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Differential regulation of the progesterone receptor (PR) by TCDD in isolated human endometrial stromal cells versus stromal-epithelial co-cultures
reported in peritoneal cavities of women with endometriosis, the macrophage content of the uterine endometrium in women with endometriosis is significantly reduced compared to normal throughout the menstrual cycle, suggesting an alternation in normal endometrial physiology. Demonstration of an association between decreased macrophage content and subnormal TNF␣ levels in eutopic endometria from women with endometriosis, would provide an explanation for the reduced apoptotic characteristics of endometrium that we have reported for women with this disease. Supported By: No support.
Monday, February 25, 2002 2:45 P.M. O-4 Activation of CD40/CD40L system in endometriotic fibroblasts is a mechanism for chronic inflammation. S. Y. Reddy, R. P. Phipps. Univ of Rochester, Rochester, NY. Objective: Endometriosis affects 15% of women of reproductive age resulting in infertility and chronic pelvic pain. Proinflammatory mediators have been found in pelvic fluid in women with endometriosis implicating involvement of the immune system. Endometriotic elements consist largely of “stromal cells” and glandular epithelium. “Stromal cells” are believed to be key cells for implantation at ectopic sites and for production of aromatase responsible for estrogen thereby contributing to the maintenance of the ectopic tissue. We have characterized “stromal cells” as fibroblasts based upon their expression of vimentin and collagen and lack of epithelial markers such as cytokeratin. We hypothesize that pure strains of endometriotic fibroblasts become activated through a receptor/ligand pair called CD40/CD40 ligand inducing key inflammatory mediators involved in the pathogenesis of endometriosis and induction of aromatase. This pathway is a central activator of the immune system. Design: Protocol was approved by the Institutional Review Board for Human Research at the University of Rochester. Written consents were obtained from patients before any surgical procedure. Tissue acquisition and processing took place at the time of laparoscopy or laparotomy. All samples were histologically confirmed. Fibroblasts were isolated using an explant technique. Glandular epithelium was isolated using separation techniques as described by Taylor, et al. Materials/Methods: IL-6 and MCP-1 cytokines were measured using ELISA assays. PGE2 levels were measured using an EIA competitive assay. Flow cytometry was used to demonstrate presence of CD40 receptors. Fibroblasts were stained with anti-CD40 or mouse IgG isotype control antibodies followed by FITC conjugated secondary antibody. For immunocytochemistry, fibroblasts were grown in chamber slides. They were fixed and stained using mouse anti-human COX-1 and COX-2 antibodies followed by a biotinylated secondary antibody and SA-HRP. Color was developed using the AEC kit. For western blotting, protein was isolated using Trireagent. Protein was denatured and separated on 12.5% SDS-PAGE. After transfer on to nitrocellulose membranes, the immunoblots were incubated with anti-aromatase antibody followed by incubation with a secondary antibody, and film exposed via chemiluminescence. Results: Endometriosis fibroblasts express CD40 significantly higher than endometriotic glandular epithelium. Stimulating endometriotic fibroblasts with rhCD40L results in the production of proinflammatory cytokines, IL-6 and MCP-1. CD40L stimulation induces cyclooxygenase-2 resulting in a significant production of the proinflammatory prostaglandin PGE2. CD40L stimulation also upregulates expression of aromatase at the protein level. Conclusions: CD40 engagement induces the key mediators found in endometriosis and is therefore a central element in the activation of endometriotic fibroblasts. We implicate fibroblasts as the major cell component contributing to the initiation of a proinflammatory environment through COX-2 expression, PGE2 production, and inflammatory cytokines. The CD40/CD40L pathway in fibroblasts also serves to maintain endometriosis through upregulation of aromatase. Supported By: This research is supported by USPHS grants DE11390 and HD01332.
