{"paper_id":"f3dc494e-40e6-4460-8ac9-e66d3d1622b0","body_text":"J OURNAL OF\nVeterinary\nScience\npISSN 1229-845X, eISSN 1976-555X\nJ. Vet. Sci. (2012), 13(2),  171-177\nhttp://dx.doi.org/10.4142/jvs.2012.13.2.171\nReceived: 6 Jul. 2011, Revised: 12 Aug. 2011, Accepted: 15 Dec. 2011\nOriginal Article\n*Corresponding author: Tel: +39-049-8272963; Fax: +39-049-8272963; E-mail: luca.aresu@unipd.it \nⓒ 2012 The Korean Society of Veterinary Science.\nT h i s  i s  a n  O p e n  A c c e s s  a r t i c l e  d i s t r i b u t ed under the terms of the Creative Commons Attribution Non-Commercial License (http://c reativecommons.org/licenses/by-nc/3.0) which permits \nunrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.\nThe role of inflammation and matrix metalloproteinases in equine \nendometriosis\nLuca Aresu1,*, Silvia Benali1, Diana Giannuzzi1, Roberto Mantovani2, Massimo Castagnaro1, Maria Elena Falomo3\n1Dipartimento di Sanità Pubblica, Patologia Comparata e Igiene Veterinaria, Facoltà di Medicina Veterinaria, Viale \ndell'Università, 16-35020-Legnaro (Padova) 049-8272963, Italy\n2Dipartimento Scienze Animali, Facoltà di Agraria, Viale dell'Università, 16-35020-Legnaro (Padova) 049-8272661, Italy\n3Dipartimento di Scienze Cliniche Veterinarie, Facoltà di Medicina Veterinaria, Viale dell'Università, 16-35020-Legnaro \n(Padova) 049-8272967, Italy\nEquine endometriosis is a multifactorial disease considered \nto be a major cause of equine infertility. The purpose of this \nstudy was to evaluate the reliability of histomorphological \ngrading for biopsy-like samples compared to entire uterine \nwall samples, to examine the association between the degree \nof endometriosis with animal age, and to investigate the role \nof inflammation in endometriosis and the expression of \ndifferent matrix metalloproteinases in equine endometrium. \nHistomorphological lesions in 35 uterine samples were \nexamined while comparing biopsy-like samples and \nentire-wall samples. Seventeen uterine samples were stained \nwith antibodies against MMP-2, MMP-9, MMP-14, and \nTIMP-2. The morphologic evaluation results of the \nbiopsy-like tissue and entire-wall samples were significantly \ncorrelated.  Endometriosis in older mares (\n＞12 years of age) \nwas more severe than in young mares (2 ∼4 years of age), \nconfirming the positive correlation between animal age and \ndisease severity, while inflammation was poorly related to the \ndegree of endometriosis. MMP-2 and MMP-14 were detected \nin stromal cells, while MMP-9 and TIMP-2 were both found \nin stromal and glandular epithelial cells. There were no \nsignificant differences in MMPs expression between the two \ngroups (young vs. old mares).  Additional studies on the \nactivity of MMPs could further define the role of these \nenzymes in equine endometriosis.\nKeywords:\n equine endometriosis, inflammation, MMPs, uterine \nbiopsy\nIntroduction\nChronic endometrial degeneration, also known as equine \nendometriosis, is a common disease in older mares (＞12 \nyears of age) and is associated with decreased fertility, \nearly embryonic death, and abortion [9]. Until now, little \nhas been known about the etiology and pathogenesis of this \nmajor cause of equine infertility [11]. Endometriosis is \na multi-factorial disease. Ag e, repeated pregnancies, \nparturition, chronic inflammation, and endocrine problems \nare all factors which seem to play a determinant role in the \nonset and severity of endometriosis [14].\nBased on histopathological findings, this disease has been \ndefined as a progressive process involving endometrial \ngland changes (cystic dilation, and atrophy or hypertrophy \nof the epithelium) associated with periglandular and/or \nstromal fibrosis [6,11,28]. In addition to this, signs of \nchronic inflammation and atrophy are frequently observed \n[13]. However, no study has actually determined the role of \ninflammation in endometriosis and extracellular matrix \n(ECM) re-modeling.