Interleukin 1a and tissue-lytic matrix metalloproteinase-1 are elevated in ectopic endometrium of patients with endometriosis

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1 Human Reproduction Vol.20, No.6 pp , 2005 Advance Access publication March 3, 2005 doi: /humrep/deh794 Interleukin 1a and tissue-lytic matrix metalloproteinase-1 are elevated in ectopic endometrium of patients with endometriosis G.Hudelist 1,2,7, H.Lass 3, J.Keckstein 2, I.Walter 4, F.Wieser 5, R.Wenzl 5, R.Mueller 6, K.Czerwenka 6, E.Kubista 1 and C.F.Singer 1 1 Department of Obstetrics and Gynecology, Division of Special Gynecology, Vienna Medical University, 1090 Vienna, 2 Department of Obstetrics and Gynaecology, LKH Villach, 9500 Villach, 3 Department of Obstetrics and Gynecology, Wilhelminenspital, 1060 Vienna, 4 Department of Histology/Embryology, University of Veterinary Medicine, 1200 Vienna, 5 Department of Obstetrics and Gynecology, Division of Endocrinology and Reproductive Medicine, 6 Department of Clinical Pathology, Division of Gynaecopathology, Vienna Medical University, 1090 Vienna, Austria 7 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, LKH Villach, Nikolaigasse 43, A 9500 Villach, Austria. gernot_hudelist@yahoo.de BACKGROUND: Matrix metalloproteinases (MMP) play an essential role in tissue remodelling and menstruation and appear to be regulated by cytokines such as interleukin-1a (IL-1a). In order to investigate their role in the pathogenesis of endometriosis, the aim of the present study was to compare the protein localization of matrix metalloproteinase-1 (MMP-1) and of its main stimulatory cytokine IL-1a in eutopic and dystopic endometrium of patients with endometriosis. METHODS: MMP-1 and IL-1a protein localization was analysed retrospectively in paired paraffin-embedded tissue biopsies obtained simultaneously from the endometrial cavity and from endometrial lesions of 37 patients with peritoneal or ovarian endometriosis and in cycling endometria from 37 women without endmetriosis. Protein localization was demonstrated by immunohistochemistry; antibody specifity was confirmed by western blot analysis. RESULTS: MMP-1 and IL-1a protein staining in women suffering from endometriosis was significantly more pronounced in endometriotic lesions than in eutopic endometrium. This held true for both epithelial MMP-1 and IL-1a staining (P <0.006 and P<0.001), and for stromal MMP-1 and IL-1a staining (P <0.001 and P<0.001). Furthermore, stromal MMP-1 and IL-1a were significantly co-expressed in dystopic endometriotic tissue (P ). Endometrial MMP-1 and IL-1a protein expression pattern in eutopic endometrium from women suffering from endometriosis, however, did not differ significantly from the pattern seen in healthy women. CONCLUSIONS: The increased expression of both matrix-degrading MMP-1 and its major stimulatory cytokine IL-1a in endometriotic lesions and the selective co-expression in the stroma of endometriotic foci clearly suggests their involvement in the pathogenic mechanisms leading to local invasion and tissue destruction. Key words: endometriosis/il-1a/mmp-1 Introduction Ten to fifteen per cent of women in reproductive age and up to 50% of infertile patients are affected by endometriosis, a disease that is characterized by the dystopic location of endometrial cells in tissues other than the uterine cavity (Goldman and Cramer, 1990). Although endometriosis is considered to be a benign gynaecological disorder, several pathophysiological aspects of endometrial lesions resemble the behaviour of malignant tissue: similar to carcinomas, endometriotic cells are able to invade surrounding tissue and to locally destruct the extracellular matrix (ECM), thereby leading to classical symptoms such as pelvic pain, dyspareunia or infertility. Although the association between endometriosis and infertility has led to extensive research efforts, the origin of endometriotic cells remains elusive. The most widely accepted theory on the aetiology of endometriosis is retrograde menstruation (Brosens and Brosens, 2000). However, besides the fact that this phenomenon has also been described in healthy women and can thus be viewed as a physiological process (Halme et al., 1984; Chung et al., 2002), several lines of evidence indicate that endometriotic lesions are biologically different from normal uterine endometrium (Kressin et al., 2001; Sillem et al., 2001; Chung et al., 2002; Mizumoto et al., 2002). One of the most prominent phenotypic features of endometriotic lesions is the expression of q The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved For Permissions, please journals.permissions@oupjournals.org

