Dose-dependent Effects Of Multiple Matrix Metalloproteinase In Human Decidual Stromal Cells And Extravillous Cytotrophoblasts By Gonadotropin Releasing Hormone Agonist In Early Pregnancy

Embryo implantation and placenta formation. It is an extra meticulous andcomplex physiological process between blastocyst and uterus. Succesuful pregnancyis actually determined by successful human placenta formation, the process ofextravillos cytotrophoblasts rapidly invading genetically dissimilar maternal tissuesand spiral artery is an important event. Normally, it requries a fine coordination withextracellular matrix degeration, angiogenesis and cytotrophoblast differentiate.Migeraion and invasion of cytotrophoblsts into the uterine wall and maternalvasculature are pivotal to the success placenta formation. Mutilpe cytokines andfactors ensure the trophoblast invasion precisely, and errors can have extremelyadverse consequences. For example, shallow invasion is associated with preeclampsiaand about half the pregnancies that are complicated by intrauterine growth retardation.Overly aggressive invasion, through the deeper portions of the myometrium isassociated with placental site tumors, choriocarcinoma, and placenta accretes.With embryo implantation and development, series of complex and elaboratephysicological changes happened in maternal-fetal interface. Early studies found preimplantation embryos and trophoblast cells secreting a variety of MMP, indicatingMMPs in the maternal-fetal dialogue playing an extremely important role. MMPs is amember of a proteolytic enzyme matrix metalloproteinase. The structure is correlatedand its activities depened on the proteolytic Zn²⁺,Ca²⁺ family by hydrolysis ofcollagen, polysaccharides and glycoproteins and other extracellular matrix proteins. Itinvolveds in cell adhesion, migration, celluar differentiation and implantation, whichis a key enzyme involved in extracellular matrix degradation. Invasion control is thekey factor in the process of embryo implantation and plays an important role invascular remodeling. They are adjusted directly or indirectly by the MMPs/TIMPsbalance, which acts as an indicator for measuring the invasion degree. Placentalhormones can regulate MMPs and control the adhesion, migration, and other intrusiveprocess. So the balance of controlling invasiong and matrix reconstruction is verycrucial in maternal-fetal interface.GnRH plays a crucial role in the regulation of reproductive function. Thisdecapeptide is released from the hypothalamus and binds to specific receptors on theanterior pituitary, resulting in stimulation of both synthesis and release of FSH andLH, which regulate gonadal hormones within gametes form and function. GnRH isunstable in vivo, while chemical structure of synthetic peptide compounds, namedGnRHa (gonadotropin-releasing-hormone-analog), is very similar to GnRH invivo, which includes GnRH agonist and GnRH antagonist. Previous studies havedemonstrated the presence of an extrahypothalamic GnRH immunologically,biologically, and chemically identical to the hypothalamic hormone in a variety oftissues. It is also a well established fact that the human placenta produces and secretesGnRH, and this immunoreactive GnRH is present in both cytotrophoblast andsyncytiotrophoblast. GnRH and its receptors are widely distributed in, endocrine,immune and digestive systems, and they are important cooperation messengers forthese systems. In central nervous system, GnRH acts as a neurotransmitter, while inthe surrounding tissue it plays a role as an autocrine or paracrine factor. The placentaproduces the highest concentrations of GnRH in early gestation, so it has beenimplicated as one of the primary regulators of the synthesis and secretion of HCG. It has also been demonstrated that GnRH receptor mRNA is expressed in bothcytotrophoblast and syncytiotrophoblast. GnRH secretion in the pregnancy period isregularly changed. There is a parallel increase early in the first trimeter and till to the17 weeks later, the secrecion is decreased, which is similar to the HCG secretion. Itindicated that there is a close relationship between HCG. GnRH played an importantrole in early embryos by affecting the synthesis and release of HCG. So GnRH is animported factor in the maintenance of pregnancy particularly. GnRH can regulate therelease of LH in vivo, while HCG is quite similat to LH in molecular structure andfunctional categories. Cytotrophoblast cells can not only synthesize GnRH, but alsoexpress GnRH receptor. The recent reseach shows that both cultured monkeyembryos, human embryonic and mouse embryo produce and secret GnRH duringimplantation process. As early as moral period of blastocyst stage GnRH secretion hasstarted. GnRH binding sites were also found in human decidual tissue