Molecular study of CYP21 gene polymorphism rs13405728 and CYP11A1 gene polymorphism rs4077582 in polycystic ovarian syndrome patients

Background PCOS is a serious endocrine-metabolic condition characterized by hyperandrogenemia, anovulation, or oligo-ovulation, and links to obesity, insulin resistance, and an elevated risk of type 2 diabetes mellitus. The pathophysiology of PCOS is thought to involve both environmental and genetic factors. PCOS etiology has been linked to genetic factors, with the CYP21 and CYP11A1 genes identified as possible candidate genes. Previous research has linked the rs13405728 polymorphism in the CYP21 gene and the rs4077582 polymorphism in the CYP11A1 gene to PCOS. However, more research is needed to confirm these connections in specific populations. The purpose of this study was to look at the role of single gene polymorphisms in PCOS, specifically the rs13405728 polymorphism in the CYP21 (LHCGR) gene and the rs4077582 polymorphism in the CYP11A1 gene. Blood was drawn from 150 PCOS patients and 150 age-and gender-matched healthy people. The phenol–chloroform procedure was used to extract DNA, and gel electrophoresis was used to quantify it. To analyze polymorphisms, researchers used polymerase chain reaction (PCR) with the allele-specific amplification refractory mutation system (ARMS-PCR) to amplify specific areas of DNA. ARMS-PCR was used to detect mutations in the CYP21 and CYP11A1 genes, followed by sequencing to examine the rs13405728 polymorphism and rs4077582 polymorphism, respectively, in 150 PCOS patients and 150 control people. ARMS-PCR polymorphism study of the CYP21 (LHCGR) and CYP11A1 genes indicated significant correlations. Results For the CYP21 gene, heterozygous (CT) carriers of the rs13405728 polymorphism had a fourfold greater incidence of PCOS (OR 4.10; CI 2.47–6.80; p = 0.0001), whereas homozygous mutant (TT) carriers had a significant connection with PCOS (OR 0.27; CI 0.16–0.45; p = 0.0001). These data imply that the CYP21 (LHCGR) gene polymorphism rs13405728 has a substantial impact on the development of polycystic ovarian syndrome. The data for the CYP11A1 gene show the SNP (rs4077582) heterozygous (CT) was associated with PCOS (OR 1.72; 95% CI 1.02–2.88; p = 0.0392). The identical SNP heterozygous (CT) raised the incidence of PCOS by up to onefold. The homozygous mutant SNP (TT) had no connection with illness onset (OR 1.377; 95% CI 0.85–2.2; p = 0.1855), while the mutant (TT) of the SNP nearly doubled the incidence of polycystic ovarian syndrome. The combined model of the same SNP (CT + TT) revealed a significant correlation with PCOS (OR 2.1905; 95% CI 1.355–3.53; p = 0.0014). The combination model (CT + TT) of the same SNP more than doubled the risk of polycystic ovarian syndrome. All the risk factors investigated had a substantial connection with PCOS.


