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Increased cell proliferation in experimental induced endometriosis in rabbits
CONCLUSIONS: Prolonged GnRHa suppression may increase the risk of IVF treatment cancellation. Hence, the claim that it improves pregnancy success without accounting for treatment cancellation remains debatable. Supported by: None.
P-318 APPLICATION OF THE CICLOOXYGENASE-2 INHIBITOR, CELECOXIB, FOR THE TREATMENT OF ENDOMETRIOSIS: AN IN-VITRO STUDY. C. N. Olivares, M. A. Bilotas, M. Borghi, C. Sueldo, M. Tesone, G. F. Meresman. Instituto de Biologıa y Medicina Experimental, Buenos Aires, Argentina; Centro de Ginecologıa y Reproduccio´n, Buenos Aires, Argentina; Hospital de Clınicas, Buenos Aires, Argentina. OBJECTIVE: Cyclooxygenase-2 (COX-2) specific inhibitors block cell growth and induce apoptosis and cell arrest in various tumour cell lines. Also, it has been shown that COX-2 inhibitors, such as celecoxib, are potent inhibitors of angiogenesis, both in-vitro and in-vivo. We have previously shown that celecoxib inhibits proliferation and induces apoptosis in cultured endometrial epithelial cells (EEC) from patients with endometriosis (EDT) and controls. In the present study we evaluated the effect of celecoxib on in-vitro vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) secretion, as well as on COX-2 expression in EEC from EDT patients. DESIGN: Experimental in vitro study. MATERIALS AND METHODS: Endometrial biopsies were taken during the proliferative phase in 13 untreated patients with EDT diagnosed by laparoscopy. Purification and culture of EEC were done according to the technique described by Meresman et al. (Fertil Steril, 2003). Cultures were treated with celecoxib in doses between 25 and 100 mM. COX-2 expression was assesed by western blot and immunocytochemistry. VEGF and PGE2 secretion were evaluated by ELISA. Statistical analysis was done by KruskalWallis nonparametric ANOVA test, and Dunn´s multiple comparisons test. RESULTS: COX-2 expression was shown by immunocytochemistry in all celecoxib treated EEC cultures and in basal conditions. Western blot analysis for COX-2 showed no statistical change in protein expression in 25–75 mM celecoxib treated EEC. However, at 100 mM there was a significant increase in COX-2 expression (P<0.05). To determine whether COX-2 activity was affected by celecoxib treatment, PGE2 production was measured in conditioned medium from EEC after celecoxib treatment (25, 50 and 100 mM). All doses of celecoxib significantly inhibited COX-2 activity (P<0.001 vs. basal). We also evaluated the levels of VEGF in EEC treated with celecoxib or vehicle (basal). Celecoxib treatment significantly reduced VEGF levels in EEC from patients with EDT (25 mM, P<0.001; 50 mM, P<0.05 and 100 mM, P<0.001 vs. basal). CONCLUSIONS: Our data suggest that removal of negative feedback by celecoxib treatment results in COX-2 induction in EEC from patients with EDT. We also showed that treatment with celecoxib reduced VEGF levels as well as PGE2 production. These findings support the investigation of COX-2 inhibitors as a treatment option in EDT. Supported by: This study was supported by Roemmers Foundation, and the National Research Council of Argentina (CONICET), Buenos Aires, Argentina.
