Endometriosis is a complex disease with systemic and topical immune system defects. Many studies have noted that changes in chemokines and immune receptors result in the development and progression of this disease in features such as increased proliferation, angiogenesis, invasion, and decreased apoptosis of ectopic cells [4]. Some essential chemokines are MCP-1, HGF, and IGF-1, which play a crucial role in the proliferation and invasion of ESCs.
In the current study, the serum and peritoneal levels of MCP-1 were higher in women with endometriosis than in control subjects, and the severity of the disease, unlike the menstrual cycle, was directly related to the concentration of this factor. These results are consistent with the findings of some previous studies [12,13,23,24], although there are studies that have shown no difference between the two groups of patients and controls [25-27]. On the other hand, Margari et al. reported remarkably lower concentrations of MCP-1 in the PF of patients with endometriosis [28]. The findings of this study showed that MCP-1 gene and protein expression in PFMCs increased more markedly in patients with endometriosis compared to controls. Also, MCP-1 gene expression was substantially higher in EESCs and PBMCs of women with endometriosis compared to EuESCs, CESCs, and PBMCs of control groups. These results are in line with other studies which showed that endometrial epithelial cells of women with endometriosis express high levels of MCP-1 [29,30], and EESCs expressed more MCP-1 than EuESCs and CESCs [31].
MCP-1 is one of the critical factors that have the potent ability not only in the infiltration of monocytes into the inflammatory site and their differentiation, but also in stimulating macrophages to secrete chemokines and cytokines. So, increased macrophage activation and recruitment into the peritoneal cavity of patients with endometriosis are considered to progress chronic inflammation and inflammatory cytokines production [32]. Increased MCP-1 secretion has been demonstrated by PF macrophages in patients with endometriosis in comparison with controls that affect monocytes and macrophages via autocrine manner [33]. In addition to macrophages, MCP-1 is secreted by endometrial, peritoneal mesothelium, mononuclear cells, endothelial cells, and fibroblasts. It can lead to the infiltration of monocytes, macrophages, eosinophils, NK, and T cells and may contribute to the shift to TH2 response [32,34]. MCP-1 directly contributes to the proliferation and survival of cancer cells [35], and the similarity of endometriosis with malignant diseases has been noted in features such as increased proliferation, angiogenesis, invasion, and decreased apoptosis of ectopic cells [36]. So, we suggest that MCP-1 may be involved in proliferation, survival, and invasion of EESCs, and it can likely increase the expression of CCR2 and MCP-1 and result in a defective cycle and more activation and recruitment of peritoneal macrophages in PF via autocrine and paracrine mechanisms.
HGF is a multi-functional and essential growth factor that, by binding to its receptor (c-Met), results in various effects, several of which are potentially related to growth and proliferation, invasion, and metastasis in cancer cells [15,37]. We observed higher concentrations of HGF in serum and PF of patients with endometriosis compared to controls, and its concentrations were higher at the late stages of endometriosis. No difference was noted regarding HGF serum levels with the menstrual cycle. Many studies have indicated increased HGF levels in PF and serum of endometriotic patients and evidenced that this elevation was significant in the late stages of the disease [12,15,17,38]. Besides, no significant difference was observed in HGF serum concentrations in endometriotic women in the follicular or luteal phases in those studies. In contrast, in one study, no significant difference was observed between the two groups of patients and controls [18]. In one study, it has been illustrated that the expression of c-Met is related to the different stages of endometriosis [39].
Our current study showed that PFMCs in women with endometriosis could produce HGF considerably more than controls, and EESCs expressed substantially high levels of HGF than EuESCs and CESCs.
So far, very few studies have been carried out regarding the production of HGF by PFMCs and PBMCs in patients with endometriosis, and some available studies have demonstrated the release of HGF by ESCs of endometriosis patients. Sugawara et al. showed increased HGF secretion by EuESCs in women with endometriosis compared with controls [40]. However, in this research, EESCs were not studied. Nasu et al. investigated HGF secretion in endometrial cell culture media and showed that HGF secretion was probably via the protein kinase C pathway [41]. According to other studies, it was revealed that HGF expression and c-Met in eutopic endometrium in patients with endometriosis increased compared to controls [42]. In line with our results, other studies have shown a significant increase in the gene expression of the HGF in EESCs compared to EuESCs and CESCs [22,43].