Background
Polycystic ovarian syndrome (PCOS) is a major endocrine-metabolic condition that causes hormonal and metabolic dysregulation in reproductive-aged women all over the world.PCOS has a complex etiology that incorporates both genetic and environmental variables.Familial aggregation analysis and investigations on numerous clinical features across different areas and races have provided evidence that several genetic and environmental variables are involved in the pathogenesis of PCOS (Ha et al., 2015).Hyperandrogenemia and anovulation/ oligo-ovulation are frequent symptoms of PCOS, as are comorbidities like obesity and insulin resistance.Insulin resistance impairs reproductive function by interfering with insulin signaling in the central nervous system and disrupting the direct insulin pathway on steroidogenesis (Sam et al., 2003;Iftikhar et al., 2023).PCOS has a wellestablished genetic component, with heredity accounting for up to 70% of the disorder's variance (Abdel-Mageed et al., 2016;Mykhalchenko et al., 2017).PCOS development and progression are influenced by both hereditary and environmental factors.Obesity, poor eating habits, and physical inactivity might aggravate PCOS symptoms in vulnerable individuals.The precise roles of environmental variables in PCOS pathogenesis, including infectious agents and toxins, are currently being investigated.Because of the syndrome's clinical variability, genetic studies of PCOS have proved difficult.While various candidate genes, including CYP11A, the insulin gene, and the follistatin gene, have been identified as potential contributions to PCOS, the greatest evidence leads to a region on chromosome 19p13.3near the insulin receptor gene.The precise gene responsible for the PCOS phenotype in this location, however, has yet to be identified (Bilal et al., 2022).PCOS is a complicated condition in which hereditary and environmental factors both play important roles.PCOS affects approximately 20-40% of females with first-degree relatives, demonstrating partial heredity and familial clustering.The prevalence and severity of PCOS presentation also vary by ethnicity.Several candidate genes have been discovered as potential factors for PCOS risk, including 7-hydroxysteroiddehydrogenase type 6 (HSD17B6).However, identifying a specific gene that causes or significantly contributes to the development of the PCOS phenotype remains challenging (Franks & Hardy, 2018).More research is needed to understand the intricate interaction of genetics and environment in the development of PCOS.
PCOS is caused by a complex and multifaceted etiology that includes both genetic and environmental variables (Ha et al., 2015).PCOS is heritable, according to family studies and genetic analysis, implying a major genetic component in its pathophysiology (Ha et al., 2015;Bhattacharya et al., 2010).PCOs are typically thought to be a combination of monthly irregularity and a greater number of androgens in females of childbearing age.PCOS is linked to alterations in several hormonal secretions, most notably insulin, and gonadotropin hormone.Later in adulthood, PCOS women develop truncal obesity, impaired glucose tolerance (IGT), T2DM, dyslipidemia, cardiovascular disease, hypertension, and partial metabolic syndrome.PCO patients exhibit symptoms of negative body image perception, low self-esteem, sadness, and decreased quality of life in addition to structural disease (Ambrose & Barua, 2004;Deeks et al., 2011).
The CYP21 (LHCGR) gene has emerged as a prospective candidate due to its involvement in steroid hormone metabolism and regulation.Polymorphisms in numerous genes have been implicated in the development and progression of PCOS, among other genetic variables.CYP21 (also known as cytochrome P45021A2) located on chromosome 6p21.3,which encodes the enzyme 21-hydroxylase involved in steroid hormone production (e.g., synthesis of cortisol and aldosterone hormone), is one such gene of relevance (Barbieri, 2014;Yariz et al., 2011).It has been proposed that genetic differences or polymorphisms in the CYP21 gene influence the chance of developing PCOS.The rs13405728 polymorphism belongs to the G-protein receptor family and has been linked to PCOS in several studies (Almawi et al., 2015;Brower et al., 2013;Yariz et al., 2011).The rs13405728 polymorphism was found in the CYP21 gene's regulatory region and has been linked to changes in gene expression and enzyme activity.The Chinese community has the most genetic variance in connection to polycystic ovarian syndrome (PCOS), according to genome-wide association studies (GWAS), with several loci found in two independent GWAS.The first GWAS discovered LHCGR and DENN1A genes to be linked to PCOS, whereas the second GWAS discovered additional loci such as FSHR, C90rf3, and INSR (Chen et al., 2011;Shi et al., 2012).The rs4077582 (C > T) polymorphism present in the 5′-upstream regulatory region, which is reported that it may alter androgen levels specifically testosterone by regulation of luteinizing hormone (LH) (Zhang et al., 2012a(Zhang et al., , 2012b)).The expression of CYP11A1 is driven by LH in the theca cells of the developing follicles because they are the main regulators of the CYP11A1.The rs4887139 (G > A) polymorphism is an upstream variant and is suggested to alter the expression of CYP11A1 and is associated with breast cancer and increased risk for its development (Yaspan et al., 2007).
PCOS is known to be associated with infertility, and LHCGR is strongly linked to the regulation of ovulation and premature ovum formation.LHCGR is expressed in PCOS patients' granulosa cells, notably in the ovaries, where it is involved in follicular maturation and ovulation maintenance (Kanamarlapudi et al., 2016;Zou et al., 2019;Azziz et al., 2019).LHCGR functions as a receptor for two glycoproteins, luteinizing hormone (LH) and human chorionic gonadotropin (HCG).LH is responsible for promoting the corpus luteum and follicular development, whereas HCG is responsible for pregnancy stabilization.FSHR, on the other hand, is a follicle-stimulating hormone (FSH) receptor that regulates LH/HCG expression and is essential for follicle growth and steroidogenesis.During the mid-cycle, LH is responsible for ovulation (Auchus & Miller, 2015;Mutharasan et al., 2013).
The rs13405728 polymorphism within the CYP21 gene and the rs4077582 polymorphism in the CYP11A1 gene have been the subject of research due to their probable link with PCOS.Several research has looked at the link between this polymorphism and PCOS susceptibility; however, the findings have been inconsistent among populations.As a result, more research is required to determine the significance of the rs13405728 polymorphism and the rs4077582 polymorphism in PCOS etiology, particularly in specific ethnic groups or populations.Understanding the molecular foundation of PCOS, as well as the genetic variables that contribute to its development, is critical for enhancing diagnostic accuracy and developing customized treatment options.Understanding the molecular underpinnings of genetic variants in the CYP21 and CYP11A1 gene and their relationship to PCOS can provide vital insights into the syndrome's underlying causes.However, research into the rs13405728 polymorphism and the rs4077582 polymorphism in PCOS patients is limited, especially in specific populations like Pakistan.As a result, the purpose of this study is to perform a molecular analysis of the CYP21 gene polymorphism rs13405728 and CYP11A1 gene polymorphism rs4077582 in PCOS patients from Pakistan to evaluate its potential association with PCOS and contribute to a better understanding of the disorder's genetic base.The CYP21 gene polymorphism rs13405728 and CYP11A1 gene polymorphism rs4077582 in PCOS patients can provide useful insights into the genetic pathways underlying the condition.It may also aid in the identification of possible biomarkers for early detection, risk assessment, and targeted therapeutic approaches.

