Genes of estrogen and progesterone receptors and characteristics of uterine damage in myoma

A uterine myoma is a hormone-dependent tumor composed of muscle and connective tissue elements [1, 2]. It holds a significant position among tumors affecting the female reproductive system, accounting for 10–20% of all gynecological pathologies, and is diagnosed in 10–30% of women of reproductive age [3, 4]. It is estimated that every fourth or fifth woman worldwide suffers from uterine fibroids. Approximately 80% of women aged 30–40 years are at risk of developing uterine myoma, with 20-25% already diagnosed, leading to an increase in surgical interventions among women of reproductive age [5, 6]. The hormonal status of a woman plays a crucial role in the development of large myomatous nodes [7, 8]. Increased expression of extracellular matrix components, along with estrogen and progesterone receptors in uterine fibroids, disrupts the balance between progesterone and estrogen levels, creating conditions conducive to the rapid growth of fibroid nodules [9–11].

The interactions of polymorphic gene variants in the development and progression of uterine fibroids have been actively discussed by researchers both domestically and internationally [12–14]. For instance, Bondagji N.S. et al. (2016) demonstrated that the polymorphic locus rs12484776 significantly contributes to the formation of uterine fibroids in women in Saudi Arabia, both independently and in interaction with the polymorphic loci rs1056836, rs2280543, and rs7913069 [15]. Rafnar T. et al. (2018) presented a meta-analysis of two genome-wide association studies of uterine fibroids among European women, involving 16,595 cases and 523,330 controls, which identified 21 variants across 16 polymorphic loci that contribute to the development of large uterine fibroids [16]. However, the interactions between estrogen and progesterone receptor genes in the development of large uterine fibroids have not yet been studied, highlighting the relevance of this research.

This study aimed to investigate the association between combined polymorphic variants of estrogen and progesterone receptor genes and the development of large uterine fibroids.

Materials and methods

The study sample included 380 women with confirmed diagnoses of uterine fibroids. Patients were selected using a continuous method in the gynecological department of the Belgorod Regional Clinical Hospital of St. Joasaph. The women included in the study were native residents of the Central Black Earth Region of Russia with no family ties to each other. Exclusion criteria were the presence of diagnosed malignant diseases of the female reproductive system and pregnancy. The study was conducted with written informed consent of the patients and was reviewed and approved by the Research Ethics Committee of Belgorod State University (Ref. No: 6 of 23.04.2021).

To assess the type of uterine damage in fibroids, the size of myomatous nodules was used as an indicator [17]. The patients (n=380) were divided into three groups. Group 1 included women with the largest nodule size greater than 60 mm (n=205, 53.94%). Group 2 included women with the largest nodule size ranging from 25 to 60 mm (n=125, 32.89%). Group 3 included women with the largest nodule size of up to 25 mm (n=50; 13.16%).

For the polymerase chain reaction (PCR), DNA was isolated from peripheral blood leukocytes using the phenol-chloroform extraction method. DNA samples were genotyped by allele-specific PCR in real-time using TaqMan probes, and amplification was conducted on a CFX96 detection system (Bio-Rad, USA) [18]. The following DNA markers were included in this study: rs2234693 ЕSR1 с453-397Т>С, rs3798577 ЕSR1 c.1029Т>С, rs9340799 ЕSR1 с.453-351А>G, rs484389 РGR c.38Т>С, and rs1042838 РGR c.1415-11113G>Т.

Statistical analysis

Statistical analysis was performed using STATISTICA 10.0. Categorical variables were presented as numbers and percentages. Differences in genotype/allele frequencies between the study groups were determined using standard methods (c2 test, 2×2 contingency table). Variables such as body mass index, patient age, and age at menarche were assessed using the Shapiro–Wilk test. Continuous variables with a normal distribution were expressed as mean (M) and standard deviation (SD). The Mann–Whitney test was used to compare continuous variables and the χ² test was used for categorical variables. The results were considered statistically significant at p<0.05. Interaction analysis of polymorphic variants of genes was performed using the AP Sampler software (https://sourceforge.net/projects/apsampler/), which uses the Monte Carlo Markov chain method and Bayesian nonparametric statistics, with the calculation of the odds ratio (OR) and its 95% confidence interval (95% CI) [19]. A permutation test (p_perm) was used to adjust for the effects of false positives in the multiple comparisons. Since the three groups were compared with each other, p_perm<0.016 (0.05/3) was considered statistically significant [20]. The functional significance of the polymorphic loci under consideration was studied using the HaploReg program (v4.1) (http://archive.broadinstitute.org/mammals/haploreg/haploreg.php). When assessing the change in interaction with transcription factors of gene structure variants, the Δ LOD Score was calculated using the formula: LOD Score of the alternative (alt) allele – LOD Score of the reference (ref) allele. If the Δ LOD Score was <0, a conclusion was made regarding the reduced affinity of DNA motifs to transcription factors; if the value was >0, a relative increase was made. The GTExportal tool (https://www.gtexportal.org/home/snp) was used to establish the role of polymorphisms in gene transcription changes.

Results and discussion

Analysis of clinical and demographic characteristics (Table 1) showed no significant differences between the study groups (p>0.05).

For all studied polymorphic loci, the observed distribution of genotypes corresponded to that expected under Hardy–Weinberg equilibrium (p>0.05).

Comparative analysis of the allele and genotype frequencies of estrogen and progesterone receptor polymorphic loci among the three groups of women with fibroids, categorized by fibroid size, found no significant differences (p>0.05) (Table 2).

