Polycystic ovary syndrome (PCOS) is defined by hyperandrogenism, metabolic and reproductive disorders and is linked to insulin resistance, which obesity aggravates, but is not a prerequisite for. PCOS is a prevalent endocrinal and metabolic condition caused by hereditary and environmental factors affecting women in the years of reproduction [1,2]. It is defined by (A) persistent anovulation, (B) biochemical and/or clinical hyperandrogenism and (C) polycystic ovarian morphology. PCOS has major medical consequences and can lead to health problems linked to hyperandrogenemia, increased insulin resistance (IR), cardiovascular diseases (CVDs), long-term inflammation, overweight or obesity and malignancies. It is the primary cause of infertility and persistent anovulation [3]. Although a lot of progress has been made in understanding PCOS's symptoms, how it works and how to treat it, problems still exist because we do not fully understand its causes and the lack of specific treatments that work for everyone [4]. PCOS is a burgeoning health concern among females due to lifestyle alterations, heightened stress levels, insufficient physical exercise and significant disruptions in menstrual cycle patterns. Currently, there is no definitive therapy for the issue, so the most efficient and important approach is to prevent its progression and facilitate early detection to avert severe long-term repercussions [5]. Numerous symptoms are associated with PCOS, including acne, hirsutism, acanthosis, seborrhoea, alopecia, insomnia, infertility and irregular menstruation [6]. Worldwide, according to 1990 NIH standards, PCOS impacts 6–10% of females or even more people according to the broader Rotterdam criteria and Androgen Excess Society (AES) criteria, which renders it one of the more prevalent human diseases and the most pervasive endocrinopathy among reproductive-age women [7].

Oxidative stress (OS) is associated with PCOS pathophysiology, which happens when free radicals and antioxidants are out of equilibrium. Inflammation and insulin resistance, two frequent characteristics of PCOS, might be made worse by elevated oxidative stress [8]. Homocysteine (HCY) was first identified in 1932.  It is chemically analogous to cysteine, hence the designation homocysteine [9]. HCY is the demethylated product of the essential amino acid methionine [10]. Hyperhomocysteinemia, a buildup of homocysteine, can be caused by deficiencies in vitamin B12 and folate since the methionine route requires the conversion of homocysteine to methionine, which is catalysed by methyltetrahydrofolate and methylcobalamin [11]. Homocysteine is distinguished by its propensity to induce oxidative stress and cellular damage. HCY-induced oxidative stress correlates with glutathione depletion, suppression of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and cellular oxidative injuries (protein inactivation, lipid peroxidation and DNA degradation), ultimately contributing to various chronic diseases, including diabetes, cardiovascular disorders and cancer [12]. Increased HCY levels in women with PCOS have been associated with diminished fertilization rates, impaired oocyte maturation and inferior embryo quality, thereby negatively impacting fertility. Moreover, elevated HCY levels are also connected to insulin resistance and obesity and worsen hyperandrogenism, a crucial characteristic of PCOS [13].

Vitamin C, referred to as 'ascorbic acid', 'ascorbate' or 'L-ascorbate', is a crucial water-soluble compound vital for human health. It was discovered in 1912 and initially synthesized in 1933 because it is not manufactured by the body itself hence people have to obtain it from their food [14,15]. Ascorbic acid functions as an antioxidant, essential for alleviating oxidative stress in various bodily tissues. Ascorbic acid safeguards different compounds in the body, including lipids, carbohydrates and nucleic acids (RNA and DNA), from the harmful impacts of free radicals and reactive oxygen species produced during cellular metabolism due to exposure to toxins and pollutants (such as cigarettes and drugs) or by cells of the immune system [16]. This study aimed to measure and compare serum homocysteine levels and vitamin C concentrations between women with PCOS versus age-matched healthy controls and to investigate the correlation between homocysteine and vitamin C levels in both groups to elucidate potential mechanistic links between oxidative stress and vitamin C deficiency in PCOS pathophysiology.

The current case-control research was carried out from April 2024 to January 2025 in the infertility centre at Al-Batool Teaching Hospital in Diyala Governorate, Iraq, by the University of Baghdad's College of Medicine's Department of Biochemistry. One hundred thirty-two women aged 18-40 years were included in this study. The participants were categorized into two groups: Sixty-six PCOS patients and 66 healthy controls. 

