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Sodium butyrate (NaBu) decreases proliferation of stromal cells derived from endometriosis and Ishikawa cells in culture
fragmentation was then assessed by means of the TUNEL assay. Sperm DNA fragmentation was correlated with the stage of the endometriosis as well as the duration of infertility. RESULTS: Sperm DNA damage showed significant association with both the stage of endometriosis and duration of infertility (see Table). TABLE.
CONCLUSIONS: Sperm DNA damage shows positive correlation with the stage of endometriosis as well as with the duration of infertility. This may explain poor fertility seen in these patients. Supported by: None.
P-295 FIBRINOLYTIC SYSTEM MULTIPLE GENE POLYMORPHISMS AND INITIATION OF ENDOMETRIOSIS A NOVEL EVIDENCE. M. A. Bedaiwy, T. Falcone, E. Mascha, R. F. Casper. Obstetrics and Gynecology, The Cleveland Clinic Foundation, Cleveland, OH; Biostatics, The Cleveland Clinic Foundation, Cleveland, OH; Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada. OBJECTIVE: To examine single gene polymorphisms in the inhibitors of fibrinolysis; the plasminogen activator inhibitor type 1 (PAI-1), and tissue activatable fibrinoloysis inhibitor (TAFI), and stimulators of fibrinolysis; tissue (tPA) and urokinase (uPA) plasminogen activator genes in endometriosis patients and controls. DESIGN: Prospective study. MATERIALS AND METHODS: Two hundred and twelve women (122 with laparoscopically confirmed endometriosis and 90 control women without endometriosis) were included. Genomic DNA was extracted from peripheral blood leukocytes PAI-1 gene 4G/5G, TAFI gene C325T, tPA gene 7351C/T, uPA-6 gene C/T and uPA 7 gene C/T polymorphisms were determined by PCR amplification of genomic DNA of all subjects. We used logistic regression to assess the associations between hypofibrinolytic 4G/4G and 4G/5G PAI-1, C325T TAFI and t-PA 7351C/T polymorphisms, respectively, and endometriosis. For uPA, we assessed associations between stage of endometriosis and the exon 6 and intron 7 polymorphisms using proportional odds logistic regression. RESULTS: We found a statistically significant difference in the distribution of PAI-1 genotypes between groups. The endometriosis group had significantly higher 4G/4G and 4G/5G combinations than the control group P<0.001, Odds Ratio ¼ 38.5 95% CI (12.2 to 122) for the 4G/4G and P<0.001, OR 8.98 95% CI (3.1 to 26) for the 4G/5G genotype. No significant differences were found in the distribution of SNPs of other genes. Using PAI1 and TAFI 325 genotype in a multivariable model predicts endometriosis fairly well. This is confirmed by the C-statistic of 0.821, which indicates good discrimination between endometriosis and controls for this dataset. Including TAFI 325 increases the C-statistic from 0.804 to 0.821, which is a noticeable improvement. There was the suggestion of a relationship between uPA6 and stage of endometriosis in a proportional odds logistic regression model (overall P¼0.056), with the odds of being in a higher stage greater for CT genotype compared to CC (odds ratio 2.3 (1.13, 4.79), P¼0.022). CONCLUSIONS: Persisting fibrin matrix may support the initiation of endometriotic lesions in the peritoneal cavity. This may explain why some women with retrograde menstruation develop endometriosis while others do not. In addition, UPA-6 polymnorphism known for a strong relationship with metastatic cancers could explain why certain women develop advanced disease while others not. Supported by: Candian institute of health research (CIHR).
