# Polymorphisms in the 3′UTR Region of ESR2 and CYP19A1 Genes and Its Influence on Allele-Specific Gene Expression in Endometriosis

Authors: Beata Smolarz; Tomasz Szaflik; Hanna Romanowicz; Krzysztof Szyłło
Journal: Disease Markers
 (2020)

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## Abstract

Objectives. Endometriosis is supported by hormonal, immunological, and environmental factors. No specific marker for endometriosis has yet been identified. ESR2 and CYP19A1 genes play a major role in the hormonal control of endometriosis women, the development of which largely depends on steroid hormones. Aim. An analysis of ESR2 and CYP19A1 allele-specific gene expressions in the context of the risk for endometriosis occurrence. Methods. The study material included paraffin-embedded tissue specimens, collected from patients (n=100) with endometriosis. Blood samples from age-matched, endometriosis-free women (n=100) served as a control. the RT-PCR technique was performed to observe the expression of ESR2 and CYP19A1 genes. Moreover, Sanger’s sequencing method was applied for polymorphism analysis. Results. A set of 4 single-nucleotide polymorphisms (SNPs) was determined; all of them most significantly associated with endometriosis: rs4986938 (G>A)(chromosome 14), rs928554 (A>G) (chromosome 14), rs10046 (C>T) (chromosome 15), and rs4646 (C>A) (chromosome 15). There were no differences in the distribution of genotypes and alleles in the studied groups, taking into account ESR2 and CYP19A1 gene expressions. Conclusion. The ESR2 and CYP19A1 polymorphisms may not be correlated with endometriosis susceptibility. Further analysis is needed to specify the role of these polymorphisms in the pathogenesis of endometriosis.


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## Body

## 1. Introduction

Endometriosis is an oestrogen-dependent disease affects 10-15% of women in the reproductive age and 35-50% of women with pains in the pelvis minor and/or with infertility [1]. Aetiology of the disease is complex. Hormonal, immunological, and environmental factors are responsible for its formation [2–4]. In recent years, special attention has been paid to the genetic mechanisms that might have a significant impact on the increased incidence of endometriosis.CYP19A1, aromatase encoding gene, is an enzyme involved in biosynthesis of oestrogens. Different expression levels of aromatase were determined in the endometriosis foci in comparison to the eutopic endometrium. The exact basis of observed changes remains unknown [5].Endometriosis lesions, unlike the eutopic endometrium, demonstrate some activity of the aromatase [6].The activity of oestrogens on target cells is possible via oestrogen receptors. Oestrogen receptors (ERs) acting as transcription factors play a significant role in the growth and differentiation processes of endometrial cells, as well as in numerous biological functions in the eutopic endometrium and endometriosis. The two following types of oestrogen receptors have thus far been identified: ERα and ERβ, encoded by two different genes (ESR1 and ESR2, respectively). ERβ is the predominant isoform in patients with endometriosis [7, 8].Different expression levels of aromatase were determined in the endometriosis foci in comparison to the eutopic endometrium. [9].Previous studies have been demonstrated that both ERs are expressed in human endometriotic tissues but it has been shown that there are significantly increased levels of oestrogen receptor ERβ and decreased levels of ERα in the endometriotic lesions in comparison with the eutopic endometrium. [10–12].The literature data showed that a number of specific SNPs in the 3′UTR regions have structural consequences that may result in the emergence of phenotypic manifestations in the form of the disease [13]. 3′UTR fragments are within the regulatory elements of genes and play an important role in the translation and distribution of RNA.The reported study was aimed at finding out whether there were any correlations between the allele-specific gene expression of theESR2 and CYP19A1 genes and the incidence of endometriosis.The goals of the study included (a) an analysis of theESR2 and CYP19A1 gene expression levels in patients with endometriosis and in a control group, (b) an analysis of the ESR2 and CYP19A1 gene polymorphisms in the 3′UTR region in patients with endometriosis and in a control group, (c) a correlation of the assayed levels of the ESR2 and CYP19A1 gene expressions with polymorphic variants, and (d) the assessment of the significance of obtained results in the context of the risk for endometriosis.


