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Concentration of osteoprotegerin (OPG) in peritoneal fluid is increased in women with endometriosis
Sa˜ o Paulo 66 women with surgically and histologically confirmed endometriosis (26 stages I-II and 40 with stages III-IV) and 38 women without the disease. The 306-base pair Alu insertion polymorphism in the intron G of the progesterone receptor gene was detected by polymerase chain reaction (PCR) and analized in a 2% agarose gel stained with Ethidium Bromide. RESULTS: After ANOVA analysis groups belonging to endometriosis stages III-IV showed statistically different incidence of PROGINS when compared to control group (A versus B ⫽ 0.001, A versus C ⫽ 0.001 and B versus C ⬍ 0.05, p-value is ⬍0.0001), see table I.
Extremely significant also by the Tukey-Kramer Multiple Comparisons Test q ⬎ 4.339 and p⬍0.05. CONCLUSION: According to our results PROGINS polymorphism is significantly associated with endometriosis in this Brazilian population. Supported by: FAPESP grant 03/04533–1 and grants from Centro Universita´ rio Fundac¸ a˜ o Santo Andre´ , Sa˜ o Paulo, Brazil. P-69 Increased proliferative activity in endometrium with endometriosis. S. S. Seo, K. J. Hwang, M. R. Kim, D. W. Park, Y. A. Kim, H.-S. Ryu. Ajou University School of Medicine, Suwon, Republic of Korea; College of Medicine, Inje University, Ilsan Paik Hospital, Ilsan, Republic of Korea. OBJECTIVE: It is well known that cellular proliferation of endometrial epithelial and stromal cells are increased in patients with endometriosis in vitro. Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. The BCL-2 family and Fas/FasL system have been extensively studied in human endometrium. Eutopic endometrium with endometriosis has some fundamental differences compared with normal endometrium without endometriosis, for examples apoptosis. The Ki-67 protein is a nuclear and nucleolar protein, which is tightly associated with somatic cell proliferation. We investigated the expression of Ki-67 and BCL-2, and measured cell proliferation in endometrium with or without endometriosis. DESIGN: Informed consent was obtained for endometrial biopsy from 2 groups of woman. The subjects were 20 samples. The study groups were 10 samples, which consisted of endometrial tissue with endometriosis. The remaining 10 samples were control group, which consisted of endometrial tissue without endometriosis. MATERIALS AND METHODS: Expression of Ki-67 and BCL-2 was immunohistochemically investigated by polyclonal antibody. Tissue samples were subjectively scored on a five-point scale by two independent observers. Evaluation of staining was performed by evaluation nomogram (Ota and Igarashi, 1993). Statistical analysis was performed using the independent t-test. P ⬍ 0.05 was considered to be statistically significant. Endometrial cells were cultured in 37°C incubator and the MTT colorimetric assay was used to detect cell proliferation after 72 h of incubation. RESULTS: In study group, expression of Ki-67 was significantly higher than that in the control group. But expression of BCL-2 was significantly lower than that in the control group. Cell proliferation was significantly increased in endometriosis group (P ⬍ 0.05). CONCLUSION: Our data may suggest that decreased BCL-2 expression may enhance proliferation of eutopic endometrial cells with endometriosis. Therefore, it was believed to correlated with pathological abnormalities in endometriosis. Supported by: None P-70 Concentration of osteoprotegerin (OPG) in peritoneal fluid is increased in women with endometriosis. M. Harada, Y. Osuga, C. Morimoto, T. Yano, O. Tsutsumi, Y. Taketani. University of Tokyo, Tokyo, Japan.