\nMatrix metalloproteinase (MMPs) are calcium- and \nzinc-dependent proteases believed to be responsible for the \ndegradation and removal of ECM from the tissue [20]. With \nthe exception of membrane MMPs, all these enzymes are \nsecreted as inactive pro-enzymes and activated in the \nextracellular space by various factors including MMPs, \nplasmin, interleukin-1 beta, tumor necrosis factor-alpha, \nand other mediators [26,27]. Tissue inhibitors of \nmetalloproteinase (TIMPs) are specific endogenous \nenzymes involved in controlling the local activities of \nMMPs in tissues [26]. MMPs play a vital role in many \nphysiological and pathological processes in humans \nincluding embryogenesis, tissue remodeling, angiogenesis, \nwound healing, and metastasis [19,26]. Among the \n\n172    Luca Aresu et al.\nTabl e  1 . Details of the antibodies used for immunohistochemical examination\nAntigen (Antibody) Source Type Dilution Antigen retrieval Manufacturer\nMMP-9 (MAB 3309)\nMMP-2 (Ab-7)\nTIMP-2 (MAB 3317)\nMMP-14 (MAB 3430)\nHuman\nHuman\nHuman\nHuman\nMouse monoclonal\nRabbit polyclonal\nMouse monoclonal\nRabbit polyclonal\n1 : 1,000\n1 : 400\n1 : 1,000\n1 : 200\n60 min at 95\noC (CC1S)\n30 min at 95oC (CC1R)\n30 min at 95oC (CC1R)\n30 min at 95oC (CC1R)\nMillipore, USA\nNeomarkers, USA\nMillipore, USA\nMillipore, USA\nMMP: matrix metalloproteinase, TIMP: tissue inhibitors of metalloproteinase, CC1R: cell conditioning 1 reduced, CC1S: cell conditioning \n1 standard.\ndifferent MMPs, MMP-2 and MMP-9 (also known as \ngelatinases A and B, respectively) are the most widely \nstudied because of their ability to degrade type IV collagen \nin the basement membrane and fibrillar collagens. Based on \ntheir ability to degrade basement membranes, various \nstudies have reported increased levels of expression and \nactivity for both MMP-2 and MMP-9 during different \nstages of organ development and under various \npathological conditions, all situations in which dynamic \ntissue remodeling takes place [1,2,20]. MMP-2 is activated \nin two steps. First, the latent MMP-2 precursor is cleaved by \nMMP-14 to produce an intermediate form of MMP-2. This \nmolecule then auto-catalytically converts itself into mature \nMMP-2 [26]. Increased expression of MMP-14 activates \nMMP-2 on the cell surface, a process that is required for cell \ninvasion when localized in the invadopodia of human \nmelanoma cells. In the ECM, TIMPs tightly regulate the \nactivity of MMPs. TIMP-2 plays a dual role in controlling \nMMP-2 activation. First, the TIMP-2/MMP-14 complex is \nnecessary for initiation of the activation process of  \npro-MMP-2. In addition, TIMP-2 binds to the active site of \nMMP-2, thereby inhibiting its activity [26].\n In horses, it has been proposed that MMPs could have a \nrole in different pathological processes. Clutterbuck et al. \n[4] have reported the involvement of MMPs in laminitis, \nosteoarthritis, recurrent airway obstru ction, skin wounds \nrepair, degenerative diseases of the central nervous system, \nulcerative keratitis, and cancer. To our knowledge, only \none study [27] has reported on the expression and activity \nof MMP-2 in equine endometriosis. \nThe objectives of the present study were to: 1) evaluate \nthe reliability grading biopsy-like samples compared to \nentire uterine wall samples; 2) examine the relationship \nbetween histomorphological grading and animal age; 3) \ndefine the role of inflammation in endometrosis, and 4) \nimmunohistochemically characterize MMP-2, MMP-9, \nMMP-14, and TIMP-2 expression in the uterus of mares \naccording to the grade of endometriosis.\nMaterials and Methods\nCase selection\nEndometrial specimens were obtained from a total of 35 \nmares of various breeds and with unknown reproductive \nhistories. The mares were slaughtered between May and \nOctober (Pantano Carni s.r.l., Italy) and divided into two \ngroups according to age: 2 to 4 years old (n = 20) and 12 \nyears or older (n = 15). From each animal two samples \nwere collected from the anterior uterine body: a \nbiopsy-like sample representative of the mucosa (10 ∼ 20 \n× 3 × 3 mm), and a larger sample (2 cm\n3) comprising of the \nentire wall. Stage of the oestrous cycle was determined by \ngross and histological examinations based on the \nmorphological criteria of endometrial differentiation [13].