{"paper_id":"f1aa7ff5-6a20-4078-bd6c-2f6a7e079b07","body_text":"Endometriosis is a common female reproductive disorder, which is characterized by the\npresence of endometrial glands and stroma outside of the uterine cavity, primarily\nin the ovaries. This condition leads to chronic abdominal pain, dysmenorrhea,\ninfertility, and other symptoms that can persist over decades or even a lifetime.\nNearly 10% to 15% women of reproductive age and 25% to 50% of infertile patients\nsuffer from this condition. 1  Conservative surgery that retains the ovaries and uterus while removing\nendometrial tissue combined with drug treatment is regarded as the gold standard\ntherapy for women with endometriosis. However, the rate of recurrence of\nendometriosis in the subsequent 5 years can be as high as 50%. 2  Despite intensive research, the pathogenesis of endometriosis and its\nrecurrence is still unclear. Furthermore, endometriotic lesions show tumor-like\ncharacteristics, despite being characterized as benign. 3\nThere is overwhelming evidence suggesting that inflammation, angiogenesis, and\ndegradation of the extracellular matrix (ECM) play major roles in the pathogenesis\nand progression of endometriosis. 4  Collette et al. 5  found that endometrial tissue can attach itself to the host tissue and then\ninvade it by obtaining its own blood supply from the local vasculature. Endometrial\ncell adhesion and proliferation are likely to be modulated by interaction between\nintegrin receptors and ECM components. 6  The epithelial cadherin (E-cadherin)–β-catenin complex is important in\nepithelial cell–cell adhesion and maintenance of tissue architecture. 7  Additionally, many studies have shown that matrix metalloproteinases (MMPs)\nplay a pivotal role in promotion of adhesion, degradation of the ECM, and\npenetration of the basement membrane during ectopic implantation of endometrial cells. 8  Based on these previous reports, we speculate that the E-cadherin–β-catenin\ncomplex and ECM-degrading enzymes, such as MMP-9, along with its inhibitor and\ninducer, may be involved in development and recurrence of endometriosis. Therefore,\nin this study, we performed a comprehensive investigation of the molecules involved\nin adhesion, invasion, and degradation of the ECM in patients with endometriosis to\nstudy their roles in the pathogenesis of this disease and it’s mechanism of\nrecurrence.\n\nThe study protocols (IRB-20200282-R) and consent forms were approved by the\nEthics Committee of the Women’s Hospital, Zhejiang University School of\nMedicine. Written informed consent was obtained from each patient before surgery\nand tissue collection. We collected endometrial tissue samples from inpatients\nof the Women’s Hospital, Zhejiang University from January 1997 to June 2009.\nForty-nine women were enrolled in the study. All of them had undergone surgery\non two separate occasions and were diagnosed with stages III to IV endometriosis\nduring the first conservative laparotomy or laparoscopy surgery according to\nrevised American Fertility Society classification. 9  Women who had undergone a second surgery for recurrent ovarian\nendometriomas were classified into the recurrent group (RE). We then\nsubclassified specimens that were collected from the RE group into group a (REa,\nsample from the first operation) and group b (REb, sample from the second\noperation). Women who were diagnosed with nonendometriosis diseases, such as\nuterine fibroids and hydrosalpinx, or those who had undergone cesarean section\nduring the second operation, were enrolled as the nonrecurrent (control) group.\nNo participants in the control group had pelvic endometriosis lesions during the\nsecondary operation. No participant was using hormones or medications known to\naffect reproductive function and showed no evidence of infection or\ninflammation.