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Gonadotropin-releasing hormone agonist (GnRH-a) induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis (EDT)
Objective: We studied the expression of three kinds of MMPs (MMP-7, MMP-9, MMP-13) in the deep lesions of endometriosis and their uterine endometrium by a immunohistochemical study to determine whether the expression of MMPs is related with the formation of endometriotic lesion. Design: Retrospective clinical analyses. Materials/Methods: We performed an immunohistochemical staining on paraffin-embedded tissue. Superficial endometriotic lesion (N ⫽ 16), deep endometriotic lesion (N ⫽ 39) and intrauterine endometrium (N ⫽ 49) of patients with endometriosis were studied. Results: Endometriosis showed statically significantly stronger immunoreactivity of MMPs compared with uterine endometrium (p ⬍ 0.01). The deep lesions of endometriosis are significantly stronger staining than superficial lesions (stroma at MMP-7, p ⬍ 0.08 and MMP-9 staining, p ⬍ 0.05; gland at MMP-9 staining, p ⬍ 0.005). Conclusions: It suggested that the endometriosis has higher capacity of degradation of extracellular matrix than uterine endometrium because of their increased expression of MMPs. Ectopic deep endometriotic lesions compared to eutopic endometrium seems to have a greater invasion capacity due to higher protease expression. Supported By: Korean Endometriosis Study Group.
P-59 Gonadotropin-releasing hormone agonist (GnRH-a) induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis (EDT). G. F. Meresman1, R. A. Buquet2, M. Bilotas1, R. I. Baran˜ ao1, C. Sueldo3, M. Tesone1. 1Inst de Biologia y Medicine Experimental (IBYME), Buenos Aires, Argentina; 2Hosp de Clinicas-UBA, Buenos Aires, Argentina; 3Inst de Ginecologia y Fertilidad (IFER), Buenos Aires, Argentina Objective: There are growing evidences that suggest a direct action of GnRH-a on endometrial growth. In previous studies we observed impaired sensitivity of endometrial tissue to spontaneous apoptosis in women with EDT. Based on this data, our purpose was to evaluate the effect of GnRH-a on the in-vitro eutopic endometrial cell growth and apoptosis. Design: Prospective and in vitro experimental study. Materials/Methods: Biopsy specimens of eutopic endometrium in proliferative phase were obtained from 16 women with untreated EDT and 8 controls. Epithelial endometrial cells were selectively isolated by enzymatic digestion and cultured for 48 h before the experiments. Percentage of apoptotic cells, by the acridine orange— ethidium bromide technique, and cell proliferation, by 3H-thymidine incorporation, were evaluated after incubation during 48 h with Leuprolide Acetate (LA) as GnRHa; Antide, as GnRH antagonist, and a combination of Antide with LA. Also, cell proliferation was measured in the presence of TGF for 48 h. Eleven endometrial cultures (5 from controls and 6 from EDT patients) were employed to evaluate cell proliferation and 13 (3 from controls and 10 from EDT patients), were used to assess apoptosis. Statistical comparisons were performed by either two or one-way ANOVA, followed by Dunn’s multiple comparison test. Only a p value less than or equal to 0.05 was considered significant. Results: LA showed an effect on endometrial growth, enhancing apoptosis in endometrial cultures from patients with EDT: 41.7 ⫾ 9.6 (LA 100 ng/ml) vs 23.9 ⫾ 8.0 (basal), (expressed as percentage of apoptotic cells, p ⬍ 0.01), as well as in cell cultures from control women: 42.5 ⫾ 14.7 (LA 100 ng/ml) vs 18.2 ⫾ 6.3 (basal), (p ⬍ 0.05). In both experiments this effect was reversible when LA was added 3 hours later than Antide. In EDT, 31.0 ⫾ 4.9 (Antide 10-7 M) vs 23.9 ⫾ 8.0 (basal) and in controls, 27.3 ⫾ 8.1 (Antide 10-7 M) vs. 18.2 ⫾ 6.3 (basal) (p ⬎ 0.05, ns). The addition of TGF 0.01, 0.1 and 1 ng/ml to endometrial cultures from patients and controls, stimulated cell proliferation in a dose dependent manner, whereas TGF 10 ng/ml inhibited the 3H-thymidine incorporation. For cell proliferation assays, TGF (0.1 ng/ml) stimulated cell cultures were considered as controls (C). In endometrial cultures from EDT patients, 3H-thymidine uptake was downregulated by LA 1 ng/ml: ⫺35.0 ⫾ 20.0, p ⬍ 0.01 vs C; LA 10 ng/ml: ⫺61.6 ⫾ 8.4, p ⬍ 0.001 vs C; and LA 100 ng/ml: ⫺64.0 ⫾ 24.6, (p ⬍ 0.001 vs. C), expressed as percentage of inhibition. Likewise this decrease in cell proliferation was observed in cell cultures from control women, LA 1 ng/ml: ⫺61.0 ⫾ 15.5, LA 10 ng/ml: ⫺46.0 ⫾ 2.8 and LA 100 ng/ml: ⫺60.0 ⫾ 12.0, (p ⬍ 0.001 vs C). The addition of Antide 10-7 M reverted this inhibition. Conclusions: Our results suggest that GnRH-a would have a local effect,
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by enhancing the apoptosis index and decreasing the cell proliferation in the endometrial cells, two of the factors possibly involved in the growth and/or recurrence of EDT. Supported By: ANPCYT (PICT 6384 BID 1201 OC-AR), UBA (01/TW05, University of Buenos Aires, Argentina) and Roemmers Foundation, Buenos Aires, Argentina.