## 2. Materials and Methods

### 2.1. Patients

100 tissue specimens, collected from patients with endometriosis (n=100) embedded into paraffin blocks (Archive of the Department of Clinical Pathomorphology, Polish Mother’s Memorial Hospital Research Institute in Lodz, Poland) provided the material for studies (Table 1). Blood samples from age-matched, endometriosis-free women (n=100) served as the control. The clinical staging was carried out in the patients by the rASRM (The Revised American Society for Reproductive Medicine classification of endometriosis 1996) (Table 1). The study protocol was approved by the Bioethical Committee of the Polish Mother’s Memorial Institute (approval number: 8/2016).Table 1
Characteristic features of the patients (n=100) and of the controls (n=100).
Patients groupControl groupAge (range) 21-53 yearsAge (range) 26-67 yearsAge (mean)34.89±7.49Age (mean)37.93±6.01Patients groupControl groupBody mass index (BMI) (kg/m2)The number (%)Body mass index (BMI) (kg/m2)The number (%)<2568 (68%)<2529 (29%)25≤BMI<3022 (22%)25≤BMI<3043 (43%)≥3010 (10%)≥3028 (28%)Deliveries>0The number (%)Deliveries>0The number (%)Yes47 (47%)Yes93 (93%)-121 (21%)-136 (36%)-2 and more26 (26%)-2 and more57 (57%)No53 (53%)No7 (7%)Spontaneous abortionsThe number (%)Spontaneous abortionsThe number (%)Yes (spontaneous)5 (5%)Yes (spontaneous)9 (9%)No95 (95%)No91 (91%)Clinical stageThe number (%)I26 (26%)II25 (25%)III17 (17%)IV32 (32%)


### 2.2. DNA Isolation

Genomic DNA was prepared, using a QIAamp DNA FFPE Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instruction. DNA was extracted from the blood, using a commercially available QIAmp DNA purification kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instruction.


### 2.3. Detection of Polymorphisms in the ESR2 and CYP19A1 Genes

A reaction mixture of the following composition was used for PCR: 2.5μl of 10x concentrated PCR buffer, 1 μl of each primer, 0.2 μl of Taq polymerase with 5 U/μl activity, 1 μl of a 200 μM mixture of nucleotides (dNTPs), and 100 ng matrix. The reaction mixture was made up to a final volume of 25 μl. The primers were designed using the Primer3 program (Table 2). For all primer pairs, the amplification reactions were carried out using the following thermal profile: pre-PCR→94°C, 4 minutes; PCR→94°C, 20 seconds; 59°C, 20 seconds; 72°C, 40 seconds (35 cycles); post-PCR→72° C, 10 minutes.Table 2
The nucleotide sequence of the amplified 3′UTR region of the ESR2 and CYP19A1 genes.
ESR2Forward 5′-GGGCAGAAAAGGCCTCTCA-3′Reverse 5′-GAAGCCTCAGCTTTCTACATTGG-3′Complete nucleotide sequencegggcagaaaaggcctctcaaacactcacctcatttggaatgaagatggagactcttttgcctgaagcaacgatggagcagtgaccctctaatcaactcggtggcctaaagaaaaatcttgggtaacattttcacttcagtttccctctgggatcattgtaatccatgaaaaaaataattttaaagaaagagttaaaatactttgaagttagttatgtggttaaaaaccaccttcctttctattatcaatccaacaatttgataactgtaaacgctaaagtgaagacggattctcttcagatggtctccttaactgcccagggcttgcagatgtctcacccatgaggggcaccaatgtagaaagctgaggcttcCYP19A1Forward 5′-CCTTGCACCCAGATGAGAC-3′Reverse 5′-CAGAGGCCAAGAGTTTGAGG-3′Complete nucleotide sequenceccttgcacccagatgagacgggagtttctggagggctgaacacgtggaggcaaacaggaaggtgaagaagaacttatcctatcaggacggaaggtcctgtgctcgggatcttccagacgtcgcgactctaaattgccccctctgaggtcaaggaacacaagatggttttggaaatgctgaacccgatacattataacatcaccagcatcgtgcctgaagccatgcctgctgccaccatgccagtcctgctcctcactggcctcaaactcttggcctctg


