Sachiko Matsuzaki

Expression of estrogen receptor alpha and beta in peritoneal and ovarian endometriosis

OBJECTIVE To quantify and compare messenger RNA (mRNA) levels of ER-alpha and ER-beta among ovarian endometriotic cysts and red and black peritoneal endometriotic lesions. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Patients with or without endometriosis. INTERVENTION(S) Samples of peritoneal (n = 33) and ovarian endometriotic lesions (n = 37) were obtained during laparoscopic surgery. Normal eutopic endometrial tissues and macroscopically normal peritoneal tissues were obtained as controls during or just after surgery. MAIN OUTCOME MEASURE(S) Expression of mRNA for ER-alpha and ER-beta, using a real-time reverse transcription (RT)-PCR assay, TaqMan RT-PCR, and nonradioactive in situ hybridization (ISH) techniques. RESULT(S) Both eutopic endometrium and endometriotic tissues showed predominantly higher levels of ER-alpha than ER-beta mRNA. Relative ratio of ER-alpha to ER-beta (ER-alpha/ER-beta) mRNA in red peritoneal lesions was significantly higher than in black lesions and ovarian endometriotic cysts. There was no significant difference in ER-alpha/ER-beta between proliferative eutopic endometrium and red peritoneal lesions. These results were confirmed by ISH analysis, which also revealed that the two estrogen receptors were localized in both epithelial and stromal cells of endometriotic tissues. CONCLUSION(S) The predominant expression of ER-alpha in both glandular epithelial and stromal cells may be essential for the development and growth of peritoneal and ovarian endometriosis.

Impaired Down-Regulation of E-Cadherin and β-Catenin Protein Expression in Endometrial Epithelial Cells in the Mid-Secretory Endometrium of Infertile Patients with Endometriosis

CONTEXT Only a few, small, human studies on E-cadherin and beta-catenin expression in normal cycling human endometrium have been reported. It remains unclear whether expression of these molecules might be altered in the endometrium of infertile patients with endometriosis. OBJECTIVES The aim of the present study was to investigate E-cadherin and beta-catenin expression in the endometrium of infertile patients with endometriosis, those with uterine fibromas, and patients with unexplained infertility. DESIGN Expression levels of E-cadherin and beta-catenin mRNA and/or protein in the endometrium of infertile patients with endometriosis (n = 151), those with uterine fibromas (n = 41), patients with unexplained infertility (n = 9), as well as healthy fertile controls (n = 57) were measured. This study utilized laser capture microdissection, real-time RT-PCR, and immunohistochemistry. RESULTS No significant differences in E-cadherin or beta-catenin mRNA expression in microdissected epithelial cells were observed among the different groups throughout the menstrual cycle. However, very low or no protein expression of E-cadherin, total beta-catenin, or dephosphorylated beta-catenin in luminal and glandular epithelial cells was detected in the mid-secretory endometrium of healthy fertile controls. E-cadherin, total beta-catenin, and dephosphorylated beta-catenin protein expression in the mid-secretory endometrium of infertile patients with endometriosis or unexplained infertility was significantly higher compared to that of healthy fertile controls in both luminal and glandular epithelial cells. CONCLUSIONS These findings suggest that impaired down-regulation of E-cadherin and beta-catenin protein expression, along with Wnt/beta-catenin signaling pathway activation during the window of implantation, might be one of the potential molecular mechanisms of infertility in patients with endometriosis.

HOXA-10 expression in the mid-secretory endometrium of infertile patients with either endometriosis, uterine fibromas or unexplained infertility

