Expression Levels of MCP-1, HGF, and IGF-1 in Endometriotic Patients Compared with Non-endometriotic Controls

Expression Levels of MCP-1, HGF, and IGF-1 in Endometriotic Patients Compared with Non-endometriotic Controls | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression Levels of MCP-1, HGF, and IGF-1 in Endometriotic Patients Compared with Non-endometriotic Controls Sahel Heidari, Sepideh Khodaverdi, Roya Kolahdouz-Mohammadi, Nader Tajik, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-605993/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2021 Read the published version in BMC Women's Health → Version 1 posted You are reading this latest preprint version Abstract To study the concentrations of monocyte chemoattractant protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) in peritoneal fluid (PF) and serum, and to evaluate their expressions by PF and peripheral blood mononuclear cells (PFMCs and PBMCs, respectively), and ectopic and eutopic endometrial stromal cells of patients with endometriosis (EESCs and EuESCs, respectively) compared with controls. The concentrations of mentioned cytokines in serum and PF, as well as their expression in PBMCs, PFMCs, EuESCs and EESCs from endometriosis patients and controls were assessed. The levels of MCP-1, HGF, and IGF-1 in serum and PF in women with endometriosis were significantly higher than the controls (P < 0.05-P < 0.001). Gene expression of MCP-1 and IGF-1 in the PFMCs, PBMCs and EESCs also showed an increased level compared to controls (P < 0.05-P < 0.0001). The protein expressions of MCP-1 and IGF-1 by PFMCs were statistically higher in endometriotic women (P < 0.05 and P < 0.01, respectively). The gene and protein expression of HGF in PFMCs and its gene expression by EESCs were significantly higher in endometriotic women compared to controls (P < 0.05-P < 0.01). The higher concentrations of mentioned cytokines in serum and PF and their higher expressions by PFMCs and EESCs in endometriosis patients may contribute to the development of endometriosis. Scientific Communication Technical Communication Endometriosis MCP-1 HGF IGF-1 PFMCs PBMCs ESCs Ectopic Figures Figure 1 Figure 2 Figure 3 1 Introduction The presence of endometrial glands and stroma outside its normal site, the uterine cavity, is defined as endometriosis. Endometriosis is a common benign inflammatory disease that causes a variety of symptoms such as chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility [1]. Prevalence of endometriosis appears to have a range between 10-15% in general population [2]. The most accepted theory for the etiology of endometriosis is Sampson's theory, which suggests endometriosis develops as a result of retrograde menstruation through the fallopian tubes [3]. However, retrograde menstruation occurs physiologically in almost 90% of healthy women, but less than one-fourth of them develop endometriosis. Studies have shown that immunological changes play a significant role in the pathogenesis of endometriosis, leading to incomplete elimination of endometrial cells and the increased ability of endometrial lesions to be created and implant in the peritoneal cavity [4]. However, the exact mechanism of endometriosis is unknown. Several changes in the number and function of various immunological components result in the increase of the volume of the peritoneal fluid (PF) in endometriotic patients. Evidence to date indicates mononuclear cells, especially macrophages, which constitute about 85% of the cells in PF, are more likely to cause inflammation and develop the disease rather than control it [5]. In addition to mononuclear cells, endometriosis may cause notable changes in the expression of different genes and proteins by eutopic endometrial stromal cells (EuESCs), and ectopic endometrial stromal cells (EESCs) [6]. Mononuclear cells, as well as EuESCs and EESCs, release cytokines and growth factors that can affect themselves and other cells, such as macrophages. These factors can promote proliferation, angiogenesis, and invasion of endometrial cells, the underlying fundamental mechanisms of the pathogenesis of endometriosis [7]. One of these factors is monocyte chemoattractant protein-1 (MCP-1). This chemokine activates and recruits macrophages and other mononuclear cells to secrete growth factors and cytokines. It also gives rise to the proliferation and maintenance of endometrial cells in ectopic sites, and so, it may be involved in the pathogenesis of endometriosis [8]. Studies in women with endometriosis showed that hepatocyte growth factor (HGF) could also have an effect on monocytes and macrophages and enhance inflammation. In addition to its growth-regulating properties, HGF has a diverse impact on epithelial and endothelial cells, such as proliferation, migration, extracellular matrix production, and tubulogenesis [9,10]. Another mitogenic factor that is secreted by macrophages and other mononuclear cells is insulin-like growth factor-1 (IGF-1). Based on recent studies, EESCs can express the IGF-1 receptor immunohistochemically [11]. HGF and IGF-1 have several physiological and pathological effects that could contribute to the survival, proliferation, and invasion of endometrial stromal cells (ESCs) associated with endometriosis. Increased concentrations of MCP-1, HGF, and IGF-1 have been reported in the PF and serum of endometriotic patients in comparison with controls in some studies [12-17]. In contrast, other studies failed to show significant differences in the concentrations of these factors between women with and without endometriosis [18-20]. The source of production of these factors is one of the controversial subjects in endometriosis, whether those originate from endometriotic lesions or are secreted by inflammatory mononuclear cells, is unknown. These unclear data on MCP-1, HGF, and IGF-1 expression, hinder the understanding of the physiologic role of signaling, in women with endometriosis and no comprehensive study has examined all of the involved cells in endometriosis concurrently. In this study, we compared the concentrations of MCP-1, HGF, and IGF-1 in serum and PF of patients with and without endometriosis. Furthermore, we evaluated the expression of MCP-1, HGF, and IGF-1 by peritoneal fluid mononuclear cells (PFMCs), peripheral blood mononuclear cells (PBMCs), and ESCs in women with endometriosis compared to controls. 2 Materials And Methods 2.1 Participants In the first step, 140 reproductive-aged women (24-40 years) took part in this study : 70 women with endometriosis (any stages of I-IV) and 70 patients with other benign gynecological disorders and without any evidence of endometriotic lesions in laparoscopy as a control. The diagnosis of endometriosis was made by laparoscopy and pathology reports, and the stage of disease was determined according to the revised American Fertility Society system [21]. Next, blood and PF samples were taken from the participants and their serum and PF stored at -70°C until protein expression. The demographic information of participants whose serum and PF samples were collected is displayed in Table 1. In the second step, 30 women with the stage III & IV of endometriosis and 15 patients with benign gynecological disorders and no evidence of endometriotic lesions in laparoscopy were selected for collection of PF, peripheral blood, and ectopic and eutopic endometrial tissues. Since some participants were virgins, the eutopic endometrial tissues were not collected from them. Demographic information of participants whose PFMC, PBMC, and ESCs were collected is displayed in Table 2 and Table 3, respectively. All subjects had a regular menstrual cycle, and patients with a history of malignancy, any acute or chronic diseases (especially autoimmune diseases), and using immunosuppressive drugs, hormones, or GnRH agonists for at least three months before sampling were excluded. We missed some samples due to the gross bloody PF, culture contamination, not obtained the desired cells, or inconsistent pathology reports. The study protocol was approved by the Ethics Committee of Medical Research of Iran University of Medical Sciences (Code: IR.IUMS.REC 1394.26098) and all methods were performed in accordance with the relevant guidelines and regulations. All subjects had written informed consent for participation in the study. The study was conducted regarding the privacy rights of all participants. 