HGF plays a significant physiological role in the proliferation of a variety of cell types. Other studies have also proven that the proliferation of ESCs and macrophages in patients with endometriosis in response to HGF in the culture medium had more significant increase than the control group. The enhanced capacity of ESCs and macrophages proliferation may reflect more co-expression between HGF and its receptor in the cells of endometriosis patients [44]. The relation between high expression of fibroblast activation protein and HGF with angiogenesis and metastasis in gastric cancer has been reported [45]. HGF also plays a crucial role in the development and progression of many tumor cells. Noguchi and colleagues showed that binding HGF to its receptor increased the angiogenesis of tumor cells, which ultimately leads to increased cell proliferation, migration, and invasion of gastric cancer cells [46].
Regarding the malignancy-like nature of endometriosis and the high concentration of HGF in the PF of patients with endometriosis, it is quite reasonable to speculate that HGF can play a role in the pathogenesis and progression of the endometriosis. Consequently, increasing the HGF secretion by PFMCs as well as EESCs leads to increased inflammation in the region, proliferation, and invasion of ESCs.
IGF-1 is another important factor involved in the growth and proliferation of ESCs, which, in combination with increased estrogen receptor B and aromatase expression, lead to the progression of endometriosis [47]. The present study also showed increased IGF-1 concentration in serum and PF of patients with endometriosis, but no correlation was found regarding the stage of disease or the phase of the menstrual cycle with serum levels of IGF-1. Previous studies consistent with ours showed increased concentrations of IGF-1 in serum and PF in patients with endometriosis compared with controls [14,48]. Although, some studies reported no significant difference in IGF-1 levels in the serum of women with endometriosis compared to control subjects [20,49]. According to our literature review, no studies have ever been done on the production of IGF-1 by PFMCs and PBMCs in patients with endometriosis. One study revealed an increased IGF-1 gene and protein expression in EESCs of patients with endometriosis [22]. Rutanen et al. showed that ESCs produced IGF-1 and IGF-1 binding protein (IGFBP), and that was associated with levels of sex hormones and the menstrual cycle [50].
Milingos and colleagues examined IGF-I isoforms in ESCs and showed that the CESCs expressed lower IGF-1 compared to EuESCs and EESCs [51].
We demonstrated that the probable source of IGF-1 is PFMCs and EESCs in women with endometriosis resulted in increased levels of this factor in PF and the uncontrolled growth of EESCs.
It has been reported that the peritoneal IGF-1 level is about 60% of its serum level. Studies have shown that IGF-1 has an effect on ESCs in the culture, and it is an influential factor in the growth and proliferation of ectopic endometrium. Therefore, IGF-1 might be one of the most critical factors in women with endometriosis [52].
So increased IGF-1 level in the peritoneum of these patients appears to be involved in the pathogenesis of endometriosis, and in particular, in infertility.
Our study revealed that the primary sources of MCP-1, HGF, and IGF-1 are probably PFMCs and EESCs, which lead to a regional inflammatory environment and, by creating a defective cycle, contribute to the progression of the disease. As based on the recent findings it has been shown that, after retrograde menstruation, refluxed endometrial cells located outside the uterus stimulate the infiltration of immune cells into lesions, which secrete inflammatory mediators (like, pro-inflammatory cytokines, and chemokines) and these factors have been shown to activate the nuclear factor kappa B (NF-κB) pathway, and NF-κB further increases transcription of multiple genes encoding pro-inflammatory cytokines, chemokines and angiogenic factors like MCP-1, HGF, and IGF-1, finally resulting in an inflammatory peritoneal microenvironment. So this cocktail of secretions in PF leads to intensification of inflammation [53,54]. In this study, despite increased expression of MCP-1 and IGF-1 in PBMCs of patients with endometriosis compared to controls, we observed no significant difference in their protein levels, and this can be due to post-transcriptional changes in mRNA and RNA degradation for various reasons [44].
This contradiction at the level of transcription and protein production require future studies.
One limitation we faced within this study was the inability to evaluate MCP-1, HGF and IGF-1 proteins in EESCs, because of the small number of EESCs that was due to the specific nature of EESCs and their difficult growth condition, so it should be examined in other studies. Furthermore, in future studies, other factors associated with growth, invasion, and angiogenesis in endometriosis and also different materials with the suppressive effect on those, could be evaluated.
Finally, we conclude that PFMCs, as well as ESCs in women with endometriosis, can express a large amount of MCP-1, HGF, and IGF-1 factors, indicating the important role of these factors in the pathology of endometriosis and their possible involvement in the development of endometrial lesions in the ectopic site.