Study subject and ethical approval
The current study was approved by the University of Lahore Sargodha campus, and its goal was to investigate the molecular aspects of polycystic ovarian syndrome (PCOS).The study included 150 patients with pathologically proven PCOS using Rotterdam criteria and 150 healthy controls.PCOS patients were recruited by ultrasound machines from several gynecology centers in the Sargodha region, while control samples were drawn at random from the persons that come to hospital for routine checkup.Persons taken as control were asked about the prior history of PCOS in them.They had normal menstrual cycles and BMI.The study excluded patients and controls who had other known familial disorders.Each patient or their attendant signed a consent form during the sample collection process, and demographic information was gathered through interviews.Blood samples of 2-3 ml were taken from each subject in 5 ml EDTA-containing vacutainers.These samples were subsequently brought to the scientific laboratory at the University of Lahore Sargodha campus's Department of Bioscience.The samples were then kept in a refrigerator at 4 °C until further processing and analysis.The systematic collecting and storage of these samples will allow for the examination of genetic and molecular components linked with PCOS, resulting in a greater knowledge of the underlying mechanisms as well as prospective diagnostic and therapeutic advances in the field.

DNA extraction
Blood samples of approximately 3-4 ml were taken from all recruited patients with polycystic ovarian syndrome (PCOS) and healthy controls for this investigation.Vacutainer tubes were used to collect the samples.Following that, the phenol-chloroform technique with slight changes was used to extract DNA.The extracted DNA was seen on a 1% agarose gel stained with ethidium bromide, allowing DNA quality and integrity to be assessed.To evaluate DNA concentration and purity, spectrophotometric measurements were taken using Nanodrop (Thermo Fischer Scientific, USA).The samples were kept at − 20 °C following the DNA extraction and quality assessment processes.These standardized processes allow the availability of high-quality DNA samples for further molecular analysis, such as genotyping or sequencing, to explore the specific genetic variations associated with polycystic ovarian syndrome, including the CYP21 gene polymorphism rs13405728.For trustworthy and precise molecular research, the adoption of proper procedures and storage conditions is critical (Bashir, 2018).