2.2 Sample collection Under sterile conditions, peritoneal ectopic endometrial patches were obtained through laparoscopic surgery, and eutopic endometrium samples were collected by uterine biopsy curettage. Endometrial tissues were placed in Dulbecco's modified Eagle's medium‐F12 (DMEM-F12) (Gibco, UK) culture medium containing 1% penicillin-streptomycin antibiotics (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Blood and PF samples were collected in EDTA-coated falcons. All samples were immediately transferred to a laboratory for analysis in the cold chain. To confirm endometriosis, parts of ectopic endometrial tissues were sent for pathologic evaluation. 2.3 Mononuclear cell culture PFMCs and PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA). About 1 × 10 6 cells/mL PFMCs or PBMCs were cultured in Roswell Park Memorial Institute medium (RPMI-1640) (Gibco, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco, UK) and 1% penicillin-streptomycin antibiotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). 2.4 ESC culture Endometrial tissues obtained from participants in both groups (endometriotic and non-endometriotic patients) were cut up into smaller pieces in dimension. Then tissue digestion was performed using collagenase-A (2 mg/mL) and DNase (300 µg/mL) (Roche, USA) for 120 minutes at 37°C in 5% CO2 atmosphere with intermittent vortexing every 15 minutes. In an attempt to remove clots and undigested tissues, the suspension was filtered through 100 µm mesh (BD Biosciences, San Jose, CA, USA). Then, cells were cultured in T25 culture flasks containing DMEM-F12 (Gibco, UK) supplemented with 10% FBS (Gibco, UK) plus 1% penicillin-streptomycin antibiotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 6 hours. Next, in order to remove non-adherent cells, they were washed twice with warm medium, and adherent stromal cells were allowed to propagate. The cells in passage three were used for flow cytometry, immunofluorescent, RNA extraction, and enzyme-linked immunosorbent assay (ELISA). Also, 3 × 10 5 cells/mL were cultured in a 24-well plate according to our previous study [22]. Flow cytometry and immunofluorescent were used for investigation of the purity of the ESCs, and these cells were identified as vimentin + , nestin + cytokeratin - , CD10 + , CD44 + , CD73 + , CD105 + , CD34 - , and CD45 - (data not shown) [22]. 2.5 Total RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time PCR reaction The RNA extraction was performed by QIAzol solution (Qiagen, Hilden, Germany) following the manufacturer's instructions. The Picodrop apparatus (Picopetol, Cambridge, UK) was applied to measure the concentration of the total RNA at 260/280 nm. RNA integrity was confirmed by electrophoresis on 1% agarose gel. For cDNA synthesis, 1 μg RNA was used, and the cDNA was synthesized according to the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) protocol. Real-time PCR was performed using the Syber premix Extaq (Biofact, Daejeon, Korea) and Rotor-Gene Q (QIAGEN, USA). Each reaction was made up of 10 μL of Syber premix (Biofact, Daejeon, Korea), 1 μL of primer pairs, 1 μL of synthesized cDNA, and 8 µL of DNase-free water with a final volume of 20 μL. The concentration of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer was equal to the amount of the same primer for each reaction. Each PCR reaction was as follows: 95°C for 15 minutes (holding step), 40 cycles of 95°C for 20 seconds and 60°C for 40 seconds (extension step), and finally, the melting step from 60°C to 99°C. To verify the real-time PCR results, melting curve analysis, and electrophoresis on 2% agarose gel were used. To increase the accuracy of the real-time PCR, all analyses were performed twice, and positive and negative controls were tested every time. The GAPDH gene was used as an internal control. The sequence of primers for GAPDH , MCP-1 , HGF , and IGF-1 genes are shown in Table 4. 2.6 ELISA procedure The method used to measure the concentrations of MCP-1, HGF, and IGF-1 in PFMCs, PBMCs, and ESCs supernatant, as well as in serum and PF was sandwich ELISA (R&D Systems, Minneapolis, MN). The detection limit for MCP-1, HGF and IGF-1 were 15.6 pg/mL, 125 pg/mL, and 31.2 pg/mL, respectively. The absorbance was measured at 570 nm using a microplate reader (Bio‐Rad, Hercules, CA, USA). 2.7 Statistical analysis All statistical analyses were carried out using GraphPad Prism software 8. The results are displayed as the mean ± standard error of the mean (SEM). The normality of distributions was evaluated by the Kolmogorov–Smirnov test. The independent t-test and Mann-Whitney U test were employed to compare two independent groups based on the normality distribution assumption and chi-square test was used to assess categorical variables. For the comparison of three groups, Kruskal-Wallis test with Dunn post hoc analysis was used. After normalization to the GAPDH control, the quantitative analysis of mRNA expression was performed using the 2 −ΔΔCt method. P -value < 0.05 was considered statistically significant. 3 Results In this study, serum and PF concentrations of MCP-1, HGF, and IGF-1 were measured in 70 endometriotic and 70 non-endometriotic participants. Demographic data of the participants whose serum and PF were collected are presented in Table 1. Based on our findings, no significant differences were observed between groups in regards to age, body mass index (BMI), marital status, infertility, abortion, and menstrual phase. Besides, the majority of patients who underwent surgery had stages III & IV of disease with deep infiltrative endometriosis, cul-de-sac obliteration, ovarian endometriosis, and dense adhesions. Superficial ovarian and peritoneal endometriosis or firm adhesions were observed in approximately 31 percent of patients who were in stages I & II. Demographic information of participants whose PFMC, PBMC, and ESCs were collected is displayed in Table 2 and Table 3, respectively. Based on our findings, no significant differences were observed between groups with regards to age, BMI, marital status, infertility, and abortion. All participants were at proliferative phase of menstrual cycle and endometriotic patients were at stage III & IV of endometriosis and had tubo-ovarian and peritoneal endometriosis (including deep infiltrating endometriosis (DIE)). The basal gene and protein expression of mentioned factors were measured in PFMCs (n=10), PBMCs (n=10), EESCs (n=8), and EuESCs (n=10) from endometriotic patients and PFMCs (n=7), PBMCs (n=10), and CESCs (n=10) from non-endometriotic women. The relative expressions of MCP-1 , HGF , and IGF-1 were measured by quantitative real-time PCR, and the protein levels of these factors were evaluated by ELISA in PFMCs, PBMCs, and ESCs samples. 