The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

International Journal of Gynecology & ObstetricsVolume 107, Issue S2 p. S137-S137 Free communication (oral) presentations O157 The membrane bound G-protein coupled receptor, GPR30, mediates the anti-invasive effect of estradiol in ovarian cancer cells B. Casslen, B. CasslenSearch for more papers by this authorE. Henic, E. HenicSearch for more papers by this author B. Casslen, B. CasslenSearch for more papers by this authorE. Henic, E. HenicSearch for more papers by this author First published: 20 November 2009 https://doi.org/10.1016/S0020-7292(09)60529-4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume107, IssueS2Abstracts of XIX FIGO World Congress of Gynecology and ObstetricsOctober 2009Pages S137-S137 RelatedInformation

International Journal of Gynecology & ObstetricsVolume 107, Issue S2 p. S699-S700 Poster presentations P1020 Cleaved forms of the upa receptor in plasma have diagnostic potential and predict postoperative survival in patients with ovarian cancer E. Henic, E. HenicSearch for more papers by this authorC. Borgfeldt, C. BorgfeldtSearch for more papers by this authorI. Christensen, I. ChristensenSearch for more papers by this authorB. Casslén, B. CasslénSearch for more papers by this authorG. Høyer-Hansen, G. Høyer-HansenSearch for more papers by this author E. Henic, E. HenicSearch for more papers by this authorC. Borgfeldt, C. BorgfeldtSearch for more papers by this authorI. Christensen, I. ChristensenSearch for more papers by this authorB. Casslén, B. CasslénSearch for more papers by this authorG. Høyer-Hansen, G. Høyer-HansenSearch for more papers by this author First published: 20 November 2009 https://doi.org/10.1016/S0020-7292(09)62506-6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume107, IssueS2Abstracts of XIX FIGO World Congress of Gynecology and ObstetricsOctober 2009Pages S699-S700 RelatedInformation

Postmenopausal patients with vaginal bleeding (n= 72) were evaluated with the combination of transvaginal ultrasonography (TVS) and analysis of the lactate dehydrogenase (LD) isoenzyme activity profile in uterine fluid aspirates. TVS evaluation of the endometrium was classified as <5 mm, ≥5 mm, or poorly defined. The LD isoenzyme activity profile was characterized as abnormal or normal. Pathologic findings were further evaluated with diagnostic curettage. TVS found the endometrium to be ≥5 mm or poorly defined in 44 patients (61%). Endometrial carcinoma was found in 6 of 72 patients (8%). They appeared in the TVS groups endometrium >5 mm (n= 2) and endometrium poorly defined (n= 4) but not in the endometrium <5 mm. The LD isoenzyme activity profile was abnormal in the six malignant cases and in ten benign cases. Thus, the need for further evaluation with hysteroscopy and curettage was reduced to 16 cases. Since TVS had 100% sensitivity but only 42% specificity, it is suitable for first-level examination in patients with postmenopausal bleeding. The second-level method should have similarly high sensitivity but much higher specificity. The LD isoenzyme activity profile in uterine fluid aspirates had 100% sensitivity and 85% specificity. Another important feature is that the method is not sensitive to endometrial thickness, amount of sample, sampling device, or dilution. Thus, it is more reliable than aspiration histology. For every hysteroscopy or curettage that can be replaced by LD analysis, the cost is reduced by approximately EUR 720 or 540, respectively.

BACKGROUND The aim of this study was to analyse the effects of an estradiol (E(2))-progesterone substitution protocol on the endometrial expression of estrogen-sensitive genes during the peri-implantation period. METHODS Peripheral blood and endometrial biopsies were obtained from 13 infertile women both in a natural cycle (NC), on days 5 and 7 after ovulation (NC5, NC7), and in an artificial (substituted) cycle (AC), on days 5 and 7 of progesterone addition (AC5, AC7). Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were assayed by immunohistochemistry. Matrix metalloproteinase-26 (MMP-26) mRNA and tissue inhibitor of metalloproteinase-4 (TIMP-4) mRNA were semiquantitatively assessed in tissue sections using in situ hybridization (ISH) and quantified in tissue extracts using real-time PCR. RESULTS Levels of both E(2) and progesterone were higher in the peripheral blood in AC than in NC. Also on day AC5, expressions of ERalpha, PR and MMP-26 mRNA (focally) were increased in the epithelium and TIMP-4 mRNA in the stroma. Expression levels of these genes dropped significantly between AC5 and AC7, but not between NC5 and NC7. Abnormally high levels in AC5 samples suggest overstimulation with E(2), and the rapid decrease between AC5 and AC7 suggests overstimulation with progesterone. CONCLUSIONS In ACs, increased levels of E(2) in the blood exaggerate the endometrial expression of estrogen-sensitive genes, whereas higher levels of progesterone in the blood in the secretory phase exaggerate the drop in expression of these genes. Dramatic variations in the gene expression may not be optimal for the implantation process.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