Primer designing for selected polymorphism
The primer for PCR amplification of selected SNP rs13405728 of gene CYP21 and rs4077582 of CYP11A1 gene was designed by WASP (Web-based allele-specific primer designing tool) and specific amplification was confirmed by BLAST and UCSC.WR stands for wild-type reverse primer, MR stands for reverse primer designed for a mutant with a mismatch and CF stands for common forward primer.Primers of all SNPs, along with product size and annealing temperature, are given in Table 1.β-actin was used as an internal control.Primer sequences specific for all gene polymorphisms are given below.

Genotyping
The polymerase chain reaction (PCR) technique was used to genotype the specified variant in both sick patients and control samples.PCR was carried out in a 10ul reaction volume with 60-90 g of isolated genomic DNA, 90 M of primers, and Solis BioDyne master mix.The PCR amplification was performed under temperature conditions.The PCR thermal cycling settings included a 60-s denaturation stage at 94 °C, followed by 35 cycles of denaturation at the same temperature for 30 s.The annealing step took 45 s at 56 °C, and the extension step took one minute at 72 °C.Following the cycling steps, a sevenminute final extension was conducted at 72 °C.These adjusted PCR thermal settings ensure that the targeted DNA region carrying the specified polymorphism is amplified.The genotyping findings from this PCR amplification will provide valuable information about the existence or absence of a specific genetic variant, rs13405728, in the CYP21 gene in both polycystic ovarian syndrome patients and healthy control individuals.The below tables show the reaction composition for the control and wild type (Tables 2 and 3).

Statistical analysis
GraphPad Prism Medclac, Forest plot, and SPSS 17.1 software were used to analyze the genetic variant rs13405728 in the CYP21 gene and rs4077582 of the CYP11A1 gene.The acquired data were structured by the software's specifications and subjected to relevant statistical testing.Following the Hardy-Weinberg Law, the allele frequencies, genotypes, and mutations of rs13405728 in the CYP21 (LHCGR) gene and rs4077582 of the CYP11A1 gene were found for both patients with polycystic ovarian syndrome and control persons.Forest plot, a statistical analysis tool, was used to perform multivariate analysis and calculate odds ratios (OR) with associated 95% confidence intervals (CI).The purpose of these studies was to determine the associations, differences, and significance of the rs13405728 CYP21 (LHCGR) and rs4077582 of the CYP11A1 gene allele frequencies, genotypes, and mutations in the patient and control groups.The P-values determined revealed the statistical significance of the observed discrepancies and were calculated by chi-square   test, one-way ANOVA, and sample t-test.The study used these statistical approaches and tools to assess any potential link between the rs13405728 polymorphism in the CYP21 gene and rs4077582 of the CYP11A1 gene and polycystic ovarian syndrome, providing vital insights into the disease's genetic underpinnings.

Polymorphism analysis of CYP21 (LHCGR) and CYP11A1 gene
The current study looked at the relationship between a single nucleotide polymorphism (SNP) in the CYP21 (LHCGR) and CYP11A1 gene and polycystic ovarian syndrome (PCOS) in both patients and healthy controls.To do so, a genotyping technology known as allele-specific ARMS-PCR (Amplification Refractory Mutant Systempolymerase Chain Reaction) was used.Using this technique on both PCOS patients and healthy controls to see if there were any significant variations in genotype and allele distribution between the two groups, this method allows researchers to look at potential links between an SNP in the CYP21 (LHCGR) gene and the development or susceptibility to PCOS.

Demographic properties of patients and controls
A total of 150 individuals with polycystic ovarian syndrome and 150 healthy controls were investigated.Table 4 summarizes the demographic characteristics of research samples and controls.The following are the major characteristics of study units:

Age
Patients and healthy controls ranged in age from 19 to 40 years.The average age of polycystic ovarian disease patients and healthy controls was 30 years.Patients with polycystic ovarian syndrome and healthy controls were separated into two age groups: 30 years and older.In the current study, 44% of PCOS patients were under 30 years old, whereas 56% were above 30 years old.In the control samples, 27% of the people were under 30 years old, while 73% were over 30.The mean age of PCOS patients and controls was found to be significantly different.

Family history
Patients with a family history of PCOS were present.
Based on their family history, the patients and healthy controls were separated into two groups.57% of patients had PCOS in their family, while 43% did not have PCOS in their family.17% of those in the control group had a family history of PCOS, while 83% of those in the healthy controls did not have a PCOS patient in their family.

