Association of vitamin D deficiency with demographic, metabolic and sex hormone levels in polycystic ovarian syndrome

Introduction: Polycystic ovarian syndrome (PCOS) is a most prevalent endocrine disorder characterized by various clinical manifestations, including menstrual irregularities, hyperandrogenism, and polycystic ovaries. Severe vitamin D deficiency is more frequent in women with PCOS than in general females and has been associated with various metabolic disorders, comprising insulin resistance, blood pressure, obesity, and menstrual abnormalities. This study aimed to investigate the correlation among deficiency of vitamin D and demographic characteristics, metabolic parameters, and sex hormone levels of PCOS women from southern India. Materials and Methods: A case-control study involving 240 individuals comprised 120 women meeting the Rotterdam criteria for PCOS and 120 healthy age-matched controls. Various parameters, including anthropometric data, fasting blood sugar (FBS) levels, hormone levels, and vitamin D status, were assessed. Results: The results showed that participants with PCOS had significant increases in BMI, FBS, insulin, and HOMAIR levels. Triglyceride levels were significantly higher according to the lipid profile when compared to the control group, additional hormonal testing on PCOS patients revealed significantly higher levels of the LH: FSH ratio and testosterone. Women with PCOS showed significant low levels of 25(OH) D when compared to healthy women. Conclusion : Our findings revealed an association between low levels of 25(OH)D and the manifestation of symptoms related to metabolic syndrome in PCOS women. Therefore, vitamin D supplementation may have the potential to improve the underlying pathophysiology of PCOS.


INTRODUCTION
olycystic ovarian syndrome (PCOS) is a clinical syndrome that predominantly impacts women during their child bearing age, with prevalence rate ranging from 9% to 18% (1)(2)(3)(4).It is a heterogeneous disorder characterized by menstrual dysfunction, infertility, increased androgen levels, polycystic ovaries, hirsutism, and/or alopecia (5,6).Moreover, PCOS is linked to a higher occurrence of factors contributing to cardiovascular disease (CVD), diabetes mellitus, dyslipidemia, hypertension and cancer (7,8).Hyperandrogenism is considered an important factor in the occurrence of PCOS, although insulin resistance and obesity contribute to both direct and indirect mechanisms involved in the excessive production of androgens in PCOS and the present pandemic obesity predicts the more occurrence of PCOS in the future (9).Due to the possible detrimental effects this could have an effect on population growth, cardiovascular disease, and death.PCOS has grown to be a significant public health issue (10).Furthermore, women with PCOS exhibited a higher likelihood of experiencing dyslipidemia compared to healthy women.The main indicators of dyslipidemia in PCOS are decreased high-density lipoprotein cholesterol (HDL-C) and heightened triglycerides (TGs) and lowdensity lipoprotein cholesterol (LDL-C) levels (11).
Vitamin D has been shown to control the growth and development of the skeleton as well as the metabolism of calcium and phosphorus.However, there are also other problems that are associated with vitamin D, including cancer, immune system disorders, T2DM, CVD and infectious diseases (9,12,13).The connection between vitamin D and metabolic variables in female PCOS patients has been a subject of continuous discussion, as well as the disparity in vitamin D levels among PCOS women and non PCOS women.Insulin resistance (IR), a common characteristic of PCOS, may significantly contribute to the long-term complications associated with the disorder (14).
Metabolic changes are notably influenced by the levels of vitamin D, particularly in maintaining calcium-phosphate (Ca-P) balance and regulating insulin secretion by the β-cells (15).A portion of P follicular development in PCOS patients may also be partially caused by abnormalities in calcium balance, which could possibly be involved in the condition's aetiology (16).Recent research suggests that insufficient levels of vitamin D can play an important role in the emergence of IR and metabolic syndrome among PCOS women.However, it is not yet clear if vitamin D is also linked to endocrine parameters and fertility in PCOS (17).In comparison to women without PCOS, several studies have found that women with PCOS had decreased serum 25-hydroxyvitamin D [25(OH) D].Additionally, it was found that hyperinsulinemia, dyslipidemia, and other metabolic risk factors were more prevalent in PCOS patients with vitamin D insufficiency (18).So, this study aimed to analyze the correlation among vitamin D levels and demographic, metabolic, and sex hormone levels in Indian PCOS women.

MATERIALS AND METHODS
A case-control study was carried out in the Department of Obstetrics and Gynecology at K.S. Hedge Charitable Hospital.After obtaining voluntary written informed consent, a total of 240 participants aged 18 to 40 years were recruited.Among these, 120 women diagnosed with PCOS and 120 age-matched controls without PCOS were enrolled in the study.Biochemical assessments were carried out at the Central Research Laboratory of the institution.The study was conducted after getting permission from the Institutional Ethical Committee, Nitte University (NU/CEC/2018/0205 dated: 12.10.2018).
The Rotterdam criteria from 2003 were used to select the PCOS-positive female participants in the study.To be eligible, a participant had to meet two of the three requirements listed below: (i) Hyperandrogenism symptoms, either biochemically or clinically.(ii) irregular menstrual periods with more than a 35-day delay between cycles.(iii) Polycystic ovaries, as determined by ultrasound imaging, which displays more than ten subcapsular follicles in a single ovary, each with a diameter of at least 10 mm.Exclusion criteria for the study comprised individuals with hyperprolactinemia, thyroid disorders, adrenal disorders, 21-hydroxylase deficiency, and androgensecreting tumors.Participants who were currently using hormone supplements such as estrogen, progesterone, combination oral contraceptives (OCPs), gonadotropins, or antiepileptic drugs (AEDs) were also excluded.Pregnant individuals and those who were unwilling to engage in the study were not enrolled.

