Introduction

Polycystic Ovary Syndrome (PCOS), a pervasive endocrine disorder, affects up to 15–20% of women in their reproductive years globally [1]. A spectrum of clinical and biochemical features marks this condition, including irregular menstrual cycles, hirsutism, obesity, insulin resistance, and hyperandrogenism [2]. The complex origins of PCOS are multifactorial, drawing on genetic, environmental, and lifestyle influences [3,4,5]. Among environmental factors, much research has been directed toward obesity and specific dietary habits known to exacerbate PCOS [6, 7]. A salient emerging factor in the environmental puzzle is exposure to endocrine-disrupting chemicals (EDCs) such as Bisphenol A (BPA) [8, 9].

BPA, an ever-present environmental contaminant, is extensively used in the production of polycarbonate plastics and epoxy resins, components commonly found in food packaging, water bottles, dental sealants, and medical devices [10]. Estrogenic effects of BPA are attributed to its ability to bind to estrogen receptors, making it a plausible factor in several endocrine disorders, including PCOS [11]. BPA's detrimental impact on the endocrine system is well-documented, with multiple studies suggesting that BPA exposure could destabilize hormonal equilibrium, paving the way for the development of PCOS [12, 13].

Numerous studies on the relationship between BPA exposure and PCOS have yielded inconclusive results. A subset of these studies finds higher BPA concentrations in PCOS patients compared to control groups [14, 15], while others find no significant differences [16]. Disputes in these findings may be attributed to disparate methodologies and the use of measurement techniques with varying degrees of precision [17]. Many past studies relied on ELISA for BPA quantification, a technique that may lack the precision required for accurate BPA detection compared to High-Performance Liquid Chromatography (HPLC). Using a more rigorous and precise measurement technique, such as HPLC, could shed more light on the role of BPA in the pathogenesis of PCOS. Currently, there is a noticeable lack of extensive research on the quantification of BPA in PCOS patients using HPLC within the Indian population [18, 19].

Motivated by this existing knowledge gap, we determined to thoroughly investigate the relationship between BPA exposure and PCOS using the HPLC method, renowned for its enhanced precision. Furthermore, in comparison to healthy controls, our study broadened its scope to investigate correlations between BPA concentrations and several demographic and biochemical parameters in females with PCOS. Our goal with this methodical investigation is to improve our understanding of the impact of environmental components like BPA on the manifestation and progression of PCOS.

Methods

Participants Recruitment and Ethical Approval

The enrollment process was meticulously planned and executed to ensure rigorous scientific standards. The figures – 130 women recruited, 80 diagnosed with PCOS, and 50 selected as controls – though seemingly rounded, are the result of our strict inclusion criteria and the actual turnout of eligible participants. These numbers directly reflect the outcome of our comprehensive screening process, outlined as follows:

Recruitment and Diagnosis

Initially, 130 women aged between 17 and 40 years were recruited for the study during January 2022 to July 2023. This recruitment took place at the Dr. Nagori Institute, Ahmedabad, a choice made for its specialization in treating endocrine disorders, particularly PCOS. The diagnosis of PCOS in 80 of these women was based on the ESHRE/ASRM’s Rotterdam Criteria, requiring the manifestation of two out of three specific features. This criterion ensured that only individuals with a clear diagnosis of PCOS were included in the study group.

Control Group Selection

The control group comprised 50 women, chosen from the initial pool of recruits based on their lack of any endocrinopathy and absence of hormonal contraceptive use. This selection process was crucial for establishing a baseline against which the effects of BPA could be accurately measured.

Ethical Considerations

Our study received approval from the Ethical Committee of Gujarat University (GU-IEC(NIV)/02/Ph.D./007), underscoring our commitment to ethical research practices and adherence to relevant guidelines and regulations.

The flow diagram provided (Fig. 1) visualizes this recruitment and selection process, offering a transparent and detailed view of how participants were systematically categorized into the study and control groups.

Fig. 1
figure 1

Flow chart showing participants recruitment in the study

Full size image

Sample Collection and Biochemical Analyses

All blood samples in our study were collected during the early follicular phase, specifically between days 2–5 of the menstrual cycle, which is a critical period for accurately assessing hormonal levels without the influence of cyclical fluctuations. This timing ensures that the measurements reflect the baseline hormonal status of the participants.

