Choline and its Metabolites Trimethylamine and Trimethylamine N-oxide Potentially Predict the Risk of Small Vulnerable Newborn and the Development of 1-Year Infants

Choline and its Metabolites Trimethylamine and Trimethylamine N-oxide Potentially Predict the Risk of Small Vulnerable Newborn and the Development of 1-Year Infants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Choline and its Metabolites Trimethylamine and Trimethylamine N-oxide Potentially Predict the Risk of Small Vulnerable Newborn and the Development of 1-Year Infants Xiaoqiu Xiao, Xiaojing Lin, Kemeng Li, Zhuyun Wang, Chang Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3970231/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The concept of Small Vulnerable Newborns (SVN), encompassing small for gestational age (SGA), low birthweight (LBW), and preterm birth (PTB) has recently proposed [1, 2]. Collectively, these births represent the main cause of neonatal mortality and specific interventions to address SVN have great potential to improve maternal and infant outcomes. Choline is an essential nutrient for many aspects of fetal development and metabolism, but despite this, upwards of 90% of pregnant women fail to achieve recommend adequate intake [3]. At present, the role of choline deficiency in pregnancy on SVN risk remains unclear. Methods: Here, we measured choline and its metabolites, TMA an TMAO by liquid chromatography-mass spectrometry (LC-MS/MS) in first (11-14 weeks), second (22-28 weeks), and third (32-34 weeks) trimester plasma of 1231 pregnant women from the Complex Lipids in Mothers and Babies (CLIMB) cohort to investigate the relationship between choline TMA, TMAO in pregnancy and the risk of SVN. We also explored the relationship of these measures with later infant developmental outcomes of SVN and non-SVN births including physical and brain development. Results: Pregnant women with SVN births showed no differences in circulating choline throughout pregnancy but had significantly lower TMAO in the first two trimesters, and higher TMA in the third trimester, relative to non-SVN births. However, TMAO levels during pregnancy were positively correlated with the risk of SVN, while TMA levels were negatively correlated with the risk of LBW and PTB in SVN. Supporting findings included positive associationbetween TMAO during pregnancy and birth weight and birth length, while negative association between TMA and birth weight and birth length. In the receiver operating characteristic (ROC) curves, inclusion of choline and its metabolites TMA and TMAO during pregnancy in the models markedly increased the predictive performance for the model which contained traditional risk factors for SVN including age, body mass index, fasting blood glucose , one-hour postprandial blood glucose, two-hour postprandial blood glucose, and placental growth factor. Moreover, during pregnancy, the TMAO was not only positively correlated with fetal development, but also with the development of postnatal offspring at six months old and 1 year old, while TMA was the opposite. Conclusions: The abnormal choline metabolism is potentially associated with the risk of SVN and the development of fetus and postnatal offspring, with particular attention paid to the opposite association between TMAO and TMA in offspring development, which may be an effective means of preventing SVN and ensuring healthy fetal development. Health sciences/Risk factors Health sciences/Biomarkers/Predictive markers Figures Figure 1 Figure 2 Introduction Small for gestational age (SGA), low birthweight (LBW), and preterm birth (PTB) are major intractable adverse birth outcomes representing considerable mortality and morbidity internationally. Recently, these pregnancy outcomes have been collectively grouped as small vulnerable newborns (SVN) [1]. In 2020, 55.3% of the 2.4 million global neonatal deaths and 1.9 million stillbirths were attributable to SVN [2]. Despite this, progress towards understanding the etiology of SVN remains less than optimal and approaches to prevent SVN with appropriate interventions remain limited. Adequate choline levels during pregnancy are important for healthy fetal development, anything requiring methyl donors or acetylcholine metabolism needs choline, particularly membrane biosynthesis, neurite myelination, cell division, tissue expansion and lipid transport [4], but supplementation does not directly alter choline concentration in peripheral blood of pregnant women and umbilical cord blood [5]. Choline supplementation during pregnancy improve offspring cognition, neurodevelopment, and placental functioning, and to protect against neural and metabolic insults [6]. The role of choline in promoting offspring development may thus be indirect, through its metabolites. Choline is metabolized by the intestinal bacteria to form trimethylamine (TMA), which is then rapidly oxidized to and trimethylamine N-oxide (TMAO) by flavin-containing monooxygenase 3 (FMO3) in the liver [7]. In order to assess the potential link between these downstream metabolites and offspring health, we determined the levels of choline, TMA, and TMAO in the plasma of 1231 pregnant women from the Complex Lipids in Mothers and Babies (CLIMB) cohort study during the first (11-14w), second (22-28w), and third (32-34w) trimesters to determine the role of choline metabolites on SVN risk and specific aspects of offspring development, including the developmental indicators of the fetus during pregnancy [(biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL)], the general infant development indicators [(weight, length, occipital frontal circumference (OFC), skin fold thickness of triceps (TSF); skin fold thickness of subscapular(SSF) and mid upper arm circumference (MUAC)] at 6 months and 1 year after birth, as well as cognitive development[mental development index (MDI) and psychomotor development index (PDI)] of 1-year-old infants. Methods Study participants One thousand five huanderds pregnant women were recruited from the CLIMB study, the pregnant women were randomized into three groups: control milk group, complex milk lipid-enhanced (CML-E) group and reference group (Chinese Clinical Trial Register number: ChiCTR-IOR-16007700). The participants were enrolled at prenatal clinics between 11 and 14 weeks gestation between September 2015 and June 2017, at the First Affiliated Hospital of Chongqing Medical University and Chongqing Health Center for Women and Children [24]. Women between 20–40 years of age and had a singleton pregnancy were included in the study, women who had a previous pregnancy with complications which resulted in delivery before 32 weeks were excluded from the study. Meanwhile, women who lost to follow-up (n=186) or miscarried (n=12), whose pregnancies were terminated (n=29), whose information were incomplete (n=42) were excluded from the analysis. A total of 1231 pregnant women were ultimately included in this study ( supplementary Fig. S1). Low birthweight (LBW) infants were defined as those weighing < 2500 g, infants born Small for gestational age (SGA) as those SGA were indicated by birth weight less than the 10th percentile for the gestational age by sex, respectively, and preterm birth (PTB) are those that occur at less than 37 weeks' gestational age [25]. This study was conducted in accordance with the principles in the Declaration of Helsinki. Ethical approval was granted by the Ethics committee of the Chongqing Medical University (No. 2014034), and written informed consent was obtained from all participants. Human subjects Maternal baseline characteristics including maternal age, smoking, alcohol consumption, education background, body mass index (BMI) and blood glucose were presented in supplementary Table S 1 . Overnight fasting maternal blood samples (11–14 weeks, 22–28 weeks, and 32-34 weeks of pregnancy) were collected and immediately stored at -80°C until analysis. The measurement of samples was performed by liquid chromatography-mass spectrometry (LC-MS/MS), with testing personnel blinded to the clinical information and pregnancy outcome of the patients. Randomly test plasma samples from 100 pregnant women in each batch during the same period. Assessment of plasma choline, TMA, and TMAO concentrations by liquid chromatography-mass spectrometry (LC-MS/MS) Added 50 μL of 1% formic acid aqueous solution into 50 μL of the plasma, vortexed for 1 min, then added 300 μL of methanol solution containing deuterated internal standard (choline-d9, concentration: 1 μg/mL) into it, further vortexed for 5 min and then centrifugation at 4°C (12700 rpm, 10 min). 