Clinical measurements
All study participants completed a pre-designed proforma with their full medical and family histories, as well as their age, the duration of their current problems, and detailed menstrual history.For every individual, standard anthropometric measurements were obtained, which comprises weight, height, waist circumference (WC), and hip circumference.The WC measurement was obtained by assessing the circumference at a midpoint between the iliac crest and the lower costal border while the participant stood upright.The hip diameter obtained from the broadest dimensions above buttocks while the individual was in a standing position.The Body Mass Index (BMI) was computed through the division of weight of the participant in kilograms by the square height of the participant in meters.Additionally, Hirsutism was measured by the revised Ferriman-Gallwey score, which assesses the extent of excessive hair growth in specific body areas.

Laboratory analysis
Blood samples were collected under fasting conditions from every enrolled participant.For the diagnosis of dyslipidemia and metabolic syndrome, blood sugar levels and the lipid levels, encompassing total cholesterol, TG, LDL, and HDL were determined via spectrophotometric analysis and shown in mg/dL.The concentrations of many hormones, including total testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin, and vitamin D, were measured using ELISA kits procured from Xema Co Ltd, Russia.The absorbance of the samples was read using an ELISA reader (Spark Tecan.).The homeostatic model assessment (HOMA-IR) was used to evaluate insulin resistance.This assessment is computed using the following formula: HOMAIR = fasting insulin (μIU/mL) * fasting glucose (mg/dL)/ divided by 405.

Statistical analysis
For parametric data, the data were presented as mean ± standard deviation (SD).In normal distribution data, the means between groups were compared using the Student's t-test.When presenting non-parametric data, the median and interquartile range (IQR) were used, and the Mann-Whitney U-test was applied to compare the medians within the groups.A linear regression analysis was performed to examine the correlation between 25(OH)D levels and metabolic parameters.Pvalue of less than 0.05 was considered as statistically significant.

RESULTS
A total of 240 subjects were enrolled, with 120 being PCOS patients and 120 without PCOS.The chief complaints among cases illustrated in Fig. 1.Among 120 PCOS women, 22% of women had complaints of 1 o and 2 o infertility, 22% of women were showing hirsutism and acne, 24% were complaining about oligo/amenorrhea, and 32% weight gain.Hirsutism and Acne are the most important features of hyperandrogenism.The distribution of case and control as percentage recruited for the study depending on BMI was shown in (Fig. 2).About 35% of women with PCOS are in the overweight category.The demographic details of the study subjects were presented in Table 1.Significant differences were seen in weight, BMI and Hip/Waist ratio between the cases (PCOS patients) and controls (normal age-matched individuals).However, there was no significance identified in the age and height between the cases and controls.Table 2. Shows the summary of statistics for the metabolic parameters of the study subjects.Patients with PCOS exhibited significantly elevated levels of Fasting Blood Sugar (FBS), TG, LDL, LDL/HDL ratio (the ratio of LDL to HDL), Insulin, and HOMA IR.Conversely, PCOS patients exhibited significantly lower HDL levels compared to the control group.The p-value was calculated using Mann-Whitney U test.(25).These discrepancies could be attributed to differences in sample size, participant characteristics, geographical location, and the timing of vitamin D assessment.In our study, we also observed that PCOS patients had significantly higher BMI, FBS, insulin, and HOMA-IR values compared to the control group.These findings highlight the metabolic disturbances commonly observed in individuals with PCOS.
Vitamin D has been implicated in enhancing insulin action by improving insulin responsiveness for glucose transport and increasing insulin receptor expression.This idea is further supported by the data from our study, which revealed a substantial correlation between vitamin D insufficiency and HOMA-IR, which in turn indicates an increased risk of obesity.According to recent findings from a study including obese and overweight women, women who have greater amounts of 25(OH)D than those who have lower levels of the vitamin react better to hypocaloric diets and shed more body fat.This finding highlights the potential role of vitamin D in weight management (26).
Furthermore, there is evidence supporting weight reduction as the most successful treatment for women with PCOS (27).Losing weight has been demonstrated to alleviate a number of PCOS-related symptoms, including hormonal imbalances, insulin resistance, and fertility issues.Therefore, focusing on weight reduction has emerged as a crucial approach in managing PCOS.

CONCLUSION
Our research supports the conclusions drawn by several studies regarding the correlation among vitamin D with metabolic, demographic and sex hormones.Insulin resistance has been shown to be a more significant independent risk factor for vitamin D deficiency among women with PCOS.The main limitation of this research was the limited sample size.So, further, more prospective studies are to be conducted to confirm the association among metabolic and sex hormones and vitamin D insufficiency.These studies will also enable us to determine whether dietary habits and way of life are associated with PCOS morbidity.

Table 4 indicates significant correlations among serum 25 (
OH)D levels and various parameters.The findings reveal a significant negative correlation between serum 25(OH)D concentration and BMI, WHR, fasting glucose, fasting insulin, HOMA-IR, TC, and testosterone (p < 0.05).Conversely, there is a significant positive correlation between serum 25(OH)D levels and HDL (p < 0.05).Notably, no significant association was there among serum 25(OH)D levels and triglycerides, LDL/HDL, estradiol (E2), and prolactin (p > 0.05).

Table 1 :
Demographic details of the subjects WHR, Waist-to-hip ratio.

Table 3 :
Hormonal parameters of study subjects

Table 4 :
Correlation of serum 25(OH)D levels with metabolic parameters in women with PCOS