For women experiencing amenorrhea or irregular menstruation, a common challenge in PCOS, our collaborating physicians at Nagori Hospital administered medication to induce a spontaneous menstrual cycle. This approach allowed us to standardize the timing of blood sample collection across all study participants, ensuring that samples were obtained during the comparable hormonal phase of the cycle.

Following centrifugation at 2500 revolutions per minute for a 15-min duration, serum samples were meticulously stored in BPA-free containers at a temperature of -80 °C, awaiting further analysis. The biochemical tests conducted included assessments of Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), and 17β-estradiol levels, alongside serum levels of thyroid-stimulating hormone (TSH) and prolactin (PRL) to exclude conditions such as hypothyroidism and hyperprolactinemia. Additionally, total serum testosterone (TST) and dehydroepiandrosterone sulfate (DHEA-S) were measured to evaluate the presence and extent of hyperandrogenemia. All hormone evaluations were performed by a certified clinical laboratory using a chemiluminescent method. Informed consent was obtained from all participants before their inclusion in the study.

Chromatographic Detection and Sample Preparation

BPA was purchased from Sigma-Aldrich. HPLC-grade chemicals including ammonium acetate, n-hexane, diethyl ether, acetonitrile, and water were purchased from Thermo Fisher. The chromatographic separation was carried out using an HPLC system (Shimadzu, Japan) consisting of a degasser, binary pump, autosampler and a column oven. The analytes were separated into the C18 column (Shimadzu; 250 mm × 4.6 mm; 5 μm). Sample preparation and chromatographic separation were carried out as described by F. Kardas et.al [20] with slight modifications to optimize the BPA quantification. The procedure was conducted using an isocratic composition of acetonitrile and water in a 7:3 volume ratio for elution. A consistent flow rate for the mobile phase was sustained at 1 mL per minute. The temperature for the column compartment was regulated and stabilized at 25 degrees Celsius while the injection volume was set at 20μLwith use of UV-visible detector.

A stock solution of BPA was prepared by dissolving 1 mg of BPA in 10 mL of acetonitrile to obtain a 100 μg/ml concentration. The stock solution was stored at -20 °C in an amber vial to minimize light exposure. Next, a series of working standard solutions were prepared by diluting the stock solution with acetonitrile. At least 5 working standard solutions were designed, with concentrations ranging from 25 to 125 ng/ml. The concentrations were chosen based on the expected concentration range of the samples. The standard working solutions were stored at 4 °C in amber vials.

Each standard solution was injected into the HPLC system and analyzed per the chromatography parameters. The peak area for each standard solution was calculated, and a calibration curve was constructed by plotting the peak area versus the corresponding concentration of BPA. A linear regression analysis was performed to determine the calibration curve equation. To confirm the accuracy and precision of the calibration curve, a quality control (QC) sample with a known concentration of BPA (25 ng/mL) was analyzed at the beginning, middle, and end of the analytical run.

50 μL of serum sample was added to fifty ul of 10 mM ammonium acetate buffer (pH 4.5), followed by 500 μL mixture of n-hexane and diethyl ether (70:30). This composite underwent vortexing for 30 s and centrifugation at 3500 rpm for 10 min. The resultant organic layer was collected, transferred to a new tube, and dried under dry nitrogen. After reconstituting with 300ul of acetonitrile and filtering through a 0.22μ syringe filter, the samples were injected into an HPLC system for analysis under the same conditions as the standard solutions. The BPA concentration within the samples was assessed by applying the calibration curve equation to the sample's peak area, with results provided in ng/mL.

Statistical Analyses

We used SPSS 20.0 for statistical analysis. The data are expressed as the mean ± SD. An unpaired 2-tailed Student’s t test was used to compare differences between groups. Pearson's correlation coefficient and a linear/multiple regression analysis were used to evaluate the relationship between BPA and clinical variables. Analyses deemed results with a P value below 0.05 as significant.