P-60 Comparative immunohistochemical studies of endometriosis lesions and endometriotic cysts. F. R. Nezhat, C. J. Cohen, J. Rahaman, H. F. Gretz, T. Kalir. Mount Sinai Medical Ctr, New York, NY. Objective: Endometriosis affects an estimated 10 million American women. While most lesions appear as solid foci, ovarian lesions are sometimes cystic, called endometriomas. There has been some question in the literature as to whether endometriomas merely represent cystic variants of endometriosis, or may indeed result from a completely different pathogenesis. To explore this question, we decided to compare immunohistochemical staining patterns in noncystic and cystic endometriosis lesions. Design: Case control study. Materials/Methods: Formalin-fixed, paraffin-embedded sections from noncystic endometriosis lesions and ovarian endometriotic cysts, were immunostained with anti-bcl-2, anti-p53, anti matrix metalloproteinase IX, and collagen VI using the striptavidin-biotin method. Fisher’s exact test was used for statistical comparison. Results: Staining for p53 was completely negative in both groups. Anti bcl-2 stained 100% (30/30) of endometriosis lesions compared to only 23% (7/30) of endometriotic cysts (p ⬍ .0001) and anti matrix metalloproteinase IX stained 85% (23/27) of endometriosis lesions and only 39% (14/36) of endometriotic cysts (p ⫽ .0003). Anti collagen VI, however stained only 6% (2/35) of endometriosis lesions and 75% (21/28) of endometriotic cyst (p ⬍ .0001). Conclusions: This is the first comparative immunohistochemical study showing that endometriotic cysts are different from endometriosis lesions with over- expression of collagen VI and under-expression of bcl-2 and matrix metalloproteinase IX. Supported By: Ovarian Cancer Research Fund Grant.
P-61 Activin-A and follistatin production by eutopic endometrium of endometriosis patients. L. J. F. Rombauts, J. F. Donoghue, P. A. W. Rogers, D. L. Healy. Ctr for Women’s Health Research, Monash Univ, Clayton, Australia. Objective: Activin-A is a growth factor influencing cellular proliferation, differentiation and tissue remodeling. It has been isolated in the peritoneal fluid of endometriosis patients and is secreted by endometrial stroma and glands and may be involved in the promotion of ectopic growth. Follistatin is an activin-binding protein neutralizing the bioactivity of activin. The purpose of this study was to compare the production of activin-A and follistatin by eutopic endometrium from patients with and without endometriosis. Design: A prospective comparative trial. Materials/Methods: Endometrial curettings from consenting women with a regular menstrual cycle undergoing laparoscopy and hysteroscopy (33 ⫾ 2 years) were dissected into 3 mm3 pieces. Explants weighing between 20 and 40 mg were cultured in 4 well plates with 500 l of conditioning media. After 24 hours, the samples were harvested, centrifuged and the media stored (⫺20°C) for activin analysis and the tissue pellet for protein analysis. Secreted activin-A was measured using an activin-A ELISA (OxfordBioinovations), follistatin was measured using a follistatin ELISA (OxfordBioinovations). The protein analysis was carried out on lysed and homogenised tissue pellets using a BCA Protein Assay kit. Results: Activin-A was detected in normal patients during the proliferative and secretory phase (64.82 ⫾ 13.3 and 50.37 ⫾ 11.56 ng/mg total protein respectively) (n ⫽ 7). Patients with endometriosis produced significantly more activin-A during the proliferative phase than the secretory phase (107.82 ⫾ 30.05 and 42.13 ⫾ 5.89 ng/mg total protein respectively) (n ⫽ 5) (p ⬍ 0.05). None of the samples tested contained measurable follistatin.
