Evaluation of the FAS and FASL Gene changes in women with premature ovarian failure: A case-control study

Abstract Background Premature ovarian failure (POF), is menopause occurring before the age of 40, affecting 1-3% of women worldwide. The risk of POF increases with altered immunological parameters such as FAS and FASL genes, which play a fundamental role in embryogenesis and cellular homeostasis. Objective The study aimed to investigate the potential role of FAS and FASL genes in POF pathogenesis. Materials and Methods In this case-control study, the polymorphisms of FAS-670A/G and FASLIVS2nt_124A/G apoptotic genes were analyzed in 51 Iranian women suffering from POF, and 61 healthy controls. Isolation of DNA was done using the salting-out method, and genotypic analysis was performed for all the subjects using the polymerase chain reaction-restriction fragment length polymorphism method. Results Our results revealed that homozygous FAS-670A/A and G/G, and heterozygous FAS-670A/G are not significantly different between cases and controls (p = 0.99). Also, in different genotyping models of FASIVS2nt_124, polymorphisms were not related to POF risk (p = 0.23). Conclusion There is no statistical association between these polymorphisms and POF risk in women referred to genetic counseling clinics.


Introduction
Infertility is a multifactorial disease that ranges from hormonal and genetic disorders to immunological changes. A gradual decrease in ovarian function is observed as menopause nears, leading to estrogen deficiency and a decrease in fertility. This process is associated with higher gonadotropin levels (1)(2)(3). Premature ovarian failure (POF), is a menopause that occurs before the age of 40, affecting 1-3% of women worldwide (4,5). The development and maturation of ovulation depend on molecular signaling pathways responding to androgens (6,7).
Estrogen is one of the 2 steroid sex hormones secreted by ovaries. Estrogen also has an important role in fetal development, the presence of secondary sexual features, the reproductive cycle, and the continuation of pregnancy. In addition, estrogen regulates the growth and differentiation of endometrial cells (8). In the reproductive cycle, implantation involves a series of events, including apoptosis in endometrial cells (9,10). Evidence suggests that apoptosis helps maintain cellular homeostasis by removing senescent cells from the functional layer of the uterine endometrium (11).
FASL acts as a mediator of apoptosis between cell differentiation and embryonic development.
It has been reported that polymorphisms in FAS and FASL have clinical worth in hormonesensitive cancers such as ovarian and breast cancer. Estrogen, one of the most important sex hormones, increases the expression of the FASL protein (12). Genetic tests also show increased FASL mRNA expression by estradiol and progesterone. It was shown that increased FASL expression may mediate apoptosis in endometrial cells (9). Therefore, it can play an important role in trophoblast invasion and consequent implantation.
FAS and FASL genes have many polymorphisms even in the gene promoter, which could be important in regulating the cell death signal (13)(14)(15)

Genotype analysis
Genomic DNA was extracted from 5 ml of whole blood by the salting out method (16). DNA was amplified through polymerase chain reaction with designed primers of FAS and FASL genes ( Table   I). The solution for polymerase chain reaction SNP: Single nucleotide polymorphism

Statistical analysis
The frequency of the alleles, genotypes and haplotype in cases and controls were compared by Chi-square test (p-value < 0.05 was significant).

Results
The genotype distribution of the 2 polymorphisms from both cases and the controls were in line with Hardy-Weinberg equilibrium (p > 0.05).

Allele frequencies
The allelic odds ratio of FAS-670A/G and FASL_124G/A were 1.004 and 1.145, respectively. No significant difference was observed between the allele frequencies of SNPs in this research (Table II). Allele frequencies within cases and controls are shown in table II. Also, odds ratio statistics with p-value for any allele were presented. Data were presented in percentages.

Genotype frequencies
Regarding the FASLIVS2nt_124, the frequency of AG genotype was higher in the controls (26.7%) than in cases (17.6%). However, the difference was not statistically significant (p = 0.23). Most POF women had AA genotype relative to FASLIVS2nt_124AG and GG genotypes (72.5%, 17.6% and 9.8%, respectively). Under the codominant model, the frequency of the FAS-670AG genotype was not significantly different between cases (47.1%) and controls (46.7%) (p = 0.99). Also, the AG genotype was the most common genotype in POF women (AG = 47.1%, GG = 31.4%, and AA = 21.6%) (Table III). No significant difference was observed in genotype frequencies of FASLIVS2nt_124 A/G and FAS-670 A/G variants between women with POF and healthy controls.

Haplotype frequency
Haplotype analysis was done for these SNPs to investigate the association between a likely combination of FASLIVS2nt_124A/G and FAS-670A/G polymorphisms with POF. Most women with POF had GA and AA haplotypes (0.45% and 0.35%, respectively) (Table IV). There was no significant difference in the frequency of haplotypes between cases and controls.  Haplotype analyses was carried out using Chi-square test and using The PLINK software. P-values < 0.05 were significant

Discussion
Apoptosis is vital for the formation of tissue structure during embryogenesis and cellular homeostasis, and it serves as a defense mechanism in facing pathogens (19,20). Apoptosis is simultaneous with the implantation window (7).

Conclusion
This study showed that the gene variants under