activity. The objective of this study was to evaluate the PAI-1 and TAFI genotypes in endometriosis patients and controls. DESIGN: Prospective study.
activity. The objective of this study was to evaluate the PAI-1 and TAFI genotypes in endometriosis patients and controls. DESIGN: Prospective study MATERIALS AND METHODS: Sixty-two women (45 with laparoscopically confirmed endometriosis and 17 control women without endometriosis) were enrolled. Genomic DNA was extracted from peripheral blood leukocytes using a Qiagen kit. The PAI-1 promoter genotype was determined by PCR amplification of genomic DNA of all subjects using the allele-specific primers and gel electrophoresis. One of the PCR products was purified and sequenced to confirm the gel electrophoresis results. The TAFI hr325Ile polymorphism was detected by PCR reaction followed by digestion with the SpeI restriction enzyme and subsequent gel electrophoresis. Each subject was classified as 4G/4G, 4G/5G, or 5G/5G for the PAI-1 genotype, and ⫹ve or -ve for the TAFI hr325Ile polymorphism. RESULTS: Demographic variables were comparable between the 2 groups. We found a statistically significant difference in the distribution of PAI-1 genotypes between groups. Forty-four out of 45 (98%) women with endometriosis had at least one 4G allele. Thirty of 45 women with endometriosis (66.7 %) were homozygous for the 4G/4G genotype, which was found in only one of 17 (5.8%) controls. In contrast, the 5G/5G genotype was found in only one of 45 (2.2%) women with endometriosis but was seen in 11 out of 17 (64.8%) controls. The TAFI hr325Ile polymorphism was observed in 3 out 4 women with endometriosis, and in 1 of 2 control subjects tested to date. The final determination of the incidence of TAFI polymorphism in this group of women is ongoing. CONCLUSIONS: The presence of the PAI-1 4G allele or the TAFI hr325Ile polymorphism, both associated with hypofibrinolysis, was found significantly more often in women with endometriosis compared to controls. The combination of both hypofibrinolytic genotypes could further impair fibrinolysis. Inhibition of fibrinolytic activity and persistence of fibrin clot in the peritoneal cavity of women with retrograde menstruation may result in the initiation of endometriotic lesions. Supported by: None
P-107 WITHDRAWN P-108 Desogestrel Suppresses Cell Proliferation and Enhances Apoptosis of Eutopic Endometrial Tissue From Patients With Endometriosis. G. Meresman, V. Abello, R. I. Baran˜ao, L. Auge, M. Tesone, C. Sueldo. Instituto de Biologia y Medicina Experimental, Ciudad de Buenos Aires, Argentina; IFER, Ciudad de Buenos Aires, Argentina; CEGYR, Ciudad de Buenos Aires, Argentina. OBJECTIVE: We demonstrated recently that combination oral contraceptives significantly diminished cell proliferation and induced apoptosis in eutopic endometrial tissue from endometriosis patients (Meresman et al. Fert Steril 77: 1141-7, 2002). The use of progestin-only compounds is an effective therapy in the treatment of painful symptoms associated with endometriosis, and showed a good cost-effectiveness balance. Also, Amezcua et al recently demonstrated that apoptosis is an early event in progestin therapy for endometrial hyperplasia (Gynecol Oncol 2000; 79: 169-76). However there is limited data evaluating the effect of desogestrel treatment on endometrial growth in endometriosis. Therefore, the purpose of this study was to assess the effect of desogestrel (Cerazette, Organon, Argentina) on cell proliferation and apoptosis in eutopic endometrial tissue from patients with endometriosis. DESIGN: Apoptosis and cell proliferation were evaluated in vitro in eutopic endometrial tissue from endometriosis patients MATERIALS AND METHODS: 12 endometriosis patients had an endometrial biopsy before and after 30 days of oral desogestrel treatment at 75 g/day (Cerazette).The assessment of cell proliferation was done by studying the nuclear protein Ki-67 by using immunohistochemical techniques before and after desogestrel treatment. Tissue sections were incubated with anti-human Ki-67 rabbit monoclonal antibody (Dako, USA) following incubation with anti-rabbit-peroxidase conjugate (Dako, USA). Binding was visualized by incubating sections with DAB, and
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Abstracts
lightly counterstaining with hematoxylin. Immunoreactivity for Ki-67 is only expressed in dividing cells. For apoptosis quantification, the same tissue sections were processed for in situ immunocytochemical localization of nuclei exhibiting DNA fragmentation, by the technique of terminal deoxynucleotidyl transferase-mediated dUTP digoxygenin nickend labeling (TUNEL), using an apoptosis detection kit (Chemicon, USA). Proliferation and apoptosis were measured as the number of positive cells/field at 1000x magnification. Comparisons among groups were performed by Kruskal-Wallis nonparametric ANOVA test, and Dunn⬘s multiple comparison test. RESULTS: Before desogestrel, endometriosis patients showed a significant increase in cell proliferation at the epithelial fraction in comparison to after treatment: 2.66 (before desogestrel) vs. 0.73 (after desogestrel) expressed as positive cells/field (p⫽0.03). There was also a significant decrease observed in apoptosis of epithelial endometrial cells, 0.37 (before desogestrel) and 1.16 (after desogestrel) (p⫽0.01) expressed as apoptotic cells/field. CONCLUSIONS: Our results showed that a progestin-only treatment with desogestrel significantly diminished cell proliferation and induced apoptosis in eutopic endometrium from endometriosis patients. This data may have clinical relevance in the prevention and recurrence of endometriosis. Supported by: Roemmers Foundation and ANPCyT, Buenos Aires, Argentina.