P-319 COMPREHENSIVE ANALYSIS OF GENE EXPRESSION ALTERATION IN RESPONSE TO GONADOTROPIN RELEASING HORMONE (GNRH) ANALOGUE IN ENDOMETRIOTIC STROMAL CELLS. Y. Tanaka, K. Kato, H. Toh, K. Hashimoto, M. Nozaki, N. Wake. Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Division of Bioinformatics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Kyushu Central Hospital, Fukuoka, Japan. OBJECTIVE: To analyze gene expression alteration comprehensively in response to GnRH analogue in endometriotic stromal cells. DESIGN: Laboratory design. MATERIALS AND METHODS: Four women of reproductive age untreated with GnRH analogue were enrolled in this study. Stromal cells were collected from endometriotic tissues, and when stromal cells reached about 80% confluency, cells were incubated in the presence or absence of 100 nM buserelin acetate for 24 hours. RNA extraction, cDNA synthesis
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and cRNA amplification were performed, and hybridized to Affymetrix Human Genome U133 plus 2.0 Arrays. The arrays were washed, stained and scanned by using a confocal scanner. Expression signals for each microarray were normalized using GeneSpring 7.3.1. software. Differentially expressed (DE) genes were identified, and with the list of DE genes with known symbols and functions, we searched Kyoto Encyclopedia of Genes and Genomes(KEGG) database to identify biological pathways in which these genes were involved. Quantitative real-time RT-PCR was performed to validate the results of microarrays. RESULTS: 52 genes whose fold changes were greater than 2 and less than 0.5 were picked up. In two-way hierarchical cluster analysis of gene expression in 8 samples and 939 genes/expression sequence tags (ESTs) that are DE, the cluster depicts two groups of genes and two groups of samples (GnRH treated and untreated). By use of KEGG database, we can identify several novel biological pathways in response to GnRH analogue, such as purine and pyrimidine metabolism and regulation of actin cytoskeleton. CONCLUSIONS: These results suggest the possibility that GnRH analogue may directly modulate genes involved in DNA synthesis and cell motility. Supported by: None.
P-320 INCREASED CELL PROLIFERATION IN EXPERIMENTAL INDUCED ENDOMETRIOSIS IN RABBITS. M. F. Silva de Sa, J. C. Rosa e Silva, S. B. Garcia, A. C. J. S. Rosa e Silva, F. J. C. dos Reis, A. A. Nogueira. Obstetrics and Gynecology, Faculty of Medicine of Ribeirao Preto- University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil; Pathology, Faculty of Medicine of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil. OBJECTIVE: To characterize the pattern of cell proliferation and apoptosis of topic and ectopic endometrium in rabbits four and eight weeks after endometrium implantation for induction of endometriotic lesions. DESIGN: Experimental study with 20 female, virgins and adult New Zeland rabbits, submitted to laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a 5mm tissue segment to the pelvic peritoneum. MATERIALS AND METHODS: The animals were divided in: Group 1 which was sacrificed after 4 weeks (4 w) of endometriosis induction and group 2 eight weeks (8 w) after. The lesion was excised for lhistological analysis together with the opposite uterine horn to verify the presence of endometrial gland and stroma. Immunohistochemistry reactions were performed in order to study cell proliferation (PCNA) and apoptosis (Fas) in the topic and ectopic endometrium. A Cell Proliferation Index (CPI) and an Apoptotic Index (AI) were calculated considering the number of marked cells per 1000 counted. The Tissue Homeostasis Index (THI) was considered to be the relation between CPI and AI. Gland and stroma were analyzed separately. RESULTS: There was an increase in the CPI of the ectopic tissue when comparing to the topic one (Table 1). However, when comparing the ectopic lesions, there was no difference between four and eight weeks of induction. Analyzing only the AI, there was no difference between the topic and the ectopic endometrium with four weeks, nevertheless there was a significant difference in the glandular tissue of the lesions with eight weeks when comparing topic and ectopic tissues (0.082 0.02 and 0.099 0.01, respectively (P¼0, 002)). Based on THI there was a tendency to cell proliferation on these tissues, always with a THI (CPI/AI) higher than 1 (Table 1). TABLE 1. CPI and THI in gland and stroma after 4 and 8 weeks of implantation
CPI Ectopic Gl 4 w St 4 w Gl 8 w St 8 w
0.25 0.04 (a) 0.20 0.05 (a) 0.22 0.04 (b) 0.20 0.04 (b)
THI Topic
Ectopic
0.18 0.04 (a) 2.3 0.83 (b) 0.14 0.04 (a) 1.68 0.61 (b) 0.17 0.03 (b) 2.95 1.14 (b) 0.16 0.03 (b) 2.33 0.85 (b)
Topic 1.66 0.53 (b) 1.26 0.57 (b) 1.75 0.49 (b) 1.75 0.50 (b)
Gl ¼ gland; St ¼ stroma; a < 0.001; b < 0.05. CONCLUSIONS: Ectopic lesions seem to have a higher cell proliferation than the topic endometrium, tending to present an uncontrolled tissue growth in the endometriotic inducted lesions. Supported by: None.