\nOvarian endometrioma samples collected by surgical removal were quickly fixed in\n10% buffered formalin and embedded in paraffin for routine histological studies\nand immunostaining. Sections of ovarian endometriomas were prepared and stained\nwith hematoxylin and eosin, and sections from each ovarian endometrioma\nunderwent histological examination. The diagnosis of endometriosis was confirmed\nhistologically in the ovarian endometrioma specimens by experienced pathologists\ndepending on evidence showing the presence of endometrial glands and stroma with\nan inflammatory response and fibrosis ( Figure 1 ). Other sections of\nendometriomas were immunostained for E-cadherin, β-catenin, and urokinase\nplasminogen activator (uPA) in the REa, REb, and control groups. Among them, 20\npatients from the REa group and 10 patients from the control group were\nimmunostained for MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-2,\nand extracellular matrix metalloproteinase inducer (EMMPRIN).\nOvarian endometrioma sections stained with hematoxylin and eosin. ×200\nmagnification.\nWe performed immunohistochemical staining procedures for examining E-cadherin,\nβ-catenin, uPA, MMP-9, TIMP-2, and EMMPRIN concentrations. Blocks were cut into\n5-μm sections and collected on glass slides. Routine deparaffinization and\nrehydration procedures were performed. The primary antibodies used were mouse\nmonoclonal antibodies against E-cadherin (Abcam, Cambridge, UK; diluted 1:150),\nβ-catenin (Abcam; diluted 1:100), MMPs (Manxin, Fuzhou, China; diluted 1:50),\nEMMPRIN (Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1:300) and the\nrabbit monoclonal antibodies against uPA (Neomarkers, Fremont, CA, USA; diluted\n1:50), and TIMP-2 (ManXin; diluted 1:80).\nImmunohistochemical staining of E-cadherin, β-catenin, and uPA was evaluated by\nusing an evaluation nomogram, without prior knowledge of the clinicopathologic\nparameters. Under standard light microscopy, each slide was examined, and 100\nglandular epithelial cells per field were counted by five stochastic\nhigh-powered fields of vision. Immunohistochemical expression staging was based\non the product of intensity and proportion scores. An intensity score of 3 was\ndetermined as positive when the samples stained strongly with a brownish\nappearance. The intensity was scored as 2 if the staining demonstrated a tannish\nappearance, and the sample was determined to be weakly positive. The intensity\nwas scored as 1 if staining was the weakest with a yellowish appearance. The\nproportion scores were 0, 1, 2, 3, or 4 when the number of positive cells in\neach section was observed to be ≤5%, 6% to 25%, 26% to 50%, 51% to 75%, or\n>76% of the total amount of cells on the slide, respectively.\nQuantification of MMP-9, TIMP-2, and EMMPRIN immunostaining was performed by\ndigital image analysis with Image-Pro Plus 6.0 software (Media Cybernetics,\nBethesda, MD, USA). In brief, areas with positive immunostaining for MMP-9,\nTIMP-2, EMMPRIN were randomly selected in each section, and the integrated\noptical density was measured. The optical density values were calculated three\ntimes in three areas per section were averaged and were used to calculate the\ngroup means.\nData were statistically analyzed with SPSS for Windows, version 12.0 (SPSS Inc.,\nChicago, IL, USA). The chi-square test and Fisher’s exact test were used to\nevaluate count data. P < 0.05 was considered statistically significant.\n\nThirty-four women (mean [ ± standard deviation] age: 30.7 ± 5.3 years, mean\nparity: 0.21 children) were classified into the RE group. Fifteen women (mean\nage: 29.1 ± 6.2 years, mean parity: 0.53 children) were enrolled into the\ncontrol group. The two groups shared similar characteristics regarding most\nvariables, including age at the first surgery, menstrual cycle, revised\nclassification of the American Fertility Society stage, cyst diameter, and\nsurgical method (adnexectomy or cystectomy). However, the postoperative\npregnancy rate was significantly lower in the RE group (20.6%) than in the\ncontrol group (53.3%, P = 0.02). After the first surgery, the proportion of\npatients who underwent a second surgery within 30 months was 41.2% in the RE\ngroup and 80% in the control group (P = 0.01).