No association between glutathione S-transferase M1 and T1 gene polymorphisms and susceptibility to endometriosis

Introduction: Endometriosis is a common gynecologic disorder influenced by genetic and environmental factors. The glutathione S-transferase family is associated with endometriosis because its main function is cellular detoxification, so the absence of those enzymes may be a factor for the development of the disorder. Objective: Investigate the relationship between polymorphisms of GSTM1 and GSTT1 genes and endometriosis, in order to gain a better understanding of the association between detoxification genes and the susceptibility to endometriosis. Material and methods: Case-control study in 132 women (49 with endometriosis and 83 of the control group). The genotype was determined using multiplex polymerase chain reaction (PCR), observed in 10% polyacrylamide gel electrophoresis stained with silver nitrate, and statistical analysis was performed. Results: There was not a significant difference between the GSTM1 and GSTT1 null genotype in the endometriosis group and the control group (p = 0.9956). The same result was observed with the combined genotype (p = 0.8129). Conclusion: In the present study, the GSTM1 and GSTT1 polymorphisms are not associated with a higher risk of endometriosis.


IntroductIon
Endometriosis is characterized by the presence of endometrial glands and stroma outside the uterine cavity. It is a common gynecologic disease that affects 5%-10% of women of reproductive age, but its incidence has been rising in recent years. The most common symptoms of endometriosis are chronic pelvic pain, dysmenorrhea and infertility (1,2) . Because of the symptoms, the condition has a negative impact on quality of life (3,4) , causing anxiety, depression (5) , and even reduced work productivity (6) .
The etiology and the pathogenesis of this disease are still unclear, however the most accepted theory is the retrograde menstruation described by Sampson in 1927 (7) . Endometriosis is considered a complex trait caused by the interaction between genetic (8)(9)(10) and environmental factors (11,12) .
The genetic factors can be related to polymorphism in some genes, like the ones that happen in the glutathione S-transferase family (GST). The role of GST polymorphisms as a risk factor for endometriosis has been researched in many studies, especially in meta-analyses (13)(14)(15)(16) . The GST family comprises enzymes of phase II conjugation, and they are involved in the detoxification of estrogen, reactive oxygen species (ROS), dioxin and products of oxidative stress, all of them associated with the development of endometriosis (16) . Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) are two candidate genes of this family. The GSTM1 gene is located on chromosome 1p13.3 (17) , and the GSTT1 gene is located on chromosome 22q11.2 (18) . In both genes there is a deletion polymorphism that the null alleles provoke, which results in the reduced activity of a functional gene product (16) . A recent study concludes that an interaction between GST genes and environmental factors plays an important role in human disease (19) .
The aim of this study was to investigate the relationship between polymorphisms of GSTM1 and GSTT1 genes and endometriosis, in order to develop a better understanding of the association between detoxification genes and susceptibility to endometriosis.

Subjects
In this case-control study, the investigated women were those treated at the Clinic of Gynecology and Obstetrics, in Universidade Federal do Triângulo Mineiro (UFTM), Brazil. The sample consisted of 49 patients with endometriosis and 83 controls that underwent laparoscopy or laparotomy. The inclusion factors were the surgical procedure, which confirmed the presence (patients) or absence (controls) of endometriosis, and the written informed consent for participation in this research. Patients were classified into two groups according to the criteria of the American Society for Reproductive Medicine (ASRM) (20) : seven patients at early-stage endometriosis (stages I-II), and 23 patients at advanced-stage endometriosis (stages III-IV). For the other 19 patients, the data were not available. The control group consisted of women who had undergone this surgical procedure, with no observed evidence of endometriosis. Ages ranged from 24 to 58 (mean: 36.14 ± 7.66) years in the endometriosis group, and from 20 to 70 (mean: 40.36 ± 10.08) years in the control group. The most common symptoms were: chronic pelvic pain, dysmenorrhea and infertility. This research was approved by the UFTM Ethics Committee (1628 protocol).