We have previously reported that endometrial mRNA expression of both tissue inhibitors of metalloproteinase-4 (TIMP-4) and matrix metalloproteinase-26 (MMP-26) peaks in the early secretory phase, which implies a role in implantation. The objective of this study was to compare the distribution of TIMP-4 and MMP-26 in endometrial tissue and uterine fluid over the menstrual cycle. Endometrial tissue was analysed with in situ hybridization and immunohistochemistry to localize mRNA and protein for TIMP-4 and MMP-26 in the same set of samples. TIMP-4 mRNA was quantified in separated stromal and epithelial cells using real-time PCR. Uterine fluid was analysed with western blotting. TIMP-4 mRNA was exclusively localized to the stroma, whereas MMP-26 mRNA was expressed by epithelial cells. TIMP-4 protein was only occasionally found in the stroma but was consistently present in granules of the apical part of luminal and glandular epithelial cells. TIMP-4, but not MMP-26, was demonstrated in uterine fluid. Thus, TIMP-4 is produced in the stroma only, secreted by stromal cells, taken up by epithelial cells, accumulated in apical granules and finally secreted to the uterine fluid. Maximal expression of MMP-26, and its strongest inhibitor TIMP-4, in the early and mid-secretory phase suggests a role during implantation. MMP-26 is stored in epithelial cells in its active form, is not released spontaneously and is controlled by TIMP-4 in both stroma and uterine fluid.

Objectives. The EGFR is expressed in malignant ovarian tumor tissue, and tissue content of EGFR has been directly associated with poor prognosis in patients with ovarian cancer. The uPA system plays a role in pericellular proteolysis, cell migration, invasion, and is over-expressed in ovarian cancer. This study explored the effects of EGF on uPAR expression in the ovarian cancer cell line OVCAR-3.Methods. We used OVCAR-3 cells and the following methods: cell migration assay, time-lapse video microscopy, real-time PCR, assays for cellular binding of I-125-uPA and cellular degradation of I-125-uPA:PAI-1 complex, biosynthetic labeling using S-35-methionin, Western blot, Northern blot, and ELISAs for uPA, PAI-1, and uPAR.Results. EGF up-regulates both protein and mRNA not only for uPAR, but also for the ligand uPA and its inhibitor PAI-1. Cell surface uPAR, in control as well as EGF-stimulated cells, is present only in the intact, not the cleaved, form. Ligand binding experiments showed an increase of endogenously occupied uPAR, whereas non-occupied receptor sites were not increased. In addition, EGF treatment resulted in decreased degradation of radiolabeled uPA:PAI-1 complex. This suggests decreased internalization of uPAR, since the complex is internalized together with uPAR. Like EGF, colchicine, which inhibits endocytosis, increased cell surface expression of uPAR. In addition, we found an immediate increase of uPAR after exposing the cells to EGF and this was accompanied by a transient increase of cell migration. The increase of cell surface uPAR in response to EGF is accompanied by increased release of the soluble form of uPAR (suPAR) to the medium as well as by increased cell migration. Both uPAR and suPAR increased in cells treated with the endocytosis inhibitor colchicine even though cell migration was inhibited, suggesting that the mechanism of uPAR shedding is not related to cell migration.Conclusion. Increased cell surface uPAR in response to EGF stimulation results from mobilization of uPAR from detergent-resistant domains, increased expression of uPAR mRNA, and decreased internalization and degradation of uPAR. Both the anti-uPAR antibody R3, which inhibits binding of uPA, and the EGFR phosphorylation inhibitor Iressa inhibited cell migration in response to uPA as well as to EGF, suggesting that EGFR and uPAR are engaged in the same multiprotein assembly on the cell surface. (C) 2005 Elsevier Inc. All rights reserved.

OBJECTIVE:To examine the expression pattern of matrix metalloproteinase-26 (MMP-26) mRNA and estrogen receptor-alpha (ER alpha) in normal, hyperplastic, premalignant and malignant endometrial tissue.DESIGN:Experimental study.SETTING:Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden, Atherosclerosis Research Unit, King Gustav V Research Institute, Karolinska Hospital, Stockholm, Sweden.METHODS:We studied MMP-26 mRNA and ER alpha in 36 normal, 7 hyperplastic, 6 premalignant and 19 malignant endometrial samples. Based on histological examination, all normal specimens were classified according to an ideal 28 day menstrual cycle as early, mid, and late proliferative phase, early, mid and late secretory phase and menstrual phase. Samples with hyperplasia were classified as simple or complex. Premalignant samples were represented by complex hyperplasia with atypia. Malignant samples were histologically classified as well, intermediately and poorly differentiated, respectively. Specimens were analyzed using in situ hybridization and real time PCR. ER alpha was localized by immunohistochemistry.RESULTS:Epithelial MMP-26 mRNA expression was highest in the early secretory phase and in endometrial hyperplasia. Expression levels were low in the late secretory and menstrual phase and in malignant samples decreased gradually with dedifferentiation. Expression pattern of MMP-26 mRNA in normal, hyperplastic, premalignant and malignant endometrial tissue strongly co-variated with that of ER alpha.CONCLUSION:Co-expression of MMP-26 and ER alpha in normal and pathological endometrial tissue suggests possible regulation of MMP-26 gene by estrogen.

