Hum. Reprod. Advance Access originally published online on December 1, 2008
Human Reproduction 2009 24(2):333-340; doi:10.1093/humrep/den392
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Menstrual activity of matrix metalloproteinases is decreased in endometrium regenerating after thermal ablation
1 Department of Gynaecology and Obstetrics, Hôpital Universitaire Pellegrin, Place Amélie-Raba-Léon, F-33076 Bordeaux, France 2 Cell Biology Unit, de Duve lnstitute, Brussels, Belgium 3 Department of Pathology, Saint-Luc University Clinics, Université catholique de Louvain, Avenue Hippocrate 10, B-1200 Bruxelles, Belgium
4 Correspondence address. E-mail:jean-luc.brun{at}chu-bordeaux.fr
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Abstract |
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BACKGROUND: Menstruation is associated with a striking increase in matrix metalloproteinase (MMP) activity. However, it is still unknown whether the level of MMP activity correlates with the amount of menstrual bleeding.
METHODS: We used histochemistry to investigate the degradation of the extracellular matrix (ECM), and immunohistochemical labelling and zymographic analysis to determine the level of expression and activity of MMP-2 and -9, and of their tissue inhibitors (TIMPs) -1, -2 and -3, in endometria sampled during menstruation in 14 women experiencing excessive menstrual bleeding and in 10 women successfully treated for menorrhagia by thermal ablation of the endometrium.
RESULTS: After thermal ablation, regenerated menstrual endometria showed reduced areas of collagen fibre lysis and increased content of TIMP-1 and TIMP-2 compared with endometria from non-treated menorrhagic women. Surprisingly, treated endometria contained more latent gelatinase A (proMMP-2) but a lower proportion of the active form of gelatinase B (MMP-9) than non-treated endometria.
CONCLUSIONS: These results suggest that ECM degradation is decreased at menstruation in the endometrium regenerated after thermal ablation, mostly because of an increased TIMP expression. This represents the first molecular explanation for the decreased amount of menstrual bleeding.
Key words: MMP/endometrial ablation/menstruation
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Introduction |
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Excessive menstrual bleeding is a major health problem that affects 20–30% of women in their reproductive age (Kjerulff et al., 1996
). In about half of the cases, no underlying pathology is present and essential menorrhagia is diagnosed (Rees, 1987).
The cellular and molecular mechanisms accounting for essential menorrhagia are poorly understood. Although down-regulation of angiopoietin-1 expression may contribute to excessive blood loss (Hewett et al., 2002), amplification of the normal menstrual mechanisms is an attractive alternative. Menstruation is characterized by haemorrhagic shedding of the superficial layer of the endometrium as a result of extracellular matrix (ECM) breakdown, associated with the expression of matrix metalloproteinases (MMPs) (Kokorine et al., 1996).
MMPs are a family of enzymes able to degrade all proteins of the ECM at neutral pH. Most MMPs are secreted as inactive zymogens (proMMPs) that need activation by cleavage of the propeptide to be able to degrade their substrates. Many MMPs are found in the human endometrium, and most of them are preferentially expressed at menstruation (reviewed by Hulboy et al., 1997; Henriet et al., 2002). Collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) and -2 (MMP-10) are almost exclusively expressed during the menstrual phase, whereas gelatinases A (MMP-2) and B (MMP-9) occur throughout the cycle, but are more abundant during the menstrual phase (Rigot et al., 2001; Vassilev et al., 2005). The tissue inhibitors of metalloproteinases (TIMPs) and 
2-macroglobulin, which inhibit the active forms of MMPs, are also produced in the human endometrium. Studies on MMP activity have been mainly carried out in vitro on explants or cell cultures. Gelatinase activities have also been localized by in situ zymography on snap-frozen sections of endometrial tissues in discrete foci of increasing number at menstruation or during dysfunctional endometrial bleeding (Zhang and Salamonsen, 2002; Galant et al., 2004). However, none of these studies reported any link between MMP activities and the amount of bleeding.
