{"paper_id":"7ce0efb5-c34c-4c11-9cde-f3fc8ed85c20","body_text":"Annals of Agricultural and Environmental Medicine 2016, Vol 23, No 4, 649–653\nwww.aaem.plORIGINAL  ARTICLE\nMatrix metalloproteinase-2 (MMP-2), MMP-9, \ntissue inhibitor  of matrix metalloproteinases \n(TIMP-1) and transforming growth factor-β2 \n(TGF-β2) expression in eutopic endometrium of \nwomen with peritoneal endometriosis\nKrzysztof Szymanowski1, Mateusz Mikołajczyk2, Przemysław Wirstlein2,  \nAnna Dera-Szymanowska3\n1 Department of Mother’s and Child’s Health, Poznań University of Medical Sciences, Poland  \n2 Department of Obstetrics and Gynecology, Division of Reproduction, Poznań University of Medical Sciences, Poland  \n3 Department of Perinatology and Gynecology, Poznań University of Medical Sciences, Poland\nSzymanowski K, Mikołajczyk M, Wirstlein P , Dera-Szymanowska A. Matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of matrix \nmetalloproteinases (TIMP-1) and transforming growth factor- b2 (TGF- b2) expression in eutopic endometrium of women with peritoneal \nendometriosis. Ann Agric Environ Med. 2016; 23(4): 649–653. doi: 10.5604/12321966.1226861\nAbstract\nIntroduction.  The prevalence of endometriosis among reproductive age women is 7–17%; however, these figures reach \n20–50% in patients suffering from infertility. Matrix metalloproteinases (MMPs) activity is thought to be particularly essential \nin the early phases of endometriosis development. Any changes in the equilibrium between MMPs activity and their \ntissue inhibitors (TIMPs) could be potentially harmful, promoting endometriosis development. The aim of this study was \nto investigate whether the MMP-2, MMP-9, TIMP-1 or TGF-B2 expression in eutopic endometrium from women with early \nendometriosis differ when compared with healthy subjects. The results were referred to the serum progesterone levels.  \nMaterials and method . Endometrial biopsy was taken from 42 patients (18 in the study group, 22 in thecontrol group) at \nthe time of hysteroscopy for routine histology and for RT-PCR procedures. Comparison of the quantity of gene products \nwas performed with a programme for densitometry and compared to GADPH product, which was a reference value.  \nResults. The obtained results did not reveal any statistical difference in endometrial expression of MMP-2, MMp-9, TIMP-1, \nand TGF-β2 or serum progesterone level between women with endometriosis and without visible signs of this illness.  \nConclusion. Despite the lack of statistical differences, it was observed that both examined metalloproteinases expressed \na tendency to higher gene expression in the eutopic endometrium of women with endometriosis. However, both TIMP-1 \nand TGF-β2 expressions had the same tendency – higher values in endometriosis patients.\nKey words\nEndometriosis, matrix metalloproteinases, tissue inhibitor of matrix metalloproteinases\nINTRODUCTION\nEndometriosis is defined as the presence of functioning \nendometrial glands and stroma outside the uterus, \npredominantly within the peritoneal cavity [1]. The prevalence \nof endometriosis is 7–17% among reproductive age women \n[2, 3, 4]; however these figures reach 20–50% in patients \nsuffering from infertility [5]. Despite intensive investigations, \nthe pathogenesis of endometriosis remains unclear. The \nadhesion of the endometrium refluxed through the fallopian \ntubes is one of the first necessary stages of the implantation \ntheory. Matrix metalloproteinases (MMPs) activity is \nthought to be particularly essential in the early phases of \nendometriosis development. Extracellular matrix remodeling \nis a common phenomenon in normal development, growth \nand tissue repair. Any change in the equilibrium between \nMMPs activity and their tissue inhibitors (TIMPs) could be \npotentially harmful.\nIt may be assumed that aberrant MMP-2, MMP-9, TIMP-1 or \nTGF-b2 activity in eutopic endometrium could contribute to \nthe endometriosis development. As might have been expected, \nUeda et  al. [6] have shown higher MMP-2, MMP-9 and \nMT1-MMP (membranous type-1 matrix metalloproteinase) \nexpression in clinically-active endometriotic foci, compared \nwith eutopic endometrium of endometriosis free patients. \nOn the other hand, Chung et al. [7] found for the very first \ntime that the expression of MMP-2 and MT1- MMP was \nhigher, and TIMP-2 was lower in the eutopic endometrium \nof women with endometriosis. They also found a higher \nMMP-9:TIMP-3 ratio in the eutopic endometrium of women \nwith endometriosis. However,these studies were performed \nexclusively on advanced endometriosis patients.