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The basement membrane in endometriosis
i
11 i' i I
Vol. 47, No.3, March 1987 Printed in U.8A.
FERTILITY AND STERILITY Copyright © 1987 The American Fertility Society
The basement membrane in endometriosis
Johannes L. H. Evers, M.D.* Dick Willebrand, M.D. t Academisch Ziekenhuis Maastricht, The University of Limburg, Maastricht, The Netherlands
Tumor invasion can be defined as the active migration of neoplastic cells out of their tissue of origin into adjacent tissue. Extracellular matrix barriers consist of basement membranes (BMs) and interstitial stroma. BMs occur in connection with epithelium, endothelium, mesothelium, smooth as well as striated muscle cells, Schwann cells, and fat cells. During cancer invasion loss of BM occurs. In benign diseases, such as fibroadenoma of the breast or adenomyosis of the uterus, intact BMs exist. 1 In lesions within the category of carcinoma in situ, such as intraductal carcinoma of the breast and Bowen's disease of the skin, BM is identical with the benign group oflesions. In micro invasive carcinoma, however, breaking, thinning, and fragmentation of the BM is found in the areas of microinvasion but not in adjacent areas. Endometriosis is a benign disease which is characterized by some malignant properties: it metastasizes, it invades and compromises surrounding tissues, it is usually nonencapsulated, and it can be rapidly growing. However, the process is always well differentiated, it has a low mitotic rate, and it seems to be of limited progression in most patients. Usually no threat of the patient's life occurs, unless a vital organ becomes mechanically obstructed. In normal endometrium, the epithelial layer is regularly delimited from the underlying stroma Received July 28, 1986; revised and accepted November 24, 1986. *Reprint requests and present address: Johannes L. H. Evers, M.D., King Faisal Specialist Hospital and Research Centre, Department of Obstetrics and Gynaecology, Reproduction Unit, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia. tDepartment of Pathology. Vol. 47, No.3, March 1987
by a sharply defined BM. In recent studies 2 it has been suggested that in some patients with endometriosis the BM between the lesion and the surrounding stroma is lacking. We used antibodies raised against BM-specific type IV collagen, one of the intrinsic components of the BM, l to examine the status of the BM in endometriosis. MATERIALS AND METHODS TISSUES
In this study a selected series of endometriosis biopsies was used. Tissue specimens of 16 patients, 4 in each stage of the revised American Fertility Society (rAFS) classification,3 were studied. Because the BM does not stain with the standard hematoxylin-eosin technique, we decided to apply an immunohistochemical staining method for collagen type IV. All tissues were fixed in a mixture of alcohol, glacial acetic acid, and formaldehyde (40%) 15/1/4 (vol/vol) overnight at room temperature and routinely paraffin embedded. Serial 4-J.Lm sections were cut and mounted on glue-coated slides. ANTISERA
Polyclonal antiserum against human placental type IV collagen was induced in rabbits by repeated subcutaneous injections of 0.5 mg of type IV collagen (Sigma Chemical Company, St. Louis, MO). Antibody titer was monitored by solid phase enzyme immunoassay (ELISA). Specificity of the antiserum was tested by ELISA against highly purified human placental type IV and type V collagen, isolated according to Rhodes and Miller, 4 and bovine type I and III Evers and Willebrand Communications-in-brief
505
collagen. Furthermore, the antiserum was assayed by Western blot analysis on electroblots of the human type IV and V collagen, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In these tests reactivity was only found against type IV collagen. In the Western blot immunoreactivity was detected against 135, 112, 97, and 55 kD bands of type IV collagen, whereas only traces of reactivity were found against type V collagen. At the dilution used for immunocytochemistry, the antiserum was specific for type IV collagen. IMMUNOHISTOCHEMISTRY
