FEATURES OF EPITHELIAL-MESENCHYMAL TRANSITION IN ECTOPIC ENDOMETRIUM IN PATIENTS WITH INTRAEPITHELIAL NEOPLASIA OF THE CERVIX

EUROPEAN JOURNAL OF MODERNMEDICINE AND PRACTICE Vol. 4 No. 11 (Nov - 2024) ISSN: 2795-921X 521 https://inovatus.es/index.php/ejmmp FEATURES OF EPITHELIAL-MESENCHYMAL TRANSITION IN ECTOPIC ENDOMETRIUM IN PATIENTS WITH INTRAEPITHELIAL NEOPLASIA OF THE CERVIX Abdieva N U. Asian International University Abstract: Endometriosis is a benign yet invasive disease marked by the ectopic presence of endometrial tissue, significantly impacting women of reproductive age. This study explores the role of epithelial-mesenchymal transition (EMT) in extragenital endometriosis by analyzing the expression of key markers, E-cadherin and vimentin, in various localizations of the disease. Conducted in Moldova and Romania between 2012 and 2017, the study involved immunohistochemical analysis of postoperative specimens from patients aged 19 to 56 years. Results revealed low membrane expression of E-cadherin and pronounced cytoplasmic expression of vimentin in invasive forms, especially in cesarean section scars and inguinal hernias. A contrasting expression pattern was noted in intestinal forms, indicating variations in invasiveness and marker expression based on localization. These findings underscore the critical involvement of EMT in the pathogenesis and invasiveness of extragenital endometriosis, particularly in diffuse-infiltrative forms. Keywords: endometriosis, epithelial-mesenchymal transition, E-cadherin, vimentin, extragenital endometriosis, cesarean section scars, immunohistochemistry, invasiveness. Introduction Endometriosis is a benign disease characterized by the presence of endometrial glands and stroma outside the uterine cavity. Endometriosis affects 6–10% (about 190 million) of women of reproductive age. The average age of women diagnosed with endometriosis is 28 years. Although various theories have been proposed to explain the development of endometriosis, its origin remains unclear. Epithelial-mesenchymal transition (EMT) is a biological process of phenotypic change of an epithelial cell with its transformation into a mesenchymal cell. This phenomenon was first described by Elizabeth Hay [4], who called it "epithelial-mesenchymal transformation" and suggested that in the early stages of embryonic development, epithelial cells can "transform" into cells with a mesenchymal phenotype. In the subsequent period of time, the term "epithelial-mesenchymal transformation" was replaced by the term EMT, which implies the reversibility of the process and does not lead to confusion with tumor transformation. In the embryo, due to EMT, cells with an epithelial phenotype acquire motor activity and colonize new territories. It is known that epithelial cells are tightly adjacent to each other due to the formation of specialized intercellular connections: nexuses, desmosomes, adhesive and locking contacts. Due to strong connections, EUROPEAN JOURNAL OF MODERN MEDICINE AND PRACTICE Vol. 4 No. 11 (Nov - 2024) EJMMP ISSN: 2795-921X EUROPEAN JOURNAL OF MODERNMEDICINE AND PRACTICE Vol. 4 No. 11 (Nov - 2024) ISSN: 2795-921X 522 epithelial tissue consists of tightly assembled immobile cells located on the basement membrane separating the epithelium from the connective tissue. The latter is formed by cells of the mesenchymal phenotype, devoid of specialized connections, torn from each other and capable of moving through the intercellular matrix by which they are surrounded. Membrane proteins, occludins and claudins, participate in the formation of these compounds. Cadherins, transmembrane proteins that provide calcium-dependent intercellular adhesion, play an important role in fixing adjacent zones. Mesenchymal embryonic cells and fibroblasts from the postnatal period are spindle-shaped cells with cytoplasmic processes (pseudopodia and filopodia), with anterior-posterior polarity ("head-tail"), capable of moving through the intercellular matrix. These cells do not form the mentioned types of intercellular junctions specific to epithelial cells and differ from them by another type of intermediate filaments of the cytoskeleton formed by the protein vimentin. In addition, mesenchymal cells contain another type of transmembrane protein on their surface - N-cadherin. Three different types of EMF The biological significance of EMF is varied, therefore it has been proposed to divide this phenomenon into three different types; the classification was adopted following discussions held within the framework of symposia devoted to EMF in Poland (2007) and the USA (2008) [14, 24]. The first type of EMT is characteristic of embryo implantation and development, formation of organs and tissues, and is manifested in the formation of cells with a common mesenchymal phenotype that do not cause fibrosis and do not spread over long distances by intravasation. Type 2 EMT is associated with the regeneration of mature tissues and organ fibrosis in chronic inflammation. Type 3 EMT is found in neoplastic epithelial cells in invasive carcinoma at the site of the primary tumor; in this case, epithelial cells acquire mesenchymal phenotypic properties and become capable of invasion, intravasation, and metastasis [25, 26]. The invasive aspect demonstrated by microscopic examination of endometriosis is due to changes in the epithelial phenotype under the influence of EMF [27, 28], in which epithelial cells lose intercellular connections, change the cytoskeleton and become mobile. The process of invasion is facilitated by metalloproteinases that affect collagen and have the ability to induce EMF [7, 29]. Thus, the significance of EMF in extragenital endometriosis remains controversial. Materials and methods The study was conducted from 2012 to 2017 in municipal clinics of Chisinau (Republic of Moldova) and Craiova (Romania), in