Association of FokI, Tru91, and ApaI vitamin D receptor gene polymorphisms with the development of polycystic ovarian syndrome: A molecular genetic study

1 Introduction

Polycystic ovary syndrome (PCOS) is prevalent as a condition among women of reproductive age and affects the reproductive, endocrine, and metabolic systems (Ganie et al. 2024). In addition to hyperandrogenism, a lack of ovulation, acne, hirsutism, irregular menstrual cycles, and alopecia (Raja et al.,2024) have a range of complications. The National Institute of Health (NIH) 1990 and Rotterdam 2003 definitions have been used to determine the global prevalence of PCOS (Jaswa et al. 2020). The combined influence of genetic and environmental factors is generally seen as vital to decide on the incidence and progression of PCOS (Mehdizadeh et al. 2017). There is a possible relationship between vitamin D concentrations, metabolism, and endocrine problems, according to research by (Tobias et al. 2023). Vitamin D is one of the main steroid hormones that helps maintain calcium levels and supports the progression of bone minerals (Skalny et al. 2024). Vitamin D is reformed into 1,25-dihydroxycholecalciferol in the liver and kidneys (Makris et al.,2020). Low vitamin D levels have previously been related to PCOS symptoms, clinical indications, and the consequences of the condition in many studies (Wehr et al., 2009; Krul-Poel et al., 2013). Vitamin D receptors (VDRs) exist in several regions of the female reproductive system (Lu et al. 2024). VDR controls the effect of the hormone 1,25(OH)2D3 on the body, which assembles a signal-transduction complex in conjunction with a heterodimer. This compound comprises a VDR coupled to 1α,25(OH)2D3 (Hendi 2023). SNPs, or single nucleotide polymorphisms, are the most frequent variations seen in nucleotides in human DNA, where the VDR gene is set at 12q13.11. The structure has eight exons that code for proteins and one untranslated exon (Baker et al. 1988). It has been found that the three most prevalent VDR variants are FokI (rs10735810 C > T), in exon 2, which are 265 bp in length. The length of the ApaI gene (rs7975232 G > T) in exon 8 is 740 bp, while that of the Tru9I gene (rs5757343 A > G), which is situated within intron 8 has a length of 331 bp. They are investigated in various studies regarding the associations between VDR and PCOS susceptibility (Shi et al. 2019). Thus, this research aims to observe the impact of the vitamin D3 together with the vitamin D receptor gene including Apa1, Fok1, and Tru9Ion, on the probability and vulnerability of PCOS. Additionally, a further goal of the present study is to look at the links relating to the impact of three genetically significant polymorphisms on anti-Mullerian hormone (AMH) levels in PCOS females.

2 Subject and Methods

3 Results

The main demographic features of the participants and controls are reported in the following Table 2.

3.2

3.2 Molecular part of the study

3.2.1

3.2.1 Correlation between the VDR (FokI) gene polymorphism and PCOS

The results of the RFLP analysis for genotypes CC, CT, and TT are illustrated in Fig. 1.

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Fig. 1

A: PCR product for the VDR gene (FokI, 265 bp). Fig. 1-B: PCR-RFLP analysis of FokI polymorphisms following digestion with FokI restriction enzymes. The 265 bp bands showed wild homozygote C/C. Both the 96 and 169 bp bands showed mutant homozygote T/T. The 265, 96, and 169 bp bands indicated the presence of heterozygote C/T.

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The FokI genotyping revealed that the homozygous CC genotype was less prevalent in PCOS women (32.5 %) than in the controls (60 %). However, this difference indicated a significant negative association with PCOS patients (OR 0.32, 95 % CI (0.13–0.02)), indicating that CC genotyping is a protective factor against this condition. The prevalence of the heterozygous CT genotype (62.5 %) was greater in PCOS patients than in the control (36 %), where this difference was significant in PCOS patients, according to an OR of 2.96 with a 95 % CI in the range of 1.16–––7.54. This suggests that the CT genotype is a strong risk factor for that condition. Moreover, the TT genotype was less prevalent in the control group (4 %) than in the PCOS group (10 %), but this difference was not significant (P-value = 0.84). Furthermore, according to the odds ratio, allele T is associated with disease (odds ratio = 2.02) among PCOS patients and controls, demonstrating that it may be associated with an increased susceptibility to PCOS (OR > 1.0). In addition, the CC-dominant model selected in this study had a protective factor, OR, of 0.32 (P = 0.01) (Table 4).

Table 4 Frequency analysis and HWE of genotypes, alleles, and genetic models for the rs10735810 SNP study among Iraqi women with PCOS and a control group.