3.1 Serum and PF concentrations of MCP-1 and its gene and protein expression by PFMCs, PBMCs, and ESCs The levels of MCP-1 in serum and PF were significantly higher in women with endometriosis than in controls ( P < 0.001 and P < 0.05, respectively) (Figures 1Aa and 1Ab). Also, the levels of MCP-1 in the serum were more remarkable in women with late-stages (III & IV) endometriosis than those with the early-stages (I & II) ( P < 0.05) (Figure 1Ac). Although, according to the menstrual phase, no significant difference in serum MCP-1 concentrations was noted in the endometriosis and control groups (Figure 1Ad). We further examined the gene and protein expressions of MCP-1 by PFMCs, PBMCs, and ESCs. There was a significantly higher MCP-1 expression at the level of mRNA and protein by PFMCs in women with endometriosis compared with non-endometriotic women ( P < 0.05) (Figures 1Ba and 1Bb). The increment in the mRNA expression of MCP-1 in PBMCs in the patient group was significant, as well ( P < 0.05) (Figure 1Bc), whilst, MCP-1 protein expression in PBMCs had no significant difference between women with and without endometriosis (Figure 1Bd). EESCs showed increased gene expression of MCP-1 compared to EuESCs and control endometrial stromal cells (CESCs) ( P < 0.0001) (Figure 1Be), whereas MCP-1 protein expression was not different between EuESCs and CESCs (Figure 1Bf). 3.2 Serum and PF concentrations of HGF and its gene and protein expression by PFMCs, PBMCs, and ESCs The results showed that the level of HGF was significantly higher in serum and PF in women with endometriosis than in controls ( P < 0.001 and P < 0.05, respectively) (Figures 2Aa and 2Ab). In addition, there was a significantly higher difference in the serum concentration of HGF in women at the stages III-IV of endometriosis compared to the patients with the stages I-II of the disease ( P < 0.01) (Figure 2Ac). No difference between the follicular or luteal phases in terms of the serum levels of HGF was detected in patient and control groups (Figure 2Ad). The expression of the HGF gene and protein by PFMCs was significantly higher in the patient's group compared to the controls ( P < 0.05 and P < 0.01, respectively) (Figures 2Ba and 2Bb). However, there was no notable difference in their expression by PBMCs in both groups (Figures 2Bc and 2Bd). HGF gene expression was significantly higher in EESCs in comparison with the EuESCs and CESCs ( P < 0.01) (Figure 2Be). However, we did not find any difference in the HGF protein expression between EuESCs and CESCs (Figure 2Bf). 3.3 Serum and PF concentrations of IGF-1 and its gene and protein expression by PFMCs, PBMCs, and ESCs Results obtained showed that the IGF-1 level in serum and PF in women with endometriosis was higher than women without endometriosis ( P < 0.05) (Figures 3Aa and 3Ab). An increase in the serum levels of IGF-1 in women with stages III-IV endometriosis was observed compared to women with stages I-II endometriosis, but it was not statistically significant (Figure 3Ac). Also, no apparent difference was detected among the IGF-1 serum concentrations in the different phases of the menstrual cycle of women with endometriosis as well as controls (Figure 3Ad). It is demonstrated that a substantial amount of IGF-1 was also produced by PFMCs at the level of mRNA and protein in women with endometriosis ( P < 0.05 and P < 0.01, respectively) (Figures 3Ba and 3Bb). IGF-1 gene expression by PBMCs in endometriosis patients was significantly higher than in women without endometriosis ( P < 0.05) (Figure 3Bc). However, there was no difference in the secretion of IGF-1 by PBMCs in the two groups (Figure 3Bd). The IGF-1 gene expression by EuESCs and CESCs were significantly lower than EESCs ( P < 0.01 and P < 0.0001, respectively) (Figure 3Be). But, the levels of IGF-1 protein expression did not significantly differ between the EuESCs and CESCs (Figure 3Bf). 4 Discussion 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. Declarations Data availability All data generated or analyzed during this study are included in this published article. Acknowledgements We would like to appreciate all patients who participated in this research and also Iran University of Medical Sciences for their financial support (Grant No. 94-02-30-26098). Author contributions S.H. and A.A.D. made substantial contributions to the conception or design of the work. S.K.H. made substantial contributions to acquisition of data. S.H. carried out the experiments. S.H., R.K.M., and A.A.D. contributed to the data analysis and interpretation of the results. S.H. and R.K.M. wrote the first draft of the manuscript. 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Serum concentrations of insulin-like growth factor-1, soluble tumor necrosis factor receptor-1 and angiogenin in endometriosis patients. Am. J. Reprod. Immunol . 51 , 166-173 (2004). Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil. Steril . 67 , 817-821 (1997). Arablou, T. et al . Resveratrol reduces the expression of insulin-like growth factor-1 and hepatocyte growth factor in stromal cells of women with endometriosis compared with nonendometriotic women. Phytother. Res . 33 , 1044-1054 (2019). Gmyrek, G. B. et al . Evaluation of monocyte chemotactic protein-1 levels in peripheral blood of infertile women with endometriosis. Eur. J. Obstet. Gynecol. Reprod. Biol. 122 , 199-205 (2005). Malutan, A. M. et al . Endometriosis-associated changes in serum levels of interferons and chemokines. Turk. J. Med. Sci . 47 , 115-122 (2017). Drosdzol-Cop, A., Skrzypulec-Plinta, V. & Stojko, R. Serum and peritoneal fluid immunological markers in adolescent girls with chronic pelvic pain. Obstet. Gynecol. Surv. 67 , 374-381 (2012). Kim, J. Y. et al . The G(-2518)A polymorphism of monocyte chemotactic protein-1 (MCP-1) and its serum and peritoneal fluid levels in Korean women with endometriosis. Eur. J. Obstet. Gynecol. Reprod. Biol. 139 , 106-110 (2008). Vodolazkaia, A. et al . Evaluation of a panel of 28 biomarkers for the non-invasive diagnosis of endometriosis. Hum. Reprod . 27 , 2698-2711 (2012). Margari, K. M. et al . Peritoneal fluid concentrations of beta-chemokines in endometriosis. Eur. J. Obstet. Gynecol. Reprod. Biol . 169 , 103-107 (2013). Akoum, A., Lemay, A., Brunet, C. & Hebert, J. Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis. Le groupe d'investigation en gynecologie. Fertil. Steril . 63 , 322-328 (1995). Ulukus, M. et al . Expression of interleukin-8 and monocyte chemotactic protein 1 in women with endometriosis. Fertil. Steril . 91 , 687-693 (2009). Kolahdouz-Mohammadi, R. et al . Resveratrol treatment reduces expression of MCP-1, IL-6, IL-8 and RANTES in endometriotic stromal cells. J. cell. Mol. Med. 25 , 1116-1127 (2021). Deshmane, S. L., Kremlev, S., Amini, S. & Sawaya, B. E. Monocyte chemoattractant protein-1 (MCP-1): an overview. J. Interferon. Cytokine. Res. 29 , 313-326 (2009). Akoum, A., Kong, J., Metz, C. & Beaumont, M. C. Spontaneous and stimulated secretion of monocyte chemotactic protein-1 and macrophage migration inhibitory factor by peritoneal macrophages in women with and without endometriosis. Fertil. Steril. 