• Inclusion Criteria: The cases were selected consecutively from the Infertility Centre after confirming the PCOS diagnosis according to the Rotterdam European Society for Human Reproduction and before starting any PCOS medication (insulin-sensitising drugs)

• Exclusion Criteria: All individuals with endocrinal or metabolic problems, including thyroid dysfunction, type 2 diabetes, high blood pressure, liver disease, chronic renal illness, premature ovarian failure and virilising adrenal or ovarian tumours, were excluded from this study. Cases with etiological factors, including prolactinoma, congenital adrenal hyperplasia and Cushing syndrome simulating PCOS, were also excluded

We aimed for 80% power to detect a significant difference. The significance level was set at 0.05. Using these parameters, we calculated the required sample size using the G*Power software (version 3.1). The specific parameters entered into the software were (1) Effect Size d: Calculated as the mean difference divided by the standard deviation; (2) Analysis: A priori power analysis; (3) Test Family: t-tests; (4) Statistical Test: Two-sample t-test (mean difference between two independent groups); (5) Type III Error Probability (alpha): 0.05; (6) Power (1–beta error probability): 0.80. The software output indicated that a sample size of 66 participants per group would be sufficient to achieve 80% power to detect a significant difference in homocysteine and vitamin C levels between the groups.

Formula for Sample Size per Group (n):

• Z_1−α/2: Critical value for the significance level α

• Z_1−β: Critical value for power 1−β

• d: Cohen’s effect size

The Ethical and Scientific Review Boards Commission of the Biochemistry Department, Faculty of Medicine, University of Baghdad gave their approval to this research. Additionally, the Scientific Research Committee of Diyala Health Directorate, Diyala, Iraq provided their ethical clearance. Participants verbally agreed before participating in this study.

Blood samples were taken from each patient and control participant. During the follicular phase (the second or third day of menstruation), five millilitres (ml) of blood were taken from a vein in the arm and allowed to coagulate for fifteen minutes. before being centrifuged for ten minutes at 4,000 rounds per minute (rpm). Laboratory testing included serum measurements of homocysteine and vitamin C, which Competitive-ELISA performed with the principle that the limited the number of antigens binding sites, forcing a target analyte and a labelled analogue to compete for antibody binding according to the manufacturer (Elabscience Company -Houston, Texas, USA). Each subject's serum was divided into two samples and then transported to a 1.5 millilitre Eppendorf tube for freezing at -80 °C until the time of the studied parameter measurements.

Statistical Analysis

Statistical analysis was done using Microsoft Excel for organised data and Statistical Package for Social Sciences (SPSS) version 26.0. which described the data using percentages, means and standard deviation (SD). Between-group comparisons were conducted using independent samples t-tests and Pearson Chi-square test for normally distributed variables. The equality of variances was evaluated using Levene's test and appropriate corrections were applied when the assumption of homogeneity of variance was violated. The Pearson correlation regression test was used to analyse the numerical data correlations. The statistical significance level was determined at p < 0.05. The ROC curve (receiver operating characteristic) and its area under the curve (AUC) have been used to evaluate the ability to diagnose each biomarker in differentiating between PCOS cases and their controls.

RESULTS

Table 1 shows that the cases and controls were not significantly different regarding age, height, weight and body mass index and were well-matched.

Table 1: The Mean±Sd Values of Demographic Variables for Patients and Controls

BMI: Body mass index; Student's t-test was used to compare the group means

The serum homocysteine concentrations were markedly elevated among the PCOS group (18.879±0.283 pmol/ml) compared to the controls (12.242±0.175 pmol/ml, p<0.001). In comparison with the control group, the PCOS group exhibited considerably lower concentrations of serum vitamin C (8356.288±160.648 ng/ml) than their controls (13842.479±566.947 ng/ml) with a p-value of<0.001, Table 2.

Table 2: Mean± Sem Values for Serum Vitamin C and Homocysteine for Patients and Controls

SEM = Standard Error of Mean; * Student's t-test was used to compare the group means

Figures 1 and 2 illustrate the correlation between study parameters and BMI.  They indicate an important positive correlation between BMI and serum homocysteine levels (r = 0.840, p = 0.0001) and a highly negative correlation between BMI and vitamin C concentrations (r = -0.741, p = 0.0001)

Figure 1: Scatter Diagram Showing the Correlation Between Serum Homocysteine Levels and BMI in PCOS Cases

Figure 2: A Scatter Diagram Demonstrating the Association Between BMI and Serum Vitamin C Levels in PCOS Subjects

Figure 3 illustrates the relationship between vitamin C concentrations and serum homocysteine concentrations among the PCOS cases. An inverse correlation was observed between vitamin C concentrations and serum homocysteine concentrations (r = -0.790, p = 0.0001).

Figure 3: A Scatter Diagram Demonstrating the Association Between Homocysteine and Serum Vitamin C Levels in PCOS Subjects

The findings of the area under the curve (AUC) and the receiver operating characteristic (ROC) analysis assessment in differentiating between PCOS and healthy groups revealed that serum homocysteine demonstrates fair diagnostic performance with an AUC of 0.994. Using a cutoff value of >15.778 (pmol/ml), it achieved 98.48% sensitivity and 100% specificity, as presented in Figure 4.