Genotyping	Frequency, n (%)		H-WE	p-value	Odds ratio	CI95%
	PCOS	Control
CC	26 (32.5 %)	15 (60 %)	P = 0.81 X2 = 0.059	0.02	0.32	0.13–0.81
CT	50 (62.5 %)	9 (36 %)		0.02	2.96	1.16––7.54
TT	4 (10 %)	1 (4 %)		0.27	1.26	0.13–11.85
Allele distribution
C	102 (63.75 %)	39 (78 %)		0.06	0.49	0.24–1.04
T	58 (36.25 %)	11 (22 %)		0.06	2.02	0.96–4.23
Genetic mode
CC	26 (32.5 %)	15 (60 %)		0.01	0.32	0.13–0.81
C/T-T/T	54 (67.5 %)	10 (40 %)
CT/CC	76 (95 %)	24 (96 %)		0.84	0.8	0.08–7.42
TT	4 (5 %)	1 (4 %)

The significant difference is considered to be ≤ 0.05. OR: odds ratio, Cl: confidence interval.

3.2.2

3.2.2 Correlation between VDR (Apal) rs7975232 gene polymorphism and PCOS risk

The genotypic frequencies of the Apal SNPs in the control group conformed to HWE. The Apa1 genotypes were positively associated with disease based on the odds ratio (1.1247; 95 % confidence interval [CI] 0.4427–2.8573 for the AA genotype, and 3.4286; 95 % CI 0.4168–28.2034 for the CC genotype). The heterozygote genotype AC showed a negative association with disease because the frequency of the AC genotype in the control group was greater than that in the PCOS group; however, this relationship was not significant (0.6341 odds ratio: Cl between 0.2546 and 1.5792). Moreover, no significant association was detected between the two alleles and PCOS patients in contrast to the control group, implying that the C allele might be considered a risk factor (OR = 1.13, 95 % CI, 0.58–2.21). Depending on the odds ratio, the recessive model could potentially be a risk factor (OR = 3.43, 95 % CI, 0.42–28.20, P-value = 0.25), as reported in Table 5 and Fig. 2.

Table 5 Frequency analysis and HWE of genotypes, alleles, and genetic models for the rs7975232 SNP among Iraqi women with PCOS and the control group.

Genotyping	Frequency, n (%)		H-WE	P-value	Odds ratio	CI95%
	PCOS	Control
AA	31 (38.75 %)	9 (36 %)	P = 0.09 X2 = 2.84	0.85	1.12	0.44–2.86
AC	39 (48.75 %)	15 (60 %)		0.33	0.63	0.44–2.86
CC	10 (12.5 %)	1 (4 %)		0.25	3.43	0.42–28.20
Alleles distribution
A	101 (63.13 %)	33 (66 %)		0.71	0.88	0.45–1.72
C	59 (36.88 %)	17 (34 %)		0.71	1.13	0.58–2.21
Genetic model
A/A	31(38.75 %)	9 (36 %)		0.80	1.12	0.44–2.86
A/C–C/C	49 (61.25 %)	16 (64 %)
A/C- A/A	70 (87.5 %)	24 (96 %)		0.25	3.43	0.42–28.20
C/C	10 (12.5 %)	1 (4 %)

Significant difference is at ≤ 0.05.

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Fig. 2

A: PCR results for the VDR gene (Apa1, 740 bp). 2-B: Electrophoretogram of DNA fragments for ApaI variations after ApaI digestion. Homozygote A/A was indicated by the bands at 740 bp. The bands at 530 and 210 bp indicated homozygote C/C. Heterozygous A/C was indicated through the bands at 740, 530, and 210 bp.

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3.2.3

3.2.3 Correlation between the VDR (Tru9I) rs757343 gene polymorphism and PCOS risk

The DNA obtained was analyzed via the PCR-RFLP technique. Three distinct genotypes (AA, AG, and GG), which are depicted in Fig. 3 (A, B).

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Fig. 3

A PCR results for the VDR gene (Tru9I, 331base pair). Fig. 3-B: Electrophoretogram of DNA segment for Tru9I variation after digestion with MesI. Homozygous wild A/A was present as a 331 bp. The homozygote mutant (G/G) was present at 178 and 153 bp. A heterozygous A/G was present as the 331, 178, and 153 bp.

