- Email: [email protected]
Development and characterization of endometriotic lesions in the chick chorioallantoic membrane model
visualization of lesion evolution after treatment in the same animal, noninvasively, reducing time, cost and number of animals involved when assessing the efficacy of drugs directed against endometriosis. The dynamic and non-invasive characteristics of this improved mouse model should make it an unprecedented tool for compound evaluation in a pre-clinical setting of endometriosis. Furthermore, this animal model could have many other applications in endometriosis research, such as evaluating the effect of tested genes on lesion implantation and regression. Supported By: PROCREA BioSciences.
Monday, February 25, 2002 4:30 P.M. O-9 Murine model of monocytic peritonitis: are glycodelin or thiazolidinedione new options in the treatment of endometriosis? D. Hornung1, V. A. Chao2, J.-L. Vigne2, D. Wallwiener1, R. N. Taylor2. 1Univ of Tuebingen, Tuebingen, Germany; 2UCSF, San Francisco, CA. Objective: Migration of macrophages into the peritoneal cavity, mediated by chemokines, is an important pathophysiological feature in women with endometriosis. Prior data from our laboratory suggest that RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) is a potential target for therapies designed to mitigate the inflammatory response associated with infertility and pain in this condition. In the current studies we characterized the inflammatory response in an in vivo murine model of peritonitis and assessed molecules whose anti-inflammatory effects we described previously in in vitro assays of endometriosis. Design: Controlled animal study. Materials/Methods: Thioglycollate medium was used to stimulate peritoneal macrophage attraction into the peritoneal cavity of C57BL/6 mice. Glycodelin, Ciglitazone or RANTES-antibodies were administered intraperitoneally. Peritoneal fluid was collected, peritoneal cells counted and cultured. After 8 days in culture, cells were stimulated with 6 nM TNF-␣ for 48 hours. RNA from cultured peritoneal macrophages was isolated and RT-PCR was performed with c-fms-specific primers. Mouse RANTES and IL-1 was quantified in the peritoneal cell culture supernatants using specific ELISAs. Results: Mice treated with thioglycollate plus ciglitazone showed a 32 ⫾ 2% reduction in cell number relative to thioglycollate alone and those treated with thioglycollate plus glycodelin had a 44 ⫾ 8% reduction compared to thioglycollate alone. Verification of macrophage phenotype was afforded by RT-PCR for mouse c-fms mRNA. Ciglitazone (54 ⫾ 10%) and glycodelin (80 ⫾ 20%) showed significant inhibition of macrophage RANTES secretion compared with thioglycollate alone. Ciglitazone (32 ⫾ 10%) and glycodelin (55 ⫾ 14%) also suppressed thioglycollate-induced IL-1 production by the peritoneal macrophages. Conclusions: Treatment with ciglitazone and glycodelin reduced monocyte infiltration in the mouse model. Both agents previously were shown to inhibit human monocytic cell migration in Boyden chamber assays. On the basis of our preclinical experiments, we suggest that these classes of molecules be evaluated as potential adjuncts to the surgical therapy of human endometriosis. Supported By: A grant from the Deutsche Forschungsgemeinschaft (DH) and the NIH/NICHD, through cooperative agreement U54-HD37321, as a part of the Special Cooperative Centers Program in Reproduction Research.
Monday, February 25, 2002 4:45 P.M. O-10 Development and characterization of endometriotic lesions in the chick chorioallantoic membrane model. A. Nap1, P. G. Groothuis2, A. Demir Weusten3, A. F. P. de Goeij4, G. A. J. Dunselman5, J. Evers5. 1Research Institute Growth and Development (GROW), Dept OB/GYN, Acad Hosp Maastricht, Maastricht, Netherlands; 2Dept of Pathology, Acad Hosp Maastricht, Maastricht, Netherlands; 3Dept OB/GYN, Univ Maastricht, Maastricht, Netherlands; 4Dept of Pathology, Univ Maastricht, Maastricht, Netherlands; 5Dept OB/GYN, Acad Hosp Maastricht, Maastricht, Netherlands.