2 G.Hudelist et al. specific matrix metalloproteinases (MMP). Constituting a group of matrix-degrading zinc enzymes, MMP are not only known to play a pivotal role in the initiation of menstrual bleeding, but have also been shown to contribute to implantation and further invasion of seeded endometriotic explants (Singer et al., 1997; Henriet et al., 2002). Chung et al. (2001, 2002); Ria et al. (2002) and Wenzl and Heinzl (1998) have detected higher levels of MMP-2 and -9 expression in eutopic and in ectopic endometria of patients with endometriosis than in corresponding healthy controls. Furthermore, Cox et al. (2001) observed elevated levels of MMP-3 in ectopic endometrial explants compared to normal uterine-derived endometrium in a rat model of endometriosis. Some reports also indicate a possible function for MMP-1 in the development and invasion of endometriosis. During the normal menstrual cycle, MMP-1 expression and activity is highly regulated and is confined to a few days before and during menstruation, where it is thought to act as the principal tissue-degrading proteolytic enzyme (Kokorine et al., 1997; Singer et al., 1997). Kokorine et al. (1997) demonstrated a direct correlation between the expression of MMP-1 and the activity of endometriotic foci. In agreement with this finding, Sillem et al. (2001) observed elevated levels of MMP-1 and MMP-3 in culture supernatants of uterine endometrial cells of patients suffering from endometriosis when compared to normal uterine endometrium. While the release of interleukin-1a (IL-1a) appears to be the principal inducer of endometrial MMP-1 expression during menstruation (Rawdanowicz et al., 1994; Singer et al., 1997; Keller et al., 2000), the mechanisms leading to the elevation and abnormal expression of MMP-1 and other MMP in endometriotic tissue are still poorly understood. Recent studies strongly suggest a link between the expression of members of the interleukin family and the altered secretion of MMP in endometriotic lesions. Although other members of this cytokine family have been shown to be elevated in peritoneal fluids of women with endometriosis (Taketani et al., 1992; Calhaz-Jorge et al., 2003; Song et al., 2003) as well as in endometriotic tissue samples (Bergqvist et al., 2001; Chegini et al., 2003), little information exists on the localization of interleukin-1a (IL-1a) in endometrial tissue of patients with endometriosis. In order to investigate the role of IL-1a and MMP-1 in the proteolytic activity and growth of endometriotic lesions, we have analysed protein staining of both components in paired eutopic and ectopic endometrial tissue of women with endometriosis and in healthy controls. Materials and methods Patients and tissue samples Paired eutopic and ectopic endometrial tissue biopsies were obtained from 37 women with endometriosis of the pelvic cavity and from 37 patients who underwent laparoscopic surgery for diseases other than endometriosis. Characteristics of patients with endometriosis are listed in Table I. Patients suffering from endometriosis who were enrolled in the study were pre-menopausal and aged between 22 and 53 years with a median age of 36.3 years. Specimens corresponding 1696 Table I. Patient characteristics Patient no. Age (years) Cycle phase Localization of endometriotic tissue 1 45 Secretory Fallopian tube 2 51 Unknown Ovary 3 40 Proliferative Peritoneum 4 26 Secretory Ovary 5 30 Secretory Peritoneum (vesical) 6 41 Secretory Peritoneum (fossa ovarica) 7 55 Proliferative Peritoneum (cavum Douglas) 8 52 Proliferative Peritoneum (cavum Douglas) 9 33 Secretory Peritoneum (cavum Douglas) Proliferative Peritoneum (fossa ovarica) Proliferative Peritoneum (fossa ovarica) Proliferative Ovary Proliferative Sacro-uterine ligament Secretory Peritoneum (cavum Douglas) Proliferative Peritoneum (vesical) Secretory Peritoneum (fossa ovarica) Secretory Sacro-uterine ligament Secretory Ovary Proliferative Peritoneum (fossa ovarica) Proliferative Ovary Proliferative Peritoneum (vesical) Unknown Ovary Secretory Ovary (endometrioma) Proliferative Peritoneum (vesical) Unknown Ovary Proliferative Sacro-uterine ligament Proliferative Peritoneum (cavum Douglas) Proliferative Peritoneum (fossa ovarica) Proliferative Ovary (endometrioma) Proliferative Peritoneum (vesical) Proliferative Ovary Proliferative Ovary Secretory Ovary Secretory Ovary (endometrioma) Secretory Peritoneum (cavum Douglas) Proliferative Sacro-uterine ligament Secretory Peritoneum (fossa ovarica) to patients with a history of pelvic inflammatory disease or malignancy, adenomyosis uteri, intake of GnRH agonists or exposure to steroids within the last 6 months prior to surgery were