| Missed abortion is a pregnancy in which there is a fetal demise (usually for anumber of weeks) but no uterine activity to expel the products of conception before20weeks gestation.The precise mechanisms of missed abortion pathogenesis remainunknown. Multiple etiologic factors, including karyotypic disorders, immunologicalfactors, hereditary thrombophilia, endocrinological disorders, Sperm DNAfragmentation, uterine abnormalities, infections, nutritional and environmental factorshave been identified for missed abortion[2-3],However, there are still about50%of thepatients with unknown etiology. In recent years, with the rising morbidity, the studyof unexplained abortion gradually caused the attention. Stat-3signalling pathwayhave been showed the important function of facilitating tumor cell proliferation andinhibiting apoptosis in the occurrence and development of malignant tumors.so wespeculate that it may play a role in trophoblast cell development.Signal transducers and activators of transcription (STAT) proteins comprise afamily of transcription factors that play important roles in cell survival, growth,proliferation, differentiation, apoptosis, metastasis, and angiogenesis[11-13].Stat3isone of the members of the family, activated by IL-6or IL-10cytokines and growthfactors[13].Once activated, Phosphorylated-Stat3(p-Stat3) formed homodimers andtranslocate to the nucleus modulate the expression of target genes[14],such as SurvivinMMP-2and VEGF. Survivin is a member of the Inhibitor of Apoptosis (IAP) genefamily and overexpressed in the majority of human and murine cancers. Survivin ispresent during normal fetal development but undetectable in differentiated adult tissues, with a dual role in apoptosis inhibition and regulation of mitosis. Matrixmetalloproteinases (MMPs) are zinc proteinases that degrade compounds of theextracellular matrix (ECM),and play a key role in cell invasion and remodelling of theextracellular matrix.MMP-2(emphasizes A) is one of the main members of thefamily of matrix metalloproteinases and play an important role in the embryoimplantation, endometrial blood vessels.We speculated that abnormal expression of the Stat3signaling pathways introphoblast might be involved in the pathogenesis of missed abortion, but littleinformation was available for human pregnancies. Hence, real-time polymerase chainreaction (RT-PCR), immunohistochemistry (IHC), and Western blot were performedto examine the expression of IL-6,Stat3, p-stat3,Survivin and MMP-2molecules inboth human first trimester Villous with missed abortion and normal controls.ObjectiveThe aims of this study were to investigate the expression pattern of IL-6,Stat-3,Phosphorylated-Stat3, Survivin,MMP-2in the first trimester human villous tissues ofnormal pregnancy and missed abortion, and to evaluate the relationship between Stat3signaling pathway and missed abortion in the first trimester villous tissues.Materials and Methods1MaterialsAfter approval by institutional review board,30women (mean age29.76, rangeyears24–40) with missed abortion and30women (mean age27.62, range years22–40) undergoing surgical termination of normally progressing pregnancies werestudied. All study subjects involved in the investigation were recruited followinginformed consent.2Methods2.1Sample collectionVillous samples from the missed-abortion group were collected by curettage or manual vacuum aspiration. Villous samples from the control group were obtained byvacuum aspiration from women undergoing elective abortion at7～10weeks ofgestation for social reasons. We picked villous tissues under inverted microscope.Each villous sample was divided into three parts: one was stored in4%formaldehydeat room temperature overnight for immunohistochemistry analysis. the other part wasstored at－80℃, and thawed only once to avoid degradation; For the current studyinvolving the analysis of villous tissues RNA and protein levels, the biopsies wereflash frozen in liquid nitrogen and stored at－80℃until the tissue was processed.Tissue was fixed in10%formalin for immunohistochemical assays.3. Statistical analysisStatistical analysis was performed in using the SPSS version17.0softwarepackage. All data were expressed as the mean±SD. Statistical differences werecalculated using Student’s t-test when the data from the two groups