Genotype-independent association between vitamin D deficiency and polycystic ovarian syndrome in Lahore, Pakistan

Both vitamin D deficiency and single nucleotide polymorphisms (SNPs) in the gene encoding the vitamin D receptor (VDR) have been widely reported to associate with susceptibility to polycystic ovarian syndrome (PCOS). A case-control study was conducted to study the influence of vitamin D status and genotpye for 24 SNPs in four genes in the vitamin D pathway (VDR, DBP, CYP27B1, CYP24A1) on PCOS. Statistical analyses were conducted to identify phenotypic and genotypic factors associated with risk of PCOS and to test for interactions between genotype and vitamin D status. PCOS was independently associated with lower age, higher body mass index, lower waist-hip ratio, vitamin D deficiency (serum 25-hydroxyvitamin D concentration <10 ng/mL), lack of outdoor exercise, increased fasting glucose and a family history of PCOS in at least one first degree relative. No statistically significant association was observed between the genotype of any SNP investigated and risk of PCOS, either as a main effect or in interaction with vitamin D status. We report a strong and independent association between vitamin D deficiency and risk of PCOS in Pakistan, that was not modified by genetic variation in the vitamin D pathway.

www.nature.com/scientificreports www.nature.com/scientificreports/ We have previously demonstrated that genetic variation in the vitamin D pathway can modify the influence of vitamin D deficiency and supplementation on clinical phenotype in the context of tuberculosis [12][13][14] . We, therefore, conducted a case control study in Lahore, Pakistan, to determine whether vitamin D deficiency and SNPs in four vitamin D pathway genes might influence the risk of PCOS either as main effects or in interaction with each other.

Results participant characteristics.
A total of 235 PCOS cases and 235 healthy controls were recruited to the study between April 2016 and April 2018. Characteristics of cases and controls are presented in Table 1. The mean age of cases vs. controls was 25.2 vs 29.7 years, respectively, and the majority (96.8%) of participants were of Punjabi ethnic origin. Married and single women were equally represented in both groups. Students were over-represented, and employed women were under-represented, in cases vs controls, but the proportion of participants at different educational levels did not differ between groups. As anticipated, acne, hair loss, hirsutism, menstrual cycle abnormalities, and pelvic ultrasound scan abnormalities were all more common among cases vs controls. The mean body mass index was higher among cases vs controls, but the mean waist-to-hip ratio was lower. Mean serum concentrations of fasting glucose and luteinizing hormone were higher in cases vs controls, but no difference was seen for mean serum concentration of the follicle-stimulating hormone. As anticipated LH/FSH ratio was higher in cases vs controls. Mean serum 25(OH)D concentration was lower in cases vs controls (17.4 vs 21.7 ng/ml, respectively; P < 0.001). Table 2 presents the results of univariable and multivariable analyses testing for associations between participant characteristics captured by the study questionnaire or found at physical examination and susceptibility to PCOS. PCOS was independently associated with lower age (adjusted odds ratio [ Association between vitamin D pathway genotype and risk of polycystic ovarian syndrome. Table 3 presents results of univariable and multivariable analyses testing for associations between SNPs in the genes encoding the vitamin D receptor (VDR), the vitamin D 1-alpha hydroxylase enzyme (CYP27B1), the vitamin D 24-hydroxylase enzyme (CYP24A1) and the vitamin D binding protein (DBP) and risk of PCOS. No statistically significant association was observed between the genotype of any polymorphism investigated and risk of PCOS.

Association between vitamin D deficiency and host genotype, stratified by vitamin D status.
We next proceeded to investigate whether genetic variation in VDR, CYP24A1, CYP27B1 and DBP modified the association between vitamin D deficiency and risk of PCOS reported above. P values for interaction for these sub-group analyses are presented in Table 3: these revealed no evidence to support the hypothesis that polymorphisms in the vitamin D pathway modify the effect of vitamin D deficiency on risk of PCOS.
Haplotype analysis. We also performed haplotype analysis to assess the cumulative impact of all