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Abstracts
Monday, February 25, 2002 3:00 P.M. O-5 Different bFGF and FGF-AS expression in eutopic endometrial stromal cells derived from patients affected by endometriosis and from controls. A. Mihalich1, M. Reina1, E. Ponti1, S. Mangioni2, M. Vignali2, A. M. Di Blasio1. 1Inst Auxologico Italy, Milano, Italy; 2Univ of Milano, Milano, Italy. Objective: Basic fibroblast growth factor (bFGF) is a heparin-binding cationic peptide, member of a family of related proteins whose basic structure has been conserved throughout evolution. In all species studied, the bFGF gene is transcribed into multiple mRNAs deriving from different polyadenilation sites. It has been demonstrated, first in the Xenopus Laevis and subsequently in human and rat tissues, that one of these mRNAs is an antisense RNA (FGF-AS) transcribed from the opposite strand of the gene, likely involved in regulating stability of the sense transcript. The nucleotide sequence of this FGF-AS mRNA is strongly conserved among species. Basic FGF mitogenic and angiogenic properties have been well characterized in endometrial physiology but their involvement in endometriosis is still to be elucidated. We have previously demonstrated that endometriosis cells derived from large cysts have increased bFGF mRNA levels. Based on the evidence in this study we investigated whether the regulatory mechanisms of bFGF expression might be altered in endometrial stromal cells derived from women affected by endometriosis. Design: To this aim bFGF and FGF-AS mRNAs levels were evaluated in samples of eutopic endometrial stromal cells obtained from 34 consecutive women undergoing laparoscopy for pelvic pain and or infertility. Patients with no laparoscopic evidence of endometriosis served as control group. Materials/Methods: Basic FGF and FGF-AS mRNA levels were quantified in primary cultures of eutopic endometrial stromal cells derived from 19 controls and 15 patients using Competitive-PCR.The amplified DNA fragments were visualized on an agarose gel and analyzed by laser densitometer. Unpaired t-student was used to evaluate differences in mRNA values among the two groups studied. Results: Both transcripts were present in all samples, however, their expression resulted to be different between the two groups of women. When the data were analyzed according to the phases of the menstrual cycle, in the central phase (days 10 –18) endometrial stromal cells derived from women affected by endometriosis showed significant higher bFGF mRNA values and significant lower FGF-AS mRNA levels compared to control samples. The ratio between bFGF mRNA and FGF-AS mRNA values was calculated and the mean ratio resulted significantly higher in endometrial cells derived from women affected by endometriosis when compared to that obtained from endometrial cells of control patients (mean ratio ⫾ S.E : 2.69 ⫾ 0.65 and 0.83 ⫾ 0.15 respectively). Conclusions: The results of this study indicate that in eutopic endometrial cells of endometriosis women the expression of bFGF and FGF-AS mRNAs is inversely correlated. This finding further support the hypothesis that FGF-AS mRNA could regulate the expression of the sense transcript. Moreover, the higher expression of bFGF mRNA observed in these cells suggest that they have an increased capacity to synthetize bFGF. Based on these data, it is tempting to speculate that an altered regulation of bFGF expression could be among the factors that favour the proliferation of endometrial cells leading to endometriosis. Supported By: No support.
Monday, February 25, 2002 3:15 P.M. O-6 Differential regulation of the progesterone receptor (PR) by TCDD in isolated human endometrial stromal cells versus stromal-epithelial cocultures. T. Igarashi1, K. Bruner-Tran2, D. Edwards3, E. Eisenberg2, K. Osteen2. 1Vanderbilt Univ, Nashville, TN; Univ of Tokyo, Tokyo, Japan; 2 Vanderbilt Univ, Nashville, TN; 3Univ of Colorado, Denver, CO. Objective: We previously reported that endometrial organ cultures exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrate a reduced ability to respond to progesterone-mediated suppression of matrix
Vol. 77, No. 2, Suppl. 1, February 2002
metalloproteinases (MMPs). MMPs are critical to normal and neoplastic matrix remodeling and have recently been implicated in the process of endometriosis. TCDD has also been linked to the development of endometriosis in both women and non-human primates, but mechanisms for this association have not been fully elucidated. In the current study, we have investigated the ability of TCDD to modulate PR in isolated endometrial stromal cells and in co-cultures of stromal and epithelial cells. Design: Proliferative phase human endometrium was acquired by biopsy during the proliferative phase (days 10 –12) from a normal donor population. Isolated stromal cells or co-cultures of stromal and epithelial cells were maintained in culture in the presence of steroids with or without TCDD. Following in vitro treatments, western analysis was performed on stromal cell lysates to determine the presence of PR while