\nImmunohistochemical analysis was performed on ectopic endometrial tissues. The\nmembranous and cytoplasmic fractions stained positive for E-cadherin, β-catenin,\nand uPA. However, these proteins were absent in the nucleus. E-cadherin was only\nexpressed in the glandular epithelium. β-catenin was primarily expressed in the\nglandular epithelium, but it was also partly expressed in stromal cells. uPA was\ntypically observed in glandular epithelial cells and stromal cells, but partial\nexpression was noted in vascular endothelial cells ( Figure 2 ). Immunohistochemical analysis\nshowed that MMP-9, TIMP-2, and EMMPRIN were mainly located in the cytoplasm of\nthe glandular epithelium. However, weak or sporadic staining was observed in the\ncytoplasm of stromal cells ( Figure 3 ).\nImmunostaining of E-cadherin, β-catenin, and uPA expression in\nendometriotic tissues. (a, d, g) Immunohistochemical staining of\nE-cadherin, β-catenin, and uPA in the REa group. (b, e, h)\nImmunohistochemical staining of E-cadherin, β-catenin, and uPA in the\nREb group. (c, f, i) Immunohistochemical staining of E-cadherin,\nβ-catenin, and uPA in the control group. ×200 magnification. E-cadherin,\nepithelial cadherin; uPA, urokinase plasminogen activator; REa,\nrecurrent a; REb, recurrent b.\nImmunostaining of MMP-9, TIMP-2, and EMMPRIN expression in endometriotic\ntissue. (a, c, e) Immunohistochemical staining of MMP-9, TIMP-2, and\nEMMPRIN expression in the REa group. (b, d, f) Immunohistochemical\nstaining of MMP-9, TIMP-2, and EMMPRIN in the control group. ×200\nmagnification. MMP-9, matrix metalloproteinase-9; TIMP-2, tissue\ninhibitor of matrix metalloproteinase-2; EMMPRIN, extracellular matrix\nmetalloproteinase inducer; REa, recurrent a.\nE-cadherin, β-catenin, and uPA were observed in ectopic endometrial samples that\nwere collected from the REa group (n = 34), REb group (n = 34), and control\ngroup (n = 15). E-cadherin expression was significantly lower in the RE group\ncompared with the control group (P < 0.05) ( Table 1 ). There was no significant\ndifference in β-catenin expression between the RE and control groups, as well as\nbetween the REa and REb groups ( Table 2 ). Expression of uPA was\nsignificantly higher in the RE group compared with the control group\n(P < 0.05). However, there was no significant difference in uPA expression\nbetween the REa and REb groups (P>0.05) ( Table 3 ).\nPositive rate of E-cadherin in the different groups.\n*Comparison between the recurrent group and the control group.\n**Comparison between the recurrent group a and the control group.\n***Comparison between the recurrent group b and the control\ngroup.\n****Comparison between the recurrent group a and the recurrent group\nb.\nE-cadherin, epithelial cadherin.\nPositive rate of β-catenin in the different groups.\n*Comparison between the recurrent group and the control group.\n**Comparison between the recurrent group a and the control group.\n**Comparison between the recurrent group b and the control group.\n****Comparison between the recurrent group a and the recurrent group\nb.\nPositive rate of uPA in the different groups.\n*Comparison between the recurrent group and the control group.\n**Comparison between the recurrent group a and the control group.\n***Comparison between the recurrent group b and the control\ngroup.\n****Comparison between the recurrent group a and the recurrent group\nb.\nuPA, urokinase plasminogen activator.\nThe immunohistochemical staining intensity of MMP-9 was significantly higher in\n20 patients from the REa group compared with that in 10 patients from the\ncontrol group with single primary ovarian endometriomas (P = 0.001). However, no\nsignificant difference was observed in TIMP-2 staining intensity between the two\ngroups. The ratio of MMP-9/TIMP-2 was significantly higher in the REa group than\nin the control group (P = 0.001). EMMPRIN expression was significantly higher in\nthe REa group than in the control group (P = 0.027) ( Table 4 ).\nImmunohistochemical staining intensities of MMP-9, TIMP-2, and EMMPRIN in\nthe different groups.