Differential expression and localization of de-novo synthesized endometriotic haptoglobin in endometrium and endometriotic lesions

Endometriosis protein-I (ENDO-I) mRNA expression and protein localization were evaluated using in-situ hybridization and immunohistochemistry in endometriotic lesions and eutopic endometrium from women with endometriosis, and in eutopic endometrium from women without endometriosis (controls). When present, ENDO-I mRNA and protein were observed in the functionalis zone of endometrial stroma and the stroma of endometriotic lesions. Expression and localization differences were scored and statistically analysed. During the secretory stage, ENDO-I mRNA expression by endometriotic lesions and eutopic endometrium from women with disease was significantly greater than ENDO-I mRNA expression by proliferative stage eutopic endometrium from women with disease or eutopic endometrium from controls, regardless of cycle stage (P < 0.001). More ENDO-I protein was localized in endometriotic lesions and eutopic endometrium from women with disease than in eutopic endometrium from controls, regardless of cycle stage (P < 0.001). Differential expression and localization of ENDO-I may help develop minimally invasive diagnostic strategies for endometriosis. Further, as ENDO-I shares nucleotide sequence and amino acid sequence with hepatic haptoglobin-which in certain disease states is immunosuppressive and angiogenic-differences in ENDO-I expression and localization in the peritoneal cavity may contribute to the pathogenesis of endometriosis and/or facilitate development of unprecedented diagnostic or therapeutic approaches for management of this enigmatic disease.     

Interleukin-6- and tumour necrosis factor alpha-mediated expression of hepatocyte growth factor by stromal cells and its involvement in the growth of endometriosis

BACKGROUND: We investigated the expression of the hepatocyte growth factor (HGF) gene and protein by the stromal cells derived from women with or without endometriosis and its regulation by interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFalpha). METHODS: Stromal cells immunoreactive to vimentin were isolated from the eutopic and ectopic endometrium of 18 infertile women with endometriosis and 12 women without endometriosis. The production of HGF in the culture media of basal and IL-6- or TNFalpha-stimulated stromal cells was examined with an enzyme-linked immunosorbent assay. The mRNA expression of HGF and its receptor c-Met in the stroma was investigated by RT-PCR. The localization of HGF and c-Met in isolated stromal cells and in intact tissue was examined by immunohistochemistry. The effect of HGF on the growth of stromal cells alone or in combination with IL-6 or TNFalpha was examined in a bromodeoxyuridine (BrdU) incorporation study. RESULTS: The production of HGF in the culture medium of stromal cells was significantly increased after single or combined treatment with either IL-6 or TNFalpha when compared with non-treated cells. The production of HGF by stromal cells derived from the eutopic endometrium of women with endometriosis was significantly higher than that of cells from women without endometriosis. This effect was paralleled by increased expression of HGF and c-Met mRNA, as demonstrated by RT-PCR. The BrdU incorporation study indicated that the addition of HGF enhanced the growth of endometrial and endometriotic stroma alone or in combination with IL-6 or TNFalpha. CONCLUSION: IL-6 and TNFalpha are involved in the production of HGF by endometrial stromal cells and may be involved in the growth of endometriosis by an autocrine mechanism.     

Urocortin 2 and urocortin 3 in endometriosis: evidence for a possible role in inflammatory response