\nOBJECTIVE\nTo the knowledge of the authors of the presented study, there \nis no clinical study comparing MMPs and tissue inhibitors \nexpression in eutopic endometrium of women with peritoneal \nendometriosis. Therefore, the aim of the present work was \nto investigate whether the MMP-2, MMP-9, TIMP-1 or \nAddress for correspondence: Anna Dera-Szymanowska, Department of \nPerinatology and Gynecology, Poznań University of Medical Sciences, Górki 15D/15, \n60–204 Poznań Poland\nE-mail: annaszerszen@wp.pl\nReceived: 03 March 2013; accepted: 22 November 2013\n\nAnnals of Agricultural and Environmental Medicine 2016, Vol 23, No 4\nKrzysztof Szymanowski, Mateusz Mikołajczyk, Przemysław Wirstlein, Anna Dera-Szymanowska  . Matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor …\nTGF-b2 expression in eutopic endometrium from women \nwith early endometriosis differ when compared to healthy \nsubjects. Furthermore, these results are to be referred to \nserum progesterone levels.\nMATERIALS AND METHOD\nThe study was performed on endometrium obtained from \n42 women treated in the Division of Reproduction at the K. \nMarcinkowski University of Medical Sciences in Poznań, \nPoland. All patients had laparoscopy and hysteroscopy within \n7–9 days after ovulation, as confirmed by ultrasonography. \nThe Human Research Committee at the University approved \nacquisition of the endometrial tissue (No: 1058/98), and \nthe patients signed an informed consent. All patients had \nregular menstrual cycles and had not taken any hormonal \ntreatment for at least 3 months prior to the endometrial \nbiopsy. Endometriosis was diagnosed by direct visualization \nof the endometrial lesions at laparoscopy, with subsequent \nhistological confirmation. Staging of the endometriosis \nwas performed according to the revised American Fertility \nSociety classification [8]. Only peritoneal endometriosis (I 0 \nand II0) was qualified for further study.\nThe study group consisted of 18 patients with endometriosis \n(I° – 4, II ° – 14). Mean age of the patients was 29.2 years \n(20–37). The indications for laparoscopy were as follows \n(in some patients more than one): primary or secondary \ninfertility (n=14; primary – 11, secondary – 3), suspicion \nof endometriosis (n=7), recurrent miscarriages (n=4) or \nbilateral occlusion of the fallopian tubes (n=2). Endometriosis \ntreatment was performed due to the protocol used at the \nDivision of Reproduction. Twenty-four patients without \nvisible endometriotic foci at laparoscopy were qualified as \nthe control group. Mean age of women in this group was \n29.6 years (24–39). The indications for laparoscopy were (in \nsome women more than one): infertility (n=16; primary – 13, \nsecondary – 3; among them, male factor – 3), occlusion of the \nfallopian tubes (n=11; bilateral – 5, unilateral – 6), uterine \nfibroids (n=5), recurrent miscarriages (n=3) or suspicion of \nendometriosis (n=1).\nIn all patients, endometrial biopsy was taken at the time \nof hysteroscopy for routine histology and for RT-PCR \nprocedures. Immediately after specimen acquisition, 30 mg \nof the endometrium was placed in 300 ml (10 ml/1 mg of the \ntissue) of RNA later Stabilization Reagent (Qiagen, Hilden, \nGermany) and frozen at -20 °C until RNA extraction. The \nRNeasy Protect Mini Kit (Qiagen, Hilden, Germany) was \nused for isolation of the total RNA. The QIAshredder columns \n(Qiagen, Hilden, Germany) were used for homogenization \nof the endometrium. Isolated total RNA was in the form of \nsuspension in 50 ml free of RNA-ases water. This suspension \nwas frozen until analysis in -20 °C. Qiagen OneStep Reverse \ntranscription and Polymerase Chain Reaction (RT-PCR) Kit \n(Qiagen, Hilden, Germany) was used for RT-PCR reaction. \nThis allowed reversed transcription and cDNA amplification \nin one thermal profile. PTC-200 thermocycler (MJ Research, \nUSA) was used for reaction.