andendometruim. The function of GnRH is defferent, because GnRH receptor expressionwas different. Meanwhile estrogen and inhibin, activinA can increase GnRH receptorexpression levels, while progesterone reduces it. Hypothesizing that GnRH may playan important role in early pregnancy by effects on the mutilple MMPs proteinexpression both in desidual cells and extravillous cytotrophblast cells. Using liquidprotein chip systems, to detect various MMPs (MMP-1, MMP-2, MMP-3, MMP-7,MMP-9) expression regulated by GnRH of decidual stromal cells and extravillouscytotrophoblasts. Discussion the function of during embryo implantation andcytotrophoblast invasion and providing molecule theory for successful prengacy.Partâ… Dose-dependent Effects of Multiple Matrix Metalloproteinase in HumanDecidual Stromal Cells by Gonadotropin Releasing Hormone Agonist in EarlyPregnancy.ObjectiveTo study the dose-dependent effects of GnRH agonist on multiple matrixmetalloproteinase (MMPs) in the decidual stromal cells in human early pregnancyand to explore the regulation of GnRH during embryo implantation and plateauformation. Materials and Methods1 Collect 10 decidual tissues from the early pregnant women from 6-9 weeks.Digested with 0.25% collagnaseâ… , Trypsin-EDTA and purified by discontinuousPercoll gradient. Immunohistochemical analysis for PRL staining was used to identifythe purity of DSC. 4×10⁵DSC were counted and cultured in 1ml FD, then change theculture metia supernatants after 24h incubation.2 Cell morphology was observed and taken photographs by inverted phase contrastmicroscope.3 GnRH agonist treatment endometrial stromal cells. Divide Decidual stromal cellsabove into four groups. After 24h incubation, removal unadherented cells and redblood cell. Add 1ml FD to a control group, then add 1 ml FD containing 0.1nmGnRH agonist, 1nm GnRH agonist and 10nm GnRH agonist to the other three groupsrespectively for 24h incubation, collected the supernatant for dictation.4 Cell population was identified by immunocytochemical staining. with cytokeratinand vimentin. The nucleus and cytoplasm were observed by staining ofstreptavidin-peroxide (SP). Positive cells were more than 95%. Cells cultured insix-well asepsis slides and treat cell to climb a basic and full, take out the slides werefixed with 4% paraformaldehyde in phosphate-buffered saline (PBS)for 30 rain atroom temperature, washed in PBS for 5 min. Immunohistochemistry was performedusing the streptavidin-peroxide from a DAB kit. Briefly, slides were treated with 3%hydrogen peroxide at room temperature for 10 min to inhibit the activity ofendogenous peroxidase, then heated for 30 min at 95℃to repair antigens and finallyrinsed in PBS. Nonspecific protein staining was blocked by goat serum.The slideswere incubated with 1:100 dilution of Cytokeratin and Vimentin at 4℃overnight. The next day, they were washed with PBS and incubated at roomtemperature with biotinylated secondary antibody for 10 min, washed extensively,incubated with the streptavidin-peroxidase complex for 10 min, and detected withDAB. For the control, the antibody was replaced by PBS. After immunoperoxidasestaining, the slides were counterstained with hemoxylin for 1 min at roomtemperature, redehydrated and mounted. The slides were observed by light microscopy. Cells were observed per view with high lnagnification. Cells showing abrown-yellow color in the cytoplasm were identified as positives.5 The levels of MMPs (MMP-1, MMP-2, MMP-3, MMP-7and MMP-9) proteinexpression regulated by GnRH agonist were detected by Luminex xMAP.6 Data of MMPs proteins expression were expressed as Mean±SD. The statisticalSignificance of differences was assessed by Repeated Measurea ANOVA test andBivariate correlation. All of the analyses were performed with SPSS13.0 software. Avalue of P＜0.05 was regarded as statistically significant.Results1. DSC normal growth in the 24 well cell culture cluster. The cell population wasidentified more than 95%, and then use to test.2 The celluler morphology was observed by inverted microscope and the nucleusand cytoplasm were observed by staining of streptavidin-peroxide. Cell morphousuniform, tighet array, fusiform shape or irregular, oviform nuclear which located incell central, transparent nucleolus, abundant cytoplasm granulation.3 The expression of MMP in the DSC supernatant and effects of GnRH agonistregualation.3.1 DSC expressed MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9, while MMP-1and MMP-3 are the highest than others3.2 Different concertrations of GnRH agonist (0.1nm, 1nm, 10nm) have the ability ofuprugulating of all MMPs in DSC, and the difference between groups is significant(F=2839.4 P=0.000), the difference within groups is also significant (F=13524.3P=0.000) (Tab. 1-1). The highest expression is at the level of 10nm GnRH agonist.3.3 MMP-1 and MMP-3 keep the advantage of higher expression thanMMP-2, MMP-7 and MMP-9. With the increasing concertration of GnRH agonist, thehigher advantage is enhanced. Concering single MMP-9 at GnRH agonistregulation, the line of MMP-1 MMP-3, MMP-2, MMP-7 is steeper, while that ofMMP-9 is parallel.