Urocortin 2 (Ucn 2) and urocortin 3 (Ucn 3) are neuropeptides expressed by human endometrium. This study evaluated (i) the expression of Ucn 2 and Ucn 3 mRNA in endometriotic lesions and in endometrium of women with endometriosis; (ii) the effect of Ucn 2 and Ucn 3 on cytokines secretion from cultured endometrial stromal cells. Endometriotic tissue was collected from endometrioma (n=39); endometrial specimens were obtained from women with (n=39) and without (n=41) endometriosis throughout menstrual cycle. Tissue specimens were analysed for Ucn 2 and Ucn 3 mRNA expression and peptide localization; the effects of Ucn 2 or Ucn 3 on tumour necrosis factor (TNF-α) and interleukin (IL-4) secretion from cultured endometrial stromal cells was studied. Ucn 2 and Ucn 3 mRNA expression and localization were assessed by RT-PCR and by immuohistochemistry, respectively; cytokines secretion were measured by ELISA. Results showed that endometriotic tissue expressed both Ucn 2 and Ucn 3, with Ucn 3 expression higher in ectopic than in eutopic endometrium. Endometrial Ucn 2 mRNA expression in controls showed peak values at early proliferative phase, while in endometriotic patients low expression and no significant changes throughout menstrual cycle were observed. Endometrial Ucn 3 mRNA expression was highest in late secretory phase in controls, while in endometriotic patients low levels and no menstrual-cycle-related changes were found. When added to cultured endometrial cell cultures, Ucn 2 significantly increased TNF-α (P<0.01) and IL-4 (P<0.001), while Ucn 3 induced an increase of IL-4 secretion (P<0.01). In conclusion, endometriotic tissue expressed and localized Ucn 2 and Ucn 3; patients with endometriosis showed Ucn 2 and Ucn 3 mRNA expression in eutopic endometrium lower than in control group, with no endometrial cycle-related changes. Ucn 2 and Ucn 3-modulated TNF-α and IL-4 secretion from culture endometrial cells. These data suggest a possible involvement of Ucn 2 and Ucn 3 in the mechanisms of endometriosis.     

Expression of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis

BACKGROUND: Endometriosis is considered a benign disease that has the ability to invade normal tissue. As in neoplastic growth, local extracellular proteolysis may take place. The aim of this study is to analyse several components of the plasminogen activator (PA) pathway and the matrix metalloproteinase (MMP) system in endometriotic tissue, endometrium and peritoneal fluid from women with and without endometriosis (controls). METHODS AND RESULTS: Thirty-nine women with endometriosis and 35 controls were studied. In eutopic endometrium of women with endometriosis, the antigenic levels of urokinase-type PA (uPA) and MMP-3 were elevated when compared with endometrium from controls. Ovarian endometriotic tissues had higher antigenic levels of PA inhibitor type 1 (PAI-1) and tissue inhibitor of metalloproteinases type 1 (TIMP-1) than endometrium. The peritoneal fluid from women with endometriosis showed a significant increase in uPA levels compared with controls. CONCLUSIONS: The increase in antigenic levels of uPA and MMP-3 in endometrium of women with endometriosis might contribute to the invasive potential of endometrial cells. Once the ovarian endometriotic cyst is developed, an increase in PAI-1 and TIMP-1 is detected and significant proteolytic activity is no longer observed. This increase in inhibitors and decrease in proteolytic activity could explain the frequent clinical finding of isolated endometriotic cyst without invasion of the surrounding ovarian tissue.     

Endometriosis results from the dislocation of basal endometrium

BACKGROUND: The hypothesis is tested that both adenomyotic and endometriotic lesions are derived from basal endometrium. METHODS: Normal uteri and uteri with adenomyosis obtained by hysterectomy, excised endometriotic lesions and menstrual blood of women with and without endometriosis were used. Estrogen receptor (ER), progesterone receptor (PR), progesterone receptor B isoform (PR(B)) and P450 aromatase (P450A) immunohistochemistry was performed with the use of specific monoclonal antibodies. RESULTS: With respect to the parameters studied there was a fundamental difference between the cyclical patterns of the basalis and the functionalis of the eutopic endometrium. The endometrium of endometriotic and adenomyotic lesions mimicked the cyclical pattern of the basalis. The peristromal muscular tissue of endometriotic and adenomyotic lesions displayed the same cyclical pattern of ER and PR expression as the archimyometrium. There was a significantly higher prevalence of fragments of shed basalis in menstrual blood of women with endometriosis than in healthy controls. CONCLUSIONS: These data suggest that ectopic endometrial lesions result from dislocation of basal endometrium. Dislocated basal endometrium has stem cell character resulting in the ectopic formation of all archimetrial components such as epithelial and stromal endometrium as well as peristromal muscular tissue.     

