Polycystic ovary syndrome (PCOS) is characterized by a wide range of clinical, biochemical, and sonographic characteristics; there is often ultrasound evidence for polycystic ovaries, and clinical signs such as oligomenorrhea, hyperandrogenism, hirsutism, and anovulation can also be categorized as biochemical hallmarks of PCOS (1). Other important complications of polycystic ovary syndrome include infertility, obesity, dark brown or black patches on the neck, male pattern baldness, and its long-term effects include an enhanced risk of type 2 diabetes, insulin resistance, hypertension, dyspepsia, lipidemia, and cardiovascular diseases (2). It has been shown that PCOS has a prevalence of 5–10% (1).
One of the main physio-pathological features of PCOS is the overproduction of ovarian androgens (3). Previous studies showed various genes, including CYP11A1, CYP17, HSD17B6, PPARγ, INSR along with CYP19 encoding necessary enzymes in androgen biosynthesis pathways (4-10). However, the findings from studies about the association of these genes are inconsistent.
CYP11A1 gene is located on chromosome 15 (15q23-24). It has 10 exons and 9 introns with an approximate length of 30 kb (11). It encodes the cholesterol side-chain cleavage enzyme, P450 scc, a member of the cytochrome P450 superfamily of enzymes, which resides in the mitochondrial inner membrane and catalyzes the conversion of cholesterol to pregnenolone, the first rate-limiting stage in steroid hormones synthesis (12). This is mainly expressed in steroidogenic tissues, including the adrenal cortex, gonads, and placenta (13). Genetic variants of CYP11A1 change its expression, causing definite hormone-related diseases, including breast cancer, prostate cancer, endometrial cancer, and polycystic ovary syndrome (14-17). Polymorphisms in this gene are proposed to have an essential role in regulating CYP11A1 expression via transcriptional up- or down- regulation resulting in enhanced or reduced androgen production (3).
A research in 2013 demonstrated a significant relation between rs6495096 polymorphism and PCOS (18). In another study performed by Zhang et al. in 2012, a relation between SNP rs4077582 in CYP11A1 and susceptibility to PCOS was reported. It changes testosterone levels by regulating LH in various genotypes in Chinese women but no association was found between rs11632698 and PCOS (19). Gao et al. showed that CYP3A4 and CYP11A1 genes might have an important impact on the stroke risk in Han Chinese women (20). Although CYP11A1 gene's role in PCOS patients is not yet fully understood, the expression of CYP11A1 gene in human ovaries has been demonstrated (21). There has been no study regarding the genotype distribution of SNP rs1484215 and rs6495096 in CYP11A1 in Iranian women (PCOS vs. normal); thus, the current research was performed to investigate the relation of CYP1A1 polymorphism and development of PCOS in Iranian women.
Study cases: In this case-control study, the population included 200 Iranian women (20-40 years) referred to infertility treatment center of ACECR of Qom in December 2019 to December 2021 (100 women with PCOS, and 100 aged-matched non-PCOS women as control with normal menstrual cycles between 28-32 days, without hirsutism, severe acne, hair loss or insulin resistance signs). Subjects suffering from PCOS were enrolled according to 2003 Rotterdam criteria which determine the disorder based on symptoms of clinical, biochemical hyperandrogenism, ovulatory dysfunction, and polycystic ovary morphology (22). The exclusion criteria of the study included infertility with male factor, endometriosis, diabetes mellitus, hypertension, acromegaly, congenital adrenal hyperplasia, androgen-secreting tumors, Cushing’s syndrome, hyperthyroidism, hyperprolactinemia, and ovarian tumor or women on oral contraceptives for the last 6 months, and smokers. In this study, people referred to this IVF selection center for sex and family balancing determination with healthy children and lack of any disorders were considered as the control group. The study was confirmed by the Institutional Review Board and its protocol was confirmed by the ethical committee of the University of Qom (Ethics code number: IR.Qom.REC. 1398.018). Moreover, all of participant in this study completed an informed consent form.
Assessment of demographics and endocrine profiles: Demographic information was registered, including height and body weight. Body mass index (BMI) was evaluated as weight (kg) divided by height (m2). For both groups, on the morning of the second or third day of the menstrual cycle after overnight fasting of at least 12 hr, blood samples were taken. The peripheral blood sample was instantly centrifuged for 10 min at 3000 rpm (Hettich, UK). The serum levels of follicle-stimulating hormone (FSH; Cat. N. DE1288), luteinizing hormone (LH; Cat. N. DE1289), prolactin (PRL; Cat. N. DE1291), estradiol (E2; Cat. N. LOT 1007843740), anti-mullerian hormone (AMH; Cat. N. LOT 001-50-N488), and total testosterone (TT; Cat. N. DE1559) of subjects were evaluated by ELISA (Demeditec Diagnostics GmbH, Germany) for the hormonal profile. The electrochemiluminescence immunoassay kit with a Roche Cobas e 411 auto-analyzer (Roche Diagnostics, Germany) was utilized to evaluate the serum level of fasting blood sugar (FBS, mg/dl).