excluded. The study was approved by the local IRB and only consenting patients were admitted into the study. Ectopic endometriotic tissues were obtained during laparoscopic or open surgical procedures for severe endometriosis or during hysterectomy for non-malignant lesions such as fibroids. Eutopic endometrial biopsies were either obtained by curettage immediately before the laparoscopic procedure, or were obtained by the pathologist immediately after removal of the organ in patients undergoing hysterectomy for reasons unrelated to endometrial pathology. Endometrial tissue from 37 healthy controls (confirmed by laparoscopic surgery) was also collected. Specimens were embedded in paraffin and in each endometrial sample the cycle phase was determined. Immunohistochemical staining of IL-1a and MMP-1 Antibody specifity and immunohistochemical detection of IL-1a in paraffin-embedded tissue has already been described elsewhere (Singer et al., 2002). In brief, 5 mm paraffin sections were deparaffinized and endogenous peroxidase was inactivated by a 15 min treatment with methanol containing 0.6% hydrogen peroxide. Additional antigen retrieval for MMP-1 staining was achieved by boiling the sections in 10 mmol/l of citrate buffer (ph 6.0) for 30 min in a microwave pressure cooker. After a short rinsing step in tap water, non-specific binding was blocked with goat serum for 30 min and anti-il-1a antibody (monoclonal mouse antibody; R&D

3 Elevated levels of IL 1a and MMP-1 in ectopic endometriosis Systems, USA) and anti-mmp-1 antibody (rabbit polyclonal antibody Ab-6; NeoMarkers, USA) were added at concentrations of 1:50. Formalin-fixed, paraffin-embedded human placenta was used as positive control for MMP-1 and IL-1a whereas primary antibodies were omitted in negative controls. All slides were incubated at 4 8C overnight, washed in PBS for 5 min, and incubated with peroxidase-labelled dextran polymers conjugated to antimouse/anti-rabbit immunoglobulins (EnVision; Dako Corporation, USA). Sections were subsequently washed with PBS and subjected either to diaminobenzidine (DAB) or to 3-amino-9-ethylcarbazole (AEC) substrate for 10 min. Following this, sections were rinsed in distilled water for 5 min. All slides were counterstained with Mayer s haematoxylin, dehydrated, and mounted with DPX mounting medium (Fluka, Switzerland). Determination of anti-mmp-1 antibody (Ab-6) specificity Lysates of HFL-1 cells (which express MMP-1) and of HT-1080 cells (which express MMP-9 but not MMP-1) were used to perform western blot analysis. Lysates were separated on a 4 20% Tris glycine polyacrylamide electrophoretic gel (PAGE; EC6025BOX, Invitrogen) at a constant voltage of 200 V for 30 min and transferred to a nitrocellulose membrane (LC2001; Novex) by electroblot analysis (20 V for 1 h). Standards (Amersham) were used as molecular weight markers. Membranes were then immersed in blocking solution [1 Tris-buffered saline (TBS) þ5% bovine serum albumin (BSA)] for 1 h at room temperature and incubated with 1 mg/ml of rabbit polyclonal anti MMP-1 Ab-6 (RB1536PO; NeoMarkers) in 1 TBS þ1% BSA at room temperature for 2 h. After three washes in 1 TBS þ 0.5% Tween 20 for 15 min, the membranes were incubated in alkaline phosphatase-coupled goat anti-rabbit antibody (Cat no. ALI4405; BioSource International) for 1 h at room temperature. Following another washing step, the membranes were incubated in 4-nitroblue tetrazolium chloride/5-bromo-4- chloro-3-indolyl phosphate (NBT/BCIP; Roche Diagnostic GmbH, Austria) developer solution for 10 min. Membranes were developed and finally rinsed in bi-distilled water. Immunostaining quantification A semiquantitative scoring system (immunoreactive score; IRS) according to Remmele and Schicketanz (1993) was used to allow for a reproducible evaluation of protein staining levels in ductal and stromal components of immunohistochemically stained tissue sections. Tissue sections were scored independently by two experienced pathologists. The IRS was calculated according to the following formula: IRS ¼ staining intensity (0 3) percentage of positive cells or nuclei (0,,10%; 1, 10 25%; 2, 26 50%; 3, 51 75%; 4, %). Possible scores ranged from a minimum of 0 to a maximum of 12. Scores of 0 2 points were considered as negative (0); 3 5 points as weak staining (þ); 6 8 points as intermediate (þþ); and 9 12 points as strong staining (þþþ). Statistical analysis SSPS version 10 (Statistical Package for Social Sciences; SSPS Inc., USA) was used for statistical analysis. Distribution of MMP-1 and IL-1a in eutopic and ectopic endometrial tissue was compared by using Kendall s tau-coefficient (categorical predictor). Wilcoxon s