77 , 989-994 (2002). Gu, L. et al . Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature . 404 , 407-411 (2000). Salcedo, R. et al . Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood. 96 , 34-40 (2000). Varma, R., Rollason, T., Gupta, J. K. & Maher, E. R. Endometriosis and the neoplastic process. Reproduction. 127 , 293-304 (2004). Gao, F., Deng, G., Liu, W., Zhou, K. & Li, M. Resveratrol suppresses human hepatocellular carcinoma via targeting HGF-c-Met signaling pathway. Oncol. Rep . 37 , 1203-1211 (2017). Osuga, Y. et al . Hepatocyte growth factor concentrations are elevated in peritoneal fluid of women with endometriosis. Hum. Reprod . 14 , 1611-1613 (1999). KhoshdelRad, N., Salehi, Z., Mashayekhi, F., Abbasi, O. & Mirzajani, E. Soluble c-Met expression in the peritoneal fluid and serum of patients with different stages of endometriosis. Arch. Gynecol. Obstet . 289 , 1107-1112 (2014). Sugawara, J., Fukaya, T., Murakami, T., Yoshida, H. & Yajima, A. Increased secretion of hepatocyte growth factor by eutopic endometrial stromal cells in women with endometriosis. Fertil. Steril . 68 , 468-472 (1997). Nasu, K. et al . Expression of hepatocyte growth factor in cultured human endometrial stromal cells is induced through a protein kinase C-dependent pathway. Biol. Reprod . 60 , 1183-1187 (1999). Khan, K. N. et al . Immunoexpression of hepatocyte growth factor and c-Met receptor in the eutopic endometrium predicts the activity of ectopic endometrium. Fertil. Steril . 79 , 173-181 (2003). Delbandi, A. A. et al . Evaluation of apoptosis and angiogenesis in ectopic and eutopic stromal cells of patients with endometriosis compared to non-endometriotic controls. BMC. Womens. Health. 20 , 3 (2020). Khan, K. N. et al . Regulation of hepatocyte growth factor by basal and stimulated macrophages in women with endometriosis. Hum. Reprod. 20 , 49-60 (2005). Gao, L. M. et al . Roles of Fibroblast Activation Protein and Hepatocyte Growth Factor Expressions in Angiogenesis and Metastasis of Gastric Cancer. Pathol. Oncol. Res. 25 , 369-376 (2019). Noguchi, E., Saito, N., Kobayashi, M. & Kameoka, S. Clinical significance of hepatocyte growth factor/c-Met expression in the assessment of gastric cancer progression. Mol. Med. Rep. 11 , 3423-3431 (2015). Zhou, Y. et al . IGF-I stimulates ERβ and aromatase expression via IGF1R/PI3K/AKT-mediated transcriptional activation in endometriosis. J. Mol. Med (Berl). 94 , 887-897 (2016). Gurgan, T., Bukulmez, O., Yarali, H., Tanir, M. & Akyildiz, S. Serum and peritoneal fluid levels of IGF I and II and insulinlike growth binding protein-3 in endometriosis. J. Reprod. Med . 44 , 450-454 (1999). Matalliotakis, I. M. et al . Serum concentrations of growth factors in women with and without endometriosis: the action of anti-endometriosis medicines. Int. Immunopharmacol . 3 , 81-89 (2003). Rutanen, E. M. Insulin-like growth factors in endometrial function. Gynecol. Endocrinol . 12 , 399-406 (1998). Milingos, D. S. et al . Insulinlike growth factor-1Ec (MGF) expression in eutopic and ectopic endometrium: characterization of the MGF E-peptide actions in vitro. Mol. Med . 17 , 21-28 (2011). Giudice, L. C., Dsupin, B. A., Gargosky, S. E., Rosenfeld, R. G. & Irwin, J. C. The insulin-like growth factor system in human peritoneal fluid: its effects on endometrial stromal cells and its potential relevance to endometriosis. J. Clin. Endocrinol. Metab. 79 , 1284-1293 (1994). Kaponis, A. et al . The role of NF-kappaB in endometriosis. Front. Biosci (Schol Ed). 4 , 1213-1234 (2012). Chapron, C., Marcellin, L., Borghese, B. & Santulli, P. Rethinking mechanisms, diagnosis and management of endometriosis. Nat. Rev. Endocrinol. 15 , 666-682 (2019). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-605993","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":32552172,"identity":"30324ea5-2eb6-43bc-b3db-f075875aae90","order_by":0,"name":"Sahel Heidari","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sahel","middleName":"","lastName":"Heidari","suffix":""},{"id":32552173,"identity":"b6f5f7e9-0002-48e2-9e67-862f733cbe3f","order_by":1,"name":"Sepideh Khodaverdi","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sepideh","middleName":"","lastName":"Khodaverdi","suffix":""},{"id":32552174,"identity":"509c8f42-716d-4c75-9a1e-739434a6e5ae","order_by":2,"name":"Roya Kolahdouz-Mohammadi","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Roya","middleName":"","lastName":"Kolahdouz-Mohammadi","suffix":""},{"id":32552175,"identity":"be78ee53-4ad6-4da5-8edf-42f4ec079732","order_by":3,"name":"Nader Tajik","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nader","middleName":"","lastName":"Tajik","suffix":""},{"id":32552176,"identity":"ba14f4f1-db95-41b4-b42b-5a321b325263","order_by":4,"name":"Ali-Akbar Delbandi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIie2PIQvCUBDHbwye5enqG+p3mBgN+yobwlsxmMSo5Ux+HTEYJoNZhllYcK/MosGmQfAmKJZNbYb3g/c4jvtx9wfQaP6R0ET6TQ7AjMx7dr3S+UIxXorp/KTQY0x8dVdjM8Xsuuy1oBbLscKFC7UoA7UqV+xkPevMk4ADl/HOx9SfcOmAl5crDo2JOkaUJcBCoRQDyhJWKHuF9q1QrAMOSXHBOn5QdgY2H1uEjIEUYyI+bLETf9ZtY8CZyPvC26Z0Z+6EVUpjE8XqhD3XsmTnfBmlVPSVulQoL9h78Y2g0Wg0mgrumWZSRJWRSdgAAAAASUVORK5CYII=","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ali-Akbar","middleName":"","lastName":"Delbandi","suffix":""}],"badges":[],"createdAt":"2021-06-09 15:14:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-605993/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-605993/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12905-021-01560-6","type":"published","date":"2021-12-01T06:49:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":10406098,"identity":"e30d5554-c6ae-4123-906a-f067e0c6cd29","added_by":"auto","created_at":"2021-06-15 18:20:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4830537,"visible":true,"origin":"","legend":"Serum and peritoneal fluid concentrations of MCP-1 and its gene and protein expression by PFMCs, PBMCs and ESCs. Serum and peritoneal fluid concentrations of MCP-1 were measured in 70 endometriotic and 70 non-endometriotic participants. The basal gene and protein expression of MCP-1 measured in PFMCs (n=10), PBMCs (n=10), EESCs (n=8), and EuESCs (n=10) from patients with endometriosis and PFMCs (n=7), PBMCs (n=10), and CESCs (n=10) from non-endometriotic women. (Aa) serum concentration of MCP-1, (Ab) peritoneal concentration of MCP-1, (Ac) serum concentration of MCP-1 in different stages of endometriosis, (Ad) serum concentration of MCP-1 in different menstrual cycles, (Ba) MCP-1 gene expression by PFMCs, (Bb) MCP-1 protein expression by PFMCs, (Bc) MCP-1 gene expression by PBMCs, (Bd) MCP-1 protein expression by PBMCs, (Be) MCP-1 gene expression by ESCs, (Bf) MCP-1 protein expression by ESCs. *P \u003c 0.05, ***P \u003c 0.001, ****P \u003c 0.0001. A and B parts analyzed by parametric and non-parametric tests, respectively.\n†P-E: Proliferative phase of endometriosis patients, S-E: Secretory phase of endometriosis patients, P-C: Proliferative phase of control group, S-C: Secretory phase of control group.