17β-雌二醇和二噁英对子宫内膜间质细胞表达趋化因子IL-8及其受体CXCR1的调控作用

解析内外环境因素对子宫内膜间质细胞表达IL-8及其自分泌作用的调控。采用免疫组化法比较子宫内膜异位症患者异位灶和在位内膜CXCR1翻译水平表达;流式细胞术分析17β-雌二醇和二噁英单独或联合作用对子宫内膜间质细胞表面CXCR1表达的调控作用;ELISA法分析17β-雌二醇和二噁英单独或联合作用对子宫内膜间质细胞分泌IL-8的影响。结果显示CXCR1在子宫内膜异位症患者异位灶组织高表达。17β-雌二醇和二噁英单独作用均抑制子宫内膜间质细胞表面CX-CR1的表达以及IL-8的分泌。二者联合作用能够上调CXCR1的表达,上调幅度与雌二醇浓度呈正相关;但进一步抑制了IL-8的分泌。雌激素与二噁英对子宫内膜间质细胞复合作用抑制其IL-8的分泌及其自分泌作用;子宫内膜异位症患者腹腔液高水平IL-8并非由内外雌激素样物质直接作用于异位灶子宫内膜间质细胞所致。     

Imbalance in the expression of the activating type I and the inhibitory type II interleukin 1 receptors in endometriosis

BACKGROUND: The ectopic establishment and progression of endometrial tissue is dependent upon its interaction with and responsiveness to the stimuli present in its new environment. Immune cell-derived cytokines, such as interleukin 1 (IL1), may alone or in concert with estrogens enhance the capability of ectopic endometrial cells to implant and develop into the host tissue. The objective of this study was to further evaluate the expression and significance of IL1 receptor type I (IL1R1), the signalling receptor that mediates cell activation by IL1, and IL1 receptor type II (IL1R2), a potent and specific down-regulator of IL1 action, in normal compared to endometriotic/endometrial tissues. METHODS: Techniques included immunohistochemistry, immunofluorescent staining, ELISA, western blotting and endometriotic cell culture transfection. RESULTS: Our study showed an imbalance in the expression of IL1R1 and IL1R2 in eutopic, and particularly in ectopic, endometrial tissues of women with endometriosis. Actually, a decreased IL1R2 expression is predominant in the eutopic and ectopic endometrium of women with endometriosis when compared with normal women, whereas a concomitant increase in IL1R1 expression occurs in ectopic endometrial tissue in comparison to eutopic endometrial tissue of normal or endometriotic women, particularly in the initial and most active implants. Transfection of endometriotic cells with a cDNA coding for IL1R2 resulted in a significant decrease in IL1-induced secretion of vascular endothelial cell growth factor and monocyte chemotactic protein 1. CONCLUSIONS: IL1R1/IL1R2 imbalance may amplify endometrial cell responsiveness to IL1 and represent a key mechanism underlying the ability of these cells to implant and develop into host tissues.     

Aromatase expression in endometriotic tissues and cell cultures of patients with endometriosis

Cytochrome P-450 aromatase is responsible for catalysing the conversion of androstendione into estrone, so its expression in endometriotic tissue could contribute to the development of endometriosis. The aims of this study were, on the one hand, to determine the presence of aromatase in eutopic and ectopic endometrium, healthy peritoneum, myometrium and leiomyomas from patients with (n = 61) and without endometriosis (n = 12) and, on the other hand, to determine the effect of peritoneal fluid (PF), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNFalpha) on aromatase activity from endometriotic stromal cells and subcutaneous adipocytes. After immunohistochemical analysis, aromatase expression was detected in the endometriotic tissue of 61% of patients, whereas the rest of the tissues, as well as those from disease-free women, were negative. Cell cultures were made to determine aromatase activity in endometriotic stromal cells and adipocytes. The addition of PF, TNFalpha and especially IL-6 (P < 0.05) stimulated the basal enzymatic activity observed in both cell types. Our findings confirm the presence of aromatase in endometriosis and probably the existence of a local estrogen production that may be stimulated by some factors such as cytokines present in the PF of these patients. Therefore, the use of aromatase inhibitors combined with immunomodulator agents could be a novel approach to be investigated in future clinical trials.     