The first-line management of essential menorrhagia is mainly based on medications. If unsuccessful, this is usually followed by endometrial curettage, which is considered relatively inefficacious, or hysterectomy, which may be too aggressive, so surgical alternatives have been proposed (Goldrath et al., 1981). The hysteroscopic ablative techniques and the non-hysteroscopic techniques, such as thermal balloon ablation, destroy the endometrium and damage the inner third of the myometrium. Both procedures aim at preventing or reducing the regeneration of the mucosa (Shah et al., 1998) and effectively reduce menorrhagia without major side effects, with success rates ranging between 70 and 90%, depending on the duration of follow-up (Lethaby et al., 2005).
Surprisingly, most treated women do not become amenorrheic despite extensive destruction of the mucosa. The reasons for persistent menstruation after endometrial ablation are unclear. A functional endometrium may differentiate from deep remnants of the mucosa in the superficial myometrium or regenerate laterally from unaltered cornual and/or isthmic endometrium. The cellular and molecular activities of this ‘new’ functional endometrium are unknown.
The present study investigated the in vivo expression, localization and activity of MMPs and TIMPs at bleeding in non-treated menorrhagic women and in women treated by endometrial ablation who recovered normal menses. It also addresses the possible relationship between MMP and TIMP expression, ECM breakdown and the results of treatment.
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Materials and Methods |
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Clinical information and tissue sampling
This prospective study was undertaken in 28 currently or previously menorrhagic women admitted to the Department of Obstetrics and Gynaecology of Bordeaux University Hospital. Menstrual bleeding was quantified by using pictorial charts, with levels >100 qualifying as menorrhagia (Higham et al., 1990
; Reid and Virtanen-Kari, 2005). None of the women received any hormonal therapy before sampling. Previously menorrhagic women were treated by endometrial ablation using thermal balloon therapy (CavatermTM, Wallsten, Morges, Switzerland). Briefly, after cervical dilation to the size of a Hegar probe Nr 9, a silicone balloon with a self-regulating heating element was introduced into the uterine cavity and filled with glycine solution. The balloon was inflated until intrauterine pressure stabilized between 180 and 220 mm Hg. A pump induced circulation of the fluid inside the catheter to distribute heat from the heating elements located in the device handle to the balloon surface. The temperature of the balloon surface was set by the manufacturer at 80°C, giving an operative temperature of 75°C. The treatment lasted 15 min (Brun et al., 2006). The local Ethical Committee approved the study on thermal balloon ablation in menorrhagia and all women gave written informed consent. Endometrial biopsies were performed using the Pipelle device or by curettage after hysteroscopy. Biopsies were sampled during the early menstrual phase: 14 in menorrhagic non-treated women and 10 in women treated by endometrial ablation. Four women who became amenorrheic after surgery were also sampled during the recruitment period.
Tissue processing
Endometrial biopsies were embedded in Tissue Tek OCT CompoundTM (Sakura, Zoeterwoude, Netherlands), frozen in liquid nitrogen and kept at –20°C until analysis. A 6-µm-thick frozen section was first examined histologically to confirm the nature of the material sampled and the absence of pathology. Five further frozen sections of 20-µm thickness were used for the biochemical analyses. Sections underwent two washes with 500 µl phosphate-buffered saline, pH 7.4, for 1 h at 4°C under agitation to remove the blood accompanying the tissue, most of the blood already being recovered in the first washing solution. The material was then solubilized in 250 µl of 50 mM Tris/HCl buffer, pH 7.5, containing 150 mM NaCl, 10 mM CaCl2, 1 µM ZnCl2, 3 mM NaN3 and 0.05% (v/v) Triton X-100. The remaining frozen tissue was fixed overnight in 4% formaldehyde and embedded in paraffin for histological and immunohistochemical analysis.
Histological and immunohistochemical analysis
Serial histological sections were stained with haematoxylin and eosin, silver (Gordon and Sweets, 1936), Alcian blue or Masson's trichrome. Silver staining was performed to highlight the network of so-called reticulinic fibres, rich in collagen III, and Masson's trichrome to indicate fibrosis of the tissue. Alcian blue stains the glycosaminoglycans of the ECM as well as the mucus. These histochemical stains allowed assessment of the relative amounts of mucus and blood, and evaluation of the stromal breakdown and lysis of the collagen fibres within the endometrial tissue. These parameters were semi-quantitatively assessed for each endometrium by estimating their proportional area on individual histological sections. Analysis was performed independently by two pathologists unaware of the clinical data and results were averaged. Variations between examiners were <20%.
