Molecular genetic determinants of adenomyosis

Adenomyosis is a common gynecological disease characterized by benign proliferation and chronic inflammation. It has significant social implications because it reduces the quality of life of women. The disease is known for its pronounced clinical manifestations and tendency to relapse even after treatment [1, 2]. The symptoms of adenomyosis include frequent aching pain in the pelvic area, irritable bowel syndrome, dysmenorrhea, painful ovulation, and dyspareunia. Despite its high prevalence among women of reproductive age, the pathogenesis of adenomyosis is not yet fully understood [3, 4].

The development of adenomyosis is attributed to hormonal imbalances, dysfunction in stem cell activity and growth factors, as well as genetic and epigenetic abnormalities [5–7]. Genetic determinants, such as genes encoding growth factors, play a role in adenomyosis formation. These genes are directly or indirectly involved in processes such as proliferation, differentiation, and cell migration and exhibit immunosuppressive properties [8–10].

This study aimed to evaluate the involvement of polymorphic loci in growth factor genes in the development of adenomyosis.

Materials and methods

To conduct a molecular genetic study, patients with adenomyosis (study group) and women from the control group were selected from the regional clinical hospital in the city of Belgorod. The study included 880 women of Russian nationality living in the Belgorod region, who were not related to each other and agreed to participate in the study. Patients with adenomyosis were included in the study group after the diagnosis of the disease was established and confirmed using clinical, clinical, instrumental, and clinical laboratory investigations. Exclusion criteria from the study group were malignant diseases of the female reproductive system and pregnancy. Women in the control group were selected from among women without reproductive system diseases. Thus, according to the above criteria, the sample for this study included 102 patients in the study group and 778 women in the control group. The study was approved by the Regional Ethics Committee of Belgorod State National Research University (Protocol 4 of October 21, 2019).

When planning the study, the following DNA markers were selected: rs4444903 EGF c.-382A>G (epidermal growth factor), rs6214 IGF1 c.*2716G>A (insulin-like growth factor 1), rs2981582 FGFR2 c.109+906T>C (factor receptor fibroblast growth), rs833061 VEGF c.-958C>T (vascular endothelial growth factor), rs1800469 TGFb1 c.-1347 T>C (transforming growth factor beta 1) [11]. For the polymerase chain reaction (PCR), DNA was isolated from peripheral blood leukocytes using phenol-chloroform extraction. Molecular genetic loci were analyzed using real-time PCR and oligonucleotide primers and probes (Syntol LLC, Russia). During molecular genetic analysis, some DNA samples were removed from the study because the level of relative fluorescence during genotyping did not allow the genotype to be determined with sufficient accuracy.

Statistical methods

The normality of the distribution was tested using the Shapiro-Wilk test. Continuous variables showing normal distribution (age, body mass index (BMI)) were expressed as mean (M) and standard deviation (SD) and compared using Student’s t-test. Allele and genotype frequencies between the study and control groups were compared using the χ² test with Yates correction for continuity. Calculations were performed using 2×2 contingency tables [12].

Associations of combinations of alleles of the analyzed genes with the development of adenomyosis were assessed using the APSampler program (https://sourceforge.net/projects/apsampler/) using the Monte Carlo Markov chain method and Bayesian nonparametric statistics, calculating the odds ratio (OR) and its 95% confidence interval (95% CI). To determine the effects of false-positive results in multiple comparisons, a permutation test (p_perm) was used. P_perm <0.05 was taken as a statistically significant level [13]. Statistical analysis was performed using STATISTICA for Windows 10.0 and Microsoft Excel 2016 software.

Results and discussion

Clinical and demographic characteristics of the study groups are presented in Table 1.

The mean age of the patients was 41.86 (4.67) years in the study group and 41.15 (4.82) years in the control group (p=0.71). Among the comorbidities, cardiovascular (29.58% and 26.86%), endocrine (9.80% and 6.33%), and genitourinary (10.78% and 14.26%) diseases were more common in both the study and control groups. There were no statistically significant differences in these parameters between the study groups (p=0.82, p=0.26, and p=0.42, respectively). It should be noted that parameters such as stress, smoking and inheritance of proliferative uterine diseases were significantly different between the study and control groups (p=0.002, p=0.007 and p=0.003, respectively).

Tables 2 and 3 show the results of the statistical analysis of the data obtained by genotyping the polymorphisms of growth factors in the control and study groups.

Thus, the GG IGF1 rs6214 genotype in the study group occurred in 52.00% (52/102) of cases and in 38.33 % (253/778) of cases in the control group (χ²=1.74, p=0.01, OR=2.64, 95% CI 1.14–2.65).

According to the literature, IGF1 in physiological concentrations regulates apoptotic processes and the action of steroid hormones, and act as mediators of cell growth, transformation, and differentiation [14].

The polymorphic A locus rs6214 IGF1 causes a decrease in the expression of the corresponding protein, whereas the G allele rs6214 IGF1, which in our study is associated with the risk of developing adenomyosis, has the opposite effect, increasing endometrial proliferation and apoptosis, which can lead to the development of adenomyosis [15].

Risk combinations of growth factor gene alleles for adenomyosis were identified (Table 4). In the study group, the combination of G rs6214 IGF1, C rs1800469 TGFb1, A rs4444903 EGF, and T rs833061 VEGF occurred in 58.82% (60/102) of patients with adenomyosis, and in the control group, in 43.12% (335/778) of women (OR=1.88, p_perm=0.0021). In addition, the combination of G rs6214 IGF1, C rs1800469 TGFb1, T rs833061 VEGF, and C rs2981582 FGFR2 was detected in 60.86% (62/102) of patients in the study group and 47.52 % (370/778) of the control group (OR=1.71, p_perm=0.003). Established combinations of polymorphic loci of growth factor genes are markers for the risk of developing adenomyosis.

According to literature, the single-nucleotide substitutions we analyzed are localized in the region of modified histones that mark promoters and enhancers in various tissues, A>G rs4444903 EGF, T>C rs2981582 FGFR2, C>T rs833061 VEGF, T>C rs1800469 TGFb1 are located in the region of open chromatin, A>G rs4444903 EGF, rs833061 VEGF, T>C rs1800469 TGFb1 are located in sites that interact with transcription factors, G>A rs6214 IGF1, T>C rs2981582 FGFR2, C>T rs833061 VEGF, T>C rs1800469 TGFb1 are located in regions of DNA regulatory motifs [16]. Thus, the polymorphic genes of growth factors included in this study are important in the body and may be involved in the etiopathogenesis of adenomyosis [17].

The data obtained on the role of polymorphic loci of growth factors in the development of adenomyosis are explained by the medical and biological effects of the protein products of these genes. According to the literature, changes in the production of growth factors stimulate differentiation and ectopic growth of the endometrium, remodeling of the extracellular matrix, and cell migration [18]. Growth factors are also involved in the pathological and physiological neovascularization of the endometrium. Increased production of growth factors in adenomyosis may contribute to its pathophysiology in a manner similar to its carcinogenic effects, causing changes in cellular metabolism, increasing cell invasion, and initiating neoangiogenesis [19, 20].

Conclusion

The gene combinations rs4444903 EGF, rs6214 IGF1, rs2981582 FGFR2, rs833061 VEGF, and rs1800469 TGFb1 are associated with the development of adenomyosis. These results indicate the importance of interlocus interactions between growth factor genes in the development of adenomyosis. In the future, the obtained data could be used in practical medicine.