\nValues are mean ± standard deviation. MMP-9, matrix\nmetalloproteinase-9; TIMP-2, tissue inhibitor of matrix\nmetalloproteinase-2; EMMPRIN, extracellular matrix metalloproteinase\ninducer.\n\nAlthough endometriosis is a benign gynecological disease, it exhibits malignant\nfeatures such as the ability of invasion, distant metastasis, and recurrence. High\nrecurrence rates among patients with endometriosis remain a significant challenge in\ntreatment of this condition. The risk of recurrence is accompanied by the need for\nreoperation, which becomes even more challenging, and may affect the ovarian\nreserve, fertility, and overall physical or mental health. A previous report showed\nthat subsequent surgery rates after the initial conservative surgical treatment for\nendometriosis were 21.6%, 46.7%, and 55.4% at 2, 5, and 7 years after the previous\nsurgery, respectively. 10  The mechanism of recurrence of endometriosis is unclear. Recently, the\nrelationship between dysfunctional cell adhesion, an abnormal extracellular matrix,\nand development of endometriosis was investigated. 11\nThis study focused on examining E-cadherin, β-catenin, and uPA in ectopic endometrium\nin recurrent and nonrecurrent ovarian endometriomas. E-cadherin is the best-studied\nmember of the cadherin family, which mediates cell-cell adhesion in a\ncalcium-dependent manner. The results regarding E-cadherin expression in patients\nwith endometriosis have been controversial. Some researchers found that E-cadherin\nexpression was decreased in endometriosis, while others observed no differences\nbetween cases and controls. 12\nLoss of E-cadherin expression in single epithelial cells within the endometrial\nglands may be essential to allow endometrial cells to detach from their primary\nsite, enabling them to adhere and invade at the implantation sites in the pelvis. 13  Our study showed significantly lower E-cadherin concentrations in the RE\ngroup, both in the REa and REb groups, compared with the control group. Therefore,\nwe speculate that loss of E-cadherin expression may be a crucial mechanism in the\npathogenesis of endometriosis and its recurrence.\nβ-catenin protein, linking E-cadherin with actin molecules, is a major component of\nadherent junctions that maintain cellular polarity and integrity, and it affects\ncellular migration and invasion. β-catenin is also an important intracellular\ntransducer in the Wnt pathway, which is associated with the majority of human\nmalignancies with aberrant activation. 14  Some studies have suggested that β-catenin concentrations are decreased in\nendometriotic lesions compared with those in the normal proliferative endometrium,\nand over-activation of the β-catenin pathway is associated with development of endometriosis. 15  We found that β-catenin staining was preserved in patients with nonrecurrent\nendometriosis and was typically present in the cytoplasm and cell membrane of\nglandular epithelial cells. We speculate that although endometriosis has malignant\nbiological behavior, such as invasion and recurrence, it is a benign disease and\nthere are no genetic mutations or unlimited proliferation of cells. The precise role\nof β-catenin in recurrent endometriosis still needs further detailed analysis.\nEctopic endometrial debris can adhere to and invade peritoneal tissue and surrounding structures. 16  This process may involve degradation of the ECM by uPA and MMPs. uPA is a\ncomponent of the plasminogen activator system and can convert plasminogen to\nplasmin. Plasmin is an active enzyme that plays a role in the degradation of a\nvariety of ECM proteins and activation of MMPs and various growth factors. 17  Our study showed that uPA was localized in glandular epithelial cells and\nstromal cells in the ectopic endometrium, and was also detected in vascular\nendothelial cells. These results are consistent with previous studies, which showed\nthat uPA had a broad range of function, such as fibrinolysis, tissue remodeling,\ninvasion, and promotion of angiogenesis in endometriosis. 