### 2.4. Sequencing of PCR Products

The PCR reaction was performed on a GeneAmp 2400 PCR System thermocycler (Perkin Elmer), and the process products were purified, precipitating them with ethanol and EDTA. Automatic sequence reading was performed using the ABI PRISM™ 377 automatic DNA sequencer (Applied Biosystems) and the Big Day ™ Terminator Cycle sequencing kit Ready Reaction Kit (Applied Biosystems) according to the manufacturer’s instructions. The results were analysed using the Sequencing Analysis Software™ ver. 3.4.1 (Applied Biosystems) and Factura ™.


### 2.5. RNA Isolation

Total RNA was isolated with the TRIzol reagent (Ambion, the USA). RNA samples were stored in the temperature of -20°C.


### 2.6. RT-PCR Reaction

A reverse transcription reaction was carried out, using a TaKaRa RNA PCR kit (AMV) ver 3.0 (Takara Bio INC, Japan). The obtained CDNA samples were stored in the temperature of -20°C.


### 2.7. PCR with an Analysis of Product Volume Growth in Real Time

The reaction mixture included 0.5μl of cDNA, 0.5 μl of 20x TaqMan® Gene Expression Assay (Applied Biosystems, USA), and 5 μl of TaqMan® Universal PCR Master Mix (Applied Biosystems, USA), containing TaqMan® DNA polymerase, dNTP, a reaction buffer, and 4 μl of water. Real-time PCR was carried out in a Mastercycler® ep realplex device (Eppendorf, Germany). The thermal profile of the reaction included a preliminary denaturation in the temperature of 95°C for 10 minutes, followed by 50 cycles of 15-second incubation in 95°C, combined with 1 minute in 60°C. The following, commercially available kits of probes and starters were applied in the real-time PCR: Hs01100353_m1 for ESR2 gene, Hs00903411_m1 for CYP19A1 gene, and Hs02800695_m1 for HPRT1 (hypoxanthine phosphoribosyltransferase 1) gene, being a reference gene. The yield and quality (260/280 optical density ratios) of the RNA products were determined using PicoDrop spectrophotometer (Picodrop Limited, Hinxton, UK) (Figure 1). The purified total RNA was immediately used for cDNA synthesis or stored at -80°C until use.Figure 1


### 2.8. Statistical Analysis

The obtained results were statistically processed by means of STATISTICA 11 software (StatSoft, Poland). The significance of differences was analysed at the level of gene expression and mRNA, using nonparametric tests (the Mann–WhitneyU test and the Kruskal-Wallis test) for a lack of distribution normality of the obtained results, as confirmed by the Shapiro-Wilk test. The R Spearman test was used to assess correlations between variables. Statistical analysis of the distribution of genotypes and alleles in the test and control group was carried out after prior confirmation that the obtained systems remain in a state of equilibrium according to the rules of Hardy and Weinberg. Wild type of the genotype and allele was the reference group. The result was considered statistically significant at the significance level p less than 0.05.