conditioned media was examined for expression of MMP-3. Materials/Methods: Isolated cells were obtained by established methodology using enzymatic digests and filter separation. Isolated stromal cells were plated on Type I collagen while epithelial cells were plated in tissue culture inserts coated with Matrigel. Cells were treated with estradiol (1 nM), estradiol plus progesterone (1 nM, 500 nM respectively) in the presence or absence of TCDD (1 nM). Following four days in culture, media was removed and stromal cell extracts were prepared in the presence of a protease inhibitor cocktail. PR and MMP-3 protein was analyzed by Western using specific antibodies. Results: Expression of PR was increased in stromal cells cultured in the presence of epithelial cells versus stromal cells cultured alone. In contrast, TCDD treatments decreased PR expression in stromal cells co-cultured with epithelial cells, but appeared to enhance PR expression in stromal cells cultured alone. Consistent with PR results, the ability of TCDD to disrupt progesterone-mediated suppression of MMP-3 in stromal cells was enhanced by the presence of the epithelial cells. Conclusions: Our results indicate a differential PR expression by human endometrial stromal cells which is dependent on the presence or absence of epithelial cells. Importantly, the ability of stromal-epithelial cell communication to affect PR expression can impact the in vitro response of stromal cells to TCDD exposure. The finding that TCDD inhibits PR expression under co-culture conditions may be important to understanding the role of this factor in the development of endometriosis. Our results may help explain several recent studies which indicate that women with endometriosis have a diminished response to progesterone and an enhanced expression of MMPs. Supported By: This work was supported through the Specialized Cooperative Centers Program in Reproduction Research (U54-HD-37321) and by grants from the U.S. Environmental Protection Agency (R826300) and The Endometriosis Association Research Program at Vanderbilt.
BASIC RESEARCH: PATHOGENESIS Monday, February 25, 2002 4:00 P.M. O-7 Pathological evaluation of rat endometriosis model. I. Uchiide1, M. Sugamata2, T. Ihara2, M. Miura3. 11st Dept of Obstetrics and Gynecology, Toho Univ Sch of Medicine, Tokyo, Japan; 2Dept of Pathology, Tochigi Institute of Clin Pathology, Tochigi, Japan; 3Dept of Pathology, Ohmori Hosp, Toho Univ Sch of Medicine, Tokyo, Japan. Objective: To clarify a pathogenesis and evaluate a therapeutic effect in a disease, it is important that experimental animal model is established and that the lesions in the model are examined pathologically. Experimental rat endometriosis has been developed by uterine autotransplantation already, and has been used in various investigations of endometriosis. However, almost all previous reports are only focused on implanted uterine tissues, therefore, little is known about morphology of the lesions in detail, specifically in peritoneum attached uterine transplants. Design: In this experiment, we induced rat endometriosis by surgical autotransplantation of uterine tissue and examined morphological alterations of uterus-attached peritoneum with light and electron microscope following the time lapse after the implantation. Materials/Methods: SLC-Sprague-Dawley Rats (8-week old) were maintained on a 12-hour (light) to 12-hour (dark) for 2 weeks, and these rats were used for induction of endometriosis models by the surgical autotransplan-
FERTILITY & STERILITY威
tation technique of uterine square (5 ⫻ 5 mm). The mesenteries were autotransplanted as a comparative control. The implanted areas of peritoneum including abdominal muscle were obtained from anesthetized rats at 4, 7, 14 days after uterine autotransplantation and were examined under a light and electron microscope. To detect DNA fragmentation, these samples were analyzed by TUNEL method. In order to examine localization of mast cells and plasma cells in each specimens, these were performed with Toluidine blue stain (for mast cells) and Methylgreen pyronin stain (for plasma cells). Results: On our light and electron microscopic observations in rat endometriosis models, stromal tissues of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophil, plasma cells, lymphocytes and macrophages. These lesions increased following time lapse after implantation, however, these infiltrated cells disappeared and the proliferation declined finally. Our findings suggest that uterine autotransplantation induced infiltration of allergic inflammatory-related cells and proliferative lesion in peritoneal stroma attached endometrial epithelium, these lesions in rat models are similar to those in endometriosis patients. Conclusions: Our findings suggest that uterine autotransplantation induced immune response, which may be hypersensitivity reaction, in peritoneal stroma attached endometrial epithelium. We believe that these morphological data of rat endometriosis are very important to clarify pathogenesis and progress of human endometriosis. Supported By: No support.
