Pathological evaluation of the rat endometriosis model. Tochigi Institute of Clinical Pathology, Tochigi, Japan

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1 REPRODUCTIVE BIOLOGY FERTILITY AND STERILITY VOL. 78, NO. 4, OCTOBER 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Pathological evaluation of the rat endometriosis model Ichiro Uchiide, M.D., Ph.D., a Tomomi Ihara, M.D., Ph.D., b and Masao Sugamata, M.D., Ph.D. b Tochigi Institute of Clinical Pathology, Tochigi, Japan Received April 2, 2002; revised and accepted May 30, Presented at the 8th World Congress on Endometriosis, San Diego, California, February 24 27, Reprint requests: Masao Sugamata, M.D., Ph.D., Department of Pathology, Tochigi Institute of Clinical Pathology, , Minamiakatsuka, Nogi, Shimotsuga-gun, Tochigi, , Japan (FAX: ; mspathol@beige.ocn.ne.jp). a Department of Obstetrics and Gynecology, Japanese Red Cross Hospital Omori, Tokyo, Japan. b Department of Pathology, Tochigi Institute of Clinical Pathology /02/$22.00 PII S (02) Objective: To observe in detail the morphology of experimental rat endometriosis, specifically in peritoneum adjacent to uterine transplants attached via autotransplantation. Design: Light and electron microscopic study. Setting: Tochigi Institute of Clinical Pathology, Japan. Animal(s): Female-SD rats maintained on a schedule of 12 hours of light and 12 hours of dark for 2 weeks. Intervention(s): Uterine transplants were attached to rat peritoneum via the surgical autotransplantation technique. The implanted area of peritoneum, including abdominal muscle, were excised from anesthetized rats at four (n 10), seven (n 10), and 14 (n 10) days after uterine autotransplantation. The mesenteries were autotransplanted as a comparative control. Main Outcome Measure(s): We examined the morphologic alterations of uterus-attached peritoneum following the time interval after the implantation. Result(s): In rat endometriosis models, the stromal tissue of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophils, plasma cells, lymphocytes, and macrophages. These lesions increased with time after implantation; however, ultimately these infiltrating cells disappeared and proliferation declined. Conclusion(s): Our findings suggest that uterine autotransplantation induces the infiltration of allergic inflammatory-related cells and proliferative lesions in peritoneal stroma attached endometrium. These data should prove useful for investigations of human endometriosis. (Fertil Steril 2002;78: by American Society for Reproductive Medicine.) Key Words: Endometriosis, rat mast cells, hypersensitivity, apoptosis Endometriosis is a disease characterized by the ectopic occurrence of endometrial tissue, commonly located on pelvic organs and the peritoneal cavity. Although the pathologic diagnosis of endometriosis is benign, the morphologic appearance is marked by proliferation, infiltration, and severe adhesions to the surrounding tissues. The progression of endometriosis shows a strong relationship to estrogen levels; the disease incidence rises sharply at menarche, then decreases in menopausal women (1). However, some theories regarding the pathogenesis of endometriosis, such as celomic metaplasia (2, 3) and transplantation of exfoliated endometrial cells (4), have been advanced based on the histopathologic features of the lesions; it also has been suggested that the immune system is abnormal in patients with endometriosis (5). To date, however, no evidence exists to support these theories. Recently, secondary to the advancement of reproductive medical science, more reports have been published on the relationship between infertility and endometriosis (6), and the clarification of the pathogenic factors of this disease have become extremely important. To address the establishment of experimental animal models of endometriosis, Vernon et al. (7) developed the surgical technique of autotransplantation of uterine tissues for induction of rat endometriosis; subsequently, the rat model has been used in various investigations of endometriosis. However, little is known about the detailed morphologic features of this model s lesions, and previous reports focused on implanted uterine tissues. In our experiment, we induced rat endometriosis by the surgical autotransplantation of uterine tissue and examined the morphologic alterations over 782

