Alterations Of Milk Glycans From Mothers With Gestational Diabetes Mellitus Affect The Gut Microbiome And Immune Balance Of Their Offspring

Gestational diabetes mellitus(GDM)is a type of diabetes mellitus with normal glucose metabolism before pregnancy or with potential glucose tolerance loss,which is diagnosed during pregnancy.More than 80% of pregnant women with diabetes are GDM,and with a significant increasing trend in China in recent years.The high incidence of early allergic diseases in GDM infants may correlate with the altered gut microbiota and immune tolerance of them.Milk glycans(including oligosaccharides,proteoglycans,etc.)are important factors in shaping the gut microbiota of infants,which has important effects on the immune development and immune balance of infants.Study had shown that compared with healthy mothers,there is no significant difference in the content of oligosaccharides in breast milk of GDM mothers,but the glycosylation level of milk N-glycans has significantly changed.However,the impact of alterations in GDM milk N-glycans on gut microbiota and immunity of neonates is yet to be studied,and the relevant mechanism needs to be revealed.Objective:Through establishing GDM maternal mouse models and using them to crossfoster different offspring mice,our aim in this study is to explore the changes of GDM breast-milk glycans and their influence on the intestinal microbiota and immune balance of the offspring,and to elucidate the underlying mechanism.Methods:1.Establishment of GDM mouse model and crossfostering of different offspring mice30 female Institute of Cancer Research(ICR)mice aged 6 weeks were randomly divided into two groups(n=15 in each group),the control(CON)group of mice were fed with standard diet,the GDM group of mice were exposed to high-fat diet for 4weeks.After that,these female mice were mated with age-matched males.During pregnancy,the GDM group of mice were intraperitoneal injected with streptozotocin(STZ)for continuous 3 days,the CON group was injected with equal dose of normal saline(NS)for 3 days.Next,three consecutive days of random blood glucose testing >11.1 proved successful modeling.After parturition,the female mice were divided into three groups(n=3)and respectively fed to their offspring:(1)CON?offspring,the CON maternal mice fed their own neonates;(2)GDM?offspring,the GDM maternal mice fed their own neonates;(3)to rule out the interference from other factors,such as genetics,and to specificly analyse the effect of GDM breast milk on intestinal microbiota and immune balance of the neonates,we set up a cross-fostering group: GDM-C?offspring,in this group,we used the GDM maternal mice to cross-feed the neonates from the CON maternal mice.Each maternal mice fed 3offspring mice(n=3).2.Mass spectrometry and other methods were used to compare and analyze the differences of oligosaccharides and N-glycans in the milk of GDM and CON maternal mice.3.16 S r RNA high-throughput sequencing was used to compare and analyze the differences of intestinal microbiota in the offspring fed by different maternal mice.4.Flow cytometry was used to compare the differences in the proportion of spleen lymocytes in the offspring fed by different maternal mice.5.Establishment of OVA immunolized mouse model and detection of serum cytokines.6.To explore the effect of Bacteroides acidifaciens ATCC800799 on mouse PPs lymphocytes in vitro.Results:1.Compared with the control group,the abundance of the free oligosaccharides in breast milk of GDM maternal mice has no significant differences,but the levels of fucosylation and sialylation of milk N-glycans were found markedly upregulated.2.Compared with the offspring mice fed by CON maternal mice(CON?offspring),theose GDM mothers-fed neonates,including the GDM?offspring and GDM-C?offspring mice showed similar altered intestinal microbial structures.The main feature was that the relative abundance of Bacteroides spp.was significantly increased(especially Bacteroides acidifaciens),while the relative abundance of Lactobacillus spp.was reduced.3.Compared with CON?offspring,the proportions of splenic B and T lymphocytes in GDM?offspring and GDM-C?offspring groups were relatively higher,and the proportion of regulatory T cells(Treg)was reduced after OVA immunization,and the balance of Th17/Treg cells was broken in these neonates.4.The metabolites of Bacteroides acidifaciens ATCC800799 can stimulate the activation of B and T cells in vitro,and affect the balance of Th1 / Th2 cells.Conclusion:The up-regulation of fucosylation and sialylation levels of milk N-glycans from maternal mice with GDM induced changes in the gut microbiome of their breast-fed offspring.The genus of Bacteroides spp.,which was significantly increased in GDM-fed neonates,could destroy the balance of Th1/Th2 and Th17/Treg cells through secondary metabolites,leading to the immune imbalance in these offspring mice.This study,for the first time,systematically analyzed the characteristics of GDM glycans in mouse models and the effects of these characteristic changes on the gut microbiota and immune balance of offspring,laid a foundation for the research and development of specific nutritional care for GDM offspring.