the gynecological and surgical departments. The study included patients aged 19 to 56 years (the average age of patients was 39.7±9.9 years; Md – 38.0, IIQ 32.0–47.0 years) with extragenital endometriosis of various localizations. The patients lived in the Republic of Moldova (n=33; 76.7%; 95% confidence interval (CI) 62.7–87.4) and Romania (n=10; 23.3%; 95% CI 12.6–37.3). A minority of patients lived in rural areas (30.2%; 95% CI 18.1–44.9), while the majority were urban residents (69.8%; 95% CI 55.1–81.9). The most common localizations of extragenital endometriosis were the scar after cesarean section - CS (20.9%; 95% CI 10.9-34.7), appendix (14.0%; 95% CI 6.0-26.5), inguinal hernia (9.3%; 95% CI 3.2-20.6), cecum (7.0%; 95% CI 2.0-17.5), diaphragm (7.0%; 95% CI 2.0-17), jejunum - 3 (7.0%; 95% CI 2.0-17.5), umbilical region (7.0%; 95% CI 2.0-17.5), rectum (7.0%; 95% CI 2.0-17.5), peritoneum (4.7%; 95% CI 1.0–14.1). Other localizations (ascending colon, descending colon, sigmoid colon, transverse colon, ileum, anterior abdominal wall, and retroperitoneal space) were detected in 2.3% (95% CI 0.3–10.4); Fig. 2. EUROPEAN JOURNAL OF MODERNMEDICINE AND PRACTICE Vol. 4 No. 11 (Nov - 2024) ISSN: 2795-921X 523 To establish the diagnosis, paraffin blocks corresponding to the postoperative material were used. Using a microtome, 3–5 μm thick sections were obtained from the blocks, which were then stained in a standard manner (hematoxylin and eosin). The stained sections were examined by two pathologists to establish and confirm the diagnosis of endometriosis. To study the expression of EMT proteins, the immunohistochemical (IHC) method was used. IHC reactions were carried out by the standard method according to the manufacturer's recommendations using 4 μm thick sections on MACH 4 MICRO-POLYMER-HRP devices (Biocare Medical; M4U534). At stage I, the expression of the E-cadherin marker (clone NCH-38; Dako) was assessed, and at stage II, the punctate cytoplasmic expression of vimentin (clone PA1-16759; ThermoFisher) was assessed. Staining of cell parts in brown (when using DAB buffer) and red (when using Vulcan Fast Red buffer), respectively, was considered positive. To assess the expression results, a semi-quantitative method was used with an assessment in points on the Allred scale according to the formula: Total score (TS) = proportion index (PS) + intensity index (IS), TS = 0–8. The proportion was assessed as follows: 1) assessment of the proportion of cells with stained nuclei: 0 – absent; 1 point – >0 to 1/100; 2 points – >1/100–1/10; 3 points – from >1/10 to 1/3; 4 points – from 1/3 to 2/3; 5 points – from 2/3 to 1); 2) intensity score (IS): 0 – no expression; 1 point – weak staining; 2 points – moderate staining; 3 points – strong staining. IHC expression in the cytoplasm (C), cell membrane (Mc) and nucleus (N) was assessed depending on the intensity: C+/Mc+/N+ (weak expression); C++/Mc++/N++ (moderate expression); C++/Mc+++/ N+++ (intense expression). For statistical analysis, we used the χ2 concordance criterion, p values <0.05 were considered significant. Results Semi-quantitative analysis of those with a positive reaction to E-cadherin showed a predominance of the result in the two cases studied, while cases demonstrating negative expression of this marker were not taken into account. Since expression was present at both membranous and cytoplasmic levels, we studied each case of endometriosis separately and tried to correlate these values with some morphological parameters. According to the obtained results, we noticed a decrease in the membrane expression of E-cadherin in endometriosis after CS, while cytoplasmic expression remained pronounced. Thus, membrane expression was highest in intestinal forms of endometriosis (Fig. 3, a). Positive cytoplasmic expression of vimentin was present in all studied samples, but was especially pronounced in cases of endometriosis in inguinal hernia (Fig. 3, b). In this study, we did not find a significant difference in the expression of mesenchymal markers in patients with endometriosis. According to the analysis, a more pronounced expression of the E-cadherin marker was recorded in endometriosis of the gastrointestinal tract and a pronounced expression of the vimentin marker was recorded in endometriosis of the anterior abdominal wall after CS, as well as in inguinal hernia. Positive expression of the E-cadherin marker was detected at both the membrane and cytoplasmic levels in 85.6% of endometriosis cases. Statistically significant expression of E-cadherin and vimentin correlated as follows: E-cadherin expression was more pronounced, and vimentin expression was less pronounced in intestinal forms of endometriosis. In cases of endometriosis of scars after CS and inguinal hernia, the level of E-cadherin expression was less pronounced, while vimentin expression was predominant, demonstrating a high potential for invasiveness. EUROPEAN JOURNAL OF MODERNMEDICINE AND PRACTICE Vol. 4 No. 11 (Nov - 2024) ISSN: 2795-921X 524 Conclusion In the vast majority of endometriosis cases, especially in diffuse-infiltrative forms, which occur in particular after CS, the phenotype of lesions is characterized by low expression of E-cadherin, while vimentin expression is at a high level. Such a phenotype proves the involvement of EMT in the pathological process. For comparison, there are differences in the expressivity of markers between superficial and deep lesions, with deep lesions being much more invasive, which is explained by the expression of the corresponding IHC markers. The results of our study confirmed the role of EMP in the pathogenesis of extragenital endometriosis and prove its invasive potential in these localizations. Literature 1. Sh, O. F., Ikhtiyarova, G. A., Xudoyqulova, F. S., & Abdieva, N. U. (2023). 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