Analysis of the interaction between the studied polymorphic loci revealed that in group 1, the combination of the ESR1 C allele (rs3798577) and the ESR1 AA genotype (rs9340799) was present in 35.12% (75/205) of patients, significantly differing from group 2 (22.40% (24/125), p_perm=0.003, OR=2.42, 95% CI 1.43–4.11) and group 3 (20.00% (10/50), p_perm=0.003, OR=2.30, 95% CI 1.09–4.88) (Table 3). This established combination of polymorphic loci serves as a marker of the risk of developing uterine fibroids with large myomatous nodules.

It was also found that in group 1, the combination of molecular genetic markers GG РGR (rs1042838), C РGR (rs484389) and G ЕSR1 (rs9340799) occurs less frequently (5.85% (12/205)) than in group 2 (18.40% (23/125), p_perm=0.009, OR=0.27, 95% CI 0.13–0.57), and in group 3 (18.00% (9/50), p_perm=0.006, OR=0.28, 95% CI 0.11–0.71). This combination of polymorphic loci indicates a minimal risk of developing large myomatous nodules. The data regarding the role of polymorphic loci of estrogen and progesterone receptors in the development of large myomatous nodules in patients with uterine myoma can be explained by the medical and biological effects of the protein products of these genes. According to the literature, myomatous nodules are sensitive to progesterone and estrogens [21, 22]. It has been shown that a significant increase in estrogens is detected in myomatous nodules, while normal myometrium has a limited response to estrogens and remains intact. Many researchers have established the stimulatory effect of progesterone on the proliferative activity of myomatous nodules [23, 24]. Progesterone influences the growth of myomatous nodules through local growth factors, extracellular matrix, and its corresponding receptors. Research indicates that sex hormones, particularly progesterone, induce the expression of Bcl-2 via their receptors, blocking apoptosis, and thereby extending the life cycle of the cell [24–26], which promotes the growth of myomatous nodules. In a study by Pechkovskij E.V. et al. [27], it was established that the T allele of rs1042838 PGR and the C allele of rs484389 PGR were associated with a decrease in the number of progesterone receptors in mammary gland cells, reducing the likelihood of a cellular response to progesterone. Progesterone has an antiproliferative effect not only in the mammary gland but also in the uterus, which helps explain the protective effects of the combination of polymorphic loci G rs1042838 PGR, C rs484389 PGR, and allele G rs9340799 ESR1 in the development of large myomatous nodules. According to Golovchenko O. et al., the latter allele is responsible for increased estrogen expression in the myometrium [28].

To evaluate the medical and biological effects of the combination of markers C rs3798577 ESR1 and AA rs9340799 ESR1, which are risk factors for the formation of large myomatous nodules, we conducted an in-silico analysis. The HaploReg database indicates that the rs9340799 polymorphism, located in the intron region of ESR1, is situated in an enhancer region and affects the affinity for transcription factors. The G allele of rs9340799 ESR1 increases the sensitivity of the DNA in this region to the transcription factors Ets (Δ LOD=4.3) and Hand1 (Δ LOD=11.8) in breast and adipose tissues, potentially regulating the expression of this gene.

The rs3798577 polymorphism, located in the 3'-UTR region of ESR1, also possesses significant regulatory potential. It is located in a region modified by histone proteins (H3K4me3 and H3K9ac), marking promoters in adipocytes, female skeletal muscle tissue, muscle satellite cell cultures, and adipose tissue precursors, among others. This polymorphic locus is also found in a region modified by histone proteins (H3K4me1 and H3K27ac), marking enhancers in over 30 different cell cultures, tissues, and organs (mesenchymal stem cells, female muscle tissue, adipocytes, ovaries, etc.) [29]. The functional effects of polymorphic loci rs3798577 and rs9340799 ESR1 confirmed their association with hormone-dependent diseases of the female reproductive system.

It should be noted that the roles of the polymorphic loci rs3798577 ESR1, rs9340799 ESR1, rs484389 PGR, and rs1042838 PGR in the development of various diseases are actively studied by various scientific teams. The PubMed Central database (https://www.ncbi.nlm.nih.gov/pmc) contains more than 190 articles related to medical and biological studies of rs9340799 ESR1, over 40 publications examining the significance of rs3798577 ESR1, and more than 50 studies on rs484389 PGR and rs1042838 PGR. These articles demonstrated associations between these polymorphic loci and the development of uterine fibroids, miscarriage, infertility, tumors of the female reproductive system, and cardiovascular diseases. [24, 25, 27, 28]. However, the literature primarily addresses the monoeffects of polymorphic variants of estrogen and progesterone receptor genes in uterine myoma or combinations of these polymorphic loci in other gynecological pathologies.

Conclusion

This study established a significant role for the genes rs3798577 ESR1, rs9340799 ESR1, rs484389 PGR, and rs1042838 PGR in the nature of uterine damage in myoma. The combination of markers C rs3798577 ESR1 and AA rs9340799 ESR1 (OR=2.30–2.42) should be considered a risk factor for the development of large myomatous nodules, whereas the combination of markers G rs9340799 ESR1, C rs484389 PGR, and GG rs1042838 PGR (OR=0.27–0.28) should be viewed as a protective factor against the development of large nodules. The combinations of polymorphic loci of estrogen and progesterone receptors identified in this study are crucial for the development of large uterine fibroids. Based on these findings, it is possible to develop preventive measures aimed at identifying patients at risk of this pathology.