Smoking status
One of the major risk factors for PCOS is smoking status.
The current study looked at the smoking habits of PCOS patients and healthy controls.Among PCOS patients, the study included 11 (7%) smokers and 139 (93%) nonsmokers.There were 49 (32%) smokers and 101 (64%) non-smokers in the control blood sample.In terms of smoking status, there was a statistically significant difference between patients and controls.(OR 0.163; 95% CI 0.08-0.32;p = 0.0001).
For the CYP11A1 gene, SNP (rs4077582) heterozygous (CT) has a strong correlation with the polycystic ovarian syndrome (CT) of SNP raising the risk of polycystic ovarian syndrome by onefold.The homozygous mutant (TT) of the same SNP had no connection with disease onset (OR 1.377; 95% CI 0.85-2.2;p = 0.1855).While the mutant (TT) of the SNP nearly doubles the incidence of polycystic ovarian syndrome, The combination model (CT + TT) of the same SNP is associated with polycystic ovarian syndrome (OR 2.1905; 95% CI 1.355-3.53;p = 0.0014).The combination model (CT + TT) of the same SNP more than doubles the risk of polycystic ovarian syndrome.

Electropherogram of cases and control for both genes
The electropherogram of cases and controls and sequence analysis of rs13405728 polymorphism of CYP21(LHCGR) are shown in Fig. 1.
The electropherogram of cases and controls and sequence analysis of rs13405728 polymorphism of the CYP11A1 gene are shown in Fig. 2.

Association of CYP21(LHCGR) and cyp11a1 gene polymorphism with age
In the CYP21 (LHCGR) age group of 30 years, heterozygous and homozygous mutants of rs13405728 demonstrate a substantial correlation with PCOS (p = 0.0002, 0.01).In the rs13405728 of CYP21 (LHCGR) age group > 30 years, heterozygous showed a significant

Association of CYP 21 and CYP11A1 polymorphisms with smoking status
In rs13405728, neither heterozygous nor homozygous mutant genotypes in smokers or non-smokers were associated with PCOS.(P = 0.60; P = 0.33; P = 0.07; P = 0.18).According to the forest plot of smokers, heterozygous non-significantly lowers the risk of PCOS by up to one and a half-fold, whereas homozygous mutant lowers the risk by up to half-fold.In non-smokers, heterozygous reduces the risk of PCOS by half, whereas homozygous increases the risk by one and a half as shown in Table 7 and figures.The CYP11A1 gene rs4077582 age group of 30 years heterozygous (CT) and homozygous mutant (TT) showed no connection with polycystic ovarian syndrome (p = 0.53 and p = 0.79, respectively).The homozygous of the age group 30 raises the risk of polycystic ovarian syndrome by onefold, and the heterozygous of the same age group increases the risk by about one and a half-fold.In other age groups > 30 years, heterozygous (CT) SNP rs4077582 is strongly associated with polycystic ovarian syndrome (OR 4.16; 95% CI 2.27-7.79;p = 0.0001).The same age group's homozygous (TT) mutant is significantly engaged in polycystic ovarian syndrome (OR 4.16; 95% CI 2.27-7.79;p = 0.0001).The homozygous of the age group > 30 is protective against polycystic ovarian syndrome, but the heterozygous of the same age group raises the risk by up to fourfold.The pertinent data are presented in Table 7 and Figures (Figs. 3,4,5,6,7,8,9 and 10).

