The Role Of Trimethylamine N-Oxide And Neutrophil Extracellular Traps In Gestational Diabetes Mellitus

Background:Gestational diabetes mellitus（GDM）is the most common metabolic abnormality during pregnancy,and more and more evidences show that GDM could bring to women and their offspring short-term or even long-term health risks.Neutrophil extracellular traps（NETs）are a new way for neutrophils to capture and kill invasive pathogens.However,the excessive and unbalanced formation of NETs will participate in the pathological process of many inflammatory diseases.The neutrophils in people with diabetes and high glucose environments are more likely to release NETs spontaneously and the activity of neutrophils in women with GDM has also changed,but the role and significance of NETs in GDM have not been reported yet.Choline is an essential nutrient required by the human body,and women during pregnancy need adequate dietary choline intake to meet the needs of normal fetal development.Trimethylamine N-oxide（TMAO）is a choline metabolite.Recent clinical studies have found that TMAO is related to the risk of GDM,and the concentration of TMAO in peripheral blood and cord blood of women with GDM is significantly higher than that in the control group.However,there is no research to explore the role and significance of TMAO in GDM.Objective:GDM is related to the adverse outcome of the fetuses and newborns.The placenta is the most direct organ connecting the mothers and the fetuses,and the changes in its development and function will affect the development of the fetuses.Therefore,the purpose of this study is to explore the effects of TMAO and NETs on the placenta,reveal the roles and significance of TMAO and NETs in GDM,and provide new perspectives and scientific basis for exploring the mechanism of adverse pregnancy outcomes caused by GDM.Methods:（1）Meta-analysis:Relevant studies from the electronic databases of Pub Med,China National Knowledge Infrastructure,VIP Journal Integration Platform and WANFANG MED were searched through December 2018.The literature included in this Meta-analysis were screened strictly according to the inclusion and exclusion criteria.The mean and standard deviation were used to calculate the weighted mean difference（WMD）to evaluate the association between plasma choline and its metabolite levels and diabetes risk.The Cochran Q and I²statistics were used to evaluate the heterogeneity between studies,and random effects models were used when P<0.10 or the I²statistics≥50%.And the Newcastle-Ottawa Quality Assessment Scale（NOS）was used to assess the quality of the literature of the included case-control studies.（2）Clinical research:Peripheral blood of 20 women with GDM and20 age-matched control pregnant women was collected during the oral glucose tolerance test（OGTT）and followed up to delivery.Cord blood and placenta of 16 women with GDM and 17 age-matched control pregnant women were collected during delivery.For peripheral plasma and cord plasma of women with GDM and control group,liquid chromatography-mass spectrometry（LC-MS）was used to detect TMAO levels,Pico Green（?）ds DNA was used to quantify cell-free double-stranded DNA（ds DNA）,and enzyme linked immunosorbent assay（ELISA）was used to detect the concentration of myeloperoxidase（MPO）,and then comparison and correlation analysis were performed.Immunofluorescence was used to detect the expression of MPO in the placenta tissue of women with GDM and control group.（3）In vitro experiment:Cell Counting Kit-8（CCK-8）,scratch test,Transwell and tube formation were used to observe the effects of TMAO and NETs on the human placental extravillous trophoblast cell line HTR-8/Svneo.Freshly isolated neutrophils from the blood of healthy donors were used to observe the effect of TMAO on NETs formation.（4）In vivo experiment:The expression of peptidylarginine deiminase4（PAD4）is very important for the formation of NETs.It can mediate histone citrullination and promote the decoagulation of chromatin necessary for the formation of NETs.Therefore,the GDM models of wild-type and PAD4^-/-mice were established,and 0.12%TMAO was added to drinking water to explore the roles of NETs and TMAO in GDM mice.The mice were randomly divided into four groups:control group,TMAO group,PAD4^-/-group and PAD4^-/-+TMAO group.Observed and recorded the weight of the mice before pregnancy and during pregnancy and tested the glucose tolerance of the mice before pregnancy.Pico Green（?）ds DNA was used to detect the content of cell-free ds DNA in mice.Observed and recorded the number of resorbed fetuses.Observed the effects of TMAO and NETs on the placenta and fetuses on gestation day（GD）14.5 and GD18.5,and recorded the number and weight.Hematoxylin-eosin staining（HE）was used to observe the effects of TMAO and NETs on placental structure.Results:（1）Compared with the control group,the levels of ds DNA and MPO in the peripheral blood and cord blood of women with GDM increased significantly,and the levels of ds DNA and MPO were correlated with the changes in the metabolism of pregnant women.（2）The immunofluorescence results showed that the expression of MPO in placenta tissue of women with GDM was significantly higher than that of the control group.（3）In vitro experiments,NETs could inhibit the viability,migration,invasion and angiogenesis of HTR-8/Svneo cells.（4）A total of 12 studies were included in the meta-analysis.Compared with the control group,the people with diabetes showed higher choline levels（WMD=0.95μmol/L,95%CI=0.03-1.86）,lower betaine levels（WMD=-2.28μmol/L,95%CI=-3.47--1.10）and higher TMAO levels（WMD=1.45μmol/L,95%CI=0.73-2.17）in plasma.Further analysis found the plasma TMAO levels of women with GDM increased significantly（WMD=0.14μmol/L,95%CI=0.09-0.20）.（5）TMAO-treated neutrophils could resist the formation of NETs stimulated by Phorbol-12-myristate-13-acetate（PMA）and lipopolysaccharide（LPS）.（6）Compared with the control group,the TMAO levels peripheral blood and cord blood of women with GDM increased significantly,and the TMAO levels were correlated with the changes in the metabolism of pregnant women.（7）The results of multiple linear stepwise regression analysis showed that the levels of TMAO in the peripheral blood of pregnant women were correlated with the placental thickness（β=0.154,P=0.001）.And the levels of TMAO in the peripheral blood（β=18.059,P=0.047）and cord blood（β=32.871,P=0.017）of pregnant women were correlated with newborn birth weight.（8）In vitro experiments,TMAO could enhance the viability,migration,invasion and angiogenesis of HTR-8/Svneo cells.（9）In vivo experiments,the placental weight of PAD4^-/-mice was significantly higher than that of control wild-type mice.TMAO feeding not only inhibited the formation of NETs in mice,but also further increased the weight of the placenta and fetuses and reduced the rate of resorbed fetuses in GDM mice,and this increase did not affect the normal structure of the placenta.Conclusion:The abnormal formation of NETs are associated with GDM and can damage the viability and biological function of the human placental extravillous trophoblast cells.The choline metabolite TMAO can not only inhibit the formation of NETs,but also promote the development of the placenta and fetuses.