(Fig.1-2)4 Bivariate correlation between the controland the regulation of GnRH agonist inhuman decidual stromal cells in early pregnancy. The correlation is significant. ( 0.814 ≤r≤0.997 P=0.000) (Tab.1-2).Conclusion1 MMP-1, -2, -3, -7, -9 were secreted by DSC, the expression of MMP-2 and MMP-9is the highest.2 Both 0.1nm GnRH agonist, 1nm GnRH agonist and 10nm GnRH agonist have theability of up-rugulating the MMP-1,-2,-3,-7,-9 proteins expression in DSC. Andthe highest effect happened at 10 nm GnRH agonist level, 1 nm GnRH agonist is thenext.3 GnRH plays an important role during the embryo implantation and placation.4 Luminex xMAP has advantage of detecting protein over other methods.,analysising all the samples of defferent factors at a very low level simultaneously.Partâ…¡Dose-dependent Effects of Multiple Matrix Metalloproteinase in HumanDecidual Stromal Cells and Extravillous Cytotrophoblasts by GonadotropinReleasing Hormone Agonist in Early Pregnancy.ObjectiveTo study the dose_dependent effects of multiple matrix metalloproteinase inextravillous cytotrophoblast in human early pregnancy and the regualation role ofGnRH on the expression of MMPs, exploring possible mechanisms on implantationand plateau formation.Materials and Methods1 Collect 10 Villi tissue from the early pregnant woman from 6-gweeks., digestedwith 0.125% collagnaseâ… , Trypsin-EDTA and purified by discontinuous Percollgradient. 4×10⁵EVCT were counted and cultured in 1ml FD, then change the culturesupernatants after 48h incubation.2 Cell morphology was observed and taken photographs by inverted phase contrastmicroscope.3 GnRH agonist treatment EVCT..Divide EVCT above into four groups. After 48h incubation, removalunadherented cells and red blood cell. Add lml FD to a control group, then add 1 mlFD containing 0.1nm GnRH agonist, 1nm GnRH agonist and 10nm GnRH agonist to the other three groups respectively for 24h, later collect the supernatant for detection.4 Cell populations were identified by immunocytochemical staining. similar to part1 methods 4.5 The levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9 protein expressionregulated by GnRH agonist were detected by Luminex xMAP.6 Data of MMP proteins expression were expressed as Mean±SD. The statisticalSignificance of differences was assessed by Repeated Measurea ANOVA test andBivariate correlation. All of the analyses were performed with SPSS13.0 software. Avalue of P＜0.05 was regarded as statistically significant.Results1 EVCT normal growth in the 24 well cell culture cluster. The cell population wasidentified more than 95%, and then use to test.2 The celluler morphology was observed by inverted microscope, the nucleus andcytoplasm were observed by staining of streptavidin-peroxide. Cell morphousuniform, tighet array, fusifonn shape or irregular, oviform nuclear which located incell central, transparent nucleolus, abundant cytoplasm granulation.3 Feature of MMPs in EVCT supernatant and effects of GnRH agonist rugulatio onthe expression of MMP (MMP-1,-2,-3,-7,-9)3.1 EVCT expressed all MMPs. And the expreesion of MMP-2 and MMP-9 ishighest than others. Different concertrations of GnRH agonist (0.1nm, 1nm, 10nm)have the ability of uprugulating of all MMP in EVCT, and the difference betweengroups is significant(F=29988.7 P＜0.001), the difference within groups is alsosignificant (F=2165.7 P＜0.001) (Tab.1-1). The highest expression is at the level of10nm GnRH agonist.3.2 MMP-2 and MMP-9 keep the advantage of higher expression than MMP-1,MMP-3 and MMP-7. With the increasing concertration of GnRH agonist, the higheradvantage is enhanced. Concering singal MMP at GnRH agonist regulation, the line ofMMP-2 and MMP-9 is steeper, while that of MMP-1 MMP-3 MMP-7 is nearlyparallel.(Fig.2-3)4 Bivariate correlation between the controland the regulation of GnRH agonist in human EVCT in early pregnancy. The correlation is significant. (0.926≤r≤1.000P＜0.001)(Tab.2-2).Conclusion1 To establish the isolation and purification model of EVCT successfully.2 MMP-1, -2, -3, -7, -9 were secreted by EVCT, expression of MMP-2 and MMP-9was particularly significant.3 Both 0.1nm GnRH agonist, 1nm GnRH agonist and 10nm GnRH agonist have theability of up-rugulating the MMP-1,-2,-3,-7,-9 proteins expression in EVCT. Andthe highest effect happened at 10 nm GnRH agonist level, 1 nm GnRH agonist is thenext.4 GnRH plays an important regulation role in extravillous cytotrophoblast invasionby up-regulated MMPs expression.