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, A9500 Villach, Austria. Email: gernot_hudelist{at}yahoo.de
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Abstract |
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Key words:
endometriosis/IL-1/MMP-1
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Introduction |
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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 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-1 (IL-1
) 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-1
(IL-1
) in endometrial tissue of patients with endometriosis. In order to investigate the role of IL-1
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.
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Materials and methods |
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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 420% Trisglycine 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 x Tris-buffered saline (TBS) +5% bovine serum albumin (BSA)] for 1 h at room temperature and incubated with 1 µg/ml of rabbit polyclonal anti MMP-1 Ab-6 (RB1536PO; NeoMarkers) in 1 x TBS +1% BSA at room temperature for 2 h. After three washes in 1 x TBS+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 = staining intensity (03)x percentage of positive cells or nuclei (0, <10%; 1, 1025%; 2, 2650%; 3, 5175%; 4, 76100%). Possible scores ranged from a minimum of 0 to a maximum of 12. Scores of 02 points were considered as negative (0); 35 points as weak staining (+); 68 points as intermediate (++); and 912 points as strong staining (+++).
Statistical analysis
SSPS version 10 (Statistical Package for Social Sciences; SSPS Inc., USA) was used for statistical analysis. Distribution of MMP-1 and IL-1 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-1
between eutopic and ectopic endometria. P<0.05 was considered statistically significant.
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Results |
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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-1 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-1
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-1 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=0.162 and P=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=0.077 and P=0.309 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<0.001 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-1
nor the expression of MMP-1 was influenced by the endometrial phase.
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Discussion |
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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-1 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)
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-1 and MMP-1, we found IL-1
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-1
autocrine loop (Yamamoto et al., 2000
). Our findings that the presence of eutopic glandular MMP-1 and IL-1
is correlated with the presence of endometriotic glandular IL-1
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-1
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 peri-menstrual 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-1 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-1
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-1
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-1
in endometriotic explants is involved in the implantation and local tissue invasion of endometriotic lesions.
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References |
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Submitted on June 29, 2004; resubmitted on December 9, 2004; accepted on January 18, 2005.
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