S160
OBJECTIVE: Osteoprotegerin (OPG) is suggested to exert an antiapoptotic effect in certain tumors. OPG, a soluble receptor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), acts as a decoy receptor and antagonizes apoptotic effect of TRAIL, which is induced by binding to Death Receptor (DR) 4 or DR5 with intracellular death domain. Anti-apoptotic environment for refluxed endometrial cells in the peritoneal cavity is proposed to be a causative factor for endometriosis. To address whether TRAIL/ OPG system in peritoneal environment is involved in the pathogenesis of endometriosis, concentrations of OPG and TRAIL in peritoneal fluids (PFs) of women with or without endometriosis were examined. DESIGN: Controlled clinical study. MATERIALS AND METHODS: PFs were collected from women with (n ⫽ 40) and without (n ⫽ 24) endometriosis. Concentrations of OPG and TRAIL in PF were measured by ELISA. Expressions of OPG and TRAIL mRNA in peritoneum, bone marrow derived cells in PF, and endometriotic tissues were analyzed by RT-PCR. Expression of DR5 mRNA in endometriotic tissues was also analyzed by RT-PCR. RESULTS: All of the PF samples contained detectable concentrations of OPG and TRAIL. OPG concentrations in PF of women with stage III/IV endometriosis were significantly higher than those without endometriosis (p ⫽ 0.006) and those with stage I/II endometriosis (p ⫽ 0.021). TRAIL/ OPG ratios were significantly lower in patients with stage III/IV endometriosis compared with controls (p ⫽ 0.032) and with stage I/II endometriosis (p ⫽ 0.015). Both OPG and TRAIL mRNA were detected in peritoneum, bone marrow derived cells in PF, and endometriotic tissues. DR5 mRNA was detected in endometriotic tissues. CONCLUSION: The increase in OPG levels with reduced TRAIL/ OPG ratios in PF of women with advanced stages of endometriosis implies that the peritoneal environment of these women may be permissive to survival of endometrial/ endometriotic cells. Supported by: None
P-71 TNF-alpha induced embryotoxicity and role of TNF-alpha blockerinfliximab on in vitro blastocyst development rate. X. Zhang, R. K. Sharma, A. Agarwal, T. Falcone. Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: TNF-␣ is required both for the establishment and maintenance of endometriosis. Evidence shows that peritoneal fluid from patients with endometriosis has elevated levels of TNF-␣ levels that may be responsible for endometriosis-associated infertility. TNF-blockers such as Remicade (Infliximab) may help neutralize the toxic effects of TNF-␣. In our earlier studies we demonstrated that infliximab is not embryotoxic at concentration of up to 400g/mL. Our study was designed to evaluate: 1) the embryotoxic effect of TNF-␣ that may be present in the peritoneal fluid of women with endometriosis, and 2) examine if infliximab could reverse the TNF-␣ induced embryotoxicity. DESIGN: Prospective in vitro study. MATERIALS AND METHODS: 150 two-cell mouse embryos were cultured in 6 different concentrations of diluted in human tubal fluid for 72 h. Blastocyst development rate (%BDR) was examined after 72 hours of incubation in the following concentrations of TNF-␣: 100, 200, 400, 500, 1000, and 2000ng/mL. Similarly, a second set containing infliximab (200g/mL) ⫹ TNF-␣(500ng/mL) was cultured under identical conditions to study if infliximab at this concentration was effective in reducing TNF-␣ induced embryotoxicity. RESULTS: A dose dependent decrease in %BDR was seen using different concentrations of TNF-␣ compared with controls (see Figure). A significant decrease in %BDR was seen at 400ng/mL compared with control (%BDR: 59% vs. 98%; P⬍0.0008), which further decreased to 21% at a concentration of 500ng/mL of TNF-␣ (P⬍0.05). Higher concentrations of TNF-␣ resulted in complete embryotoxicity (%BDR ⫽ 0). Infliximab alone had 100% BDR and was comparable with controls. Infliximab, in the presence of TNF-␣, completely neutralized the embryotoxic effect of TNF-␣ and %BDR increased significantly from 55% to 100% (P⬍0.05).
Vol. 82, Suppl. 2, September 2004
Extremely significant also by the Tukey-Kramer Multiple Comparisons Test q ⬎ 4.339 and p⬍0.05. CONCLUSION: According to our results PROGINS polymorphism is significantly associated with endometriosis in this Brazilian population. Supported by: FAPESP grant 03/04533–1 and grants from Centro Universita´ rio Fundac¸ a˜ o Santo Andre´ , Sa˜ o Paulo, Brazil. P-69 Increased proliferative activity in endometrium with endometriosis. S. S. Seo, K. J. Hwang, M. R. Kim, D. W. Park, Y. A. Kim, H.-S. Ryu. Ajou University School of Medicine, Suwon, Republic of Korea; College of Medicine, Inje University, Ilsan Paik Hospital, Ilsan, Republic of Korea. OBJECTIVE: It is well known that cellular proliferation of endometrial epithelial and stromal cells are increased in patients with endometriosis in vitro. Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. The BCL-2 family and Fas/FasL system have been extensively studied in human endometrium. Eutopic endometrium with endometriosis has some fundamental differences compared with normal endometrium without endometriosis, for examples apoptosis. The Ki-67 protein is a nuclear and nucleolar protein, which is tightly associated with somatic cell proliferation. We investigated the expression of Ki-67 and BCL-2, and measured cell proliferation in endometrium with or without endometriosis. DESIGN: Informed consent was obtained for endometrial biopsy from 2 groups of woman. The subjects were 20 samples. The study groups were 10 samples, which consisted of endometrial tissue with endometriosis. The remaining 10 samples were control group, which consisted of endometrial tissue without endometriosis. MATERIALS AND METHODS: Expression of Ki-67 and BCL-2 was immunohistochemically investigated by polyclonal antibody. Tissue samples were subjectively scored on a five-point scale by two independent observers. Evaluation of staining was performed by evaluation nomogram (Ota and Igarashi, 1993). Statistical analysis was performed using the independent t-test. P ⬍ 0.05 was considered to be statistically significant. Endometrial cells were cultured in 37°C incubator and the MTT colorimetric assay was used to detect cell proliferation after 72 h of incubation. RESULTS: In study group, expression of Ki-67 was significantly higher than that in the control group. But expression of BCL-2 was significantly lower than that in the control group. Cell proliferation was significantly increased in endometriosis group (P ⬍ 0.05). CONCLUSION: Our data may suggest that decreased BCL-2 expression may enhance proliferation of eutopic endometrial cells with endometriosis. Therefore, it was believed to correlated with pathological abnormalities in endometriosis. Supported by: None P-70 Concentration of osteoprotegerin (OPG) in peritoneal fluid is increased in women with endometriosis. M. Harada, Y. Osuga, C. Morimoto, T. Yano, O. Tsutsumi, Y. Taketani. University of Tokyo, Tokyo, Japan.