## 2.1. Patients

100 tissue specimens, collected from patients with endometriosis (n=100) embedded into paraffin blocks (Archive of the Department of Clinical Pathomorphology, Polish Mother’s Memorial Hospital Research Institute in Lodz, Poland) provided the material for studies (Table 1). Blood samples from age-matched, endometriosis-free women (n=100) served as the control. The clinical staging was carried out in the patients by the rASRM (The Revised American Society for Reproductive Medicine classification of endometriosis 1996) (Table 1). The study protocol was approved by the Bioethical Committee of the Polish Mother’s Memorial Institute (approval number: 8/2016).Table 1
Characteristic features of the patients (n=100) and of the controls (n=100).
Patients groupControl groupAge (range) 21-53 yearsAge (range) 26-67 yearsAge (mean)34.89±7.49Age (mean)37.93±6.01Patients groupControl groupBody mass index (BMI) (kg/m2)The number (%)Body mass index (BMI) (kg/m2)The number (%)<2568 (68%)<2529 (29%)25≤BMI<3022 (22%)25≤BMI<3043 (43%)≥3010 (10%)≥3028 (28%)Deliveries>0The number (%)Deliveries>0The number (%)Yes47 (47%)Yes93 (93%)-121 (21%)-136 (36%)-2 and more26 (26%)-2 and more57 (57%)No53 (53%)No7 (7%)Spontaneous abortionsThe number (%)Spontaneous abortionsThe number (%)Yes (spontaneous)5 (5%)Yes (spontaneous)9 (9%)No95 (95%)No91 (91%)Clinical stageThe number (%)I26 (26%)II25 (25%)III17 (17%)IV32 (32%)


## 2.2. DNA Isolation

Genomic DNA was prepared, using a QIAamp DNA FFPE Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instruction. DNA was extracted from the blood, using a commercially available QIAmp DNA purification kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instruction.


## 2.3. Detection of Polymorphisms in the ESR2 and CYP19A1 Genes

A reaction mixture of the following composition was used for PCR: 2.5μl of 10x concentrated PCR buffer, 1 μl of each primer, 0.2 μl of Taq polymerase with 5 U/μl activity, 1 μl of a 200 μM mixture of nucleotides (dNTPs), and 100 ng matrix. The reaction mixture was made up to a final volume of 25 μl. The primers were designed using the Primer3 program (Table 2). For all primer pairs, the amplification reactions were carried out using the following thermal profile: pre-PCR→94°C, 4 minutes; PCR→94°C, 20 seconds; 59°C, 20 seconds; 72°C, 40 seconds (35 cycles); post-PCR→72° C, 10 minutes.Table 2
The nucleotide sequence of the amplified 3′UTR region of the ESR2 and CYP19A1 genes.
ESR2Forward 5′-GGGCAGAAAAGGCCTCTCA-3′Reverse 5′-GAAGCCTCAGCTTTCTACATTGG-3′Complete nucleotide sequencegggcagaaaaggcctctcaaacactcacctcatttggaatgaagatggagactcttttgcctgaagcaacgatggagcagtgaccctctaatcaactcggtggcctaaagaaaaatcttgggtaacattttcacttcagtttccctctgggatcattgtaatccatgaaaaaaataattttaaagaaagagttaaaatactttgaagttagttatgtggttaaaaaccaccttcctttctattatcaatccaacaatttgataactgtaaacgctaaagtgaagacggattctcttcagatggtctccttaactgcccagggcttgcagatgtctcacccatgaggggcaccaatgtagaaagctgaggcttcCYP19A1Forward 5′-CCTTGCACCCAGATGAGAC-3′Reverse 5′-CAGAGGCCAAGAGTTTGAGG-3′Complete nucleotide sequenceccttgcacccagatgagacgggagtttctggagggctgaacacgtggaggcaaacaggaaggtgaagaagaacttatcctatcaggacggaaggtcctgtgctcgggatcttccagacgtcgcgactctaaattgccccctctgaggtcaaggaacacaagatggttttggaaatgctgaacccgatacattataacatcaccagcatcgtgcctgaagccatgcctgctgccaccatgccagtcctgctcctcactggcctcaaactcttggcctctg


## 2.4. Sequencing of PCR Products

The PCR reaction was performed on a GeneAmp 2400 PCR System thermocycler (Perkin Elmer), and the process products were purified, precipitating them with ethanol and EDTA. Automatic sequence reading was performed using the ABI PRISM™ 377 automatic DNA sequencer (Applied Biosystems) and the Big Day ™ Terminator Cycle sequencing kit Ready Reaction Kit (Applied Biosystems) according to the manufacturer’s instructions. The results were analysed using the Sequencing Analysis Software™ ver. 3.4.1 (Applied Biosystems) and Factura ™.