70 μL of the supernatant was transferred to an auto-sampler vial for detection. After sample pretreatment, the supernatants (5 µL) were injected onto a silica column (InfinityLab Poroshell 120 HILIC-Z, 2.1 × 100 mm, 2.7 µm) and analyzed at a flow rate of 0.3 mL/min. Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were mixed to generated a discontinuous gradient to resolve the analytes at different ratios starting from 10% A linearly to 50% A from 0 min to 5 min, then linearly to 10% A from 5min to 5.6 min, held for 5.4 min and then stopped. Mass spectrometry conditions: Ion mode: AJS ESI, positive ion mode; Drying gas temperature: 120°C; drying gas flow rate: 13 L/min; Nebulizer pressure: 30 psi; sheath gas temperature: 300°C; sheath gas flow rate: 12 L/min; capillary voltage (+): 2500 V. The parameters for the ion monitoring were as follows: choline (104.11 m/z, 58.2 m/z, 45V, 2.63 min), TMA (60.08 m/z, 45.1 m/z, 10V, 2.38 min), TMAO (152.11 m/z, 76 m/z, 9V, 2.42 min), d9-choline (113.17 m/z, 45.1 m/z, 45V, 2.63 min). Agilent 1290 Infinity II High-Speed Pump (G7120A) with an Agilent 6495 Triple Quadrupole LC/MS System (G6495C) was used to perform LC-MS/MS. Agilent MassHunter LC/MS was used to collect data and Agilent MassHunter was used to conduct qualitative analysis. Statistical analysis Data were analyzed by SPSS 25.0, R 4.3.0 and GraphPad Prism 8.3.0; A two-sided test were used, where applicable. If the data showed a Gaussian distribution, Student's t-test was used to compare two groups, otherwise, the nonparametric was adopted. Poisson regression was used to estimate the relative risk between choline and its metabolites TMA and TMAO and SVN, if the model was over-dispersion, negative Binomial regression analysis was used for estimation. The areas under the receiver operating characteristic (ROC) curves (AUC) were calculated to check whether the inclusion of choline and its metabolites TMA and TMAO was able to would increase the AUCs from a model which only included the traditional risk factors. Multiple linear regression was used to analyze the association of the measurement data. Multivariate mdel was adjusted for body mass index, age, blood glucose, complex lipids and gestational week. False discovery rates (FDR) were used to account for multiple comparisons, and the parameter desired FDR was set to the 0.05. The set of p-values produced across choline, TMA, or TMAO and serum glucose levels were FDR corrected with the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Multiple imputation and logistic regression were used to reanalyze the main results to test the sensitivity of our results. Results Characteristics of the study participants The characteristics of the CLIMB study participants are shown in supplementary Table S1. Of 1231 pregnancies investigated in the current study, a total of 143 resulted in SVN births (comprising 92 SGA, 31 LBW and 53 PTB, of which 10 were both SGA and LBW). There were generally no differences between SVN and non SVN pregnancies in terms of gestation, maternal age, education, BMI smoking, or OGTT results, despite SGA pregnancies showing slightly elevated 1 and 2 hour glucose levels. Mothers of SVN had significantly lower concentrations of TMAO in the first and second trimester, but had higher concentrations of TMA in the third trimester, as compared to mothers of infant who were not SVN ( Table 1 ). Choline and its metabolites TMA and TMAO and the risk of SVN Choline, TMA, and TMAO levels were grouped according to quartiles into Q (quartile) 1, Q2, Q3, and Q4, with the Q4 group as reference. The relative risks (RRs) estimated by Poisson regression indicated that the lower TMAO of the first trimester (Q3: adjusted RR = 1.897, 95% CI: 1.127-3.193; Q2: adjusted RR = 1.838, 95% CI: 1.091-3.097) and the third trimester (Q1: adjusted RR = 1.797, 95% CI: 1.092-2.956) were significantly associated with a higher risk of SVN (Fig. 1 ). Because SVN includes SGA, LBW and PTB, we further calculated the RRs between choline and its metabolites TMA and TMAO and SGA or LBW or PTB separately. We found that lower TMA during pregnancy were associated with a lower risk of these pregnancy outcomes, which were opposite to TMAO (supplementary table S2) . Birthweight is a measure of vulnerability [1, 2], we analyzed the association between choline and its metabolites TMA and TMAO and birthweight and birth length of newborn in the total population and SVN, respectively, and we found opposite effects of TMA and TMAO as before. In the total population, TMAO levels in the third trimester were positively correlated with birthweight (β = 0.066, P = 0.030, q = 0.032) and birth length (β = 0.071, P = 0.023, q = 0.032), while TMA levels in the third trimester were negatively correlated with birthweight (β = -0.076, P = 0.016, q = 0.017) and birth length (β = -0.092, P = 0.004, q = 0.008). In SVN, TMAO levels in the first trimester (β = 0.275, P = 0.002, q = 0.002) and the third trimester (β = 0.223, P = 0.024, q = 0.025) were positively correlated with birthweight (Table 2) . Predictive values of choline and its metabolites TMA and TMAO for SVN In order to assess the potential predictive ability of choline and its metabolites TMA and TMAO in SVN, we calculated the receiver operating characteristic (ROC) curves (AUCs) (Fig 2 and Supplementary Table S3) . The traditional model contented the traditional risk factors of SVN, including age, body mass index (BMI), fasting blood glucose (FBG), one-hour postprandial blood glucose (1h-PBG), two-hour postprandial blood glucose (2h-PBG), and placental growth factor (PLGF). Inclusion of choline and its metabolites TMA and TMAO in the first trimester (CTT1) or the second trimester (CTT2) or the third trimester (CTT3) in the models markedly increased the AUCs for the traditional model. The AUC for SVN was increased from 0.646 to 0.763, 0.753, and 0.854. The AUC for SGA was increased from 0.692 to 0.871, 0.866, and 0.719. The AUC of LBW was increased from 0.625 to 0.756, 0.769, and 0.923. The AUC of PTB was increased from 0.603 to 0.804, 0.705, and 0.920. Choline and its metabolites TMA and TMAO and fetal development during pregnancy Early screening and intervention are key to ensuring fetal health and should be undertaken throughout pregnancy [8], so we analyzed the association between choline and its metabolites TMA and TMAO at all three pregnancy trimesters and subsequent fetal development indicators in the whole cohort and SVN group separately. Our prospective cohort study found that after adjusting for age, BMI, FBG, 1h-PBG, 2h-PBG, complex lipids and gestational week, TMAO levels in the first trimester were positively correlated with the BPD (β = 0.098, P = 0.003, q = 0.006), HC (β = 0.104, P = 0.002, q = 0.006), AC (β = 0.093, P = 0.005, q = 0.007), and