Unraveling the interplay between vitamin D deficiency, VDR polymorphisms, and polycystic ovary syndrome

Unraveling the interplay between vitamin D deficiency, VDR polymorphisms, and polycystic ovary syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unraveling the interplay between vitamin D deficiency, VDR polymorphisms, and polycystic ovary syndrome Sanchari Chakraborty, Randrita Pal, Farzana Begum, Tapan Kumar Naskar, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5417644/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Polycystic ovary syndrome (PCOS) is a complex and emerging heterogeneous disorder in reproductive-aged women and teenagers. Vitamin D deficiency (VDD) and genetic variations in the vitamin D receptor ( VDR ) pronouncedly influence its manifestations. The interplay between VDD and VDR polymorphisms has an umbrella effect on the endocrine and metabolic milieu of PCOS, underscoring the importance of VD in its management. This study tried to find out: How how VDD and single-nucleotide polymorphisms (SNPs) in the VDR gene influence the pathophysiology of PCOS, and how do these associations vary across different ethnic groups? Methods: A case-control study was conducted involving 80 PCOS women (ages 17–36 years) and 100 of their gender, and age-matched healthy controls (HC) belonging to the ethnicity of West Bengal, India. VDD and VDR polymorphisms [BsmI (rs1544410) and FokI (rs2228570)] were estimated by biochemical assessment and PCR-RFLP, respectively. Bioelectrical impedance and structured questionnaires were used for evaluation of anthropometric indices, sunlight (UVB) exposure, and nutritional status, respectively. Results: This study finds significant correlations between VDR variants and insulin resistance, hyperandrogenism, inflammatory markers, and obesity indices. Mutant VDR genotypes (BsmI-bb/Bb, FokI-ff/Ff) influence metabolic and cutaneous features, suggesting a genetic basis for VD-related disturbances in PCOS. Conclusions: The study accentuates the need for personalised therapeutic strategies, particularly VD supplementation, based on genetic profiles to manage PCOS and its associated metabolic disturbances. Key Message: VDD, a genetic predisposition related to VDR -SNPs, combined with limited sun exposure and poor dietary choices, exacerbates PCOS symptoms, impacting metabolic and endocrine homeostasis. Vitamin D deficiency Vitamin D receptor polymorphism Polycystic ovary syndrome PCR-RFLP BsmI FokI Figures Figure 1 Figure 2 Figure 3 Introduction Polycystic ovary syndrome (PCOS) is a complex, polygenic disorder affecting reproductive-aged women, characterized by hyperandrogenism, menstrual irregularities, and PCO morphology (PCOM), leading to infertility and miscarriage [ 1 – 3 ]. There is a complex interplay between genetic predisposition and environmental factors in manifestation of PCOS and its prevalence varies from 5–10% globally, 4–20% in India and 28–75% in West Bengal [ 1 – 4 ]. Research shows a high incidence of vitamin D deficiency (VDD) in women with PCOS, affecting 67–85% of cases, and correlating with insulin resistance (IR) and metabolic syndrome (MetS) [ 5 , 6 ]. Vitamin D (VD) plays a crucial role in reproductive health, influencing processes like follicle maturation, anti-müllerian hormone (AMH) signaling, follicle-stimulating hormone (FSH) sensitivity, progesterone secretion, and insulin sensitivity [ 3 , 6 ]. It also helps to manage dyslipidaemia and reduces the bioavailability of TGF-β1, improving ovarian function and mitigating clinical hyperandrogenism [ 3 ]. Moreover, VDD impairs glucose transporter (GLUT4) function, exacerbating IR in PCOS [ 3 ]. Emerging evidence links VDD to long-term risks of MetS and cardiovascular diseases (CVDs) in PCOS patients, highlighting the need for further research into VD's broader impact on the syndrome [ 3 , 7 ]. These findings underscore the potential therapeutic value of VD supplementation in managing PCOS and its associated metabolic and reproductive complications. VD exerts its biological effects through the vitamin D receptor (VDR), a ligand-activated transcription factor encoded by the VDR gene on chromosome 12q13.11. This gene spans 2776 genomic positions and translates a 427-amino-acid protein. VDR influences the expression of 229 genes, or about 3% of the human genome, across various tissues, including the hypothalamus-pituitary-ovarian (HPO) axis, granulosa cells, theca cells, endometrium, and placenta [ 3 , 5 ]. These interactions are crucial for maintaining reproductive health. The regulation of VD metabolism primarily occurs through genetic variations in VDR , vitamin D binding protein ( VDBP/GC ), and cytochrome P450 ( CYP ) family genes. Key enzymes involved include 25-hydroxylase ( CYP2R1 , VD to 25-hydroxyvitamin D), 1-hydroxylase ( CYP27B1 , activates 25-hydroxyvitamin D to calcitriol), and 24-hydroxylase ( CYP24A1 , inactivates VD metabolites) [ 7 , 8 ]. VDR plays a central role by binding to calcitriol [1,25(OH)2D3] and activating gene transcription that regulates these enzymes [ 6 , 7 ]. VD and VDR interactions are also significant for ovarian steroidogenesis where VDR upregulates the mRNA expression of CYP19 (aromatase) and downregulates CYP17 , both of which are involved in the production of estrogen [ 9 , 10 ]. Furthermore, CYP11A1 , a regulator of steroidogenesis, can metabolize vitamin D3 in the placenta and adrenal glands [ 11 ]. In uterine stromal cells, 1,25(OH)2D3-VDR interactions increase the mRNA expression of HOXA10 , a gene crucial for implantation and embryonic development [ 12 ]. Variations in VDR gene expression, such as single nucleotide polymorphisms (SNPs), can affect VD binding, leading to VDD and disrupted metabolic and endocrine functions, contributing to PCOS and related complications [ 3 , 5 , 13 ]. The prevalence of VDD varies significantly, ranging from 20–60% worldwide, 34–99% across India and 51–93% in West Bengal [ 14 , 15 ]. Factors contributing to VDD include low solar ultraviolet-B (UVB) exposure due to indoor preferences, a sedentary lifestyle, inadequate dietary intake of VD, and a preference to use sunscreen cosmetics [ 16 ]. The prolonged existence of these habits may exacerbate symptoms of multifaceted conditions like PCOS [ 13 ]. Despite residing in a tropical climate and having sufficient exposure to sunlight along with dietary intake of VD, a notable number of individuals are found to be VDD, suggesting the impact of predisposed genetic factors, particularly polymorphisms in the VDR , ( VDBP/GC ), and CYP family, such as CYP2R1 , CYP24A1 , and CYP11A1 [ 3 , 8 , 16 ]. SNPs of VDR predominantly regulate the metabolism, circulating levels, and activities of VD [ 6 , 7 , 17 ]. The SNPs in introns could potentially generate alternate splicing, skipping of exons, modulation of nuclear export, transcription rate, and stability of the transcript, while exonic SNPs can cause non-synonymous changes that may modify the protein structure [ 6 ]. It was reported that the ApaI (rs7975232) and TaqI (rs731236) SNPs in VDR have been linked to insulin signalling deregulation and IR in Iranian PCOS populations [ 18 , 19 ]. In Egyptian PCOS populations, TaqI variants in untranslated regions (UTRs) have been associated with PCOM, ovulatory dysfunction, and hyperandrogenism, impacting ovarian steroidogenesis [ 20 , 21 ]. VDD may also contribute to obesity, IR, and CVDs in PCOS patients [ 3 , 22 ]. A cross-sectional study of VDR suggests that ApaI (rs7975232) polymorphism is associated with hypertriglyceridemia and MetS, while the variants of TaqI (rs731236) and BsmI (rs1544410) seem to affect high-density lipoprotein cholesterol (HDL-C), and the VDR- FokI (rs2228570) variants affect the systolic blood pressure, which is ambiguous in the Chinese population [ 23 ]. It was reported that VDR -BsmI (rs1544410) polymorphism can trigger CVD, an alarming feature of PCOS in later age [ 2 , 23 – 25 ]. A study report from Hyderabad, India, stated that VDR polymorphisms of ApaI (rs7975232) and TaqI (rs731236) are associated with PCOM, hyperandrogenism, ovarian dysfunction, and obesity in PCOS individuals [ 22 ]. Variants of BsmI are associated with similar symptoms, including obesity and IR across various regions [ 6 , 25 ]. Additionally, in PCOS patients, VDR- FokI (rs10735810) polymorphism is associated with infertility, alopecia, hirsutism (India), high insulin levels and IR (Iran), and increased total testosterone (TT, Korea) [ 18 , 22 , 26 , 27 ]. Studies on different ethnic populations indicate the association of VDR polymorphisms (BsmI and FokI) with VDR . SNPs of the GC gene (rs4588 and rs7041) and Cdx2 (rs11568820), which are often found to be interlinked with altered VDR expression, leading to VDD-associated differential phenotypic manifestations and metabolic alterations of PCOS [ 22 , 28 , 29 ]. Overall, diverse manifestations of PCOS appear to be influenced by specific VDR polymorphisms across different ethnic populations. West Bengal, the eighth-most populous region globally and the fourth-most populous state in India, is celebrated for its diverse cultures and traditional cuisines. The predominant ethnic group, the Bengalis, primarily consists of Indo-Aryans with a unique genetic heritage influenced by Iranian hunter-gatherers, Central Asian pastoralists, and South Asian populations [ 30 ]. This genetic diversity may affect the prevalence of certain health conditions and responses to treatments [ 31 ], particularly in conditions like PCOS. According to the National Family Health Survey, approximately 30% of women in Kolkata are classified as obese [ 31 ]. Data from the West Bengal Health Statistical database highlight a concerning link between PCOS and major health issues such as diabetes and CVD. However, research on VDD and insufficiency (VDI) and their impact on PCOS within West Bengal's ethnic communities are limited. This lacuna has prompted the current study, which aims to explore the association between specific polymorphisms of the VDR -namely, BsmI in intron 8 (rs1544410, A > G, B > b) and FokI in exon 2 (rs2228570, C > T, F > f)—and the pathophysiology of PCOS in this population. The research could yield insights for developing personalised therapeutic strategies and optimising VD supplementation based on genetic profiles. Materials and Methods Study Design and Sample Size The case-control study enrolled 80 women with PCOS (PCOS, N=80), aged 17 to 36 years, experiencing menstrual and fertility complications, along with 100 gender- and age-matched healthy control participants (HC, N=100) in the ethnic population of West Bengal. Approved by the Human Ethics Committee of Medical College and Hospital, Kolkata (MC/KOL/IEC/NON-SPON/1275/02/22) and the Calcutta University Institutional Ethics Committee (CUIEC/03/40/2022-23), the research took place in the outpatient department of gynaecology and obstetrics of Medical College and Hospital, from September 2022 to June 2023. All participants were informed of the study's societal and personal relevance, and written consent was obtained from each volunteer using structured trilingual consent forms. Study Subject Selection Case group Inclusion criteria The Rotterdam criteria, 2003 [European Society of Human Reproduction and Embryology (ESHRE )/ American Society for Reproductive Medicine (ASRM)], were used to incorporate PCOS women in the study. The syndrome was diagnosed based on two of the following three criteria: (a) an intermenstrual interval of ≥35 days (oligomenorrhea) or >3 months (amenorrhea); (b) gynaecological ultrasound of PCOM (size: 2-9 mm, number: >12, and ovarian volume: >10 cm 3 ); and (c) clinical and/or biochemical hyperandrogenism [2, 3, 32]. Exclusion criteria Women having physical and/or intellectual disabilities, pregnancy, and lactation were excluded [4]. Congenital adrenal hyperplasia (CAH), prolactinoma, virilizing tumours, and Cushing syndrome mimicking PCOS were also disregarded as potential etiologies [2, 30]. Medical history of oncological patients, those previously or presently undergoing treatment, such as hormonal medication and contraceptives (progesterone) injections prior to the screening, and implausible energy intake (≤500 or ≥4500 kcal/day) were also ruled out [2, 3]. Control group The participating healthy control individuals had regular menstruation, no evidence of clinical and/or biochemical manifestations of hyperandrogenism, no abnormality in their endocrine profile, no PCOM on ultrasonography, and neither a history of drug ingestion nor an unbalanced diet [2, 3]. Assessment of Sunlight Exposure A detailed questionnaire was designed to record time (between 06:00 AM-11:00 AM, 11:00 AM-03:00 PM and 03:00 PM-05:00 PM) and duration [(120) minutes per day] of sunlight exposure [16]. Cutaneous Manifestation Hirsutism was evaluated using the modified Ferriman-Gallwey (F-G) score, and alopecia was assessed by the Norwood scale [4]. Velvety and pigmented skin in the neck and antecubital fossa region were categorized as acanthosis nigricans (AN), while nodules in the face, neck, and back were classified as acne [4]. Assessment of Nutritional Status Dietary assessment Indian Council of Medical Research (ICMR) guidelines were followed to evaluate the nutritional status of the participants [13, 33, 34]. The food frequency questionnaire (FFQ) method was applied to assess the nutritional status of the study population [13, 33-35]. Categorization of food items Nine food items, including millets and cereals (n=2), wheat (n=2), vegetables (green leafy and others, n=91), grain legumes (n=25), roots and tubes (n=19), fish (freshwater and marine, n=101), poultry (n=6), animal meat (n=13), egg (n=1), fruits (n=68), nuts and oil seeds (n=4), milk (n=1), and food ingredients, such as condiments and spices (dry and fresh, n=26), and cooking oil (n=1) were taken to be analyzed [13, 33-35]. Quantifying common measures Different-sized bowls and spoons were used to measure the weight of different food items. The average of the food components was evaluated [13, 33-35]. Evaluation of Anthropometric Indices The height (cm) of the barefoot participant was measured using a portable ultrasonic-based handheld digital stature height measuring scale (make: EasyCare EC1800, India). Body mass index (BMI, kg/m^2), visceral fat, and the percentage of skeletal muscle mass in the whole-body of the least-clothed individuals were estimated using a body composition monitor (Model: OMRON-HBF-375, Karada Scan, Kyoto, Japan) based on bioelectrical impedance analysis [2]. The digit 2D:4D ratio was obtained by dividing the length (mm) of the index finger [second (2nd) digit] by the ring fingers [fourth (4th) digit], which were measured from the fingertip to the midpoint of the basal crease on the dorsal surface of the left hand using digital vernier callipers (make: ZHART, Rajasthan, India) [4] . Biochemical Assay Collection of saliva The volunteers were requested to rinse their mouths with water and refrain from any food or beverage (except water) for 1 hour before their saliva collection. After that, every individual was requested to sit in an upright, comfortable position and tilt their head forward to collect whole saliva samples within the same time interval of day by the passive drool method in an ice-chilled graduated cryovial (1.80 ml) through a saliva collection aid (Salimetrics, Item No. 5016.02; Carlsbad, United States). Immediately the saliva-filled cryovials were transferred to a mini-cooler and subsequently stored in a -20°C freezer for further analysis [2]. Enzyme-linked immunosorbent assay of interleukin 6 The salivary interleukin-6 (IL-6) concentration was determined using the sandwich-enzyme linked immunosorbent assay (ELISA) principle (FineTest, EH0201, Wuhan, China). The reference range (pg/ml) is 4.1-17.5, and the intra- and inter-assay CV% were <8 and <10, respectively. Firstly, the saliva samples were centrifuged at 1000×g at 4 ° C for 20 minutes, and the supernatants were used for further steps of the assay. The salivary IL-6 was evaluated spectrophotometrically of absorbance at the 450 nm wavelength in a microplate reader [make: Bio-rad, Model No.: iMark (Microplate) reader, SL No. 10095, California, United States]. Blood parameters After a 12-hour fast, blood samples from PCOS patients were taken by venipuncture in the early morning hours of the second or third day of menstruation (early follicular phase). Serum 25(OH)D concentration of the participants [reference interval (ng/ml): 10-20 (VDD), 20-30 (VDI/suboptimal) and 30-50 (sufficient/optimal)] was estimated by the Alinityi (Abbott) system and the chemiluminescent microparticle immunoassay (CMIA) method [assay CV% (intra=3.66 to 6.56 and inter=4.19 to 7.01), fasting insulin (FI) and postprandial insulin (PPI) by [system: Alinityi (Abbott), method: chemiluminescent microparticle immunoassay (CMIA)]; fasting glucose (hexokinase; automated biochemistry analyzer-DXC 700AU/COBAS 501); TT, luteinizing hormone (LH) and FSH by (system: COBAS e 411, method: electrochemiluminescence); HDL, LDL, and triglyceride [enzymatic colour, and GPO-POD methods, automated biochemistry analyser (AU 680/COBAS 501)], C-reactive protein by (CRP, Nephelometric immunoassay Batman coulter image/immuno turbidimetric), calcium, and phosphate by [automated biochemistry analyser-DXC 700AU/COBAS 501); Arsenazo III method, Inorganic, Molybdate UV method, respectively] parathyroid hormone by (PTH, enzyme-linked fluorescent assay, Mini Vidas system) were estimated from the drawn blood samples. Homeostatic model assessment for IR (HOMA-IR), quantitative insulin-sensitivity check index (QUICKI), fasting glucose to insulin ratio (FGIR), and triglyceride glucose (TyG) index were evaluated by applying the standard formulae [3, 4]. Study of VDR Polymorphism Collection of buccal swabs Swab samples were collected from the buccal cavities of the participants using sterile cotton swab sticks. The subjects were advised to abstain from eating or drinking anything (except water) for one hour before the collection of the swab. To collect the swab in the cotton head region of the swab stick, the handles of the sticks were held in the subject's wide-open mouth, and the cheek was rubbed in a twisting motion for 60 seconds [36]. The swab containing the head area of the stick was carefully transferred to an ice-chilled cryovial (1.80 ml) without touching any surfaces by discarding the tail part using a sterile scissor. After that, the cryovial was instantly transferred to a minicooler and then stored in a -20°C freezer. The stored swabs were further used for deoxyribonucleic acid (DNA) isolation. Isolation of genomic DNA DNA from the buccal swab was isolated using the phenol-chloroform (PCI) organic extraction method [37]. The quality was defined by using 1% agarose gel electrophoresis, and the quantity was estimated by UV absorbance at 260 nm in a UV spectrophotometer (make: SHIMADZU, UV-1780, Kyoto 604-8511, Japan) of the isolated genomic DNA [36, 37]. SNPs of VDR The compendium analysis of VDR has been performed using GeneCards and the HUGO gene nomenclature committee (Tables 1.a and b). The details of restriction endonuclease (BsmI and FokI) have been comprehended from New England Biolabs (Table 1.c). The primers were designed based on the primer design tool, primer-BLAST (Gene ID: 7421, >NC_000012.12:c47904994-47841537 Homo sapiens chromosome 12, GRCh38.p14 Primary Assembly, NCBI, Bethesda, USA). The VDR was amplified [polymerase chain reaction (PCR)] using isolated genomic DNA samples (Table 1.d) and then checked for two SNPs [BsmI (rs1544410) and FokI (rs2228570), make: New England Biolabs, Ipswich, Massachusetts, United States] by the PCR-restriction fragment length polymorphism (PCR-RFLP) method to generate distinct polymorphic fragments (Table 1.e) [22, 37-39]. The amplified (PCR) and digested (PCR-RFLP) products were visualised by 2% and 3% agarose gel electrophoresis, respectively. The obtained order of the nucleotides of Sanger sequencing (Applied Biosystems® Sanger Sequencing 3500 Genetic Analysers, Foster City, USA) was determined by chromatogram (Sequence Scanner 1.0; Life Technology Corporation, Waltham, Massachusetts, United States). Software and Data Analysis Sample size In the current case-control study, the sample size [odds ratio (OR): 4 and confidence interval (CI): 0.95] was determined based on EPITOOLS, Ausvet, epidemiological web-based calculators (Fremantle, Australia) [3]. Metabolic profile The collected data was analysed in statistical software for the social sciences (SPSS, IBM Corp., version-20, Chicago, Illinois, United States). The Shapiro-Wilk (S-W) normality test was performed to determine the VDR -polymorphism (BsmI and FokI: mutant)-based 25(OH)D distribution pattern. Simple boxplot [dispersion of 25(OH)D among VDR polymorphisms (wild types and mutants) and dietary profile], independent samples-t test [comparisons of dietary intake (between case-control group) and IR status (between different genotypes of BsmI and FokI in VDR ], bivariate Pearson correlation [estimation of the association between 25(OH)D with TT and HDL, and adiposity indices], and a stacked bar chart (sunlight exposure-time and duration) were applied. The statistical significance was considered at P<0.05 and P<0.01. The technical error was within the limit [2]. Genetic polymorphism Frequencies of allele and genotype in VDR (BsmI and FokI), exact test for Hardy-Weinberg equilibrium (HWE) of case (PCOS) and controls, OR, and 95% CI of dominant model were statistically analysed using SNPStats (Institut Catala d'Oncologia , Spain), a web tool (https://www.snpstats.net/start.htm) [40, 41]. Results Genetic polymorphisms of VDR , levels of 25(OH)D and hyperandrogenic parameters (TT, LH:FSH ratio, and 2D:4D ratio), indices of the metabolic panel (PTH, calcium, phosphate, LDL-HDL ratio, and TyG index), inflammatory markers (CRP and IL-6), anthropometric indices (visceral fat and skeletal muscle mass whole body), and cutaneous manifestations were illustrated in PCOS patients and healthy controls. However, a few participants were dropped from the survey midway through the study due to a loss of willingness and were unable to provide blood, saliva and swab samples. Fig. 1 [a] Comparative study of vitamin D status - [a.1] time range and [a.2] time duration of sunlight exposure [a.3] vitamin D levels in blood, [a.4] dietary intake of vitamin D; [b] Comparative analysis of nutritional status of [b.1] calcium, [b.2] phosphorus, [b.3] carbohydrate, [b.4] fat, and [b.5] protein between the case-control group. Fig. 1.a.1 indicated that PCOS patients (n=86.25%) were less exposed to sunlight (time: 11:00 AM to 03:00 PM) compared to the control individuals (n=73%). Furthermore, 85% of PCOS patients showed indoor preference (sunlight exposure time duration: <30 mins. to 120 mins.), whereas 78% of control individuals had this preference ( Fig. 1.a.2 ). It was observed that VD status in blood [25(OH)D, P=0.650] and nutritional profile (combination of ergocalciferol-D2, cholecalciferol D3 and calcifediol, P=0.820) was lower in the PCOS patients than the control group ( Fig. 1 . a.3 and 4 ). Furthermore, dietary intake of carbohydrate (P=0.003), fat (P=0.169), protein (P=0.198) and phosphorus (P=0.074) was found to be higher in the PCOS group than in control individuals, whereas calcium (P=0.516) intake reflected the inverse relationship ( Fig. 1.b.3-5 ). Fig. 2 [a.1] Genomic location, [a.2] structure of VDR gene; [b] Representative structure of vitamin D3 receptor protein; [c] Ethidium bromide (EtBr)-stained agarose gel (PCR: 2% and PCR-RFPL: 3%) analysis of [c.1] PCR-amplified undigested VDR gene: BsmI (360 bp: lane 2 to 5) and FokI (265 bp: lane 6 to 9), [c.2] PCR-RFLP digested VDR -FokI: Ff heterozygous genotype (265 bp, 169 bp and 96 bp) in lane 2 and 3, FF wild type genotype (265 bp) in lane 4 and 6 and ff homozygous genotype (169 bp and 96 bp): lane 5; and VDR -BsmI: bb homozygous genotype (191 bp and 169 bp) in lane 10 and 11, BB wild type genotype (360 bp) in lane 12 and 13 and Bb heterozygous genotype (360 bp, 191 bp and 169 bp) in lane 14; [d] DNA Sanger sequencing analysis: [d.1] VDR -BsmI: BB genotype, [ d . 2 ] VDR -BsmI : Bb genotype, [d.3] VDR -BsmI : bb genotype, [d.4] VDR -FokI : FF genotype, [d.5] VDR -BsmI : Ff genotype and [d . 6] VDR -FokI : ff genotype. BB=AA, Bb=AG, bb=GG, FF=CC, Ff=CT, ff=TT. Nucleotide base: “A” (adenine, green peak), “G” (guanine, black peak), “C” (cytosine, blue peak) and “T” (thymine, red peak). The positions of FokI-rs2228570 (in exon 2) and BsmI-rs1544410 (in intron 8) of VDR on chromosome 12q13.11 were illustrated ( Fig. 2.a. 1 and 2 ). The representative three dimensional vitamin D3 receptor structure was depicted ( Fig. 2.b ). The PCR amplified product ( Fig. 2.c.1 ), PCR-RFLP of transition mutations of BsmI (A>G, B>b) and FokI (C>T, F>f) ( Fig. 2.c.2 ), and order of nucleotides were illustrated ( Fig. 2.d.1 to 6 ). Fig. 3 [a] Comparison of 25(OH)D between mutant variants of BsmI (bb+Bb) and FokI (ff+Ff), [b] Distribution pattern of 25(OH)D in vitamin D receptor ( VDR ) polymorphisms (wild-type and mutants: BsmI and FokI) in PCOS patients (S-W=Shapiro-Wilk normality test, P<0.01 and 0.05). BB=AA, Bb=AG, bb=GG, FF=CC, Ff=CT, ff=TT. 25(OH)D was found to be asymmetrically (S-W=0.000) distributed in both mutant [BsmI (bb+Bb) and FokI (ff+Ff)] types of VDR ( Fig. 3 . a ). Furthermore, 25(OH)D was slightly (P=0.763) higher in mutant (bb+Bb) BsmI than the mutant (ff+Ff) FokI ( Fig. 3 . a ). Additionally, 25(OH)D level (ng/ml) was higher (16.00±7.30) in wild type (FF) relative to mutant types [ff (13.02±6.70) and Ff (13.00±5.31)] of FokI, and wild type [BB (12.62±4.93)] and mutant types [bb (12.65±7.30) and Bb (13.00±6.45) of BsmI ( Fig. 3 . b ). It was found that PCOS group was deviated (P=0.019) from HWE in VDR -FokI polymorphism ( Table 2.a ). However, control (P=0.055) and all participants (P=0.73) were within HWE in VDR -FokI polymorphism ( Table 2.a ). Furthermore, in VDR -BsmI, PCOS, control and all individuals were in HWE (P>0.05). In the dominant model of VDR -FokI (C/C, C/T-T/T) polymorphism OR (95% CI) was 0.23 (0.08-0.71), P=0.0085 and VDR -BsmI (A/A, A/G-G/G) polymorphism OR (95% CI) was 0.86 (0.30-2.48), P=0.77 ( Table 2.b ). In the PCOS study population, 25(OH)D had a direct (P>0.05) association with HDL, while an inverse (P>0.05) relationship was found with TT, visceral fat, and BMI ( Table 3.a ). Comparative analysis of the association of IR with the VDR- BsmI polymorphism in the PCOS study group depicted that HOMA-IR was higher (P=0.042) and FGIR was lower (P=0.021) in the mutant types (bb+Bb) relative to the wild type (BB) of the VDR- BsmI polymorphism ( Table 3.b ). Interestingly, mutant types (ff+Ff) showed lower (P=0.977) HOMA-IR and higher (P=0.837) QUICKI values compared to wild type (FF) in VDR- FokI polymorphism ( Table 3.b ). In the PCOS individuals, the cutaneous manifestations of hyperandrogenism were 98.75%, and alopecia predominated (96.20%) over AN (74.68%), hirsutism (69.62%), and acne (36.71%) ( Table 3.c.1 ). The phenotypes AN (77.78%), alopecia (76.47%), hirsutism (72.41%), and acne (64.29%) were prevalently expressed in PCOS patients with heterogeneous (Ff) mutants of the VDR -FokI polymorphism ( Table 3.c.2 ). In the VDR (BsmI and FokI) polymorphism-based comparative study, it was found that TT (ng/ml) was predominant (0.34±0.22) in BB-BsmI, where high LH:FSH ratio and low 2D:4D ratio were prevalent in Ff-FokI (1.36±1.31) and ff-FokI (0.92±0.04), respectively ( Table 4.a ). It was also found that high PTH (pg/ml, 46.10±15.01), calcium (mg/dl, 9.78±0.42), phosphate (mg/dl, 4.05±0.52) and TyG index (4.57±0.27) were prevalent in bb-BsmI, whereas high LDL:HDL ratio (2.16±0.7) was predominantly distributed in ff-FokI (Table 4.B). In Table 4.c , visceral fat (7.93±5.02) was highest in ff-FokI, and the skeletal muscle mass-whole body(%) was lowest (24.18±2.27) in BB-BsmI polymorphism ( Table 4.c ). The inflammatory markers like CRP (mg/dl, 0.64±0.44) and IL-6 (pg/ml, 14.00±5.06) were highest in bb-BsmI and FF-FokI, respectively ( Table 4.d ). Discussion 7-dehydrocholesterol in the skin absorbs UVB radiation (290–315 nm) from sunlight, converting it to provitamin D3, which is then isomerised into vitamin D3, the sunshine vitamin [ 42 ]. However, avoidance of sun exposure, sedentary lifestyles, and poor dietary choices can lead to obesity and vitamin D deficiency (VDD) [ 16 , 42 ]. Excess adipose tissue in obese individuals stores fat-soluble vitamin D (VD), dispersing it over a larger volume and resulting in lower serum concentrations [ 2 , 12 , 15 ] In this study, 86.25% of women with polycystic ovary syndrome (PCOS) reported avoiding UVB exposure, and 85% preferred staying indoors (Fig. 1 . a.1 and 2 ). Compared to controls, PCOS patients had lower (P > 0.05) VD levels and dietary quality (Fig. 1 . a.3 and 4 ). Notably, carbohydrate intake was higher (P = 0.003) in PCOS individuals, while no significant differences were found in fat, protein, phosphorus, or calcium intake (Fig. 1 . b.1 to 5 ). This excessive carbohydrate consumption may disrupt stearoyl-CoA desaturase activity, leading to triacylglycerol accumulation and contributing to obesity-related depletion of the ovarian reserve, a key characteristic of PCOS [ 3 , 13 , 43 ]. These findings underscore the interconnectedness of lifestyle, diet, and the pathophysiology of PCOS. Hardy-Weinberg equilibrium (HWE) is used to analyse allele frequencies in populations, with deviations indicating genotyping errors or consanguinity [ 44 ]. Heterozygote excess (HetExc) may signal natural selection highlighting heterozygous variations, especially in disease contexts [ 44 ], however, in our study In our study, heterogeneity is a disadvantageous condition. Parker et al. suggested that ancient PCOS genetic polymorphisms provided a survival advantage in prehistoric environments, but these same variants may reduce fitness in today's obesogenic context [ 45 ]. The study found that VDR -FokI polymorphism deviated (P = 0.019) from HWE in the PCOS population (Table 2 . a ), with heterozygous mutants (CT-TT) showing a significant association (P = 0.0085) with disease manifestation (Table 2 . b ). However, similar mutant variations in controls did not lead to the syndrome, suggesting protective mechanisms like a shielding effect during post-translational modifications and non-genomic interactivity (Table 2 ). VDD is predominantly (65–90%) contributed by genetic polymorphisms such as BsmI (rs1544410) and FokI (rs2228570) of the VDR [ 22 , 39 ]. VDR polymorphisms regulate circulatory calcifediol [25(OH)D] levels by feedback mechanism [ 6 , 22 , 39 , 46 , 47 ]. The SNP of VDR -BsmI can trigger a silent mutation, which leads to a reduction in the stability and expression of VDR without altering the amino acid sequence of the encoded protein [ 48 ]. Calero et al. reported that silent polymorphism can affect mRNA structure and ligand interaction specificity, which seems to modulate translation kinetics, tRNA-mediated post-transcriptional modifications, and protein folding [ 48 ]. Multiple studies on Indians, Chinese, Japanese, Caucasians, and Americans reported that the SNP of VDR -BsmI is in strong linkage disequilibrium (LD) with a polyadenosine [poly(A)] microsatellite repeat in the VDR- 3'UTR, which may affect either stability or translational activity of its mRNA [ 46 , 49 , 50 ]. Polymorphisms of FokI in the 5'-region of VDR can alter the nucleotide sequence of the start codon from ATG to ACG, resulting in a reduction of translational protein size by three amino acids relative to its normal counterpart, 424 from 427 amino residues [ 6 ]. The mutated variant f (T allele) of VDR weakly attaches with calcitriol in comparison to the longer VDR protein variant coded by the wild-type F allele (C allele), which often imitates the condition of VDD [ 6 ]. Various investigations on different ethnic populations such as India, China, Poland, and Australia reported substantial associations between FokI polymorphism, VDD, and the expression of PCOS-associated manifestations such as deregulated ovarian cell-intertwined hyperandirgenism and obesity [ 3 , 47 , 51 , 52 ]. VDD boosts PI3K/Akt pathway activity and androgen receptor (AR) expression in ovarian tissue by lowering ARA70 protein levels [ 51 , 53 , 54 ]. VD is essential in monitoring obesity by regulating lipid metabolism through stimulation of lipolysis, fat distribution patterns like visceral fat, and the adiposity indicator BMI [ 24 , 53 ]. Studies suggest that VDD inhibits fatty acid oxidation-associated lipolysis intertwined with declining Ca2 + levels, increased cAMP activity, and dephosphorylation of hormone-sensitive lipase [ 24 , 53 ]. Table 3 . a indicated that VD exhibited a positive correlation with HDL and an inverse relationship with obesity (visceral fat and BMI) and hyperandrogenism (TT). Ethnicity-based studies on PCOS hypothesised that VDR- BsmI and VDR- FokI polymorphisms are associated with VDD-induced IR by altering calcium flux and phosphorylation-mediated inhibition of GLUT4, inflammation by increased levels of TNFα, and hyperandrogenism by changing AR expression [ 3 , 8 , 53 ]. VDD-associated obesity leads to the release of proinflammatory cytokines (IL-6, CRP, IL-18 and TNF-α), which downregulates insulin signalling by altering suppressor of cytokine signalling (SOCS) and JUN N-terminal kinase (JNK) and leads to IR [ 3 , 8 , 22 , 55 , 56 ]. This abnormality often increases pustule secretion of LH, an alarming cause of hypeandrogenism [ 3 , 8 , 22 , 55 , 56 ]. The order of nucleotides of VDR (FokI and BsmI) polymorphism of the present study was illustrated in Fig. 2 . The asymmetrically (P = 0.000) distributed serum VD was lower in mutant (ff and Ff) types relative to wild-type (FF) in VDR- FokI polymorphisms (Fig. 3 ). IR was prevalent (P < 0.05) in mutants (bb + Bb) VDR- BsmI in the PCOS study group (Table 3 . b ). Alopecia was found to be the predominant (96.20%) cutaneous manifestation of hyperandrogenism in the PCOS patients (Table 3 . c.1 ). The findings of Table 3 . c.2 indicated that AN (77.78%), alopecia (76.47%), hirsutism (72.41%), and acne (64.29%) were prevalently expressed in heterogeneous (Ff) mutant of the VDR -FokI polymorphism. It was found that HA (high TT and LH:FSH ratio) was more frequent in the BB-BsmI and Ff-FokI of VDR , respectively, whereas in the 2D:4D ratio, no fascinating difference was observed between the wild-type and mutant variants of VDR polymorphism (Table 4 . a ). Elevated TT at the molecular level affects in multiple ways: (i) binding with ARs, particularly in target tissues such as the hair follicles, skin, and, sebaceous glands, and activating signalling pathways that can lead to increased hair follicle sensitivity, sebum production, and alters in keratinization, contributing to hirsutism and acne; (ii) it can influence the hypothalamic-pituitary-gonadal (HPG) axis by negative feedback mechanism that affects LH:FSH ratio imbalance often seen in PCOS, promoting further androgen production from the ovaries; (iii) increased TT can inhibit follicular development, leading to anovulation and the formation of cysts; through impairing granulosa cell functionality, (iv) elevated TT levels are often associated with IR, that can induce ovarian androgen production, creating a vicious cycle of hyperandrogenism and metabolic dysfunction. This interaction further complicates the clinical picture and can exacerbate symptoms; (v) elevated levels of TT can trigger the expression of genes involved in hair growth (e.g., androgen-responsive genes), leading to phenotypic alternations such as hirsutism [ 2 , 3 , 56 – 59 ]. Furthermore, it can impact the expression of enzymes like aromatase, which transforms androgens to oestrogens, abnormally altering the hormonal balance [ 2 , 3 , 56 – 59 ]. Though the potential link between the 2D:4D ratio and PCOS is under the scanner, some studies reported a significant link between a lower 2D:4D ratio and higher prenatal testosterone exposure [ 2 , 3 , 56 – 59 ]. All these three parameters—hyperandrogenism, LH:FSH ratio, and 2D:4D—showed an influential indication in the PCOS population under study. However, the result is not consistent with all aspects of HA with homo (bb/ff) and heterozygous (Bb/Ff) mutant genotypes, which implies further study with a larger sample size. Panidis et al. suggest that elevated levels of PTH may contribute to hyperandrogenism associated with PCOS, particularly in conjunction with obesity and reduced concentrations of VD metabolites [ 60 ]. The present findings indicate that VD is influencing the impact on insulin sensitivity, which could potentially alleviate the metabolic and reproductive complications linked to PCOS. PTH was higher than the normal reference range [reference interval (pg/ml): 9.2–44.6 (normal) and > 44.6 (high), PCOS PTH = 46.10 ± 15.01] in the homozygous (bb) mutant type of VDR -BsmI (Table 4 . b ). In VDR- FokI, the homozygous (ff) mutant type had a high LDL:HDL ratio, while the TyG index could not achieve any difference between homo and heterozygosity (Table 4 . b ). Table 4 . c indicated that the Bb, ff, and Ff might be associated with high levels of visceral fat, but the result of skeletal muscle-whole body is inconclusive. In addition to these, findings indicate that CRP was higher in the homozygous (bb) mutants of VDR -BsmI but not IL-6 (Table 4 . d ). IL-6 is a pro-inflammatory cytokine, and CRP is an acute-phase protein. Both are interconnected in the inflammatory response, where IL-6 can stimulate the liver to produce CRP [ 57 ]. In chronic inflammation like PCOS, metabolic dysregulation like IR, obesity, and MetS, or increased androgens, may influence and contribute to elevated CRP without significantly affecting IL-6 levels [ 57 ]. This important finding highlights the systemic inflammation in PCOS without affecting specific immune cells to produce IL-6. To comprehend better, further research involving other inflammatory markers like TNF-alpha and IL-10 needs to be carried out. Strengths and Limitations of the Study The study provides a comprehensive analysis of PCOS, exploring metabolic, hormonal, and cutaneous aspects alongside genetic factors, particularly VDR polymorphisms (BsmI and FokI). It identifies key correlations with insulin resistance, hyperandrogenism, and obesity, suggesting personalized vitamin D supplementation as a potential therapeutic approach. The study has several limitations, including a small sample size, cross-sectional design, and potential biases from participant dropouts, which affect generalizability. It lacks intervention data, doesn't account for environmental factors, and focuses mainly on VDR polymorphisms, missing other genetic factors in PCOS. Future research with larger sample sizes and further exploration of inflammatory markers and their role in PCOS will be crucial for better understanding and treatment of this complex syndrome. Conclusion By identifying specific VDR polymorphisms, such as BsmI and FokI, that influence vitamin D (VD) metabolism and related pathways, the study provides a clearer understanding of how genetic factors contribute to the diverse manifestations of PCOS. The findings suggest that vitamin D deficiency (VDD), influenced by both genetic and environmental factors, is prevalent in PCOS patients and correlates with metabolic and reproductive complications, including insulin resistance (IR), obesity, hyperandrogenism, and infertility. Additionally, these polymorphisms influence key metabolic pathways and hormonal imbalances, exacerbating conditions like hirsutism, acne, and anovulation. Furthermore, the findings suggest that correcting VDD could alleviate key metabolic disturbances in PCOS patients, potentially improving insulin sensitivity, regulating androgen levels, and addressing symptoms like hirsutism and anovulation. This could pave the way for more targeted interventions, where treatment plans could be tailored based on genetic predispositions, such as using personalised vitamin D supplementation regimens for those with specific VDR mutations. Declarations Funding Statement Department of Science and Technology and Biotechnology, Government of West Bengal (DSTBT-WB, Grant no. ST/P/S&T/9G-5/2018) Conflicts of Interest All authors (Sanchari Chakraborty, Randrita Pal, Farzana Begum, Tapan Kumar Naskar, Nilansu Das, Barnali Ray Basu) declare that there is no competing interest. The authors are solely responsible for the content and writing of the manuscript. Author Contribution(s): All authors read and approved the final manuscript. Conceptualization: [Barnali Ray Basu]; Data curation: [Sanchari Chakraborty], [Randrita Pal]; Formal analysis: [Sanchari Chakraborty], [Randrita Pal]; Funding acquisition: [Barnali Ray Basu], Investigation: [Sanchari Chakraborty], [Randrita Pal], [Farzana Begum]; Methodology: [Sanchari Chakraborty], [Randrita Pal], [Farzana Begum]; Project administration: [Barnali Ray Basu]; Software: [Sanchari Chakraborty]; Resources: [Barnali Ray Basu]; Supervision: [Barnali Ray Basu], [Tapan Kumar Naskar], [Nilansu Das]; Validation: [Barnali Ray Basu]; Visualisation: [Barnali Ray Basu], [Nilansu Das]; Writing—original draft: [Sanchari Chakraborty], [Randrita Pal]; Writing—review & editing: [Barnali Ray Basu], [Nilansu Das]. Funding Organization: Department of Science and Technology and Biotechnology, Government of West Bengal (DSTBT-WB, Grant no. ST/P/S&T/9G−5/2018) Funding Statement Department of Science and Technology and Biotechnology, Government of West Bengal (DSTBT-WB, Grant no. ST/P/S&T/9G-5/2018) Compliance with Ethical Standards Human Ethics Committee of Medical College and Hospital, Kolkata (MC/KOL/IEC/NON-SPON/1275/02/22) and the Calcutta University Institutional Ethics Committee (CUIEC/03/40/2022−23) Acknowledgement People and Technical Support: The authors are thankful to all the volunteer women in this study. The authors are also grateful to InBOL Healthcare educational Centre and Scientific Clinical Laboratory Pvt. Ltd. for their technical support. Data Availability Data will be made available to the corresponding author of the paper for review or query upon request. References Khan, M.J., Ullah, A., Basit, S. Genetic basis of polycystic ovary syndrome (PCOS): current perspectives. Appl. Clin. Genet. 