Endometriosis is an enigmatic disease of unknown origin.

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1 The Depolarized Expression of the Alpha-6 Integrin Subunit in the Endometria of Women With Endometriosis María del Mar Vernet-Tomás, MD, PhD, Carlos Tomás Pérez-Ares, MD, PhD, Núria Verdú, MD, María Teresa Fernández-Figueras, MD, PhD, José Luis Molinero, MD, and Ramón Carreras, MD, PhD OBJECTIVE: The current study sought to compare the endometrial localization of the integrin subunit alpha-6 in women with endometriosis and women without the disease. Alpha-6 integrins have an important function, not only in the attachment of cells to the extracellular matrix and laminin, but they also serve as inductors of cell migration and invasion, depending on their pattern of expression in the cell membrane. METHODS: The endometriosis group consisted of 32 women with a confirmed diagnosis of endometriosis by laparoscopy or laparotomy. The control group consisted of 20 women not having endometriosis or any other gynecologic disease at laparoscopy. Endometria were obtained by biopsy. Immunohistochemical techniques were used to assess alpha-6 localization. In each section, the percentage of positive cells and the localization of expression were evaluated. RESULTS: All glandular cells expressed alpha-6 in all of the samples but presented two different patterns, either only in the basal side of the cells (polarized) or also in other sides of the cells (depolarized). The percentage of samples showing depolarized expression was significantly higher in the endometriosis group (66.6% vs 15.8%, , P.001). CONCLUSIONS: The endometria of women with endometriosis more frequently show a depolarized expression of integrin subunit alpha-6, a characteristic usually found in highly proliferating cells with migrating and invasive abilities. (J Soc Gynecol Investig 2006;13:292 6) Copyright 2006 by the Society for Gynecologic Investigation. KEY WORDS: Endometriosis, integrin alpha-6, endometrium, depolarization. From the Department of Obstetrics and Gynecology, Hospital Universitari del Mar, Barcelona, Spain; and the Departments of Obstetrics and Gynecology, and Pathology, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain. Supported by the Fondo de Investigación Sanitaria of the Spanish Ministry of Health (grant number 94/609). The authors thank Marta Pulido, MD, for editing the manuscript and editorial assistance. Address correspondence and reprint requests to: Maria del Mar Vernet-Tomás, MD, PhD, Department of Obstetrics and Gynecology Hospital Universitari del Mar, Passeig Marítim 25 29, E Barcelona, Spain. mvernet@imas.imim.es Endometriosis is an enigmatic disease of unknown origin. The most accepted hypothesis is the one formulated by Sampson in 1927, in which the endometrium reaches the peritoneum through retrograde menstruation. 1 Several investigators have proposed that molecular differences between the endometria of women with endometriosis and endometria of women without endometriosis could explain why not all women with retrograde menstruation develop the illness. 2 In this respect, the expression of integrins in the endometrium, one of the families of cell adhesion molecules, has been investigated. Some differences have been found, for instance the lack of expression of alpha-v beta-3 in the endometria of women with endometriosis during the period of uterine receptivity, suggesting a deficient implantation in these women. 2,3 Interestingly, integrins have a crucial function in the attachment of cells to the extracellular matrix and in the promotion of cell migration and invasion. In the last few years, alpha-6 integrins have been the focus of attention of many investigators, 4 10 and not only because of their attachment functions. They also have roles as important transducers of signals from the extracellular matrix to the intracellular medium and act as stimulators of some growth factors. 8 Signaling through alpha-6 beta-4 promotes cell migration and invasion more effectively than through other integrins. 9 Importantly, changes in the pattern of expression of the alpha-6 subunit are associated with highly proliferating cells. 5,10 The loss of basal localization to other sides of the cell is associated with acquisition of an invasive phenotype. 6,7 The expression of alpha-6 integrin subunit in normal endometria has been previously reported, but there are a few data about the expression of alpha-6 in the endometria of women with endometriosis. 13,18 The aim of the current study was to compare the expression and localization of integrin subunit alpha-6 in the endometria of women with and without endometriosis. METHODS The endometriosis group consisted of 32 patients operated on in our Department because of pelvic pain, sterility, menstrual Copyright 2006 by the Society for Gynecologic Investigation /06/$32.00 Published by Elsevier Inc. doi: /j.jsgi