FL (β = 0.081, P = 0.015, q = 0.016) of fetuses in 11-14 weeks in the total population ( Table 3) . In SVN, TMAO levels in the first trimester were positively correlated with the BPD of fetuses before delivery (β = 0.551, P = 0.002, q = 0.006). TMAO levels in the third trimester were positively correlated with BPD (β = 0.549, P = 0.013, q = 0.014) and AC (β = 0.500, P = 0.005, q = 0.011) of fetuses before delivery ( Table 4 ). But we found that TMA levels in the second trimester were negatively correlated with the BPD (β = -0.208, P = 0.033, q = 0.042), HC (β = -0.242, P = 0.012, q = 0.032), and AC (β = -0.199, P = 0.040, q = 0.042) of fetuses in 32-34 weeks ( Table 5 ), once again proposed the adverse effects of TMA on offspring development. Choline and its metabolites TMA and TMAO and postnatal offspring development More and more evidence supports that maternal metabolic changes during pregnancy not only affect fetal development but also play an important role in the development of offspring after birth, so we analyzed the association between choline and its metabolites TMA and TMAO and development in 6-month-old and 1-year-old infants. In the total population, choline levels in the third trimester were positively correlated with weight (β = 0.108, P = 0.002, q = 0.011) and TMAO levels in the third trimester were positively correlated with the weight (β = 0.099, P = 0.003, q = 0.006) and length (β = 0.107, P = 0.002, q = 0.006), while TMA levels in the third trimester were negatively correlated with the weight (β = -0.137, P = <0.001, q = <0.001), length (β = -0.124, P = <0.001, q = <0.001) and OFC (β = -0.098, P = 0.009, q = 0.009) of 6-month-old infants (Table 6) . TMAO levels in the third trimester were positively correlated with the MUAC (β = 0.095, P = 0.005, q = 0.026), while TMA levels in the third trimester were negatively correlated with the MUAC (β = -0.096, P = 0.005, q = 0.026) of 1-year-old infants. Also, we found the positive association between TMAO levels in the second trimester and the OFC (β = 0.089, P = 0.008, q = 0.041) of 1-year-old infants (Table 7) . We observed a positive association between TMAO and fetal BPD and OFC of 1-year-old infants, while a negative association between TMA and fetal BPD, which sparked our interest in their relationship with offspring brain development (Table 3, 4, 5) . Therefore, we explore the association between choline and its metabolites TMA and TMAO with the MDI and PDI, and we were surprised to find the positive association between TMAO levels in the second trimester and MDI (β = 0.084, P = 0.013, q = 0.014) of 1-year-old infants (Table 8) . Discussion In this prospective cohort study, we investigated for the first time the relationship between maternal choline and its metabolites TMA and TMAO and the risk of SVN and the development of fetus and early postnatal offspring. Compared with non-SVN mothers , SVN mothers had significantly lower concentrations of TMAO in the first and second trimester, while higher concentrations of TMA in the third trimester. We found that TMAO levels during pregnancy were negatively correlated with the risk of SVN, while TMA levels were positively correlated with the risk of LBW and PTB in SVN. Supporting findings included positive correlations between TMAO during pregnancy and birth weight and birth length, while negative correlations between TMA and birth weight and birth length. Moreover, during pregnancy, the TMAO levels was not only positively correlated with fetal development, but also with the development of postnatal offspring, while TMA was the opposite. TMAO and TMA are two important products of choline metabolism. At present, a large number of studies have focused on the importance of TMAO and cardiovascular disease, diabetes, obesity and other metabolic syndrome, and very limited studies have been reported as their levels during pregnancy. Our prospective cohort study found for the first time that TMAO levels were negatively correlated with SVN risk, after adjusting for confounding factors, TMAO levels in the first trimester were positively correlated with the BPD, HC, AC, and FL of fetuses in 11-14 weeks in the total population. In SVN, TMAO levels in the first trimester were positively correlated with the BPD of fetuses before delivery. TMAO levels in the third trimester were positively correlated with BPD and AC of fetuses before delivery. The placenta is the most direct organ connecting the mother and the fetus, possessing extensive endocrine and transportation functions, and the developmental and functional changes of the placenta can affect the development of the fetus and offspring after birth. Therefore, we introduced PLGF, the indicator for evaluating placental growth and development, and found a positive association between TMAO levels and PLGF in SVN (supplementary table S4) . The disruption of the homeostasis of the placental ER and the increase in ER stress are important reasons that affect placental development and lead to fetal dysplasia [9, 10]. TMAO is a permeable substance that plays a crucial role in adapting cells to osmotic and hydrostatic pressures. TMAO can play the role of a protein chaperone in stabilizing protein structure, stabilizing the structure of peptide regions, and thus protecting proteins from damage caused by hydration. TMAO can promote protein folding and reduce UPR, thereby reducing endoplasmic reticulum (ER) stress and reducing cell apoptosis [11-13]. Our previous study also demonstrated that TMAO can enhance the biological function of HTR-8/Svneo cells and promote the development of placenta and fetuses of mice with GDM [14]. TMA is the most important intermediate in choline and its metabolites TMA and TMAO. However, the majority of studies have focused on TMAO rather than TMA, thus the role and mechanism of TMA are still unclear. Only in the 1990s did Guest et al [15, 16] discovered that TMA could cause selective growth inhibition and developmental toxicity in progeny of mice, but subsequent studies have generally not focused on TMA and offspring development. We found that TMA levels in the second trimester were negatively correlated with the BPD and AC of fetuses in 32-34 weeks, once again proposed the adverse effects of TMA on offspring development. Similarly, we also introduced PLGF and found a negative association between TMA levels and PLGF in SVN ( supplementary table S4) . Some studies have found that as the concentration of TMA increases, ROS production increases. TMA induced oxidative stress can lead to DNA damage and compromise cell membrane integrity, leading to cell death [17, 18]. Secondly, the intracellular calcium mobilization induced by TMA in the endoplasmic reticulum (ER) calcium stores also contributes to ER stress [19-21]. The placenta is a tissue rich in mitochondria, which inevitably produces a large amount of reactive oxygen species (ROS). Oxidative stress can be generated upon disruption of the balance between ROS formation and detoxification, inducing inflammatory responses and damage the cellular system, leading to premature placental aging and degenerative changes, reducing placental function, and leading to abnormal pregnancy outcomes [22]. TMAO levels in the third trimester were positively correlated with birthweight and birth length, while TMA levels in the third trimester were negatively correlated with birthweight and birth length in all tested subjects. In SVN, TMAO levels in the first trimester and the third trimester were positively correlated with birthweight. Birthweight and birth length are the two most basic indicators for evaluating pregnancy outcomes, emphasizing once again the importance of choline and its metabolites TMA