Further serial histological sections were immunolabelled for MMPs, TIMPs, CD15 (a marker of neutrophils) and CD68 (a marker of macrophages). Mouse monoclonal antibodies against MMP-2 (IgG1, clone 42-5D11; 0.7 µg/ml), MMP-9 (IgG1, clone 56-2A4; 0.5 µg/ml) and TIMP-3 (IgG1, clone 136-13H4; 1.0 µg/ml) were from Calbiochem (Darmstadt, Germany). Those against TIMP-1 (IgG1, clone 102D1; 8.0 µg/ml) and TIMP-2 (IgG2a, clone 3A4; 4.0 µg/ml) were from Lab Vision Corporation (Fremont, CA, USA); against CD15 (IgM, clone MMA; 1.0 µg/ml) from BD PharMingen (Erembodegem, Belgium) and against CD68 (IgG1, clone KP1; 8.1 µg/ml) from Dako (Glostrup, Denmark). All these antibodies were specific and did not react with other MMPs, TIMPs or CDs, as demonstrated in previous studies (Hanjan et al., 1982; Pulford et al., 1989; Fujimoto et al., 1993; Fariss et al., 1997; Rigot et al., 2001; Kim et al., 2006;). An anti-adrenocorticotropin mouse monoclonal IgG1 (clone 02A3; Dako; 8.1 µg/ml) used as negative control yielded no staining.
Immunolabelling of all proteins except TIMP-1 and -2 was carried out after removal of paraffin and inactivation of endogenous peroxidases with 0.3% (v/v) H2O2 for 30 min at room temperature. Slides were immersed in 0. M citrate buffer, pH 5.8, and incubated in a water bath at 98°C for 75 min to retrieve the antigenic sites, except for CD68. Non-specific reactions were blocked by incubating the slides for 30 min in 50 mM Tris–HCl, pH 7.4, containing 10% (v/v) normal goat serum and 1% (w/v) bovine serum albumin. The histological sections were then incubated with the primary antibody overnight at 4°C. After washes in 50 mM Tris–HCl, pH 7.4, specific binding was detected with peroxidase-conjugated polymer backbone carrying secondary antibody molecules (EnvisionTM, Dako). Peroxidase activity was revealed by incubating the slides during 10 min at room temperature with 0.5 mg/ml diaminobenzidine in the Tris–HCl buffer. Sections were then washed in tap water and lightly counterstained with haematoxylin. TIMP-1 and -2 immunolabelling were performed by the Ventana Nexes automated immunohistochemistry system (Ventana Medical Systems, Tucson, AZ, USA). This procedure is based on an indirect biotin–avidin system with a biotinylated secondary antibody, diaminobenzidine as substrate, and haematoxylin as counterstain. In addition to the automated procedure, a Ventana amplification kit was used.
Immunolabelling was semi-quantitatively assessed by three examiners by scoring the number of immunostained stromal or epithelial cells as 0 (no cell), 1 (few cells), 2 (moderate number of cells) or 3 (many cells). The three examiners gave identical scores in most cases. The median score was used in the remaining few cases, in which the score of one examiner varied by at most one unit from the score of the other two examiners.
Zymographies and assay of DNA content
Latent and active forms of gelatinases (MMPs-2 and -9) were analysed by gelatine-substrate zymography (Marbaix et al., 1992). Samples were submitted to 2% sodium dodecylsulfate (SDS) electrophoresis on 8 or 12% polyacrylamide (Serva, Heidelberg, Germany) slab gels co-polymerized with 0.5 mg/ml gelatine (Sigma, St Louis, MO, USA). After migration, SDS was removed by rinsing with 2.5% Triton X-100, and gels were incubated for 18 h at 35°C, stained with Serva blue G and destained with acetic acid. MMPs are dissociated from their inhibitors during the electrophoresis, and their refolding after removal of SDS allows the measurement of the activity not only of their active forms but also of their artificially activated latent forms (proMMPs).