Monday, February 25, 2002 4:15 P.M. O-8 Establishment of an improved mouse model of endometriosis allowing the non-invasive assessment of implantation, progression and regression of human endometriotic lesions. A.-M. Steff1, M. Fortin1, B. Massie2, P. Hugo1. 1PROCREA BioSciences, Montre´ al, QC, Canada; 2Biotech Research Institute, Montre´ al, QC, Canada. Objective: To develop an improved mouse model of endometriosis to allow the non-invasive and dynamic monitoring of human endometriotic lesion implantation and regression. Design: Develop a murine non-invasive experimental model for endometriosis by implanting green fluorescent protein (GFP) expressing fragments of human endometrium into immunocompromised mice. Materials/Methods: We developed an improved, non-invasive animal model for endometriosis. To this end, we introduced the GFP gene into 1–2 mm3 fragments of proliferative phase endometrium, by adenoviral infection in vitro. Ten fragments of GFP-expressing endometrial tissue were then injected either subcutaneously or intra-peritoneally into ovariectomized Nude mice, previously implanted with an estradiol-releasing pellet. Living transplanted mice were periodically examined using a light emitting box allowing GFP excitation. Image analysis allowed quantification of fluorescence intensity and size of endometriotic lesions. In a second set of experiments, monitoring of lesion evolution in untreated or progesterone-treated animals was performed by serial in vivo imaging of animals. Number of lesions, their size and fluorescence intensity, as well as time of complete disappearance were quantified and compared between groups. Results: One day after adenoviral infection, endometrial tissue was shown to be highly fluorescent both by microscopic observation and flow cytometric analysis. GFP-expressing endometrial tissue transplanted to Nude mice was able to form endometriotic lesions. Importantly, fluorescent endometriotic lesions were readily observed non-invasively in subcutaneous and intra-peritoneal sites. Lesion evolution could be followed in the same animal over a 3 week period, and their size and fluorescence intensity could be measured. These two parameters spontaneously decreased with time, likely due to GFP dilution among dividing cells. We postulated that decrease in size and fluorescence intensity of lesions would be more pronounced in mice undergoing endometriosis medication. To make the proof of concept that this model could indeed be used for the pre-clinical testing of new drugs targeting endometriosis, we treated Nude mice exhibiting fluorescent lesions with progesterone. Mice treated with progesterone had a faster and more intense decrease in the number and size of lesions, as assessed by non-invasive imaging. Conclusions: The easier identification of human endometriotic lesions in animals is a major strength of this improved model. Moreover, it allows the
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Monday, February 25, 2002 2:45 P.M. O-4 Activation of CD40/CD40L system in endometriotic fibroblasts is a mechanism for chronic inflammation. S. Y. Reddy, R. P. Phipps. Univ of Rochester, Rochester, NY. Objective: Endometriosis affects 15% of women of reproductive age resulting in infertility and chronic pelvic pain. Proinflammatory mediators have been found in pelvic fluid in women with endometriosis implicating involvement of the immune system. Endometriotic elements consist largely of “stromal cells” and glandular epithelium. “Stromal cells” are believed to be key cells for implantation at ectopic sites and for production of aromatase responsible for estrogen thereby contributing to the maintenance of the ectopic tissue. We have characterized “stromal cells” as fibroblasts based upon their expression of vimentin and collagen and lack of epithelial markers such as cytokeratin. We hypothesize that pure strains of endometriotic fibroblasts become activated through a receptor/ligand pair called CD40/CD40 ligand inducing key inflammatory mediators involved in the pathogenesis of endometriosis and induction of aromatase. This pathway is a central activator of the immune system. Design: Protocol was approved by the Institutional Review Board for Human Research at the University of Rochester. Written consents were obtained from patients before any surgical procedure. Tissue acquisition and processing took place at the time of laparoscopy or laparotomy. All samples were histologically confirmed. Fibroblasts were isolated using an explant technique. Glandular epithelium was isolated using separation techniques as described by Taylor, et al. Materials/Methods: IL-6 and MCP-1 cytokines were measured using ELISA assays. PGE2 levels were measured using an EIA competitive assay. Flow cytometry was used to demonstrate presence of CD40 receptors. Fibroblasts were stained with anti-CD40 or mouse IgG isotype control antibodies followed by FITC conjugated secondary antibody. For immunocytochemistry, fibroblasts were grown in chamber slides. They were