and TMAO in fetal development during pregnancy. Previous studies have showed that the physiologically relevant concentrations of TMAO can enhance blood-brain barrier (BBB) integrity and protected it from inflammatory insult, and long-term exposure to TMAO can protect murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. In contrast, TMA impaired BBB function and disrupted tight junction integrity [23]. Similarly, our observation of a positive association between TMAO and fetal BPD, while a negative association between TMA and fetal BPD, sparked our interest in their relationship with offspring brain development. It is surprised to find the positive association between TMAO levels in the second trimester and the OFC and the MDI of 1-year-old infants. It suggests that the level of TMAO during pregnancy is related to fetal brain development and the effect may even persist after birth. In the sensitivity analysis, we used multiple imputation and logistic regression to recalculate the RRs after adjusting for BMI, age, gestational week, blood glucose and complex lipids, we found no significant changes in the results, which means our findings are stable and reliable ( supplementary Table S5 and S6 ). One limitation of the present study is that our cohort study only includes participants in one region, and the applicability of our study results in other regions needs further verification. Secondly, due to the unreliable blood pressure measurements of pregnant women in the cohort, we cannot adjust for this confounding factor. Due to the lack of pre-pregnancy BMI data, the confounding factor of weight gain during pregnancy cannot be adjusted. Multi-center and multi-racial studies are needed to further validate our results, and we hope that TMA and TMAO can become biomarkers for predicting SVN in the future. In summary, our report proposes for the first time that choline and its metabolites TMA and TMAO during pregnancy can not only predict fetal development during pregnancy, but can even extend to one year after birth. Especially the opposite association between TMAO and TMA in offspring development may be key to ensuring healthy fetal development, which suggests that it is more necessary to focus on the transformation of metabolites TMA and TMAO while paying attention to the adequacy of choline during pregnancy in women. Declarations Acknowledgments Author Contributions X.L. and K.L. contributed to the statistical analysis, interpretation of data, and manuscript writing. Z.W., X.H., Y.X. and C.C. contributed to sample measurement and data management. All authors contributed to results interpretation. P.N.B. established CLIMB cohort. X.X., P.N.B. and R.S. reviewed and edited manuscript. X.X., H.Z. and H.Q. contributed to the conception, design of the study, and study supervision. X.X. and H.Z. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conflict of Interest statement All authors read and approved the final manuscript. The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (82071734, 82271715, and 82301929) Data availability statement The data for our sample testing is available, since the individual patient data contain confidential information, it can be supplied only in an anonymised format to suitably qualified researchers who can make appropriate institutional commitments relating to data security and confidentiality. The data of participants in CLIMB cohort can be applied for from the creator PNB according to reasonable requirements. References Ashorn P, Ashorn U, Muthiani Y, Aboubaker S, Askari S, Bahl R, Black RE, Dalmiya N, Duggan CP, Hofmeyr GJ, Kennedy SH, Klein N, Lawn JE, Shiffman J, Simon J, Temmerman M, UNICEF–WHO Low Birthweight Estimates Group. Small vulnerable newborns-big potential for impact. Lancet. 2023, 401(10389):1692-1706. 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Contractile effect of trimethylamine and trimethylamine-n-oxide on isolated human umbilical arteries. J Obstet Gynaecol Res. 2023, 49: 1736-1742. Chhibber-Goel J, Gaur A, Singhal V, Parakh N, Bhargava B, Sharma A. The complex metabolism of trimethylamine in humans: endogenous and exogenous sources. Expert Rev Mol Med. 2016, 18: e8. Joo EH, Kim YR, Kim N, Jung JE, Han SH, Cho HY. Effect of Endogenic and Exogenic Oxidative Stress Triggers on Adverse Pregnancy Outcomes: Preeclampsia, Fetal Growth Restriction, Gestational Diabetes Mellitus and Preterm Birth. Int J Mol Sci. 2021, 22(18):10122. Hoyles L, Pontifex MG, Rodriguez-Ramiro I, Anis-Alavi MA, Jelane KS, Snelling T, Solito E, Fonseca S, Carvalho AL, Carding SR, Müller M, Glen RC, Vauzour D, McArthur S. Regulation of blood-brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide. Microbiome. 202, 9(1):235. Huang S, Mo T, Norris T, Sun S, Zhang T, Han T, Rowan A, Xia Y, Zhang H, Qi H, Baker PN. The CLIMB (Complex Lipids In Mothers and Babies) study: protocol for a multicentre, three-group, parallel randomised controlled trial to investigate the effect of supplementation of complex lipids in pregnancy, on maternal ganglioside status and subsequent cognitive outcomes in the offspring. BMJ Open. 2017, 7(10):e016637. Chen YT, Zhang T, Chen C, Xia YY, Han TL, Chen XY, He XL, Xu G, Zou Z, Qi HB, Zhang H, Albert BB, Colombo J, Baker PN. Associations of early pregnancy BMI with adverse pregnancy outcomes and infant neurocognitive development. Sci Rep. 2021 Feb 15;11(1):3793. Tables Tables 1 to 8 are available in the Supplementary Files section Additional Declarations There is NO Competing Interest. Supplementary Files nreditorialpolicychecklist.pdf Article File - Editorial Policy Checklist SupplementaryMaterials.pdf Supplementary Materials nrreportingsummary.pdf Article File - Reporting Summary Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3970231","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275678175,"identity":"a33b0bfa-1ebf-4303-99f4-701f88415e3c","order_by":0,"name":"Xiaoqiu Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACAwYeIFkhwcPG3tj48APxWs7YyPHxHG42liBaC2NbmrGcRHqbAA8xWsz5zx78zMN2OLFN8mEbgwSDnZxuAwEtljPykqV5eIBapBPbHhQwJBubHSDksBs8BtI5EmAt7QYSDAcStxHUcv6M8e8cA5DDDrZJ8BCl5UCOmXROQpoxmwQjkVosZ+SYWf85YCPHxpMIDGQDIvxizn/G+ObMfxI88u3HHz78UGEnR1ALujtJUz4KRsEoGAWjAAcAAJl5P9D9rI5/AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4568-8772","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoqiu","middleName":"","lastName":"Xiao","suffix":""},{"id":275678176,"identity":"c2f0893e-7cb2-4d78-ac31-01995c66e2e4","order_by":1,"name":"Xiaojing Lin","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Lin","suffix":""},{"id":275678177,"identity":"e749e5ab-a473-4d4b-83d0-7862c49847df","order_by":2,"name":"Kemeng Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kemeng","middleName":"","lastName":"Li","suffix":""},{"id":275678178,"identity":"6af258fe-5b96-44bc-ab9c-61130fac6af4","order_by":3,"name":"Zhuyun Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuyun","middleName":"","lastName":"Wang","suffix":""},{"id":275678179,"identity":"c14d6802-f326-4d62-aa2a-0e934d125fc0","order_by":4,"name":"Chang Chen","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Chen","suffix":""},{"id":275678180,"identity":"ae944e35-16cd-4b68-a1ec-673d1c72afac","order_by":5,"name":"Xiaoling He","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"He","suffix":""},{"id":275678181,"identity":"c833e7a1-ed0f-4437-ae3e-3edb8343824f","order_by":6,"name":"Hongbo Qi","email":"","orcid":"","institution":"Women and Children's Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Qi","suffix":""},{"id":275678182,"identity":"9ed46f74-0ec2-453d-8d20-0ae976210032","order_by":7,"name":"Philip Baker","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Baker","suffix":""},{"id":275678183,"identity":"be166203-cbf6-4f09-9794-58556c3f3a0b","order_by":8,"name":"Richard Saffery","email":"","orcid":"https://orcid.org/0000-0002-9510-4181","institution":"Murdoch Childrens Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Saffery","suffix":""},{"id":275678184,"identity":"3a95444a-09ab-4e50-a0c3-f40e5d6e32f1","order_by":9,"name":"Hua Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-02-19 14:50:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3970231/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3970231/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52036742,"identity":"abb50555-a946-4831-8873-3884927a653d","added_by":"auto","created_at":"2024-03-05 17:10:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative risks and 95% confidence interval of SVN incidence by choline, TMA, or TMAO.