2 FIGURE 1 Histopathologic features of autotransplantation-performed peritoneum. (A), Four days after uterine autotransplantation. (B), Seven days after uterine autotransplantation. (C), Fourteen days after uterine autotransplantation. (D), Seven days after mesentery autotransplantation. (All views: am Abdominal muscle; magnification: 40.) The proliferative lesions in the peritoneum adjacent to the implanted uterine tissue were more pronounced at 7 days after uterine autotransplantation (B) than at 4 days after autotransplantation (A); subsequently, the proliferation declined at 14 days after autotransplantation (C). None the peritoneum implanted with mesentery showed any appreciable change (D). time after the implantation. We present the unique findings in the peritoneum adjacent to attached uterine tissue specifically in the stromal tissue, not in the uterine transplants. MATERIALS AND METHODS Rat Endometriosis Induced by Uterine Autotransplantation and Sampling of Tissues For the induction of endometriosis models, SLC-Sprague- Dawley Rats (8 weeks old) were purchased from Sankyo Labo Service (Shizuoka, Japan) and maintained on a schedule of 12 hours of light and 12 hours of dark for 2 weeks. The surgical technique of autotransplanting 5 mm 5mmof uterine tissue has been previously described in detail (7); we performed the same technique with minor modifications. The implanted peritoneal tissue, including abdominal muscle, was obtained from anesthetized rats at 4 (n 10), 7 (n 10), 14 (n 10) days after uterine autotransplantation. The mesenteries were autotransplanted as a comparative control (at 4 days (n 5), 7 days (n 5); and 14 days (n 5) after autotransplantation). Light Microscopic Analysis The tissue samples from the implanted area were fixed with 10% buffered formalin and, after routine dehydration, were embedded in paraffin. Sections 5- m thick were stained with hematoxylin and eosin and examined under a light microscope. To examine localization of mast cells and plasma cells in each specimen, 5- m-thick paraffin sections of these tissue samples were stained with toluidine blue (for mast cells) and methylgreen pyronin (for plasma cells). Electron Microscopic Analysis These tissue samples were fixed immediately with 1% glutaraldehyde and 4% formalin for 6 hours at 4 C and rinsed in 0.1 M cacodylate buffer overnight. The tissues were postfixed with 1% osmium tetraoxide and were embedded in Epon 812 resin. Ultrathin sections were prepared with FERTILITY & STERILITY 783

3 FIGURE 2 Localization of mast cells and plasma cells in the peritoneal stroma adjacent to implanted uterine tissue at 7 days after autotransplantation. (A), Toluidine blue stain. Arrow indicates mast cells (violet color; magnification: 200). (B), Methylgreen pyronin stain. Arrow indicates plasma cells (red color; magnification: 200). Infiltrations of numerous mast cells and plasma cells are observed diffusely. RESULTS Light Microscopic Analysis Histopathologically, from 4 to 7 days after uterine autotransplantation, the progression of proliferative lesions in the peritoneum adjacent to uterine tissue increased; subsequently, at 14 days after autotransplantation the proliferation declined (Figs. 1A through 1C). Infiltrations of mast cells and plasma cells were observed at 4 and 7 days after autotransplantation (Figs. 2A and 2B); however, these infiltrating cells disappeared at 14 days after autotransplantation. All mesenteric tissue-implanted peritoneum as a comparative control showed no significant change (Fig. 1D). Electron Microscopic Analysis At 4 days after uterine autotransplantation, mast cells, eosinophils, and plasma cells were observed in the peritoneal stroma; some of the mast cells showed degranulation, and some apoptotic eosinophils were observed (Figs. 3A through 3C). In addition, lymphocytes and macrophages were observed at 7 days after autotransplantation. Furthermore, apoptosis of eosinophils was induced and some macrophages phagocytosed the apoptotic bodies (Figs. 3D and 3E). At 14 days after implantation, the stromal peritoneal tissues showed only apoptotic eosinophils and macrophages, and the infiltrating mast cells, plasma cells, and lymphocytes had disappeared (Fig. 3F). DISCUSSION an Ultratome Nova (LKB, Bromma, Sweden), doublestained with uranyl acetate and lead citrate, and examined under an electron microscope (Model 1200EX; JEOL, Tokyo, Japan). Apoptotic appearance was evaluated based on the characteristics of the ultrastructural process in the apoptotic body, as described in our previous report (8). In our study, the ultrastructural apoptotic process is divided into four stages, and characteristic findings can be observed at each stage. One of the findings is the spaces described as crescentshaped spaces (CSS). From an early stage of the morphologic process of apoptotic formation, CSS appear around the nuclear membrane of the cell; this is apparently normal, but may have been triggered by apoptotic induction. With light and electron microscopic analysis of rat endometriosis, the stromal tissues of the peritoneum adjacent to implanted uterine tissue showed the infiltration of mast cells, eosinophils, plasma cells, lymphocytes, and macrophages. This suggests that the uterine autotransplantation induced a hypersensitivity reaction (an allergic reaction) in the peritoneal stroma attached to the endometrial epithelium. The appearance of the mast cells is pathognomonic for the development of hypersensitivity reaction, and the granules are an eosinophil chemotactic factor of anaphylaxis. Ultrastructurally, we observed the degranulation of mast cells, and it is assumed that this degranulation induced the infiltration of eosinophils. Recently, it has been noted that human endometriosis correlates with allergic disorders epidemiologically (9). We have found infiltration and degranulation of numerous mast cells in the stromal proliferative lesion areas of human endometriosis (unpublished observations), and a previous report also described the appearance of mast cells in human peritoneal endometriosis (10). Therefore, our findings indicate that the rat endometriosis model closely approximates human endometriosis. Plasma cells and B lymphocytes play an important role in the activation of mast cells and eosinophils in hypersensitivity reactions (11). Some reports suggest that no significant increase in B lymphocytes is exhibited by the lesion areas of 784 Uchiide et al. Pathological evaluation of rat endometriosis model Vol. 78, No. 4, October 2002

