The Association Between Polycystic Ovary Syndrome and Vitamin D Deficiency in Infertile Women: A Case-Control Study.

Fatemeh Janani : conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript. Fatemeh Yari and Parasto Baharvand: designed the data collection instruments, collected data, carried out the initial analyses, and reviewed and revised the manuscript. Ali Momeni: coordinated and supervised data collection, and critically reviewed the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript. Fatemeh Janani had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.

Among the 66 women with PCOS, 59.1% presented with secondary infertility, and clinical records indicated that 72.7% (48/66) had oligo‐ or anovulation, 63.6% (42/66) exhibited clinical hyperandrogenism, and 81.8% (54/66) had polycystic ovarian morphology on ultrasound. Due to the retrospective nature of the study, detailed phenotype stratification was not available. A total of 66 patients and 66 women without polycystic ovaries were examined. The mean age in the patient group was 30.47 ± 6.51 years, with an age range of 16 to 44 years, while the mean age in the control group was 32.02 ± 6.43 years, with an age range of 18 to 44 years (Table  1 ). Absolute and relative frequency distribution of demographic characteristics by study group and comparison of their significance levels. Test: χ . 2 The most common age group in both cohorts was 26–35 years (48.5% in patients and 53% in the control group), with no significant age difference between the two groups ( p  > 0.05). n = ( Z 1 − α / 2 . 2 P ¯ ( 1 − P ¯ ) + Z 1 − β . P 1 ( 1 − P 1 ) + P 2 ( 1 − P 2 ) ) 2 d 2 Regarding education, the most frequent educational level in both groups was university education (48.5% in each group), with no significant difference between the two groups ( p  > 0.05). There was a significant difference in socioeconomic status between the two groups ( p   0.05), with the majority being homemakers (81.8% in patients vs. 80.3% in controls). Secondary infertility was present only in the patient group (59.1%). The most common BMI category in both groups was 25–29.9 (50% in patients vs. 43.9% in controls), with a significant difference in BMI between the two groups ( p  < 0.05). As shown in Table  2 , the most common duration of infertility among patients was 1 year (24.2%), followed equally by 2 years and 5 years (15.2% each). Frequency distribution of infertility duration (years) in patients with polycystic ovaries. The unadjusted (crude) analysis revealed a significant association between PCOS and vitamin D deficiency. Sixty‐eight percent (45/66) of the PCOS group had vitamin D deficiency, compared to 27.3% (18/66) of the control group ( p  < 0.001). The crude odds ratio indicated that women with PCOS had 2.37 times higher odds of being vitamin D deficient than women without PCOS (OR = 2.37, 95% CI 1.59–3.46, p  = 0.001) (Figure  1 ). Frequency of vitamin D deficiency by study group. Among women with PCOS under 26 years of age, 7 individuals (23.3%) had severe vitamin D deficiency, while 6 (40%) showed moderate deficiency. In the 26‐35 age group, these figures were 53.3% and 33.3% respectively. For those over 35 years, severe deficiency was observed in 23.3% and moderate deficiency in 26.7% of cases. However, these differences between age groups were not statistically significant ( p  = 0.714) (Table  3 ). Vitamin D deficiency status by age and BMI in patients with polycystic ovary syndrome ( n  = 66). Note: p  = 0.714 (nonsignificant age association). Test: χ. 2 p  = 0.001 (significant BMI association). Vitamin D deficiency by BMI in women with polycystic ovaries showed distinct patterns. Among women with polycystic ovaries, no vitamin D deficiency was observed in those with a BMI < 18.6. In the BMI 18.6–24.9 group, 38.9% had severe deficiency and 11.1% had moderate deficiency. The BMI 25–29.9 group demonstrated higher rates, with 42.4% showing severe deficiency and 30.3% having moderate deficiency. Most strikingly, in the BMI ≥ 30 group, 64.3% exhibited severe deficiency while 21.4% had moderate deficiency. These progressive increases in deficiency prevalence across BMI categories were statistically significant ( p  < 0.05) (Table  3 ). However, given the significant differences in BMI and socioeconomic status between the groups (Table  1 ), a multivariable logistic regression was performed to adjust for these and other potential confounders. After adjustment for BMI, age, socioeconomic status, and season of blood draw, the association between PCOS and vitamin D deficiency, while attenuated, remained statistically significant.