\nHistopathologic examination\nSamples were fixed in 4% buffered formalin, embedded \nin paraffin, cut into 4- μm sections, and stained with \nhematoxylin and eosin. Endometrial fibrosis in the \nbiopsies was identified in sections stained with Masson’s \ntrichrome according to the grading system proposed by \nKenney and Doig [13] that includes four categories: I, IIa, \nIIb, and III. Inflammation was quantified according to the \ntotal number of inflammatory cells in 10 high power fields \n(HPF) (×400). The samples were graded as follows: grade \nI, less then 100 inflammatory  cells; grade II A, between \n100 and 150 cells; grade II B, between 150 and 200 cells; \nand grade III more than 200  inflammatory cells. \nAdditionally, severity of th e score was upgraded by one \nstage when the degree of exocytosis was greater then 15 in \n10 HPF (×400). \nImmunohistochemical examination\nSamples of the entire uterine wall were evaluated. Serial \nparaffin sections were cut (4-μm thick) and placed onto slides \n(SuperFrost Plus; Menzel-Gläser, Germany). The slides were \nthen incubated at 37\noC for 30 min before immunostaining \nwas performed with an automatic immunostainer (Ventana \nBenchmark XT; Roche, Switzerland). The immunostainer \nused a secondary antibody with a horseradish peroxidase- \nconjugated polymer to enhance the signal (UltraViews \n\nEquine endometriosis: the role of inflammation and matrix metalloproteinases     173\nFig. 1. Histopathology of the equine endometrial specimens. A1 \nand 2: grade I, B1 and 2: grade IIA, C1 and 2: grade IIB, D1 and\n2: grade III. Specimens were classified according to Kenney and \nDoig [13] classification of endometriosis. Hematoxylin and \neosin stain (A1, B1, C1, and D1) and Masson’s trichrome stain \n(A2, B2, C2, and D2), ×40. \nFig. 2. Immunohistochemical staining of endometrial specimens \nfrom older mares with anti bodies against MMP-2, MMP-9, \nMMP-14, and TIMP-2. (A) MMP- 2 staining in inflammatory \ncells. (B) MMP-9 immunolabeling in glandular epithelium and \nstromal cells. (C) MMP-14 immunostaining in stromal cells. (D) \nTIMP immunostaining in glandular epithelium and periglandular\nstromal cells. A and C: ×200, B and D: ×400.\nUniversal DAB; Ventana Medical System, USA). All \nreagents are automatically dispensed except for the primary \nantibody, which was dispensed by hand. The slides were \nincubated with antibodies against MMP-2, MMP-9, \nMMP-14, and TIMP-2 for 32 min at 42\noC (Table 1). Negative \ncontrols were incubated with diluent instead of primary \nantibody; plasma cells in the tissue controls were used as \ninternal positive control. The immunohistochemical staining \nresults were divided into the following four categories based \non staining intensity: negative, 0 = no stained cells; 1 = weak \nstaining; 2 = moderate staining; and 3 = intense staining. \nImmunostaining was blindly scored by two independent \nobservers (L.A. and S.B.), and discordant scores were \nre-evaluated. The consensus scores were used for further \nanalysis.\nThe extent and distribution of immunoreactivity specific \nfor MMP-2, MMP-9, and MMP- 14 were quantified as \nfollows. At first, the number of stromal cells in 10 HPF \n(×400) in two horses from each group (control and older \nmares) was measured. Subsequently, a group mean value \nwas obtained. Immunoreactivity specific for the different \nmarkers was expressed as number of labeled cells/the \nmean number of stromal cells.  Finally, the data were \nexpressed as a percentage. For TIMP-2, evaluation of \nimmunolabeling was only qualitative.\nStatistical analysis\nAll histochemical and immunohistochemical data were \nanalyzed with an ANOV A using the GLM procedure and a \ncorrelation analysis with the CORR procedure of SAS (ver. \n9.1; SAS, USA). A Chi-square test was used to compare the \nproportions of the histochemical data between the two \ndifferent age groups and groups in different phases of the \nreproductive cycle. p-values less than 0.05 were \nconsidered to be significant for any statistical test \nperformed.