fixed and stained using mouse anti-human COX-1 and COX-2 antibodies followed by a biotinylated secondary antibody and SA-HRP. Color was developed using the AEC kit. For western blotting, protein was isolated using Trireagent. Protein was denatured and separated on 12.5% SDS-PAGE. After transfer on to nitrocellulose membranes, the immunoblots were incubated with anti-aromatase antibody followed by incubation with a secondary antibody, and film exposed via chemiluminescence. Results: Endometriosis fibroblasts express CD40 significantly higher than endometriotic glandular epithelium. Stimulating endometriotic fibroblasts with rhCD40L results in the production of proinflammatory cytokines, IL-6 and MCP-1. CD40L stimulation induces cyclooxygenase-2 resulting in a significant production of the proinflammatory prostaglandin PGE2. CD40L stimulation also upregulates expression of aromatase at the protein level. Conclusions: CD40 engagement induces the key mediators found in endometriosis and is therefore a central element in the activation of endometriotic fibroblasts. We implicate fibroblasts as the major cell component contributing to the initiation of a proinflammatory environment through COX-2 expression, PGE2 production, and inflammatory cytokines. The CD40/CD40L pathway in fibroblasts also serves to maintain endometriosis through upregulation of aromatase. Supported By: This research is supported by USPHS grants DE11390 and HD01332.
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Abstracts
Monday, February 25, 2002 3:00 P.M. O-5 Different bFGF and FGF-AS expression in eutopic endometrial stromal cells derived from patients affected by endometriosis and from controls. A. Mihalich1, M. Reina1, E. Ponti1, S. Mangioni2, M. Vignali2, A. M. Di Blasio1. 1Inst Auxologico Italy, Milano, Italy; 2Univ of Milano, Milano, Italy. Objective: Basic fibroblast growth factor (bFGF) is a heparin-binding cationic peptide, member of a family of related proteins whose basic structure has been conserved throughout evolution. In all species studied, the bFGF gene is transcribed into multiple mRNAs deriving from different polyadenilation sites. It has been demonstrated, first in the Xenopus Laevis and subsequently in human and rat tissues, that one of these mRNAs is an antisense RNA (FGF-AS) transcribed from the opposite strand of the gene, likely involved in regulating stability of the sense transcript. The nucleotide sequence of this FGF-AS mRNA is strongly conserved among species. Basic FGF mitogenic and angiogenic properties have been well characterized in endometrial physiology but their involvement in endometriosis is still to be elucidated. We have previously demonstrated that endometriosis cells derived from large cysts have increased bFGF mRNA levels. Based on the evidence in this study we investigated whether the regulatory mechanisms of bFGF expression might be altered in endometrial stromal cells derived from women affected by endometriosis. Design: To this aim bFGF and FGF-AS mRNAs levels were evaluated in samples of eutopic endometrial stromal cells obtained from 34 consecutive women undergoing laparoscopy for pelvic pain and or infertility. Patients with no laparoscopic evidence of endometriosis served as control group. Materials/Methods: Basic FGF and FGF-AS mRNA levels were quantified in primary cultures of eutopic endometrial stromal cells derived from 19 controls and 15 patients using Competitive-PCR.The amplified DNA fragments were visualized on an agarose gel and analyzed by laser densitometer. Unpaired t-student was used to evaluate differences in mRNA values among the two groups studied. Results: Both transcripts were present in all samples, however, their expression resulted to be different between the two groups of women. When the data were analyzed according to the phases of the menstrual cycle, in the central phase (days 10 –18) endometrial stromal cells derived from women affected by endometriosis showed significant higher bFGF mRNA values and significant lower FGF-AS mRNA levels compared to control samples. The ratio between bFGF mRNA and FGF-AS mRNA values was calculated and the mean ratio resulted significantly higher in endometrial cells derived from women affected by endometriosis when compared to that obtained from endometrial cells of control patients (mean ratio ⫾ S.E : 2.69 ⫾ 0.65 and 0.83 ⫾ 0.15 respectively). Conclusions: The results of this study indicate that in eutopic endometrial cells of endometriosis women the expression of bFGF and FGF-AS mRNAs is inversely correlated. This finding further support the hypothesis that FGF-AS mRNA could regulate the expression of the sense transcript. Moreover, the higher expression of bFGF mRNA observed in these cells suggest that they have an increased capacity to synthetize bFGF. Based on these data, it is tempting to speculate that an altered regulation of bFGF expression could be among the factors that favour the proliferation of endometrial cells leading to endometriosis. Supported By: No support.