\nStarters used are listed in the table below (Tab. 1)\nThe thermal profile applied in the experiment was as \nfollows:\n50 deg C – 30 min\n95 deg C – 15 min\n94 deg C – 1 min\n55 deg C – 1 min × 25 cycles\n72 deg C – 1 min\n72 deg C – 10 min\nReactive mixture was prepared according to manufacturer’s \ninstructions. In the next step, 12 ml of the 6x Mass Loading \nDye Solution (Fermentas, Vilnius, Lithuania) was added \nto the reaction products. Final products were plated in \n10 ml aliquots on 0.8% agarose gel ( Prona Agarose, Spain) \nwith 5 ml/100 ml of ethidine bromide (Sigma, USA). To \none of the pockets 10 ml of MassRuller DNA ladder, Low \nRange (Fermentas, Vilnius, Lithuania) was added. Finally, \ngel electrophoresis was performed in TBE buffer 1x. Gel \nvisualization was performed with UV light. Comparison \nof the quantity of gene products was performed with a \nprogramme for densitometry. This quantity was compared \nto GADPH which was the reference value.\nPrior to the anaesthesia induction, a blood sample was \ntaken for progesterone assessment (ELISA). The values were \nexpressed in ng/ml.\nFor statistical analysis, the students T-test and Pearson`s \ntest were used. A p<0.05 was considered significant.\nRESULTS\nResults of the MMP-2, MMP-9, TIMP-1, TGF- b2, and \nGADPH (as a reference) are presented in Figures 1 and 2.\nNo statistical differences were found in the expression \nof MMP-2, MMP-9, TIMP-1 and TGF- b2 gene products \nin endometrium between the group of women with and \nwithout endometriosis. Mean values of the gene products \nare presented in the Table 2.\nAs a next step, a search was made for possible correlations \nbetween the assessed genes. For the entire group of 42 women \nfollowing positive correlations were found:\n1. MMP-2 and TIMP-1 (p<0.001);\n2. MMP-9 and TIMP-1 (p<0.05);\n3. MMP-9 and MMP-2 (p<0.05);\n4. TGF-β2 and MMP-9 (p<0.01).\nIn the group of women with endometriosis (n=18) the \nfollowing correlations were found:\n1. MMP-2 and TIMP-1 (p<0.001);\n2. MMP-9 and TIMP-1 (p<0.05);\n3. MMP-9 and MMP-2 (p<0.05).\nTable 1. Primers for the examined gene (9, 10, 11, 12)\nGene Primer 5’ Primer 3’\nSize of \nproduct \n(bp)\nMMP2 CCACGTGACAAGCCCATGG GCAGCCTAGCCAGTCGG 486\nMMP9 TGGGCAAGGGCGTCGTGG-\nTTC\nTGGTGCAGGCGGAGTAG-\nGATT 276\nTIMP1 TGCACCTGTGTCCCACCC GGCTATCTGGGACCGCAG 552\nTGF-β2 TCCGCACCCGAGACTGAC AGGCTGAGCGCGACCGTG 441\nGAPDH\n(control)\nTGAAGGTCGGAGTCAACGG-\nATTTGGT\nCATGTGGGCCATGAGGTC-\nCAC CAC 983\n650\n\nAnnals of Agricultural and Environmental Medicine 2016, Vol 23, No 4\nKrzysztof Szymanowski, Mateusz Mikołajczyk, Przemysław Wirstlein, Anna Dera-Szymanowska  . Matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor …\nIn the group of women without endometriosis (n=24) \nonly a correlation between MMP-2 and IMP-1 was found \n(p<0.001).\nNo statistical difference was found in serum progesterone \nlevels between the groups of women with and without \nendometriosis. There were no correlations between serum \nlevels of progesterone and MMP-2, MMP-9, TIMP-1 or \nTGF-b2 endometrial gene expressions.\nDISCUSSION\nClinical trials in vivo regarding early endometriotic foci \ndevelopment, are impossible to perform for obvious ethical \nreasons. Therefore, knowledge of endometriosis development \narises from experimental works, animal studies, as well as the \nobservations of women with minimal or mild endometriosis. \nThe results of the studies analyzing relationship between \nMMPs expression and endometriosis are difficult to compare \nsince different techniques are utilized, different selection of \nMMPs are used, and furthermore, different study and control \ngroups are enrolled. The results of such studies are still \nconflicting. Although the expression of a number of MMPs \nhave been described, information about the function of these \nenzymes in the initial steps of endometriosis development \nis lacking.