\nResults\nHistopathology\nThis part of the study evaluated a total of 35 animals. \nAccording to the histomorphological features, 25 of the \nmares were in oestrus while and 10 were in diestrus. \nAccording to the classification of endometriosis established \nby Kenney and Doig, 19 of the 20 specimens from young \nmares were classified as grade I (Fig. 1, A1 and 2) and one \nanimal was grade IIA (Fig. 1, B1 and 2). Among the older \n\n174    Luca Aresu et al.\nTabl e  2 .  Results of MMP and TIMP-2 immunohistochemical analysis and endometriosis grade \nCase* Age\n(years) Breed Grade †\nNumber (%) of positive stromal cells (10 HPF) (A) Staining intensity (B) A × B × 100\nMMP-2 MMP-9 MMP-14 MMP-2 MMP-9 MMP-14 TIMP-2 MMP-2 MMP-9 MMP-14\n1\n2\n3\n4\n5\n6\n7\n8\n9\n10\n11\n12\n13\n14\n15\n16\n17\n3\n3\n13\n17\n17\n16\n19\n14\n13\n13\n14\n13\n16\n15\n13\n13\n13\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nPolish Draft Horse\nI\nI\nII A\nII A\nII B\nII B\nII B\nII B\nII B\nII B\nIII\nIII\nIII\nIII\nIII\nIII\nIII\n26 (0.0099)\n24 (0.0092)\n10 (0.0043)\n231 (0.0990)\n2 (0.0009)\n11 (0.0047)\n4 (0.0017)\n8 (0.0034)\n7 (0.0030)\n17 (0.0072)\n6 (0.0026)\n12 (0.0052)\n6 (0.0025)\n4 (0.0017)\n251 (0.1070)\n8 (0.0034)\n4 (0.0017)\n7 (0.0027)\n11 (0.0042)\n3 (0.0012)\n214 (0.092)\n9 (0.0038)\n7 (0.0030)\n3 (0.0012)\n2 (0.0009)\n3 (0.0012)\n3 (0.0012)\n3 (0.0012)\n8 (0.0034)\n6 (0.0025)\n1 (0.0004)\n221 (0.095)\n6 (0.0025)\n4 (0.0017)\n10 (0.0038)\n1 (0.0004)\n9 (0.0038)\n222 (0.0950)\n2 (0.0009)\n1 (0.0004)\n1 (0.0004)\n5 (0.0021)\n3 (0.0012)\n3 (0.0012)\n1 (0.0004)\n10 (0.0043)\n0 (0.0000)\n5 (0.0021)\n218 (0.0935)\n19 (0.0081)\n2 (0.0009)\n2\n2\n2\n3\n1\n1\n1\n1\n1\n2\n1\n3\n1\n1\n3\n2\n1\n2\n2\n1\n3\n2\n1\n2\n1\n1\n1\n1\n2\n1\n2\n3\n2\n2\n1\n0\n1\n3\n1\n0\n1\n1\n0\n2\n0\n1\n0\n1\n3\n2\n1\n3\n1\n3\n3\n3\n2\n2\n3\n3\n2\n3\n3\n2\n3\n3\n2\n3\n1.98\n1.84\n0.86\n29.7\n0.09\n0.47\n0.17\n0.34\n0.30\n1.44\n0.26\n1.56\n0.25\n0.17\n32.10\n0.68\n0.17\n0.54\n0.84\n0.12\n27.6\n0.76\n0.30\n0.24\n0.09\n0.12\n0.12\n0.12\n0.68\n0.25\n0.08\n28.5\n0.50\n0.34\n0.38\n0.00\n0.38\n28.5\n0.09\n0.00\n0.04\n0.21\n0.00\n0.24\n0.00\n0.43\n0.00\n0.21\n28.05\n1.62\n0.09\n*1∼2: control cases, 3∼17: older mares. \n†Grade according to the system proposed by Kenney and Doig [13] that includes four categories: I, IIa, IIb, and III. \nHPF: high power field.\nmares (n = 15), two cases were graded as IIA, six cases were \ngrade IIB (Fig. 1, C1 and 2), and seven cases were grade III \n(Fig. 1, D1 and 2). \nIn young mares, the endometrial glands had a normal to \nslightly degenerated appearance with few multifocal fibrotic \nperiglandular nests. Glandu lar ectasia was moderate. \nMultifocal inflammation was detected in the sub-epithelial \nlayer, and was mainly characterized by the presence of \nlymphocytes and plasma cells with occasional eosinophils \nand neutrophils. In older mares, the glands were multifocally \nectatic and surrounded by a thick layer of connective tissue. \nGlandular fibrotic nests were haphazardly distributed with \nnumbers ranging from 1 to 10 per HPF and increasing under \nmore severe fibrotic conditi ons. Moderate to severe \nmultifocal lymphoplasmacytic infiltrate was found in the \nlamina propria. No differences in exocytosis were observed \nbetween the two groups. \nMorphologic evaluation and grading of endometriosis for \nthe biopsy-like tissue and entire-wall samples were highly \ncorrelated (r = 0.92; p ＜ 0.001). Inflammation grade was \npoorly correlated with the severity of endometriosis for the \nentire-wall and biopsy-like samples ( r\n2 = 0.47 and r2 = \n0.39, respectively).\nImmunohistochemistry\nThe expression patterns of MMP-2, MMP-9, MMP-14, and \nTIMP-2 were evaluated with immunohistochemistry; \nlocalization and staining intensity were observed. Data for \nthis study are shown in Table 2. All specimens showed a \nhighly granular staining pattern for MMP-9 in the cytoplasm \nof epithelial cells in the uterine glands and rarely in the \nluminal epithelium (Fig. 2B). The number of positive stromal \ncells was low ( ＜ 1%) and staining intensity was low or \nmoderate except for two cases (5 and 13) that showed 9.5 and \n9.2% of positive cells with high intensity staining. No \ndifferences were observed in the expression of MMP-9 \naccording to reproductive