P-109 Antioxidants Increase Apoptosis in Eutopic Endometrium of Subjects With Endometriosis. M. Song, C. E. Dominguez, S. Parthasarathy, A. A. Murphy. Emory University School of Medicine, Atlanta, GA; Louisiana State University, New Orleans, LA. OBJECTIVE: To determine if continuous antioxidants regulate apoptosis in eutopic and ectopic endometrium of subjects with endometriosis. DESIGN: Randomized placebo control trial of antioxidants with endometrial biopsy obtained at the time of surgery. MATERIALS AND METHODS: Eutopic endometrium was obtained from ten subjects with endometriosis randomized to vitamin E 1000 IU and Vitamin C 1000mg supplementation or placebo for two months. Normal endometrium was obtained from subjects without endometriosis. An endometrial biopsy in the proliferative phase of the cycle was obtained. In Situ Death Detection Kit by Roche was used. HSCORE (Fertil Steri 69(6): 1092,1998) was used to semi-quantify TUNEL assay levels. RESULTS: Positive staining for TUNEL was noted in the nuclei of endometrial glandular epithelial cells and stromal cells. Using HSCORE, the endometrium from endometriosis subjects on placebo (1.69⫾0.66) showed a statistically significant decrease when compared to that from normal subjects (3.10⫾0.76; P⬍0.01). Staining for TUNEL in the endometrium from endometriosis subjects on Vitamin E and C (2.72⫾1.01; P⬍0.05) showed a statistically significant increase when compared to that from endometriosis subjects on placebo (1.69⫾0.66). There were no significant difference between the staining from the subjects on Vitamin E, C and normal subjects (P⬎0.05). The staining in ectopic endometrium did not show significant change in the subjects on Vitamin E and C compared to the subjects on placebo. Statistical evaluation was performed by multi-variate ANOVA and post hoc testing with the Bonferroni corrected T-test. CONCLUSIONS: The results demonstrate that Vitamin E and C increases apoptotic levels in the eutopic endometrium of subjects with endometriosis. How might this finding with these antioxidants be reconciled with the fact that oxidative stress has also been shown to decrease Bcl-2 and increase apoptosis, albeit in other cell systems? Although this question has not yet been answered, some clues may be provided by the paradoxical effects of some antioxidants being able to act as pro-oxidants under certain conditions. For example, our group has shown that estradiol appears to act as a pro-oxidant in a milieu with increased oxidative stress (PNAC 1992;89: 10628).To this end, the endometrium is rich in peroxidases that metabolize estradiol into the estradiol/phenoxy radical, catechol-estrogens and the quinone metabolites of estradiol and estrone. Estradiol also induces myeloperoxidase (MPO) and becomes a pro-oxidant. We have described the phenomenon of phenolic antioxidants, such as Vitamin E, as proxidants in the presence of peroxidases (JCI.1995; 95: 2594). Thus, our finding of increased apoptosis in the endometrium may be explained by this paradoxical effect of estradiol and Vitamin E. More importantly, it suggests that
Vol. 84, Suppl 1, September 2005