Results

A variety of demographic and biochemical factors were carefully compared between the two groups (Table 1). PCOS women were older compared with the controls (29.16 ± 4.15 years Vs 24.34 ± 5.11 years; p = 0.010). Their average BMI also exceeded that of the control group (26.84 ± 5.49 kg/m2 Vs 22.87 ± 4.78 kg/m2; p < 0.0001).

Table 1 Demographic and Biochemical parameters of study participants
Full size table

In terms of biochemical and hormonal factors, testosterone levels were higher in PCOS women than in controls (29.99 ± 20.42 ng/dl Vs 21.51 ± 26.40 ng/dl; p = 0.042). However, estradiol levels showed no significant difference between the groups (75.38 ± 50.15 pg/ml Vs 71.79 ± 27.88 pg/ml; p = 0.64). The LH levels were greater in PCOS women compared to the control group (7.38 ± 4.28 mIU/ml Vs 5.64 ± 4.81 mIU/ml; p = 0.03). The FSH levels were similar in both groups (8.65 ± 6.78 mIU/ml Vs 9.02 ± 6.38 mIU/ml; p = 0.75). The LH to FSH ratio in PCOS was higher than in the control group, but not significantly (1.13 ± 0.98 Vs 0.80 ± 1.01; p = 0.06). PRL levels were similar for both groups (15.11 ± 12.43 ng/ml Vs 15.63 ± 9.11 ng/ml; p = 0.80), as were the TSH levels (1.96 ± 0.98 uIU/ml Vs 1.93 ± 1.08 uIU/ml; p = 0.86). The DHEA-S levels were nearly identical in both groups (141.28 ± 63.41 ug/dl Vs 151.38 ± 50.70 ug/dl; p = 0.33). Bisphenol A levels were significantly higher in the PCOS group than in the control group (102.15 ± 0.1 ng/ml Vs 61.35 ± 50.13 ng/ml; p < 0.0001).

A meticulous and precise high-performance liquid chromatography (HPLC) method was designed to quantify BPA concentration in serum samples. Figure 2 presents a BPA Chromatogram with a 4.14 ± 0.4-min retention time using a UV detector at 233 nm wavelength. With a high correlation coefficient of 0.999 within the 25 to 125 ng/mL test concentration range, the calibration curve served as a reliable BPA quantification tool (Refer Fig. 3a). Decreasing standard concentrations corresponded to decreasing peak height and area (Refer Fig. 3b). The method demonstrated high precision, with a relative standard deviation (RSD) of peak areas under 5% for all concentrations tested. Sensitivity for BPA detection was indicated by the method's LOD and LOQ, established at 0.01 ng/mL and 0.04 ng/mL, respectively. Following the method's validation, spiked recoveries were performed to assess accuracy. Spiked serum samples with BPA at concentrations of 15, 70, and 120 ng/mL demonstrated recovery rates between 95–98%, indicating excellent accuracy. Precision was further affirmed by relative standard deviations (RSD) for these recoveries, which were consistently below 5%. In this study, the HPLC method demonstrated exceptional intraday precision with an RSD of 4.6% and interday precision with an RSD of 5.2%, ensuring reliable and consistent BPA quantification across different time frames. These results highlight the method's reliability and suitability for accurate BPA quantification in serum samples. Figure S1(a) represents the chromatogram of positive serum sample and Figure S1(b) shows the chromatogram of spiked sample (5 ng/ml BPA spiked in serum sample).

Fig. 2
figure 2

The chromatogram shows a sharp peak at a retention time of 4.14 ± 0.4 min, corresponding to a standard solution of BPA at a concentration of 5 ng/ml

Full size image
Fig. 3
figure 3

Standard chromatogram and calibration curve of Bisphenol A (BPA) analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. a The calibration curve was constructed by plotting the chromatographic peak area against the concentration of BPA, using standard solutions of BPA at various concentrations ranging from 25 to 125 ng/ml. The high R2 value of 0.999 indicates the method's strong correlation and reliability. b The chromatogram also shows the elution profile of BPA at different concentrations ranging from 25 to 125 ng/mL, demonstrating the linearity of the response

Full size image

The study also discerned a significant discrepancy in Bisphenol A levels, with the PCOS group presenting a considerably higher mean of 102.15 ± 0.1 ng/ml compared to 61.35 ± 50.13 ng/ml in the control group (p < 0.0001) (Table 1) (Fig. 4a).

