Histopathologic evaluation of the inflammatory factors and stromal cells in the endometriosis lesions: A case-control study

Abstract Background Endometriosis is a multifaceted gynecological disorder defined as a benign estrogen-dependent chronic inflammatory process in which endometrial glands and stroma-like tissues are located outside the uterine cavity. It affects around 2-10% of all women during their reproductive years. Objective This study aimed to evaluate the traffic of mesenchymal stem cells and inflammatory factors toward the lesions. Materials and Methods Ten samples of normal endometrium and eutopic endometrium were studied as a control group and 10 ectopic samples were considered as a case group. Hematoxylin and eosin staining was used to evaluate stromal cells and inflammatory cells. Immunohistochemical staining was performed to show the presence of proliferating cell nuclear antigen in the lesions. The cells were digested and cultured in the laboratory to study cell proliferation. The number of cells and vessels were counted with Image J software, and data analysis was performed with Prism software. Results Data analysis showed that the number of stromal cells and vessels in ectopic tissue were significantly higher than the control group (p < 0.001). Also, the number of inflammatory cells, including neutrophils, monocytes, lymphocytes, and macrophages, in the ectopic group was much higher than in the control group (p < 0.005). Conclusion By expanding the number of blood vessels, blood flow increases, and cell migration to tissues is facilitated. The accumulation of inflammatory cells, especially macrophages, stimulates the growth of stem cells and helps implant cells by creating an inflammatory process.


Introduction
Endometriosis is a benign disease of the female reproductive tract defined by the presence of endometrial glands and stroma-like structures outside the uterine cavity (1, 2). 2-10% within the general female population and up to 50% with infertility were found to have endometriosis (3). This disease may be a major issue in female health, which causes a decreased quality of life (4,5).
Endometriosis's etiology and pathophysiology remain unknown. The classic and broadly acknowledged speculation for endometriosis, first proposed by Sampson in 1927, was the retrograde hypothesis. It was stated that the menstrual tissue contains endometrial glands and stroma appearing from the fallopian tubes reaching the peritoneal cavity through retrograde menstruation. It is implanting in areas such as fallopian tubes and various parts of the pelvic cavity (2). The lesions found exterior to the pelvis, such as the peripheral nervous system superior organs in the abdomen which are surrounded by viscera as well as border organs between thorax and abdomen wall cannot be justified by retrograde bleeding (6). As a result, additional mechanisms must contribute to endometriosis development. Meyer's theory of cellular metaplasia assumes that visceral and parietal peritoneal cells undergo metaplastic transformation into endometriosis lesions. Induction theory states that an endogenous (unspecified) biochemical agent can stimulate undifferentiated peritoneal cells to become endometrial tissue (7).
Adult stem cells are undifferentiated cells while maintaining their regenerative capacity is defined by their ability to produce different types of tissues distinct from the originated tissue (8). The endometrium contains stem/progenitor cells that play an important role in endometrial physiology, regeneration, repair, and endometriosis prognosis (9,10). The recent focus of most studies was that endometriosis might originate from retrograde menstruation or the hematogenous/lymphatic spread of this stem cell population, and the source of endometriosis lesions may be stem cells that are circulating during menstruation (11,12). Proliferating cell nuclear antigen (PCNA) is a factor for cell propagation expressing in the cell nucleus during the DNA synthesis (13). PCNA is a DNA polymerase δ side protein that takes part in amplifying, replicating, and repairing DNA strands, chromatin structure maintenance, chromosome segregation, and cell cycle progression and can be considered as a particular marker for the S stage of the cell cycle. Its expression level can reflect the grade of cell proliferation, so it can be considered as a common factor for cell proliferation activity (14).
In the last few decades, many studies have shown the role of immune system imbalances and immune responses in the etiology and pathophysiology of endometriosis (15,16). In this regard, changes in the innate and acquired immune  Immunity against invading agents is the responsibility of the innate immune system and the adaptive immune system. Macrophages do not act as scavengers to remove the ectopic cells of endometriosis. But, activated peritoneal macrophages and monocytes in the circulation of affected women can suppress the disease by secreting growth factors and cytokines that stimulate the proliferation of ectopic endometrium and inhibit the scavenger function of macrophages. Speed up cytokines and growth factors accelerate the implantation and growth of the ectopic endometrium by facilitating the attachment of ectopic tissue to the surface of the peritoneum and stimulating proliferation and angiogenesis. Various studies show a decrease in the activity of cytotoxic T cells, NK cells, cytokine secretion by T helper cells, and the production of autoantibody compounds by B lymphocytes in women with endometriosis. Women with endometriosis after biopsy of ectopic endometriosis tissue and pathological examination of women whose endometriosis was not confirmed were excluded from the study. Ten samples of normal endometrium and eutopic endometrium were studied as a control group and 10 ectopic samples were studied as a case group.

