycystic

ovarian morphology.² The Rotterdam criteria, introduced in 2003, remain the most widely accepted diagnostic standard, requiring the presence of at least two of the following features: oligo- or anovulation, clinical or biochemical hyperandrogenism, and polycystic ovaries on ultrasonography.³ PCOS is a major cause of infertility and menstrual irregularities and has substantial implications for women's reproductive, metabolic, and psychological health.⁴

From a gynecological perspective, PCOS is associated with irregular menstrual cycles, amenorrhea, oligomenorrhea, infertility, recurrent pregnancy loss, endometrial hyperplasia, and an increased risk of endometrial carcinoma due to prolonged unopposed estrogen exposure.⁵ Clinical manifestations such as hirsutism, acne, androgenic alopecia, and obesity often negatively affect body image and quality of life. Early identification and appropriate management are essential to reduce reproductive complications and improve long-term health outcomes.⁶

Physiologically, PCOS is characterized by disturbances in the hypothalamic-pituitary-ovarian (HPO) axis. Increased pulsatile secretion of gonadotropin-releasing hormone (GnRH) leads to preferential secretion of luteinizing hormone (LH) over follicle-stimulating hormone (FSH), resulting in an elevated LH/FSH ratio.⁷ Excess LH stimulates ovarian theca cells to produce androgens, while relatively low FSH levels impair follicular maturation, preventing normal ovulation and promoting the formation of multiple immature follicles.⁸ Additionally, insulin resistance, present in approximately 50–70% of women with PCOS, further enhances ovarian androgen production and decreases hepatic synthesis of sex hormone-binding globulin (SHBG), thereby increasing circulating free testosterone levels.⁹

Biochemically, oxidative stress has emerged as a critical contributor to the pathogenesis of PCOS.¹⁰ Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the body's antioxidant defense mechanisms, leading to cellular damage.¹¹ Elevated levels of oxidative stress biomarkers such as malondialdehyde (MDA), advanced oxidation protein products (AOPPs), and reactive oxygen metabolites have been consistently reported in women with PCOS. Conversely, antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) are often diminished, reflecting impaired antioxidant capacity.¹² These biochemical alterations contribute to chronic inflammation, endothelial dysfunction, insulin resistance, and impaired ovarian function.

Insulin resistance is one of the central metabolic abnormalities in PCOS and plays a pivotal role in disease progression.¹³ Hyperinsulinemia not only promotes androgen synthesis by ovarian theca cells but also suppresses SHBG production by the liver, resulting in increased bioavailability of androgens.¹⁴ This interaction between insulin resistance and hyperandrogenism creates a vicious cycle that exacerbates reproductive dysfunction and metabolic disturbances.¹⁵ Furthermore, dyslipidemia, impaired glucose tolerance, type 2 diabetes mellitus, hypertension, and metabolic syndrome are significantly more prevalent among women with PCOS than in the general population.¹⁶

Endothelial dysfunction represents an important physiological consequence of oxidative stress and chronic low-grade inflammation in PCOS.¹⁷ Reduced nitric oxide (NO) bioavailability, increased inflammatory cytokines, elevated C-reactive protein (CRP), and vascular oxidative injury contribute to impaired endothelial-dependent vasodilation.¹⁸ These alterations increase the long-term risk of cardiovascular disease, making cardiovascular assessment an important component of PCOS management.¹⁹ Flow-mediated dilation (FMD) and circulating endothelial biomarkers have therefore become valuable tools for evaluating vascular health in affected women.²⁰

Recent evidence suggests that oxidative stress, hormonal imbalance, insulin resistance, and chronic inflammation are closely interconnected in the pathophysiology of PCOS. Understanding these interactions is essential for identifying biomarkers that may aid in early diagnosis, disease monitoring, and therapeutic decision-making. Comprehensive evaluation of biochemical, physiological, and gynecological parameters may provide a better understanding of disease severity and the mechanisms underlying reproductive dysfunction.