Fig. 4
figure 4

Comparison of Bisphenol A (BPA) levels and correlation analysis of metabolic markers in women with polycystic ovary syndrome (PCOS) and healthy controls. a Box and whisker plot showing significantly higher levels of BPA in PCOS group compared to controls (p < 0.0001), suggesting a potential link between environmental exposure to BPA and the development of PCOS. b A positive correlation observed between BPA and luteinizing hormone (LH) in PCOS women (r = 0.23, p = 0.039). c A negative correlation was found between BPA and thyroid stimulating hormone (TSH) among PCOS group (r = -0.33, p = 0.002). d A positive correlation was found between Homeostatic Model Assessment of Insulin Resistance (HOMA IR) and body mass index (BMI) among women with PCOS (r = 0.46, p = 0.0002), highlighting the importance of weight loss and improved insulin sensitivity in the management of PCOS

Full size image

Table 2 shows the correlation analysis to explore the relationships between Bisphenol A concentrations and an array of biochemical parameters among individuals in the PCOS and control groups. In the PCOS group, Bisphenol A levels showed no meaningful relationship with BMI, (r = 0.01, p = 0.94). A notable positive relationship emerged between Bisphenol A and luteinizing hormone (LH) levels (r = 0.23, p = 0.03) (Fig. 4b). A significant negative correlation appeared between Bisphenol A and thyroid-stimulating hormone (TSH) levels (r = -0.33, p = 0.002) (Fig. 4c). No significant correlations existed between Bisphenol A levels and other parameters, such as testosterone, estradiol, follicle-stimulating hormone (FSH), prolactin (PRL), LH/FSH ratio, and dehydroepiandrosterone sulfate (DHEA-S), in the PCOS group.

Table 2 Correlations between Bisphenol A and Biochemical Parameters in the Study Group
Full size table

Contrarily, the control group showed no meaningful associations between Bisphenol A levels and the examined biochemical parameters. An upward trend was apparent between Bisphenol A levels and BMI (r = 0.25, p = 0.07), yet it failed to hit the threshold of statistical significance. Moreover, Bisphenol A correlations with biochemical parameters such as testosterone, estradiol, LH, FSH, PRL, LH/FSH ratio, DHEA-S, and TSH were negligible and statistically insignificant. Furthermore, the correlation matrix revealed a substantial positive association between the body mass index (BMI) and the homeostatic model assessment of insulin resistance (HOMA IR) among the PCOS affected women (r = 0.46, p = 0.0002) (Fig. 4d).

Discussion

Our study probed the effects of environmental exposure to Bisphenol A (BPA) on women with and without PCOS. The wide prevalence of BPA, a synthetic compound in plastics and epoxy resins, coupled with its endocrine-disrupting properties, have made it a subject of concern. Through our study, we revealed significant ties between BPA exposure and various hormonal and some metabolic parameters in women with PCOS.

Given the observation of an age difference between the two groups, it is imperative to address concerns regarding age-related BPA accumulation. Extensive research, including studies by Kono et al. (2017) [21] and Tillett (2009) [22], suggests that BPA exposure is widespread and not significantly impacted by age. These findings affirm the multifactorial nature of BPA exposure, indicating that the age disparity observed does not directly correlate with the higher BPA levels found in PCOS patients. This context underscores the significance of our results, which contribute critical insights into the relationship between BPA exposure and PCOS, independent of the age difference.

One of the core findings of this research was the significantly elevated serum levels of BPA in subjects with PCOS compared to the control group. This aligns with prior studies indicating BPA, with its endocrine-disrupting capacity, might amplify hormonal discrepancies tied to PCOS [15]. Our data illustrated a strong positive correlation between serum BPA levels and luteinizing hormone (LH) in the PCOS cohort. Interestingly, Liang et al. observed that exposure to BPA was associated with increased serum levels of LH and FSH in male smokers [23]. As BPA is known to interact with estrogen receptors, this finding supports the hypothesis that it may modulate the hypothalamic-pituitary-ovarian axis, affecting the secretion of gonadotropins such as LH [24]. Elevated LH levels, a defining feature of PCOS, are well known to be associated with ovarian dysfunction [25].