TIMPs were identified by reverse zymography, after 15% polyacrylamide-gelatine zymograms had been incubated in 20 ml buffer supplemented with 1 ml of medium conditioned by cultured mouse calvaria (Marbaix et al., 1992). This medium contains high levels of (pro)gelatinases, which were fully activated after treatment for 2 h at 25°C with 0.4 mM aminophenylmercuric acetate prior to addition to the incubation buffer. Gelatine degradation by the soluble gelatinases is inhibited by TIMPs at their site of migration, resulting in dark bands on a diffusely cleared gel. Because of the stoichiometric inhibition of one molecule of gelatinase by one molecule of TIMP, the inhibitory activity of TIMPs is correlated to the amount of TIMP protein.
MMP and TIMP activities were quantified by gel densitometry using the NIH image 1.62 software. The activities were normalized according to those contained in a culture medium conditioned by endometrial explants, used as standard in each gel.
For each endometrium, MMPs and TIMPs were assayed in the tissue lysate and the two washing solutions. Because similar levels of activities were predominantly recovered in the tissue lysate and in the first washing and because many dissociated cells and small cell aggregates were found at histology in the blood surrounding the tissue pieces, the activity of each form of (pro)enzyme or inhibitor was assessed by adding up the three measures. Each activity was normalized to the DNA content of the tissue lysate for a quantitative comparison. DNA was assayed by adding 100 µl of the lysate to 100 µl of 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma D9542), as described (Brunk et al., 1979). The fluorescence enhancement of DAPI intercalated into DNA was measured using a FluoroCountTM (Packard, Downers Grove, IL, USA), and referred to a standard curve established with salmon sperm DNA (Calbiochem).
Statistics
Quantitative and semi-quantitative data were compared by the two-tailed Wilcoxon rank-sum test. P-values at or below 0.05 were considered significant.
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Clinical information
Clinical characteristics of the 28 women are reported in Table I. The pictorial chart established according to the last bleeding episode confirmed that the non-treated women were menorrhagic. In contrast, the treated women who were previously menorrhagic had levels <100, indicating that thermal balloon therapy was successful. The median delay between endometrial ablation and sampling was 4.5 months (range, 2–12).
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Histological and immunohistochemical analysis
Endometrial biopsy in women menstruating after thermocoagulation confirmed that a functional mucosa grew again. Biopsies from amenorrheic women showed necrotic or scar tissue (Fig. 1) or endocervical mucus and tissue, and pieces of proliferative endometrium in two of the four patients. Therefore, the following investigations were performed only in the endometrium of bleeding women.
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Foci of stromal breakdown, characterized by tissue collapse, fragmentation and shedding, as well as clustering of stromal cells into ‘blue balls’, were present in all menstruating women. Stromal breakdown tended to be less extensive in treated women but the difference from non-treated women did not reach significance (Fig. 1; Table II). Furthermore, the lysis of the argyrophilic fibres was significantly lower in women treated by thermotherapy than in non-treated women. There was no difference in the abundance of neutrophils (CD15+) and macrophagic cells (CD68+) between both groups. Although blood tended to be more abundant in menstrual tissues of non-treated women, the difference was not significant. No difference was disclosed regarding the amount of fibrosis or endocervical mucus contamination.
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MMP-9 was immunolabelled in foci of stromal breakdown, whereas MMP-2 was diffusely immunolabelled in the stroma with a stronger staining in areas of stromal breakdown (Fig. 2). Both MMPs were immunolabelled in the stromal cells with a quite distinct pattern. MMP-9 immunolabelling produced a scattered granular signal and MMP-2 immunolabelling was diffuse in the cytoplasm with an increased signal at the cell periphery. There was no difference in the number of stromal cells immunolabelled for MMPs between non-treated and treated women (Table II).
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TIMP-1, -2 and -3 were immunolabelled in both epithelial and stromal cells. TIMP-1 and -2 immunolabelling produced granular signals in the cytoplasm and the nuclei. TIMP-3 was diffusely immunolocalized in the cytoplasm of decidual-like stromal cells in only a few cases. There was no difference in the number of epithelial or stromal cells immunolabelled for any TIMP between treated and non-treated women (Fig. 2; Table II).