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) RR and 95% CI of SVN incidence by TMAO in the first trimester.\u003c/p\u003e\n\u003cp\u003eB) RR and 95% CI of SVN incidence by TMAO in the third trimester.\u003c/p\u003e\n\u003cp\u003eC) RR and 95% CI of SGA incidence by TMAO in the first trimester.\u003c/p\u003e\n\u003cp\u003eD) RR and 95% CI of LBW incidence by TMAO in the second trimester.\u003c/p\u003e\n\u003cp\u003eE) RR and 95% CI of LBW incidence by TMA in the third trimester.\u003c/p\u003e\n\u003cp\u003eF) RR and 95% CI of LBW incidence by TMAO in the third trimester.\u003c/p\u003e\n\u003cp\u003eG) RR and 95% CI of PTB incidence by choline in the first trimester.\u003c/p\u003e\n\u003cp\u003eH) RR and 95% CI of PTB incidence by TMA in the third trimester.\u003c/p\u003e\n\u003cp\u003eThe dashed line represented that the RR values of quartile 1 that equal to 1. Triangles, circles, and squares represented RR values and 95% CI.\u003c/p\u003e\n\u003cp\u003eMultivariate model was adjusted for body mass index, age, blood glucose, complex lipids and gestational week.\u003c/p\u003e\n\u003cp\u003eAbbreviations: TMA, trimethylamine; TMAO, trimethylamine-N-oxide; SVN, small vulnerable newborn; SGA, small for gestational age; LBW, low birthweight; PTB, preterm birth.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05: \u003csup\u003e⁎\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01: \u003csup\u003e⁎⁎\u003c/sup\u003e, \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001: \u003csup\u003e⁎⁎⁎\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/648790e36d3da4fc8766591a.png"},{"id":52037043,"identity":"29d50ffa-a558-4dfd-8e61-6e32337e86af","added_by":"auto","created_at":"2024-03-05 17:18:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":862046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReceiver operating characteristic (ROC) curves for SVN.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) ROC curve for SVN\u003c/p\u003e\n\u003cp\u003eB) ROC curve for SGA\u003c/p\u003e\n\u003cp\u003eC) ROC curve for PTB\u003c/p\u003e\n\u003cp\u003eD) ROC curve for LBW\u003c/p\u003e\n\u003cp\u003eAbbreviations: SVN, small vulnerable newborn; SGA, small for gestational age; LBW, low birthweight; PTB, preterm birth. CTT1, choline, trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) in the first trimester; CTT2, choline, trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) in the second trimester; CTT3, choline, trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) in the third trimester.\u003c/p\u003e\n\u003cp\u003eTraditional risk factors included age, BMI, fasting blood glucose (FBG), one-hour postprandial blood glucose (1h-PBG), two-hour postprandial blood glucose (2h-PBG) and placental growth factor (PLGF).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/2ed80cdba923d1f35ba2e454.png"},{"id":53035139,"identity":"aeafd733-0d3f-4964-a90c-6cca9ab113cf","added_by":"auto","created_at":"2024-03-19 21:01:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1289548,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/0680d73c-c557-445c-993e-a686fa1fa6fd.pdf"},{"id":52036745,"identity":"c0523754-53e3-4b7d-8e3e-2e0b86c4de03","added_by":"auto","created_at":"2024-03-05 17:10:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1682334,"visible":true,"origin":"","legend":"Article File - Editorial Policy Checklist","description":"","filename":"nreditorialpolicychecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/cf61c298644d657ee6159bb9.pdf"},{"id":52036747,"identity":"e324dd03-e4c8-4d3d-977f-d64c946399da","added_by":"auto","created_at":"2024-03-05 17:10:49","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":211409,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/d08f4cea91afdff353e7acb1.pdf"},{"id":52037044,"identity":"5010198f-cf07-4278-a4e9-b3ac2b2c39be","added_by":"auto","created_at":"2024-03-05 17:18:49","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1666132,"visible":true,"origin":"","legend":"Article File - Reporting Summary","description":"","filename":"nrreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/6144ff7db4c90054bc733462.pdf"},{"id":52036743,"identity":"ca820b46-42ce-432d-9884-24a119630038","added_by":"auto","created_at":"2024-03-05 17:10:49","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":100159,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3970231/v1/4cd6623015aaae025ce1ea62.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Choline and its Metabolites Trimethylamine and Trimethylamine N-oxide Potentially Predict the Risk of Small Vulnerable Newborn and the Development of 1-Year Infants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSmall for gestational age (SGA), low birthweight (LBW), and preterm birth (PTB) are major intractable adverse birth outcomes representing considerable mortality and morbidity internationally. Recently, these pregnancy outcomes have been collectively grouped as small vulnerable newborns (SVN) [1]. In 2020, 55.3% of the 2.4 million global neonatal deaths and 1.9 million stillbirths were attributable to SVN [2]. Despite this, progress towards understanding the etiology of SVN remains less than optimal and approaches to prevent SVN with appropriate interventions remain limited.\u003c/p\u003e\n\u003cp\u003eAdequate choline levels during pregnancy are important for healthy fetal development, anything requiring methyl donors or acetylcholine metabolism needs choline, particularly membrane biosynthesis, neurite myelination, cell division, tissue expansion and lipid transport [4], but supplementation does not directly alter choline concentration in peripheral blood of pregnant women and umbilical cord blood [5]. Choline supplementation during pregnancy improve offspring cognition, neurodevelopment, and placental functioning, and to protect against neural and metabolic insults [6]. The role of choline in promoting offspring development may thus be indirect, through its metabolites. Choline is metabolized by the intestinal bacteria to form trimethylamine (TMA), which is then rapidly oxidized to and trimethylamine N-oxide (TMAO) by flavin-containing monooxygenase 3 (FMO3) in the liver [7]. In order to assess the potential link between these downstream metabolites and offspring health, we determined the levels of choline, TMA, and TMAO in the plasma of 1231 pregnant women from the Complex Lipids in Mothers and Babies (CLIMB) cohort study during the first (11-14w), second (22-28w), and third (32-34w) trimesters to determine the role of choline metabolites on SVN risk and specific aspects of offspring development, including the developmental indicators of the fetus during pregnancy [(biparietal diameter (BPD),\u0026nbsp;head circumference (HC),\u0026nbsp;abdominal circumference (AC)\u0026nbsp;and\u0026nbsp;femur length (FL)], the general infant development indicators [(weight, length, occipital frontal circumference (OFC),\u0026nbsp;skin fold thickness of triceps (TSF); skin fold thickness of subscapular(SSF) and\u0026nbsp;mid upper arm circumference (MUAC)] at 6 months and 1 year after birth, as well as cognitive development[mental development index (MDI) and psychomotor development index (PDI)] of 1-year-old infants.