S160
OBJECTIVE: Osteoprotegerin (OPG) is suggested to exert an antiapoptotic effect in certain tumors. OPG, a soluble receptor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), acts as a decoy receptor and antagonizes apoptotic effect of TRAIL, which is induced by binding to Death Receptor (DR) 4 or DR5 with intracellular death domain. Anti-apoptotic environment for refluxed endometrial cells in the peritoneal cavity is proposed to be a causative factor for endometriosis. To address whether TRAIL/ OPG system in peritoneal environment is involved in the pathogenesis of endometriosis, concentrations of OPG and TRAIL in peritoneal fluids (PFs) of women with or without endometriosis were examined. DESIGN: Controlled clinical study. MATERIALS AND METHODS: PFs were collected from women with (n ⫽ 40) and without (n ⫽ 24) endometriosis. Concentrations of OPG and TRAIL in PF were measured by ELISA. Expressions of OPG and TRAIL mRNA in peritoneum, bone marrow derived cells in PF, and endometriotic tissues were analyzed by RT-PCR. Expression of DR5 mRNA in endometriotic tissues was also analyzed by RT-PCR. RESULTS: All of the PF samples contained detectable concentrations of OPG and TRAIL. OPG concentrations in PF of women with stage III/IV endometriosis were significantly higher than those without endometriosis (p ⫽ 0.006) and those with stage I/II endometriosis (p ⫽ 0.021). TRAIL/ OPG ratios were significantly lower in patients with stage III/IV endometriosis compared with controls (p ⫽ 0.032) and with stage I/II endometriosis (p ⫽ 0.015). Both OPG and TRAIL mRNA were detected in peritoneum, bone marrow derived cells in PF, and endometriotic tissues. DR5 mRNA was detected in endometriotic tissues. CONCLUSION: The increase in OPG levels with reduced TRAIL/ OPG ratios in PF of women with advanced stages of endometriosis implies that the peritoneal environment of these women may be permissive to survival of endometrial/ endometriotic cells. Supported by: None
P-71 TNF-alpha induced embryotoxicity and role of TNF-alpha blockerinfliximab on in vitro blastocyst development rate. X. Zhang, R. K. Sharma, A. Agarwal, T. Falcone. Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: TNF-␣ is required both for the establishment and maintenance of endometriosis. Evidence shows that peritoneal fluid from patients with endometriosis has elevated levels of TNF-␣ levels that may be responsible for endometriosis-associated infertility. TNF-blockers such as Remicade (Infliximab) may help neutralize the toxic effects of TNF-␣. In our earlier studies we demonstrated that infliximab is not embryotoxic at concentration of up to 400g/mL. Our study was designed to evaluate: 1) the embryotoxic effect of TNF-␣ that may be present in the peritoneal fluid of women with endometriosis, and 2) examine if infliximab could reverse the TNF-␣ induced embryotoxicity. DESIGN: Prospective in vitro study. MATERIALS AND METHODS: 150 two-cell mouse embryos were cultured in 6 different concentrations of diluted in human tubal fluid for 72 h. Blastocyst development rate (%BDR) was examined after 72 hours of incubation in the following concentrations of TNF-␣: 100, 200, 400, 500, 1000, and 2000ng/mL. Similarly, a second set containing infliximab (200g/mL) ⫹ TNF-␣(500ng/mL) was cultured under identical conditions to study if infliximab at this concentration was effective in reducing TNF-␣ induced embryotoxicity. RESULTS: A dose dependent decrease in %BDR was seen using different concentrations of TNF-␣ compared with controls (see Figure). A significant decrease in %BDR was seen at 400ng/mL compared with control (%BDR: 59% vs. 98%; P⬍0.0008), which further decreased to 21% at a concentration of 500ng/mL of TNF-␣ (P⬍0.05). Higher concentrations of TNF-␣ resulted in complete embryotoxicity (%BDR ⫽ 0). Infliximab alone had 100% BDR and was comparable with controls. Infliximab, in the presence of TNF-␣, completely neutralized the embryotoxic effect of TNF-␣ and %BDR increased significantly from 55% to 100% (P⬍0.05).
Vol. 82, Suppl. 2, September 2004