\n","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-605993/v1/1f6a683db6d67ff2c98e8129.jpg"},{"id":10406508,"identity":"a7702c99-379b-4b31-9ebe-32ef135d3fdc","added_by":"auto","created_at":"2021-06-15 18:23:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5196671,"visible":true,"origin":"","legend":"Serum and peritoneal fluid concentrations of HGF and its gene and protein expression by PFMCs, PBMCs and ESCs. Serum and peritoneal fluid concentrations of HGF were measured in 70 endometriotic and 70 non-endometriotic participants. The basal gene and protein expression of HGF was measured in PFMCs (n=10), PBMCs (n=10), EESCs (n=8), and EuESCs (n=10) from patients with endometriosis and PFMCs (n=7), PBMCs (n=10), and CESCs (n=10) from non-endometriotic women. (Aa) serum concentration of HGF, (Ab) peritoneal concentration of HGF, (Ac) serum concentration of HGF in different stages of endometriosis, (Ad) serum concentration of HGF in different menstrual cycles, (Ba) HGF gene expression by PFMCs, (Bb) HGF protein expression by PFMCs, (Bc) HGF gene expression by PBMCs, (Bd) HGF protein expression by PBMCs, (Be) HGF gene expression by ESCs, (Bf) HGF protein expression by ESCs. *P \u003c 0.05, **P \u003c 0.01, ***P \u003c 0.001. A and B parts analyzed by parametric and non-parametric tests, respectively.\n†P-E: Proliferative phase of endometriosis patients, S-E: Secretory phase of endometriosis patients, P-C: Proliferative phase of control group, S-C: Secretory phase of control group.\n","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-605993/v1/0d4d2356b92ab1aefe94de97.jpg"},{"id":10406100,"identity":"42d41879-4c9a-4c9f-93b1-56a206ac19a8","added_by":"auto","created_at":"2021-06-15 18:20:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5208320,"visible":true,"origin":"","legend":"Serum and peritoneal fluid concentrations of IGF-1 and its gene and protein expression by PFMCs, PBMCs and ESCs. Serum and peritoneal fluid concentrations of IGF-1 were measured in 70 endometriotic and 70 non-endometriotic participants. The basal gene and protein expression of IGF-1 was measured in PFMCs (n=10), PBMCs (n=10), EESCs (n=8), and EuESCs (n=10) from patients with endometriosis and PFMCs (n=7), PBMCs (n=10), and CESCs (n=10) from non-endometriotic women. (Aa) serum concentration of IGF-1, (Ab) peritoneal concentration of IGF-1, (Ac) serum concentration of IGF-1 in different stages of endometriosis, (Ad) serum concentration of IGF-1 in different menstrual cycles, (Ba) IGF-1 gene expression by PFMCs, (Bb) IGF-1 protein expression by PFMCs, (Bc) IGF-1 gene expression by PBMCs, (Bd) IGF-1 protein expression by PBMCs, (Be) IGF-1 gene expression by ESCs, (Bf) IGF-1 protein expression by ESCs. *P \u003c 0.05, **P \u003c 0.01, ****P \u003c 0.0001. A and B parts analyzed by parametric and non-parametric tests, respectively.\n†P-E: Proliferative phase of endometriosis patients, S-E: Secretory phase of endometriosis patients, P-C: Proliferative phase of control group, S-C: Secretory phase of control group.\n","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-605993/v1/d4cfa32895d6297e459e794c.jpg"},{"id":16601850,"identity":"90a2c192-c639-4886-81ce-21f4aa37277a","added_by":"auto","created_at":"2021-12-20 06:49:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":810609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-605993/v1/37244496-2a60-4aea-91e9-0d874a66d23e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eExpression Levels of MCP-1, HGF, and IGF-1 in Endometriotic Patients Compared with Non-endometriotic Controls\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe presence of endometrial glands and stroma outside its normal site, the uterine cavity, is defined as endometriosis. Endometriosis is a common benign inflammatory disease that causes a variety of symptoms such as chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility [1]. Prevalence of endometriosis appears to have a range between 10-15%\u0026nbsp;in general population [2].\u003c/p\u003e\n\u003cp\u003eThe most accepted theory for the etiology of endometriosis is Sampson\u0026apos;s theory, which suggests endometriosis develops as a result of retrograde menstruation through the fallopian tubes [3]. However, retrograde menstruation occurs physiologically in almost 90% of healthy women, but less than one-fourth of them develop endometriosis. Studies have shown that immunological changes play a significant role in the pathogenesis of endometriosis, leading to incomplete elimination of endometrial cells and the increased ability of endometrial lesions to be created and implant in the peritoneal cavity [4]. However, the exact mechanism of endometriosis is unknown. Several changes in the number and function of various immunological components result in the increase of the volume of the peritoneal fluid (PF) in endometriotic patients. Evidence to date indicates mononuclear cells, especially macrophages, which constitute about 85% of the cells in PF, are more likely to cause inflammation and develop the disease rather than control it [5]. In addition to mononuclear cells, endometriosis may cause notable changes in the expression of different genes and proteins by eutopic endometrial stromal cells (EuESCs), and ectopic endometrial stromal cells (EESCs) [6]. Mononuclear cells, as well as EuESCs and EESCs, release cytokines and growth factors that can affect themselves and other cells, such as macrophages. These factors can promote proliferation, angiogenesis, and invasion of endometrial cells, the underlying fundamental mechanisms of the pathogenesis of endometriosis [7]. One of these factors is monocyte chemoattractant protein-1 (MCP-1). This chemokine activates and recruits macrophages and other mononuclear cells to secrete growth factors and cytokines. It also gives rise to the proliferation and maintenance of endometrial cells in ectopic sites, and so, it may be involved in the pathogenesis of endometriosis [8].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Studies in women with endometriosis showed that hepatocyte growth factor (HGF) could also have an effect on monocytes and macrophages and enhance inflammation. In addition to its growth-regulating properties, HGF has a diverse impact on epithelial and endothelial cells, such as proliferation, migration, extracellular matrix production, and tubulogenesis [9,10]. Another mitogenic factor that is secreted by macrophages and other mononuclear cells is insulin-like growth factor-1 (IGF-1). Based on recent studies, EESCs can express the IGF-1 receptor immunohistochemically [11].\u003c/p\u003e\n\u003cp\u003eHGF and IGF-1 have several physiological and pathological effects that could contribute to the survival, proliferation, and invasion of endometrial stromal cells (ESCs) associated with endometriosis.\u003c/p\u003e\n\u003cp\u003eIncreased concentrations of MCP-1, HGF, and IGF-1 have been reported in the PF and serum of endometriotic patients in comparison with controls in some studies [12-17].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eIn contrast, other studies failed to show significant differences in the concentrations of these factors between women with and without endometriosis [18-20].\u003c/p\u003e\n\u003cp\u003eThe source of production of these factors is one of the controversial subjects in endometriosis, whether those originate from endometriotic lesions or are secreted by inflammatory mononuclear cells, is unknown. These unclear data on MCP-1, HGF, and IGF-1 expression, hinder the understanding of the physiologic role of signaling, in women with endometriosis and no comprehensive study has examined all of the involved cells in endometriosis concurrently. In this study, we compared the concentrations of MCP-1, HGF, and IGF-1 in serum and PF of patients with and without endometriosis. Furthermore, we evaluated the expression of MCP-1, HGF, and IGF-1 by peritoneal fluid mononuclear cells (PFMCs), peripheral blood mononuclear cells (PBMCs), and ESCs in women with endometriosis compared to controls.