12, 249–260 (2019). https://doi.org/10.2147/TACG.S200341 . Basu, B.R., Chowdhury, O., Saha, S.K. Possible link between stress-related factors and altered body composition in women with polycystic ovarian syndrome. J. Hum. Reprod. 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Indian Dermatol Online J. 10, 97–105 (2019). https://doi.org/10.4103/idoj.IDOJ_249_17 . Rudnicka, E., Suchta, K., Grymowicz, M., Ksepka A. C., Smolarczyk, K., Duszewska, A. M., et al. Chronic Low Grade Inflammation in Pathogenesis of PCOS. Int J Mol Sci. 22, 1–12 (2021). https://doi.org/10.3390/ijms22073789 . Wu, S., Yu, K., Lian, Z., Deng, S. Molecular regulation of androgen receptors in major female reproductive system cancers. Int J Mol Sci. 23, 1–19 (2022). https://doi.org/10.3390/ijms23147556 . Yan, X., Zhu, A., Li, Y., Yang, Z., Wang, Y., Liu, L., et al. Systematical assessment of digit ratio in a female masculinization disease: polycystic ovary syndrome. Front Endocrinol (Lausanne). 14:1–9 (2023). https://doi.org/10.3389/fendo.2023.1146124 . Panidis, D., Balaris, C., Farmakiotis, D., Rousso, D., Kourtis, A., Balaris, V., et al. Serum parathyroid hormone concentrations are increased in women with polycystic ovary syndrome. Clin Chem. 51, 1691–1697 (2005). https://doi.org/10.1373/clinchem.2005.052761 . Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1VDDVDRPCOSBasuetal.Endocrine.docx Table2VDDVDRPCOSBasuetal.Endocrine.docx Table3VDDVDRPCOSBasuetal.Endocrine.docx Table4VDDVDRPCOSBasuetal.Endocrine.docx STROBEChecklistVDDVDRPCOSBasuetal.Endocrine.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5417644","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376248133,"identity":"19996254-af36-4108-a122-95340a6941c2","order_by":0,"name":"Sanchari Chakraborty","email":"","orcid":"","institution":"Surendranath College","correspondingAuthor":false,"prefix":"","firstName":"Sanchari","middleName":"","lastName":"Chakraborty","suffix":""},{"id":376248134,"identity":"dd8ca8d7-778a-4868-bed8-de7c1a59c81f","order_by":1,"name":"Randrita Pal","email":"","orcid":"","institution":"Surendranath College","correspondingAuthor":false,"prefix":"","firstName":"Randrita","middleName":"","lastName":"Pal","suffix":""},{"id":376248135,"identity":"53c21ea4-51c9-471c-a8cc-6b28d8b16bfd","order_by":2,"name":"Farzana Begum","email":"","orcid":"","institution":"Surendranath College","correspondingAuthor":false,"prefix":"","firstName":"Farzana","middleName":"","lastName":"Begum","suffix":""},{"id":376248136,"identity":"3bec261b-50b7-486d-ac5a-bfc89d04133e","order_by":3,"name":"Tapan Kumar Naskar","email":"","orcid":"","institution":"Medical College","correspondingAuthor":false,"prefix":"","firstName":"Tapan","middleName":"Kumar","lastName":"Naskar","suffix":""},{"id":376248137,"identity":"9eae5220-bf3e-4e23-bd2f-1dce6d731da0","order_by":4,"name":"Nilansu Das","email":"","orcid":"","institution":"Surendranath College","correspondingAuthor":false,"prefix":"","firstName":"Nilansu","middleName":"","lastName":"Das","suffix":""},{"id":376248138,"identity":"624d9aae-daa0-4506-aba4-485f12e947e1","order_by":5,"name":"Barnali Ray Basu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACAyBmBjH4QURCASlaJBtAWgxI0WJwAMYlBMylDz+TLqipS9x8fnXihwcGDPL8Ygfwa7HsSzOTnnHscOK2G283SwAdZjhzdgIBh51hMJPmYTsA1HJ2A0hLgsFtglrYv0nz/AM6bMbZzT+I1MJjJs3bxpy4gb93G3G2WPbwFFvP7DtsPOMG7zaLBAMJwn4x52HfeLvgW51sf//ZzTd/VNjI80sT0AIELBJgSgKsUoKgchBg/gCm+A8QpXoUjIJRMApGIAAAGyFEM0tPtYUAAAAASUVORK5CYII=","orcid":"","institution":"Surendranath College","correspondingAuthor":true,"prefix":"","firstName":"Barnali","middleName":"Ray","lastName":"Basu","suffix":""}],"badges":[],"createdAt":"2024-11-08 15:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5417644/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5417644/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71505064,"identity":"4eae047f-c403-4bde-8a81-66a10faae6ee","added_by":"auto","created_at":"2024-12-16 09:39:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[a]\u003c/strong\u003eComparative study of vitamin D status - \u003cstrong\u003e[a.1]\u003c/strong\u003etime range and \u003cstrong\u003e[a.2]\u003c/strong\u003e time duration of sunlight exposure \u003cstrong\u003e[a.3] \u003c/strong\u003evitamin D levels in blood, \u003cstrong\u003e[a.4]\u003c/strong\u003e dietary intake of vitamin D; \u003cstrong\u003e[b] \u003c/strong\u003eComparative analysis of\u003cstrong\u003e \u003c/strong\u003enutritional status of \u003cstrong\u003e[b.1]\u003c/strong\u003e calcium, \u003cstrong\u003e[b.2]\u003c/strong\u003e phosphorus, \u003cstrong\u003e[b.3]\u003c/strong\u003ecarbohydrate, \u003cstrong\u003e[b.4]\u003c/strong\u003e fat, and \u003cstrong\u003e[b.5]\u003c/strong\u003e protein between the case-control group.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/5c8d6daa120860d03045e10b.jpg"},{"id":71505058,"identity":"32bb6a97-7ced-48f6-bb99-af3b45d7c11d","added_by":"auto","created_at":"2024-12-16 09:39:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470993,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[a.1]\u003c/strong\u003e Genomic location, \u003cstrong\u003e[a.2]\u003c/strong\u003e structure of \u003cem\u003eVDR\u003c/em\u003e gene; \u003cstrong\u003e[b]\u003c/strong\u003e Representative structure of vitamin D3 receptor protein; \u003cstrong\u003e[c]\u003c/strong\u003e Ethidium bromide (EtBr)-stained agarose gel (PCR: 2% and PCR-RFPL: 3%) analysis of \u003cstrong\u003e[c.1]\u003c/strong\u003e PCR-amplified undigested \u003cem\u003eVDR\u003c/em\u003e gene: BsmI (360 bp: lane 2 to 5) and FokI (265 bp: lane 6 to 9), \u003cstrong\u003e[c.2]\u003c/strong\u003e PCR-RFLP digested \u003cem\u003eVDR\u003c/em\u003e-FokI: Ff heterozygous genotype (265 bp, 169 bp and 96 bp) in lane 2 and 3, FF wild type genotype (265 bp) in lane 4 and 6, and ff homozygous genotype (169 bp and 96 bp): lane 5; and \u003cem\u003eVDR\u003c/em\u003e-BsmI: bb homozygous genotype (191 bp and 169 bp) in lane 10 and 11, BB wild type genotype (360 bp) in lane 12 and 13 and Bb heterozygous genotype (360 bp, 191 bp and 169 bp) in lane 14; \u003cstrong\u003e[d]\u003c/strong\u003e DNA Sanger sequencing analysis: \u003cstrong\u003e[d.1]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-BsmI:\u003cem\u003e \u003c/em\u003eBB genotype, [\u003cstrong\u003ed\u003c/strong\u003e.\u003cstrong\u003e2\u003c/strong\u003e] \u003cem\u003eVDR\u003c/em\u003e-BsmI\u003cem\u003e: \u003c/em\u003eBb genotype, \u003cstrong\u003e[d.3]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-BsmI\u003cem\u003e: \u003c/em\u003ebb\u003cem\u003e \u003c/em\u003egenotype, \u003cstrong\u003e[d.4]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-FokI\u003cem\u003e: \u003c/em\u003eFF genotype, \u003cstrong\u003e[d.5]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-BsmI\u003cem\u003e: \u003c/em\u003eFf genotype\u003cem\u003e \u003c/em\u003eand \u003cstrong\u003e[d\u003c/strong\u003e.\u003cstrong\u003e6]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-FokI\u003cem\u003e: \u003c/em\u003eff genotype. BB=AA, Bb=AG, bb=GG, FF=CC, Ff=CT, ff=TT. Nucleotide base: “A” (adenine, green peak), “G” (guanine, black peak), “C” (cytosine, blue peak) and “T” (thymine, red peak).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/6827aa0e3b7cc69ff7bc0773.jpg"},{"id":71505055,"identity":"36a9bcba-45bf-466b-980e-cd88b433570b","added_by":"auto","created_at":"2024-12-16 09:39:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[a] \u003c/strong\u003eComparison of 25(OH)D between mutant variants of BsmI (bb+Bb) and FokI (ff+Ff), \u003cstrong\u003e[b] \u003c/strong\u003eDistribution pattern of 25(OH)D in vitamin D receptor (\u003cem\u003eVDR\u003c/em\u003e) polymorphisms (wild-type and mutants: BsmI and FokI) in PCOS patients (S-W=Shapiro-Wilk normality test, P\u0026lt;0.01 and 0.05). BB=AA, Bb=AG, bb=GG, FF=CC, Ff=CT, ff=TT.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/e74caaaa3dfa263bb76f1b9e.jpg"},{"id":71505628,"identity":"7e154783-32eb-48b7-adcc-9044ba5b5847","added_by":"auto","created_at":"2024-12-16 09:47:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1639735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/4f8cab56-4e95-4c2b-83e3-caf1347b0b7a.pdf"},{"id":71505053,"identity":"d82d8cc9-927e-4eb8-a12d-6e2c409b139e","added_by":"auto","created_at":"2024-12-16 09:39:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20127,"visible":true,"origin":"","legend":"","description":"","filename":"Table1VDDVDRPCOSBasuetal.Endocrine.docx","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/6f52b93b47de9945a8583c8e.docx"},{"id":71505059,"identity":"730d3d6c-fa71-4012-b43b-46f95ebc3752","added_by":"auto","created_at":"2024-12-16 09:39:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18289,"visible":true,"origin":"","legend":"","description":"","filename":"Table2VDDVDRPCOSBasuetal.Endocrine.docx","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/ec96d0917594496ce48a5aca.docx"},{"id":71505065,"identity":"50706092-433f-4867-a702-9fcaa62895ff","added_by":"auto","created_at":"2024-12-16 09:39:40","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18653,"visible":true,"origin":"","legend":"","description":"","filename":"Table3VDDVDRPCOSBasuetal.Endocrine.docx","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/fa614f9e16d3987712fcd787.docx"},{"id":71505063,"identity":"42907853-ad65-48d8-8d3a-6849c7335a6b","added_by":"auto","created_at":"2024-12-16 09:39:39","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18458,"visible":true,"origin":"","legend":"","description":"","filename":"Table4VDDVDRPCOSBasuetal.Endocrine.docx","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/55fc6d0a5f4c394e09f81124.docx"},{"id":71505060,"identity":"5f22a7aa-631e-4097-9b0a-723f3f8ae79d","added_by":"auto","created_at":"2024-12-16 09:39:37","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":93184,"visible":true,"origin":"","legend":"","description":"","filename":"STROBEChecklistVDDVDRPCOSBasuetal.Endocrine.doc","url":"https://assets-eu.researchsquare.com/files/rs-5417644/v1/ca9734f059b5c22b255b2dcc.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unraveling the interplay between vitamin D deficiency, VDR polymorphisms, and polycystic ovary syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS) is a complex, polygenic disorder affecting reproductive-aged women, characterized by hyperandrogenism, menstrual irregularities, and PCO morphology (PCOM), leading to infertility and miscarriage [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. There is a complex interplay between genetic predisposition and environmental factors in manifestation of PCOS and its prevalence varies from 5\u0026ndash;10% globally, 4\u0026ndash;20% in India and 28\u0026ndash;75% in West Bengal [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Research shows a high incidence of vitamin D deficiency (VDD) in women with PCOS, affecting 67\u0026ndash;85% of cases, and correlating with insulin resistance (IR) and metabolic syndrome (MetS) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Vitamin D (VD) plays a crucial role in reproductive health, influencing processes like follicle maturation, anti-m\u0026uuml;llerian hormone (AMH) signaling, follicle-stimulating hormone (FSH) sensitivity, progesterone secretion, and insulin sensitivity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It also helps to manage dyslipidaemia and reduces the bioavailability of TGF-β1, improving ovarian function and mitigating clinical hyperandrogenism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, VDD impairs glucose transporter (GLUT4) function, exacerbating IR in PCOS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Emerging evidence links VDD to long-term risks of MetS and cardiovascular diseases (CVDs) in PCOS patients, highlighting the need for further research into VD's broader impact on the syndrome [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These findings underscore the potential therapeutic value of VD supplementation in managing PCOS and its associated metabolic and reproductive complications.