This single‐center, case‐control study provides local evidence of a significant association between vitamin D deficiency and PCOS among women in Lorestan province. It is important to frame these findings within the context of the existing literature. While large meta‐analyses of RCTs (e.g., Miao 2020; Yang 2023) provide strong evidence for the causal effects of vitamin D supplementation on metabolic parameters in PCOS, our study offers complementary, regional prevalence data [ 22 , 23 ]. Our findings align with the hypothesis that vitamin D deficiency may be a contributing factor in PCOS, potentially through its role in insulin secretion and resistance [ 24 ]. However, as a retrospective observational study, it cannot establish causality. PCOS is the most common cause of oligo‐ovulation and anovulation, affecting 4%–6% of the general population, particularly infertile women [ 17 ]. Recent studies suggest a potential role of vitamin D in insulin secretion and the improvement of insulin resistance [ 15 ]. Furthermore, vitamin D deficiency appears to be a possible contributing factor in the pathogenesis of PCOS [ 6 ]. Given the impact of insulin resistance on elevated androgen levels—and consequently [ 10 ], the disruption of developing follicles in PCOS patients vitamin D supplementation may prove beneficial in metabolic improvement, androgen reduction, and even the restoration of ovulation in individuals with PCOS [ 11 ]. Among the various processes influenced by vitamin D, its vital role in reproductive physiology is particularly significant [ 20 ]. Based on available data, vitamin D is considered one of the key components in achieving successful fertility. Therefore, vitamin D deficiency is proposed as a pathophysiological mechanism that can compromise fertility [ 7 ]. The results of the present study show that infertile individuals with polycystic ovary syndrome (PCOS), according to the presented tables and data, did not differ significantly from the control group in terms of age, education level, or employment status. However, BMI in the case group was significantly higher than in the control group. The findings also indicate no significant difference in education level between the two groups. The distribution of employment status between the two groups was not statistically different; thus, no association can be established between vitamin D deficiency and employment status in these individuals. In the study by Rashidi et al. [ 25 ], the mean serum vitamin D level in employed women was higher than in housewives, but this difference was not statistically significant. These results are consistent with the findings of the present study, which also showed no statistically significant association between employment status and vitamin D deficiency. In the present study, among women with PCOS, those with higher BMI showed a more severe deficiency in serum vitamin D levels. This difference between the study groups was statistically significant. The findings of the present study are consistent with those of Rahimi et al. [ 24 ], who investigated vitamin D deficiency and found a significant association between serum vitamin D levels and BMI [ 26 , 27 , 28 ]. Results from various studies indicate that increased BMI and obesity are associated with insulin resistance and vitamin D deficiency, while vitamin D deficiency, in turn, reduces cellular sensitivity to insulin [ 29 , 30 ]. In fact, vitamin D influences glucose transport into cells by regulating the expression of insulin receptors and insulin secretion [ 31 , 32 ]. While our study establishes a significant association between PCOS and vitamin D deficiency independent of BMI, it does not address the critical question of how this deficiency influences clinical outcomes. Our findings should be interpreted in light of interventional meta‐analyses that have demonstrated that vitamin D supplementation in women with PCOS can lead to improvements in insulin sensitivity, lipid profiles, and menstrual regularity (Zhang 2023; Gao 2024), and may enhance fertility treatment outcomes [ 33 ]. The high prevalence of deficiency observed in our cohort (68.2%) suggests that a substantial number of women with PCOS in this population may have a modifiable risk factor for poorer metabolic and reproductive health. Therefore, our data on prevalence provide a strong rationale for future prospective studies in this region to investigate whether systematic screening and correction of vitamin D deficiency translates into tangible improvements in ovulation rates, metabolic health, and success rates of fertility treatments (Chu 2018; Zhou 2022) [ 33 , 34 ]. Statistical analysis showed that, out of all patients, 45 individuals (68.2%) had vitamin D deficiency, whereas in the control group only 18 individuals (27.3%) had vitamin D deficiency. This difference was statistically significant ( p  < 0.05). The odds of having abnormal serum vitamin D levels in women with polycystic ovary syndrome (patients) were approximately 2.37 times higher than in the control group. These findings are consistent with those of Irani et al. [ 35 ], Mohammadbeigi et al. [ 36 ], and Nestler et al. [ 37 ], in which the relationship and role of vitamin D in reproductive function among women with PCOS were examined. These studies indicated that low serum vitamin D levels contribute to unsuccessful ovulation induction in women with PCOS, aligning with the present study's results. In contrast, in the study by Kim et al. [ 38 ] on vitamin D deficiency in women with PCOS, no significant difference in vitamin D levels was found between patients and controls. The discrepancy between their results and those of the present study may be due to differences in the characteristics of the study populations. The 2023 International Evidence‐Based PCOS Guideline emphasizes the importance of phenotype stratification to better understand the heterogeneity of PCOS. While our study confirmed PCOS diagnoses using the Rotterdam criteria, detailed phenotype distribution (e.g., hyperandrogenic vs. non‐hyperandrogenic phenotypes) was not recorded in the medical records. This limitation restricts our ability to analyze phenotype‐specific associations with vitamin D deficiency. However, the high prevalence of oligo‐ovulation (72.7%) and hyperandrogenism (63.6%) in our cohort suggests a predominance of classic PCOS phenotypes, consistent with previous studies reporting higher metabolic risk in these groups [ 1 ]. A key limitation of this study is the lack of insulin resistance markers, such as HOMA‐IR, due to the retrospective design and the absence of routine insulin measurements at the study center. This precludes a direct assessment of the role of insulin resistance in the observed association between PCOS and vitamin D deficiency. Future research should incorporate metabolic profiling, including fasting glucose, insulin, and HOMA‐IR, to align with the 2023 International Evidence‐Based PCOS Guideline recommendations.

This study provides important local data indicating a high prevalence of vitamin D deficiency in women with PCOS in Lorestan province, Iran, which is significantly associated with higher BMI. While this associative finding cannot support a causal inference, it underscores the value of screening for and addressing vitamin D deficiency in this patient population. The results generate a hypothesis for this specific region and support the rationale for designing a future randomized controlled trial within this population to directly investigate the efficacy of vitamin D supplementation as an adjuvant treatment to improve metabolic and reproductive outcomes. Alongside any potential supplementation, lifestyle modification remains a cornerstone of PCOS management. In addition, alongside vitamin D supplementation, lifestyle modification and increased physical activity are recommended for women with PCOS to help control and reduce BMI. We will reframe the narrative to highlight that our study's strength lies not in being the first, but in providing detailed, confounder‐adjusted local data that confirms the high burden of deficiency and its strong BMI‐dependent gradient in this specific population. This directly serves the “service value for local public health planning” as the reviewer aptly suggested.

