The Mechanisms Of Assisted Reproductive Technology On Abnormal Growth And Glycogen Accumulation In Placenta

Because of different physiological environments between in vitro and in vivo,assisted reproductive technologies(ART),as a kind of iatrogenic stress,can lead to a number of adverse pregnancy outcomes.The placenta,as a specific organ during pregnancy,plays a defining role in keeping balance of nutrition distribution between maternal and fetal,which related to many chronic diseases after birth.Glucose,as the important nutrient for fetal growth and development,is stored as glycogen in the placenta.Many studies have showed that ART might lead to the placenta overgrowth and abnormal structure,but the mechanisms are still unclear.Our study used a mouse in vitro fertilization(IVF)model to explore the effects on placenta growth,glycogen accumulation,transportation of glucose and the possible mechanisms of the phenomena.We also studied on the clinical samples to explore the epigenetics mechanism on the abnormal growth of the placenta.Part 1 The effects of IVF on the growth of placenta and related mechanismsObjective: To explore the effect of IVF on fetal and placenta growth,and detect the expression level of the Phlda2 and down stream Akt/mTor signaling pathway to find the possible mechanisms of abnormal growth.Methods: We used Kunming mouse to build model.The blastocysts form in vitro fertilization were transplanted into day3.5 pseudopregnancy mouse,and we called the day 18.5 fetal and placenta as IVF group.We named the fetal and placenta from natural pregnancy mouse as negative control(NC)group.We weighted and compared the fetal weight,placenta weight and placental efficiency between the two groups.We used qRT-PCR to detect the expression of paternal imprinted gene Phlda2,used western blot to detect the protein expression level of Akt,p-Akt,mTor,p-mTor in the placenta.HE staining was used to analyse the morphology of placenta of the two groups.Results:(1)IVF resulted a higher placental weight and a lower placental efficiency(P<0.01),but the fetal weight had no statistical difference.(2)Compared with the NC group,the mRNA expression level of Phlda2 is significantly reduced in IVF group(P<0.05).(3)Compared with the NC group,protein expression level of p-Akt and the ratio between p-Akt and Akt,between p-mTor and mTor were significantly induced.(4)Compared with the control group,the placental area and the percentage of labyrinth area of the IVF group increased significantly(P<0.05),and the spongiotrophoblast layer was separatedfrom the labyrinth area abnormally.Conclusion: ART influenced the growth and the development of mice placenta,and the overgrowth and low efficiency might be related to the induced activation of Akt/mTor signaling pathway mediated by reduced Phlda2.Part 2 The effects of IVF on glycogen accumulation in placenta and related mechanismsObjective: To investigate whether IVF can cause changes in the accumulation of glycogen in mouse placenta and to ascertain the mechanism underlying these changes.Methods: A mouse model was used to study the glycogen in placenta by using a glycogen quantitation kit.And we used qRT-PCR to detect mRNA expression level of genes related to glycogen metabolism,and used western blot to monitor the protein expression level of GSK3?,p-GSK3?(Ser9),p-GSK3?(Y216),Glycogen Synthase and PYGL.Immunofluorescence staining was performed to determine histological localization of p-Akt,p-GSK3?(Ser9),GS and PYGL.Results:(1)IVF resulted an uncliear separation between the spongetrophoblastic layer and the labyrinth layer,and a significant increase of glycogen accumulation in placenta(P<0.05).(2)Many glycogen synthesis-related genes were significantly up-regulated,while decomposition related genes and metabolic regulation genes were significantly down-regulated(P<0.05).(3)The Akt/GSK3? pathway was activated by IVF,the protein expression level of p-GSK3?(Ser9)and the ratio between p-GSK3?(Ser9)and GSK3? were significantly increased(P<0.05).The expression of Glycogen Synthase and PYGL were both significantly decreased(P<0.05).(5)All of the p-Akt,p-GSK3?(Ser9),GS and PYGL were expressed in the cytoplasm of spongiotrophoblast cells.Conclusion: ART can lead to morphological abnormalities and the excessive glycogen accumulation in the placenta,and the enhanced activation of Akt/GSK3? pathway and abnormal expression of genes,Glycogen Synthase and PYGL may be related to this phenomenon.Part 3 The effects of IVF on glucose transporters in placentaObjective: To explore the effect of IVF on the expression level of glucose transporters in placenta and to ascertain the possible mechanism of low glucose transfer capacity and low placental efficiency.Methods: We used qRT-PCR and western blot to detect the expression level of Glut1 and Glut3.Immunofluorescence staining was performed to determine histological localization of Glut1 and Glut3.Results:(1)Compared with the NC group,the mRNA expression of Glut3 in IVF group was significantly reduced(P<0.00),but Glut1 had no significant difference(P>0.05).(2)IVF resulted to a significantly decreased in protein expression level of both Glut1 and Glut3(P<0.05).(3)Glut1 was expressed in the cell membrane of the labyrinth,and Glut3 was expressed in the cytoplasm of spongiotrophoblast cells..Conclusion: ART leaded to a decreased glucose transporters,which might be related to abnormal glucose transport function and low placental efficiency.