Alpha-6 Integrin Depolarization in Endometriosis J Soc Gynecol Investig Vol. 13, No. 4, May 2006 293 disturbances, or adnexal masses, and in whom the only diagnosis was endometriosis.

2 Alpha-6 Integrin Depolarization in Endometriosis J Soc Gynecol Investig Vol. 13, No. 4, May disturbances, or adnexal masses, and in whom the only diagnosis was endometriosis. The control group consisted of 20 women who underwent laparoscopy for tubal sterilization, pelvic pain, or sterility, and in whom no evidence of endometriosis or any other gynecologic disease was found. Patients with history of immune disease, malignancy, hormone therapy, or immune therapy were excluded from the study. Samples of endometrium were obtained by biopsy. In the endometriosis group, excision of endometriosis implants was performed during laparoscopy or laparotomy. The hospital committees on human research approved the protocol, and written informed consent was obtained from all participants. Both groups were compared for age, the percentage of nulliparous women, and the percentage of samples in proliferative and secretory phases of the cycle. The endometriotic tissue was embedded in paraffin and stained with hematoxilin and eosin to histologically confirm endometriosis. The samples of endometrium from both groups were cut into blocks of approximately 1 cm 3. Tissue aliquots were embedded in paraffin and stained with hematoxilin and eosin for histologic study. Other aliquots were immediately frozen in liquid nitrogen for immunohistochemical analysis. The monoclonal antibody used for alpha-6 staining was CA- 956M (Labgen, Labclinics, Barcelona, Spain) diluted 1/10 (antigen retriever was not used). The peroxidase-antiperoxidase method was used, by which representative 6 m thick cryostat sections were cut and mounted on slides. They were fixed in acetone, then washed three times for 5 minutes in phosphatebuffered saline (PBS) and immersed in 0.3% hydrogen peroxide to block endogenous peroxidase activity. The sections were washed again three times for 5 minutes in PBS and then pre-incubated with diluted normal serum for 15 minutes. Incubation with primary antibodies was performed for 1 hour at 37C. After washing three times for 10 minutes in PBS, the sections were incubated for 30 minutes with a biotin-labeled immunoglobulin, IgG. After washing in PBS, the sections were incubated with a streptavidin-biotin-peroxidase complex for 30 minutes. After washing the sections three times for 10 minutes with PBS, they were stained with diaminobenzidine and hydrogen peroxide. Negative controls for immunostaining were prepared by replacing the first antibody with non-immune serum IgG. In addition, tissue samples of a case of squamous cell carcinoma of the vulva were used as negative external control (Figure 1). Frozen sections of lymph nodes and tissue samples of carcinoma of the colon (Figure 2) were used as positive external controls. Endothelia cells were used as positive internal controls. Samples were examined by two independent observers who were blinded to the study group. Evaluation of the staining was made by examining ten non-overlapping fields per biopsy with a magnification of 400, counting a total of 300 to 400 cells per case. The percentage of positive glandular cells and the localization of expression were evaluated in each section. It was considered that alpha-6 localization was polarized when expression was exhibited only on the basal side of the cell. When expression was observed in the basal side, as well as in Figure 1. Squamous cell carcinoma of the vulva were used as negative external control. Original magnification 400. any other side of the cell, alpha-6 localization was considered depolarized. The control and endometriosis groups were compared, first considering all of the samples together, and then comparing samples in the proliferative phase and samples in the secretory phase separately. In the endometriosis group, the expression of alpha-6 between in samples of the proliferative phase and samples of the secretory phase was compared. Statistical analysis was performed using the SPSS computer program (SPSS Inc, Chicago, IL). Categorical data were compared with the chi-square for normally distributed variables or the Fisher exact test when the distribution of variables departed from normality. Continuous data were compared with the Student t test. RESULTS Nineteen samples in the control group and 30 samples in the endometriosis group were found to be valid for the study. One sample in the control group and two samples in the endometriosis group were illegible probably due to deficiencies in the freezing process. In the control group, the mean (SD) age was (3.36) (range, 27 to 41 years). Twelve women were parous and seven were nulliparous. The 19 patients underwent a laparoscopy (tubal sterilization, n 11; sterility, n 3; pelvic pain, n 5). Four patients were in the proliferative phase and 15 in the secretory phase. The mean age in the endometriosis group was 32.8 (7.11) (range, 20 to 50 years). Thirteen patients were parous and 17 were nulliparous. Thirteen patients underwent a laparoscopy, while 17 underwent laparotomy (pelvic pain, n 12; sterility, n 8; adnexal mass, n 6; menstrual disturbances, n 4). Fifteen patients were in the proliferative phase and 15 in the secretory phase. Following the revised American Fertility Society Classification, two patients had endometriosis stage I, two endometriosis stage II, 11 endometriosis stage III, and 15 endometriosis stage IV. The group with endometriosis and the control group were comparable in terms of mean age (t test