Therefore, the present study was designed to evaluate oxidative stress biomarkers, antioxidant status, reproductive hormone profiles, insulin resistance, endothelial function, and physiological parameters in women with Polycystic Ovary Syndrome compared with healthy controls. The findings may provide valuable insights into the biochemical and physiological mechanisms of PCOS and contribute to the development of more effective diagnostic and therapeutic strategies for improving reproductive and metabolic health.

Study Design and Study Setting: A hospital-based comparative cross-sectional study was conducted to evaluate oxidative stress biomarkers, reproductive hormone profile, endothelial function, and physiological parameters in women with Polycystic Ovary Syndrome (PCOS). The study was carried out in the Departments of Gynecology, Physiology, and Biochemistry of a tertiary  care teaching hospital over a period of 12 months. All laboratory analyses were performed in the institutional clinical biochemistry laboratory using standardized operating procedures and calibrated analytical instruments.

Study Population and Sample Size: The study included a total of 120 women aged between 18 and 35 years. Sixty women diagnosed with PCOS constituted the study group, while sixty age-matched healthy women with regular menstrual cycles and no evidence of endocrine or metabolic disorders served as the control group. Participants were recruited consecutively from the gynecology outpatient department after obtaining informed written consent. The sample size was determined to provide adequate statistical power for detecting significant differences in oxidative stress biomarkers, hormonal profile, and endothelial function between the two groups at a 95% confidence level and 80% study power.

Diagnostic Criteria for PCOS: Women were diagnosed with PCOS according to the revised Rotterdam criteria (2003), requiring the presence of at least two of the following three features after exclusion of related endocrine disorders: (1) oligo-ovulation or anovulation manifested by oligomenorrhea or amenorrhea; (2) clinical or biochemical evidence of hyperandrogenism; and (3) polycystic ovarian morphology identified by transvaginal or transabdominal ultrasonography. Other causes of hyperandrogenism and menstrual irregularities, including congenital adrenal hyperplasia, Cushing's syndrome, thyroid dysfunction, hyperprolactinemia, and androgen-secreting tumors, were excluded before enrollment.

Inclusion and Exclusion Criteria Women aged 18–35 years with newly diagnosed or previously confirmed PCOS according to the Rotterdam criteria were included in the study. Healthy women of similar age with regular ovulatory menstrual cycles, normal ovarian morphology, and no clinical evidence of endocrine abnormalities were recruited as controls. Pregnant or lactating women, women receiving hormonal contraceptives, anti-androgen therapy, insulin-sensitizing agents, lipid-lowering drugs, antioxidant supplements, or corticosteroids within the previous three months were excluded. Participants with diabetes mellitus, hypertension, cardiovascular disease, chronic kidney disease, liver disease, autoimmune disorders, acute or chronic inflammatory diseases, malignancy, smoking, alcohol abuse, or any other endocrine disorders were also excluded from the study.

Clinical and Anthropometric Assessment A detailed clinical history was obtained from all participants using a structured questionnaire. Information regarding age, menstrual history, infertility, family history of PCOS or diabetes mellitus, medication use, and lifestyle characteristics was recorded. Physical examination included measurement of height, weight, waist circumference, hip circumference, systolic blood pressure (SBP), and diastolic blood pressure (DBP). Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m²), while waist-to-hip ratio (WHR) was calculated by dividing waist circumference by hip circumference. Clinical manifestations of hyperandrogenism, including hirsutism, acne, and androgenic alopecia, were assessed, and hirsutism was graded using the modified Ferriman–Gallwey scoring system.

Blood Sample Collection Following an overnight fast of 10–12 hours, approximately 10 mL of venous blood was collected from each participant between the third and fifth day of the menstrual cycle or following progesterone-induced withdrawal bleeding in women with amenorrhea. Blood samples were divided into ethylenediaminetetraacetic acid (EDTA) tubes and plain serum tubes. Serum was separated by centrifugation at 3000 rpm for 10 minutes and stored at −80°C until biochemical analysis. All analyses were completed within three months of sample collection to minimize biochemical degradation.