Figure 4: Receiver Operating Characteristic (Roc) Curve for Distinguishing Between (PCOS) and Control Groups. the Curve Illustrates the Diagnostic Performance of the Homocysteine, with Sensitivity Plotted Against 1-Specificity

Serum vitamin C demonstrates strong diagnostic performance (AUC: 0.823). Using a cutoff value of ≤10938.1 (ng/mL), with 92.42% sensitivity and 78.79% specificity, as shown in Figure 5, indicated a very high discriminatory ability between these two groups.

Figure 5: Receiver Operating Characteristic (ROC) Curve for Distinguishing Between (PCOS) and Control Groups. The Curve Illustrates the Diagnostic Performance of the Vitamin C, with Sensitivity Plotted Against 1-Specificity

The results of the current study demonstrated that the PCOS group had a much higher mean homocysteine value than the control group, probably indicating oxidative damage and increased free radical production among PCOS subjects, which agrees with previous studies which demonstrated that hyperhomocysteinemia (Hhcy) may be considered as one of the features of PCOS [17]. Furthermore, Feng et al. discovered that two mutations in homocysteine-related genes, including MTHFR A1298C and MTRR A66G, had been linked to the risk of PCOS and the correlations occurred via changing the amount of homocysteine [18]. Another study showed that women with PCOS have higher mean serum homocysteine concentrations. When considering the majority of factors affecting homocysteine concentration, the elevated homocysteine levels in these patients may be due to hyperinsulinemia. In women with PCOS, high homocysteine levels may increase the risk of cardiovascular disease in conjunction with other risk factors such as dyslipidaemia or hyperinsulinemia [19]. We suggested elevated homocysteine levels are associated with impaired methylation processes and increased cardiovascular risk. This finding points to the potential for long-term cardiovascular complications in PCOS patients and reiterates the importance of monitoring and managing homocysteine levels.

Vitamin C or ascorbic acid, is a water-soluble antioxidant which functions as a cofactor for many biological processes and is involved in a broad range of enzymatic and non-enzymatic interactions [20]. It is the inaugural antioxidant employed throughout the peroxidation of lipids. Vitamin C is a crucial element in cellular defence versus oxidative damage and peroxidation of lipids resulting from free radicals in the body [21]. In the current research, the PCOS group's serum vitamin C concentrations were substantially lower than those of the control group, as shown in Table 2. 

This aligns with the research conducted by Mahmud et al. and Oyebanji and Asaolu [22,23]. The substantial reduction may be attributed to the depletion of vitamin C across the neutralization of free radicals [23].

The result of this study also found significant positive correlations between homocysteine levels and BMI in the patient group, as shown in Figure 1. This finding aligns with another study that demonstrated significant increases in mean serum homocysteine levels among cases of PCOS. The increase was more pronounced with an increase in BMI and waist. Obese cases showed higher levels compared to normal-weight cases and controls [24].

The findings of the present research indicated a noteworthy negative relationship between concentrations of vitamin C and BMI within the PCOS group, as illustrated in Figure 2. This finding aligns with a previous study on chronic wounds. We found no significant previous study about the correlation between vitamin C status and BMI in PCOS [25]. This association has been elucidated through prior research: a considerable number of women with PCOS exhibit dietary patterns that are notably deficient in vital nutrients, including omega-3 fatty acids, fibre, magnesium, calcium, zinc, vitamin C, folic acid, vitamin D and vitamin B12. Conversely, their diet typically comprises excessive quantities of salt, sugar, cholesterol, total fats and saturated fatty acids; these diets lead to an increase in the BMI [26].

The findings of this research additionally revealed substantial negative correlations between serum homocysteine and vitamin C levels in the patient group, as illustrated in Figure (3), in accordance with previous research which showed that vitamin C removes free radicals generated by homocysteine and may be related to plasma homocysteine levels [27]. The study explored the relationship between homocysteine levels and vitamin C in women with PCOS. Key findings included significantly higher homocysteine levels in the PCOS group, indicating elevated oxidative stress and significantly lower vitamin C levels, highlighting a prevalent deficiency. A strong negative correlation was observed between homocysteine and vitamin C, suggesting that vitamin C deficiency may exacerbate oxidative stress in PCOS. Additionally, higher BMI correlated with increased homocysteine and decreased vitamin C levels, further linking obesity to oxidative stress and a deficient level of vitamin C in PCOS.

Acknowledgements

The authors express gratitude to all participants in this study and their relatives. We also would like to thank all the staff at the infertility center of Al-Batool Teaching Hospital, Diyala Governorate, for their help and valuable support throughout the research project.

Conflict of Interest 

We declare that there is no conflict of interests regarding the publication of this manuscript.

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