The Association Of Maternal Gut Microbiota And Its Metabolite TMAO With Congenital Heart Disease In Offspring

Background: Congenital heart disease(CHD)is the most common neonatal congenital disease and the main cause of neonatal death due to congenital malformations.If effective treatments are not taken in time,about 1/3 of the children suffer from severe complications within the first year of life,and the prognosis for patients with congenital heart disease is poor.However,risk factors for congenital heart disease have not been fully elucidated and known risk factors include maternal diabetes and hypertension.Studies in recent years have shown that the differences in the composition of maternal gut microbiota and the resulting changes in metabolite levels have a certain impact on the growth and development of the offspring’s embryos and juveniles.In addition,the differences in the composition of maternal gut microbiota and the resulting changes in metabolite levels are related to the occurrence and development of diseases in offspring such as infection,asthma and nervous system developmental disorders.Trimethylamine N-oxide(TMAO)is a small molecule gut microbiota metabolite.Plasma TMAO concentrations are positively correlated with various cardiovascular diseases and chronic diseases such as diabetes and hypertension,and which are expected to be an independent risk factor and biomarker for atherosclerosis and cardiovascular disease.The role of maternal gut microbiota and its metabolite TMAO in the occurrence and development of congenital heart disease in offspring has not been reported until now.Objectives: This study used the Next generation sequence(NGS)and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)to explore the correlation between CHD in offspring and maternal gut microbiota and its metabolite TMAO to provides a theoretical basis for further exploration of the risk factors of CHD and the clinical prevention and treatment of CHD.Methods: The study received approval from the ethics committee of Xiangya School of Public Health Central South University(No: XYGW-2018-36)and was conducted based on the Helsinki declaration.The study protocol has been registered with the Chinese Clinical Trial Registry(No:Chi CTR1800018492).We obtained written informed consent from all mothers.A total of 159 respondents were included in this study,all of whom were women who had given birth for more than one year and whose offspring were treated at Hunan Children’s Hospital.Mothers of infants with congenital heart disease were the case group(MCHD),and mothers of infants without any congenital defects were the control group(MCon).There were 81 cases in the MCHD group and 78 cases in the MCon group.The basic information of the respondents and their offspring and eating habits of respondents were collected by questionnaires.At the same time,statistical analysis was carried out on these data to find out the indicators with statistical differences.The bacterial 16 Sr DNA gene was sequenced by NGS to detect stool samples,and the expression profiles of gut microbiota in the MCHD and MCon groups were obtained for the following analysis:(1)Alpha-diversity and Beta-diversity were different between case group and control group;(2)Between-group differences in gut microbiota at the phylum and genus levels;(3)The previous literature were reviewed to identify the bacteria associated with TMAO production and to compare the differences of relative abundance of these bacteria between groups.UPLC-MS/MS technology was used to detect the contents of TMAO,Choline,Carnitine and Betaine in plasma samples.The data obtained is analyzed as follows:(1)Differences between groups in plasma levels of TMAO,choline,betaine,and carnitine;(2)Indicators with statistically significant differences between case and control groups were included in the models,and these models were developed to analyze their correlation with the risk of congenital heart disease by logistic regression.Model 1was not adjusted for any influencing factors;Model 2 was adjusted for the frequency of weekly consumption of fruits,aquatic products,eggs,milk and dairy products,beans and soy products,and fried foods(≥ 6times/week,3-5 times/week,1-2 times/week,< 1 times/week);Model 3was further adjusted for negative life events(yes or no)and residence(rural or country)on the basis of Model 2.Logistic regression was used to investigate whether maternal plasma levels of TMAO,choline,betaine and carnitine were associated with CHD risk in offspring.Results:(1)There were statistical differences in the following indicators in the basic information and dietary survey of the subjects of the case group and the control group: the mother’s residence before pregnancy,whether she experienced negative life events before pregnancy,and weekly intake frequency of fruits,aquatic products,egg foods,milk and dairy products,beans,soy products and fried foods;(2)Compared with mothers of infants without congenital heart disease,the chao index,ace index,shannon index of mothers of infants with congenital heart disease increased and the simpson index decreased,indicated a decrease diversity and richness in case group.The results of the beta-diversity diversity analysis showed that the composition of gut microbiota was different in the mothers of infants with congenital heart disease compared with those whose infants without congenital heart disease;(3)Compared with the mothers of infants without congenital heart disease,the relative abundance of Enterobacter and Fusobacterium that related to TMAO production in the gut microbiota of mothers of infants with congenital heart disease increased;(4)Compared with mothers of infants without congenital heart disease,the plasma levels of TMAO,choline and betaine in mothers of infants with congenital heart disease were increased(P<0.05)and the level of carnitine was decreased(P=0.92);(5)Logistic regression analysis showed the following results: in model 1,maternal plasma TMAO(OR=1.51),choline(OR=1.68)and betaine(OR=1.52)levels were positively associated with the risk of CHD in offspring(P<0.05);In model 2,maternal plasma TMAO(OR=1.93),choline(OR=1.67)and betaine(OR=1.65)levels were positively associated with the risk of CHD in the offspring(P<0.05);In model 3,maternal plasma TMAO(OR=1.83),choline(OR=1.57)and betaine(OR=1.67)levels were positively associated with the risk of CHD in the offspring(P<0.05).Conclusion:(1)There was significant difference in the composition of the gut microbiota of mothers of infants with CHD compared to mothers of infants without CHD,and the relative abundance of Enterobacter and Fusobacterium which associated with TMAO production were increased.(2)Plasma levels of TMAO,choline,and betaine levels of mothers of infants with CHD were significantly increased,and plasma TMAO,choline,and betaine levels were positively associated with the risk of CHD in offspring.