Introduction

Endometriosis is a steroid-dependent condition recognized as the most common cause of female infertility [1, 2]. The disease is defined by the presence of endometrial glands and/or stroma outside the uterine cavity.

The estrogen-dependent growth of endometrial tissue is mediated by aromatase, that is the key of local estrogenic biosynthesis, promoting the conversion of androstenedione to estrone and from testosterone to estradiol [3].

Genetic polymorphisms in genes associated to the estrogen synthesis pathway, as receptors and metabolizing enzymes of the hormone have been associated to interindividual variation in the levels of circulating estrogen [4].

CYP2C19 is an important gene of cytochrome p450 family and encodes an aromatase associated by the estrogen metabolism, including the conversion of estradiol in estrone and the 2α- e 16α metabolites of the hydroxilate of estradiol and estrone4. Besides, the HSD17B1 gene (17β-hydroxysteroid dehydrogenase typo 1), 17β-HSD reductase dehydrogenase is a gene of the short chain superfamily (SDR) [5], produce the enzyme that catalyzes the final step of the estradiol biosynthesis, in other words, the conversion of the estrone in estradiol, which is also expressed in the endometrium [6, 7].

Some studies point to the association between the development of endometriosis and genetic polymorphisms [8–11]. This study aimed to evaluate the frequency of the polymorphisms 85952 A/G (rs 11592737) and 937 A/G (rs 605059) of the CYP2C19A1 and HSD17B1 genes respectively, previously associated with endometriosis in other population, in Brazilian women with endometriosis and in the control group and correlate the clinical and genetics findings with the risk of endometriosis, in a search for molecular biomarkers of this popular disease.

Material and methods

Patients

Five hundred women with endometriosis (mean age: 34.3 ± 4.0 y) from the Endometriosis Outpatient Clinic of the Human Reproduction Service of Faculdade de Medicina do ABC (FMABC) were studied. Women with endometriosis diagnosed by laparoscopy and with histological confirmation of the disease were selected and classified according to the American Society for Reproductive Medicine (ASRM, 1997) [12]. In the endometriosis group, stage of the disease was found to be minimal/mild (stage I and II) and moderate/severe (stage III and IV).

To compose the control group, five hundred of fertile women (mean age: 32.0 ± 4.0 y) previously submitted to tubal ligation and with confirmed absence of endometriosis were called, from the Outpatient Clinic of familial planning from FMABC.

Clinical data and peripheral blood samples were collected only after explaining the objectives of the study and obtaining signed informed consent, as approved by the Research Ethics Committee of Faculdade de Medicina do ABC.

Molecular analysis

Peripheral blood was collected from each patient and control in an EDTA-containing tube. Genomic DNA was extracted from peripheral blood lymphocytes according to Lahiri and Nurnberger (1991) protocol [13]. DNA samples were quantified and diluted to 50 ng/μL. CYP2C19 85952 A/G (rs115927737) and HSD17B1 937 A/G (rs605059) polymorphisms were identified by Real time PCR (TaqMan® Assay - C__1329162-10, CYP2C19 gene and C__2350902_10, HSD17B1 gene), performed in the thermo cycler Step One Real Time PCR System (Applied Biosystems, Carlsbad, California, USA).

Statistical analyses

Statistical analyses were carried out using SPSS for Windows 11.0 (SPSS, Inc., Chicago, IL). The chi-square was used to compare allele and genotype frequencies between groups and to estimate the Hardy-Weinberg equilibrium. The odds ratio (OR) and range with 95 % confidence interval (CI) were calculated for the presence of the reference genotype using a logistic regression model. All p-values were two-tailed, and 95 % confidence intervals (CIs) were calculated. A p-value < 0.05 was considered statistically significant.

Results

Genotypes and allelic distribution of CYP2C19 85952 A/G and HSD17B1 937 A/G polymorphisms in women with endometriosis and controls are summarized in Table 1.

Table 1 Frequency between the genotypes and alleles for the CYP2C19 and HSD17B1 for the endometriosis group in women with endometriosis stage I and II and women with endometriosis stage III and IV, comparing with a control group
Full size table

The AA, AG and GG genotype frequencies of the CYP2C19 85952 polymorphism in the endometriosis group were 328 (65.6 %), 141 (28.2 %) and 31 (6.2 %) respectively. In the controls we observed 345 (69.0 %), 142 (28.4 %) and 13 (2.6 %). When endometriosis group was divided according to endometriosis stage, the genotypes frequencies were 145 (68.7 %), 51 (24.1 %) and 15 (7.1 %) among women with minimal/mild endometriosis and 183 (63.3 %), 90 (31.1 %) and 16 (5.6 %) considering the women with moderate/severe endometriosis (Table 1).