Biochemical Analysis Fasting blood glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using automated enzymatic colorimetric methods on a fully automated clinical chemistry analyzer. Serum fasting insulin concentration was determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. Insulin resistance was estimated using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), calculated as:

HOMA-IR = [Fasting Insulin (µIU/mL) × Fasting Glucose (mg/dL)] / 405

Hormonal Profile Assessment Serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, and total testosterone were measured using chemiluminescent immunoassay (CLIA) or ELISA-based methods. The LH/FSH ratio was subsequently calculated for each participant as an indicator of hypothalamic-pituitary-ovarian axis dysfunction. All hormonal analyses were performed in duplicate following the manufacturers' quality control recommendations.

Assessment of Oxidative Stress Biomarkers Oxidative stress status was evaluated by measuring serum malondialdehyde (MDA) as an indicator of lipid peroxidation using the thiobarbituric acid reactive substances (TBARS) assay. Antioxidant defense capacity was assessed by measuring the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) using commercially available assay kits based on spectrophotometric methods. All assays were performed according to the manufacturers' protocols, and internal quality controls were included in each analytical run.

Assessment of Inflammatory and Endothelial Biomarkers High-sensitivity C-reactive protein (hs-CRP) was measured using an immunoturbidimetric assay as a marker of systemic inflammation. Serum nitric oxide (NO) concentration was estimated indirectly by measuring stable nitrate and nitrite metabolites using the Griess reaction. Endothelial function was assessed non-invasively by measuring brachial artery flow-mediated dilation (FMD) using high-resolution B-mode ultrasonography. Baseline brachial artery diameter was recorded after 10 minutes of rest, followed by inflation of a blood pressure cuff to 50 mmHg above systolic pressure for five minutes. The maximum arterial diameter after cuff release was measured, and FMD was expressed as the percentage increase from baseline diameter.

Quality Control All laboratory investigations were performed by trained laboratory personnel who were blinded to participants' clinical status. Analytical instruments were calibrated regularly, and both internal and external quality assurance procedures were followed throughout the study. All biochemical measurements were performed in duplicate, and the average value was used for statistical analysis to improve analytical reliability and reproducibility.

Statistical Analysis Data were entered into Microsoft Excel and analyzed using Statistical Package for the Social Sciences (SPSS) version 26.0. Continuous variables were expressed as mean ± standard deviation (SD), while categorical variables were presented as frequencies and percentages. Normality of data distribution was assessed using the Shapiro–Wilk test. Comparisons between the PCOS and control groups were performed using the independent samples Student's t-test for normally distributed variables and the Mann–Whitney U test for non-normally distributed variables. Pearson's correlation coefficient was used to evaluate relationships among oxidative stress markers, reproductive hormones, insulin resistance, inflammatory biomarkers, and endothelial function. Multiple linear regression analysis was performed to identify independent predictors of endothelial dysfunction after adjusting for potential confounding variables. A p-value of less than 0.05 was considered statistically significant.

purposes.

Table 1. Demographic, Anthropometric, and Clinical Characteristics of Study Participants

*Data are expressed as Mean ± SD. p < 0.05 indicates statistical significance.

Table 2. Reproductive Hormonal Profile of the Study Participants

Table 3. Metabolic and Lipid Profile

Table 4. Oxidative Stress Biomarkers and Antioxidant Enzyme Activity

Table 5. Endothelial Function Assessment

Table 6. Pearson Correlation Analysis in Women with PCOS

Table 7. Multiple Linear Regression Analysis for Predictors of Endothelial Dysfunction (Dependent Variable: FMD)

Model statistics: R² = 0.61, Adjusted R² = 0.58, F = 17.84, p < 0.001.

