Study On The Difference Of Polysialic Acid And Its Related Proteins In Placenta Of Pregnant Women With Assisted Reproductive Pregnancy And Natural Pregnancy

Background:Sialic acid(Sia)is a natural class of sugar acids that are widely found in living organisms.There are currently more than 50 natural derivatives of sialic acid,of which the more common three derivatives are N-acetylneuraminic acid(Neu5Ac),N-hydroxyacetylneuraminic acid(Neu5Gc)and 5-position.KDN(2-keto-deoxy-D-glycero-D-galacto-nonulosonic acid).Sialic acid is often present in the form of short-chain residues at the ends of glycoproteins,glycolipids,and the like,for exanmple,by galactose by a-2,3,a-2,6,a-2,8 glycosidic linkages,respectively.(Gal),N-acetylgalactosamine(GalNAc)and other sialic acid linkages.The sialylation of cell surface glycoproteins and glycolipids plays an important role in many physiological processes.As an important bioinformatic transfer molecule,sialic acid is irnvolved in many physiological processes such as cell adhesion,antigen recognition and signal transduction.In addition,tumor infiltration and metastasis are also closely related to the expression of sialic acid on the cell membrane.Assessed reproductive technology(ART)refers to the method of assisting infertile couples to conceive through medical aids.In 1978,the world's first test-trube baby was born.So far,tens of thousands of test-tube babies have been born every year in the world.At present,assisted reproductive technology has become an important auxiliary means for treating infertility.However,manual manipulation of gametes,zygotes,and embryos may cause damage to the embryo to some extent.Early investigations showed that the birth defect rate of ART progeny did not exceed that of natural gestational offspring,but many epidemiological studies in recent years have shown that ART may cause adverse pregnancy outcomes,such as gestational comorbidity,premature delivery,low birth weight,perinatal Increased morbidity and mortality,neurodevelopmental abnormalities,epigenetic related diseases,etc.Therefore,the research on ART safety has become a hot spot in clinical reproductive med:icine research.However,there are no studies on sialicc acid and assisted reproductive pregnancy.In this study,human placenta derived from IVF-ET and placenta of natural preg:nancy were studied.The ART-derived placenta was studied from the gene and protein levels by RNA-seq,Western Blot,Realtime-PCR,ELISA,etc.Differences in functional gene expression in the placenta.On this basis,combined with the expression of polysialic acid and its carrier protein,the invasion of placental trophoblast cells may be related to the entry point,and the expression difference of sialic acid and corresponding vector is detected to understand the intrauterine development environment of ART.Potential changes to further evaluate ART safety.Objectives:1.If ART technology can cause changes in embryonic gene levels;2.To Prove the presence of polysialic acid in the placenta and identify the type of carrier protein;3.Depending on the pregnancy method and pregnancy outcome,we detected polysialic acid and its related genes and proteins in placenta,the expression changes in different groups were analyzed;4.The effect of different pregnancy methods on fetal weight/placental ratio;5.Exploring the correlation between the levels of polysialic acid and blood glucose in pregnant women with GDM,Exploring the correlation between the expression levels of related genes and proteins and blood glucose in pregnant women with GDM.Methods:(1)From September 2018 to March 2019,pregnant women who were hospitalized and delivered after regular check-ups at the Xiamen University affiliated hospital were recruited.According to the different methods of conception,the study group was pregnant women with assisted reproductive technology(ART group),and the control group)was pregnant women with natural pregnancy(NC group);with the follow-up study,according to whether pregnant women have gestational diabetes mellitus(GDIM),The ART group was divided into a healthy group(group A)and a disease group(A GDM group),and the NC group was divided into a healthy group(group N)and a disease group(N GDM group).A total of 52 cases of ART-derived placenta and 60 cases of natural pregnancy-derived placenta were collected in this study.The general information and pregnancy outcomes were investigated by general data survey method.RNA-seq was used to compare the changes in gene expression caused by different pregnancy methods and pregnancy outcomes.The presence of polysialic acid in the placenta was confirmed by western blot and the carrier protein was confirmed.The expression difference of polysialic between different groups was compared.Real time PCR,Western