19 More recently, alpha-6 integrins, including alpha-6 beta-1 and alpha-6 beta-4, have been implicated as a determinant factor in cell migration capacity 6 and in the progression of neoplastic cells.

3 294 J Soc Gynecol Investig Vol. 13, No. 4, May 2006 Vernet-Tomás et al functions in cell adhesion, cell migration, and cell invasion. Classically, this integrin subunit has been studied as a receptor for most of the known laminins and its altered expression explains some skin diseases. 19 More recently, alpha-6 integrins, including alpha-6 beta-1 and alpha-6 beta-4, have been implicated as a determinant factor in cell migration capacity 6 and in the progression of neoplastic cells. 7 Alpha-6 beta-4 is functionally associated with the epidermal growth factor (EGF) receptor ErbB-2 4 and stimulates translation of vascular endothelial growth factor (VEGF). 6 Marked stimulation of migra- Figure 2. Tissue samples of carcinoma of the colon were used as positive external controls. Original magnification , P.451) and parity ( , P.176) but not for the number of samples in the proliferative phase, which was significantly higher in the endometriosis group (Fisher test, P.026). In both groups, all of the samples showed alpha-6 in 100% of the endothelial cells in the basal sides of the cells (polarized). The endometrial stromal cells were negative. In the control group, glandular cells were 100% positive in all of the samples, but they showed two different localizations for alpha-6 integrin: polarized in 16 of 19 patients (84.2%, Figure 3A and 3B) and depolarized in three of 19 patients (15.8%). In the endometriosis group, glandular cells were 100% positive in all of the samples, also presenting the two different patterns of molecular localization described: polarized in 10 of 30 patients (33.3%) and depolarized in 20 of 30 patients (66.6%, Figure 4A and 4B). In all cases it was observed that for each patient, expression of alpha-6 integrin was consistently of one type, that is, polarized or depolarized. Mixed patterns were not found. There was a significantly higher percentage of depolarized expression of alpha-6 in the endometriosis group than in controls ( , P.001). In the subset of samples in the proliferative phase (controls, n 4; endometriosis, n 15), the difference remained significant (more depolarized samples in the endometriosis group) 10 of 15 versus zero of four in controls (Fisher test, P.033). Similar results were obtained in the subset of samples in the secretory phase (controls, n 15; endometriosis, n 15), with more depolarized samples in the endometriosis group (10/15 vs 3/15, , P.011). In the endometriosis group, however, no differences were found in the percentage of samples with depolarized expression of alpha-6 integrin when samples in the proliferative (10 of 15) and secretory (10 of 15) phases were compared. DISCUSSION A member of the huge integrin family, alpha-6 is a molecule that holds the attention of investigators due to its important Figure 3. Polarized alpha-6 expression in a patient from the control group corresponding to an endometrium of the eighth day of the menstrual cycle. Alpha-6 integrin expression is exclusively limited to the basal side of the cells. A, original magnification 200; B, original magnification 400.