S206
Abstracts
P-296 SODIUM BUTYRATE (NaBu) DECREASES PROLIFERATION OF STROMAL CELLS DERIVED FROM ENDOMETRIOSIS AND ISHIKAWA CELLS IN CULTURE. A. Demiroglu-Zergeroglu, B. Pelin, R. Attar, E. Karatekelioglu, E. Attar, S. E. Bulun. Molecular Biology, Gebze High Technology Institute, Kocaeli, Turkey; Obstetrics and Gynecology, Yeditepe University, Istanbul, Turkey; Obstetrics and Gynecology, Istanbul University, Istanbul, Turkey; IVF and Women’s Health, Dr. Pakize Tarzi Hospital, Istanbul, Turkey; Obstetrics and Gynecology, Northwestern University, Chicago, IL. OBJECTIVE: NaBu exerts potent positive effects on growth arrest and cell differentiation and induces apoptosis in vitro in various malignant tumor cell lines including breast cancer cell lines. Some derivatives of NaBu had been clinically evaluated in a phase I study after oral administration in patients with solid tumors. We have previously shown that NaBu has a promoter-specific inhibitory effect on aromatase expression in endomeriotic cells and taurine transporter (TauT) is a target of NaBu. In this study we have shown that NaBu inhibits endometriotic and Ishikawa cell growth in vitro. DESIGN: Prospective experimental study. MATERIALS AND METHODS: Human endometriotic tissue samples were obtained at the time of surgery from women undergoing laparascopy (n ¼ 3). Primer cell cultures were grown in DMEM/F12 containing mg/ml L-glutamine. A cell titer 96 Aqueous One Solution Cell Proliferation Assay kit was used to measure cell proliferation rate. Subculture cells were trypsinized and harvested. For each group assay 1 103 cells in 100 ml volumes were aliquoted into 96 well plates in duplicate, and then incubated for 24h. Cells were exposed to NaBu at 37 C for 0–96 h. To each well, 20 ml of MTS compound was added and the plate was incubated for 2 hours before reading. The average of the duplicate wells for each sample was calculated. RESULTS: NaBu inhibits endometritic and Ishicawa cell proliferation in a dose and time dependent manner. The effect of NaBu on cell proliferation was first observed at 24 h of treatment, and gradually increased at 48, 72 and 96 hours. The effect of NaBu was concentration dependent in both human endometriotic and Ishikawa cells. Endometriotic and Ishikawa cell proliferation were significantly decreased at the dose of 10 mM/mL and higher concentrations (P<0.05). CONCLUSIONS: We had previously provided the evidence that NaBu reduced the total P450 aromatase activity in endometriotic cells. Now, we have shown that it also inhibits the proliferation of endometriotic and Ishikawa cells. The observation that NaBu exerts potent effects on estrogen synthesis, and growth arrest of endometriotic cells and tumor cells has established its use as a novel therapeutic strategy in endometriosis and estrogen dependent adenoid cancers. The results presented here are encouraging to warrant an in vivo study and future clinical evaluation of the potential therapeutic benefits of NaBu in endometriosis and endometrial tumors. Supported by: NIH grant HD 36891 and a grant from Friends of Prentice.
P-297 EFFECT OF MATRIX METALLOPROTEINASE (MMP) INHIBITOR – DOXYCYCLINE ON THE INDUCTION OF ENDOMETRIOSIS IN AN EXPERIMENTAL RAT MODEL. H. B. Zeyneloglu, P. Akkaya, G. Onalan, P. Dursun. Obstetrics and Gynecology, Baskent University, Ankara, Turkey. OBJECTIVE: Doxycycline (DOX) a member a tetracycline family, have a number of nonantibiotic properties. DOX has been approved for treatment of periodontal disease via a mechanism of action linked to the inhibition of MMP activity. We aimed to assess the effect of DOX in a rat endometriosis model with prospective randomized study. DESIGN: Prospective randomized study. MATERIALS AND METHODS: Endometriosis was surgically induced in 40 rats by transplanting an autologous fragment of endometrial tissue onto the inner surface of the abdominal wall. After allowing 3 weeks for tissue growth, repeat laparotomies were performed on the animals to check the implants. After the animals were randomized into 4 groups; Group I: (controls) no medication; Group II: leuprolide acetate 1 mg/kg single dose, sc; Group III: low-dose DOX (5 mg/kg/d p.o.) and Group IV: high-dose doxycyclin (40 mg/kg/d p.o.). The treatment was initiated on the day of surgery and continuing for 3 weeks, was administered to the study groups. Three weeks later rats were killed and implants were evaluated morphologically. The immunohistochemical examination of the implants is underway at the time of writing of this abstract.