Expression of interleukin-8 receptors in endometriosis

BACKGROUND: Although the etiology of endometriosis is not well understood, chemokines and their receptors are believed to play a role in its pathogenesis. Therefore, we aimed to investigate the expression and localization of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 in eutopic and ectopic endometrial tissues of women with endometriosis, and in endometrium of women without endometriosis. METHODS: Ectopic (n = 27) and homologous eutopic endometrium (n = 25) from women with endometriosis and endometrium from women without endometriosis (n = 27) were used for immunohistochemical analysis of CXCR1 and CXCR2. RESULTS: In normal endometrium, epithelial CXCR1 and CXCR2 immunostaining intensities were similar in the proliferative and secretory phase. Stromal CXCR1 expression was less then epithelial expression and did not show cyclical difference. No stromal CXCR2 expression was observed. In eutopic endometrium of women with endometriosis compared to endometrium of women without endometriosis, there was a significant increase in both proliferative and secretory phases for epithelial CXCR2 expression, and in proliferative phase for CXCR1 expression (P < 0.05). Both receptor immunoreactivities were significantly increased in the epithelial cells of ectopic endometrial tissues compared to that of normal endometrium (P < 0.05). CONCLUSIONS: These findings suggest that IL-8 and its receptors may be involved in the pathogenesis of endometriosis.     

GnRH II as a possible cytostatic regulator in the development of endometriosis

BACKGROUND: GnRH II is the second form of GnRH and is widely distributed in peripheral tissues of the female reproductive tract as well as in the central nervous system. In the present study, we studied the possible implication of GnRH II in endometriosis. METHODS: Effects of GnRH II on 5-bromo-2'-deoxyuridine (BrdU) uptake by cultured endometriotic stromal cells were examined. Effects of GnRH II on interleukin (IL)-1beta-induced expression of cyclooxygenase (COX)-2 and IL-8 were also studied. mRNA levels of GnRH I, GnRH II, type I GnRH receptor and type II GnRH receptor were determined by real-time quantitative RT-PCR in endometrial tissues of women with or without endometriosis and in endometriotic tissues. RESULTS: GnRH II dose-dependently suppressed BrdU uptake by endometrial stromal cells. Treatment with IL-1beta markedly increased mRNA levels of COX-2 and IL-8 in endometrial stromal cells and IL-8 protein secretion by these cells, while these increments were significantly suppressed by supplementation with GnRH II. The mRNA levels of GnRH II were lower in endometrial and endometriotic tissues of women with endometriosis than in endometrial tissues of women without endometriosis, both in the proliferative phase and the secretory phase. In addition, as for GnRH I, type I GnRH receptor and type II GnRH receptor, the mRNA levels were lower in endometrial tissues of women with endometriosis than in those without endometriosis in the secretory phase. CONCLUSIONS: In the light of the demonstrated antiproliferative and anti-inflammatory effects of GnRH II on endometrial stromal cells, the lower expression of GnRH II in eutopic and ectopic endometrium of women with endometriosis suggests that endogenous GnRH II-mediated cytostatic regulation may be impaired in the development of endometriosis.     

Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9

BACKGROUND: For the implantation of endometrium in ectopic locations, remodelling of the extracellular matrix (ECM) is necessary. Many studies have shown an increased expression of various proteases in the ectopic endometrium of women with endometriosis. Few, however, have addressed possible changes in protease expression in the eutopic endometrium. METHODS AND RESULTS: Herein, we reveal an increased release of proteolytic activity by the eutopic endometrium of women with endometriosis compared with normal women (P < 0.01). Using zymography and western blotting, we identified matrix metalloproteinase (MMP)-2 and MMP-9 in the culture medium, and further found that MMP-9 secretion, as assessed by zymography and enzyme-linked immunosorbent assay (ELISA), was elevated in women with endometriosis compared with normal women (P < 0.05). No statistically significant difference in MMP-2 secretion between women with and without endometriosis was noted. However, a significant difference in the levels of the tissue inhibitor of metalloproteinases (TIMP)-1, a known MMP-9 inhibitor, was found (P < 0.05). CONCLUSION: The endometriosis-associated increase in proteolysis and imbalance between the secretion of MMP-9 and that of its natural inhibitor, TIMP-1, revealed in the culture medium of endometrial tissue may reflect in vivo the enhanced capacity of this tissue to break down the ECM in host tissues, thereby favouring its ectopic implantation and development.     