signed rank test was used for the comparison of quantitative differences in the staining of MMP-1 and IL-1a between eutopic and ectopic endometria. P, 0.05 was considered statistically significant. Results MMP-1 and IL1-a protein staining in eutopic and ectopic endometrium Table II shows staining intensities of MMP-1 and IL-1a in eutopic and ectopic endometria of patients with endometriosis: ectopic endometriotic glands exhibited strong MMP-1 staining in four cases (11%), moderate signals in eight cases (22%), weak intensities in 11 samples (29%), and lacked MMP-1 protein in 14 specimens (38%). In ectopic endometriotic stroma, MMP-1 was strongly expressed in four samples (11%), moderately in eight (22%), weakly in 16 (43%), and was absent in nine samples (24%). In contrast, glands in uterine endometrium of patients with endometriosis lacked strong MMP-1 staining. Moderate intensities were only observed in five cases (14%) and weak staining was seen in nine specimens (24%). Eutopic glandular tissue did not express MMP-1 protein in 23 cases (62%). Similarly, strong or moderate MMP-1 staining was absent in stromal components. A weak stromal MMP-1 staining was only observed in three samples (8%), and remained undetectable in all of the other 34 cases (92%). When paired eutopic and ectopic tissues were compared, glandular presence of MMP-1 in uterine samples significantly correlated with the presence of MMP-1 in glands of ectopic lesions (P ¼ 0.001, Kendall s tau test). Additionally, ectopic lesions in patients with endometriosis expressed significantly higher levels of MMP-1 when compared to corresponding eutopic tissue, regarding both glandular and stromal components (P ¼ and P, respectively, Figure 1 and 2). When the staining intensities of IL-1a in uterine and ectopic endometrium of patients with endometriosis were analysed (Table II), ectopic glands exhibited strong staining in three cases (8%), moderate in six cases (16%), weak in 19 samples (52%), and no staining in nine samples (24%). In ectopic endometriotic stroma, IL-1a lacked strong staining in any of the samples and was moderately detected in two tissues (5%), weakly in 12 (33%), and absent in 23 samples (62%). Weak stromal IL-1a protein was only detected in one uterine endometrial sample (3%) but was absent in all the other 36 biopsies obtained from endometrial curettages (97%). None of eutopic glandular components strongly expressed IL-1a (0%). Table II. Matrix metalloproteinase-1 (MMP-1) and interleukin-1a (IL-1a) protein staining intensities in ectopic and uterine endometrium of patients with endometriosis Lesion gland Lesion stroma Eutopic gland Eutopic stroma MMP-1 Strong 4 (11) 4 (11) 0 (0) 0 (0) Intermediate 8 (22) 8 (22) 5 (14) 0 (0) Weak 11 (29) 16 (43) 9 (24) 3 (8) Absent 14 (38) 9 (24) 23 (62) 34 (92) IL-1a Strong 3 (8) 0 (0) 0 (0) 0 (0) Intermediate 6 (16) 2 (5) 2 (5) 0 (0) Weak 19 (52) 12 (33) 12 (33) 1 (3) Absent 9 (24) 23 (63) 23 (63) 36 (97) Values in parentheses are percentages. Protein levels were assessed by using a semi-quantitative scoring system. 1697

4 G.Hudelist et al. Figure 1. (A) Uterine endometrium from a patient with endometriosis (magnification 100), and (B) negative for interleukin-1a (IL-1a) after staining with an anti-il-1a-specific antibody ( 200). In contrast, IL-1a is weakly expressed in stromal and strongly expressed epithelial components (C: 100; D: 200) of ectopic endometriotic tissue (peritoneal lesion) deriving from the same patient. Figure 2. (A) Uterine endometrium from a patient with endometriosis (magnification 100), and (B) negative for matrix metalloproteinase-1 (MMP-1) after staining with an anti-mmp-1-specific antibody ( 200). In contrast, MMP-1 is weakly expressed in stromal and strongly expressed in epithelial components (C: 100; D: 200) of ectopic endometriotic tissue (peritoneal lesion) deriving from the same patient. Moderate staining was observed in two samples (5%), weak staining in 12 samples (32%), whereas 23 cases failed to express stromal IL-1a (63%). Glandular localization of IL-1a protein staining in uterine endometrium significantly correlated with glandular IL-1a staining of corresponding ectopic lesions (P, 0.001, Kendall s tau). Similar to MMP-1, uterine glandular and stromal endometrial cells expressed significantly lower 1698 levels of IL-1a when compared to ectopic endometrial tissue (P, and P, respectively, Kendall s tau). In addition, stromal IL-1a protein expression was significantly coexpressed with stromal MMP-1 in dystopic endometriotic tissue (P ¼ 0.045). However, no such correlation was found for the epithelial or epithelial stromal expression pattern of these two compounds.