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There were significant differences in the distribution of genotypes and allele frequencies between the control group and the PCOS patients at which the prevalence of the homozygous AA genotype was 65 %, while in healthy women was 40 % (OR = 2.78; 95 % CI; 1.11–7.01), indicating a strong positive correlation. The significant genotype heterozygous AG was present in 23.75 % of PCOS patients and 56 % of the controls, showing a negative association with PCOS (OR = 0.2447; 95 % CI; 0.2447–7.0089). Furthermore, the frequency distributions of the homozygous GG genotyping in the PCOS patients and the control group were 11.25 % and 4 %, respectively with positive risk factors (OR = 3.0423; 95 % CI; 0.37–25.27). Moreover, the A allele demonstrated a positive correlation in PCOS women (OR = 1.56; 95 % CI; 0.78–3.15). Additionally, the distribution of Tru91 SNPs was significant under AA-dominant genetic models and high-risk factors among Iraqi women with PCOS (OR = 2.79; 95 % CI; 1.11–7.01) (Table 6).

Table 6 Frequency analysis and HWE of genotypes, alleles, and genetic models for the rs757343 SNP in Iraqi women with PCOS and in controls.

Genotypes	Frequency, n (%)			p-value	Odds ratio	CI95%
	PCOS	Control
AA	52 (65 %)	10(40 %)	P = 0.15 X2 = 2.06	0.03	2.78	1.11–7.01
AG	19 (23.75 %)	14(56 %)		0.00	0.24	0.09––7.01
GG	9(11.25 %)	1(4 %)		0.30	3.04	0.37–25.28
Alleles distribution
A	123 (76.88 %)	34 (68 %)		0.21	1.57	0.78–3.15
G	37 (23.13 %)	16 (32 %)		0.21	0.64	0.31–1.28
Genetic model
A/A	52 (65 %)	10 (40 %)		0.03	2.78	1.11–7.01
A/G-G/G	28 (35 %)	15 (60 %)
A/G- A/A	71 (88.75 %)	24 (30 %)		0.30	0.33	0.04–2.73
G/G	9 (11.3 %)	1 (4 %)

The significant difference is at ≤ 0.05.

4 Discussion

The demographic data of the study patients and healthy individuals revealed that the age was not significantly different between the two study groups. These results were in agreement with the findings of a previous study conducted by (Ganie et al. 2020). The mean BMI ± SD of women with PCOS did not differ significantly from that of healthy controls. These results are consistent with those reported by (Dumesic et al. 2021), who suggested there were no significant differences in the mean BMI of PCOS women compared with that of the healthy control (P > 0.05). However, these results were in disagreement with another study that found a statistically significant difference in the BMIs of PCOS patients and their control group (P < 0.05) (Almukhtar and Almohaidi 2019). Additionally, this study found that those with PCOS had a notably higher occurrence of hirsutism (72.5 %) and acne (62.5 %). This is in comparison to a previous study by (Özdemir et al. 2010) Which reported that 53 % of patients with PCOS had acne and that hirsutism was prevalent in 73.9 % of patients. This could be attributed to hormonal abnormalities. In the current study, 92.5 % of women had irregular menstrual cycles, while 7.5 % had regular; however, all healthy individuals had regular menstrual cycles. Our study agreed with Iraqi research that revealed an obvious increase in irregular menstruation patterns in women with PCOS (78.8 %) compared with a control group (0.00 %) (Al-Quraishy et al. 2022). In addition, 44(55 %) were married and 36 (45 %) were single women in cases whereas 5(20 %) were married, 20(80 %) were single women in control. Out of 44 (55 %) married patients, 35 (79.5 %) had infertility disorders, whereas 9 (20.5 %) had normal fertility while the control group represented 5 (100 %) married women who had normal fertility (P < 0.05) as reflected in Table 2. Our study agreed with that of (Joham et al. 2015), who reported that women with PCOS were significantly more likely to be infertile (72 %, n = 224) than women without PCOS (16 %, n = 747) (p < 0.05) due to elevated ovarian androgen and LH to FSH ratios (Louwers and Laven 2020).

The results of vitamin D, free testosterone, and anti-Mullerian hormone analyses showed that PCOS patients had higher levels of free testosterone and anti-Mullerian hormone. Vitamin D deficiency (< 20 ng/mL) was significantly more common among PCOS patients. This result was in agreement with (Omran et al. 2020) while other studies in Iraq concluded that serum vitamin D3 levels in women who suffer from PCOS are significantly lower than those in the associated control groups (Quasi et al., 2022). On the other hand, other research revealed there is no link between low vitamin D levels and an increased risk of PCOS (Moini et al. 2015). A significantly low level of vitamin 25(OH)D3 in PCOS patients could potentially indicate that vitamin D plays a vital role in regulating estrogen production (Yang et al. 2023).