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P-59 Gonadotropin-releasing hormone agonist (GnRH-a) induces apoptosis and reduces cell proliferation in eutopic endometrial cultures from women with endometriosis (EDT). G. F. Meresman1, R. A. Buquet2, M. Bilotas1, R. I. Baran˜ ao1, C. Sueldo3, M. Tesone1. 1Inst de Biologia y Medicine Experimental (IBYME), Buenos Aires, Argentina; 2Hosp de Clinicas-UBA, Buenos Aires, Argentina; 3Inst de Ginecologia y Fertilidad (IFER), Buenos Aires, Argentina Objective: There are growing evidences that suggest a direct action of GnRH-a on endometrial growth. In previous studies we observed impaired sensitivity of endometrial tissue to spontaneous apoptosis in women with EDT. Based on this data, our purpose was to evaluate the effect of GnRH-a on the in-vitro eutopic endometrial cell growth and apoptosis. Design: Prospective and in vitro experimental study. Materials/Methods: Biopsy specimens of eutopic endometrium in proliferative phase were obtained from 16 women with untreated EDT and 8 controls. Epithelial endometrial cells were selectively isolated by enzymatic digestion and cultured for 48 h before the experiments. Percentage of apoptotic cells, by the acridine orange— ethidium bromide technique, and cell proliferation, by 3H-thymidine incorporation, were evaluated after incubation during 48 h with Leuprolide Acetate (LA) as GnRHa; Antide, as GnRH antagonist, and a combination of Antide with LA. Also, cell proliferation was measured in the presence of TGF for 48 h. Eleven endometrial cultures (5 from controls and 6 from EDT patients) were employed to evaluate cell proliferation and 13 (3 from controls and 10 from EDT patients), were used to assess apoptosis. Statistical comparisons were performed by either two or one-way ANOVA, followed by Dunn’s multiple comparison test. Only a p value less than or equal to 0.05 was considered significant. Results: LA showed an effect on endometrial growth, enhancing apoptosis in endometrial cultures from patients with EDT: 41.7 ⫾ 9.6 (LA 100 ng/ml) vs 23.9 ⫾ 8.0 (basal), (expressed as percentage of apoptotic cells, p ⬍ 0.01), as well as in cell cultures from control women: 42.5 ⫾ 14.7 (LA 100 ng/ml) vs 18.2 ⫾ 6.3 (basal), (p ⬍ 0.05). In both experiments this effect was reversible when LA was added 3 hours later than Antide. In EDT, 31.0 ⫾ 4.9 (Antide 10-7 M) vs 23.9 ⫾ 8.0 (basal) and in controls, 27.3 ⫾ 8.1 (Antide 10-7 M) vs. 18.2 ⫾ 6.3 (basal) (p ⬎ 0.05, ns). The addition of TGF 0.01, 0.1 and 1 ng/ml to endometrial cultures from patients and controls, stimulated cell proliferation in a dose dependent manner, whereas TGF 10 ng/ml inhibited the 3H-thymidine incorporation. For cell proliferation assays, TGF (0.1 ng/ml) stimulated cell cultures were considered as controls (C). In endometrial cultures from EDT patients, 3H-thymidine uptake was downregulated by LA 1 ng/ml: ⫺35.0 ⫾ 20.0, p ⬍ 0.01 vs C; LA 10 ng/ml: ⫺61.6 ⫾ 8.4, p ⬍ 0.001 vs C; and LA 100 ng/ml: ⫺64.0 ⫾ 24.6, (p ⬍ 0.001 vs. C), expressed as percentage of inhibition. Likewise this decrease in cell proliferation was observed in cell cultures from control women, LA 1 ng/ml: ⫺61.0 ⫾ 15.5, LA 10 ng/ml: ⫺46.0 ⫾ 2.8 and LA 100 ng/ml: ⫺60.0 ⫾ 12.0, (p ⬍ 0.001 vs C). The addition of Antide 10-7 M reverted this inhibition. Conclusions: Our results suggest that GnRH-a would have a local effect,
FERTILITY & STERILITY威
by enhancing the apoptosis index and decreasing the cell proliferation in the endometrial cells, two of the factors possibly involved in the growth and/or recurrence of EDT. Supported By: ANPCYT (PICT 6384 BID 1201 OC-AR), UBA (01/TW05, University of Buenos Aires, Argentina) and Roemmers Foundation, Buenos Aires, Argentina.