Vol. 88, Suppl 1, September 2007
CONCLUSIONS: Sperm DNA damage shows positive correlation with the stage of endometriosis as well as with the duration of infertility. This may explain poor fertility seen in these patients. Supported by: None.
P-295 FIBRINOLYTIC SYSTEM MULTIPLE GENE POLYMORPHISMS AND INITIATION OF ENDOMETRIOSIS A NOVEL EVIDENCE. M. A. Bedaiwy, T. Falcone, E. Mascha, R. F. Casper. Obstetrics and Gynecology, The Cleveland Clinic Foundation, Cleveland, OH; Biostatics, The Cleveland Clinic Foundation, Cleveland, OH; Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada. OBJECTIVE: To examine single gene polymorphisms in the inhibitors of fibrinolysis; the plasminogen activator inhibitor type 1 (PAI-1), and tissue activatable fibrinoloysis inhibitor (TAFI), and stimulators of fibrinolysis; tissue (tPA) and urokinase (uPA) plasminogen activator genes in endometriosis patients and controls. DESIGN: Prospective study. MATERIALS AND METHODS: Two hundred and twelve women (122 with laparoscopically confirmed endometriosis and 90 control women without endometriosis) were included. Genomic DNA was extracted from peripheral blood leukocytes PAI-1 gene 4G/5G, TAFI gene C325T, tPA gene 7351C/T, uPA-6 gene C/T and uPA 7 gene C/T polymorphisms were determined by PCR amplification of genomic DNA of all subjects. We used logistic regression to assess the associations between hypofibrinolytic 4G/4G and 4G/5G PAI-1, C325T TAFI and t-PA 7351C/T polymorphisms, respectively, and endometriosis. For uPA, we assessed associations between stage of endometriosis and the exon 6 and intron 7 polymorphisms using proportional odds logistic regression. RESULTS: We found a statistically significant difference in the distribution of PAI-1 genotypes between groups. The endometriosis group had significantly higher 4G/4G and 4G/5G combinations than the control group P<0.001, Odds Ratio ¼ 38.5 95% CI (12.2 to 122) for the 4G/4G and P<0.001, OR 8.98 95% CI (3.1 to 26) for the 4G/5G genotype. No significant differences were found in the distribution of SNPs of other genes. Using PAI1 and TAFI 325 genotype in a multivariable model predicts endometriosis fairly well. This is confirmed by the C-statistic of 0.821, which indicates good discrimination between endometriosis and controls for this dataset. Including TAFI 325 increases the C-statistic from 0.804 to 0.821, which is a noticeable improvement. There was the suggestion of a relationship between uPA6 and stage of endometriosis in a proportional odds logistic regression model (overall P¼0.056), with the odds of being in a higher stage greater for CT genotype compared to CC (odds ratio 2.3 (1.13, 4.79), P¼0.022). CONCLUSIONS: Persisting fibrin matrix may support the initiation of endometriotic lesions in the peritoneal cavity. This may explain why some women with retrograde menstruation develop endometriosis while others do not. In addition, UPA-6 polymnorphism known for a strong relationship with metastatic cancers could explain why certain women develop advanced disease while others not. Supported by: Candian institute of health research (CIHR).