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne thousand five huanderds pregnant women were recruited from the CLIMB study, the pregnant women were randomized into three groups: control milk group, complex milk lipid-enhanced (CML-E) group and reference group (Chinese Clinical Trial Register number: ChiCTR-IOR-16007700). The participants were enrolled at prenatal clinics between 11 and 14 weeks gestation between September 2015 and June 2017, at the First Affiliated Hospital of Chongqing Medical University and Chongqing Health Center for Women and Children [24]. Women between 20–40 years of age and had a singleton pregnancy were included in the study, women who had a previous pregnancy with complications which resulted in delivery before 32 weeks were excluded from the study. Meanwhile, women who lost to follow-up (n=186) or miscarried (n=12), whose pregnancies were terminated (n=29), whose information were incomplete (n=42) were excluded from the analysis. A total of 1231 pregnant women were ultimately included in this study \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003esupplementary Fig. S1).\u003c/strong\u003e Low birthweight (LBW) infants were defined as those weighing \u0026lt; 2500 g, infants born Small for gestational age (SGA) as those SGA were indicated by birth weight less than the 10th percentile for the gestational age by sex, respectively, and preterm birth (PTB) are those that occur at less than 37 weeks' gestational age [25]. This study was conducted in accordance with the principles in the Declaration of Helsinki. Ethical approval was granted by the Ethics committee of the Chongqing Medical University (No. 2014034), and written informed consent was obtained from all participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman\u003c/strong\u003e\u003cstrong\u003esubjects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaternal\u0026nbsp;baseline\u0026nbsp;characteristics\u0026nbsp;including\u0026nbsp;maternal age, smoking, alcohol consumption,\u0026nbsp;education background,\u0026nbsp;body mass index (BMI)\u0026nbsp;and blood glucose were\u0026nbsp;presented in\u003cstrong\u003esupplementary\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Overnight fasting maternal blood samples\u0026nbsp;(11–14\u0026nbsp;weeks,\u0026nbsp;22–28\u0026nbsp;weeks, and 32-34 weeks\u0026nbsp;of pregnancy)\u0026nbsp;were collected\u0026nbsp;and\u0026nbsp;immediately stored at -80°C until analysis. The measurement of samples was performed by liquid chromatography-mass spectrometry (LC-MS/MS), with testing personnel blinded to the clinical information and pregnancy outcome of the patients. Randomly test plasma samples from 100 pregnant women in each batch during the same period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of plasma choline, TMA, and TMAO concentrations by liquid chromatography-mass spectrometry (LC-MS/MS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdded 50 μL of 1% formic acid aqueous solution into 50 μL of the plasma, vortexed for 1 min, then added 300 μL of methanol solution containing deuterated internal standard (choline-d9, concentration: 1 μg/mL) into it, further vortexed for 5 min and then centrifugation at 4°C (12700 rpm, 10 min). 70 μL of the supernatant was transferred to an auto-sampler vial for detection.\u003c/p\u003e\n\u003cp\u003eAfter sample pretreatment, the supernatants (5 µL) were injected onto a silica column (InfinityLab Poroshell 120 HILIC-Z, 2.1 × 100 mm, 2.7 µm) and analyzed at a flow rate of 0.3 mL/min. Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were mixed to generated a discontinuous gradient to resolve the analytes at different ratios starting from 10% A linearly to 50% A from 0 min to 5 min, then linearly to 10% A from 5min to 5.6 min, held for 5.4 min and then stopped.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMass spectrometry conditions: Ion mode: AJS ESI, positive ion mode; Drying gas temperature: 120°C; drying gas flow rate: 13 L/min; Nebulizer pressure: 30 psi; sheath gas temperature: 300°C; sheath gas flow rate: 12 L/min; capillary voltage (+): 2500 V. The parameters for the ion monitoring were as follows: choline (104.11 m/z, 58.2 m/z, 45V, 2.63 min), TMA (60.08 m/z, 45.1 m/z, 10V, 2.38 min), TMAO (152.11 m/z, 76 m/z, 9V, 2.42 min), d9-choline (113.17 m/z, 45.1 m/z, 45V, 2.63 min).\u003c/p\u003e\n\u003cp\u003eAgilent 1290 Infinity II High-Speed Pump (G7120A) with an Agilent 6495 Triple Quadrupole LC/MS System (G6495C) was used to perform LC-MS/MS. Agilent MassHunter LC/MS was used to collect data and Agilent MassHunter was used to conduct qualitative analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed by SPSS 25.0, R 4.3.0 and GraphPad Prism 8.3.0; A two-sided test were used, where applicable. If the data showed a Gaussian distribution, Student's t-test was used to compare two groups, otherwise, the nonparametric was adopted. Poisson regression was used to estimate the relative risk between choline and its metabolites TMA and TMAO and SVN, if the model was over-dispersion, negative Binomial regression analysis was used for estimation. The areas under the receiver operating characteristic (ROC) curves (AUC) were calculated to check whether the inclusion of choline and its metabolites TMA and TMAO was able to would increase the AUCs from a model which only included the traditional risk factors. Multiple linear regression was used to analyze the association of the measurement data. Multivariate mdel was adjusted for body mass index, age, blood glucose, complex lipids and gestational week. False discovery rates (FDR) were used to account for multiple comparisons, and the parameter desired FDR was set to the 0.05. The set of p-values produced across choline, TMA, or TMAO and serum glucose levels were FDR corrected with the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Multiple imputation and logistic regression were used to reanalyze the main results to test the sensitivity of our results.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacteristics of the study participants\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe characteristics of the CLIMB study participants are shown in\u0026nbsp;\u003cstrong\u003esupplementary Table S1.