P-60 Comparative immunohistochemical studies of endometriosis lesions and endometriotic cysts. F. R. Nezhat, C. J. Cohen, J. Rahaman, H. F. Gretz, T. Kalir. Mount Sinai Medical Ctr, New York, NY. Objective: Endometriosis affects an estimated 10 million American women. While most lesions appear as solid foci, ovarian lesions are sometimes cystic, called endometriomas. There has been some question in the literature as to whether endometriomas merely represent cystic variants of endometriosis, or may indeed result from a completely different pathogenesis. To explore this question, we decided to compare immunohistochemical staining patterns in noncystic and cystic endometriosis lesions. Design: Case control study. Materials/Methods: Formalin-fixed, paraffin-embedded sections from noncystic endometriosis lesions and ovarian endometriotic cysts, were immunostained with anti-bcl-2, anti-p53, anti matrix metalloproteinase IX, and collagen VI using the striptavidin-biotin method. Fisher’s exact test was used for statistical comparison. Results: Staining for p53 was completely negative in both groups. Anti bcl-2 stained 100% (30/30) of endometriosis lesions compared to only 23% (7/30) of endometriotic cysts (p ⬍ .0001) and anti matrix metalloproteinase IX stained 85% (23/27) of endometriosis lesions and only 39% (14/36) of endometriotic cysts (p ⫽ .0003). Anti collagen VI, however stained only 6% (2/35) of endometriosis lesions and 75% (21/28) of endometriotic cyst (p ⬍ .0001). Conclusions: This is the first comparative immunohistochemical study showing that endometriotic cysts are different from endometriosis lesions with over- expression of collagen VI and under-expression of bcl-2 and matrix metalloproteinase IX. Supported By: Ovarian Cancer Research Fund Grant.
P-61 Activin-A and follistatin production by eutopic endometrium of endometriosis patients. L. J. F. Rombauts, J. F. Donoghue, P. A. W. Rogers, D. L. Healy. Ctr for Women’s Health Research, Monash Univ, Clayton, Australia. Objective: Activin-A is a growth factor influencing cellular proliferation, differentiation and tissue remodeling. It has been isolated in the peritoneal fluid of endometriosis patients and is secreted by endometrial stroma and glands and may be involved in the promotion of ectopic growth. Follistatin is an activin-binding protein neutralizing the bioactivity of activin. The purpose of this study was to compare the production of activin-A and follistatin by eutopic endometrium from patients with and without endometriosis. Design: A prospective comparative trial. Materials/Methods: Endometrial curettings from consenting women with a regular menstrual cycle undergoing laparoscopy and hysteroscopy (33 ⫾ 2 years) were dissected into 3 mm3 pieces. Explants weighing between 20 and 40 mg were cultured in 4 well plates with 500 l of conditioning media. After 24 hours, the samples were harvested, centrifuged and the media stored (⫺20°C) for activin analysis and the tissue pellet for protein analysis. Secreted activin-A was measured using an activin-A ELISA (OxfordBioinovations), follistatin was measured using a follistatin ELISA (OxfordBioinovations). The protein analysis was carried out on lysed and homogenised tissue pellets using a BCA Protein Assay kit. Results: Activin-A was detected in normal patients during the proliferative and secretory phase (64.82 ⫾ 13.3 and 50.37 ⫾ 11.56 ng/mg total protein respectively) (n ⫽ 7). Patients with endometriosis produced significantly more activin-A during the proliferative phase than the secretory phase (107.82 ⫾ 30.05 and 42.13 ⫾ 5.89 ng/mg total protein respectively) (n ⫽ 5) (p ⬍ 0.05). None of the samples tested contained measurable follistatin.
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