The present comparative cross-sectional study evaluated oxidative stress, reproductive hormone profile, insulin resistance, endothelial function, and physiological parameters in women with Polycystic Ovary Syndrome (PCOS). The findings demonstrated that women with PCOS exhibited significant hormonal imbalance, increased oxidative stress, impaired antioxidant defense, insulin resistance, dyslipidemia, chronic low-grade inflammation, and endothelial dysfunction compared with

healthy controls. These observations support the concept that PCOS is a multisystem endocrine-metabolic disorder involving complex interactions between reproductive, biochemical, and cardiovascular abnormalities.

One of the major findings of the present study was the significantly higher body mass index (BMI), waist-to-hip ratio (WHR), and blood pressure among women with PCOS. Central obesity is a common clinical feature of PCOS and contributes substantially to insulin resistance and chronic inflammation. Increased visceral adiposity promotes the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which impair insulin signaling and increase oxidative stress. The higher anthropometric indices observed in this study are consistent with previous reports demonstrating that obesity aggravates both reproductive and metabolic abnormalities in PCOS.

The hormonal profile revealed significantly elevated luteinizing hormone (LH), LH/FSH ratio, and serum testosterone levels, while follicle-stimulating hormone (FSH), estradiol, and progesterone were significantly lower in women with PCOS. These findings reflect dysregulation of the hypothalamic-pituitary-ovarian axis, a hallmark of PCOS. Increased pulsatile gonadotropin-releasing hormone (GnRH) secretion preferentially stimulates LH release, resulting in excessive androgen production by ovarian theca cells. Reduced FSH levels impair follicular maturation and ovulation, leading to chronic anovulation and the formation of multiple immature ovarian follicles. Hyperandrogenism further contributes to menstrual irregularities, infertility, acne, hirsutism, and metabolic dysfunction. These hormonal alterations agree with numerous clinical studies reporting elevated LH/FSH ratios and increased circulating testosterone concentrations in women with PCOS.

Insulin resistance was another prominent finding of the present investigation. Women with PCOS demonstrated significantly higher fasting insulin concentrations and HOMA-IR values than healthy controls. Hyperinsulinemia is recognized as one of the primary pathogenic mechanisms in PCOS because insulin acts synergistically with LH to stimulate ovarian androgen synthesis while simultaneously suppressing hepatic production of sex hormone-binding globulin (SHBG). Consequently, circulating free testosterone increases, further worsening hyperandrogenism and reproductive dysfunction. The strong positive correlation between HOMA-IR and oxidative stress observed in this study emphasizes the close relationship between metabolic abnormalities and redox imbalance. Similar associations have been consistently reported in previous investigations, suggesting that insulin resistance contributes substantially to disease progression and long-term metabolic complications.

The present study also demonstrated significant dyslipidemia characterized by elevated total cholesterol, triglycerides, LDL-cholesterol, and reduced HDL-cholesterol. These lipid abnormalities are commonly observed in women with PCOS and largely result from insulin resistance and obesity. Altered lipid metabolism promotes endothelial injury and accelerates atherosclerotic processes, thereby increasing future cardiovascular risk. The coexistence of dyslipidemia with insulin resistance further supports the concept that PCOS should be considered not only a reproductive disorder but also an important metabolic disease requiring long-term cardiovascular monitoring.

A major objective of this study was to evaluate oxidative stress. Serum malondialdehyde (MDA), a marker of lipid peroxidation, was significantly elevated in women with PCOS, indicating excessive production of reactive oxygen species (ROS). At the same time, antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were markedly reduced, reflecting depletion of endogenous antioxidant defenses. These findings suggest that oxidative stress is an important component of PCOS pathophysiology. Increased ROS production may damage cellular lipids, proteins, DNA, and ovarian tissues, impair follicular development, and reduce oocyte quality. Furthermore, oxidative stress activates inflammatory pathways, including nuclear factor-kappa B (NF-κB), which promotes cytokine production and perpetuates chronic inflammation. The present findings are consistent with previous studies that have demonstrated elevated lipid peroxidation together with reduced antioxidant enzyme activity in women with PCOS.