Hematoxylin and eosin staining
Sample from each group were transferred to formalin to prepare paraffin blocks. After Finally, inflammatory cells, including neutrophils, macrophages, lymphocytes, and monocytes, were counted.

Immunohistochemical staining of tissue sections
To confirm the presence of the PCNA + Blocking performed with 10% rabbit serum and then primary and secondary PCNA antibody staining followed by hematoxylin counterstained.
Hundred cells were counted at x10 magnification, and the percentage of the number of nuclei with positive PCNA was calculated.

Cell culture
The endometrial cells were cultured after confirming the tissue type, so their images were evaluated under a microscope. The tissue was washed with PBS solution, and after homogenizing,

Ethical considerations
This study was conducted in the growth and  Data were analyzed using Prism software.

Student's t test, Mann-Whitney U test, and
Chi-square test were applied for data analysis.
A p-value ≤ 0.05 was considered statistically significant.

Results
The average weight was 68.

Stromal cells, inflammatory cells, and blood vessels
The histological evaluation of ectopic tissue demonstrated presence of stromal cells, including fibroblast and mesenchymal cells, of which the larger portion belongs to MSCs (Figure 2). The Image J analysis represents the higher incidence of these cells compared to eutopic and control endometrium tissues (p < 0.0005). The number of stromal cells in the eutopic group also showed a significant difference compared to both control groups in the proliferative and secretory phases (p < 0.005) ( Table I). As illustrated in figure  3, the ectopic group had more blood vessels than the other groups (p < 0.0001). In the H&E study, the total number of inflammatory cells, including neutrophils, lymphocytes, macrophages, and plasmocytes were considerably greater in ectopic endometrial tissue than in the control group during secretory and proliferative phases, and in the eutopic endometrial group (p < 0.005) ( Table I). Fibrous cells were seen in a higher magnification of ectopic tissue along with these inflammatory cells (Figure 4).

Cells culture
Following cell culture, the endometrial tissue cells grew as spherical cells. However, in the cells of endometriosis ectopic tissues, due to many stem cells next to the fibroblast cells of the endometrium, golden-colored colonies of stem cells were observed. These colonies were not seen as eutopic and controlled endometrial groups ( Figure 5).

PCNA
By studying immunohistochemistry on ectopic, eutopic, and normal endometrial tissues in the proliferative phase using PCNA antibody to determine the rate of cell proliferation in each group, the number of cells expressing this antibody was counted using Image J software and shown in table I. Data analysis with Prism software showed that antibody expression was higher in the ectopic group with a higher level than in the 2 eutopian and the control groups (p < 0.01) ( Figure 6).