For HSD17B1 937 polymorphism the genotype frequencies of AA, AG and GG in the endometriosis group were 134 (26.8 %), 221 (44.2 %) and 145 (29.0 %) respectively. In the controls we observed 121 (24.2 %), 257 (51.4 %) and 122 (24.4 %). When patients were divided according to endometriosis stage, the genotypes frequencies found were 54 (24.8 %), 100 (46.1 %) and 63 (29.0 %) among women with minimal/mild endometriosis and 80 (28.3 %), 121 (42.7 %) and 82 (29.0 %) considering the women with moderate/severe endometriosis (Table 1).

Discussion

Endometriosis is an estrogen-dependent disease. Estradiol, which reaches endometriosis by circulation or is locally produced, acts regulating endometriotic tissue’s growth. Circumstantial and laboratory evidence strongly support the idea that estradiol is a key hormone in the growth and persistence of endometriotic tissue as well as inflammation and pain associated to endometriosis [14]. Estrogen levels observed in endometriosis lesions are highly correlated with the levels of steroidogenic enzyme aromatase cytochrome P450 including the abnormal expression of enzymes [15].

Trabert et al. (2011) [16] assessed the metabolic pathway genes of sex hormones and their correlation with endometriosis risk. CYP19A1 and HSD17B1, from the aromatase group, were the ones for what a positive correlation endometriosis risk was found. HSD17B1 was in the borderline for statistical significance as much as in our present study.

Other study, conducted by Painter et al. (2011) [4], evaluated 3223 women with endometriosis, 1190 women without endometriosis and 7060 people from regular population found similar results; CYP19A1 showed to be statically associated to the disease, corroborating Trabert’s and our results.

CYP19A1 and HSD17B1 encode important enzymes in the metabolic estradiol pathway. A reasonable hypothesis for the observed association is that estradiol and related hormones’ levels are largely dependent of genetic control [17]. Recent conflicting studies in the literature [4, 18–20] relate the presence of polymorphisms in the cytochrome p450 gene family and HSD17B1 to infertility. HSD17B1 has a wider tissue distribution, which enhances estrogen production in target tissues, thus increasing the estradiol/estrone thereof, thereby providing an increased concentration for estrogen receptors (ESR1 and ESR2) [21]. In normal tissues, HSD17B1 is expressed in placenta, ovaries, follicles, uterus and mammary glands; it is also expressed in breast cancer and leiomyoma and may be involved in disease processes in these tissues, due to a local increase of estradiol [22, 23].

Tsuchyia et al. (2005) [18], in a study conducted in Japan, with 79 cases and 59 controls, found association of HSD17B1 polymorphism with most severe endometriosis stages compared to the control group (p < 0.01). In our study, we found not a strong association of HSD17B1 polymorphism but a tendency of association with the stages III and IV of endometriosis (p = 0.0687). Despite of not achieving statistically significant results, Hardy-Weinberg equilibrium observation showed that HSD17B1 mutated allele is more frequent in the case group, especially in stages III and IV.

Considering CYP2C19, we found a statistically significant difference between the different genotypes and alleles (normal as being in common) of the 85952 A/G polymorphism, demonstrating its association with endometriosis (p = 0.0203). Hardy-Weinberg equilibrium evaluation showed that there is a disequilibrium concerning the CYP2C19 mutated allele in the case population, more frequent in stages I/II, reinforcing the role of the allele in disease pathogenesis. The risk associated to the allele presence is around 0.3.

Corroborating the association of CYP19 with endometriosis, Bukulmez et al., (2008) [24] demonstrated increased levels CYP19A1 mRNA in cultured human endometrial explants and stromal cells, and in those specimens the expression correlated well with the inflammatory stage of endometriosis.

The identification of genes involved in the biological process of endometriosis may have implications in the diagnosis, identifying risk groups and therapeutic targets [25]. Literature already confirmed a genetic background for endometriosis development; however environmental factors and immunological factors play an important role for disease establishment. An aggregation of different pathways is necessary for the appropriate understanding of the disease.

Conclusions

We observed that patients with endometriosis compared to the control group have a difference in the incidence of the genotypes and alleles of CYP2C19 polymorphism, more evident in the stages I and II of endometriosis. Considering HSD17B1 gene polymorphisms, no statistical differences were observed. According to the results, we infer that in our sample, the CYP2C19 gene polymorphism is associated with the presence of endometriosis and can be considered a potential biomarker of the disease.