Inflammation also appeared to play a central role in disease progression. Women with PCOS exhibited significantly higher concentrations of high-sensitivity C-reactive protein (hs-CRP), supporting the presence of chronic low-grade systemic inflammation. The positive correlation between hs-CRP and MDA suggests that oxidative stress and inflammation reinforce one another in PCOS. Persistent inflammatory activation contributes to endothelial injury, insulin resistance, ovarian dysfunction, and cardiovascular complications. These findings support previous reports indicating that inflammatory biomarkers are significantly elevated in women with PCOS independent of obesity.

Endothelial dysfunction was another important finding of the present study. Flow-mediated dilation (FMD) was significantly reduced in women with PCOS, while nitric oxide (NO) levels were also markedly decreased. Nitric oxide is the principal endothelial-derived vasodilator responsible for maintaining vascular tone, inhibiting platelet aggregation, and preventing vascular inflammation. Excessive oxidative stress reduces nitric oxide bioavailability through direct interaction with superoxide radicals, leading to impaired endothelial-dependent vasodilation. The positive correlation between antioxidant enzymes and FMD observed in this study indicates that preservation of antioxidant defenses contributes to improved vascular function. Conversely, the inverse association between insulin resistance and endothelial function highlights the adverse vascular effects of metabolic dysfunction.

Correlation analysis further demonstrated that MDA was positively associated with insulin resistance, testosterone levels, and inflammatory markers, while antioxidant enzyme activity showed significant positive relationships with endothelial function. These findings emphasize the close interaction among oxidative stress, endocrine abnormalities, metabolic dysfunction, and vascular impairment. Multiple linear regression analysis identified HOMA-IR and MDA as the strongest independent predictors of endothelial dysfunction after adjustment for other variables. These observations suggest that insulin resistance and oxidative stress are major determinants of vascular injury in women with PCOS and may represent important therapeutic targets.

The clinical implications of these findings are substantial. Routine evaluation of oxidative stress biomarkers, insulin resistance, reproductive hormones, inflammatory markers, and endothelial function may help identify women at increased risk of infertility, metabolic syndrome, type 2 diabetes mellitus, and cardiovascular disease. Lifestyle modification, including weight reduction, regular physical activity, and dietary interventions, together with insulin-sensitizing agents and antioxidant supplementation, may improve metabolic control, restore ovulatory function, and reduce oxidative damage. Early identification of high-risk patients could facilitate individualized management and prevent long-term complications.

The present study has several strengths. It comprehensively evaluated biochemical, hormonal, physiological, inflammatory, and vascular parameters within the same population, allowing a more integrated understanding of PCOS pathophysiology. However, several limitations should be acknowledged. The cross-sectional design precludes establishing causal relationships between oxidative stress and disease progression. The relatively small sample size and single-center setting may limit the generalizability of the findings. Furthermore, additional biomarkers such as adiponectin, leptin, advanced oxidation protein products, total antioxidant capacity, and inflammatory cytokines were not assessed. Future multicenter prospective studies with larger populations and longer follow-up are recommended to confirm these findings and evaluate the effects of antioxidant and insulin-sensitizing therapies on reproductive and cardiovascular outcomes.

Women with Polycystic Ovary Syndrome (PCOS) exhibit significant oxidative stress, hormonal imbalance, insulin resistance, chronic low-grade inflammation, dyslipidemia, and impaired endothelial function compared with healthy women. These interconnected biochemical, physiological, and gynecological abnormalities contribute to reproductive dysfunction and increase the risk of long-term metabolic and cardiovascular complications. Early assessment of oxidative stress biomarkers, reproductive hormones, and endothelial function, together with appropriate lifestyle and therapeutic interventions, may improve disease monitoring, optimize reproductive outcomes, and reduce future cardiometabolic risk in women with PCOS.

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