PCOS is a heterogeneous endocrine disorder affecting 5%–10% of women of reproductive age, characterized by a spectrum of reproductive and metabolic abnormalities [ 1 ]. The Rotterdam criteria (2003) are widely used for diagnosis, requiring at least two of three features: oligo‐ or anovulation, hyperandrogenism, or polycystic ovarian morphology, with exclusion of other causes [ 2 ]. The 2023 International Evidence‐Based PCOS Guideline emphasizes the need for clear diagnostic criteria, phenotype stratification, and metabolic profiling, including insulin resistance markers, to guide clinical management and research. Vitamin D deficiency, prevalent in women of reproductive age, has been implicated in PCOS pathogenesis, potentially through its effects on insulin sensitivity and androgen metabolism [ 3 ]. Long‐term complications of this syndrome include high blood pressure, cardiovascular disease, and type 2 diabetes [ 4 ]. Additionally, women with PCOS who have not received treatment may develop endometrial hyperplasia and endometrial cancer, highlighting the need for special medical attention [ 5 ]. Infertility is a significant and often forgotten part of reproductive health. The World Health Organization (WHO) identifies infertility as a disease that affects 13% to 17% of couples globally [ 6 ]. It ranks fourth among stressful life events, following the death of a mother, the death of a father, and spousal infidelity [ 7 ]. The WHO estimates that one in four couples in developing countries is affected by infertility [ 8 ]. In Iran, the overall infertility rate is 13.2%. Of these cases, 39.9% are due to male factors, 40.3% to female factors, and 10.1% are of unknown cause [ 9 ]. Factors such as diet, obesity, lifestyle, and physical activity all impact fertility [ 10 ]. Studies have shown that people with a healthy diet and lifestyle can have a sixfold increase in fertility [ 11 ]. Vitamin D is a crucial micronutrient known for maintaining calcium homeostasis and skeletal health [ 12 ]. However, its functions extend beyond this, as nearly all cells in the body have vitamin D receptors [ 13 ]. Vitamin D is recognized as a prohormone because the body synthesizes it in the skin from a precursor, dehydrocholesterol, under the influence of ultraviolet B radiation [ 14 ]. Despite the body's ability to produce vitamin D, lower than normal levels are a widespread problem [ 15 ]. Women of reproductive age are at a particularly high risk of deficiency [ 16 ]. Maternal vitamin D deficiency is common, especially in developing countries, and it can cause health problems in children that last into adulthood [ 17 ]. Physiologically, there are two active forms of vitamin D:Vitamin D2 (ergocalciferol) is produced by plants and Vitamin D3 (cholecalciferol) is synthesized in the skin from 7‐dehydrocholesterol under the influence of ultraviolet (UV) radiation [ 17 ]. Both D2 and D3 undergo a metabolic process in which they are converted in the liver and kidneys to their active form, 1,25‐dihydroxyvitamin D (also known as calcitriol). The primary method for diagnosing a deficiency is by measuring serum 25(OH)D levels. While there is no universal consensus on what constitutes a normal level, a level of 20 ng/mL or higher is generally considered sufficient [ 18 ]. We will refine the rationale to more explicitly state that the study aims to provide the necessary local epidemiological foundation for targeting a modifiable risk factor (vitamin D deficiency), which is a prerequisite for designing effective local management strategies. Given the importance of this micronutrient and the influence of geographical and ethnic factors on vitamin D status, there is a need for local prevalence data. While large meta‐analyses of randomized trials provide high‐level evidence for the effects of supplementation, local observational studies are crucial for understanding the burden of deficiency in specific patient populations. Therefore, this study was designed to provide provincial data by determining and comparing the serum vitamin D levels in infertile patients with PCOS versus healthy controls visiting the Jahad Daneshgahi Infertility Center in Lorestan Province.

The lead author Fatemeh Janani affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

The authors declare no conflicts of interest.