Discussion
We report findings from one of the largest and most detailed studies conducted to date to investigate the influence of vitamin D deficiency and genetic variation in the vitamin D pathway on susceptibility to PCOS -and the first such study to be conducted in Pakistan. Uniquely, in addition to investigating SNPs in VDR and DBP, we also explored the influence of variants in CYP24A1 (the gene encoding the major enzymes of 25(OH)D catabolism) and CYP27B1 (the gene encoding the enzyme responsible for converting 25[OH]D to its active metabolite 1,25[OH] 2 D) on risk of disease. Our major findings were that lower vitamin D status was independently associated with susceptibility to PCOS, but that genetic variation in VDR, DBP, CYP24A1, and CYP27B1 was not. Moreover, we found no evidence of any interaction between serum 25(OH)D levels and genetic variation in the vitamin D pathway on disease risk.
Our finding that vitamin D deficiency associates independently with PCOS risk in women in Pakistan is consistent with reports from other settings including the Netherlands 10 , Iran 16 , Egypt 17 and the USA 18 , as well as with findings from a meta-analysis including these and other studies 19 , showing that mean 25(OH)D level was lower in women with PCOS than controls. The high prevalence of vitamin D deficiency seen among participants in the current study is in keeping with our previous study in Lahore showing a high prevalence of vitamin D deficiency among women of reproductive age 20 . These and other studies reporting high rates of vitamin D deficiency in other groups 21 , highlight that vitamin D deficiency is a major public health problem in Pakistan 22 . The adjusted odds ratio for the association between vitamin D deficiency at the pre-specified 25(OH)D cut-off of 10 ng/ml was higher than has been reported elsewhere; this primarily reflects the very low prevalence of 25(OH)D values <10 ng/ml among controls in the current study. Exploratory analyses investigating 25(OH)D cut-offs of 15 ng/ml (aOR 3.41, 95% CI 1.77 to 6.57), 20 ng/ml (aOR 1.79, 95% CI 0.96 to 3.37), or analyzing 25(OH)D as a continuous variable (aOR 0.98, 95% CI 0.96 to 1.01) showed consistent inverse associations between 25(OH)D level and PCOS risk. Our findings with regard to other factors associated with PCOS, such as higher BMI, reduced physical exercise, higher fasting glucose levels and positive family history are also in keeping with the literature [22][23][24][25] .
In contrast to these positive findings, the lack of association seen between polymorphisms in VDR in our study contrasts with findings of a recent meta-analysis reporting that the VDR ApaI (rs7975232) polymorphism associated with susceptibility to PCOS in Asian populations (aOR for allelic model, C vs A, 1.19; 95% CI 1.07 to 1.34) 6 . The lack of association seen for SNPs in DBP is consistent with findings of both the other studies that have investigated polymorphisms in this gene 26,27 .
Our study has several strengths. Cases comprised a broad range of PCOS phenotypes, including obese and lean, fertile and infertile, and those with and without hyper-androgenic characteristics. We collected detailed information on potential confounders of the association between vitamin D status and PCOS risk, minimizing the potential for residual/unmeasured confounding to explain our findings. We also investigated SNPs in a wider range of vitamin D pathway genes than previously investigated, and applied a stringent correction for multiple testing. By deseasonalizing 25(OH)D data, we were able to calculate individuals average 25(OH)D level throughout the year, which represents their vitamin D status more effectively than a season-specific 'snapshot' provided by a single unadjusted reading 28 .  www.nature.com/scientificreports www.nature.com/scientificreports/ Our study also has some limitations. Due to the case-control design, reverse causality and/or confounding cannot be excluded as explanations for the associations observed. Although large by comparison with others in the field, our power to detect modest genetic effects, and gene-environment interactions, was limited. 25(OH)D was measured by ELISA rather than with Liquid Chromatography Tandem Mass Spectrometry, which is the gold standard methodology; however, this should not have introduced bias, since the same assay was used to measure vitamin D status in cases and controls. Moreover, the assay we used detects both 25(OH)D 2 and 25(OH)D 3 .

conclusion
In conclusion, this large case-control study-the first of its kind to be conducted in Pakistan-reports that low vitamin D status associates independently with increased susceptibility to PCOS. However, no statistically significant association between polymorphisms in vitamin D pathway genes and risk of PCOS was demonstrated.