Endometriosis: absence of recurrence in patients after endometrial ablation.

BACKGROUND: The present study was undertaken to evaluate differences between patients with and without eutopic endometrium in the recurrence of ectopic endometriotic implants. METHODS: Endometrial ablation (EA) was carried out in 14 women out of 28 laparoscopically treated for endometriosis and recurrence of the disease was evaluated 24 months later. Data were compared using paired Student's t-test and chi2 test. RESULTS: Patients undergoing EA procedures did not exhibit recurrence of endometriosis while nine patients without that procedure had recurrence of the disease (P < 0.001). The endometrial cells found in the debris of the cul de sac of eight patients who did not undergo EA were both stromal and epithelial cells. No blood or blood cells were found in the cul de sac of patients undergoing EA. CONCLUSIONS: The present study supports a role of eutopic endometrium in the recurrence of endometriosis through tubal dissemination of endometrial debris and implantation of endometrial cells into the abdomen.     

Transcriptional expression of survivin and its splice variants in endometriosis

Survivin is a novel inhibitor of apoptosis (IAP), and the two splice variants of survivin (survivin-2B and survivin-EX3) have been identified. Gene expression levels of survivin, survivin-2B and survivin-EX3 in 56 ectopic (16 peritoneal red and 16 peritoneal black lesions and 24 ovarian endometriomata) and 13 eutopic endometrial tissues surgically obtained from 42 women with endometriosis (group A) were compared with those in 16 control eutopic endometrium from 16 women without endometriosis (group B) by quantitative RT-PCR analysis. Survivin mRNA expression levels in ectopic endometriotic tissues were significantly higher than those in eutopic endometrium of groups A and B over the whole cycle. Red peritoneal lesions had higher gene expression levels of survivin than black lesions. In contrast, all tissue samples examined showed relatively lower gene expression levels of survivin-2B and survivin-EX3. No cyclic variation was found in survivin and the two splice variants, both in ectopic and in eutopic endometrium. Although there was no significant difference in the ratio of survivin-2B/survivin between ectopic and eutopic endometrium, the ratio of survivin-EX3/survivin in peritoneal endometriotic lesions was significantly higher than that of eutopic endometrium of groups A and B. These results suggest that survivin and survivin-EX3 may be closely linked to escape from apoptosis and the development of endometriosis.     

17β-雌二醇和二噁英对子宫内膜间质细胞表达趋化因子IL-8及其受体CXCR1 的调控作用

解析内外环境因素对子宫内膜间质细胞表达IL-8及其自分泌作用的调控。采用免疫组化法比较子宫内膜异位症患者异位灶和在位内膜CXCRI翻译水平表达;流式细胞术分析17β-雌二醇和二嗯英单独或联合作用对子宫内膜间质细胞表面CXCRI表达的调控作用;ELISA法分析17β-雌二醇和二嗯英单独或联合作用对子宫内膜间质细胞分泌IL-8的影响。结果显示CXCRI在子宫内膜异位症患者异位灶组织高表达。17β-雌二醇和二嗯英单独作用均抑制子宫内膜间质细胞表面CX—CR1的表达以及IL-8的分泌。二者联合作用能够上调CXCR1的表达,上调幅度与雌二醇浓度呈正相关;但进一步抑制了IL-8的分泌。雌激素与二嗯英对子宫内膜间质细胞复合作用抑制其IL-8的分泌及其自分泌作用;子宫内膜异位症患者腹腔液高水平IL-8并非由内外雌激素样物质直接作用于异位灶子宫内膜间质细胞所致。     