Discussion
PCOS is a serious endocrine condition with a complicated etiology involving both genetic and environmental variables (Goodarzi et al., 2006).PCOS shows itself in a variety of ways, including delayed ovulation, irregular menstrual cycles, and infertility, mostly because of the creation of ovarian cysts caused by the retention  of premature ova in the follicular layer (Krysiak et al., 2006).PCOS is believed to affect 5-10% of women in underdeveloped nations, posing a significant burden on both individuals and society (Jones et al., 2004).The prevalence of PCOS is much higher in Pakistan, reaching 52% (Rotterdam 2003).PCOS is linked to several health problems, including a sevenfold increase in the risk of type 2 diabetes and hormonal and metabolic abnormalities (Sam et al., 2003).Furthermore, PCOS patients have a higher risk of cardiovascular diseases than females who cycle normally (Battaglia et al., 2008;Dahlgren et al., 1992).Cigarette smoking has been connected to PCOS and has been shown to affect steroid hormone production, metabolism, and clearance (Shiverik et al., 1999;Soldin et al., 2011).In contrast, our study found that certain genotypes of the CYP21  (LHCGR) gene polymorphism, specifically heterozygous and homozygous mutants of rs13405728, significantly reduced the incidence of PCOS in smokers.The relationship between genetic variables and PCOS has received a lot of attention, and the gene map of the LHCGR (2p16.3)has been connected to PCOS susceptibility (Chen et al., 2011).
In our study, we looked at the polymorphism of the SNP rs13405728 in the CYP21 (LHCGR) gene and polymorphism of the SNP rs4077582 in the CYP11A1 gene and discovered that it was associated with PCOS.Because SNPs are numerous forms of human DNA sequence variants, they were chosen.Blood samples were collected from several gynae facilities in Sargodha from 150 healthy and 150 sick people for gene polymorphism analysis, together with personal and disease-related questionnaires.The absence of a history of PCOS was an essential requirement for the individuals in the control group to be included; therefore, it was established that they did not have polycystic ovarian syndrome (PCOS).All the controls were also of reproductive age.Those patients and controls with other known familial illnesses were specifically excluded from the study.The control subjects were chosen at random from the general population, with an emphasis on the importance of having a control group free of the condition under study to establish a baseline for comparison with the PCOS patient group.The heterozygous genotype (CT) (OR 4.10; 95% CI 2.47-6.80;p = 0.0001) and homozygous mutant genotype (TT) of rs13405728 indicate a protective effect (OR 0.27; 95% CI 0.16-0.45;p = 0.0001), according to our findings.Our study also took age into account, with the average age of PCOS patients being 30 years.Surprisingly, the age group over 30 years old had the highest number of PCOS patients.The homozygous mutant genotype of rs13405728 was related to a 2.5-fold increased risk in the age group under 30 years, and a onefold increased risk in the age group over 30 years.These findings contrast with those found in the Dutch population, where the average age of PCOS patients was 38.7 years (Elting et al., 2001).In our investigation, family history was found to considerably enhance the risk of PCOS.Only 17% of healthy controls had a family history of PCOS, whereas 57% of PCOS patients reported having a family history of PCOS.This research backs up previous findings of PCOS clustering among families (Bhattacharya et al., 2010;Petermann et al., 2012;Diamanti-Kandarakis, 2007;Urbanek et al., 1999).Finally, PCOS is a complicated illness that is influenced by both genetic and environmental factors.PCOS is extremely common in Pakistan, and family history plays a key influence in its development.Our research found a link between the CYP21 (LHCGR) gene polymorphism rs13405728 and PCOS, with specific genotypes conferring a higher risk.These findings add to our understanding of the genetic basis of PCOS rs4077582 of CYP11A1 was discovered to be strongly linked with the etiology of PCOS in our current investigation, indicating that rs4077582 impacts the P450scc enzyme activity.The genetic distribution of SNP rs4077582 of CYP11A1 in PCOS was significantly different from the control in our investigation.Previous research in China has found significant differences in the genetic distribution of the CYP11A1 SNP rs4077582 (Zhang et al., 2012a(Zhang et al., , 2012b)).Another study on Chinese women was also undertaken, but no conclusive results were obtained.Tan and Chen found no link between this polymorphism and PCOS in other Chinese studies with a small sample size (Tan et al., 2016;Chen et al., 2008).In Egypt, researchers discovered a strong link between the genotypic difference of polymorphism rs4077582 of gene CYP11A1 in PCOS patients and controls (Beni et al., 2016).In this study, the CT heterozygous genotype of rs4077582 showed a significant association by increasing risk by nearly two folds in PCOS, demonstrating that women with the CT genotype are more prone to PCOS.In this investigation, they also discovered that the level of LH in this genotype is higher than in others (Beni et al., 2016).The CYP11A1 polymorphism is substantially related to breast cancer; however, the mechanism is different.Estrogen is linked to breast cancer, while androgens are linked to PCOS via a CYP11A1 polymorphism (Goa et al., 2010).The strong connection of rs4077582 with altered testosterone and LH levels was examined in Chinese women (Cheng et al., 2015).
Furthermore, for a complete understanding of PCOS, relationships with many genes and factors must be investigated.While this study concentrated on a single gene and SNP, future research should investigate other genes and variants.Considering clinical and demographic variables in addition to genetic considerations may provide a more comprehensive picture of PCOS, unraveling the intricate connections between genetic predisposition and environmental circumstances.

