International Journal of

Introduction
 
Endometriosis is defined as the presence of endometrial glands and stroma outside their normal locations (1, 2). This disease is one of the main causes of infertility and chronic disorders among women, affecting approximately 7-10% of women in their fertility age and 5-50% of infertile women (3-6). It has a wide range of symptoms of which pelvic pain, dysmenorrhea, dyspareunia, and abnormal uterus bleeding are the most important ones (7). Endometriosis is a complex trait. Some of studies have suggested the role of immunological, endocrine, environmental and genetics factors in the pathogenesis of this disease. Moreover, it has been revealed that endometriosis is more common in first degree relatives of endometriosis patients compared to first degree relatives of unaffected individuals; reflecting the involvement of genetics factors (3-5). Increasing evidences have shown that multiple gene loci interact with each other and with the environment to produce endometriosis (6, 7). So far, the relationship between endometriosis and different genes has been investigated. Among them, genes involved in detoxification systems such as Cytochrome (CYP) P450 superfamily are the most important ones  and possibly a good link for interactions of genetic and environmental factors (8, 9). Enzymes of this family are participated in detoxification process of a wide range of environmental pollutants and carcinogens (10).
CYP1A1, a member of P450 cytochrome superfamily, is located on chromosome 15 (15q 24.1) (11). CYP1A1 rs4646903 (T>C) polymorphism, downstream from the polyadenylation site at the 3´ end of exon 7, is one of the most important polymorphisms of the gene. Substitution of T to C at this position formed a target site for MspI enzyme, so this polymorphism is also named MspI  or M1 (11, 12). It has been reported that this polymorphism is correlated with CYP1A1 enzyme inducibility phenotype. There are some studies reported the relationship between MspI polymorphism and the risk of endometriosis (13, 14).
The goal of this study was investigation of the association between MspI polymorphism of CYP1A1 gene and endometriosis in women referred to two infertility centers in Yazd, Iran who were from central and southern parts of Iran.
 
Materials and methods
 
In this case-control study, 93 women with endometriosis (age range 20-42 yr) referred to two infertility centers in Yazd, Iran: Madar Hospital and Yazd Research and Clinical Center for Infertility were enrolled in this study as the case group. Endometriosis was diagnosed by an experienced gynecologist through laparoscopy. Guidelines of American Society for Reproductive Medicine (ASRM) was used to classify patients (ASRM) into 4 stages; minimal (stage I), mild (stage II), moderate (stage III), and severe (stage IV) (15).
The control group consisted of 139 unrelated healthy premenopausal women (age range: 18-50 yr) undergoing Cesarean section or hysterectomy at the same centers with no history of endometriosis and without any lesion suggesting endometriosis during Cesarean section or hysterectomy. Women with a history of autoimmune disease and cancer were excluded from the research. Clinical characteristics of all the participants were collected personally using a designed questionnaire.
 
Genotype determination
Genomic DNA was extracted from 5 mL of EDTA anticoagulated whole blood using AccuPrep® Genomic DNA Extraction Kit (Bioneer, South Korea) based on manufacturer's manual. To study MspI polymorphism of CYP1A1 gene, PCR-based restriction fragment length polymorphism (RFLP) method was used. The specific primers for the target region (forward primer of 5'-TAGGAGTCTTGTCTCATGCC-3' and reverse primer of 5'-CAGTGAAGAGGTGT AGCCGC-3') was designed by Gene Runner (version 5.1) software and synthesized by Macrogen (South Korea). PCR reaction mixture was contained 1X PCR buffer, 2 mM MgCl₂, 2.5 unit of Taq DNA Polymerase (Cinnagen, Iran), 0.5 mM dNTP, 5 pM of each primer, and 30 ng of pattern DNA up to the volume of 25 μl of sterile distilled water. Amplification was performed as follows: initial denaturation at 95^oC for 5 min, then 30 cycles of denaturation at 94^oC for 30 s, annealing at 60^oC for 45 s, extension at 72^oC for 60 s and final extension at 72^oC for 10 min.
The amplification products (343 bp) were digested with MspI (Fermentas, Germany) for 16 hr at 37ºC. Then MspI restriction digestion products were loaded on 2% agarose gel. The genotypes were characterized as TT when the PCR products remained uncut (343 bp), as CC when the restriction site was present, and as TC when the digestion produced a pattern of three DNA fragments sized 343 bp, 136 bp, and 207 bp.
 
Ethical consideration
The Islamic Azad University, Yazd Branch Ethics Committee approved this study (IR.IAU.YAZD.REC.1396,23). Written informed consent was obtained from all participants.
 