\u003c/strong\u003e Of 1231 pregnancies investigated in the current study, a total of 143 resulted in SVN births (comprising 92 SGA, 31 LBW and 53 PTB, of which 10 were both SGA and LBW). There were generally no differences between SVN and non SVN pregnancies in terms of gestation, maternal age, education, BMI smoking, or OGTT results, despite SGA pregnancies showing slightly elevated 1 and 2 hour glucose levels. Mothers of SVN had significantly lower concentrations of TMAO in the first and second trimester, but had higher concentrations of TMA in the third trimester, as compared to mothers of infant who were not SVN (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCholine and its metabolites TMA and TMAO and the risk of SVN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCholine, TMA, and TMAO levels were grouped according to quartiles into Q (quartile) 1, Q2, Q3, and Q4, with the Q4 group as reference. The relative risks (RRs) estimated by Poisson regression indicated that the lower TMAO of the first trimester (Q3: adjusted RR = 1.897, 95% CI: 1.127-3.193; Q2: adjusted RR = 1.838, 95% CI: 1.091-3.097) and the third trimester (Q1: adjusted RR = 1.797, 95% CI: 1.092-2.956) were significantly associated with a higher risk of SVN\u0026nbsp;\u003cstrong\u003e(Fig. 1\u003c/strong\u003e). Because SVN includes SGA, LBW and PTB, we further calculated the RRs between choline and its metabolites TMA and TMAO and SGA or LBW or PTB separately. We found that lower TMA during pregnancy were associated with a lower risk of these pregnancy outcomes, which were opposite to TMAO\u003cstrong\u003e\u0026nbsp;(supplementary table S2)\u003c/strong\u003e. Birthweight is a measure of vulnerability [1, 2], we analyzed the association between choline and its metabolites TMA and TMAO and birthweight and birth length of newborn in the total population and SVN, respectively, and we found opposite effects of TMA and TMAO as before. In the total population, TMAO levels in the third trimester were positively correlated with birthweight (β = 0.066, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.030, \u003cem\u003eq\u003c/em\u003e = 0.032) and birth length (β = 0.071, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.023, \u003cem\u003eq\u0026nbsp;\u003c/em\u003e= 0.032), while TMA levels in the third trimester were negatively correlated with birthweight (β = -0.076, \u003cem\u003eP\u003c/em\u003e = 0.016, \u003cem\u003eq\u003c/em\u003e = 0.017) and birth length (β = -0.092, \u003cem\u003eP\u003c/em\u003e = 0.004, \u003cem\u003eq\u003c/em\u003e = 0.008). In SVN, TMAO levels in the first trimester (β = 0.275,\u003cem\u003e\u0026nbsp;P\u003c/em\u003e = 0.002,\u003cem\u003e\u0026nbsp;q\u003c/em\u003e = 0.002) and the third trimester (β = 0.223, \u003cem\u003eP\u003c/em\u003e = 0.024, \u003cem\u003eq\u003c/em\u003e = 0.025) were positively correlated with birthweight\u0026nbsp;\u003cstrong\u003e(Table 2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePredictive values of choline and its metabolites TMA and TMAO for SVN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to assess the potential predictive ability of choline and its metabolites TMA and TMAO in SVN, we calculated the receiver operating characteristic (ROC) curves (AUCs)\u0026nbsp;\u003cstrong\u003e(Fig 2 and Supplementary Table S3)\u003c/strong\u003e. The traditional model contented the traditional risk factors of SVN, including age, body mass index (BMI), fasting blood glucose (FBG), one-hour postprandial blood glucose (1h-PBG), two-hour postprandial blood glucose (2h-PBG), and placental growth factor (PLGF). Inclusion of choline and its metabolites TMA and TMAO in the first trimester (CTT1) or the second trimester (CTT2) or the third trimester (CTT3) in the models markedly increased the AUCs for the traditional model. The AUC for SVN was increased from 0.646 to 0.763, 0.753, and 0.854. The AUC for SGA was increased from 0.692 to 0.871, 0.866, and 0.719. The AUC of LBW was increased from 0.625 to 0.756, 0.769, and 0.923. The AUC of PTB was increased from 0.603 to 0.804, 0.705, and 0.920.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCholine and its metabolites TMA and TMAO and fetal development during pregnancy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEarly screening and intervention are key to ensuring fetal health and should be undertaken throughout pregnancy [8], so we analyzed the association between choline and its metabolites TMA and TMAO at all three pregnancy trimesters and subsequent fetal development indicators in the whole cohort and SVN group separately. Our prospective cohort study found that after adjusting for age, BMI, FBG, 1h-PBG, 2h-PBG, complex lipids and gestational week, TMAO levels in the first trimester were positively correlated with the BPD (β = 0.098, \u003cem\u003eP\u003c/em\u003e = 0.003, \u003cem\u003eq\u003c/em\u003e = 0.006), HC (β = 0.104, \u003cem\u003eP\u003c/em\u003e = 0.002, \u003cem\u003eq\u003c/em\u003e = 0.006), AC (β = 0.093, \u003cem\u003eP\u003c/em\u003e = 0.005, \u003cem\u003eq\u003c/em\u003e = 0.007), and FL (β = 0.081, \u003cem\u003eP\u003c/em\u003e = 0.015, \u003cem\u003eq\u003c/em\u003e = 0.016) of fetuses in 11-14 weeks in the total population (\u003cstrong\u003eTable 3)\u003c/strong\u003e. In SVN, TMAO levels in the first trimester were positively correlated with the BPD of fetuses before delivery (β = 0.551, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.002, \u003cem\u003eq\u0026nbsp;\u003c/em\u003e= 0.006). TMAO levels in the third trimester were positively correlated with BPD (β = 0.549, \u003cem\u003eP\u003c/em\u003e = 0.013, \u003cem\u003eq\u003c/em\u003e = 0.014) and AC (β = 0.500, \u003cem\u003eP\u003c/em\u003e = 0.005, \u003cem\u003eq\u003c/em\u003e = 0.011) of fetuses before delivery (\u003cstrong\u003eTable 4\u003c/strong\u003e). But we found that TMA levels in the second trimester were negatively correlated with the BPD (β = -0.208, \u003cem\u003eP\u003c/em\u003e = 0.033,\u003cem\u003e\u0026nbsp;q\u0026nbsp;\u003c/em\u003e= 0.042), HC (β = -0.242, \u003cem\u003eP\u003c/em\u003e = 0.012, \u003cem\u003eq\u0026nbsp;\u003c/em\u003e= 0.032), and AC (β = -0.199, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.040, \u003cem\u003eq\u0026nbsp;\u003c/em\u003e= 0.042) of fetuses in 32-34 weeks (\u003cstrong\u003eTable 5\u003c/strong\u003e), once again proposed the adverse effects of TMA on offspring development. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCholine and its metabolites TMA and TMAO and postnatal offspring development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;More and more evidence supports that maternal metabolic changes during pregnancy not only affect fetal development but also play an important role in the development of offspring after birth, so we analyzed the association between choline and its metabolites TMA and TMAO and development in 6-month-old and 1-year-old infants. In the total population, choline levels in the third trimester were positively correlated with weight (β = 0.108, \u003cem\u003eP\u003c/em\u003e = 0.002, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.011) and TMAO levels in the third trimester were positively correlated with the weight (β = 0.099, \u003cem\u003eP\u003c/em\u003e = 0.003, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.006) and length (β = 0.107, \u003cem\u003eP\u003c/em\u003e = 0.002, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.006), while TMA levels in the third trimester were negatively correlated with the weight (β = -0.137, \u003cem\u003eP\u003c/em\u003e = \u0026lt;0.001, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e\u0026lt;0.001), length (β = -0.124, \u003cem\u003eP\u003c/em\u003e = \u0026lt;0.001, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e\u0026lt;0.001) and OFC (β = -0.098, \u003cem\u003eP\u003c/em\u003e = 0.009, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.009) of 6-month-old infants\u0026nbsp;\u003cstrong\u003e(Table 6)\u003c/strong\u003e. TMAO levels in the third trimester were positively correlated with the MUAC (β = 0.095, \u003cem\u003eP\u003c/em\u003e = 0.005, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.026), while TMA levels in the third trimester were negatively correlated with the MUAC (β = -0.096, \u003cem\u003eP\u003c/em\u003e = 0.005, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.026) of 1-year-old infants. Also, we found the positive association between TMAO levels in the second trimester and the OFC (β = 0.089, \u003cem\u003eP\u003c/em\u003e = 0.008, \u003cem\u003eq =\u0026nbsp;\u003c/em\u003e0.041) of 1-year-old infants\u0026nbsp;\u003cstrong\u003e(Table 7)\u003c/strong\u003e. We observed a positive association between TMAO and fetal BPD and OFC of 1-year-old infants, while a negative association between TMA and fetal BPD, which