| Object:Women with gestational diabetes mellitus(GDM)have a higher risk of diseases such as overweight and obesity in their offspring,and breastfeeding is an important measure to reduce the risk of these diseases.In this study,we collected,analyzed and compared the differences in breast milk macronutrients,metabolites and microbial diversity between women with GDM and normal pregnancy at different lactation stages after delivery to explore the possible effects of gestational diabetes on breast milk macronutrients,metabolites and microbial diversity at different lactation stages and provide a theoretical basis for guiding reasonable breastfeeding.Method:Baseline data of 45 lactating mothers with GDM and 90 normal pregnant lactating mothers who met the study inclusion criteria from May 2019 to September 2020 were collected by questionnaire.And collected breast milk at three stages(colostrum,42-day and 3-month mature milk).The macronutrients of breast milk(including fat,total protein,true protein,lactose,dry matter and total energy)were used a Miris human milk analyzer.~1H-NMR was used to detect human milk metabolic and 16S rDNA microbial diversity sequencing was used to detect the milk microbiota,including 15 samples in each of the three stages in the NGT group,15 samples in each of colostrum and 42-day mature milk in the GDM group,and 7 samples of 3-months.Result:1.From the analysis of lactation stage,the total protein and true protein contents decreased continuously with the extension of lactation time in both groups(P<0.05).The fat,lactose and dry matter contents of 42-day mature milk were significantly increased in both groups compared with the colostrum(P<0.01).The fat and dry matter content of 42-day mature milk was significantly increased compared with 3-month mature milk in the GDM group(P<0.01),but the fat and dry matter of the NGT group were not significantly different in the two mature milk stages(P>0.05).2.The dry matter and total energy of the GDM group at the 42-day mature milk stage were significantly higher than those of the NGT group(P<0.05),while the macronutrients of each breast milk at the colostrum and 3-month mature milk stages were not significantly different between the two groups(P>0.05).3.~1H-NMR-based analysis was used to analyze the metabolites of breast milk in different lactation stages.In NGT group,compared with colostrum,the relative content of butyrate was significantly increased in 42-day mature milk,and the relative content of16 metabolites including citrate,dimethylamine and formate was significantly decreased with lactation time(P<0.05);compared with 42-day mature milk,the relative content of6 metabolites including acetic acid,alanine and glutamic acid was significantly increased(P<0.05),and the relative content of 7 metabolites including carnitine,citrate and dimethylamine was significantly decreased(P<0.05)in the 3-month mature milk.In GDM,compared with colostrum,the relative content of 5 metabolites including carnitine,glutamate,acetone was significantly increased(P<0.05),and the relative content of 6metabolites including 6-SL,galactose and creatinine was significantly decreased(P<0.05)in 42-day mature milk;there was no significant difference in metabolites between 42-day and 3-month mature milk stages.4.~1H-NMR-based analysis was used to analyze the milk metabolic between the GDM group and the NGT group.No significant differential metabolites were found in the two groups during the colostrum phase.At the 42-day mature milk stage,there were11 differential metabolites between the two groups.Compared with the NGT group,the GDM group had lower relative contents of 3-FL and LNDFH II in breast milk(P<0.05),while the relative contents of 9 metabolites including 2-FL+LNFP I,galactose and carnitine were higher(P<0.05).At the 3-month mature milk stage,there were 17differential metabolites between the two groups.Compared with the NGT group,the GDM group had lower relative contents of 12 metabolites including LNDFH I,alanine and taurine in breast milk(P<0.05),whereas the formate,urea,2-FL+LNFP I and Fuc-α1,4Glc NAc metabolites had higher relative contents(P<0.05).5.The 16S rDNA sequencing technology was used to analyze the microbial diversity of breast milk at different lactation stages.In the NGT group,the colostrum compared with 42 days mature milk,at the phylum classification level,the relative abundance of Firmicutes was higher in colostrum,while the relative abundance of Proteobacteria,Bacteroidota,and Bdellovibrionota was higher in 42-day mature milk.At the genus classification level,the relative abundances of Ralstonia,Burkholderia-Caballeronia-Paraburkholderia,and Rhodococcus were higher in 42-day mature milk than in colostrum(P<0.05),and the relative abundances of Streptococcus,Staphylococcus,and Gemella were higher in colostrum than