\u0026nbsp;\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003ch2\u003e2.1 Participants\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;In the first step, 140 reproductive-aged women (24-40 years) took part in this study\u003cspan dir=\"RTL\"\u003e:\u003c/span\u003e 70 women with endometriosis (any stages of I-IV) and 70 patients with other benign gynecological disorders and without any evidence of endometriotic lesions in laparoscopy as a control. The diagnosis of endometriosis was made by laparoscopy and pathology reports, and the stage of disease was determined according to the revised American Fertility Society system [21].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, blood and PF samples were taken from the participants and their serum and PF stored at -70\u0026deg;C until protein expression. The demographic information of participants whose serum and PF samples were collected is displayed in Table 1. In the second step, 30 women with the stage III \u0026amp; IV of endometriosis and 15 patients with benign gynecological disorders and no evidence of endometriotic lesions in laparoscopy were selected for collection of PF, peripheral blood, and ectopic and eutopic endometrial tissues. Since some participants were virgins, the eutopic endometrial tissues were not collected from them.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDemographic information of participants whose PFMC, PBMC, and ESCs were collected is displayed in Table 2 and Table 3, respectively. All subjects had a regular menstrual cycle, and patients with a history of malignancy, any acute or chronic diseases (especially autoimmune diseases), and using immunosuppressive drugs, hormones, or GnRH agonists for at least three months before sampling were excluded. We missed some samples due to the gross bloody PF, culture contamination, not obtained the desired cells, or inconsistent pathology reports. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Ethics Committee of Medical Research of Iran University of Medical Sciences (Code: IR.IUMS.REC 1394.26098) and all methods were performed in accordance with the relevant guidelines and regulations. All subjects had written informed consent for participation in the study. The study was conducted regarding the privacy rights of all participants.\u003c/p\u003e\n\u003ch2\u003e2.2 Sample collection\u003c/h2\u003e\n\u003cp\u003eUnder sterile conditions, peritoneal ectopic endometrial patches were obtained through laparoscopic surgery, and eutopic endometrium samples were collected by uterine biopsy curettage. Endometrial tissues were placed in Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium‐F12 (DMEM-F12) (Gibco, UK) culture medium containing 1% penicillin-streptomycin antibiotics (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). \u0026nbsp;Blood and PF samples were collected in EDTA-coated falcons. All samples were immediately transferred to a laboratory for analysis in the cold chain. To confirm endometriosis, parts of ectopic endometrial tissues were sent for pathologic evaluation.\u003c/p\u003e\n\u003ch2\u003e2.3 Mononuclear cell culture\u003c/h2\u003e\n\u003cp\u003ePFMCs and PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA). About 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL PFMCs or PBMCs were cultured in Roswell Park Memorial Institute medium (RPMI-1640) (Gibco, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco, UK) and 1% penicillin-streptomycin antibiotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.4 ESC culture\u003c/h2\u003e\n\u003cp\u003eEndometrial tissues obtained from participants in both groups (endometriotic and non-endometriotic patients) were cut up into smaller pieces in dimension. Then tissue digestion was performed using collagenase-A (2 mg/mL) and DNase (300 \u0026micro;g/mL) (Roche, USA) for 120 minutes at 37\u0026deg;C in 5% CO2 atmosphere with intermittent vortexing every 15 minutes. In an attempt to remove clots and undigested tissues, the suspension was filtered through 100 \u0026micro;m mesh (BD Biosciences, San Jose, CA, USA). Then, cells were cultured in T25 culture flasks containing DMEM-F12\u0026nbsp;(Gibco, UK) supplemented with 10% FBS (Gibco, UK) plus 1% penicillin-streptomycin antibiotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 6 hours. Next, in order to remove non-adherent cells, they were washed twice with warm medium, and adherent stromal cells were allowed to propagate. The cells in passage three were used for flow cytometry, immunofluorescent, RNA extraction, and enzyme-linked immunosorbent assay (ELISA). Also, 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL were cultured in a 24-well plate according to our previous study [22].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Flow cytometry and immunofluorescent were used for investigation of the purity of the ESCs, and these cells were identified as vimentin\u003csup\u003e+\u003c/sup\u003e, nestin\u003csup\u003e+\u003c/sup\u003e cytokeratin\u003csup\u003e-\u003c/sup\u003e, CD10\u003csup\u003e+\u003c/sup\u003e, CD44\u003csup\u003e+\u003c/sup\u003e, CD73\u003csup\u003e+\u003c/sup\u003e, CD105\u003csup\u003e+\u003c/sup\u003e, CD34\u003csup\u003e-\u003c/sup\u003e, and CD45\u003csup\u003e-\u0026nbsp;\u003c/sup\u003e(data not shown) [22].\u003c/p\u003e\n\u003ch2\u003e2.5 Total RNA extraction, complementary DNA (cDNA) synthesis, and quantitative real-time PCR reaction\u003c/h2\u003e\n\u003cp\u003eThe RNA extraction was performed by QIAzol solution (Qiagen, Hilden, Germany) following the manufacturer\u0026apos;s instructions. The Picodrop apparatus (Picopetol, Cambridge, UK) was applied to measure the concentration of the total RNA at 260/280 nm. RNA integrity was confirmed by electrophoresis on 1% agarose gel. For cDNA synthesis, 1 \u0026mu;g RNA was used, and the cDNA was synthesized according to the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) protocol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eReal-time PCR was performed using the Syber premix Extaq (Biofact, Daejeon, Korea) and Rotor-Gene Q (QIAGEN, USA). Each reaction was made up of 10 \u0026mu;L of Syber premix (Biofact, Daejeon, Korea), 1 \u0026mu;L of primer pairs, 1 \u0026mu;L of synthesized cDNA, and 8 \u0026micro;L of DNase-free water with a final volume of 20\u0026nbsp;\u0026mu;L. The concentration of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer was equal to the amount of the same primer for each reaction. Each PCR reaction was as follows: 95\u0026deg;C for 15 minutes (holding step), 40 cycles of 95\u0026deg;C for 20 seconds and 60\u0026deg;C for 40 seconds (extension step), and finally, the melting step from 60\u0026deg;C to 99\u0026deg;C. To verify the real-time PCR results, melting curve analysis, and electrophoresis on 2% agarose gel were used. To increase the accuracy of the real-time PCR, all analyses were performed twice, and positive and negative controls were tested every time.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe \u003cem\u003eGAPDH\u003c/em\u003e gene was used as an internal control. The sequence of primers for \u003cem\u003eGAPDH\u003c/em\u003e, \u003cem\u003eMCP-1\u003c/em\u003e, \u003cem\u003eHGF\u003c/em\u003e, and \u003cem\u003eIGF-1\u003c/em\u003e genes are shown in Table 4.