BACKGROUND The aim of this study was to investigate HOXA-10 expression in endometrium from infertile patients with different forms of endometriosis; with uterine fibromas, or with unexplained infertility and from normal fertile women. METHODS Expression levels of HOXA-10 mRNA and protein in endometrium were measured during the mid-secretory phase. This study utilized laser capture microdissection, real-time RT-PCR and immunohistochemistry. RESULTS HOXA-10 mRNA and protein expression levels in endometrial stromal cells were significantly lower in infertile patients with different types of endometriosis (deep infiltrating endometriosis, ovarian endometriosis and superficial peritoneal endometriosis), with uterine myoma, and unexplained infertility patients as compared with healthy fertile controls. HOXA-10 mRNA expression levels of microdissected glandular epithelial cells were significantly lower than those of microdissected stromal cells, without significant differences among the different groups. No protein expression was detected in glandular epithelial cells. The percentage of patients with altered protein expression of HOXA-10 in stromal cells were significantly higher in patients with only superficial peritoneal endometriosis (100%, 20/20, P < 0.05) compared with the other infertile groups (deep infiltrating endometriosis: 72.7%, 16/22; ovarian endometriosis: 70.0%, 14/20; uterine myoma: 68.8%, 11/16; unexplained infertility: 55.6%, 5/9). CONCLUSION The present findings suggested that altered expression of HOXA-10 in endometrial stromal cells during the window of implantation may be one of the potential molecular mechanisms of infertility in infertile patients, particularly in patients with only superficial peritoneal endometriosis. One of the underlying causes of infertility in patients with only superficial endometriosis may be altered expression of HOXA-10 in endometrial stromal cells.

Oxidative stress status in normal ovarian cortex surrounding ovarian endometriosis

Expression levels of 8-hydroxydeoxyguanosine, a sensitive indicator of DNA damage resulting from oxidative stress, were significantly higher in samples of normal ovarian cortex surrounding endometriotic cysts when compared with ovarian cortex surrounding dermoid and serous ovarian cysts. These findings suggest that the normal ovarian cortex surrounding endometriotic tissues is more severely affected by oxidative stress than ovarian cortex adjacent to other benign ovarian cysts.

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified. Objectives The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice. Methods Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice. Results Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis. Conclusion Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.

Angiogenesis in endometriosis

We evaluated angiogenesis in eutopic endometrium and ectopic endometrium from patients with endometriosis. Microvessels were identified by immunohistochemistry using anti-von-Willebrand factor antibody and vascular parameters were measured using a computerized image analysis system. No relationship was observed between vascular density and type of endometriotic lesion. Heterogeneity in vascular density was observed in different lesions within the same patient. Between red and black peritoneal lesions a significant difference was observed in the frequency distribution of luminal diameter. In black lesions the variability of luminal diameter was decreased and most microvessels were less than 20 mm in diameter. We suggest that the frequency distribution of microvessel luminal diameter of red and black peritoneal lesions may be different and should be important when studying the regulation of angiogenesis in endometriotic implants.

Analysis of risk factors for the removal of normal ovarian tissue during laparoscopic cystectomy for ovarian endometriosis

BACKGROUND The aim of this study was to identify risk factors for the removal of normal ovarian tissue during laparoscopic cystectomy for endometriosis. METHODS A total of 121 patients who had histologically confirmed ovarian endometriosis and 56 control patients who had other histologically confirmed benign cysts were included for the present analysis. The blocks of removed tissue were sectioned at 120 microm intervals and a total of five sections were analyzed for each ovarian cyst. Eight variables (age, pre-operative medical treatment, previous surgery for ovarian endometriosis, single or multiple cysts, size of the largest cyst, side of cyst, co-existence of deep endometriosis, revised American Society for Reproductive Medicine classification) were evaluated using a generalized linear modeling analysis to identify major factors associated with the removal of normal ovarian tissue. RESULTS Normal ovarian tissue adjacent to the cyst wall was detected in 71 patients (58.7%) with endometriosis, whereas normal ovarian tissue was removed from only three patients (5.4%) with other benign cysts. A significant factor that was independently associated with the removal of normal ovarian tissue with ovarian endometriosis was pre-operative medical treatment. CONCLUSIONS The present retrospective, controlled study suggests that pre-operative medical treatment might be a risk factor for the removal of normal ovarian tissue during laparoscopic cystectomy for ovarian endometriosis.