in 42-day mature milk(P<0.05).In GDM,the relative abundance of Proteobacteria was lower in the colostrum and Firmicutes was higher in colostrum than in 42-day mature milk(P<0.05).At the genus classification level,the relative abundances of Ralstonia,unclassified＿f＿Xanthobacteraceae,and Rhodococcus in colostrum of the GDM group were lower than those in 42-day mature milk,and the relative abundances of Cutibacterium were higher than those in 42-day mature milk(P<0.05).There was no significant difference in the microbial composition of all species between 42-day mature milk and 3-month mature milk in the GDM and NGT groups at either the phylum classification level or the genus classification level(P<0.05).6.The 16S rDNA sequencing technology was used to analyze the diversity of breast milk between the GDM group and the NGT group,and the results showed that there was no significant difference in the microbial composition between the two groups at the colostrum stage.At the 42-day mature milk stage,the relative abundances of Bdellovibrionota and Patescibacteria in the GDM group at the phylum taxonomic level were lower than those in the NGT group(P<0.05);at the genus taxonomic level,the relative abundances of unclassified＿f＿Xanthobacteraceae of GDM were higher(P<0.001),and the relative abundances of Rhodococcus and Acinetobacter were lower(P<0.05).At the 3-month mature milk stage,the relative abundances of Bacteroidota and Bdellovibrionota at the phylum taxonomic level were lower in the GDM group compared with the NGT group(P<0.05);at the genus taxonomic level,the relative abundances of unclassified＿f＿Xanthobacteraceae were higher(P<0.001)and the relative abundances of Burkholderia-Caballera-Paraburkeria and Acinetobacter were lower(P<0.05)in the Flavobacteraceae family in the GDM group.7.There is a correlation between small molecule metabolites in breast milk and microorganisms.In the colostrum stage,five phyla including Firmicutes,Actinobacteriota,and Cyanobacteria at the phylum classification level were significantly correlated with breast milk small molecule metabolites(P<0.05)and positively or negatively correlated according to different metabolites;at the genus classification level,Cutibacterium,Rhodococcus,Streptococcus,and Escherichia-Shigella were significantly correlated with breast milk small molecule metabolites(P<0.05).At the 42-day mature milk stage,seven phyla including Proteobacteria,Actinobacteriota,and Acidobacteriota at the phylum taxonomic level were significantly correlated with breast milk small molecule metabolites(P<0.05);at the genus taxonomic level,ten species including Streptococcus,Ralstonia,and Rhodococcus were significantly correlated with breast milk small molecule metabolites(P<0.05).At the 3-month mature milk stage,seven phyla including Firmicutes,Actinobacteriota,and Bacteroidota at the phylum taxonomic level were significantly correlated with breast milk small molecule metabolites(P<0.05);at the genus taxonomic level,nine species including Rhodococcus,Corynebacterium,and Ralstonia were significantly correlated with breast milk small molecule metabolites(P<0.05).Conclusion:The lactation stage had a significant effect on the macronutrients of breast milk.There were significant differences in macronutrients in the three lactation stages in the GDM group,and there were differences between colostrum and 42-day mature milk in the NGT group,while there were no significant changes in macronutrients after 42-day mature milk in the NGT group.The lactation stage has a significant effect on the small molecular metabolites of breast milk.There were significant differences in the contents of small molecule metabolites in the three lactation stages in the NGT group,but there were significant changes in the contents of small molecule metabolites in colostrum and42-day mature milk in the GDM group.The lactation stage has a significant effect on the microbial diversity of breast milk.The influence of the NGT group and GDM group on the microbial diversity of breast milk was similar.The microbial diversity of breast milk at colostrum stage was significantly different from that at a 42-day maturation stage,and a stable microbial community gradually formed from 42-day to 3-month stage.The effects of GDM on breast milk macronutrients at different stages were shown as higher dry matter content and total energy in 42-day mature milk,but had no effect on breast milk macronutrients at other stages.GDM affected the relative content of small molecule metabolites and microbial diversity of breast milk after the 42-day maturation stage,and the relative content of small molecule metabolites and microbial diversity of breast milk at the colostrum stage were no different from that of normal pregnant lactating mothers. |
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