Alpha-6 Integrin Depolarization in Endometriosis J Soc Gynecol Investig Vol. 13, No. 4, May 2006 295 Figure 4.

4 Alpha-6 Integrin Depolarization in Endometriosis J Soc Gynecol Investig Vol. 13, No. 4, May Figure 4. Depolarized alpha-6 expression in a patient from the endometriosis group corresponding to an endometrium of the 24th day of the menstrual cycle. In contrast to Figure 3, alpha-6 integrin expression is found in the lateral sides and occasionally in the apical portion of glandular cells. A, original magnification 200; B, original magnification 400. tion and invasion is observed in response to alpha-6 beta-4 signaling, and, importantly, in these migrating cells alpha-6 loses its basal localization (polarization). 7,9 Migration of alpha-6 from the basal side to other sides of the cells would mean a switch from its attachment function to a signaling function. 6 In this study, a depolarized localization of alpha-6 integrin was more frequently found in the endometria of women with endometriosis than in controls. As described previously, in other epithelium depolarization is associated with highly proliferating cells 5,10,20 that lose their attachment to the basal membrane through hemidesmosomes. This study may suggest an interesting hypothesis that endometrial glandular cells of women with endometriosis may exhibit characteristics corresponding to rapidly proliferating cells and with migration capacity. New studies are needed to confirm this hypothesis. Actually, similarities between the endometria of women with endometriosis and carcinoma cells have already been described by Zeitvogel et al., who found cadherins expressed in a pattern that was reminiscent of the expression in tumoral cells. 21 It should be noted that there was a considerable heterogeneity among the samples. The control group and the endometriosis group were not equivalent for samples in the proliferative and the secretory phases of the cycle, as there was a higher percentage of samples in the secretory phase in the endometriosis group. This is a limitation of the study because molecular expression may vary in the different phases of the menstrual cycle. However, the majority of investigators who have studied endometrial expression of alpha-6 integrin have not documented changes in expression of the alpha-6 integrin subunit during the menstrual cycle. 11,12,15,22 Furthermore, the higher depolarized localization found in the endometriosis group compared with controls was also statistically significant when samples in the proliferative and secretory phases were analyzed separately. However, within the endometriosis group, the percentage of samples showing depolarized localization was exactly the same in both phases of the cycle. These data suggest that the greater percentage of depolarized localization in the endometriosis group seems to be independent of hormonal factors. The fact that endometrial samples were taken at any time of the cycle is another limitation of the study. It will be of interest to confirm the present findings in menstrual endometrium samples. Up to the present time there are a few studies of alpha-6 expression in patients with endometriosis. 13,18 In none of these studies, however, was a difference in alpha-6 expression between endometriosis and controls found. Moreover, depolarization of this molecule was not studied. Further research should be carried out to confirm the importance of alpha-6 depolarization in the endometrium. Our study showed interesting data for defining the molecular alterations in the endometria of women with endometriosis. These molecular alterations might contribute to explain the greater ability to migrate and adhere to the peritoneum, when these endometrial cells reach the abdominal cavity during retrograde menstruation. REFERENCES 1. Sampson J. Metastatic or embolic endometriosis due to menstrual dissemination of endometrial tissue into venous circulation. Am J Pathol 1927;3: Sharpe-Timms KL. Endometrial anomalies in women with endometriosis. Ann N Y Acad Sci 2001;943: Lessey BA, Castelbaum AJ, Sawin SW, et al. Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab 1994;79:643 9.