Objectives. We have previously shown that high activity of lactate dehydrogenase (LD) isoenzymes 4 and 5 in uterine aspirates is a marker for endometrial carcinoma. The purpose of this study was to identify an abnormal activity profile of LD2-5 using LD1 as an internal standard, thereby being able to dispense with measurement of the total LD activity. The profile was subsequently tested for diagnostic power in clinical settings.Methods. We used data from 11 cases of endometrial cancer. Each isoenzyme was estimated relative to the activity of LD I (LD1= 1.0). Based on the lowest level found for each of LD2 - 5 in the I I cases, the cut-off levels for an "abnormal profile" were identified. The abnormal profile was subsequently tested for diagnostic power in a group of postmenopausal women at risk for endometrial cancer, that is, they had experienced vaginal bleeding (n = 100). A second group of asymptomatic postmenopausal women (n = 366) had endouterine aspiration performed as part of a regular gynecologic check up to evaluate the prevalence of an abnormal LD isoenzyme profile.Results. In the group of postmenopausal women who had experienced vaginal bleeding, abnormal profile was found in 27 cases: 14 with adenocarcinoma and 13 with benign histology. All cases with normal profile had benign histology. Thus, sensitivity as well as the negative predictive value was 100%. In the group of asymptomatic women, abnormal LD isoenzyme profile was found in 15 cases (4.1%). All had benign histology.Conclusions. The LD isoenzyme profile detects endometrial malignancy with high accuracy, and equally important, a normal profile excludes malignancy. The profile, which uses relative rather than absolute activity levels, based on LD1 as an internal standard, has the great advantage of being independent of both the dilution factor and the aspiration technique. Larger studies comparing the LD isoenzyme activity profile with ultrasonographic evaluation and biopsy histology are needed. (C) 2004 Elsevier Inc. All rights reserved.

OBJECTIVE:To examine the endometrial expression pattern of messenger RNA (mRNA) for selected matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases-1 (TIMP-1).DESIGN:Experimental study.SETTING:Department of Obstetrics and gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obsterics and Gynecology, University Hospital, Luna, Sweden.METHODS:We studied MMP-1, -3, -7, -10, -11, -12, -13, -14, -16, -26 and TIMP-1 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. mRNA extracted from frozen tissue samples was quantitated using real time PCR.RESULTS:Four distinct patterns of MMP mRNA expression were detected in cycling endometrium. MMP-1, -3, -10, and -12 were expressed predominantly in perimenstrual period, MMP-7 and -11 had highest levels in proliferative phase, MMP-13, -14 and -16 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period. Levels of TIMP-1 mRNA remained unchanged during the cycle.CONCLUSION:The specific endometrial expression profiles of MMPs during menstrual cycle point to their specific biologic roles during the cycle. MMP-26 exhibits a unique expression pattern.

OBJECTIVETo examine the expression pattern of some novel matrix metalloproteinases (MMPs) in cycling endometrium.DESIGNExperimental study.SETTINGDepartment of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden.METHODSWe studied MMP-12, -16, -17, -19 and -26 mRNA in 39 normal endometrial samples obtained across the menstrual cycle. Based on histological examination, all specimens were classified according to an ideal 28 day menstrual cycle as early (n=8), mid (n=6) and late (n=7) proliferative phase, early (n=4), mid (n=4) and late (n=8) secretory phase and menstrual (n=3) phase. Cycle variation was examined in frozen samples using in situ hybridization.RESULTSThree distinct pattern of MMP mRNA expression were detected in cycling endometrium. MMP-12 was expressed predominantly in perimenstrual period, MMP-16, -17 and 19 were expressed throughout the cycle and MMP-26 was found to be maximal in periovulatory period.CONCLUSIONDifferent endometrial expression patterns of novel MMPs during menstrual cycle may indicate their specific roles for menstruation, endometrial growth and remodelling and implantation.

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.

OBJECTIVE:To examine possible estrogen dependent endometrial expression of MMP-26 in vitro.DESIGN:Experimental study.SETTING:Department of Obstetrics and Gynecology of the Palacky University Medical School and University Hospital, Olomouc, Czech Republic, Department of Obstetrics and Gynecology, University Hospital, Lund, Sweden.METHODS:We studied MMP-26 mRNA in 14 normal endometrial samples obtained from the proliferative phase of the menstrual cycle. Samples were cultured for five days either with estradiol alone or in combination with progesterone. Samples cultured with ethanol represented control groups. MMP-26 mRNA expression was examined in frozen samples using in situ hybridization. Immunohistochemistry was used to study the presence of estrogen and progesterone receptors in endometrial explants.RESULTS:MMP-26 mRNA expression was highest in fresh (non cultured) samples. Signal intensity decreased during the first two days of culture and was negligable in the following days. Nuclear intensity for estrogen and progesterone receptor was high after five days of culture.CONCLUSION:We did not find MMP-26 mRNA in vitro expression to be directly estrogen dependent.