Vol. 88, Suppl 1, September 2007
P-318 APPLICATION OF THE CICLOOXYGENASE-2 INHIBITOR, CELECOXIB, FOR THE TREATMENT OF ENDOMETRIOSIS: AN IN-VITRO STUDY. C. N. Olivares, M. A. Bilotas, M. Borghi, C. Sueldo, M. Tesone, G. F. Meresman. Instituto de Biologıa y Medicina Experimental, Buenos Aires, Argentina; Centro de Ginecologıa y Reproduccio´n, Buenos Aires, Argentina; Hospital de Clınicas, Buenos Aires, Argentina. OBJECTIVE: Cyclooxygenase-2 (COX-2) specific inhibitors block cell growth and induce apoptosis and cell arrest in various tumour cell lines. Also, it has been shown that COX-2 inhibitors, such as celecoxib, are potent inhibitors of angiogenesis, both in-vitro and in-vivo. We have previously shown that celecoxib inhibits proliferation and induces apoptosis in cultured endometrial epithelial cells (EEC) from patients with endometriosis (EDT) and controls. In the present study we evaluated the effect of celecoxib on in-vitro vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) secretion, as well as on COX-2 expression in EEC from EDT patients. DESIGN: Experimental in vitro study. MATERIALS AND METHODS: Endometrial biopsies were taken during the proliferative phase in 13 untreated patients with EDT diagnosed by laparoscopy. Purification and culture of EEC were done according to the technique described by Meresman et al. (Fertil Steril, 2003). Cultures were treated with celecoxib in doses between 25 and 100 mM. COX-2 expression was assesed by western blot and immunocytochemistry. VEGF and PGE2 secretion were evaluated by ELISA. Statistical analysis was done by KruskalWallis nonparametric ANOVA test, and Dunn´s multiple comparisons test. RESULTS: COX-2 expression was shown by immunocytochemistry in all celecoxib treated EEC cultures and in basal conditions. Western blot analysis for COX-2 showed no statistical change in protein expression in 25–75 mM celecoxib treated EEC. However, at 100 mM there was a significant increase in COX-2 expression (P<0.05). To determine whether COX-2 activity was affected by celecoxib treatment, PGE2 production was measured in conditioned medium from EEC after celecoxib treatment (25, 50 and 100 mM). All doses of celecoxib significantly inhibited COX-2 activity (P<0.001 vs. basal). We also evaluated the levels of VEGF in EEC treated with celecoxib or vehicle (basal). Celecoxib treatment significantly reduced VEGF levels in EEC from patients with EDT (25 mM, P<0.001; 50 mM, P<0.05 and 100 mM, P<0.001 vs. basal). CONCLUSIONS: Our data suggest that removal of negative feedback by celecoxib treatment results in COX-2 induction in EEC from patients with EDT. We also showed that treatment with celecoxib reduced VEGF levels as well as PGE2 production. These findings support the investigation of COX-2 inhibitors as a treatment option in EDT. Supported by: This study was supported by Roemmers Foundation, and the National Research Council of Argentina (CONICET), Buenos Aires, Argentina.