18  Higher uPA concentrations in the endometrium might result in endometrial\nfragments with high degradation potential of the ECM following implantation at\nectopic sites. 19  Our study showed high uPA concentrations in ectopic endometrial tissues\ncollected from the REa and REb groups, which suggested that uPA might contribute to\nrecurrence of endometriosis.\nProteolysis of MMPs can generate space for cells to migrate and regulate the tissue\narchitecture by exerting effects on the ECM and the intercellular junctions, along\nwith the activation, deactivation, or modification of the activity of signaling\nmolecules directly and indirectly. Increased concentrations of MMPs have been\ndetected in a wide range of cancers and highly correlated with tumor invasion and metastasis. 20  MMP activity is thought to be particularly essential in the early phases of\ndevelopment of endometriosis. In murine and chicken chorioallantoic membrane models,\nNap et al. 21  showed prevention of early endometriotic lesion formation when MMP activity\nwas blocked. MMP-9 has the largest molecular weight among the members of the MMP\nfamily. EMMPRIN, also known as CD147, stimulates production of MMPs that digest the\nECM to facilitate cell migration. 22  Studies have shown that human uterine epithelial cells secrete intact EMMPRIN\nto stimulate MMPs, 23  including MMP-1, -2, -3, -9, and -14. Our study showed that MMP-9 and EMMPRIN\nwere primarily located in the cytoplasm of glandular epithelial cells, while weak or\nsporadic staining was also observed in the cytoplasm of stromal cells. We found\nhigher MMP-9 and EMMPRIN concentrations in the REa group compared with the control\ngroup. Blocking expression of the EMMPRIN gene can reduce aggressiveness and\nsensitivity of tumors to chemotherapy, which might be useful for preventing\nrecurrence of ovarian cysts.\nTIMPs are local endogenous inhibitors that bind to MMPs with 1:1 stoichiometry.\nPrevious studies have indicated that TIMPs inhibit invasiveness of tumors but on the\nother hand over-concentration of TIMPs is related to a high invasiveness. 24  Any changes in the equilibrium between MMP activity and TIMPs could be\npotentially harmful, promoting development of endometriosis. In this study, we did\nnot observe any significant difference in TIMP-2 expression between the REa and the\ncontrol groups. This finding indicated that TIMP-2 acts as the primary inhibitor of\nMMP-2, while also leading to indirect inhibition of MMP-9. Analysis of the\nassociation between MMP-9 and TIMP-2 expression has shown interesting results as\nfollows. Endometrioid tissue and ovarian endometriomas show high levels of MMPs\nalong with an increased ratio of MMP/TIMP. 8  In this study, we found that MMP-9 levels and the MMP-9/TIMP-2 ratio were\nsignificantly higher in the REa group than in the control group, which might be\nassociated with recurrence of endometriosis.\n\nOur study shows that decreased E-cadherin concentrations, increased uPA, MMP-9, and\nEMMPRIN concentrations, and an imbalanced MMP-9/TIMP-2 ratio may play a pivotal role\nin recurrence of endometriosis. These results suggest that abnormal expression and\nregulation of cell adhesion molecules and ECM metalloproteinases may contribute to\ndevelopment of recurrent ovarian endometriosis. Intervention of these pathways may\nenable development of novel therapeutic approaches for preventing recurrence of\nendometriosis to a certain extent. We hope that the conclusions of this study will\nbe helpful in larger prospective studies. However, there are some limitations\nassociated with this study. This was a retrospective study with a small sample size.\nFurthermore, only immunohistochemical analysis was performed for evaluation. For a\nquantitative research method, western blotting should be used in future studies. The\nnext stage of research should be  in vitro  cell culture to\ninvestigate the presence of upstream- and downstream-associated molecules and other\nmechanisms to further study the pathogenesis of endometriosis.","source_license":"CC0","license_restricted":false}