Monday, February 25, 2002 3:15 P.M. O-6 Differential regulation of the progesterone receptor (PR) by TCDD in isolated human endometrial stromal cells versus stromal-epithelial cocultures. T. Igarashi1, K. Bruner-Tran2, D. Edwards3, E. Eisenberg2, K. Osteen2. 1Vanderbilt Univ, Nashville, TN; Univ of Tokyo, Tokyo, Japan; 2 Vanderbilt Univ, Nashville, TN; 3Univ of Colorado, Denver, CO. Objective: We previously reported that endometrial organ cultures exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrate a reduced ability to respond to progesterone-mediated suppression of matrix
Vol. 77, No. 2, Suppl. 1, February 2002
metalloproteinases (MMPs). MMPs are critical to normal and neoplastic matrix remodeling and have recently been implicated in the process of endometriosis. TCDD has also been linked to the development of endometriosis in both women and non-human primates, but mechanisms for this association have not been fully elucidated. In the current study, we have investigated the ability of TCDD to modulate PR in isolated endometrial stromal cells and in co-cultures of stromal and epithelial cells. Design: Proliferative phase human endometrium was acquired by biopsy during the proliferative phase (days 10 –12) from a normal donor population. Isolated stromal cells or co-cultures of stromal and epithelial cells were maintained in culture in the presence of steroids with or without TCDD. Following in vitro treatments, western analysis was performed on stromal cell lysates to determine the presence of PR while conditioned media was examined for expression of MMP-3. Materials/Methods: Isolated cells were obtained by established methodology using enzymatic digests and filter separation. Isolated stromal cells were plated on Type I collagen while epithelial cells were plated in tissue culture inserts coated with Matrigel. Cells were treated with estradiol (1 nM), estradiol plus progesterone (1 nM, 500 nM respectively) in the presence or absence of TCDD (1 nM). Following four days in culture, media was removed and stromal cell extracts were prepared in the presence of a protease inhibitor cocktail. PR and MMP-3 protein was analyzed by Western using specific antibodies. Results: Expression of PR was increased in stromal cells cultured in the presence of epithelial cells versus stromal cells cultured alone. In contrast, TCDD treatments decreased PR expression in stromal cells co-cultured with epithelial cells, but appeared to enhance PR expression in stromal cells cultured alone. Consistent with PR results, the ability of TCDD to disrupt progesterone-mediated suppression of MMP-3 in stromal cells was enhanced by the presence of the epithelial cells. Conclusions: Our results indicate a differential PR expression by human endometrial stromal cells which is dependent on the presence or absence of epithelial cells. Importantly, the ability of stromal-epithelial cell communication to affect PR expression can impact the in vitro response of stromal cells to TCDD exposure. The finding that TCDD inhibits PR expression under co-culture conditions may be important to understanding the role of this factor in the development of endometriosis. Our results may help explain several recent studies which indicate that women with endometriosis have a diminished response to progesterone and an enhanced expression of MMPs. Supported By: This work was supported through the Specialized Cooperative Centers Program in Reproduction Research (U54-HD-37321) and by grants from the U.S. Environmental Protection Agency (R826300) and The Endometriosis Association Research Program at Vanderbilt.