S4
Abstracts
Objective: According to Sampson’s theory on the pathogenesis of endometriosis, retrogradely shed viable endometrial cells can adhere to, and subsequently invade and proliferate in the peritoneum. Using the chicken embryo chorioallantoic membrane (CAM) to study invasion, we observed that grafting of biopsied cyclic endometrium onto the CAM results in endometriosis-like lesions containing human endometrial glands and stroma in the mesenchymal layer of the CAM 72 hours after grafting. In contrast, enzymatically dispersed cyclic endometrial cells and shed menstrual endometrium were not able to accomplish the same. In this study we attempt to clarify how these endometriosis-like lesions originate, and we study the invasive potential of biopsied menstrual endometrium. Design: Transplantation of human endometrial tissue on the chick chorioallantoic membrane. Materials/Methods: We collected endometrium in the menstrual (cycle day 2) and periovulatory (cycle day 14) phase from women undergoing laparoscopy for benign conditions using a probet endometrial sampling device. After collection, endometrium was divided into fragments of 2 mm3 that were grafted individually onto the CAM. After 48 (n ⫽ 3), 53 (n ⫽ 3), 65 (n ⫽ 3), 70 (n ⫽ 3) and 72 hours (n ⫽ 3) CAMs were fixed in formalin. Sections were immunostained for expression of matrix metalloproteinases (MMP) 3 and 7, the proliferation marker Ki 67, and estrogen receptor (ER). Results: After 72 hours lesions were observed in all CAMs transplanted with both cycle days 2 and 14 endometrium. During incubation, MMP3 and MMP7 expression was maintained, whereas nuclear ER and Ki67 expression decreased precipitously. As early as 48 hours after grafting, large numbers of invading cells interrupting the CAM epithelium were seen. Strands of epithelial cells accompanied by stromal cells were seen to extend from the graft into the CAM mesenchyme, forming a connection with endometriotic-like lesions. From 65 hours onwards, vessels filled with nucleated erythrocytes were present in the lesions. A few hours later, large numbers of small blood vessels appeared to separate the graft and the lesion. Conclusions: In this study, we show that biopsied menstrual endometrium, in contrast to shed menstrual endometrium, is able to invade and form endometriosis-like lesions in the CAM model. Lesions obtain vascular supply from the surrounding tissue, which allows them to survive. The limited expression of Ki67 suggests that the lesion formation is mostly a result of the migration and organization of endometrial cells, rather than increased proliferation. The maintained expression of MMPs supports the assumption that these regulators of the extracellular matrix play an important role in the implantation of endometrial tissue. The lack of nuclear ER suggests that for the development of the lesions in the CAM, estrogens are not required. Supported By: No support.
Monday, February 25, 2002 5:00 P.M. O-11 Implantation of human menstrual endometrium into nude mice. F. Casanas-Roux1, A. Van Langendonckt1, M. Nisolle1, J. Donnez2. 1Catholic Univ of Louvain, Bruxelles, Belgium; 2Catholic Univ of Louvain, Brussels, Belgium. Objective: In the present study, human menstrual endometrium was transplanted into 66 nude mice in order to 1) examine the early stages of evolution of the endometriotic lesion, 2) to test whether the cellular, seric and red blood cell fractions from the menstrual effluent may have an influence on the development of endometriotic lesions in this animal model and 3) to investigate whether adhesion occurs on intact peritoneum or at the site of incision. Design: In a first series of experiments (n ⫽ 10), the timing of adhesion of menstrual endometrial tissue was analyzed by removal of the transplant on days 1, 3 and 5. In a second series (n ⫽ 25), 5 different fractions of menstrual effluent were transplanted by minilaparotomy performed on the ventral midline: 1) effluent as a whole, 2) seric fraction, 3) cellular ⫹ seric fractions 4) cellular ⫹ red blood cell fractions and 5) cellular fraction only. In a third series (n ⫽ 31), the same fractions, except the seric, were deposited by an incision on the ventral right side in order to test if the site of adhesion is influenced by the healing area. Materials/Methods: Menstrual effluent was obtained from women on day 2 or 3 of the cycle, by aspiration. The effluent was centrifuged and the seric fraction collected. Endometrial tissue was separated from red blood cells