## 2.5. RNA Isolation

Total RNA was isolated with the TRIzol reagent (Ambion, the USA). RNA samples were stored in the temperature of -20°C.


## 2.6. RT-PCR Reaction

A reverse transcription reaction was carried out, using a TaKaRa RNA PCR kit (AMV) ver 3.0 (Takara Bio INC, Japan). The obtained CDNA samples were stored in the temperature of -20°C.


## 2.7. PCR with an Analysis of Product Volume Growth in Real Time

The reaction mixture included 0.5μl of cDNA, 0.5 μl of 20x TaqMan® Gene Expression Assay (Applied Biosystems, USA), and 5 μl of TaqMan® Universal PCR Master Mix (Applied Biosystems, USA), containing TaqMan® DNA polymerase, dNTP, a reaction buffer, and 4 μl of water. Real-time PCR was carried out in a Mastercycler® ep realplex device (Eppendorf, Germany). The thermal profile of the reaction included a preliminary denaturation in the temperature of 95°C for 10 minutes, followed by 50 cycles of 15-second incubation in 95°C, combined with 1 minute in 60°C. The following, commercially available kits of probes and starters were applied in the real-time PCR: Hs01100353_m1 for ESR2 gene, Hs00903411_m1 for CYP19A1 gene, and Hs02800695_m1 for HPRT1 (hypoxanthine phosphoribosyltransferase 1) gene, being a reference gene. The yield and quality (260/280 optical density ratios) of the RNA products were determined using PicoDrop spectrophotometer (Picodrop Limited, Hinxton, UK) (Figure 1). The purified total RNA was immediately used for cDNA synthesis or stored at -80°C until use.Figure 1


## 2.8. Statistical Analysis

The obtained results were statistically processed by means of STATISTICA 11 software (StatSoft, Poland). The significance of differences was analysed at the level of gene expression and mRNA, using nonparametric tests (the Mann–WhitneyU test and the Kruskal-Wallis test) for a lack of distribution normality of the obtained results, as confirmed by the Shapiro-Wilk test. The R Spearman test was used to assess correlations between variables. Statistical analysis of the distribution of genotypes and alleles in the test and control group was carried out after prior confirmation that the obtained systems remain in a state of equilibrium according to the rules of Hardy and Weinberg. Wild type of the genotype and allele was the reference group. The result was considered statistically significant at the significance level p less than 0.05.