5 Elevated levels of IL 1a and MMP-1 in ectopic endometriosis Histological dating confirmed that 19 patients were in the proliferative phase and 14 were in the secretory phase. In four patients, the phase of the menstrual cycle could not be determined. In our patient collective, the presence of IL-1a and MMP-1 was not influenced by the endometrial phase in eutopic and ectopic endometrial samples. Furthermore, the location of the endometriotic lesions (ovarian endometriosis versus peritoneal lesions) did not yield significant differences in MMP-1 or IL-1a expression. We therefore evaluated the results of immunohistochemical detection of both parameters irrespective of endometriotic location and menstrual cycle. We then looked at the IL-1a protein staining in endometria from women without endometriosis undergoing comparable cycle phases (Table III). We found an intermediate and weak epithelial expression in only three cases each (8% and 8% respectively) and a weak stromal expression in only one case (3%), which is similar to the expression pattern we observed in the endometrium of endometriotic patients (P ¼ and P ¼ 0.368, Kendall s tau). By analogy, in comparable women without endometriosis, epithelial MMP-1 expression was detected in 12 cases (one case of moderate expression, 11 cases of weak expression, 3% and 30%) and stromal expression in 19 cases (two cases of moderate, 17 cases of weak expression, 5% and 46%) which is again similar to the distribution pattern we observed in the endometrium of endometriotic patients (P ¼ and P ¼ Kendall s tau). Stromal MMP-1 in uterine endometrium of healthy controls was found to be elevated when compared to eutopic endometrium of patients with endometriosis (P, respectively). No further quantitative differences could be found between healthy controls and uterine endometrium of patients with endometriosis. Twenty-two patients were in the proliferative phase and 15 were in the secretory phase. Again, statistical analysis of the two subgroups revealed that neither the presence of IL-1a nor the expression of MMP-1 was influenced by the endometrial phase. Table III. Matrix metalloproteinase-1 (MMP-1) and interleukin-1a (IL-1a) protein staining intensities in normal endometrium of women without endometriosis Glandular Stromal MMP-1 Strong 0 (0) 0 (0) Intermediate 1 (3) 2 (5) Weak 11 (30) 17 (46) Absent 25 (68) 18 (49) IL-1a Strong 0 (0) 0 (0) Intermediate 3 (8) 0 (0) Weak 3 (8) 1 (3) Absent 31 (84) 36 (97) Protein levels were assessed by using a semi-quantitative scoring system. Figure 3. Western blot demonstrating the specifity of anti-mmp-1 antibody (NeoMarkers Ab-6; 57 kda, lane 2). To exclude cross-reactivity with MMP-9, cell lysate of MMP-9 expressing cell lines (HT- 1080) was loaded on lane 3 lacking reactivity with anti-mmp-1 Ab6. Lane 1: molecular weight markers (97, 66, 45, 30 and 20 kda). Specifity of anti-mmp-1 antibody Ab-6 by western blot analysis Figure 3 shows the results of western blot analysis performed on MMP-1-expressing HFL-1 cells (lane 2) and MMP-9- expressing HT-1080 cells (lane 3). Lane 1 shows molecular weight markers. The anti-mmp-1 antibody used for immunohistochemistry specifically detects MMP-1 (lane 2, band at 57 kda) but does not recognize MMP-9 (lane 3, band at 92 kda). Discussion It is now well established that MMP play an essential role in tissue remodelling and menstruation. In regularly cycling endometrium, the expression of MMP-1 is induced by the peri-menstrual fall of ovarian steroids and appears to be mediated by interleukins, most importantly by IL-1a. Rawdanowicz et al. (1994) were able to stimulate the secretion of MMP-1, -3 and -9 in cultured human endometrial cells by adding IL-1a to culture media. Using endometrial cells and tissue explants, Singer et al. (1997) demonstrated a correlation between the epithelial release of IL-1a and the stromal production of MMP-1 which could be suppressed by anti-il-1a antibodies and sex steroids. Similarly, in vitro data by Keller et al. (2000) provide evidence for the IL-1a-mediated stimulation of MMP-3, which could be blocked by progesterone in a dose-dependent manner. Besides a possible paracrine function of IL-1a in the stimulation of MMP, a study conducted on dermal fibroblasts (Yamamoto et al., 2000) also suggested a mechanism by which expression of MMP-1 is mediated by IL-1a via an autocrine loop. In the present study, we hypothesized that the peri-menstrual rise of these two compounds also could play a role in the implantation and growth of ectopic endometrium. Indeed, we observed that ectopic endometrial tissue obtained from patients with endometriosis expressed significantly higher amounts of IL-1a and MMP-1 compared to corresponding eutopic tissue deriving from the same patients. Interestingly, the presence of MMP-1 was not exclusively confined to stromal components but could also be detected in eutopic and ectopic epithelial cells. This is in contrast to studies by Kokorine et al. (1996) 1699