4 FIGURE 3 Ultrastructural images of infiltrated cells in the peritoneal stroma adjacent to implanted uterine tissue after autotransplantation. (A), Four days after autotransplantation (M mast cell). (B), Four days after autotransplantation. (E apoptotic eosinophil). Fragmentation of nucleus with condensed chromatin is observed (*). (C), Four days after autotransplantation (P plasma cells). (D), Seven days after autotransplantation (M mast cell; m macrophage). The macrophage contains some phagosomes (*). (E), Seven days after autotransplantation (E apoptotic eosinophils; L lymphocytes). Fragmentation eosinophil s nucleus with condensed chromatin is observed (*). (F), At 14 days after implantation. Eosinophils (E) and some macrophages (m) are observed. Some of the eosinophils show nuclear fragmentation (*). (A E: scale bar 3 m; F: scale bar 4 m.) Four days after autotransplantation, mast cells, eosinophils, and plasma cells are observed; some apoptotic eosinophils also were observed (A through C). Lymphocytes and macrophages, which phagocytose some apoptotic bodies, are observed at 7 days after autotransplantation (D and E). The stromal peritoneal tissue at 14 days after autotransplantation shows only apoptotic eosinophils and macrophages (F). endometriosis patients (12, 13); conversely, B lymphocytes increase in peripheral blood and peritoneal fluid (14 16). However, there are no data on plasma cells, which are the mature element of B lymphocytes. It is possible that the increase of B lymphocytes in the lesion area cannot be identified because plasma cells predominate, as seen in the rat model. The stromal peritoneal tissues at 14 days after autotransplantation showed only apoptotic eosinophils and macrophages; ultimately, mast cells, plasma cells and lymphocytes disappeared, and the proliferative lesion declined. Macrophages play a role in the clearance of apoptotic cells and are observed at the final stage of the apoptotic process (8). We propose that the apoptosis of the nearly infiltrated allergic inflammatory cells was induced, and that these apoptotic cells were subsequently phagocytosed by macrophages. In FERTILITY & STERILITY 785

5 short, the reaction against the uterine transplants was complete at this phase. These results suggest that progression of the lesions in rat endometriosis models peaks between 4 and 7 days after uterine autotransplantation. Further study is necessary to maintain the lesions in models for an extended period after autotransplantation. In summary, we made the following observations of endometriosis in the rat model. Secondary to the attachment of endometrial epithelium to the peritoneum, the peritoneal stromal tissue showed proliferation and infiltration of cells related to allergic inflammation; these cells included mast cells, eosinophils, plasma cells, and macrophages. The findings in the rat model are similar to those in human endometriosis; however, in the rat model, the lesions in the peritoneal stroma declined at 14 days after autotransplantation. We believe that this information will be helpful in further investigations to clarify the pathogenesis and progression of human endometriosis. References 1. Houston DE, Noller KL, Melton LJ 3d, Selwyn BJ. The epidemiology of pelvic endometriosis. Clin Obstet Gynecol 1988;31: Iwanoff NS. Drüsiges cystinhaltiges uterusfibromyom kompliziert durch Sarcom und Carcinom. Monatschr Gebrutsh u Gyneak 1898;7: Fujii S. Secondary mullerian system and endometriosis. Am J Obstet Gynecol 1991;165: Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissues into the peritoneal cavity. Am J Obstet Gynecol 1927;14: Gleicher N, El-Roeiy A, Confino E, Friberg J. Is endometriosis an autoimmune disease? Obstet Gynecol 1987;70: Dmownski WP, Braum D, Gebel HM. Endometriosis: genetics and immunologic aspects. In: Current Concepts in Endometriosis. New York: Alan R Liss, 1990: Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44: Ihara T, Yamamoto T, Sugamata M, Okumura H, Ueno Y. The process of ultrastructural changes from nuclei to apoptotic body. Virch Arch 1998;433: Nichols TR, Lamb K, Arkins JA. The association of atopic diseases with endometriosis. Ann Allergy 1987;59: Matsuzaki S, Canis M, Darcha C, Fukaya T, Yajima A, Brauhat MA. Increased mast cell density in peritoneal endometriosis compared with eutopic endometrium with endometriosis. Am J Reprod Immunol 1998; 40: Roitt I, Brosoff J, Male D. Imunology. 6th ed. London: Mosby International, 2001: Badawy SZA, Cuenca V, Stitzel A, Tice D. Immune rosettes of T and B lymphocytes in infertile women with endometriosis. J Reprod Med 1987;32: Badawy SZA, Stitzel A, Cuenca V, Thompson M, Kaufman L. The regulation of immunoglobulin production by B cells in patients with endometriosis. Fertil Steril 1989;51: Witz CA, Montoya IA, Dey TD, Schenken RS. Characterization of lymphocyte subpopulations and T cell activation in endometriosis. Am J Reprod Immunol 1994;32: Klentzeris LD, Bulmer JN, Liu DT, Morrison L. Endometrial leukocyte subpopulations in women with endometriosis. Eur J Obstet Gynecol 1995;63: Ho H-N, Wu M-Y, Yang Y-S. Peritoneal cellular immunity and endometriosis. Am J Reprod Immunol 1997;38: Uchiide et al. Pathological evaluation of rat endometriosis model Vol. 78, No. 4, October 2002