were normallydistributed or a nonparametric Mann-Whitney U test if the data distribution of anygroup was skewed. A P-value <0.05was considered statistically significant.Results1. Clinical Data between missed abortion patients and normalcontrolsWe compared variables (such as age, gestational weeks, gestation sac) betweenthe two groups,found there was no significant association between the patients for themissed abortion and control groups, nor the occurrence of missed abortion with theage of women experiencing missed abortion.2. The IL-6,Stat3,Survivin and MMP-2mRNA expression in thevillous tissues of women with missed abortion and normal controls2.1The IL-6mRNA expression in villous tissues of the missed abortionand normal control groupOur results indicated that IL-6mRNA expression could be detected in both the missed abortion and normal control groups. The IL-6mRNA expression wasdecreased in the missed abortion group compared with the control group (P<0.05).2.2The Stat3mRNA expression in villous tissues of the missed abortionand normal control groupThe Stat3mRNA expression could be detected in both the two groups. There isno significant difference was observed in Stat3expression between the missedabortion group and the control group (P＞0.05).2.3The Survivin mRNA expression in villous tissues of the missedabortion and normal control groupThe Survivin mRNA expression could be detected in both the two groups.TheSurvivin mRNA expression was decreased in the missed abortion group comparedwith the control group (P<0.05).2.4The MMP-2mRNA expression in villous tissues of the missedabortion and normal control groupThe MMP-2mRNA expression could be detected in both the two groups.TheMMP-2mRNA expression was significantly increased in the missed abortion groupcompared with the control group.(P<0.05)3. Immunostaining of IL-6, Stat3, p-stat3, Survivin and MMP-2invillous tissues of the twe GroupsThe sections were examined by hematoxylin and eosin (H&E) staining beforeIHC analysis. Immunoreactivity for IL-6, Stat3and MMP-2were found in thecytomembrane and cytoplasm of cells on the villous syncytiotrophoblast and thecytotrophoblast, p-Stat3was found staining in the nuclear on the villouscytotrophoblast,The immunohistochemical staining for Survivin was primarily innuclear and cytoplasmic in both the syncytiotrophoblast and cytotrophoblasts. TheH&E staining was shown in Figure3.3A. Image of tissue section stained with PBSwas shown in Figure3.3B.The immunostaining were examined in tissue sections from 60villous (30for each group). Stat3showed a hardly changed staining intensity inthe control group than in the missed abortion group.(P＞0.05) However, IL-6, p-Stat3and Survivin showed a weaker staining intensity in the missed abortion group than inthe control group, MMP-2showed a stronger staining intensity in the missed abortiongroup.(P＜0.05)4. The IL-6, Stat3, p-Stat3, Survivin and MMP-2protein expressionin villous tissues of the two groups4.1The IL-6protein expression in villous tissues of the missed abortionand normal control groupThe IL-6protein expression was significantly decreased in the missed abortiongroup compared with the normal control group(P<0.05).4.2The Stat3protein expression in villous tissues of the missed abortionand normal control groupThe Stat3expression was showed a hardly changed between the two groups(P＞0.05).4.3The p-Stat3protein expression in villous tissues of the missedabortion and normal control groupThe p-Stat3protein expression was significantly decreased in the missedabortion group compared with the normal control group (P<0.05).4.4The Survivin protein expression in villous tissues of the missedabortion and normal control groupThe Survivin protein expression was significantly decreased in the missedabortion group compared with the normal control group (P<0.05).4.5The MMP-2protein expression in villous tissues of the missedabortion and normal control groupThe MMP-2expression was significantly increased in the missed abortion group compared with the normal control group (P<0.05).ConclusionsIL-6, Stat3, p-Stat3, Survivin and MMP-2were detected in human third trimestervillous tissues, proper expressions of IL-6, Stat3, p-Stat3, Survivin and MMP-2in thevillus tissue play a key role in the maintenance of early pregnancy. |
PDF Full Text Request