\u003c/p\u003e\n\u003ch2\u003e2.6 ELISA procedure\u003c/h2\u003e\n\u003cp\u003eThe method used to measure the concentrations of MCP-1, HGF, and IGF-1 in PFMCs, PBMCs, and ESCs supernatant, as well as in serum and PF was sandwich ELISA (R\u0026amp;D Systems, Minneapolis, MN). The detection limit for MCP-1, HGF and IGF-1 were 15.6 pg/mL, 125 pg/mL, and 31.2 pg/mL, respectively. The absorbance was measured at 570 nm using a microplate reader (Bio‐Rad, Hercules, CA, USA).\u003c/p\u003e\n\u003ch2\u003e2.7 Statistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAll statistical analyses were carried out using GraphPad Prism software 8. The results are displayed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The normality of distributions was evaluated by the Kolmogorov\u0026ndash;Smirnov test. The independent t-test and Mann-Whitney U test were employed to compare two independent groups based on the normality distribution assumption and chi-square test was used to assess categorical variables. For the comparison of three groups, Kruskal-Wallis test with Dunn post hoc analysis was used.\u003c/p\u003e\n\u003cp\u003eAfter normalization to the GAPDH control, the quantitative analysis of mRNA expression was performed using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method. \u0026nbsp;\u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003eIn this study, serum and PF concentrations of MCP-1, HGF, and IGF-1 were measured in 70 endometriotic and 70 non-endometriotic participants. Demographic data of the participants whose serum and PF were collected are presented in Table 1. Based on our findings, no significant differences were observed between groups in regards to age, body mass index (BMI), marital status, infertility, abortion, and menstrual phase. \u0026nbsp;Besides, the majority of patients who underwent surgery had stages III \u0026amp; IV of disease with deep infiltrative endometriosis, cul-de-sac obliteration, ovarian endometriosis, and dense adhesions. Superficial ovarian and peritoneal endometriosis or firm adhesions were observed in approximately 31 percent of patients who were in stages I \u0026amp; II. Demographic information of participants whose PFMC, PBMC, and ESCs were collected is displayed in Table 2 and Table 3, respectively. Based on our findings, no significant differences were observed between groups with regards to age, BMI, marital status, infertility, and abortion. All participants were at proliferative phase of menstrual cycle and endometriotic patients were at stage III \u0026amp; IV of endometriosis and had tubo-ovarian and peritoneal endometriosis (including deep infiltrating endometriosis (DIE)). The basal gene and protein expression of mentioned factors were measured in PFMCs (n=10), PBMCs (n=10), EESCs (n=8), and EuESCs (n=10) from endometriotic patients and PFMCs (n=7), PBMCs (n=10), and CESCs (n=10) from non-endometriotic women. The relative expressions of \u003cem\u003eMCP-1\u003c/em\u003e, \u003cem\u003eHGF\u003c/em\u003e, and \u003cem\u003eIGF-1\u003c/em\u003e were measured by quantitative real-time PCR, and the protein levels of these factors were evaluated by ELISA in PFMCs, PBMCs, and ESCs samples.\u003c/p\u003e\n\u003ch2\u003e3.1 Serum and PF concentrations of MCP-1 and its gene and protein expression by PFMCs, PBMCs, and ESCs\u003c/h2\u003e\n\u003cp\u003eThe levels of MCP-1 in serum and PF were significantly higher in women with endometriosis than in controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, respectively) (Figures 1Aa and 1Ab). Also, the levels of MCP-1 in the serum were more remarkable in women with late-stages (III \u0026amp; IV) endometriosis than those with the early-stages (I \u0026amp; II) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure 1Ac). Although, according to the menstrual phase, no significant difference in serum MCP-1 concentrations was noted in the endometriosis and control groups (Figure 1Ad). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe further examined the gene and protein expressions of MCP-1 by PFMCs, PBMCs, and ESCs. There was a significantly higher MCP-1 expression at the level of mRNA and protein by PFMCs in women with endometriosis compared with non-endometriotic women (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figures 1Ba and 1Bb). The increment in the mRNA expression of \u003cem\u003eMCP-1\u003c/em\u003e in PBMCs in the patient group was significant, as well (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure 1Bc), whilst, MCP-1 protein expression in PBMCs had no significant difference between women with and without endometriosis (Figure 1Bd). EESCs showed increased gene expression of \u003cem\u003eMCP-1\u003c/em\u003e compared to EuESCs and control endometrial stromal cells (CESCs) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001) (Figure 1Be), whereas MCP-1 protein expression was not different between EuESCs and CESCs (Figure 1Bf).\u003c/p\u003e\n\u003ch2\u003e3.2 Serum and PF concentrations of HGF and its gene and protein expression by PFMCs, PBMCs, and ESCs\u003c/h2\u003e\n\u003cp\u003eThe results showed that the level of HGF was significantly higher in serum and PF in women with endometriosis than in controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, respectively) (Figures 2Aa and 2Ab). In addition, there was a significantly higher difference in the serum concentration of HGF in women at the stages III-IV of endometriosis compared to the patients with the stages I-II of the disease (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Figure 2Ac). No difference between the follicular or luteal phases in terms of the serum levels of HGF was detected\u0026nbsp;in patient and control groups\u0026nbsp;(Figure 2Ad).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The expression of the HGF gene and protein by PFMCs was significantly higher in the patient\u0026apos;s group compared to the controls (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, respectively) (Figures 2Ba and 2Bb). However, there was no notable difference in their expression by PBMCs in both groups (Figures 2Bc and 2Bd). \u003cem\u003eHGF\u003c/em\u003e gene expression was significantly higher in EESCs in comparison with the EuESCs and CESCs (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Figure 2Be). However, we did not find any difference in the HGF protein expression between EuESCs and CESCs (Figure 2Bf).\u003c/p\u003e\n\u003ch2\u003e3.3 Serum and PF concentrations of IGF-1 and its gene and protein expression by PFMCs, PBMCs, and ESCs\u003c/h2\u003e\n\u003cp\u003eResults obtained showed that the IGF-1 level in serum and PF in women with endometriosis was higher than women without endometriosis (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figures 3Aa\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eand 3Ab). An increase in the serum levels of IGF-1 in women with stages III-IV endometriosis was observed compared to women with stages I-II endometriosis, but it was not statistically significant (Figure 3Ac). Also, no apparent difference was detected among the IGF-1 serum concentrations in the different phases of the menstrual cycle of women with endometriosis as well as controls (Figure 3Ad).