子宫内膜及异位灶趋化因子受体转录在子宫内膜异位症发病中的作用

目的：探讨子宫内膜异位症患者在位内膜及异位灶18种趋化因子受体的转录特征，以揭示趋化因子受体及其配体在子宫内膜异位症发生发展中的作用。方法：以正常子宫内膜为对照，半定量RT-PCR检测子宫内膜异位症患者在位内膜及异位灶18种趋化因子受体mRNA的表达水平，并比较其差异。结果：与正常子宫内膜相比，子宫内膜异位症患者在位子宫内膜CCR6、CCR8、CCR9、CX3CR1表达明显升高（P＜0．05）。与在位内膜相比，异位灶CCR4、CCR8、CCR9、CXCR1表达显著升高（P＜0．05）。结论：在位子宫内膜CCR6、CCR8、CCR9、CX3CR1高表达，可能参与子宫内膜异位症的发生；异位灶CCR4、CCR8、CCR9、CXCR1高表达，可能参与子宫内膜异位症的进一步发展。     

Correlation between decreased type-II interleukin-1 receptor and increased monocyte chemotactic protein-1 expression in the endometrium of women with endometriosis

PROBLEM: Monocyte chemotactic protein-1 (MCP-1), a potent inducer of macrophage recruitment and activation, is overexpressed in the eutopic endometrium of women with endometriosis. Eutopic endometrial cells of women with endometriosis secrete higher levels of MCP-1 than those of normal women, following stimulation with interleukin-1 (IL-1). The aim of this study was to examine whether there is any correlation between the expression of IL-1 receptor type II (IL-IRII), a specific downregulator of IL-1 activity, and that of MCP-1 in the eutopic endometrium of women with endometriosis. METHOD OF STUDY: Endometrial biopsies of 46 women with endometriosis and 22 healthy women were evaluated simultaneously for IL-1RII and MCP-1 expression by immunohistochemistry. RESULTS: Our study revealed a highly significant correlation between the decreased expression of IL-1RII and the increased expression of MCP-1 in the endometrial tissue of women with endometriosis (Spearman correlation coefficient r = -0.377, P = 0.002), particularly in the initial stages of the disease (stages I and II; r = - 0.368, P = 0.020 and r = -0.480, P = 0.002, respectively). Furthermore, this correlation was observed in the proliferative (r = -0.366, P = 0.047) and the secretory phases (r = -0.321, P = 0.049) of the menstrual cycle. CONCLUSIONS: These results suggest that the reduced capability of endometrial tissue to downregulate IL-1 proinflammatory effects may be involved in the increased expression of MCP-1 in the endometrium of women with endometriosis and the establishment of an inflammatory state. The results also indicate a sustained process of cell activation throughout the menstrual cycle.     

mRNA analysis of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis using a real-time quantitative RT-PCR assay

BACKGROUND: The plasminogen activator (PA) and matrix metalloproteinase (MMP) systems are implicated in the establishment of endometriosis. The mechanisms by which these systems are involved in the pathogenesis of this disease are not well defined and controversial results have been published. The aim of this study was to analyse mRNA and protein levels of several components of the PA and MMP systems in endometriotic tissue and endometrium from women with and without endometriosis. METHODS and RESULTS: Real-time quantitative RT-PCR assays were developed to quantify mRNA levels of these components in 57 women with endometriosis and 32 controls. Endometrium of women with endometriosis showed higher mRNA and antigenic levels of urokinase type-PA (uPA) and MMP-3 than endometrium from controls. In these patients, ovarian endometriotic tissue had higher mRNA and antigenic levels of PA inhibitor type 1 (PAI-1) and MMP inhibitor type 1 (TIMP-1) than endometrium. CONCLUSIONS: The increase in mRNA and protein levels of uPA and MMP-3 observed in endometrium of women with endometriosis may facilitate the attachment of endometrial tissue to the peritoneum and ovarian surface, as well as the invasion of the extracellular matrix. This process would lead to the formation of early endometriotic lesions. Once the ovarian endometriotic cyst is developed, PAI-1 and TIMP-1 would increase which could explain the frequent clinical finding of an endometrioma without invasion of the adjacent ovarian tissue.