\nMatrix metalloproteinases activity in the eutopic \nendometrium has been a topic of publications within the \nlast few years, although there have not been many clinical \ntrials undertaken. Among them these concerning women \nwith endometriosis are scarce [10, 11, 12, 13]. The authors of \nthe current study did not find any data on MMPs activity \nin eutopic endometrium of women with minimal or mild \nendometriosis. The obtained results did not reveal any \nstatistical difference in the endometrial expression of MMP-\n2, MMP-9,TIMP-1, and TGF-b2 or serum progesterone level \nbetween women with endometriosis and without visible \nsigns of this illness. Despite the lack of statistical differences, \nthe authors observed interesting trends in the results. Both \nexamined metalloproteinases expressed tendency to higher \ngene expression in eutopic endometrium of women with \nendometriosis. However, both TIMP-1 expression and \nTGF-b2 expressions had the same tendency m – higher \nvalues in endometriosis patients. It is probable that larger \ngroups will allow the acquisition of statistical differences. \nBut the presently observed tendencies seem to show a sort \nof readiness of the eutopic endometrium in women with \nendometriosis for implantation.\nThe afore-mentioned works of Chung et al. [7] revealed \nhigher activity of MMPs than their inhibitors. The authors \nsuggested that eutopic endometrium of women with \nendometriosis possess higher proteolytic activity, thereby \nenabling implantation in the peritoneal cavity. This study \nwas performed on women with advanced endometriosis (III° \nand IV °; rAFS classification), and the authors suggest that \nthe expression of MMP-2 and MT1-MMP changed after the \ninitial stage of endometriosis, so that after the development \nof ovarian endometriotic cysts, enzyme activity was no \nlonger significant. The authors of the presented study do not \nconsider that such a conclusion may be drawn on the basis \nof that study. The final suggestion of Chung et al. was that \neutopic endometrium in women with endometriosis is more \nprone to peritoneal implantation than the endometrium from \nwomen without endometriosis. Furthermore, they speculated \nthat evaluation of the MT1-MMP and MMP-2 system may \nbe used as a prognostic marker of endometrial invasion.\nHowever, the main question remains: what is the primary \nphenomenon? Is endometriosis caused by different properties \nof eutopic endometrium, or does the growth of endometriotic \nimplants change characteristic of the eutopic endometrium? \nIn previous animal experiments, the authors of the current \nstudy showed that apoptosis could induce changes in the \nproperties of the eutopic endometrium [13]. However, these \n                                         18159_29530 AAEM_2015 / Corrected\nTIMP1\nMMP2\n         \nMMP9\nTGF 2 β\nGADPH\n                                 \nFigure 1. TIMP-1, MMP-2, MMP-9, TGF-β2 and GADPH expression in endometrium (patients 1-19)\n750bp\n500\n250\n600\n500\n400\n500\n400\n300\n600\n500\n400\n300\n2kb\n1kb\n750bp\nTIMP1\nMMP2\nMMP9\nTGF 2 β\nGADPH\n                                \nFig.  2. TIMP-1, MMP-2, MMP-9, TGF-β2 i GADPH in endometrium (patients 20-42) \n750bp\n500\n250\n600\n500\n400\n500\n400\n300\n600\n500\n400\n300\n2kb\n1kb\n750bp\nFigure 1. TIMP-1, MMP-2, MMP-9, TGF-β2 and GADPH expression in endometrium \n(patients 1–19)\nFigure 2. TIMP-1, MMP-2, MMP-9, TGF-β2 i GADPH in endometrium (patients 20–42)\nTable 2. Means and standard deviations of the expression of studiem \ngenes in women with and without endometriosis\nGroup Studied gene Product of studied\ngene/GAPDH\nStandard \ndeviation P\nWith endometriosis MMP2 1,67 0,25 NS\nWith endometriosis MMP9 0,30 0,12 NS\nWith endometriosis TIMP1 1,62 0,26 NS\nWith endometriosis TGF-β2 0,62 0,15 NS\nWithout endometriosis MMP2 1,56 0,26 NS\nWithout endometriosis MMP9 0,25 0,07 NS\nWithout endometriosis TIMP1 1,54 0,33 NS\nWithout endometriosis TGF-β2 0,55 0,13 NS\nP value corresponds to the expression of each gene in women with and without endometriosis.\n651\n\nAnnals of Agricultural and Environmental Medicine 2016, Vol 23, No 4\nKrzysztof Szymanowski, Mateusz Mikołajczyk, Przemysław Wirstlein, Anna Dera-Szymanowska  . Matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor …\nanimal studies also revealed that endometriosis development \nin the abdominal cavity provoked further changes in the \nintegrin pattern [14, 15]. Whether a similar situation exists \nwith regard to metalloproteinases expression and their \nactivity in eutopic endometrium remains a mystery.