S206
Abstracts
P-296 SODIUM BUTYRATE (NaBu) DECREASES PROLIFERATION OF STROMAL CELLS DERIVED FROM ENDOMETRIOSIS AND ISHIKAWA CELLS IN CULTURE. A. Demiroglu-Zergeroglu, B. Pelin, R. Attar, E. Karatekelioglu, E. Attar, S. E. Bulun. Molecular Biology, Gebze High Technology Institute, Kocaeli, Turkey; Obstetrics and Gynecology, Yeditepe University, Istanbul, Turkey; Obstetrics and Gynecology, Istanbul University, Istanbul, Turkey; IVF and Women’s Health, Dr. Pakize Tarzi Hospital, Istanbul, Turkey; Obstetrics and Gynecology, Northwestern University, Chicago, IL. OBJECTIVE: NaBu exerts potent positive effects on growth arrest and cell differentiation and induces apoptosis in vitro in various malignant tumor cell lines including breast cancer cell lines. Some derivatives of NaBu had been clinically evaluated in a phase I study after oral administration in patients with solid tumors. We have previously shown that NaBu has a promoter-specific inhibitory effect on aromatase expression in endomeriotic cells and taurine transporter (TauT) is a target of NaBu. In this study we have shown that NaBu inhibits endometriotic and Ishikawa cell growth in vitro. DESIGN: Prospective experimental study. MATERIALS AND METHODS: Human endometriotic tissue samples were obtained at the time of surgery from women undergoing laparascopy (n ¼ 3). Primer cell cultures were grown in DMEM/F12 containing mg/ml L-glutamine. A cell titer 96 Aqueous One Solution Cell Proliferation Assay kit was used to measure cell proliferation rate. Subculture cells were trypsinized and harvested. For each group assay 1 103 cells in 100 ml volumes were aliquoted into 96 well plates in duplicate, and then incubated for 24h. Cells were exposed to NaBu at 37 C for 0–96 h. To each well, 20 ml of MTS compound was added and the plate was incubated for 2 hours before reading. The average of the duplicate wells for each sample was calculated. RESULTS: NaBu inhibits endometritic and Ishicawa cell proliferation in a dose and time dependent manner. The effect of NaBu on cell proliferation was first observed at 24 h of treatment, and gradually increased at 48, 72 and 96 hours. The effect of NaBu was concentration dependent in both human endometriotic and Ishikawa cells. Endometriotic and Ishikawa cell proliferation were significantly decreased at the dose of 10 mM/mL and higher concentrations (P<0.05). CONCLUSIONS: We had previously provided the evidence that NaBu reduced the total P450 aromatase activity in endometriotic cells. Now, we have shown that it also inhibits the proliferation of endometriotic and Ishikawa cells. The observation that NaBu exerts potent effects on estrogen synthesis, and growth arrest of endometriotic cells and tumor cells has established its use as a novel therapeutic strategy in endometriosis and estrogen dependent adenoid cancers. The results presented here are encouraging to warrant an in vivo study and future clinical evaluation of the potential therapeutic benefits of NaBu in endometriosis and endometrial tumors. Supported by: NIH grant HD 36891 and a grant from Friends of Prentice.
P-297 EFFECT OF MATRIX METALLOPROTEINASE (MMP) INHIBITOR – DOXYCYCLINE ON THE INDUCTION OF ENDOMETRIOSIS IN AN EXPERIMENTAL RAT MODEL. H. B. Zeyneloglu, P. Akkaya, G. Onalan, P. Dursun. Obstetrics and Gynecology, Baskent University, Ankara, Turkey. OBJECTIVE: Doxycycline (DOX) a member a tetracycline family, have a number of nonantibiotic properties. DOX has been approved for treatment of periodontal disease via a mechanism of action linked to the inhibition of MMP activity. We aimed to assess the effect of DOX in a rat endometriosis model with prospective randomized study. DESIGN: Prospective randomized study. MATERIALS AND METHODS: Endometriosis was surgically induced in 40 rats by transplanting an autologous fragment of endometrial tissue onto the inner surface of the abdominal wall. After allowing 3 weeks for tissue growth, repeat laparotomies were performed on the animals to check the implants. After the animals were randomized into 4 groups; Group I: (controls) no medication; Group II: leuprolide acetate 1 mg/kg single dose, sc; Group III: low-dose DOX (5 mg/kg/d p.o.) and Group IV: high-dose doxycyclin (40 mg/kg/d p.o.). The treatment was initiated on the day of surgery and continuing for 3 weeks, was administered to the study groups. Three weeks later rats were killed and implants were evaluated morphologically. The immunohistochemical examination of the implants is underway at the time of writing of this abstract.
Vol. 88, Suppl 1, September 2007