Zymographies
Figure 3 and Table III show the activities of MMPs (both in latent and active forms) and TIMPs that could be extracted from frozen sections of menstrual endometria from non-treated and treated menorrhagic women. The latter showed higher levels of latent gelatinase A (proMMP-2) (P < 0.05), a trend towards a higher expression of latent gelatinase B (proMMP-9) (P = 0.076) but with a lower ratio of active on total (latent + active) forms (P < 0.05), and much higher levels for both TIMP-1 and -2 (P < 0.05). No difference was found for the other forms of MMPs. TIMP-3 was not detected in most endometria.
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Discussion |
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All menorrhagic women treated by thermal balloon ablation in our study declared a pattern of significant reduction of bleeding, which may be explained by quantitative and qualitative changes in their regenerated mucosa. Indeed, this new functional endometrium showed reduced lysis of the collagen-rich argyrophilic fibres at menstruation, despite increased expression of MMP-2. We suggest that limited fibre lysis could reflect increased inhibition of MMPs by TIMP-1 and -2 and likely explains the decrease in blood loss.
So far, most investigations carried out on the uterus after endometrial ablation have used imaging and hysteroscopy, providing indirect information on the regenerated endometrium, or have been performed on hysterectomy specimens after thermal ablation treatment. Post-operative sonography showed that dimensions of the uterus and endometrial thickness were lower than before therapy, whereas Doppler indices revealed a decreased vascularization at the levels of the uterine, arcuate and spiral arteries, which may be related to fibrosis in the uterine cavity (Khastgir et al., 1993; Bahçeci et al., 1998; Järvelä et al., 2001, 2002). Hysteroscopy showed intrauterine adhesions, complete obliteration of the cavity, diffuse fibrosis or a normal uterine cavity, in various proportions (Taskin et al., 2002; Leung et al., 2003). Focal endometrial regenerative areas were confirmed to be located in the fundus and the tubal ostia (Taskin et al., 2002). Although complete intrauterine adhesion is classically associated with amenorrhea, the relationship between menstrual outcome and post-ablation appearance remains controversial. Histological patterns of uterine tissue destruction, healing and repair, and endometrial regeneration correlated with the post-ablation interval (Silvernagel et al., 1997; Davis et al., 1998; Colgan et al., 1999). Necrosis dominated the first month, followed by regeneration with chronic inflammation characterized by fibrosis and foreign body giant cells reacting towards necrotic eschar, remnants of which were present for up to 4 years. Intact endometrium regenerated in 30–60% of uteri (Davis et al., 1998; Tresserra et al., 1999). However, these histological data may reflect the endometrium only partially when the outcome is successful, since most of the studied hysterectomy specimens were related to ablation failures.
Very few studies have addressed the histological appearance of endometrium in women responding to endometrial ablation. Random endometrial biopsies performed 3 years after thermal balloon ablation revealed diminished endometrial glands with necrosis and scarring (Taskin et al., 2002). The number of endometrial glands and their histopathologic features were not correlated with the amount of post-operative bleeding. Endothelial cells proliferated more abundantly in the endometrium of menorrhagic women than in controls with normal menses and in endometria after thermal ablation, suggesting an altered angiogenesis in the endometrium of menorrhagic women, which normalized after thermal ablation (Kooy et al., 1996).
Besides changes in uterus appearance and histological data of the endometrium, MMPs and TIMPs may be involved in the post-ablation bleeding pattern. Indeed, MMPs are able to degrade the ECM of the endometrium and their activity is largely increased at menstruation and during irregular bleeding episodes (Marbaix et al., 1996; Galant et al., 2004). Surprisingly, high levels of latent gelatinases were observed in the menstrual endometrium of treated women, but only proMMP-2 was more abundant than in non-treated women. ProMMP-9 is known to be stored in neutrophils and activated leukocytes are likely inducers of MMPs through the release of cytokines (Salamonsen, 2003). Neutrophils participate in the detersion of the damaged mucosa, in the cicatrization and the regeneration of the new endometrium. Although large amounts of inflammatory cells have been reported in the endometrium of women after ablation (Taskin et al., 2002; Mishra et al., 2003), we found no increase in neutrophils or macrophages in our study.