\u003c/p\u003e \u003cp\u003eVD exerts its biological effects through the vitamin D receptor (VDR), a ligand-activated transcription factor encoded by the \u003cem\u003eVDR\u003c/em\u003e gene on chromosome 12q13.11. This gene spans 2776 genomic positions and translates a 427-amino-acid protein. \u003cem\u003eVDR\u003c/em\u003e influences the expression of 229 genes, or about 3% of the human genome, across various tissues, including the hypothalamus-pituitary-ovarian (HPO) axis, granulosa cells, theca cells, endometrium, and placenta [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These interactions are crucial for maintaining reproductive health. The regulation of VD metabolism primarily occurs through genetic variations in \u003cem\u003eVDR\u003c/em\u003e, vitamin D binding protein (\u003cem\u003eVDBP/GC\u003c/em\u003e), and cytochrome P450 (\u003cem\u003eCYP\u003c/em\u003e) family genes. Key enzymes involved include 25-hydroxylase (\u003cem\u003eCYP2R1\u003c/em\u003e, VD to 25-hydroxyvitamin D), 1-hydroxylase (\u003cem\u003eCYP27B1\u003c/em\u003e, activates 25-hydroxyvitamin D to calcitriol), and 24-hydroxylase (\u003cem\u003eCYP24A1\u003c/em\u003e, inactivates VD metabolites) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. VDR plays a central role by binding to calcitriol [1,25(OH)2D3] and activating gene transcription that regulates these enzymes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. VD and VDR interactions are also significant for ovarian steroidogenesis where \u003cem\u003eVDR\u003c/em\u003e upregulates the mRNA expression of \u003cem\u003eCYP19\u003c/em\u003e (aromatase) and downregulates \u003cem\u003eCYP17\u003c/em\u003e, both of which are involved in the production of estrogen [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Furthermore, \u003cem\u003eCYP11A1\u003c/em\u003e, a regulator of steroidogenesis, can metabolize vitamin D3 in the placenta and adrenal glands [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In uterine stromal cells, 1,25(OH)2D3-VDR interactions increase the mRNA expression of \u003cem\u003eHOXA10\u003c/em\u003e, a gene crucial for implantation and embryonic development [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Variations in \u003cem\u003eVDR\u003c/em\u003e gene expression, such as single nucleotide polymorphisms (SNPs), can affect VD binding, leading to VDD and disrupted metabolic and endocrine functions, contributing to PCOS and related complications [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe prevalence of VDD varies significantly, ranging from 20\u0026ndash;60% worldwide, 34\u0026ndash;99% across India and 51\u0026ndash;93% in West Bengal [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Factors contributing to VDD include low solar ultraviolet-B (UVB) exposure due to indoor preferences, a sedentary lifestyle, inadequate dietary intake of VD, and a preference to use sunscreen cosmetics [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The prolonged existence of these habits may exacerbate symptoms of multifaceted conditions like PCOS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite residing in a tropical climate and having sufficient exposure to sunlight along with dietary intake of VD, a notable number of individuals are found to be VDD, suggesting the impact of predisposed genetic factors, particularly polymorphisms in the \u003cem\u003eVDR\u003c/em\u003e, (\u003cem\u003eVDBP/GC\u003c/em\u003e), and \u003cem\u003eCYP\u003c/em\u003e family, such as \u003cem\u003eCYP2R1\u003c/em\u003e, \u003cem\u003eCYP24A1\u003c/em\u003e, and \u003cem\u003eCYP11A1\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSNPs of \u003cem\u003eVDR\u003c/em\u003e predominantly regulate the metabolism, circulating levels, and activities of VD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The SNPs in introns could potentially generate alternate splicing, skipping of exons, modulation of nuclear export, transcription rate, and stability of the transcript, while exonic SNPs can cause non-synonymous changes that may modify the protein structure [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It was reported that the ApaI (rs7975232) and TaqI (rs731236) SNPs in \u003cem\u003eVDR\u003c/em\u003e have been linked to insulin signalling deregulation and IR in Iranian PCOS populations [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In Egyptian PCOS populations, TaqI variants in untranslated regions (UTRs) have been associated with PCOM, ovulatory dysfunction, and hyperandrogenism, impacting ovarian steroidogenesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. VDD may also contribute to obesity, IR, and CVDs in PCOS patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A cross-sectional study of \u003cem\u003eVDR\u003c/em\u003e suggests that ApaI (rs7975232) polymorphism is associated with hypertriglyceridemia and MetS, while the variants of TaqI (rs731236) and BsmI (rs1544410) seem to affect high-density lipoprotein cholesterol (HDL-C), and the \u003cem\u003eVDR-\u003c/em\u003eFokI (rs2228570) variants affect the systolic blood pressure, which is ambiguous in the Chinese population [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It was reported that \u003cem\u003eVDR\u003c/em\u003e-BsmI (rs1544410) polymorphism can trigger CVD, an alarming feature of PCOS in later age [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A study report from Hyderabad, India, stated that \u003cem\u003eVDR\u003c/em\u003e polymorphisms of ApaI (rs7975232) and TaqI (rs731236) are associated with PCOM, hyperandrogenism, ovarian dysfunction, and obesity in PCOS individuals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Variants of BsmI are associated with similar symptoms, including obesity and IR across various regions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additionally, in PCOS patients, \u003cem\u003eVDR-\u003c/em\u003eFokI (rs10735810) polymorphism is associated with infertility, alopecia, hirsutism (India), high insulin levels and IR (Iran), and increased total testosterone (TT, Korea) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies on different ethnic populations indicate the association of \u003cem\u003eVDR\u003c/em\u003e polymorphisms (BsmI and FokI) with \u003cem\u003eVDR\u003c/em\u003e. SNPs of the \u003cem\u003eGC\u003c/em\u003e gene (rs4588 and rs7041) and \u003cem\u003eCdx2\u003c/em\u003e (rs11568820), which are often found to be interlinked with altered \u003cem\u003eVDR\u003c/em\u003e expression, leading to VDD-associated differential phenotypic manifestations and metabolic alterations of PCOS [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Overall, diverse manifestations of PCOS appear to be influenced by specific \u003cem\u003eVDR\u003c/em\u003e polymorphisms across different ethnic populations.\u003c/p\u003e \u003cp\u003eWest Bengal, the eighth-most populous region globally and the fourth-most populous state in India, is celebrated for its diverse cultures and traditional cuisines. The predominant ethnic group, the Bengalis, primarily consists of Indo-Aryans with a unique genetic heritage influenced by Iranian hunter-gatherers, Central Asian pastoralists, and South Asian populations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This genetic diversity may affect the prevalence of certain health conditions and responses to treatments [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], particularly in conditions like PCOS. According to the National Family Health Survey, approximately 30% of women in Kolkata are classified as obese [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Data from the West Bengal Health Statistical database highlight a concerning link between PCOS and major health issues such as diabetes and CVD. However, research on VDD and insufficiency (VDI) and their impact on PCOS within West Bengal's ethnic communities are limited. This lacuna has prompted the current study, which aims to explore the association between specific polymorphisms of the \u003cem\u003eVDR\u003c/em\u003e-namely, BsmI in intron 8 (rs1544410, A\u0026thinsp;\u0026gt;\u0026thinsp;G, B\u0026thinsp;\u0026gt;\u0026thinsp;b) and FokI in exon 2 (rs2228570, C\u0026thinsp;\u0026gt;\u0026thinsp;T, F\u0026thinsp;\u0026gt;\u0026thinsp;f)\u0026mdash;and the pathophysiology of PCOS in this population. The research could yield insights for developing personalised therapeutic strategies and optimising VD supplementation based on genetic profiles.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eStudy Design and Sample Size\u003c/h2\u003e\n\u003cp\u003eThe case-control study enrolled 80 women with PCOS (PCOS, N=80), aged 17 to 36 years, experiencing menstrual and fertility complications, along with 100 gender- and age-matched healthy control participants (HC, N=100) in the ethnic population of West Bengal. Approved by the Human Ethics Committee of Medical College and Hospital, Kolkata (MC/KOL/IEC/NON-SPON/1275/02/22) and the Calcutta University Institutional Ethics Committee (CUIEC/03/40/2022-23), the research took place in the outpatient department of gynaecology and obstetrics of Medical College and Hospital, from September 2022 to June 2023. All participants were informed of the study\u0026apos;s societal and personal relevance, and written consent was obtained from each volunteer using structured trilingual consent forms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudy Subject Selection\u003c/p\u003e\n\u003cp\u003eCase group\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eInclusion criteria\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe Rotterdam criteria, 2003 [European Society of Human Reproduction and Embryology (ESHRE\u003cem\u003e)/\u003c/em\u003eAmerican Society for Reproductive Medicine (ASRM)], were used to incorporate PCOS women in the study. The syndrome was diagnosed based on two of the following three criteria: (a) an intermenstrual interval of \u0026ge;35 days (oligomenorrhea) or \u0026gt;3 months (amenorrhea); (b) gynaecological ultrasound of PCOM\u0026nbsp;(size: 2-9 mm, number: \u0026gt;12, and ovarian volume: \u0026gt;10 cm\u003csup\u003e3\u003c/sup\u003e); and (c) clinical and/or biochemical hyperandrogenism [2, 3, 32].\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eExclusion criteria\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWomen having physical and/or intellectual disabilities, pregnancy, and lactation were excluded [4]. Congenital adrenal hyperplasia (CAH), prolactinoma, virilizing tumours, and Cushing syndrome mimicking PCOS were also disregarded as potential etiologies [2, 30]. Medical history of oncological patients, those previously or presently undergoing treatment, such as hormonal medication and contraceptives (progesterone) injections prior to the screening, and implausible energy intake (\u0026le;500 or \u0026ge;4500 kcal/day) were also ruled out [2, 3].\u003c/p\u003e\n\u003cp\u003eControl group\u003c/p\u003e\n\u003cp\u003eThe participating healthy control individuals had regular menstruation, no evidence of clinical and/or biochemical manifestations of hyperandrogenism, no abnormality in their endocrine profile, no PCOM on ultrasonography, and neither a history of drug ingestion nor an unbalanced diet [2, 3].\u003c/p\u003e\n\u003cp\u003eAssessment of Sunlight Exposure\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA detailed questionnaire was designed to record time (between 06:00 AM-11:00 AM, 11:00 AM-03:00 PM and 03:00 PM-05:00 PM) and duration [(\u0026lt;30, 30-120, and \u0026gt;120) minutes per day] of sunlight exposure [16].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCutaneous Manifestation\u003c/p\u003e\n\u003cp\u003eHirsutism was evaluated using the modified Ferriman-Gallwey (F-G) score, and alopecia was assessed by the Norwood scale [4]. Velvety and pigmented skin in the neck and antecubital fossa region were categorized as acanthosis nigricans (AN), while nodules in the face, neck, and back were classified as acne [4].\u003c/p\u003e\n\u003cp\u003eAssessment of Nutritional Status\u003c/p\u003e\n\u003cp\u003eDietary assessment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIndian Council of Medical Research (ICMR) guidelines were followed to evaluate the nutritional status of the participants [13, 33, 34]. The food frequency questionnaire (FFQ) method was applied to assess the nutritional status of the study population [13, 33-35].\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCategorization of food items\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eNine food items, including millets and cereals (n=2), wheat (n=2), vegetables (green leafy and others, n=91), grain legumes (n=25), roots and tubes (n=19), fish (freshwater and marine, n=101), poultry (n=6), animal meat (n=13), egg (n=1), fruits (n=68), nuts and oil seeds (n=4), milk (n=1), and food ingredients, such as condiments and spices (dry and fresh, n=26), and cooking oil (n=1) were taken to be analyzed [13, 33-35].\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eQuantifying common measures\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eDifferent-sized bowls and spoons were used to measure the weight of different food items. The average of the food components was evaluated [13, 33-35].