## 3. Results

The performed analysis was aimed at determining the significance of new genetic variants as endometriosis risk factors. PCR analysis identifies statistically significant correlations among SNPs localised on chromosomes 14 and 15 (Table3). A pool of 4 SNPs mostly correlated with endometriosis risk was determined: rs4986938 (G>A) (chromosome 14), rs928554 (A>G) (chromosome 14), rs10046 (C>T) (chromosome 15), and rs4646 (C>A) (chromosome 15).Table 3
The chromosomal coordinates of the four studied SNPs. (https://www.ncbi.nlm.nih.gov/genome/guide/human/).
LocationReferencers4986938Chromosome 14, NC_000014.9 (64226707..64338631, complement)GRCh38 38.1/141rs928554Chromosome 14, NC_000014.9 (64226707..64338631, complement)GRCh38.p12rs10046Chromosome 15, NC_000015.10 (51208057..51338596, complement)GRCh38 38.1/141rs4646Chromosome 15, NC_000015.10 (51208057..51338596, complement)GRCh38 38.1/141In the females with endometriosis, GG, GA, and AA genotypes withinESR2 rs4986938 SNP were observed in 73% (73/100), 25% (25/100), and 2% (2/100) of the studied individuals, respectively (Table 4). In the case of rs928554, AA, GA, and GG genotypes were found in 48% (48/100), 52% (52/100), and 0% (0/100) of the studied patients, respectively (Table 5).Table 4
Relative ESR2 gene expression with regard to the polymorphism rs4986938.
ESR2Median25th percentile75th percentilep valueaGG (73%)201.27112.42365.320.46GA (25%)169.80104.73214.31AA (2%)———p trendb0.35aKruskal-Wallis test. bTesting additive genetic model (Cochran-Armitage test for trend).Table 5
Relative ESR2 gene expression with regard to the polymorphism rs928554.
ESR2Median25th percentile75th percentilep valueaAA (48%)601.95151.41772.820.11AG (52%)179.73159.26557.43GG (0%)———p trendb0,10aMann–Whitney U test. bTesting additive genetic model (Cochran-Armitage test for trend).Taking into account the nonendometriosis control females, the GG, GA, and AA genotypes in rs4986938 polymorphism were found in 61% (61/100), 32% (32/100), and 7% (7/100) of the patients, respectively. Regarding rs928554, the AA, AG, and GG genotypes were observed in 51 (51/100), 49% (49/100), and 0% (0/100) of the studied individuals, respectively.In the females with endometriosis, CC, CT, and TT genotypes withinCYP19A1 rs10046 SNP were observed in 37% (37/100), 63% (63/100), and 0% (0/100) of the studied individuals, respectively (Table 6). In the case of rs4646, CC, CA, and AA genotypes were found in 48% (48/100), 51% (51/100), and 1% (1/100) of the studied patients, respectively (Table 7). Taking into account the nonendometriosis control females, the CC, CT, and TT in rs10046 polymorphism were found in 41% (41/100), 59% (59/100), and 0% (0/100) of the patients, respectively. Regarding rs4646, the CC, CA, and AA were observed in 56% (56/100), 44% (44/100), and 0% (0/100) of the studied individuals, respectively.Table 6
Relative CYP19A1gene expression with regard to polymorphism rs10046.
CYP19A1Median25th percentile75th percentilep valueaCC (37%)299.24155.661089.460.23CT (63%)514.13254.23614.31TT (0%)———p trendb0.27aMann–Whitney U test. bTesting additive genetic model (Cochran-Armitage test for trend).Table 7
Relative CYP19A1gene expression with regard to polymorphism rs4646.
CYP19A1Median25th percentile75th percentilep valueaCC (48%)174.13151.41572.820.18CA (51%)209.73159.26557.43AA (1%)———p trendc0.62aMann–Whitney U test. bTesting additive genetic model (Cochran-Armitage test for trend).However, the current study failed to show any correlation of analysed SNPs with the expression of theESR2 and CYP19A1 genes.The genotype frequency of theESR2 rs4986938 polymorphism in relation to its expression is summarized in Table 2. There were no statistically significant differences in the ESR2 gene expression in relation to the occurrence of rs4986938 polymorphism (Kruskal-Wallis test p=0.46).No statistically significant differences were observed in genotype frequencies ofESR2 rs928554 polymorphism and ESR2 expression (Mann–Whitney test p=0.11) (Table 3).There were no statistically significant differences in theCYP19A1 gene expression in relation to the occurrence of rs10046 polymorphism genotypes (Mann–Whitney test p=0.23) (Table 4).Our data did not demonstrate any statistically significant correlation betweenCYP19A1 rs4646 polymorphisms and CYP19A1 expression (Mann–Whitney test p=0.18) (Table 5).No relationship was revealed betweenESR2 and CYP19A1 polymorphisms and rASRM (revised American Society for Reproductive Medicine) classification scores in the study group. The SNPs in the study group did not correlate with either the age of the patients or with their BMI, menopausal status, or with the number of pregnancies in history.