Blot,ELISA were used to compare the expression of NRP2 CADMI ST8siaIV in different groups of genes and protein levels,and the data were correlated with blood glucose levels.Results:(1)Comparing the healthy ART group)(group A)and the healthy natural pregnancy group(group N),the gene expression of the placenta was indeed different.We screened 1051 statistically significant differential genes between group A and group N,among which Group A had 382 up-regulated genes and 669 down-regulated genes in group N.There were also differences in placental gene expression between different pregnant women in ART.Between the AG group and the A group,we screened 681 differential genes.Among them,the AG group had 310 up-regulated genes and 371 down-regulated genes.Compared with group N,group A was significantly up-regulated with genes involved in intracellular protein metabolism,transcriptional processes,and ribosome structural components;regulation of cell gro,wth,insulin-like growth factor synthesis,binding to growth factors,and regulation of intracellular component synthesis The gene's genes are significantly downregulated.There were no significant differences in biological processes,cellular components,and molecular functions between AG and Group A.Compared with the N grouq,the genes that are up-regulated in group A are significantly enriched in the pathway in which the ribosomal subunit is formed;the genes that are down-regulated are infected with S.aureus,primary immunodeficiency,graft-versus-host disease,Significant enrichment in the immune system-related pathways such as the complement pathway.Compared with the placenta of the different pregnancy outcomes in the ART group,the AG group was significantly more enriched in the ribosome formation pathway than the A group,and the enrichment in other pathways was significantly lower.(2)Polysialic acid is present in placenta,and its carrier protein is NRP2 and CADM1,which is not a common NCAM carrier,polysialic acid in the placenta binds to the carrier in an N-linked form(3)The expression of NRP2 in ART group was higher than that in NC group(P<0.05).After being divided into four groups,at the gene and protein levels,group A placenta was higher than group N placenta,but it was not statistically significant.(4)The CADM1 expression at gene level of ART group was higher than that of NC group(P<0.05).There was no difference between the two groups at the protein level.After being divided into four groups,there was no difference between group A placenta and group N placenta at the gene level.The placenta of the A GDM group was higher than that of the N GDM group;at the protein level,there was no difference between the placenta of group A and the placenta of group N.(5)ST8siaIV in ART group was higher than NC group in gene and protein expression level(P<0.05).After being divided into four groups,at the genetic level,the placenta of group A was slightly higher than that of group N,but there was no statistical significance.The placenta of group A GDM was higher than that of group N GDM(P<0.05);at the protein level,group A Placenta was higher than N group placenta(P<0.05).(6)The expression level of polysialic in ART group was higher than that in NC group(P<0.05).After being divided into four groups,placenta of group A was higher than that of healthy group N(P<0.05).(7)The placental weight of the ART group was higher than that of the NC group(P<0.05).There was no difference in fetal weight between the two groups.The fetal/placental ratio of the ART group was lower than that of the NC group(P<0.05).(8)Both CADM1 and ST8sia?were positively correlated with blood glucose levels in pregnant women(P<0.05),and polysialic expression levels were negatively correlated with blood glucose in pregnant women(P<0.05).Conclusions:The operation of embryos by assisted reproductive pregnancy techniques can result in changes in the genetic level of the embryo.The polysialic in the placenta of the ART group was higher than that in the NC group.The NRP2 carrier and ST8siaIV in the ART group were higher than the NC group in the gene and protein levels.The CADM1 of ART group was higher in the gene level than the NC group,and the protein expression level was no difference in the two groups.The placental weight of the ART group was higher than that of the NC group,and the fetal weight was no difference in the two groups.The fetal/placental ratio of the ART group was lower than that of the NC group.The expression level of CADM1 gene and protein was positively correlated with blood glucose of pregnant women;the expression level of ST8siaIV gene and protein was positively correlated with blood glucose of pregnant women;the expression level of polysialic was negatively correlated with blood glucose of pregnant women.