Tissue sections were deparaffinized and rehydrated. For blocking of endogeneous peroxidase activity the sections were exposed to 0.5% hydrogen peroxide in methanol for 15 minutes, at room temperature. After washing the sections were treated with 0.1% pepsin (Sigma) in 0,5-M acetic acid (pH 2.6,2 hours, 37°C). After washing with tris/saline buffer (TSB) the sections were incubated with a 115 dilution of normal swine serum (Dako, Copenhagen, Denmark) for 10 minutes to reduce background staining. Subsequently the tissue sections were incubated with primary antibody (dilution 11750, 1 hour, room temperature). After washing with TSB the tissue sections were incubated with swine antirabbit IgG (dilution 111000, 30 minutes). The sections were again washed in TSB and incubated for 30 minutes with rabbit peroxidase-antiperoxidase complexes (dilution 11500, 30 minutes). After washing the sections were finally treated with a diaminobenzidine substrate solution, counterstained with hematoxylin and mounted. Specificity of the obtained immunoreaction was assessed by staining of parallel sections with preimmune serum or with immune serum preincubated with the appropriate antigen. In these sections immunoreactivity was not detected. RESULTS
All endometriosis lesions in all patients were found to be surrounded by a continuous, intact BM, irrespective oftheir localization (Fig. 1). The staining pattern of collagen type IV was homogeneous, and similar in lesions from different patients with various stages of endometriosis, scored according to the AFS classification system. 3 506
Evers and Willebrand Communications-in-brief
Figure 1 Endometriosis lesion from the peritoneum showing invasion in the surrounding tissue without 19ss of continuity of the BM (large arrow). Also, the BM of the vessel wall shows immunoreactivity to collagen type IV (small arrow).
In one patient, both endometriosis and endometrioid adenocarcinoma were found in the same ovary. Although a normal uninterrupted BM was found in the epithelial-stromal interface of the endometriosis lesions, in the adjacent tissue of the endometrioid carcinoma disruptions of the BM were observed, with a lack of immunoreactivity for collagen type IV around the invading tissues (Fig. 2). DISCUSSION
BM-type proteins have been described in benign and malignant tumors, notably those of the mammary gland, where they have been associated with tumor behavior and differentiation. Application of immunohistochemistry has shown that benign and in situ lesions are surFertility and Sterility
tions varying from the ovary and the peritoneum to the skin. The BM was homogeneous and continuous in all endometriotic lesions we investigated. We conclude that the demonstration of immunoreactivity to collagen type IV in endometriotic lesions offers no advantage in determining the prognosis of the disease and its possible response to therapy. SUMMARY
Disruption of the BM can be found in metastasizing neoplasms. It has been suggested that also in some patients with endometriosis the BM is lacking, depending on the severity of disease. 2 Thus studying the aspect of the BM would enable characterization of the disease and possibly have a prognostic value with respect to (medical) treatment. We studied the aspect of the BM in 16 patients with endometriosis, 4 in each of the stages of the rAFS classification,3 and demonstrated all endometriosis lesions in all patients to be surrounded by a continuous and intact BM. We conclude that studying the BM in endometriosis offers no advantage in determining the prognosis of the disease and its possible response to therapy. Figure 2 Lack of immunoreactivity to collagen type IV in tissue specimen from an endometrioid adenocarcinoma.
REFERENCES 1. Bosman FT, Havenith M, Cleutjens JPM: Basement
rounded by intact BMs, whereas the majority of invasive carcinomas appear to lack immunoreactivity to collagen type IV.1 No study has been published yet regarding the behavior of the BM in endometriosis, although it has been suggested that in some patients with endometriosis the BM between the lesion and the surrounding stroma is lacking. 2 We could not confirm this in 16 patients, 4 in each rAFS stage ofthe disease, and with localiza-
membranes in cancer. Ultrastruct Pathol 8:291, 1985 2. Mettler L, Semm K: Three-step therapy of genital endometriosis in cases of human infertility with lynestrenol, danazol or gestrinone administration in the second step. In Medical Management of Endometriosis, Edited by J-P Raynaud, T Ojasoo, L Martini. New York, Raven Press, 1984, p 237 3. American Fertility Society: Revised American Fertility Society Classification of Endometriosis: 1985. Fertil Steril 43:351, 1985 4. Rhodes RK, MillerEJ: Physicochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 17:3442, 1978
Vol. 47, No.3, March 1987
Evers and Willebrand Communications-in-brief
507
11 i' i I
Vol. 47, No.3, March 1987 Printed in U.8A.