Vol. 77, No. 2, Suppl. 1, February 2002
Monday, February 25, 2002 4:30 P.M. O-9 Murine model of monocytic peritonitis: are glycodelin or thiazolidinedione new options in the treatment of endometriosis? D. Hornung1, V. A. Chao2, J.-L. Vigne2, D. Wallwiener1, R. N. Taylor2. 1Univ of Tuebingen, Tuebingen, Germany; 2UCSF, San Francisco, CA. Objective: Migration of macrophages into the peritoneal cavity, mediated by chemokines, is an important pathophysiological feature in women with endometriosis. Prior data from our laboratory suggest that RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) is a potential target for therapies designed to mitigate the inflammatory response associated with infertility and pain in this condition. In the current studies we characterized the inflammatory response in an in vivo murine model of peritonitis and assessed molecules whose anti-inflammatory effects we described previously in in vitro assays of endometriosis. Design: Controlled animal study. Materials/Methods: Thioglycollate medium was used to stimulate peritoneal macrophage attraction into the peritoneal cavity of C57BL/6 mice. Glycodelin, Ciglitazone or RANTES-antibodies were administered intraperitoneally. Peritoneal fluid was collected, peritoneal cells counted and cultured. After 8 days in culture, cells were stimulated with 6 nM TNF-␣ for 48 hours. RNA from cultured peritoneal macrophages was isolated and RT-PCR was performed with c-fms-specific primers. Mouse RANTES and IL-1 was quantified in the peritoneal cell culture supernatants using specific ELISAs. Results: Mice treated with thioglycollate plus ciglitazone showed a 32 ⫾ 2% reduction in cell number relative to thioglycollate alone and those treated with thioglycollate plus glycodelin had a 44 ⫾ 8% reduction compared to thioglycollate alone. Verification of macrophage phenotype was afforded by RT-PCR for mouse c-fms mRNA. Ciglitazone (54 ⫾ 10%) and glycodelin (80 ⫾ 20%) showed significant inhibition of macrophage RANTES secretion compared with thioglycollate alone. Ciglitazone (32 ⫾ 10%) and glycodelin (55 ⫾ 14%) also suppressed thioglycollate-induced IL-1 production by the peritoneal macrophages. Conclusions: Treatment with ciglitazone and glycodelin reduced monocyte infiltration in the mouse model. Both agents previously were shown to inhibit human monocytic cell migration in Boyden chamber assays. On the basis of our preclinical experiments, we suggest that these classes of molecules be evaluated as potential adjuncts to the surgical therapy of human endometriosis. Supported By: A grant from the Deutsche Forschungsgemeinschaft (DH) and the NIH/NICHD, through cooperative agreement U54-HD37321, as a part of the Special Cooperative Centers Program in Reproduction Research.
Monday, February 25, 2002 4:45 P.M. O-10 Development and characterization of endometriotic lesions in the chick chorioallantoic membrane model. A. Nap1, P. G. Groothuis2, A. Demir Weusten3, A. F. P. de Goeij4, G. A. J. Dunselman5, J. Evers5. 1Research Institute Growth and Development (GROW), Dept OB/GYN, Acad Hosp Maastricht, Maastricht, Netherlands; 2Dept of Pathology, Acad Hosp Maastricht, Maastricht, Netherlands; 3Dept OB/GYN, Univ Maastricht, Maastricht, Netherlands; 4Dept of Pathology, Univ Maastricht, Maastricht, Netherlands; 5Dept OB/GYN, Acad Hosp Maastricht, Maastricht, Netherlands.