6 G.Hudelist et al. and Salamonsen and Woolley (1996) who detected MMP-1 expression exclusively in stromal components of normal endometrial tissue. However, both groups only examined the presence of MMP-1 in eutopic endometrium but did not extend their investigations to endometriotic lesions. Epithelial expression of MMP-1 was also shown by Mizumoto et al. (2002) using immunohistochemical techniques, thereby supporting our observation that MMP-1 is secreted by both stromal and epithelial endometrial cells. Although the present work does not provide evidence for a mechanistic link between IL-1a and MMP-1, we found IL-1a to be significantly co-expressed with MMP-1 in the stroma of dystopic endometriotic tissue. This observation is in accordance with previous studies indicating that the secretion of MMP-1 in endometriotic tissue could be cytokine-mediated, possibly via an IL-1a autocrine loop (Yamamoto et al., 2000). Our findings that the presence of eutopic glandular MMP-1 and IL-1a is correlated with the presence of endometriotic glandular IL-1a and MMP-1 protein suggests a biochemical similarity of eutopic and ectopic endometrial tissue. We therefore aimed to investigate paired eutopic and ectopic endometrial samples from patients with endometriosis, in contrast to previous works investigating endometrial tissues obtained from patients with endometriosis and healthy controls. Nevertheless, we also looked at the expression of IL-1a and MMP-1 in endometrial samples of healthy controls. We found MMP-1 levels to be elevated in uterine stroma of women without disease when compared to uterine stroma of patients with endometriosis during both the secretory and proliferative phases. This somewhat contradicts previous studies demonstrating that significant endometrial MMP secretion is confined to the comparatively short perimenstrual phase. Although the reason for and significance of the decreased uterine MMP-1 expression in women with endometriosis are unclear, it is quite possible that it is the side-effect of a physiological systemic feedback regulation by which the body attempts to down-regulate elevated MMP in endometriotic foci. Some in vitro studies have already pointed out a possible correlation between the altered expression of MMP-1 and IL- 1a and the ability of endometrial cells to infiltrate their local environment. Recently, Wolber et al. (2003) found a significant increase of MMP-1 mrna concentrations of endometrial explants after culture on a model of endometriosis using chorioallantoic membranes of chick embryos (CAM). Apart from several studies reporting on altered cytokine levels in endometriotic tissue, our own laboratory was able to detect a direct correlation between the expression of IL-1a and the invasive properties of malignant endometrial epithelium (Singer et al., 2002). To our knowledge, this is the first report describing elevated levels of IL-1a and MMP-1 protein in endometriotic lesions when compared to the corresponding eutopic endometrium from patients with endometriosis. We therefore hypothesize that the increased expression of MMP-1 and its stimulatory cytokine IL-1a in endometriotic explants is involved in the implantation and local tissue invasion of endometriotic lesions References Bergqvist A, Bruse C, Carlberg M and Carlstrom K (2001) Interleukin 1beta, interleukin-6, and tumor necrosis factor-alpha in endometriotic tissue and in endometrium. Fertil Steril 75, Brosens IA and Brosens JJ (2000) Endometriosis. Eur J Obstet Gynecol Reprod Biol 90, Calhaz-Jorge C, Costa AP, Santos MC and Palma-Carlos ML (2003) Peritoneal fluid concentrations of interleukin-8 in patients with endometriosis depend on the severity of the disorder and are higher in the luteal phase. 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