stage. Differences in the staining \npatterns for MMP-2 were observed between older mares and \nthe control animals (young mares). In normal endometrium \n(control), stromal cells in the stratum compactum were \nmoderately stained. In the older mares, stromal cells in both \nthe stratum compactum and stratum spongiosum showed \nvariable immunoreactivity (Table 2 and Fig. 2A). Glandular \nepithelium, stromal cells, and occasionally luminal \nepithelium were positive for MMP-14 with weak to moderate \nintensity in 16 cases (Fig. 2C). No statistical significant \ndifferences were observed among animals with different \ngrades of endometriosis and expression of MMP-14. \nImmunoreactivity against TIMP-2 was found in 16 cases. \nStromal cells along with glandular and luminal epithelium \nhad moderately to strongly stained cytoplasm (Fig. 2D).\n\nEquine endometriosis: the role of inflammation and matrix metalloproteinases     175\nDiscussion\nEndometriosis is recognized as a multifactorial disease [9] \nand many studies have shown that advanced age is one of the \nmain predisposing factors [12,13,22]. Uterine biopsy is a \nroutine test to evaluate the grade of endometriosis and mare \nfertility [13]. Despite the widespread use of this technique, \nrecent studies have questioned the efficacy of such methods \ndue to the low reliability of a single endometrial biopsy to \naccurately assess the grade of  endometriosis [7,8]. Our \nresults demonstrated that the grading systems established \nfor biopsies and entire-wall sa mples coincide. Therefore, \nresults from this study confirmed that biopsy can be an \nindispensable clinical diagnostic tool for evaluating the \nfertility of mares. In agreement with the literature, our study \nalso demonstrated the existence of a relationship between \nanimal age and the grade of endometriosis [6,22]. When \nevaluating fertility, the most predictive factors seem to be \nthe number of fibrotic nests and fibrosis grade. Our study \nshowed that these two parameters are positively correlated \nwith animal age (p ＜ 0.001).\nWe found that inflammation is poorly correlated with the \ngrade of endometriosis. Endometrial degeneration and \nfibrosis are known to originate from repetitive inflammatory \ninjuries [8,24]. Damage from chronic inflammation triggers \na complex tissue reaction resulting in ECM deposition and \naccumulation within the interstitium. Fibrosis continues to \nprogress even after the inflammatory process has ended [3]. \nTherefore, progression of the fibrotic process after a critical \npoint becomes independent of inflammation despite the \npresence of inflammation preceding fibrosis that affects the \npathogenesis of endometriosis. This explains the low \ncorrelation between inflammation and endometriosis grade \nobserved in the present study. Our finding is concordant with \nthe results of a previous report showing that endometritis is \nindependent of the degree of periglandular fibrosis [11]. It \nhas been reported that induced endometritis is associated \nwith fibrotic stromal cells activation 5 days post-infection, \nbut over the 2-year period of the experiment the degree of \nendometriosis did not change [11]. In our investigation, \nstatistical analysis showed that the degree of inflammation \npresents little correlation with endometrosis (p ＜ 0.05), but \nit remains an indispensable parameter for the diagnosis.\nWe also analyzed the expression and distribution of \ndifferent MMPs using immunohistochemistry.  To the best \nof our knowledge, this is the first study to investigate \ndifferent MMPs in equine endometrium. MMP-9, also \nknown as gelatinase B, principally degrades collagen IV , \nthe main component of basement membranes. In humans, \nMMP-9 has been reported to be expressed in inflammatory \ncells as well as glandular and periglandular stromal cells in \nthe endometrium [17,23]. In our study, control luminal \nepithelial cells were positive for MMP-9 in normal equine \nendometrium. With different grades of endometriosis, \nincreasing amounts of collagen IV are deposed around the \nendometrial glands and fibrotic nests [28]. In our study, we \nhypothesized that