P-319 COMPREHENSIVE ANALYSIS OF GENE EXPRESSION ALTERATION IN RESPONSE TO GONADOTROPIN RELEASING HORMONE (GNRH) ANALOGUE IN ENDOMETRIOTIC STROMAL CELLS. Y. Tanaka, K. Kato, H. Toh, K. Hashimoto, M. Nozaki, N. Wake. Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Division of Bioinformatics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Kyushu Central Hospital, Fukuoka, Japan. OBJECTIVE: To analyze gene expression alteration comprehensively in response to GnRH analogue in endometriotic stromal cells. DESIGN: Laboratory design. MATERIALS AND METHODS: Four women of reproductive age untreated with GnRH analogue were enrolled in this study. Stromal cells were collected from endometriotic tissues, and when stromal cells reached about 80% confluency, cells were incubated in the presence or absence of 100 nM buserelin acetate for 24 hours. RNA extraction, cDNA synthesis
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Abstracts
and cRNA amplification were performed, and hybridized to Affymetrix Human Genome U133 plus 2.0 Arrays. The arrays were washed, stained and scanned by using a confocal scanner. Expression signals for each microarray were normalized using GeneSpring 7.3.1. software. Differentially expressed (DE) genes were identified, and with the list of DE genes with known symbols and functions, we searched Kyoto Encyclopedia of Genes and Genomes(KEGG) database to identify biological pathways in which these genes were involved. Quantitative real-time RT-PCR was performed to validate the results of microarrays. RESULTS: 52 genes whose fold changes were greater than 2 and less than 0.5 were picked up. In two-way hierarchical cluster analysis of gene expression in 8 samples and 939 genes/expression sequence tags (ESTs) that are DE, the cluster depicts two groups of genes and two groups of samples (GnRH treated and untreated). By use of KEGG database, we can identify several novel biological pathways in response to GnRH analogue, such as purine and pyrimidine metabolism and regulation of actin cytoskeleton. CONCLUSIONS: These results suggest the possibility that GnRH analogue may directly modulate genes involved in DNA synthesis and cell motility. Supported by: None.
P-320 INCREASED CELL PROLIFERATION IN EXPERIMENTAL INDUCED ENDOMETRIOSIS IN RABBITS. M. F. Silva de Sa, J. C. Rosa e Silva, S. B. Garcia, A. C. J. S. Rosa e Silva, F. J. C. dos Reis, A. A. Nogueira. Obstetrics and Gynecology, Faculty of Medicine of Ribeirao Preto- University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil; Pathology, Faculty of Medicine of Ribeirao Preto - University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil. OBJECTIVE: To characterize the pattern of cell proliferation and apoptosis of topic and ectopic endometrium in rabbits four and eight weeks after endometrium implantation for induction of endometriotic lesions. DESIGN: Experimental study with 20 female, virgins and adult New Zeland rabbits, submitted to laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a 5mm tissue segment to the pelvic peritoneum. MATERIALS AND METHODS: The animals were divided in: Group 1 which was sacrificed after 4 weeks (4 w) of endometriosis induction and group 2 eight weeks (8 w) after. The lesion was excised for lhistological analysis together with the opposite uterine horn to verify the presence of endometrial gland and stroma. Immunohistochemistry reactions were performed in order to study cell proliferation (PCNA) and apoptosis (Fas) in the topic and ectopic endometrium. A Cell Proliferation Index (CPI) and an Apoptotic Index (AI) were calculated considering the number of marked cells per 1000 counted. The Tissue Homeostasis Index (THI) was considered to be the relation between CPI and AI. Gland and stroma were analyzed separately. RESULTS: There was an increase in the CPI of the ectopic tissue when comparing to the topic one (Table 1). However, when comparing the ectopic lesions, there was no difference between four and eight weeks of induction. Analyzing only the AI, there was no difference between the topic and the ectopic endometrium with four weeks, nevertheless there was a significant difference in the glandular tissue of the lesions with eight weeks when comparing topic and ectopic tissues (0.082 0.02 and 0.099 0.01, respectively (P¼0, 002)). Based on THI there was a tendency to cell proliferation on these tissues, always with a THI (CPI/AI) higher than 1 (Table 1). TABLE 1. CPI and THI in gland and stroma after 4 and 8 weeks of implantation
CPI Ectopic Gl 4 w St 4 w Gl 8 w St 8 w
0.25 0.04 (a) 0.20 0.05 (a) 0.22 0.04 (b) 0.20 0.04 (b)
THI Topic
Ectopic
0.18 0.04 (a) 2.3 0.83 (b) 0.14 0.04 (a) 1.68 0.61 (b) 0.17 0.03 (b) 2.95 1.14 (b) 0.16 0.03 (b) 2.33 0.85 (b)
Topic 1.66 0.53 (b) 1.26 0.57 (b) 1.75 0.49 (b) 1.75 0.50 (b)
Gl ¼ gland; St ¼ stroma; a < 0.001; b < 0.05. CONCLUSIONS: Ectopic lesions seem to have a higher cell proliferation than the topic endometrium, tending to present an uncontrolled tissue growth in the endometriotic inducted lesions. Supported by: None.
Vol. 88, Suppl 1, September 2007