Our study also revealed an inverse association between BPA levels and thyroid-stimulating hormone (TSH) within the PCOS group. Chevrier et al. reported that male neonatal TSH negatively associated with maternal BPA urine concentrations during pregnancy [26]. Park et al. found a negative correlation between BPA and TSH in adult Korean population [27]. A study on adult women in Cyprus and Romania found a significant association between urinary BPA and serum TSH [28]. This inverse relationship evidence consideration as thyroid function is integral to reproductive physiology. The ability of BPA to disrupt thyroid hormone pathways could possibly result in an exacerbated adverse effect on ovarian function in women with PCOS [29].

Regarding metabolic parameters, our study did not find any significant correlation between BPA and BMI in the PCOS group. This finding contrasts with the classification of BPA as an obesogen, which increases insulin resistance and fat accumulation [30, 31]. we observed a significant positive correlation between BMI and insulin resistance (HOMA IR) among women with PCOS. This is in line with the widespread understanding of the interplay between obesity and insulin resistance in PCOS [32]. The lack of a direct association between BPA and BMI or insulin resistance in our study could be indicative of the complex interactions between environmental and genetic factors in the pathogenesis of PCOS. However, the study by IA Kawa et al. found high levels of BPA in women with PCOS compared to controls and an association between BPA levels and biochemical abnormalities in PCOS, such as fasting blood glucose, triglycerides, and HOMA-IR [15].

One notable aspect of our study was to quantify serum BPA levels through high-performance liquid chromatography (HPLC) rather than traditional enzyme-linked immunosorbent assays (ELISA) method. ELISA has been criticized for its susceptibility to cross-reactivity and matrix interferences, which raises concerns about measurement accuracy [33]. In contrast, HPLC provides a more sophisticated and precise method for separating, identifying, and quantifying compounds due to its high resolution and specificity [17, 34]. The use of HPLC in our study not only improved the robustness of data, but also served as a pioneering approach, establishing a benchmark for future studies in environmental endocrinology. This methodological refinement is crucial in accurately quantifying BPA, which is known for its potent endocrine-modulating properties. Further, our study stands out as one of the few that uses the power of HPLC to quantify BPA levels in women with Polycystic Ovary Syndrome (PCOS) in Indian population. A thorough search of databases, including PubMed, Scopus, and Web of Science, employing 'PCOS' and 'Bisphenol A' as search terms, surprisingly revealed a considerable dearth of analogous studies conducted within India. The existing studies, such as study by Prabhu et. al 2023 [18] and Kawa I.A et. al 2019 [15], provide insights into the BPA-PCOS connection, but a significant research gap remains in the Indian context.

While our findings provide valuable insights, it is critical to acknowledge the limitations, which include a relatively small sample size and a cross-sectional design. Furthermore, determining BPA levels at a single point in time may not reflect long-term exposure, and variation in BPA concentrations may have significant consequences on hormonal activities. The extensive use of BPA and the high prevalence of PCOS among Indian women, the absence of aligned studies shows an urgent need for more exploration. Despite its limitations, this study established the path for future research by indicating a potential BPA-PCOS relationship in the Indian context.

Conclusion

In conclusion, our study reveals that women with PCOS have higher levels of BPA than healthy controls, suggesting that BPA exposure may play a role in developing PCOS. The HPLC technique provides a reliable and sensitive method for assessing BPA exposure in women with PCOS. The findings highlight the need for further research to explore the relationship between BPA exposure and the development of PCOS, as well as the potential mechanisms underlying this association. Moreover, our study underscores the importance of reducing environmental exposure to BPA and implementing lifestyle modifications, such as weight loss and improved insulin sensitivity to prevent and manage PCOS in women. These insights have significant implications for public health and can aid in developing strategies to reduce BPA exposure and prevent PCOS development and its associated comorbidities.