The most striking finding in this study was the increase in TIMP-1 and -2 levels after thermal ablation. During the normal menstrual cycle, TIMP levels are rather stable with only a slight increase during the late secretory and menstrual phases (Zhang and Salamonsen, 1997; Goffin et al., 2003; Vassilev et al., 2005; Graesslin et al., 2006). TIMP-1 and -2 are expressed by epithelial and stromal cells, whereas TIMP-3 is preferentially expressed by decidualized stromal cells (Zhang and Salamonsen, 1997; Hickey et al., 2006). Absence of decidual cells in most samples may account for the limited detection of TIMP-3 in the present study. TIMP-1 and -2 were immunolocalized in the cytoplasm and in the nuclei of stromal and epithelial cells, as previously reported in cultured human fibroblasts and in breast carcinoma cells (Zhao et al., 1998; Ritter et al., 1999). TIMP-1 and -2 were strongly immunolabelled in endothelial and vascular smooth muscle cells and are thought to be primarily responsible for maintaining the integrity of the endometrial vessels (Zhang and Salamonsen, 1997). Consistent with such a role, no strong immunolabelling of TIMP-1 or -2 was disclosed in the wall of vessels in the biopsies we sampled during menstrual bleeding. TIMP-1 activity was also considerably decreased in the endometrium of metrorrhagic women, when sampled at the time of dysfunctional bleeding but not outside of the bleeding episode (Galant et al., 2004). These previous results together with the present findings suggest that high TIMP-1 activities may contribute to the control of excessive menstrual bleeding by inhibiting active forms of MMPs. In striking contrast, a higher number of TIMP-1-immunolabelled stromal cells was found at bleeding episodes than in the absence of bleeding in menopaused women upon hormonal replacement therapy (Hickey et al., 2006). This apparent discrepancy may be due to a less quantitative assessment of the amount of TIMP-1 by immunohistochemistry than by zymography in particular, because the TIMP signal may fall below the detection level by immunohistochemistry after secretion in the ECM, but can still be detected by zymography, a very sensitive method.
To conclude, the present study suggests that the causes of decreased bleeding in menorrhagic women successfully treated by endometrial ablation are multifactorial. In addition to a lower amount of endometrial tissue and its decreased vascularization after regeneration as suggested by hysteroscopic and ultrasonographic studies, alterations in the balance between MMP and TIMP activities also contribute to reducing bleeding at menstruation. The cellular and molecular mechanisms responsible for these alterations remain unclear and deserve further studies.
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Author's contribution |
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J.L.B. contributed to study conception and design, patient recruitment, acquisition and interpretation of data and drafted the manuscript. C.G. contributed to study design, acquisition and interpretation of data and drafted the manuscript. D.D. contributed to laboratory investigations, acquisition and interpretation of data and revised the manuscript. P.L. contributed to laboratory investigations, acquisition and interpretation of data and revised the manuscript. P.H. supervised the progression of the study, revised the article critically and finally approved the version to be published. P.J.C. supervised the progression of the study, revised the article critically and finally approved the version to be published. E.M. contributed to study conception and design, interpretation of data and drafted the manuscript.
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Funding |
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This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale, the InterUniversity attraction Poles of the French Community of Belgium and the Concerted Research Actions of the Université Catholique de Louvain.
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Acknowledgements |
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We are grateful to all patients who agreed to participate in this study. The technical skill of D. Dubois and A. Rodenas is gratefully acknowledged. We are thankful to Dr M. Pellegrin de Villeneuve, Pathology Department of the Pellegrin University Hospital, for her help in sending biopsies. Critical reading of the manuscript by H. Gaide Chevronnay, C. Pretto and C. Selvais was much appreciated.
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Footnotes |
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Both authors contributed equally to the study 
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References |
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Submitted on July 5, 2008; resubmitted on September 29, 2008; accepted on October 8, 2008.
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