\u003c/p\u003e\n\u003cp\u003eEvaluation of Anthropometric Indices\u003c/p\u003e\n\u003cp\u003eThe height (cm) of the barefoot participant was measured using a portable ultrasonic-based handheld digital stature height measuring scale (make: EasyCare EC1800, India). Body mass index\u0026nbsp;(BMI, kg/m^2), visceral fat, and the percentage of skeletal muscle mass in the whole-body of the least-clothed individuals were estimated using a body composition monitor (Model: OMRON-HBF-375, Karada Scan, Kyoto, Japan) based on bioelectrical impedance analysis [2]. The digit 2D:4D ratio was obtained by dividing the length (mm) of the index finger [second (2nd) digit] by the ring fingers [fourth (4th) digit], which were measured from the fingertip to the midpoint of the basal crease on the dorsal surface of the left hand using digital vernier callipers (make: ZHART, Rajasthan, India) [4]\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003ch2\u003eBiochemical Assay\u003c/h2\u003e\n\u003cp\u003e\u003cu\u003eCollection of saliva\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe volunteers were requested to rinse their mouths with water and refrain from any food or beverage (except water) for 1 hour before their saliva collection. After that, every individual was requested to sit in an upright, comfortable position and tilt their head forward to collect whole saliva samples within the same time interval of day by the passive drool method in an ice-chilled graduated cryovial (1.80 ml) through a saliva collection aid (Salimetrics, Item No. 5016.02; Carlsbad, United States). Immediately the saliva-filled cryovials were transferred to a mini-cooler and subsequently stored in a -20\u0026deg;C freezer for further analysis [2].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEnzyme-linked immunosorbent assay of interleukin 6\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe salivary interleukin-6 (IL-6) concentration was determined using the sandwich-enzyme linked immunosorbent assay (ELISA) principle (FineTest, EH0201, Wuhan, China). The reference range (pg/ml) is 4.1-17.5, and the intra- and inter-assay CV% were \u0026lt;8 and \u0026lt;10, respectively. Firstly, the saliva samples were centrifuged at 1000\u0026times;g at 4\u003cstrong\u003e\u003cem\u003e\u0026deg;\u003c/em\u003e\u003c/strong\u003eC for 20 minutes, and the supernatants were used for further steps of the assay. The salivary IL-6 was evaluated spectrophotometrically of absorbance at the 450 nm wavelength in a microplate reader [make: Bio-rad, Model No.: iMark (Microplate) reader, SL No. 10095, California, United States].\u003c/p\u003e\n\u003cp\u003eBlood parameters\u003c/p\u003e\n\u003cp\u003eAfter a 12-hour fast, blood samples from PCOS patients were taken by venipuncture in the early morning hours of the second or third day of menstruation (early follicular phase). Serum 25(OH)D concentration of the participants [reference interval (ng/ml): 10-20 (VDD), 20-30 (VDI/suboptimal) and 30-50 (sufficient/optimal)] was estimated by the Alinityi (Abbott) system and the chemiluminescent microparticle immunoassay (CMIA) method [assay CV% (intra=3.66 to 6.56 and inter=4.19 to 7.01), fasting insulin (FI) and postprandial\u0026nbsp;insulin (PPI) by [system: Alinityi (Abbott), method: chemiluminescent microparticle immunoassay (CMIA)]; fasting glucose (hexokinase; automated biochemistry analyzer-DXC 700AU/COBAS 501); TT, luteinizing hormone\u0026nbsp;(LH) and FSH by (system: COBAS e 411, method: electrochemiluminescence); HDL, LDL, and triglyceride [enzymatic colour, and GPO-POD methods, automated biochemistry analyser (AU 680/COBAS 501)], C-reactive protein by\u0026nbsp;(CRP, Nephelometric immunoassay Batman coulter image/immuno turbidimetric), calcium, and phosphate by [automated biochemistry analyser-DXC 700AU/COBAS 501); Arsenazo III method, Inorganic, Molybdate UV method, respectively] parathyroid\u0026nbsp;hormone by (PTH, enzyme-linked fluorescent assay, Mini Vidas system) were estimated from the drawn blood samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHomeostatic model assessment for IR (HOMA-IR), quantitative insulin-sensitivity check index\u0026nbsp;(QUICKI), fasting glucose to insulin ratio (FGIR), and triglyceride glucose (TyG) index were evaluated by applying the standard formulae [3, 4].\u003c/p\u003e\n\u003cp\u003eStudy of \u003cem\u003eVDR\u0026nbsp;\u003c/em\u003ePolymorphism\u003c/p\u003e\n\u003cp\u003eCollection of buccal swabs\u003c/p\u003e\n\u003cp\u003eSwab samples were collected from the buccal cavities of the participants using sterile cotton swab sticks. The subjects were advised to abstain from eating or drinking anything (except water) for one hour before the collection of the swab. To collect the swab in the cotton head region of the swab stick, the handles of the sticks were held in the subject\u0026apos;s wide-open mouth, and the cheek was rubbed in a twisting motion for 60 seconds [36]. The swab containing the head area of the stick was carefully transferred to an ice-chilled cryovial (1.80 ml) without touching any surfaces by discarding the tail part using a sterile scissor. After that, the cryovial was instantly transferred to a minicooler and then stored in a -20\u0026deg;C freezer. The stored swabs were further used for deoxyribonucleic acid (DNA)\u0026nbsp;isolation. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIsolation of genomic DNA\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNA from the buccal swab was isolated using the phenol-chloroform (PCI) organic extraction method [37]. The quality was defined by using 1% agarose gel electrophoresis, and the quantity was estimated by UV absorbance at 260 nm in a UV spectrophotometer (make: SHIMADZU, UV-1780, Kyoto 604-8511, Japan) of the isolated genomic DNA [36, 37].\u003c/p\u003e\n\u003cp\u003eSNPs of \u003cem\u003eVDR\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe compendium analysis of \u003cem\u003eVDR\u003c/em\u003e has been performed using GeneCards\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003ethe HUGO gene nomenclature committee (Tables 1.a and b). The details of restriction endonuclease (BsmI and FokI) have been comprehended from New England Biolabs (Table 1.c). The primers were designed based on the primer design tool, primer-BLAST (Gene ID: 7421,\u0026nbsp;\u0026gt;NC_000012.12:c47904994-47841537 Homo sapiens chromosome 12, GRCh38.p14 Primary Assembly,\u0026nbsp;NCBI, Bethesda, USA). The \u003cem\u003eVDR\u003c/em\u003e was amplified [polymerase chain reaction (PCR)] using isolated genomic DNA samples (Table 1.d) and then checked for two SNPs [BsmI (rs1544410) and FokI (rs2228570), make: New England Biolabs, Ipswich, Massachusetts, United States] by the PCR-restriction fragment length polymorphism (PCR-RFLP) method to generate distinct polymorphic fragments (Table 1.e) [22, 37-39]. The amplified (PCR) and digested (PCR-RFLP) products were visualised by 2% and 3% agarose gel electrophoresis, respectively. The obtained order of the nucleotides of Sanger sequencing (Applied Biosystems\u0026reg; Sanger Sequencing 3500 Genetic Analysers,\u0026nbsp;Foster City, USA) was determined by chromatogram\u0026nbsp;(Sequence Scanner 1.0;\u0026nbsp;Life Technology Corporation, Waltham, Massachusetts, United States).\u003c/p\u003e\n\u003ch2\u003eSoftware and Data Analysis\u003c/h2\u003e\n\u003cp\u003eSample size\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the current case-control study, the sample size [odds ratio (OR): 4 and confidence interval (CI): 0.95] was determined based on EPITOOLS, Ausvet, epidemiological web-based calculators (Fremantle, Australia) [3].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMetabolic profile\u003c/p\u003e\n\u003cp\u003eThe collected data was analysed in statistical software for the social sciences (SPSS, IBM Corp., version-20, Chicago, Illinois, United States). The Shapiro-Wilk (S-W) normality test was performed to determine the \u003cem\u003eVDR\u003c/em\u003e-polymorphism (BsmI and FokI: mutant)-based 25(OH)D distribution pattern. Simple boxplot [dispersion of 25(OH)D among \u003cem\u003eVDR\u003c/em\u003e polymorphisms (wild types and mutants) and dietary profile], independent samples-t test [comparisons of dietary intake (between case-control group) and IR status (between different genotypes of BsmI and FokI in \u003cem\u003eVDR\u003c/em\u003e], bivariate Pearson correlation [estimation of the association between 25(OH)D with TT and HDL, and adiposity indices], and a stacked bar chart (sunlight exposure-time and duration) were applied. The statistical significance was considered at P\u0026lt;0.05 and P\u0026lt;0.01. The technical error was within the limit [2].\u003c/p\u003e\n\u003cp\u003eGenetic polymorphism\u003c/p\u003e\n\u003cp\u003eFrequencies of allele and genotype in \u003cem\u003eVDR\u003c/em\u003e (BsmI and FokI), exact test for Hardy-Weinberg equilibrium (HWE) of case (PCOS) and controls, OR, and 95% CI of dominant model were statistically analysed using SNPStats (Institut Catala d\u0026apos;Oncologia\u003cem\u003e,\u003c/em\u003e Spain), a web tool (https://www.snpstats.net/start.htm) [40, 41].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eGenetic polymorphisms of \u003cem\u003eVDR\u003c/em\u003e, levels of 25(OH)D and hyperandrogenic parameters (TT, LH:FSH ratio, and 2D:4D ratio), indices of the metabolic panel (PTH, calcium, phosphate, LDL-HDL ratio, and TyG index), inflammatory markers (CRP and IL-6), anthropometric indices (visceral fat and skeletal muscle mass whole body), and cutaneous manifestations were illustrated in PCOS patients and healthy controls. However, a few participants were dropped from the survey midway through the study due to a loss of willingness and were unable to provide blood, saliva and swab samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1 [a]\u003c/strong\u003e Comparative study of vitamin D status - \u003cstrong\u003e[a.1]\u003c/strong\u003e time range and \u003cstrong\u003e[a.2]\u003c/strong\u003e time duration of sunlight exposure \u003cstrong\u003e[a.3]\u0026nbsp;\u003c/strong\u003evitamin D levels in blood, \u003cstrong\u003e[a.4]\u003c/strong\u003e dietary intake of vitamin D; \u003cstrong\u003e[b]\u0026nbsp;\u003c/strong\u003eComparative analysis of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003enutritional status of \u003cstrong\u003e[b.1]\u003c/strong\u003e calcium, \u003cstrong\u003e[b.2]\u003c/strong\u003e phosphorus, \u003cstrong\u003e[b.3]\u003c/strong\u003e carbohydrate, \u003cstrong\u003e[b.4]\u003c/strong\u003e fat, and \u003cstrong\u003e[b.5]\u003c/strong\u003e protein between the case-control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1.a.1\u003c/strong\u003e indicated that PCOS patients (n=86.25%) were less exposed to sunlight (time: 11:00 AM to 03:00 PM) compared to the control individuals (n=73%). Furthermore, 85% of PCOS patients showed indoor preference (sunlight exposure time duration: \u0026lt;30 mins. to 120 mins.), whereas 78% of control individuals had this preference (\u003cstrong\u003eFig. 1.a.2\u003c/strong\u003e). It was observed that VD status in blood [25(OH)D, P=0.650] and nutritional profile (combination of ergocalciferol-D2, cholecalciferol D3 and calcifediol, P=0.820) was lower in the PCOS patients than the control group (\u003cstrong\u003eFig. 1\u003c/strong\u003e.\u003cstrong\u003ea.3\u003c/strong\u003e and \u003cstrong\u003e4\u003c/strong\u003e). Furthermore, dietary intake of carbohydrate (P=0.003), fat (P=0.169), protein (P=0.198) and phosphorus (P=0.074) was found to be higher in the PCOS group than in control individuals, whereas calcium (P=0.516) intake reflected the inverse relationship (\u003cstrong\u003eFig. 1.b.3-5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2\u003c/strong\u003e \u003cstrong\u003e[a.1]\u003c/strong\u003e Genomic location, \u003cstrong\u003e[a.2]\u003c/strong\u003e structure of \u003cem\u003eVDR\u003c/em\u003e gene; \u003cstrong\u003e[b]\u003c/strong\u003e Representative structure of vitamin D3 receptor protein; \u003cstrong\u003e[c]\u003c/strong\u003e Ethidium bromide (EtBr)-stained agarose gel (PCR: 2% and PCR-RFPL: 3%) analysis of \u003cstrong\u003e[c.1]\u003c/strong\u003e PCR-amplified undigested \u003cem\u003eVDR\u003c/em\u003e gene: BsmI (360 bp: lane 2 to 5) and FokI (265 bp: lane 6 to 9), \u003cstrong\u003e[c.2]\u003c/strong\u003e PCR-RFLP digested \u003cem\u003eVDR\u003c/em\u003e-FokI: Ff heterozygous genotype (265 bp, 169 bp and 96 bp) in lane 2 and 3, FF wild type genotype (265 bp) in lane 4 and 6 and ff homozygous genotype (169 bp and 96 bp): lane 5; and \u003cem\u003eVDR\u003c/em\u003e-BsmI: bb homozygous genotype (191 bp and 169 bp) in lane 10 and 11, BB wild type genotype (360 bp) in lane 12 and 13 and Bb heterozygous genotype (360 bp, 191 bp and 169 bp) in lane 14; \u003cstrong\u003e[d]\u003c/strong\u003e DNA Sanger sequencing analysis: \u003cstrong\u003e[d.1]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-BsmI:\u003cem\u003e\u0026nbsp;\u003c/em\u003eBB genotype, [\u003cstrong\u003ed\u003c/strong\u003e.\u003cstrong\u003e2\u003c/strong\u003e] \u003cem\u003eVDR\u003c/em\u003e-BsmI\u003cem\u003e:\u0026nbsp;\u003c/em\u003eBb genotype, \u003cstrong\u003e[d.3]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-BsmI\u003cem\u003e:\u0026nbsp;\u003c/em\u003ebb\u003cem\u003e\u0026nbsp;\u003c/em\u003egenotype, \u003cstrong\u003e[d.4]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-FokI\u003cem\u003e:\u0026nbsp;\u003c/em\u003eFF genotype, \u003cstrong\u003e[d.5]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-BsmI\u003cem\u003e:\u0026nbsp;\u003c/em\u003eFf genotype\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cstrong\u003e[d\u003c/strong\u003e.\u003cstrong\u003e6]\u003c/strong\u003e \u003cem\u003eVDR\u003c/em\u003e-FokI\u003cem\u003e:\u0026nbsp;\u003c/em\u003eff genotype. BB=AA, Bb=AG, bb=GG, FF=CC, Ff=CT, ff=TT. Nucleotide base: \u0026ldquo;A\u0026rdquo; (adenine, green peak), \u0026ldquo;G\u0026rdquo; (guanine, black peak), \u0026ldquo;C\u0026rdquo; (cytosine, blue peak) and \u0026ldquo;T\u0026rdquo; (thymine, red peak). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe positions of FokI-rs2228570 (in exon 2) and BsmI-rs1544410 (in intron 8) of \u003cem\u003eVDR\u0026nbsp;\u003c/em\u003eon chromosome 12q13.11 were illustrated (\u003cstrong\u003eFig. 2.a. 1 and 2\u003c/strong\u003e). The representative three dimensional vitamin D3 receptor structure was depicted (\u003cstrong\u003eFig. 2.b\u003c/strong\u003e). The PCR amplified product (\u003cstrong\u003eFig. 2.c.1\u003c/strong\u003e), PCR-RFLP of transition mutations of BsmI (A\u0026gt;G, B\u0026gt;b) and FokI (C\u0026gt;T, F\u0026gt;f) (\u003cstrong\u003eFig. 2.c.2\u003c/strong\u003e), and order of nucleotides were illustrated (\u003cstrong\u003eFig. 2.d.1 to 6\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3 [a]\u0026nbsp;\u003c/strong\u003eComparison of 25(OH)D between mutant variants of BsmI (bb+Bb) and FokI (ff+Ff), \u003cstrong\u003e[b]\u0026nbsp;\u003c/strong\u003eDistribution pattern of 25(OH)D in vitamin D receptor\u0026nbsp;(\u003cem\u003eVDR\u003c/em\u003e) polymorphisms (wild-type and mutants: BsmI and FokI) in PCOS patients (S-W=Shapiro-Wilk normality test, P\u0026lt;0.01 and 0.05). BB=AA, Bb=AG, bb=GG, FF=CC, Ff=CT, ff=TT. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e25(OH)D was found to be asymmetrically (S-W=0.000) distributed in both mutant [BsmI (bb+Bb) and FokI (ff+Ff)] types of \u003cem\u003eVDR\u003c/em\u003e (\u003cstrong\u003eFig. 3\u003c/strong\u003e.\u003cstrong\u003ea\u003c/strong\u003e). Furthermore, 25(OH)D was slightly (P=0.763) higher in mutant (bb+Bb) BsmI than the mutant (ff+Ff) FokI (\u003cstrong\u003eFig. 3\u003c/strong\u003e.\u003cstrong\u003ea\u003c/strong\u003e). Additionally, 25(OH)D level (ng/ml) was higher (16.00\u0026plusmn;7.30) in wild type (FF) relative to mutant types [ff (13.02\u0026plusmn;6.70) and Ff (13.00\u0026plusmn;5.31)] of FokI, and wild type [BB (12.62\u0026plusmn;4.93)] and mutant types [bb (12.65\u0026plusmn;7.30) and Bb (13.00\u0026plusmn;6.45) of BsmI (\u003cstrong\u003eFig. 3\u003c/strong\u003e.\u003cstrong\u003eb\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIt was found that PCOS group was deviated (P=0.019) from HWE in \u003cem\u003eVDR\u003c/em\u003e-FokI polymorphism (\u003cstrong\u003eTable 2.a\u003c/strong\u003e). However, control (P=0.055) and all participants (P=0.73) were within HWE in \u003cem\u003eVDR\u003c/em\u003e-FokI polymorphism (\u003cstrong\u003eTable 2.a\u003c/strong\u003e). Furthermore, in \u003cem\u003eVDR\u003c/em\u003e-BsmI, PCOS, control and all individuals were in HWE (P\u0026gt;0.05). In the dominant model of \u003cem\u003eVDR\u003c/em\u003e-FokI (C/C, C/T-T/T) polymorphism OR (95% CI) was 0.23 (0.08-0.71), P=0.0085 and \u003cem\u003eVDR\u003c/em\u003e-BsmI (A/A, A/G-G/G) polymorphism OR (95% CI) was 0.86 (0.30-2.48), P=0.77 (\u003cstrong\u003eTable 2.b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the PCOS study population, 25(OH)D had a direct (P\u0026gt;0.05) association with HDL, while an inverse (P\u0026gt;0.05) relationship was found with TT, visceral fat, and BMI (\u003cstrong\u003eTable 3.a\u003c/strong\u003e). Comparative analysis of the association of IR with the\u003cem\u003e\u0026nbsp;VDR-\u003c/em\u003eBsmI polymorphism in the PCOS study group depicted that HOMA-IR was higher (P=0.042) and FGIR was lower (P=0.021) in the mutant types (bb+Bb) relative to the wild type (BB) of the \u003cem\u003eVDR-\u003c/em\u003eBsmI polymorphism (\u003cstrong\u003eTable 3.b\u003c/strong\u003e). Interestingly, mutant types (ff+Ff) showed lower (P=0.977) HOMA-IR and higher (P=0.837) QUICKI values compared to wild type (FF) in \u003cem\u003eVDR-\u003c/em\u003eFokI polymorphism (\u003cstrong\u003eTable 3.b\u003c/strong\u003e). In the PCOS individuals, the cutaneous manifestations of hyperandrogenism were 98.75%, and alopecia predominated (96.20%) over AN (74.68%), hirsutism (69.62%), and acne (36.71%) (\u003cstrong\u003eTable 3.c.1\u003c/strong\u003e). The phenotypes AN (77.78%), alopecia (76.47%), hirsutism (72.41%), and acne (64.29%) were prevalently expressed in PCOS patients with heterogeneous (Ff) mutants of the \u003cem\u003eVDR\u003c/em\u003e-FokI polymorphism (\u003cstrong\u003eTable 3.c.2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eVDR\u003c/em\u003e (BsmI and FokI) polymorphism-based comparative study, it was found that TT (ng/ml) was predominant (0.34\u0026plusmn;0.22) in BB-BsmI, where high LH:FSH ratio and low 2D:4D ratio were prevalent in Ff-FokI (1.36\u0026plusmn;1.31) and ff-FokI (0.92\u0026plusmn;0.04), respectively (\u003cstrong\u003eTable 4.a\u003c/strong\u003e). It was also found that high PTH (pg/ml, 46.10\u0026plusmn;15.01), calcium (mg/dl, 9.78\u0026plusmn;0.42), phosphate (mg/dl, 4.05\u0026plusmn;0.52) and TyG index (4.57\u0026plusmn;0.27) were prevalent in bb-BsmI, whereas high LDL:HDL ratio (2.16\u0026plusmn;0.7)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas predominantly distributed in ff-FokI (Table 4.B). In \u003cstrong\u003eTable 4.c\u003c/strong\u003e, visceral fat (7.93\u0026plusmn;5.02) was highest in ff-FokI, and the skeletal muscle mass-whole body(%) was lowest (24.18\u0026plusmn;2.27) in BB-BsmI polymorphism (\u003cstrong\u003eTable 4.c\u003c/strong\u003e). The inflammatory markers like CRP (mg/dl, 0.64\u0026plusmn;0.44) and IL-6 (pg/ml, 14.00\u0026plusmn;5.06) were highest in bb-BsmI and FF-FokI, respectively (\u003cstrong\u003eTable 4.d\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e7-dehydrocholesterol in the skin absorbs UVB radiation (290\u0026ndash;315 nm) from sunlight, converting it to provitamin D3, which is then isomerised into vitamin D3, the sunshine vitamin [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, avoidance of sun exposure, sedentary lifestyles, and poor dietary choices can lead to obesity and vitamin D deficiency (VDD) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Excess adipose tissue in obese individuals stores fat-soluble vitamin D (VD), dispersing it over a larger volume and resulting in lower serum concentrations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] In this study, 86.25% of women with polycystic ovary syndrome (PCOS) reported avoiding UVB exposure, and 85% preferred staying indoors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003ea.1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e). Compared to controls, PCOS patients had lower (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) VD levels and dietary quality (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003ea.3\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e). Notably, carbohydrate intake was higher (P\u0026thinsp;=\u0026thinsp;0.003) in PCOS individuals, while no significant differences were found in fat, protein, phosphorus, or calcium intake (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003eb.1 to 5\u003c/b\u003e). This excessive carbohydrate consumption may disrupt stearoyl-CoA desaturase activity, leading to triacylglycerol accumulation and contributing to obesity-related depletion of the ovarian reserve, a key characteristic of PCOS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These findings underscore the interconnectedness of lifestyle, diet, and the pathophysiology of PCOS.\u003c/p\u003e \u003cp\u003eHardy-Weinberg equilibrium (HWE) is used to analyse allele frequencies in populations, with deviations indicating genotyping errors or consanguinity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Heterozygote excess (HetExc) may signal natural selection highlighting heterozygous variations, especially in disease contexts [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], however, in our study In our study, heterogeneity is a disadvantageous condition. Parker et al. suggested that ancient PCOS genetic polymorphisms provided a survival advantage in prehistoric environments, but these same variants may reduce fitness in today's obesogenic context [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The study found that \u003cem\u003eVDR\u003c/em\u003e-FokI polymorphism deviated (P\u0026thinsp;=\u0026thinsp;0.019) from HWE in the PCOS population (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003cb\u003ea\u003c/b\u003e), with heterozygous mutants (CT-TT) showing a significant association (P\u0026thinsp;=\u0026thinsp;0.0085) with disease manifestation (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003cb\u003eb\u003c/b\u003e). However, similar mutant variations in controls did not lead to the syndrome, suggesting protective mechanisms like a shielding effect during post-translational modifications and non-genomic interactivity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVDD is predominantly (65\u0026ndash;90%) contributed by genetic polymorphisms such as BsmI (rs1544410) and FokI (rs2228570) of the \u003cem\u003eVDR\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. \u003cem\u003eVDR\u003c/em\u003e polymorphisms regulate circulatory calcifediol [25(OH)D] levels by feedback mechanism [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The SNP of \u003cem\u003eVDR\u003c/em\u003e-BsmI can trigger a silent mutation, which leads to a reduction in the stability and expression of \u003cem\u003eVDR\u003c/em\u003e without altering the amino acid sequence of the encoded protein [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Calero et al. reported that silent polymorphism can affect mRNA structure and ligand interaction specificity, which seems to modulate translation kinetics, tRNA-mediated post-transcriptional modifications, and protein folding [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Multiple studies on Indians, Chinese, Japanese, Caucasians, and Americans reported that the SNP of \u003cem\u003eVDR\u003c/em\u003e-BsmI is in strong linkage disequilibrium (LD) with a polyadenosine [poly(A)] microsatellite repeat in the \u003cem\u003eVDR-\u003c/em\u003e3'UTR, which may affect either stability or translational activity of its mRNA [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Polymorphisms of FokI in the 5'-region of \u003cem\u003eVDR\u003c/em\u003e can alter the nucleotide sequence of the start codon from ATG to ACG, resulting in a reduction of translational protein size by three amino acids relative to its normal counterpart, 424 from 427 amino residues [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The mutated variant f (T allele) of \u003cem\u003eVDR\u003c/em\u003e weakly attaches with calcitriol in comparison to the longer VDR protein variant coded by the wild-type F allele (C allele), which often imitates the condition of VDD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Various investigations on different ethnic populations such as India, China, Poland, and Australia reported substantial associations between FokI polymorphism, VDD, and the expression of PCOS-associated manifestations such as deregulated ovarian cell-intertwined hyperandirgenism and obesity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. VDD boosts PI3K/Akt pathway activity and androgen receptor (AR) expression in ovarian tissue by lowering ARA70 protein levels [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. VD is essential in monitoring obesity by regulating lipid metabolism through stimulation of lipolysis, fat distribution patterns like visceral fat, and the adiposity indicator BMI [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Studies suggest that VDD inhibits fatty acid oxidation-associated lipolysis intertwined with declining Ca2\u0026thinsp;+\u0026thinsp;levels, increased cAMP activity, and dephosphorylation of hormone-sensitive lipase [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003cb\u003ea\u003c/b\u003e indicated that VD exhibited a positive correlation with HDL and an inverse relationship with obesity (visceral fat and BMI) and hyperandrogenism (TT). Ethnicity-based studies on PCOS hypothesised that \u003cem\u003eVDR-\u003c/em\u003eBsmI and \u003cem\u003eVDR-\u003c/em\u003eFokI polymorphisms are associated with VDD-induced IR by altering calcium flux and phosphorylation-mediated inhibition of GLUT4, inflammation by increased levels of TNFα, and hyperandrogenism by changing AR expression [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. VDD-associated obesity leads to the release of proinflammatory cytokines (IL-6, CRP, IL-18 and TNF-α), which downregulates insulin signalling by altering suppressor of cytokine signalling (SOCS) and JUN N-terminal kinase (JNK) and leads to IR [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This abnormality often increases pustule secretion of LH, an alarming cause of hypeandrogenism [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The order of nucleotides of \u003cem\u003eVDR\u003c/em\u003e (FokI and BsmI) polymorphism of the present study was illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The asymmetrically (P\u0026thinsp;=\u0026thinsp;0.000) distributed serum VD was lower in mutant (ff and Ff) types relative to wild-type (FF) in \u003cem\u003eVDR-\u003c/em\u003eFokI polymorphisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). IR was prevalent (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in mutants (bb\u0026thinsp;+\u0026thinsp;Bb) \u003cem\u003eVDR-\u003c/em\u003eBsmI in the PCOS study group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003cb\u003eb\u003c/b\u003e). Alopecia was found to be the predominant (96.20%) cutaneous manifestation of hyperandrogenism in the PCOS patients (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003cb\u003ec.1\u003c/b\u003e). The findings of Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003cb\u003ec.2\u003c/b\u003e indicated that AN (77.78%), alopecia (76.47%), hirsutism (72.41%), and acne (64.29%) were