\u003c/p\u003e\n\u003cp\u003eIt is demonstrated that a substantial amount of IGF-1 was also produced by PFMCs at the level of mRNA and protein in women with endometriosis (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, respectively) (Figures 3Ba and 3Bb). \u003cem\u003eIGF-1\u003c/em\u003e gene expression by PBMCs in endometriosis patients was significantly higher than in women without endometriosis (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (Figure 3Bc). However, there was no difference in the secretion of IGF-1 by PBMCs in the two groups (Figure 3Bd). The \u003cem\u003eIGF-1\u003c/em\u003e gene expression by EuESCs and CESCs were significantly lower than EESCs (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, respectively) (Figure 3Be). But, the levels of IGF-1 protein expression did not significantly differ between the EuESCs and CESCs (Figure 3Bf).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eEndometriosis 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn 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\u0026nbsp;[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].\u0026nbsp;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, \u003cem\u003eMCP-1\u003c/em\u003e 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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMCP-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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHGF 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].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eWe 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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur 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.\u003c/p\u003e\n\u003cp\u003eSo 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 \u003cem\u003eHGF\u003c/em\u003e in EESCs compared to EuESCs and CESCs [22,43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHGF 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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding 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.\u003c/p\u003e\n\u003cp\u003eIGF-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\u0026nbsp;[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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMilingos and colleagues examined \u003cem\u003eIGF-I\u003c/em\u003e isoforms in ESCs and showed that the CESCs expressed lower \u003cem\u003eIGF-1\u003c/em\u003e compared to EuESCs and EESCs [51].\u003c/p\u003e\n\u003cp\u003eWe 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.\u003c/p\u003e\n\u003cp\u003eIt 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].\u003c/p\u003e\n\u003cp\u003eSo increased IGF-1 level in the peritoneum of these patients appears to be involved in the pathogenesis of endometriosis, and in particular, in infertility.\u003c/p\u003e\n\u003cp\u003eOur 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-\u0026kappa;B) pathway, and NF-\u0026kappa;B 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 \u003cem\u003eMCP-1\u003c/em\u003e and \u003cem\u003eIGF-1\u003c/em\u003e 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].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis contradiction at the level of transcription and protein production require future studies. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eFinally, 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cspan dir=\"LTR\"\u003eData availability\u003c/span\u003e\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe would like to appreciate all patients who participated in this research and also Iran University of Medical Sciences for their financial support (Grant No. 94-02-30-26098).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eS.H. and A.A.D. made substantial contributions to the conception or design of the work. S.K.H. made substantial contributions to acquisition of data. S.H. carried out the experiments. S.H., R.K.M., and A.A.D. contributed to the data analysis and interpretation of the results. S.H. and R.K.M. wrote the first draft of the manuscript. S.K.H., R.K.M., N.T., and A.A.D. critically reviewed the manuscript. A.A.D. supervised the project. All authors read and approved the submitted version of the manuscript. All authors have agreed both to be personally accountable for the author\u0026apos;s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eAll authors report no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBulun, S. E. 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Rethinking mechanisms, diagnosis and management of endometriosis. \u003cem\u003eNat. Rev. Endocrinol.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 666-682 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, MCP-1, HGF, IGF-1, PFMCs, PBMCs, ESCs, Ectopic","lastPublishedDoi":"10.21203/rs.3.rs-605993/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-605993/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"To study the concentrations of monocyte chemoattractant protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) in peritoneal fluid (PF) and serum, and to evaluate their expressions by PF and peripheral blood mononuclear cells (PFMCs and PBMCs, respectively), and ectopic and eutopic endometrial stromal cells of patients with endometriosis (EESCs and EuESCs, respectively) compared with controls. The concentrations of mentioned cytokines in serum and PF, as well as their expression in PBMCs, PFMCs, EuESCs and EESCs from endometriosis patients and controls were assessed. The levels of MCP-1, HGF, and IGF-1 in serum and PF in women with endometriosis were significantly higher than the controls (P \u003c 0.05-P \u003c 0.001). Gene expression of MCP-1 and IGF-1 in the PFMCs, PBMCs and EESCs also showed an increased level compared to controls (P \u003c 0.05-P \u003c 0.0001). The protein expressions of MCP-1 and IGF-1 by PFMCs were statistically higher in endometriotic women (P \u003c 0.05 and P \u003c 0.01, respectively). The gene and protein expression of HGF in PFMCs and its gene expression by EESCs were significantly higher in endometriotic women compared to controls (P \u003c 0.05-P \u003c 0.01). The higher concentrations of mentioned cytokines in serum and PF and their higher expressions by PFMCs and EESCs in endometriosis patients may contribute to the development of endometriosis.","manuscriptTitle":"Expression Levels of MCP-1, HGF, and IGF-1 in Endometriotic Patients Compared with Non-endometriotic Controls","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-06-15 18:20:07","doi":"10.21203/rs.3.rs-605993/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9de651b2-9f89-4c1a-8a7a-f6090e1167e9","owner":[],"postedDate":"June 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":5038996,"name":"Scientific Communication"},{"id":5038997,"name":"Technical Communication"}],"tags":[],"updatedAt":"2021-12-20T06:49:14+00:00","versionOfRecord":{"articleIdentity":"rs-605993","link":"https://doi.org/10.1186/s12905-021-01560-6","journal":{"identity":"bmc-womens-health","isVorOnly":false,"title":"BMC Women's Health"},"publishedOn":"2021-12-01 06:49:14","publishedOnDateReadable":"December 1st, 2021"},"versionCreatedAt":"2021-06-15 18:20:07","video":"","vorDoi":"10.1186/s12905-021-01560-6","vorDoiUrl":"https://doi.org/10.1186/s12905-021-01560-6","workflowStages":[]},"version":"v1","identity":"rs-605993","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-605993","identity":"rs-605993","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}