Discussion
The present study represented that the number of stromal cells, especially mesenchymal stem cells in ectopic tissue, is more numerous than endometrial tissue in both control and eutopic tissue. This may indicate that these cells are composed of endometrial stem cell aggregation (17,18). This can be accompanied by an increase in the expression of genes involved in the function of stem cells (19). Still, some recent studies have shown the presence of bone marrow-derived mesenchymal stem cells in ectopic tissues, which may indicate that the ectopic endometrium can recruit stem cells from different parts of the body (20,21). On the other hand, the observation of many blood vessels in ectopic tissue in this study indicates the blood supply needed for migration, transplantation, replacement, and growth of stem cells. The expression of vascular endothelial growth factor, a key mediator of local angiogenesis that stimulates the proliferation of vascular endothelial cells in ectopic tissue, was considerably higher (22,23). This study showed that an increase in the expression of PCNA in the presence of a large number of stromal cells, especially mesenchymal stem cells in ectopic tissue, increases the rate and rate of cell growth. Ectopic cells culture in the in vitro environment and high growth rate, caused golden-colored colonies of mesenchymal stem cells.
Our study also showed that the number of inflammatory cells, including lymphocytes, monocytes, neutrophils, and especially macrophages, within ectopic tissue increased dramatically.
Increasing the number of inflammatory cells in ectopic tissue and peritoneal fluid can stimulate the growth of endometriosis. Laboratory studies have shown that peritoneal macrophages in women with endometriosis express cytokines IL-6, IL-1B, and tumor necrosis factor more than in women with benign abnormalities (24). Kempuraj and colleagues showed that the number of highly active mast cells increased in peritoneal endometrial lesions compared to the eutopic endometrium (25). Failure in immune response at the cellular level will result in impaired removal of endometrial cells from peritoneal cavity and ectopic cell. So far it has been confirmed that the activity status of the cytotoxic T cells, NK cells, cytokine secretion by helper T cells, and production of autoantibodies by B lymphocytes will be reduced in patients with endometriosis and it will increase the inflammation and illness, especially with severe pain during menstrual bleeding (26,27). The use of nonsteroidal anti-inflammatory medicines frequently alleviates pain and other symptoms associated with inflammatory indicators (28,29).
Some studies have shown that PCNA is associated with tumor differentiation, penetration, recurrence and lymph node, or organ metastasis (30,31). PCNA expression levels are known to be elevated in endometrial hyperplasia and endometriosis. Decreased size of endometriosis lesions is associated with decreased PCNA levels, and PCNA levels are directly related to the size of endometriosis lesions (32).
It may be possible to search for the missing link in the etiology of endometriosis in the basal immunity of endometriosis, which plays an important role in developing and growing the disease (33)(34)(35). Although ectopic endometrial cells are recognized by the cells of the peritoneal cavity immune system, they are, as a rule, not devastated and elude the immune system. Therefore, the weakening or modulation of the local immune system in the peritoneal cavity is one of the main factors in the growth of endometrial parts in the peritoneal cavity of women with endometriosis. The susceptibility of implantation and growth of endometrial cells may International Journal of Reproductive BioMedicine Cell trafficking in endometriosis lesions be due to the lack of regulation of the immune system clearance mechanism (36,37).
Macrophages constitute the majority of peritoneal leukocytes, and based on existing research, they constitute about 90% of peritoneal cells (38). Macrophages do not act as scavengers to remove the abnormal endometriosis cells, rather it activates the peritoneal macrophages and circulating monocytes in endometriosis women by secreting growth factors and cytokines that stimulate ectopic endometrial proliferation and inhibit our scavenger function (39,40).

Conclusion
Endometriosis is a common disease in women at fertility age as a social and welfare problem. Classical theories in the pathophysiology of this disease alone cannot justify all types and variations in disease severity. Our histopathological examination showed that stem cells in ectopic tissue and high expression of PCNA increases the growth rate of this tissue. On the other hand, increased angiogenesis in ectopic tissue, and increasing blood flow to these tissues, allow stem cells to migrate from other parts of the body to this site.
The presence of many inflammatory cells can also stimulate the angiogenic factors in these tissues. Identification of a large number of these inflammatory cells, especially macrophages in ectopic tissue, shows that these cells not only perform their main function as xenophagous and destroy foreign cells but also themselves as a stimulus for the growth of these cells. Increased inflammation can facilitate the implantation of migrating cells and retrograde blood cells, so it is necessary to characterize the type of these cells to design the therapeutic targets. Identifying and blocking these immune factors with a pharmaceutical approach can shed light on future potential treatments.