endometriosis increased the production \nof MMP-9 as a cellular reaction to degraded collagen IV . \nHowever, the expression of MMP-9 did not significantly \ndiffer between mares with endometriosis and the control \nhorses. MMP-9 is reported to be involved in inflammatory \nprocesses and seems to be more involved in damage \noccurring during the early stages than chronic conditions \n[4]. In horses as well as humans, MMP-9 is principally \nproduced by leucocytes and increased presence of this \nenzyme during acute inflammation is possibly associated \nwith inflammatory cell migration during the earliest stages \n[5,15,16]. No previous studies have compared the \nexpression of MMP-9 in healthy and fibrotic equine \nendometrial tissue. On one hand, MMP-9 expression and \nactivity could not increase with endometriosis, leading to \nECM accumulation and periglandular fibrosis. On the \nother hand, it is possible that MMP-9 is a marker of \ninflammation rather than fibrosis since expression of this \nenzyme is mainly associated with inflammatory processes \nrather than fibrotic conditions. \nIn the present study, MMP-2 (gelatinase A) expression \nwas not significantly increased in diseased horses. Walter \net al. [27] reported that the overall MMP-2 expression in \nnormal or mildly affected endometrium was one third of \nthat observed in severely affected tissues. Moreover, they \nused gelatine zymography to determine that up to 58% of \nMMP-2 is active in healthy specimens whereas that this \nvalue increased to 76% in affected samples. Likewise, \nWalter et al. [27] noted a difference in MMP-2 expression \nrelative to progesterone plasma concentrations. More \nspecifically, they observed increased expression of the \nprotein during di-oestrus. In the present study, the oestrus \ncycle did not influence MMP expression but further \ninvestigations are required to elucidate the interaction \nbetween steroid hormone production and MMP activity.\nMMP-14 is a trans-membrane protease capable of \ndegrading different ECM components such as collagen \ntype I, II, and III as well as fibronectin and laminin [10].  \nThe main interest in this enzyme is due to its ability to \nactivate different proteases, particularly MMP-2 and \nMMP-9 [21]. Our study was designed to measure MMP-14 \nexpression relative to MMP-2 and MMP-9 in equine \nendometrium. We observed immunostaining specific for \nMMP-14 only occasionally. Few reports on MMP-14 \nexpression in the reproductive system have been published, \nand most are related to pregnancy and placentation [18,25]. \nMore studies are necessary to explain the role of MMP-14 \nin equine endometriosis.\nSimilarly, no information about TIMP-2 in horse \nendometrium exists in the literature. In our study, TIMP-2 \nwas highly expressed in all samples regardless age or \nuterine condition. This is consistent with reports in humans \n\n176    Luca Aresu et al.\nshowing that expression of TIMP-2 does not change during \ndifferent phases of the menstrual cycle [29]. TIMP-2 has a \ndual role. At low concentrations it binds to MMP-14 and \nindirectly activates MMP-2. At high concentrations, \nactually inhibits MMP-2 [20,26]. A quantitative study \ncould better identify the different conditions under which \nTIMP-2 acts as an inhibitor or activator. \nIn conclusion, our result indicated that immunohistochemistry \nappears to not be useful for evaluating equine fertility in \nclinical practice. However, further studies are necessary to \nbetter understand the role of the metalloproteinases in the \npathogenesis of endometriosis. It is of particular interest to \nmeasure the different concentrations of active and latent \nforms of MMPs through gelatine zimography and evaluation \nof gene expression.\nReferences\n1. Aresu L, Benali S, Garbisa S, Gallo E, Castagnaro M. \nMatrix metalloproteinases and their role in the renal \nepithelial mesenchymal transition. Histol Histopathol 2011, \n26, 307-313.\n2. Aresu L, Giantin M, Morello E, Vascellari M, \nCastagnaro M, Lopparelli R, Zancanella V, Granato A, \nGarbisa S, Aric\nò A, Bradaschia A, Mutinelli F, Dacasto \nM. 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