Genotyping of SNP rs1484215 and rs6495096: The data on the human CYP11A1 gene were gathered from Entrez Gene on National Center for Biotechnology Information (NCBI) website. The SNP ID-info of this gene was received from the NCBI’s single-nucleotide polymorphism database (dbSNP) at http://www.ncbi.nlm.nih. gov/snp/. SNPs of the CYP11A1 gene with minor allele frequency (MAF) >0.05 in the HapMap Asian population were obtained from previously published polymorphism studies related to PCOS and were applied to an initial screening. Genomic DNA was extracted from peripheral leukocytes via salting out procedure (23). For DNA extraction, 3 ml of peripheral blood were obtained from the coagulant in sterile tubes with EDTA anticoagulants and stored at -70°C.
Polymerase chain reaction (PCR): The DNA extraction process was done by the GeneAll Exgene TM kit (105-101) and according to the relevant instructions. DNA purity along with quantity was assessed by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) at 260/280 nm. The quality of bands from DNA derived by agarose electrophoresis was studied. Genotypes were determined by the amplification-refractory mutation system (ARMS) technique. Tetra-primer amplification refractory mutation system-polymerase chain (T-ARMS-PCR) was optimized to find rapid, efficient, and economic laboratory method for detecting SNPs or mutations. The reaction of T-ARMS-PCR was run in a single tube and in one PCR step, and genotype variation from SNPs of interest could be visualized directly using common agarose gel electrophoresis (24). To amplify the gene in specific SNPs, specific primers were designed using primer design for tetra-primer ARMS-PCR (PRIMER1). The characteristics of the primers along with the length of the reproduced piece are given in table 1.
Initial denaturation at 94°C for 5 min was performed by 35 cycles at 94°C for 60 s, at 56°C for 40 s and extension at 72°C for 40 s continued with a final extension at 72°C for 10 min. Subsequently, the PCR products were separated by 2% agarose gel electrophoresis and visualized under UV illuminator following ethidium staining (AlphaImager™ Gel Imaging System, Alpha Innotech, USA) (Figure 1).
Statistical analysis: Mann–Whitney U test and Chi-square test for categorical variables and the student’s t-test for continuous variables were utilized to measure the differences in demographic characteristics of both groups and for analysis of the genotype distribution in our case–control study using SPSS software vs. 23.0 (SPSS Inc., USA). All P values were two-tailed, and those less than 0.05 were determined statistically significant. Moreover, 95% confidence intervals (CIs) and odds ratios (ORs) were measured. Genotype frequencies were checked for Hardy–Weinberg equilibrium in both PCOS and control groups.
Characteristics of the study participants: The demographic, biochemical, and hormonal parameters in both groups were compared (Table 2). BMI, the waist-to-hip ratio (WHR), and infertility duration significantly differed in both groups (p<0.01 ). also, hormonal as well as biochemical parameters (lh, lh sh ratio, total testosterone, fasting blood sugar (fbs), prolactin, amh, and estradiol) illustrated that both groups had significant differences (p<0.01). nonetheless, fsh and age did not significantly differ in both groups.
Association of SNPs with PCOS: Table 3 shows the genotype distribution of SNPs rs1484215 along with rs6495096 of CYP11A1 gene and allele frequencies. The genotype "CG" of rs6495096 was associated with enhanced PCOS risk (OR=0.336, 95%CI=0.171-0.66, p=0.005) as analyzed by the co-dominant model (Table 3). No difference was found in rs1484215 (Table 3).
Distributions of the genotypic frequencies satisfied Hardy-Weinberg equilibrium in both groups. According to the Hardy-Weinberg test, the frequency of genotypes in rs1484215 and rs6495096 is not in balance and varies from generation to generation (p0.001). The frequency distributions of CYP11A1 rs6495096 alleles in PCOS were 1.14 for C and 0.86 for G compared with 1.34, 0.66 for C and G in the control group, respectively (p=0.05) (OR=0.653, 95% CI=0.435-0.98). The frequency distribution of CYP11A1 rs1484215 alleles in PCOS were 0.97 for T and 1.03 for C compared with 1.05, 0.95 for T and C in the control group, respectively (p=0.484) (OR=0.852, 95%CI=0.576-1.261) (Table 3).
Associations between different demographic, biochemical, hormonal, and sonographic characteristics with different genotypes among 100 women with PCO were studied. The waist-to-hip ratio (WHR) and infertility duration were significantly higher among women with genotype GG of rs6495096 compared to genotypes CC and CG (p0.01). Other biochemical and hormonal parameters showed insignificant differences among different genotypes of rs6495096 (Table 4). As for rs1484215, no difference was identified in all biochemical and hormonal parameters among the three genotypes (Table 4).