Study design.
We conducted a case control study. Cases were patients aged 14 to 49 years diagnosed with PCOS and recruited from out-patient clinics at the Jinnah Hospital and the Lady Willingdon Hospital, Lahore. A total of 235 women were selected according to Rotterdam criteria, 2003 29 . Patients with thyroid and adrenal diseases and androgen-secreting tumours were excluded. 235 healthy controls were selected from the Citi lab and Research centre, Lahore, on the basis of having no history of infertility, no evidence of clinical hyperandrogenism and normal menstrual cycles. Informed consent was taken from all participants who fulfilled eligibility criteria. Written informed consent was taken from the parents or guardians of the participants who were under 18 years of age. Participants completed a detailed questionnaire including details of age, weight, height, sociodemographic status, dietary habits, physical exercise, sun exposure, androgenic features (acne, hirsutism and patchy hair loss), menstrual cycle history, family history of PCOS and fertility details for married participants. 5 mL of blood was drawn on the 2 nd or 3 rd day of menstrual cycle 30 , from a median cubital vein; 2 mL were transferred into vials containing EDTA and frozen at −20 °C for subsequent DNA extraction, and 3 mL was added to serum vials and sent to the laboratory within two hours of collection, where serum was isolated from clotted blood by centrifugation and stored at −20 °C for subsequent determination of 25(OH)D concentration.

Serum 25(OH)D assay. Serum 25(OH) D concentration was determined by ELISA (Calbiotech, EI Cajon
U.S.A). Calibrators and controls for the assay were run in duplicates. Interassay CV for serum 25(OH)D for our samples was 14%. Season-adjusted (deseasonalized) values for 25(OH)D were calculated for each participant from their individual standardized 25(OH)D concentration and date of blood sample collection, using a sinusoidal model with values derived from standardized values for all participants as previously described 28,31 . Vitamin D deficiency was defined using a pre-specified 25(OH)D cut-off of 10 ng/ml; this cut-off was selected a priori on the basis that it is widely used by Public Health bodies 32 and that deficiency at this level has been shown to associate most strongly with PCOS 11 and other pathologies attributable to vitamin D deficiency 33-35 . Genotyping. Genomic DNA was extracted from whole blood and quantified using a nanodrop spectrophotometer as previously described 36   www.nature.com/scientificreports www.nature.com/scientificreports/ of nuclease-free water. 3 µL of sample pre-mix and 3 µL of assay mix into each assay inlet of the 192.24 arrays.
Pressure fluid was then pipetted into the appropriate wells. Statistical analysis. Statistical analyses were done with Stata IC (version 15.1). Frequencies of alleles and genotypes were compared using chi-square tests; all were found to be in Hardy-Weinberg equilibrium. Baseline characteristics of cases vs controls were compared using unpaired Student's t-tests and chi-square tests for continuous and categorical variables, respectively. Chi-square tests were used to test for associations between independent variables and risk of PCOS in univariate analysis. Binary logistic regression was used for multivariate analysis of phenotypic determinants of PCOS risk, with adjustment for factors found to associate with PCOS with P < 0.05 in the univariate analysis of phenotypic determinants. Binary logistic regression was also used to test for association between genotype and risk of PCOS, using an additive model and adjusting for phenotypic factors found to associate independently with PCOS risk (age, body mass index, waist-to-hip ratio, deseasonalized 25(OH)D <10 vs. ≥10 ng/ml, outdoor exercise and fasting glucose). Sub-group analyses were performed to determine whether genetic variation in the vitamin D pathway modified effects of vitamin D status on susceptibility to PCOS by repeating primary efficacy analysis with the inclusion of a term for the interaction between vitamin D status and genotype, using an additive model. Haplotype analysis was performed using the Clark method. Odds ratios are presented with 95% confidence intervals and P values. The Benjamini-Hochberg procedure for multiple testing correction 37 was applied to genetic analyses to control the false discovery rate (FDR) at 5%. power and sample size. Assuming the risk of vitamin D deficiency (serum 25[OH]D <10 ng/ml) in the control arm to be 56% 38 , we calculated that 235 cases and 235 controls would need to be recruited in order to detect an odds ratio for the association between vitamin D deficiency and risk of PCOS of ≥1.86 with 80% power and an alpha of 5%.

Data availability
The primary data for this study is available from the corresponding author on direct request.