Immunohistochemical analysis of the role of angiogenic status in the vasculature of peritoneal endometriosis

OBJECTIVE To investigate the angiogenic status of the vasculature in peritoneal endometriosis based on macroscopic appearance. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Patients with peritoneal endometriosis. INTERVENTION(S) During laparoscopy, 25 samples of predominantly red peritoneal lesions and 27 samples of predominantly black peritoneal lesions were collected from a total of 31 patients with endometriosis. Eutopic endometrium from 25 patients with endometriosis was collected by curettage during laparoscopy or just after surgery. MAIN OUTCOME MEASURE(S) A proliferating endothelial cell index (PCI) was determined by calculating the percentage of microvessels that contained proliferating endothelial cells. A vessel maturation index (VMI) was determined by calculating the percentage of microvessels showing colocalization of CD34- and alpha-SMA-positive staining. RESULT(S) Peritoneal endometriotic tissues had extremely low or null PCI. The VMI of red peritoneal lesions was significantly lower than that of black ones. Vessel maturation index of red lesions was higher that that of proliferative eutopic endometrium and similar to that of secretory eutopic endometrium. CONCLUSION(S) Compared with the case of black peritoneal lesions, red lesions had a much higher fraction of immature vessels.

Analysis of estrogen receptor α and β in endometrial carcinomas : Correlation with ERβ and clinicopathologic findings in 45 cases

Estrogens play important roles in the pathogenesis of the great majority of endometrial endometrioid adenocarcinoma. Recently, a novel estrogen receptor (ER), ERβ, has been characterized, but little is known about the status of ERβ in endometrial carcinoma. We therefore examined expression of both ERa and ERβ in 45 cases of endometrioid endometrial adenocarcinoma using mRNA in situ hybridization, reverse transcription and polymerase chain reaction (RT-PCR), and immunohistochemistry. We also correlated the findings with various clinicopathologic parameters in these cases to examine their possible biologic significance. Accumulation of mRNA hybridization signals for both ERa and ERβ was detected predominantly in the cytoplasm of carcinoma cells, and to a lesser extent in some stromal cells. ERβ mRNA was detected in 16/45 cases (35.6%), and ERa mRNA hybridization signals were detected in 36/45 cases (80.0%). Among the 16 ERp positive cases, 15 cases also had ERa mRNA hybridization signals. In the cases that expressed both ERα and ERβ, ERa mRNA hybridization signals were more widely distributed than ERp mRNA. In 21 cases, carcinoma cells had ERa mRNA hybridization signals but not ERβ mRNA. There was a statistically significant positive correlation between the results of mRNA in situ hybridization and semiquantitative RT-PCR or immunohistochemistry for both ERa and ERβ. There were no significant correlations between ERβ mRNA expression and PR labeling index, Ki67 LI, age, or histologic grade. The results from our study indicate that ERβ is coexpressed with ERa, and that the estrogenic effects occur predominantly through ERa in endometrial carcinomas.

Quantitative analysis of estrogen receptor alpha and beta messenger ribonucleic acid levels in normal endometrium and ovarian endometriotic cysts using a real-time reverse transcription-polymerase chain reaction assay

OBJECTIVE To quantify messenger RNA (mRNA) levels of the two estrogen receptor isoforms, estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) in the eutopic endometrium and ovarian endometriotic cysts. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Patients with endometriosis and patients with uterine leiomyoma or carcinoma in situ. INTERVENTION(S) Gonadotropin-releasing hormone agonist (GnRH-a)-treated (n = 12) or untreated (n = 24) endometriotic cysts were obtained from 36 patients during laparoscopic cystectomy. Eutopic endometrium tissues were obtained from 24 patients during or immediately after surgery. MAIN OUTCOME MEASURE(S) ER-alpha and ER-beta mRNA levels, using a real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay, TaqMan RT-PCR. RESULT(S) Eutopic endometrium and ovarian endometriotic cysts showed predominantly higher levels of ER-alpha mRNA than ER-beta mRNA. Although ER-alpha and ER-beta mRNA levels in the eutopic endometrium were affected by a cyclic change in ovarian hormones, ovarian endometriotic cysts were less affected. Moreover, a long-term hypoestrogenic state induced by GnRH-a especially decreased ER-alpha mRNA levels in endometriotic cysts. Consequently, the relative ratios of ER-alpha to ER-beta mRNA levels in both GnRH-a-treated and untreated endometriotic cysts were significantly lower than those in the eutopic endometrium. CONCLUSION(S) The results suggest that the principal and regulatory effects of estrogens may be mediated mainly via ER-alpha rather than ER-beta in both the eutopic endometrium and endometriotic cysts.