Statistical analysis
The Pearson χ² or Fisher's exact test was used to compare genotype distributions between patients and controls. The statistical modeling using logistic regression was used to calculate the relative risk (odds ratio; OR) of genotypes for case-control study. Odds ratios were expressed together with the 95% confidence interval (16). p≤0.05 was considered statistically significant. The Hardy-Weinberg equilibrium analysis was conducted by comparing observed versus expected genotype frequencies using a Chi-square test.
 
Results
 
The mean age of the case group was 28.91±5.18 yr and 29.85±8.77 yr in control group (p=0.356). The mean body mass index (BMI) was 24.30±3.69 in the case group and 26.14±4.82 in controls. Statistical analysis showed that BMI of the cases is significantly less than control group (p=0.003) (Table I). Stage II of the disease has the highest frequency (40.20%). Totally, 51 patients (55.40%) had primary stages of disease (I, II) and 41 of them (44.60%) were in the advanced stages of endometriosis (III, IV). Regarding both controls and patients; the distribution of MspI genotypes was in Hardy-Weinberg equilibrium.
The most frequent genotype in two groups was TT. The frequencies of this genotype in patients and control group were 73.1% and 74.1%, respectively. Data analysis by χ² test revealed that there is no significant difference between the two groups (p=0.961). Using the TT homozygote as a reference group, the OR of TC heterozygotes (OR, 1.016; 95% CI, 0.54 to 1.93) and CC homozygotes was (OR, 1.204; 95% CI, 0.31-4.6). Allele frequencies of MspI polymorphism in the case and control groups are shown in table II. Comparison between the alleles by χ² test indicates no significant difference between two groups (p=0.804). In all stages of endometriosis, CC genotype has the lowest frequency of 7.2%, 5.4%, 0.0%, and 4.5% for stages I to IV, respectively (Table III). Statistical analysis of the frequencies of MspI genotypes by Fisher's exact test indicated that there is no significant difference between the genotypes and different stages of endometriosis (p=0.491). In addition, the patients with advanced stages were compared with the primary stages and control group; there was no significant difference between two groups (p=0.427 and p=0.507, respectively).
 

Table I. General characteristics of the case and control groups




Table II. Genotype and allele frequencies of MspI polymorphism in the caseand control groups


Table III. Genotype frequencies of MspI polymorphism in different stages of endometriosis


Discussion
 
The role of products of CYP1A1 gene in metabolism of estrogen and PAH has been well-defined (9, 17). It was observed that CYP1A1 was implicated in metabolism of dioxin, which may increase the severity of endometriosis in rhesus monkeys (18). Therefore, considering that endometriosis is an estrogen-dependent disease (19), CYP1A1 may be associated with this disease. The level of gene expression or mRNA stability can be altered by the CYP1A1 MspI polymorphism which resulting in a highly inducible activity of the enzyme (20).
In the present study, results showed that there is no statistical significant difference between the frequencies of the genotypes in two groups (p=0.961). Previous reports on association of MspI polymorphism of CYP1A1 gene with endometriosis are controversial. In consistency with our findings, there are some studies in different populations such as in UK (p=0.11) (15), China (p>0.05) (21), India (p>0.05) (22, 23), Austria (p=0.6) (24), Taiwan (p>0.05) (1), USA (p=0.65) (14) and Brazil (p=0.087) (12), which did not found any correlation between the polymorphism and endometriosis. In contrast, Arvanitis and colleagues examined Greek population and noticed that TT genotype will result in 38% decrease in endometriosis risk (OR, 0.62; 95% CI, 0.440 to 0.883) (13). In our study, there was no significant difference between the allele frequencies of two groups (p=0.804). The same results were reported by some other studies (1, 14, 15, 21-24). In contrast, Barbosa and colleagues suggested that the frequencies of C allele was higher in Brazilian women with endometriosis as compared to the control group (p=0.006). Diversity in genetics and environmental background, sample size and selection of control group might be reason for differences among results. In our research, no significant difference was observed between age and cause of infertility of patient and control groups. However, mean BMI of the patients was less than the control group and this difference was statistically significant (p=0.003). These findings confirmed the results of other studies which were reported decrease of BMI is associated with endometriosis (1, 14, 25, 26).

Conclusion
 
In conclusion, this study showed that CYP1A1 MspI polymorphism was not associated with endometriosis risk in our population. More studies with larger sample size and especially, in other populations with different ethnicity can provide a better understanding of the relationship between population-specific environmental variables and genetic backgrounds.
 
Acknowledgments
 
This article was financially and technically supported by the Islamic Azad University, Ashkezar Branch. The authors are grateful to the Madar Hospital and Yazd Research and Clinical Center for Infertility for helping us.
 
Conflict of interest
 
The authors report no conflicts of interest.