FERTILITY AND STERILITY Copyright © 1987 The American Fertility Society
The basement membrane in endometriosis
Johannes L. H. Evers, M.D.* Dick Willebrand, M.D. t Academisch Ziekenhuis Maastricht, The University of Limburg, Maastricht, The Netherlands
Tumor invasion can be defined as the active migration of neoplastic cells out of their tissue of origin into adjacent tissue. Extracellular matrix barriers consist of basement membranes (BMs) and interstitial stroma. BMs occur in connection with epithelium, endothelium, mesothelium, smooth as well as striated muscle cells, Schwann cells, and fat cells. During cancer invasion loss of BM occurs. In benign diseases, such as fibroadenoma of the breast or adenomyosis of the uterus, intact BMs exist. 1 In lesions within the category of carcinoma in situ, such as intraductal carcinoma of the breast and Bowen's disease of the skin, BM is identical with the benign group oflesions. In micro invasive carcinoma, however, breaking, thinning, and fragmentation of the BM is found in the areas of microinvasion but not in adjacent areas. Endometriosis is a benign disease which is characterized by some malignant properties: it metastasizes, it invades and compromises surrounding tissues, it is usually nonencapsulated, and it can be rapidly growing. However, the process is always well differentiated, it has a low mitotic rate, and it seems to be of limited progression in most patients. Usually no threat of the patient's life occurs, unless a vital organ becomes mechanically obstructed. In normal endometrium, the epithelial layer is regularly delimited from the underlying stroma Received July 28, 1986; revised and accepted November 24, 1986. *Reprint requests and present address: Johannes L. H. Evers, M.D., King Faisal Specialist Hospital and Research Centre, Department of Obstetrics and Gynaecology, Reproduction Unit, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia. tDepartment of Pathology. Vol. 47, No.3, March 1987
by a sharply defined BM. In recent studies 2 it has been suggested that in some patients with endometriosis the BM between the lesion and the surrounding stroma is lacking. We used antibodies raised against BM-specific type IV collagen, one of the intrinsic components of the BM, l to examine the status of the BM in endometriosis. MATERIALS AND METHODS TISSUES
In this study a selected series of endometriosis biopsies was used. Tissue specimens of 16 patients, 4 in each stage of the revised American Fertility Society (rAFS) classification,3 were studied. Because the BM does not stain with the standard hematoxylin-eosin technique, we decided to apply an immunohistochemical staining method for collagen type IV. All tissues were fixed in a mixture of alcohol, glacial acetic acid, and formaldehyde (40%) 15/1/4 (vol/vol) overnight at room temperature and routinely paraffin embedded. Serial 4-J.Lm sections were cut and mounted on glue-coated slides. ANTISERA
Polyclonal antiserum against human placental type IV collagen was induced in rabbits by repeated subcutaneous injections of 0.5 mg of type IV collagen (Sigma Chemical Company, St. Louis, MO). Antibody titer was monitored by solid phase enzyme immunoassay (ELISA). Specificity of the antiserum was tested by ELISA against highly purified human placental type IV and type V collagen, isolated according to Rhodes and Miller, 4 and bovine type I and III Evers and Willebrand Communications-in-brief
505
collagen. Furthermore, the antiserum was assayed by Western blot analysis on electroblots of the human type IV and V collagen, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In these tests reactivity was only found against type IV collagen. In the Western blot immunoreactivity was detected against 135, 112, 97, and 55 kD bands of type IV collagen, whereas only traces of reactivity were found against type V collagen. At the dilution used for immunocytochemistry, the antiserum was specific for type IV collagen. IMMUNOHISTOCHEMISTRY
