The role of interleukin-8 (IL-8) and polymorphism of IL-8 gene in formation of external genital endometriosis in patients of reproductive age

External genital endometriosis (EGE) is one of the most important problems of gynecology. In the structure of gynecological pathology, EGE takes the third place and affects up to 50% of women with preserved menstrual function [1–5]. EGE is characterized by dysmenorrhea, dyspareunia, chronic pelvic pain and infertility, often negatively affecting the psychoemotional state of women and overall quality of life [6, 7].

The pronounced negative impact of EGE on the quality of life of patients, their reproductive function, impaired adaptation of patients in society, and growing costs of treatment determine the social significance of the disease and the importance of studying it [8, 9].

Despite numerous theories describing the pathogenesis of endometriosis, there is a lack of knowledge about the development of the disease in a woman's body [10–14]. Considerable attention is currently being paid to the role of the immune system in the development of endometriosis. In patients with genital endometriosis, significant changes are observed in both local immunity and immunological components in circulating blood [15, 16]. There is few data on the role of interleukin (IL) -8 and its gene polymorphism in IGE [17].

In this regard, the aim of the research was to study the production of IL-8 and the polymorphism 251T> A of the IL-8 gene in patients of reproductive age with EGE.

Materials and methods

The study included 71 patients with EGE, who were divided into two groups: Group 1 – patients with stages I–II EGE (n=31); Group 2 – patients with stages III– IV EGE (n=40).

The control group consisted of 24 patients who underwent diagnostic laparoscopy with no evidence of EGE in the Department of Operative Gynecology of the Research Institute of Obstetrics and Pediatrics, Rostov State Medical University, Ministry of Health of Russia, who underwent the criteria for the inclusion of patients in the study: reproductive age 18–49 years, infertility and/or pelvic pain, body mass index 18.5–25 kg/m2, normal body temperature, laparo- and hysteroscopy with morphological verification of the diagnosis of EGE.

Exclusion criteria of the study: pubertal and perimenopausal age of patients, malignant neoplasms, severe decompensated extragenital pathology, acute infections forms.

Determination of IL-8 content in blood serum and peritoneal fluid was carried out by enzyme immunoassay using Bendermedsystems kits (Austria).

Allelic variants of IL-8 genes were determined by polymerase chain reaction followed by restriction analysis using a commercial test system for molecular genetic analysis developed by GosNIIgenetics (Russia).

Statistical analysis

To create a database and conduct a statistical study, we used the capabilities of an Excel 2006 spreadsheet processor and application packages (Megastat and Statisticapp 6.0). When determining the differential validity between the study groups, Mann – Whitney test was used with the Bonferroni correction, in which the traditional level of type 1 error is divided by the number of comparisons. The number of comparison pairs was calculated using the formula m=n(n–1)/2, where n is the number of groups; therefore, the critical value of the significance level (p) in our study was 0.02. Data are presented as median (Me) and quartiles Q1 and Q3. Determination of statistically significant differences between the groups of genotypes and studied genes is conducted using the criterion or χ2 according to the formula.

Results and Discussion

At laparoscopy, ovarian endometriomas was found in 66.2% of patients, providing evidence on the late diagnosis of the diseas. Superficial foci of endometriosis on the ovaries were found in 23.9% of cases. Pelvic peritoneal lesions were found: in the rectal-uterine pouch in 71.8% of cases; on the uterosacral ligaments in 57.7%; in the vesicouterine space – in 40.8% of cases.

During hysteroscopy, the endometrial hyperplasia was found with similar incidence (70%) in both clinical groups with EGE and was histologically confirmed. In the results of the morphological study, simple hyperplasia without atypia prevailed.

Histological examination of endometrioid lesions removed during surgery revealed that the presence of fibrosis and sclerosis, which were considered the outcomes of inflammatory process was statistically significantly higher in patients of the Group 2 (with III-IV stages of EGE) than in patients of the Group 1 (with I–II stages EGE) – 28/40 (70%) versus 4/31 (12.9%), p=0.01, which is apparently the. Hemosiderosis and angiomatosis were also more frequent in the advanced stages of the disease 31/40 (77.5%) and 15/40 (37.5%) versus 4/31 (12.9%) and 1/31 (3.2%), respectively (p=0.01), and were considered as the signs of the disease progression.