Our study revealed increased levels of free testosterone among women with PCOS. This finding was in agreement with the studies performed in Iraq (Abdalqader and Hussein 2020), who focused on the fact that increased free testosterone was significantly greater in PCOS patients than in the controls. In a cross-sectional study performed at Qatar University, 126 female students were subjected to hormonal analysis, demonstrating a significant increase in free testosterone (P-value < 0.0001) (Sharif et al. 2016). Our results revealed that women with PCOS had an AMH concentration > 4 ng/mL. A previous Iraqi study reported that serum AMH levels were elevated in PCOS patients compared with controls (P-value <0.05) (Alfatlawi 2017). A study in Saudi Arabia also demonstrated that the control and PCOS groups had significantly different AMH levels. The additional group revealed that AMH levels in the case group were twice as high as those in the healthy group (Moursi et al. 2023). When AMH production increases, follicles become less sensitive to FSH at the receptor level, which is crucial for their growth. This led to a subsequent rise in the number of antral follicles but a decrease in their size, leading to an increase in the number of small antral follicles (size 2–5 mm) (Kristensen et al. 2022). The PCR-RFLP study revealed that the most common genotype in PCOS women was the CT of Fok1 (rs2228510), which is heterozygous for the VDR gene. Furthermore, allele T has been identified as a risk factor in making women more likely to develop this condition, as based on the OR. Our results are in agreement with those of the Iraqi study in Karbala, demonstrating a statistically significant association between the Fok1 heterozygous genotype with a one-fold increase in the incidence of PCOS (OR = 2.04, 95 % CI; 2–4.2, P < 0.05) (Zahra and Altu’ma 2019). However, our results disagreed with those of a study in Poland that reported that there was no correlation between the distribution of FokI polymorphisms among PCOS patients and healthy individuals (Jedrzejuk et al. 2015). This suggests there may be other affecting factors such as genetic heterogeneity, environmental factors, or sample size. The PCR-RFLP of Apa1 (rs7975232) PCOS patients were more likely to have CC than the controls, and the OR was three-fold greater in PCOS patients than in the controls, whilst AC was more frequent in the controls than in the patients. Furthermore, the presence of allele C showed a positive correlation with PCOS according to the OR. In this research, we noted the impact of the homozygote CC genotype of the VDR gene SNP (rs7975232) in intron 8 on susceptibility to PCOS development amongst Iraqi women.

The genotyping according to the OR revealed that recessive CC homozygotes have greater risk factors for VDR gene polymorphisms as represented in Fig. 4. Our study was in agreement with an Iranian study (Mahmoudi 2009) which revealed that the VDR Apa-I AC genotype (Aa in the Iranian study) was linked to a lower risk of PCOS, while the CC genotype (aa in the Iranian study) was linked to a higher risk of PCOS. The ApaI polymorphisms are located in intron 8, specifically at the 3′ terminus of the VDR gene. The 3′ untranslated region (UTR) of genes plays a significant role in regulating gene expression, especially about mRNA stability (Fang et al. 2005). Among PCOS patients, the most prevalent genotype for the Tru91 variation in the VDR gene was AA GG. Additionally, allele A has been strongly implicated as a basic cause of the disease based on the observed OR. Regarding the OR, our study was somewhat in agreement with (Zadeh-Vakili et al. 2013), who found that the presence of the A allele is associated with a 74 % increase in risk of developing severe PCOS (OR, 1.74; 95 % CI; 1.07–2.82), whilst the combined genotype (GA + AA) was associated with the severity of clinical features of PCOS, including severe hirsutism and oligo-amenorrhea. However, our study disagreed with the literature about the association between the VDR Tru9I rs757343 (G > A) variant and susceptibility to PCOS. The study focused on populations of Asian descent, and indeed a further study conducted for Asian populations did not identify any significant association between the VDR Tru9I rs757343 (G > A) polymorphism and susceptibility to PCOS (Zadeh-Vakili et al. 2013).

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Fig. 4

DNA sequencing of Fok1 (CC, CT, and TT) in the upper section, Tru91 (GG, GA, and AA) in the middle, and Apa1 (CC, AC, and AA) in the lower portion.

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The study concludes that increases in free testosterone and anti-Mullerian hormone levels, and decreases in vitamin D, are predictive of PCOS. Furthermore, this meta-analysis provides statistical evidence that VDR polymorphisms correlate with PCOS risk across all genetic models, including VDR FokI polymorphisms under the dominant model and VDR ApaI polymorphisms under the recessive model. VDR Tru91 is dominantly inherited. More research with a larger sample size and the examination of additional confounding variables is necessary to draw firm conclusions.

CRediT authorship contribution statement

Walaa Talal Abdul-Lateef: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Mushtak T.S. Al-Ouqaili: Writing – review & editing, Supervision, Project administration, Investigation, Conceptualization. Rafal M. Murshid: Validation, Supervision, Project administration, Investigation.