Tissue sections were deparaffinized and rehydrated. For blocking of endogeneous peroxidase activity the sections were exposed to 0.5% hydrogen peroxide in methanol for 15 minutes, at room temperature. After washing the sections were treated with 0.1% pepsin (Sigma) in 0,5-M acetic acid (pH 2.6,2 hours, 37°C). After washing with tris/saline buffer (TSB) the sections were incubated with a 115 dilution of normal swine serum (Dako, Copenhagen, Denmark) for 10 minutes to reduce background staining. Subsequently the tissue sections were incubated with primary antibody (dilution 11750, 1 hour, room temperature). After washing with TSB the tissue sections were incubated with swine antirabbit IgG (dilution 111000, 30 minutes). The sections were again washed in TSB and incubated for 30 minutes with rabbit peroxidase-antiperoxidase complexes (dilution 11500, 30 minutes). After washing the sections were finally treated with a diaminobenzidine substrate solution, counterstained with hematoxylin and mounted. Specificity of the obtained immunoreaction was assessed by staining of parallel sections with preimmune serum or with immune serum preincubated with the appropriate antigen. In these sections immunoreactivity was not detected. RESULTS
All endometriosis lesions in all patients were found to be surrounded by a continuous, intact BM, irrespective oftheir localization (Fig. 1). The staining pattern of collagen type IV was homogeneous, and similar in lesions from different patients with various stages of endometriosis, scored according to the AFS classification system. 3 506
Evers and Willebrand Communications-in-brief
Figure 1 Endometriosis lesion from the peritoneum showing invasion in the surrounding tissue without 19ss of continuity of the BM (large arrow). Also, the BM of the vessel wall shows immunoreactivity to collagen type IV (small arrow).
In one patient, both endometriosis and endometrioid adenocarcinoma were found in the same ovary. Although a normal uninterrupted BM was found in the epithelial-stromal interface of the endometriosis lesions, in the adjacent tissue of the endometrioid carcinoma disruptions of the BM were observed, with a lack of immunoreactivity for collagen type IV around the invading tissues (Fig. 2). DISCUSSION
BM-type proteins have been described in benign and malignant tumors, notably those of the mammary gland, where they have been associated with tumor behavior and differentiation. Application of immunohistochemistry has shown that benign and in situ lesions are surFertility and Sterility
tions varying from the ovary and the peritoneum to the skin. The BM was homogeneous and continuous in all endometriotic lesions we investigated. We conclude that the demonstration of immunoreactivity to collagen type IV in endometriotic lesions offers no advantage in determining the prognosis of the disease and its possible response to therapy. SUMMARY
Disruption of the BM can be found in metastasizing neoplasms. It has been suggested that also in some patients with endometriosis the BM is lacking, depending on the severity of disease. 2 Thus studying the aspect of the BM would enable characterization of the disease and possibly have a prognostic value with respect to (medical) treatment. We studied the aspect of the BM in 16 patients with endometriosis, 4 in each of the stages of the rAFS classification,3 and demonstrated all endometriosis lesions in all patients to be surrounded by a continuous and intact BM. We conclude that studying the BM in endometriosis offers no advantage in determining the prognosis of the disease and its possible response to therapy. Figure 2 Lack of immunoreactivity to collagen type IV in tissue specimen from an endometrioid adenocarcinoma.
REFERENCES 1. Bosman FT, Havenith M, Cleutjens JPM: Basement
rounded by intact BMs, whereas the majority of invasive carcinomas appear to lack immunoreactivity to collagen type IV.1 No study has been published yet regarding the behavior of the BM in endometriosis, although it has been suggested that in some patients with endometriosis the BM between the lesion and the surrounding stroma is lacking. 2 We could not confirm this in 16 patients, 4 in each rAFS stage ofthe disease, and with localiza-
membranes in cancer. Ultrastruct Pathol 8:291, 1985 2. Mettler L, Semm K: Three-step therapy of genital endometriosis in cases of human infertility with lynestrenol, danazol or gestrinone administration in the second step. In Medical Management of Endometriosis, Edited by J-P Raynaud, T Ojasoo, L Martini. New York, Raven Press, 1984, p 237 3. American Fertility Society: Revised American Fertility Society Classification of Endometriosis: 1985. Fertil Steril 43:351, 1985 4. Rhodes RK, MillerEJ: Physicochemical characterization and molecular organization of the collagen A and B chains. Biochemistry 17:3442, 1978
Vol. 47, No.3, March 1987
Evers and Willebrand Communications-in-brief
507