5 296 J Soc Gynecol Investig Vol. 13, No. 4, May 2006 Vernet-Tomás et al 4. Falcioni R, Antonini A, Nistico P, et al. Alpha 6 beta 4 and alpha 6 beta 1 integrins associate with ErbB-2 in human carcinoma cell lines. Exp Cell Res 1997;236: Lussier C, Basora N, Bouatrouss Y, Beaulieu JF. Integrins as mediators of epithelial cell-matrix interactions in the human small intestinal mucosa. Microsc Res Tech 2000;51: Mercurio AM, Rabinovitz I, Shaw LM. The alpha 6 beta 4 integrin and epithelial cell migration. Curr Opin Cell Biol 2001; 13: Mercurio AM, Bachelder RE, Chung J, et al. Integrin laminin receptors and breast carcinoma progression. J Mammary Gland Biol Neoplasia 2001;6: Chung J, Bachelder RE, Lipscomb EA, Shaw LM, Mercurio AM. Integrin (alpha 6 beta 4) regulation of eif-4e activity and VEGF translation: A survival mechanism for carcinoma cells. J Cell Biol 2002;158: Giannelli G, Astigiano S, Antonaci S, et al. Role of the alpha3beta1 and alpha6beta4 integrins in tumor invasion. Clin Exp Metastasis 2002;19: Sashiyama H, Shino Y, Sakao S, et al. Alteration of integrin expression relates to malignant progression of human papillomavirus-immortalized esophageal keratinocytes. Cancer Lett 2002;177: Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA. Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992;90: Tabibzadeh S. Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod 1992;7: Bridges JE, Prentice A, Roche W, Englefield P, Thomas EJ. Expression of integrin adhesion molecules in endometrium and endometriosis. Br J Obstet Gynaecol 1994;101: Lanteri E, Pistritto M, Bartoloni G, Cordaro S, Stivala F, Montoneri C. Expression of alpha6 and beta4 integrin subunits on human endometrium throughout the menstrual cycle and during early pregnancy. Fertil Steril 1998;69: Murray MJ, Zhang J, Lessey BA. Expression of alpha6 and beta4 integrin subunits throughout the menstrual cycle: no correlation with uterine receptivity. Fertil Steril 1999;72: Koks CA, Groothuis PG, Dunselman GA, de Goeij AF, Evers JL. Adhesion of menstrual endometrium to extracellular matrix: The possible role of integrin alpha(6)beta(1) and laminin interaction. Mol Hum Reprod 2000;6: Park KR, Inoue T, Ueda M, et al. CD9 is expressed on human endometrial epithelial cells in association with integrins alpha(6), alpha(3) and beta(1). Mol Hum Reprod 2000;6: Regidor PA, Vogel C, Regidor M, Schindler AE, Winterhager E. Expression pattern of integrin adhesion molecules in endometriosis and human endometrium. Hum Reprod Update 1998;4: Hogg N, Bates PA. Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biol 2000;19: De Luca M, Pellegrini G, Zambruno G, Marchisio PC. Role of integrins in cell adhesion and polarity in normal keratinocytes and human skin pathologies. J Dermatol 1994;21: Zeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an invasive, N-cadherin-expressing epithelial cell type in endometriosis using a new cell culture model. Am J Pathol 2001;159: van der Linden PJ, de Goeij AF, Dunselman GA, Erkens HW, Evers JL. Expression of cadherins and integrins in human endometrium throughout the menstrual cycle. Fertil Steril 1995;63:

Cadherin expression in gastrointestinal tract endometriosis: possible role in deep tissue invasion and development of malignancy

38 & 2010 USCAP, Inc. All rights reserved 0893-3952/10 $32.00 Cadherin expression in gastrointestinal tract endometriosis: possible role in deep tissue invasion and development of malignancy Katy Van Patten,