Receiver operating characteristic (ROC) analysis was used as a scale to measure the ability to separate a model with the largest area that indicates better predictive ability. It was applied to compare the performance of models. In this study, the value of the area under the ROC curve for the conditional logistic regression (95%, CI:0.96-1) was obtained with a sensitivity of 99% and a specificity of 97%. The results of the analysis of the conditional logistic regression showed that among all the variables in table 2, the level of testosterone, FSH, and AMH variables demonstrated the greatest effect (Figure 2).
The complex nature of polycystic ovary syndrome (PCOS) generates a need to investigate genetic and environmental factors contributing to its progression in Iranian women. In the present study, rs6495096 was found to be associated with infertility duration and waist-to-hip ratio (WHR) showed significant association with PCOS, indicating that rs6495096 may affect the P450scc enzyme activity and thus androgen production. The statistical results of the distribution of genotypes of rs6495096 indicate that individuals with CG genotype are at higher risk of PCOS when compared to CC and GG genotype groups; however, in control group, carriers of CC and GG consist the majority of cases in the group. Consequently, "C" allele of rs6495096 in PCOS cases was less frequent than the control group. Therefore, carriers of allele "G" are more susceptible to developing PCOS. Also, the current study showed that waist-to-hip ratio (WHR) and infertility duration were higher among women with PCOS who carried the genotype GG in comparison to genotypes CG and CC.
While PCOS is considered a heterogeneous disorder, its genetic base is supported via accumulated evidence from twin concordance and familial aggregation (3). In the light of lacking universally accepted criteria for PCOS, researchers could not propose a pathogenic gene for triggering PCOS, although earlier studies showed the potential influence of SNPs of CYP11A1 gene in different populations (13, 25, 26). This study investigated the potential pathogenic role of CYP11A1, especially SNPs rs1484215 and rs6495096, and the association of several genetic variants of rs1484215 and rs6495096 with PCOS predisposition in Iranian women. Results from the study indicated that significant genotypic and allelic frequencies for SNP rs6495096 significantly differ in both groups.
In line with our results, Shan et al. discovered four SNPs (rs12917295, rs11632698, rs1484215, and rs6495096) which constructed four haplotypes "(CACG, CATC, CACC, GGCC)" in the CYP11A1. There was no significant relation between haplotypes and the risk of PCOS (13). In Chinese women, four-, six-, and eight-repeat alleles are related to breast cancer (27) and polycystic ovary syndrome (28) are the key variants. The mechanism was different while CYP11A1 polymorphism was related to breast cancer and POCS. Estrogen is associated with breast cancer. Androgen is related to PCOS (13).
Earlier researches on the relation of CYP11A1 with PCOS dealt with (tttta)n microsatellite polymorphism in the CYP11A1 promoter (9, 14, 29). In a research by Zhang et al., rs4077582 was related to PCOS pathogenesis, showing that rs4077582 can influence the P450scc enzyme activity and affect androgen production (19). P450scc catalyzes the first stage of androgen and estrogen biosynthesis and changes cholesterol into pregnenolone, an androgen precursor (30). Thecal cells cultured from PCOS samples indicate enhanced androgen production or enhanced CYP11A mRNA expression compared to samples from control women (31). In particular, PCOS individuals with the GG genotype of rs6495096 showed a significantly higher waist-to-hip ratio (WHR) and infertility duration compared to that of CC and CG genotypes, though no difference was found in the waist-to-hip ratio (WHR) and infertility duration in the CC and CG genotype. These bits of evidence demonstrated that normal women with GG genotype of rs6495096 were more likely to experience PCOS, which was supported by the fact that PCOS patients had a greater proportion of GG genotype as compared to controls.
This research is the first to report the relation of rs6495096 in CYP11A1 gene with PCOS in Iranian population. Some limitations have constrained the replication of the current research. Firstly, the sample size was relatively small, and further studies are required to identify the possible relation of CYP11A1 gene with PCOS. Secondly, only two SNPs causing incomplete coverage of the gene variations were selected. Large and more genome-wide association researches among PCOS cases will be essential to determine new candidate genes as well as proteins responsible for PCOS risk.
Based on the findings of this study, it was revealed that polymorphism rs6495096 of CYP11A1 gene may increase the risk of PCOS in Iranian women. However, PCOS cannot be associated with a single factor of this gene, and more research should be conducted to discover other risk factors for PCOS; moreover, the capacity of CYP11A1 gene must be studied in future research.
We appreciate the assistance of members of the Infertility Research Center at ACECR, Qom.
Funding: This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.
Conflict of Interest
The authors declare that there is no conflict of interest.