S4
Abstracts
Objective: According to Sampson’s theory on the pathogenesis of endometriosis, retrogradely shed viable endometrial cells can adhere to, and subsequently invade and proliferate in the peritoneum. Using the chicken embryo chorioallantoic membrane (CAM) to study invasion, we observed that grafting of biopsied cyclic endometrium onto the CAM results in endometriosis-like lesions containing human endometrial glands and stroma in the mesenchymal layer of the CAM 72 hours after grafting. In contrast, enzymatically dispersed cyclic endometrial cells and shed menstrual endometrium were not able to accomplish the same. In this study we attempt to clarify how these endometriosis-like lesions originate, and we study the invasive potential of biopsied menstrual endometrium. Design: Transplantation of human endometrial tissue on the chick chorioallantoic membrane. Materials/Methods: We collected endometrium in the menstrual (cycle day 2) and periovulatory (cycle day 14) phase from women undergoing laparoscopy for benign conditions using a probet endometrial sampling device. After collection, endometrium was divided into fragments of 2 mm3 that were grafted individually onto the CAM. After 48 (n ⫽ 3), 53 (n ⫽ 3), 65 (n ⫽ 3), 70 (n ⫽ 3) and 72 hours (n ⫽ 3) CAMs were fixed in formalin. Sections were immunostained for expression of matrix metalloproteinases (MMP) 3 and 7, the proliferation marker Ki 67, and estrogen receptor (ER). Results: After 72 hours lesions were observed in all CAMs transplanted with both cycle days 2 and 14 endometrium. During incubation, MMP3 and MMP7 expression was maintained, whereas nuclear ER and Ki67 expression decreased precipitously. As early as 48 hours after grafting, large numbers of invading cells interrupting the CAM epithelium were seen. Strands of epithelial cells accompanied by stromal cells were seen to extend from the graft into the CAM mesenchyme, forming a connection with endometriotic-like lesions. From 65 hours onwards, vessels filled with nucleated erythrocytes were present in the lesions. A few hours later, large numbers of small blood vessels appeared to separate the graft and the lesion. Conclusions: In this study, we show that biopsied menstrual endometrium, in contrast to shed menstrual endometrium, is able to invade and form endometriosis-like lesions in the CAM model. Lesions obtain vascular supply from the surrounding tissue, which allows them to survive. The limited expression of Ki67 suggests that the lesion formation is mostly a result of the migration and organization of endometrial cells, rather than increased proliferation. The maintained expression of MMPs supports the assumption that these regulators of the extracellular matrix play an important role in the implantation of endometrial tissue. The lack of nuclear ER suggests that for the development of the lesions in the CAM, estrogens are not required. Supported By: No support.
Monday, February 25, 2002 5:00 P.M. O-11 Implantation of human menstrual endometrium into nude mice. F. Casanas-Roux1, A. Van Langendonckt1, M. Nisolle1, J. Donnez2. 1Catholic Univ of Louvain, Bruxelles, Belgium; 2Catholic Univ of Louvain, Brussels, Belgium. Objective: In the present study, human menstrual endometrium was transplanted into 66 nude mice in order to 1) examine the early stages of evolution of the endometriotic lesion, 2) to test whether the cellular, seric and red blood cell fractions from the menstrual effluent may have an influence on the development of endometriotic lesions in this animal model and 3) to investigate whether adhesion occurs on intact peritoneum or at the site of incision. Design: In a first series of experiments (n ⫽ 10), the timing of adhesion of menstrual endometrial tissue was analyzed by removal of the transplant on days 1, 3 and 5. In a second series (n ⫽ 25), 5 different fractions of menstrual effluent were transplanted by minilaparotomy performed on the ventral midline: 1) effluent as a whole, 2) seric fraction, 3) cellular ⫹ seric fractions 4) cellular ⫹ red blood cell fractions and 5) cellular fraction only. In a third series (n ⫽ 31), the same fractions, except the seric, were deposited by an incision on the ventral right side in order to test if the site of adhesion is influenced by the healing area. Materials/Methods: Menstrual effluent was obtained from women on day 2 or 3 of the cycle, by aspiration. The effluent was centrifuged and the seric fraction collected. Endometrial tissue was separated from red blood cells
Vol. 77, No. 2, Suppl. 1, February 2002