prevalently expressed in heterogeneous (Ff) mutant of the \u003cem\u003eVDR\u003c/em\u003e-FokI polymorphism. It was found that HA (high TT and LH:FSH ratio) was more frequent in the BB-BsmI and Ff-FokI of \u003cem\u003eVDR\u003c/em\u003e, respectively, whereas in the 2D:4D ratio, no fascinating difference was observed between the wild-type and mutant variants of \u003cem\u003eVDR\u003c/em\u003e polymorphism (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003cb\u003ea\u003c/b\u003e). Elevated TT at the molecular level affects in multiple ways: (i) binding with ARs, particularly in target tissues such as the hair follicles, skin, and, sebaceous glands, and activating signalling pathways that can lead to increased hair follicle sensitivity, sebum production, and alters in keratinization, contributing to hirsutism and acne; (ii) it can influence the hypothalamic-pituitary-gonadal (HPG) axis by negative feedback mechanism that affects LH:FSH ratio imbalance often seen in PCOS, promoting further androgen production from the ovaries; (iii) increased TT can inhibit follicular development, leading to anovulation and the formation of cysts; through impairing granulosa cell functionality, (iv) elevated TT levels are often associated with IR, that can induce ovarian androgen production, creating a vicious cycle of hyperandrogenism and metabolic dysfunction. This interaction further complicates the clinical picture and can exacerbate symptoms; (v) elevated levels of TT can trigger the expression of genes involved in hair growth (e.g., androgen-responsive genes), leading to phenotypic alternations such as hirsutism [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Furthermore, it can impact the expression of enzymes like aromatase, which transforms androgens to oestrogens, abnormally altering the hormonal balance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Though the potential link between the 2D:4D ratio and PCOS is under the scanner, some studies reported a significant link between a lower 2D:4D ratio and higher prenatal testosterone exposure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. All these three parameters\u0026mdash;hyperandrogenism, LH:FSH ratio, and 2D:4D\u0026mdash;showed an influential indication in the PCOS population under study. However, the result is not consistent with all aspects of HA with homo (bb/ff) and heterozygous (Bb/Ff) mutant genotypes, which implies further study with a larger sample size.\u003c/p\u003e \u003cp\u003ePanidis et al. suggest that elevated levels of PTH may contribute to hyperandrogenism associated with PCOS, particularly in conjunction with obesity and reduced concentrations of VD metabolites [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The present findings indicate that VD is influencing the impact on insulin sensitivity, which could potentially alleviate the metabolic and reproductive complications linked to PCOS. PTH was higher than the normal reference range [reference interval (pg/ml): 9.2\u0026ndash;44.6 (normal) and \u0026gt;\u0026thinsp;44.6 (high), PCOS PTH\u0026thinsp;=\u0026thinsp;46.10\u0026thinsp;\u0026plusmn;\u0026thinsp;15.01] in the homozygous (bb) mutant type of \u003cem\u003eVDR\u003c/em\u003e-BsmI (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003cb\u003eb\u003c/b\u003e). In \u003cem\u003eVDR-\u003c/em\u003eFokI, the homozygous (ff) mutant type had a high LDL:HDL ratio, while the TyG index could not achieve any difference between homo and heterozygosity (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003cb\u003eb\u003c/b\u003e). Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003cb\u003ec\u003c/b\u003e indicated that the Bb, ff, and Ff might be associated with high levels of visceral fat, but the result of skeletal muscle-whole body is inconclusive. In addition to these, findings indicate that CRP was higher in the homozygous (bb) mutants of \u003cem\u003eVDR\u003c/em\u003e-BsmI but not IL-6 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003cb\u003ed\u003c/b\u003e). IL-6 is a pro-inflammatory cytokine, and CRP is an acute-phase protein. Both are interconnected in the inflammatory response, where IL-6 can stimulate the liver to produce CRP [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In chronic inflammation like PCOS, metabolic dysregulation like IR, obesity, and MetS, or increased androgens, may influence and contribute to elevated CRP without significantly affecting IL-6 levels [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This important finding highlights the systemic inflammation in PCOS without affecting specific immune cells to produce IL-6. To comprehend better, further research involving other inflammatory markers like TNF-alpha and IL-10 needs to be carried out.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStrengths and Limitations of the Study\u003c/h2\u003e \u003cp\u003eThe study provides a comprehensive analysis of PCOS, exploring metabolic, hormonal, and cutaneous aspects alongside genetic factors, particularly \u003cem\u003eVDR\u003c/em\u003e polymorphisms (BsmI and FokI). It identifies key correlations with insulin resistance, hyperandrogenism, and obesity, suggesting personalized vitamin D supplementation as a potential therapeutic approach.\u003c/p\u003e \u003cp\u003eThe study has several limitations, including a small sample size, cross-sectional design, and potential biases from participant dropouts, which affect generalizability. It lacks intervention data, doesn't account for environmental factors, and focuses mainly on \u003cem\u003eVDR\u003c/em\u003e polymorphisms, missing other genetic factors in PCOS.\u003c/p\u003e \u003cp\u003eFuture research with larger sample sizes and further exploration of inflammatory markers and their role in PCOS will be crucial for better understanding and treatment of this complex syndrome.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBy identifying specific \u003cem\u003eVDR\u003c/em\u003e polymorphisms, such as BsmI and FokI, that influence vitamin D (VD) metabolism and related pathways, the study provides a clearer understanding of how genetic factors contribute to the diverse manifestations of PCOS. The findings suggest that vitamin D deficiency (VDD), influenced by both genetic and environmental factors, is prevalent in PCOS patients and correlates with metabolic and reproductive complications, including insulin resistance (IR), obesity, hyperandrogenism, and infertility. Additionally, these polymorphisms influence key metabolic pathways and hormonal imbalances, exacerbating conditions like hirsutism, acne, and anovulation. Furthermore, the findings suggest that correcting VDD could alleviate key metabolic disturbances in PCOS patients, potentially improving insulin sensitivity, regulating androgen levels, and addressing symptoms like hirsutism and anovulation. This could pave the way for more targeted interventions, where treatment plans could be tailored based on genetic predispositions, such as using personalised vitamin D supplementation regimens for those with specific \u003cem\u003eVDR\u003c/em\u003e mutations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Statement\u003c/h2\u003e\n\u003cp\u003eDepartment of Science and Technology and Biotechnology, Government of West Bengal (DSTBT-WB, Grant no. ST/P/S\u0026amp;T/9G-5/2018)\u003c/p\u003e\n\u003ch2\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eConflicts of Interest\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAll authors (Sanchari Chakraborty, Randrita Pal, Farzana Begum, Tapan Kumar Naskar, Nilansu Das, Barnali Ray Basu) declare that there is no competing interest. The authors are solely responsible for the content and writing of the manuscript.\u003c/span\u003e\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution(s):\u003c/h2\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eConceptualization: [Barnali Ray Basu]; Data curation: [Sanchari Chakraborty], [Randrita Pal]; Formal analysis: [Sanchari Chakraborty], [Randrita Pal]; Funding acquisition: [Barnali Ray Basu], Investigation: [Sanchari Chakraborty], [Randrita Pal], [Farzana Begum]; Methodology: [Sanchari Chakraborty], [Randrita Pal], [Farzana Begum]; Project administration: [Barnali Ray Basu]; Software: [Sanchari Chakraborty]; Resources: [Barnali Ray Basu]; Supervision: [Barnali Ray Basu], [Tapan Kumar Naskar], [Nilansu Das]; Validation: [Barnali Ray Basu]; Visualisation: [Barnali Ray Basu], [Nilansu Das]; Writing\u0026mdash;original draft: [Sanchari Chakraborty], [Randrita Pal]; Writing\u0026mdash;review \u0026amp; editing: [Barnali Ray Basu], [Nilansu Das].\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eOrganization: Department of Science and Technology and Biotechnology, Government of West Bengal (DSTBT-WB, Grant no. ST/P/S\u0026amp;T/9G\u0026minus;5/2018)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Science and Technology and Biotechnology, Government of West Bengal (DSTBT-WB, Grant no. ST/P/S\u0026amp;T/9G-5/2018)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eHuman Ethics Committee of Medical College and Hospital, Kolkata (MC/KOL/IEC/NON-SPON/1275/02/22) and the Calcutta University Institutional Ethics Committee (CUIEC/03/40/2022\u0026minus;23)\u003c/span\u003e\u003c/p\u003e\n\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003ePeople and Technical Support: The authors are thankful to all the volunteer women in this study. The authors are also grateful to InBOL Healthcare educational Centre and Scientific Clinical Laboratory Pvt. Ltd. for their technical support.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData will be made available to the corresponding author of the paper for review or query upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhan, M.J., Ullah, A., Basit, S. Genetic basis of polycystic ovary syndrome (PCOS): current perspectives. Appl. Clin. Genet. 12, 249\u0026ndash;260 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/TACG.S200341\u003c/span\u003e\u003cspan address=\"10.2147/TACG.S200341\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasu, B.R., Chowdhury, O., Saha, S.K. Possible link between stress-related factors and altered body composition in women with polycystic ovarian syndrome. J. Hum. Reprod. 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Clin Chem. 51, 1691\u0026ndash;1697 (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1373/clinchem.2005.052761\u003c/span\u003e\u003cspan address=\"10.1373/clinchem.2005.052761\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vitamin D deficiency, Vitamin D receptor polymorphism, Polycystic ovary syndrome, PCR-RFLP, BsmI, FokI","lastPublishedDoi":"10.21203/rs.3.rs-5417644/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5417644/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003ePolycystic ovary syndrome (PCOS) is a complex and emerging heterogeneous disorder in reproductive-aged women and teenagers. Vitamin D deficiency (VDD) and genetic variations in the vitamin D receptor (\u003cem\u003eVDR\u003c/em\u003e) pronouncedly influence its manifestations. The interplay between VDD and \u003cem\u003eVDR\u003c/em\u003e polymorphisms has an umbrella effect on the endocrine and metabolic milieu of PCOS, underscoring the importance of VD in its management. This study tried to find out: How how VDD and single-nucleotide polymorphisms (SNPs) in the VDR gene influence the pathophysiology of PCOS, and how do these associations vary across different ethnic groups?\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A case-control study was conducted involving 80 PCOS women (ages 17–36 years) and 100 of their gender, and age-matched healthy controls (HC) belonging to the ethnicity of West Bengal, India. VDD and \u003cem\u003eVDR\u003c/em\u003epolymorphisms [BsmI (rs1544410) and FokI (rs2228570)] were estimated by biochemical assessment and PCR-RFLP, respectively. Bioelectrical impedance and structured questionnaires were used for evaluation of anthropometric indices, sunlight (UVB) exposure, and nutritional status, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e This study finds significant correlations between \u003cem\u003eVDR\u003c/em\u003e variants and insulin resistance, hyperandrogenism, inflammatory markers, and obesity indices. Mutant \u003cem\u003eVDR\u003c/em\u003egenotypes (BsmI-bb/Bb, FokI-ff/Ff) influence metabolic and cutaneous features, suggesting a genetic basis for VD-related disturbances in PCOS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eThe study accentuates the need for personalised therapeutic strategies, particularly VD supplementation, based on genetic profiles to manage PCOS and its associated metabolic disturbances.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKey Message:\u003c/strong\u003eVDD, a genetic predisposition related to \u003cem\u003eVDR\u003c/em\u003e-SNPs, combined with limited sun exposure and poor dietary choices, exacerbates PCOS symptoms, impacting metabolic and endocrine homeostasis.\u003c/p\u003e","manuscriptTitle":"Unraveling the interplay between vitamin D deficiency, VDR polymorphisms, and polycystic ovary syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-16 09:39:21","doi":"10.21203/rs.3.rs-5417644/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c41b4570-bad4-47b8-a453-bde279f0a04b","owner":[],"postedDate":"December 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T09:39:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-16 09:39:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5417644","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5417644","identity":"rs-5417644","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}