Proinflammatory cytokines are the main mediators and communicators of the immune system, initiate and enhance inflammatory responses to infection or injury, and are able to recruit immune cells to the sites of injury and stimulate them to synthesize additional cytokines.

Disregulation of cytokines can be considered as an important aspect of the pathogenesis of endometriosis. However, their role in the survival of endometriotic lesions remains poorly understood. This is in part due to the fact that these modulators are pleiotropic proteins that have a variety of functions.

Chemokines, in particular IL-8, are important factors involved in inflammation associated with endometriosis. IL-8 induces chemotaxis of neutrophils and other immune cells, and is also a potent angiogenic agent, involved in all processes of disease development: adhesion, invasion, implantation of ectopic tissue. In addition, IL-8 plays a role in the growth and maintenance of endometrial ectopic tissue, directly affecting the proliferation of endometrial cells. IL-8 can also protect ectopic cells from death as a result of apoptosis [18, 19].

The results of studying the median values of IL-8 in the study groups are presented in the table.

In women in Group 1 (in the early stages of endometriosis), compared with the control and Group 2, there was a statistically significant increase in the IL-8 content in the peritoneal fluid, (p=0.01), moreover, the indicators in Group 1 exceeded the indicators in Group 2 1.5 times, (p=0.01) (Table 1). There were no statistically significant differences between the groups in IL-8blood serum levels.

According to modern concepts, the pathogenesis of endometriosis can be associated with the expression of inflammatory mediators, the induction of an immune response, and the level of their expression depends on the allelic variant of the gene in a genotype. Of particular importance is the study of genes encoding signaling molecules and causing the coordinated work of various systems and organs [20–22].

Since IL-8 promotes endometrial cell proliferation, adhesion and angiogenesis, an excess of this cytokine can lead to the growth of endometriotic foci and local neovascularization. In this regard, it was interesting to study the dependence of the risk of developing endometriosis in the presence of polymorphism in the IL-8 gene. The results of studying the frequency of polymorphism 251T> A of the IL-8 gene in patients are shown in the Figure.

When studying the distribution of the polymorphism 251T> A of the IL-8 gene, we did not find statistically significant differences between the patients of Groups 1 and 2.

The T/T genotype of the IL-8 gene was found 9.3 and 10.7 times more often in Groups 1 and 2, respectively, compared with the control group, χ2 =8.94 (p=0.01) and χ2 =8.94 (p=0.01).

In polymorphism 251T> A, the incidence of allele A in patients with EGE was lower, and the incidence of allele T was higher than in the control group (χ2 =7.52 at p=0.007, OR=2.9 (95% CI 1.35; 6.5) and χ2 =15.23 at p=0.01, OR=4.4 (95% CI 2.05; 9.45), respectively (Fig. 1).

EGE is one of the most common gynecological diseases affecting women of reproductive age, associated with an increase in the level of proinflammatory cytokines in the peritoneal cavity.

We found that the level of IL-8 was increased in the peritoneal fluid of women, mainly with stage I–II endometriosis. Our results indicate local IL-8 disregulation in the early stages of endometriosis. An increase in the level of IL-8 can lead to the development of proliferation of endothelial cells, and to inhibition of apoptosis processes [23]. IL-8 can act as an autocrine growth factor in the endometrium and promote a vicious cycle of endometrial cell attachment, which can lead to a transformation of acute inflammation to chronic. The data obtained indicate the decisive participation of IL-8 in the formation of endometrioid lesions. Since IL-8 promotes endometrial cells proliferation, adhesions development and angiogenesis, excess of these cytokines can promote growth and local neovascularization. We suggested that the IL-8 gene polymorphism associated with increased production of this cytokine may lead to the growth and maintenance of ectopic endometrial tissue, determining a more pronounced inflammatory response.

The data obtained show that the distribution of allelic variants of IL-8 genes among patients with different stages of EGE is characterized by a predominance of T/T genotypes of polymorphism 251T> A. Thus, the detection of 251T> A polymorphism of the IL-8 gene may serve as a marker of an increased risk of endometriosis in patients of reproductive age.

Conclusion

An increase in the level of IL-8 in the peritoneal fluid, the presence of the polymorphic marker 251T> A of the IL-8 gene is associated with the risk of EGE development in patients of reproductive age.