BASIC RESEARCH: PATHOGENESIS Monday, February 25, 2002 4:00 P.M. O-7 Pathological evaluation of rat endometriosis model. I. Uchiide1, M. Sugamata2, T. Ihara2, M. Miura3. 11st Dept of Obstetrics and Gynecology, Toho Univ Sch of Medicine, Tokyo, Japan; 2Dept of Pathology, Tochigi Institute of Clin Pathology, Tochigi, Japan; 3Dept of Pathology, Ohmori Hosp, Toho Univ Sch of Medicine, Tokyo, Japan. Objective: To clarify a pathogenesis and evaluate a therapeutic effect in a disease, it is important that experimental animal model is established and that the lesions in the model are examined pathologically. Experimental rat endometriosis has been developed by uterine autotransplantation already, and has been used in various investigations of endometriosis. However, almost all previous reports are only focused on implanted uterine tissues, therefore, little is known about morphology of the lesions in detail, specifically in peritoneum attached uterine transplants. Design: In this experiment, we induced rat endometriosis by surgical autotransplantation of uterine tissue and examined morphological alterations of uterus-attached peritoneum with light and electron microscope following the time lapse after the implantation. Materials/Methods: SLC-Sprague-Dawley Rats (8-week old) were maintained on a 12-hour (light) to 12-hour (dark) for 2 weeks, and these rats were used for induction of endometriosis models by the surgical autotransplan-
FERTILITY & STERILITY威
tation technique of uterine square (5 ⫻ 5 mm). The mesenteries were autotransplanted as a comparative control. The implanted areas of peritoneum including abdominal muscle were obtained from anesthetized rats at 4, 7, 14 days after uterine autotransplantation and were examined under a light and electron microscope. To detect DNA fragmentation, these samples were analyzed by TUNEL method. In order to examine localization of mast cells and plasma cells in each specimens, these were performed with Toluidine blue stain (for mast cells) and Methylgreen pyronin stain (for plasma cells). Results: On our light and electron microscopic observations in rat endometriosis models, stromal tissues of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophil, plasma cells, lymphocytes and macrophages. These lesions increased following time lapse after implantation, however, these infiltrated cells disappeared and the proliferation declined finally. Our findings suggest that uterine autotransplantation induced infiltration of allergic inflammatory-related cells and proliferative lesion in peritoneal stroma attached endometrial epithelium, these lesions in rat models are similar to those in endometriosis patients. Conclusions: Our findings suggest that uterine autotransplantation induced immune response, which may be hypersensitivity reaction, in peritoneal stroma attached endometrial epithelium. We believe that these morphological data of rat endometriosis are very important to clarify pathogenesis and progress of human endometriosis. Supported By: No support.
Monday, February 25, 2002 4:15 P.M. O-8 Establishment of an improved mouse model of endometriosis allowing the non-invasive assessment of implantation, progression and regression of human endometriotic lesions. A.-M. Steff1, M. Fortin1, B. Massie2, P. Hugo1. 1PROCREA BioSciences, Montre´ al, QC, Canada; 2Biotech Research Institute, Montre´ al, QC, Canada. Objective: To develop an improved mouse model of endometriosis to allow the non-invasive and dynamic monitoring of human endometriotic lesion implantation and regression. Design: Develop a murine non-invasive experimental model for endometriosis by implanting green fluorescent protein (GFP) expressing fragments of human endometrium into immunocompromised mice. Materials/Methods: We developed an improved, non-invasive animal model for endometriosis. To this end, we introduced the GFP gene into 1–2 mm3 fragments of proliferative phase endometrium, by adenoviral infection in vitro. Ten fragments of GFP-expressing endometrial tissue were then injected either subcutaneously or intra-peritoneally into ovariectomized Nude mice, previously implanted with an estradiol-releasing pellet. Living transplanted mice were periodically examined using a light emitting box allowing GFP excitation. Image analysis allowed quantification of fluorescence intensity and size of endometriotic lesions. In a second set of experiments, monitoring of lesion evolution in untreated or progesterone-treated animals was performed by serial in vivo imaging of animals. Number of lesions, their size and fluorescence intensity, as well as time of complete disappearance were quantified and compared between groups. Results: One day after adenoviral infection, endometrial tissue was shown to be highly fluorescent both by microscopic observation and flow cytometric analysis. GFP-expressing endometrial tissue transplanted to Nude mice was able to form endometriotic lesions. Importantly, fluorescent endometriotic lesions were readily observed non-invasively in subcutaneous and intra-peritoneal sites. Lesion evolution could be followed in the same animal over a 3 week period, and their size and fluorescence intensity could be measured. These two parameters spontaneously decreased with time, likely due to GFP dilution among dividing cells. We postulated that decrease in size and fluorescence intensity of lesions would be more pronounced in mice undergoing endometriosis medication. To make the proof of concept that this model could indeed be used for the pre-clinical testing of new drugs targeting endometriosis, we treated Nude mice exhibiting fluorescent lesions with progesterone. Mice treated with progesterone had a faster and more intense decrease in the number and size of lesions, as assessed by non-invasive imaging. Conclusions: The easier identification of human endometriotic lesions in animals is a major strength of this improved model. Moreover, it allows the
S3