Conclusions
The investigation of single nucleotide polymorphisms (SNPs) in this study included 150 patients with polycystic ovarian syndrome (PCOS) and 150 healthy controls.The SNP rs13405728 in the CYP21 (LHCGR) and rs4077582 of gene CYP11A1 genes were specifically studied, and it was discovered to have a strong connection with PCOS.The study's findings demonstrated that this specific SNP, rs13405728, and rs4077582 had a substantial connection with both the family history of PCOS and the age of the individuals affected.The presence of the rs13405728 variation was found to enhance the chance of developing PCOS.The allele-specific ARMS-PCR (Amplification Refractory Mutant System-polymerase Chain Reaction) technology was used for the analysis.This study's findings add to our understanding of the genetic factors involved in the development of PCOS.The substantial link of SNP rs13405728, and rs4077582 with PCOS, as well as its relationship with family history and age, suggests that this SNP may play a role in PCOS pathogenesis.More studies are needed to determine the functional consequences of this SNP and its possible application in the diagnosis and treatment of PCOS.

Recommendations
As it is the first time in Pakistan to elaborate on the causes and genetic bases on a molecular level, there are some recommendations, for example, molecular bases of PCOS study should be conducted on a large population.The association should be studied on more than one gene and on more variables and for a complete understanding of disease, and mRNA expression study should be conducted.

Limitations
There are certain limitations to every research work.As in this study, the population size was limited as per university criteria and shortage of time.So, the population size can be extended up to 1000 for better and more accurate results.Also, some factors were missing in the research like BMI, menstrual cycle duration, etc.

Fig. 1
Fig. 1 Electropherogram of cases and controls.In A: 2% Agarose gel of polycystic ovarian syndrome patients.P1w shows patient 1 with wild genotype, and p1m shows patient 1 with mutant genotype.In B: 2% Agarose gel of polycystic ovarian syndrome patients.c1w showing patient 1 with wild genotype, c1m showing patient 1 with mutant genotype.LDR stands for DNA ladder of 197 bp (Fermentas)

Fig. 3
Fig. 3 Electropherogram of cases and controls of rs4077582 polymorphism of CYP11A1 gene.In A: 2% agarose gel of PCOS patients.p1w shows patients with wild genotype, and p1m shows patients with mutant genotype.In B: 2% agarose gel of controls, in this c1w showing control 1 with wild genotype and c1m showing control 1 with mutant genotype.LDR stands for DNA ladder of 100 bp (Fermentas)

Fig. 4
Fig. 4 Allele and genotype frequencies of selected SNPs of CYP11A1inpatients and controls

Table 1
Primer designing

Table 2
PCR reaction mix composition (each reaction) for wild type

Table 3
PCR reaction mix composition (each reaction) for mutant

Table 4
Demographic table for controls and cases AOR (adjusted odds ratio), CI (confidence interval), P = χ2-test, Chi-square test, CI, and p-value calculated by regression analysis

Table 5
Allele and genotype frequencies SNPs of CYP21 and CYP11A1 in patients and controls OR (odds ratio), CI (confidence interval), OR CI, and p-value calculated by regression analysis

Table 6
Association of CYP21 and CYP11A1 polymorphisms with ageOR (odds ratio), CI (confidence interval), OR CI, and p-value calculated by regression analysis

Table 7
Association of CYP 21 and CYP11A1 polymorphisms with smoking status OR (odds ratio), CI (confidence interval), OR CI, and p-value calculated by regression analysis