Deoxyribonucleic acid (DNA) analysis
The DNA was extracted from peripheral blood according to the technique described by Miller et al. (1988) (21) . Quality and quantity of extracted DNA were checked by agarose gel electrophoresis 1% and spectrophotometer, respectively.
Polymorphisms of GSTM1 and GSTT1 genes were evaluated by multiplex polymerase chain reaction (PCR). The GSTM1 primers were forward (F): 5' GAA CTC CCT GAA AAG CTA AAG C 3' and reverse (R): 5'GTT GGG CTC AAA TAT ACG GTG G3' (219 bp); the GSTT1 primers were F: 5' TTC CTT ACT GGT CCT CAC ATC TC 3' and R: 5' TCA CCG GAT CAT GGC CAG CA 3' (480 bp). The CYP1A1 primers were F: 5 'CTG GAA CTG TCT CCA AGC CTT 3' and R: 5 'CAG CTG TTG GAA CAT GTC CTC 3' (312 bp); and this gene was used as an internal positive control reaction. Multiplex PCR was performed in 25 µl reaction volume containing 100 ng of genomic DNA, 0.1 µm of GSTM1, GSTT1 and CYP1A1 primers, 2.5 mM MgCl2, 0.2 µm deoxy-nucleotide triphosphate, 2.5 µl of 1× PCR buffer, and 1U Taq DNA polymerase. Amplification was performed with initial denaturation at 95ºC, followed by 35 cycles at 94ºC for 2 min, 59ºC for 1 min, and 72ºC for 1 min, and a final extension at 72ºC for 7 min. The PCR products were analyzed in polyacrylamide gel electrophoresis and stained with silver nitrate. The null genotype indicated that both alleles were deleted. The GSTT1 and GSTM1 genes were identified by the absence of the fragments of 480 bp and 219 bp, respectively. The presence of the 312 bp fragment corresponding to the CYP1A1 gene showed a positive amplification in all samples (Figure).

Statistical analysis
The statistical analysis used the chi-square test, considering the significance of p < 0.05. The Hardy-Weinberg equilibrium analyses were performed to compare the observed and expected genotype frequencies.
results In order to investigate whether GSTM1 and GSTT1 polymorphisms were associated with endometriosis in the Brazilian female population, a comparison was drawn among 132 Brazilian women (49 with endometriosis and 83 of the control group). Table 1 shows the distribution of GSTT1 and GSTM1 genotypes in both cases and controls.
These gene polymorphisms were also searched in Italy and Tunisia. In Italy, the study conducted by Vichi et al. (2012) (32) found a negative association with both genes as our study, while the one from Tunisia, searched by Henidi et al. (2015) (33) , showed a positive association with both genes.
Three studies were conducted in Brazil (34)(35)(36) . The first one was carried out in Goiás, by Frare et al. (2013) (35) , and found a positive association with GSTM1 and a negative association with GSTT1 in 50 cases and 46 controls. Another research also conducted in Goiás reported positive association with both genes (36) . The last study conducted in Brazil was carried out in Cuiabá, in the state of Mato Grosso, by Kubiszeski et al. (2015) (34) , who found a negative association with GSTM1 and a positive one with GSTT1 in 121 cases and 97 controls.
Results found in those studies are very different, with a few of them showing the same results. Similarly to the results in our research, we observed that only two studies which investigated both genes also reported no association (24,32) . So, the difference between the populations' eating habits and environmental pollution can be one of the reasons influencing different results found in the studies. The environmental pollution and food intake are related to endometriosis because some compounds can affect endometrial function; such compounds are difficult to break down, have long half-lives and can be accumulated in various tissues (11,12) . Therefore, diet and environmental pollution have an influence on the pathogenesis and progression of endometriosis.
Another reason influencing the difference of the found results is the genetic background among ethnic groups in these populations.
So, a limitation of our study is that our sample was small, what happened because the diagnostic method was invasive. Another limitation is that because of the lack of information about some patients we were unable to classify them according to the ASRM (20) criteria, and due to this we were also unable to compare the stages with the genotypes.

conclusIon
In conclusion, the present study shows that the GSTM1 and GSTT1 null polymorphisms are not associated with an increased risk of endometriosis in our population.

conFlIct oF Interest
None of the authors has any conflict of interest to disclose.

dIscussIon
The aim of this study was to evaluate whether the polymorphisms in the glutathione S-transferase genes, especially GSTM1 and GSTT1, could be associated with susceptibility to endometriosis. This study showed that GSTM1 and GSTT1 null genotypes were not associated with an increased risk for endometriosis. In addition, no significant difference was observed for the combined genotype, again showing the lack of association between endometriosis and the investigated polymorphisms.
The GST family plays an important role in the detoxification of environmentally toxic compounds and products of oxidative stress, and these are related to an increased susceptibility to endometriosis. The presence of unbalanced ROS can cause cellular damage and change cellular functions because they regulate the protein activity and gene expression, which can lead to severe effects (22) .
The GSTM1 and GSTT1 gene polymorphisms have been searched in the association with endometriosis in different populations, most of them from Asia (13)(14)(15)(16)(23)(24)(25)(26)(27)(28)(29)(30)(31) . In that population, five studies (15,(26)(27)(28)(29) investigated only GSTM1, and three of them found a positive association (15,27,29) . The other eight studies searched both genes, GSTM1 and GSTT1. Similarly to the results found in this research, a study conducted in Japan, by Matsuzaka et al. (2012) (24) , found a negative association. A study from Korea (31) found a negative association with GSTM1 and a positive association with GSTT1; and three studies, one from Turkey, one from Iran and one from Taiwan showed a positive association with GSTM1 gene and a negative association with GSTT1. Three meta-analyses from China reported positive association with both genes (13,14,16) .