## 4. Discussion

Our research addressed the role of allele-specific gene expression ofESR2 and CYP19A1 genes as a risk factor for endometriosis. Performed genetic analyses were based on the rs4986938, rs928554, rs10046, and rs4646 polymorphisms of the ESR2 and CYP19A1 genes.The allele-specific gene expression was assayed in patients with endometriosis vs. a healthy control group, while also studying their effects on the increased prevalence of endometriosis among Polish women.Disorders at the level of theESR2 and CYP19A1 gene expressions, observed in endometriosis as related to the types and sites of lesions, associated with the disease, are confirmed by world literature reports [14–20]. Several different studies are conducted in order to evaluate the risk of endometriosis in association with different genes polymorphism: CYP17A1, CYP19A1, ESR1, ESR2, PGR, HSD17B1, and HSD17B2 [21–26].Regarding studies ofCYP17 and ESR2 gene polymorphisms, there are few reports concerning the 3′UTR region, which is important, because it attaches to the miRNA, which regulates gene expression. This region is therefore important for phenotypic changes that may trigger development of endometriosisWhile SNPs inCYP19A1 have been associated with sex-steroid hormone levels and other oestrogen-dependent diseases including endometriosis, the results of previous studies of the associations between various CYP19A1 polymorphisms and endometriosis have been inconsistent up to a certain degree. Many studies of CYP19A1 polymorphisms in relation to endometriosis risk have been published; however, their findings were not confirmed by any additional researches on affected populations [27, 28].Tempfer et al. reported that theERβ gene is associated with increased risk of stage IV endometriosis in Japanese women, while we found polymorphism in all stages of classification of endometriosis of the patients (classification of endometriosis according to the American Society for Reproductive Medicine) [29].In the other studies, it was found that the allelic frequency ofRsaI polymorphism of the ERβ gene in AG is about nine times higher in patients with endometriosis in comparison to control groups [30].However, when comparing fertile and infertile subgroups, no significant differences were stated. The study of ERβ and its correlation with the risk of endometriosis could help in the explanation of disease’s genetic aetiology.Szaflik et al. identified statistically significant correlations between new SNP, rs4986938 and rs928554, not described earlier, and endometriosis. In the case of rs4986938, the AA genotype decreased the risk of endometriosis. A similar effect was demonstrated in the case of rs928554 polymorphism of the AG genotype. The results, obtained during that analysis, demonstrated that rs4986938 and rs928554 polymorphisms of theESR2 gene are associated with the occurrence of endometriosis [31].In the present work, a pool of 4 SNPs was determined, all of them most importantly associated with endometriosis: rs4986938 (G>A) (chromosome 14), rs928554 (A>G) (chromosome 14), rs10046 (C>T) (chromosome 15), and rs4646 (C>A) (chromosome 15).The study involved 100 endometriosis patients. A control group consisted of 100 nonendometriosis women. In the patients with endometriosis, GG, GA, and AA genotypes withinESR2 rs4986938 SNP were observed in 73% (73/100), 25% (25/100), and 2% (2/100) of the studied individuals, respectively (Table 4). In the case of rs928554, AA, AG, and GG genotypes were found in 48% (48/100), 52% (52/100), and 0% of the studied patients, respectively (Table 5). In the females with endometriosis, CC, CT, and TT genotypes within CYP19A1 rs10046 SNP were observed in 37% (37/100), 63% (63/1000, and 0% of the studied individuals, respectively (Table 6). In the case of rs4646, CC, CA, and AA genotypes were found in 48% (48/100), 51% (51/100), and 1% (1/100) of the studied patients, respectively (Table 7). Neither were any relationships demonstrated between the ESR2/CYP19A1 gene expression levels and rs4986938, rs928554, rs10046, and rs4646 polymorphisms (p>0.05) (Figures 2–5).Figure 2Figure 3Figure 4Figure 5We did not find a relationship between allele-specific genes expression and risk of endometriosis. This study is limited by the sample sizes, and it is possible statistical power may be not enough to detect smaller expression differences among the comparison groups. The study results will be useful for further study design with larger samples sizes.Therefore, further studies would be justified and needed to reveal the common involvement mechanisms ofESR2 and CYP19A1 SNPs in endometriosis formation.However the results, obtained in the study, contribute to better knowledge of and information on the molecular mechanisms which support the development of endometriosis.


## 5. Conclusions

There are no statistically significant differences in the ESR2 and CYP19A1 allele-specific gene expressions based on the comparisons of the 100 cases vs. the 100 controls, which lead us to conclude that the two genes do not play significant roles in the pathogenesis of endometriosis.



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