\nThe observed tendency for higher gene expression of TGF-b2 \nin eutopic endometrium in women with endometriosis could \npossibly provoke the above-mentioned changes. Chegini \n[16] has shown an increase of the metalloproteinases tissue \ninhibitors and a decrease of metalloproteinases expression \nafter TGF-b2 application. Pizzo et al. [17] have shown truly \nhigher levels of TGF- b2 in the peritoneal fluid of women \nwith endometriosis, compared to endometriosis-free \npatients (p<0.001). This cytokine is also responsible for \nMMPs expression, being a mediator of progesterone action \n[18]. In the meantime, TGF- b2 was shown to provoke an \nincrease in TIMP-1 expression in the stromal cells of human \nendometrium [19]. With this in mind, the lack of correlation \nbetween the examined MMPs and TIMP-1 expressions seems \nsurprising.\nAnalysis of the correlations between MMP-2, MMP-\n9, TIMP-1 and TGF- b2 expression also revealed very \ninteresting results. All these data may emphasize the role \nof the meticulous balance between extracellular matrix \nreabsorbtion and stabilization for homeostasis.\nThe obtained data does not permit the use of MMPs and \ntheir inhibitors expression in eutopic endometrium in the \ndiagnostic process of peritoneal endometriosis. Nevertheless, \nthis study encourages the authors to continue investigations \nin an attempt to further explain the pathogenesis of \nendometriosis.\nCONCLUSIONS\nProgesterone directly or indirectly stimulates MMP-9 \nexpression in both the glandular and stromal cells [20]. The \nresults of experimental studies regarding the progesterone \nand TGF–β [18] suggest their complex action on the \nexpression of MMPs. Increased activity of MMPs was shown \nin endometriotic lesions which subsided during treatment. \nThe blocking of the TGF–β action removes the endometrial \nmetalloproteinases supression caused by progesterone. \nDefects in the TGF–β make the action of progesterone upon \nthe endometriotic lesions more difficult. Therefore, very \ncrucial points remain to be explained.\nIt is worth mentioning that estimation of MMPs mRNA \ndoes not reveal what the true activity of the enzymes will be. \nThis is only the first step in the authors’ considerations. At \nthe moment they searching for aberrant activity of MMPs \nthrough active and inactive forms, a problem presented \nby Laird et al. [21], who suggested that the bands obtained \nby zymography with molecular masses of 73 and 67 kDa \ncorresponded to inactive and active forms of MMP-2, \nrespectively, and that at 98 and 90 kDa could correspond \nto inactive and active forms of MMP-9. Furthermore, \nconcentrations of MMP-9 in individual uterine flushings \ndetected by ELISA showed an excellent correlation with the \n98K/90K activity bands seen by zymography.\nRecently, Bruner et  al. [18, 22] and Nap et  al. [23] \ndemonstrated in murine and chicken chorioallantoic \nmembrane models the prevention of early endometriotic \nlesions formation when MMPs activity was blocked. These \nresults allow the assumption that imbalance between \nMMPs and their inhibitors is the immanent feature of the \neutopic endometrium, thus provoking ectopic implantation \nin some women. Furthermore, changing MMPs activity \nlocally may  be effective in endometriosis prophylaxis, or \neven treatment.\nStudies by the authors on endometriosis are still in progress, \nand it hoped that the acquisition of much more interesting \ndata with statistical power concerning the role of the MMPs \nand their inhibitors will result through bigger study groups, \nand newly-programmed animal studies.\nREFERENCES\n1. Burney RO, Giudice LC. Pathogenesis and pathophysiology of \nendometriosis. Fertil Steril 2012; 98(3): 511–9.\n2. D`Hooghe T, Xiao L, Hill J. Cytokine profiles in autologous pertioneal \npluid and peripheral blood of women with deep and superficial \nendometriosis. Arch Gynecol Obstet. 2001; 265: 40–44.\n3. Crosignani P, Olive D, Bergqvist A, Luciano A. Advances in the \nmanagement of endometriosis. Hum Reprod Update 2006; 12(12): \n179–89.\n4. Senapati S, Barnhart K. Managing endometriosis – associated infertility. \nClin Obstet Gynecol. 2011; 54(4): 720–6.\n5. 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