This case‐control study compared vitamin D levels between infertile women with PCOS and healthy controls. The case group consisted of 66 patients aged 18 to 45 with PCOS, diagnosed according to the Rotterdam criteria (2003). This required the presence of at least two of the following features: oligo‐ or anovulation, clinical or biochemical hyperandrogenism, or polycystic ovarian morphology on ultrasound, after the exclusion of other etiologies such as congenital adrenal hyperplasia, androgen‐secreting tumors, or hyperprolactinemia. A gynecologist at the Jahad Daneshgahi Infertility Center of Lorestan province, where the patients were recruited, confirmed the diagnosis based on clinical evaluation and ultrasound findings. The control group included 66 healthy women without PCOS. The study was conducted using data from patients who visited the center during of 2020. Blood samples for serum 25‐hydroxyvitamin D measurement were collected throughout this 12‐month period to account for seasonal variation. Blood samples for serum 25‐hydroxyvitamin D [25(OH)D] measurement were collected throughout the year (January to December 2020). Serum 25(OH)D levels were measured using a Assay Method enzyme‐linked immunosorbent assay(ELISA) kit manufactured by (Pishtaz Teb Zaman Diagnostics, Tehran, Iran). The precision of the assay, determined by the coefficient of variation (CV), was for intra‐assay variability and for inter‐assay variability. All laboratory procedures were performed according to the manufacturer's instructions [ 19 ]. Vitamin D status was classified using categories aligned with international guidelines [ 20 ]. For the primary analysis, vitamin D deficiency was defined as a serum 25(OH)D level < 20 ng/mL. For descriptive purposes, levels were also categorized as follows: severe deficiency ( 100 ng/mL). Inclusion Criteria: Women with a diagnosis of infertility and a confirmed diagnosis of PCOS based on the Rotterdam criteria. Exclusion Criteria: Women with PCOS were excluded if their medical records indicated an additional, confirmed cause of female infertility, such as tubal factor (e.g., diagnosed by hysterosalpingography), endometriosis, diminished ovarian reserve (e.g., based on anti‐Müllerian hormone levels or antral follicle count), or uterine abnormalities. This was to ensure that PCOS was the primary and likely cause of infertility in the case group. Inclusion Criteria: Healthy, fertile women with a history of at least one prior pregnancy and no clinical or ultrasonographic features of PCOS. Exclusion Criteria: Same as for the case group, plus any history of infertility. Diagnosis of PCOS: PCOS was diagnosed by a gynecologist at the center according to the Rotterdam criteria (2003) [ 2 ], requiring at least two of the following: oligo‐ or anovulation, clinical or biochemical hyperandrogenism, or polycystic ovarian morphology on ultrasound, after the exclusion of other related etiologies such as congenital adrenal hyperplasia, androgen‐secreting tumors, and hyperprolactinemia. The sample size was calculated for a case‐control study using the formula for comparing two proportions. The calculation was based on findings from a previous study by (Irani et al.) [ 21 ], which reported a prevalence of vitamin D deficiency of 58% (p₁ = 0.58) in women with PCOS and 36% (p₂ = 0.36) in healthy controls. n = ( Z 1 − α / 2 . 2 P ¯ ( 1 − P ¯ ) + Z 1 − β . P 1 ( 1 − P 1 ) + P 2 ( 1 − P 2 ) ) 2 d 2 P ¯ The =the average of two proportions (0.46). d  = The difference between two proportions (0.22). 1 − β = % 80 p1 = 36 and p2 = 58 The initial calculation yielded approximately 77 participants per group. However, anticipating a potential attrition rate of approximately 15% due to incomplete medical records, the sample size was adjusted upwards. The final target sample size was set at 90 per group. During the data collection period (January to December 2020), a total of 66 eligible patients with PCOS and 66 eligible controls who had complete data were identified. A post hoc power calculation confirmed that with the achieved sample size of 66 per group, an observed proportion of 68.2% in cases and 27.3% in controls, an alpha of 0.05, and a 1:1 ratio, the study power exceeded 99%. Data were analyzed using SPSS software version 22. Descriptive statistics were presented as mean ± standard deviation, frequencies, and percentages. Group comparisons for categorical variables were made using the χ 2 test, and for continuous variables using the independent t ‐test. The association between PCOS and vitamin D deficiency was first assessed using a crude logistic regression model, yielding a crude odds ratio (OR) with a 95% confidence interval (CI). To control for potential confounding, a multivariable logistic regression analysis was performed. The model included PCOS status as the primary independent variable and vitamin D deficiency (dichotomized as deficient/insufficient vs. normal) as the dependent variable. The model was adjusted for variables known to be associated with both PCOS and vitamin D status: (BMI, as a continuous variable), age (as a continuous variable), socioeconomic status (categorical), and season of blood draw (categorical). The results of the adjusted model are presented as an adjusted odds ratio (aOR) with a 95% CI. A two‐sided p‐value of < 0.05 was considered statistically significant. Incomplete files and lack of cooperation from some patients. This study was approved by the Research Ethics Board of Lorestan University of Medical Sciences (IR.LUMS. REC.1400.113). The study was conducted by the STROBE guidelines.