sparked our interest in their relationship with offspring brain development\u0026nbsp;\u003cstrong\u003e(Table 3, 4, 5)\u003c/strong\u003e. \u0026nbsp;Therefore, we explore the association between choline and its metabolites TMA and TMAO with the MDI and PDI, and we were surprised to find the positive association between TMAO levels in the second trimester and MDI (β\u0026nbsp;= 0.084, P = 0.013, q = 0.014) of 1-year-old infants\u0026nbsp;\u003cstrong\u003e(Table 8)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this prospective cohort study, we investigated for the first time the relationship between maternal choline and its metabolites TMA and TMAO and the risk of SVN and the development of fetus and early postnatal offspring. Compared with non-SVN mothers , SVN mothers had significantly lower concentrations of TMAO in the first and second trimester, while higher concentrations of TMA in the third trimester. We found that TMAO levels during pregnancy were negatively correlated with the risk of SVN, while TMA levels were positively correlated with the risk of LBW and PTB in SVN. Supporting findings included positive correlations between TMAO during pregnancy and birth weight and birth length, while negative correlations between TMA and birth weight and birth length. Moreover, during pregnancy, the TMAO levels was not only positively correlated with fetal development, but also with the development of postnatal offspring, while TMA was the opposite.\u003c/p\u003e\n\u003cp\u003eTMAO and TMA are two important products of choline metabolism. At present, a large number of studies have focused on the importance of TMAO and cardiovascular disease, diabetes, obesity and other metabolic syndrome, and very limited studies have been reported as their levels during pregnancy. Our prospective cohort study found for the first time that TMAO levels were negatively correlated with SVN risk, after adjusting for confounding factors, TMAO levels in the first trimester were positively correlated with the BPD, HC, AC, and FL of fetuses in 11-14 weeks in the total population. In SVN, TMAO levels in the first trimester were positively correlated with the BPD of fetuses before delivery. TMAO levels in the third trimester were positively correlated with BPD and AC of fetuses before delivery. The placenta is the most direct organ connecting the mother and the fetus, possessing extensive endocrine and transportation functions, and the developmental and functional changes of the placenta can affect the development of the fetus and offspring after birth. Therefore, we introduced PLGF, the indicator for evaluating placental growth and development, and found a positive association between TMAO levels and PLGF in SVN\u0026nbsp;\u003cstrong\u003e(supplementary table S4)\u003c/strong\u003e. The disruption of the homeostasis of the placental ER and the increase in ER stress are important reasons that affect placental development and lead to fetal dysplasia [9, 10]. TMAO is a permeable substance that plays a crucial role in adapting cells to osmotic and hydrostatic pressures. TMAO can play the role of a protein chaperone in stabilizing protein structure, stabilizing the structure of peptide regions, and thus protecting proteins from damage caused by hydration. TMAO can promote protein folding and reduce UPR, thereby reducing endoplasmic reticulum (ER) stress and reducing cell apoptosis [11-13]. Our previous study also demonstrated that TMAO can enhance the biological function of HTR-8/Svneo cells and promote the development of placenta and fetuses of mice with GDM [14].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTMA is the most important intermediate in choline and its metabolites TMA and TMAO. However, the majority of studies have focused on TMAO rather than TMA, thus the role and mechanism of TMA are still unclear. Only in the 1990s did Guest et al [15, 16] discovered that TMA could cause selective growth inhibition and developmental toxicity in progeny of mice, but subsequent studies have generally not focused on TMA and offspring development. We found that TMA levels in the second trimester were negatively correlated with the BPD and AC of fetuses in 32-34 weeks, once again proposed the adverse effects of TMA on offspring development. Similarly, we also introduced PLGF and found a negative association between TMA levels and PLGF in SVN (\u003cstrong\u003esupplementary table S4)\u003c/strong\u003e. Some studies have found that as the concentration of TMA increases, ROS production increases. TMA induced oxidative stress can lead to DNA damage and compromise cell membrane integrity, leading to cell death [17, 18]. Secondly, the intracellular calcium mobilization induced by TMA in the endoplasmic reticulum (ER) calcium stores also contributes to ER stress [19-21]. The placenta is a tissue rich in mitochondria, which inevitably produces a large amount of reactive oxygen species (ROS). Oxidative stress can be generated upon disruption of the balance between ROS formation and detoxification, inducing inflammatory responses and damage the cellular system, leading to premature placental aging and degenerative changes, reducing placental function, and leading to abnormal pregnancy outcomes [22]. TMAO levels in the third trimester were positively correlated with birthweight and birth length, while TMA levels in the third trimester were negatively correlated with birthweight and birth length in all tested subjects. In SVN, TMAO levels in the first trimester and the third trimester were positively correlated with birthweight. Birthweight and birth length are the two most basic indicators for evaluating pregnancy outcomes, emphasizing once again the importance of choline and its metabolites TMA and TMAO in fetal development during pregnancy.\u003c/p\u003e\n\u003cp\u003ePrevious studies have showed that the physiologically relevant concentrations of TMAO can enhance blood-brain barrier (BBB) integrity and protected it from inflammatory insult, and long-term exposure to TMAO can protect murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. In contrast, TMA impaired BBB function and disrupted tight junction integrity [23]. Similarly, our observation of a positive association between TMAO and fetal BPD, while a negative association between TMA and fetal BPD, sparked our interest in their relationship with offspring brain development. It is surprised to find the positive association between TMAO levels in the second trimester and the OFC and the MDI of 1-year-old infants. It suggests that the level of TMAO during pregnancy is related to fetal brain development and the effect may even persist after birth.\u003c/p\u003e\n\u003cp\u003eIn the sensitivity analysis, we used\u0026nbsp;multiple imputation and logistic regression to recalculate the RRs after adjusting for BMI, age, gestational week, blood glucose and complex lipids, we found no significant changes in the results, which means our findings are stable and reliable (\u003cstrong\u003esupplementary Table S5 and S6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eOne limitation of the present study is that our cohort study only includes participants in one region, and the applicability of our study results in other regions needs further verification. Secondly, due to the unreliable blood pressure measurements of pregnant women in the cohort, we cannot adjust for this confounding factor. Due to the lack of pre-pregnancy BMI data, the confounding factor of weight gain during pregnancy cannot be adjusted. Multi-center and multi-racial studies are needed to further validate our results, and we hope that TMA and TMAO can become biomarkers for predicting SVN in the future.\u003c/p\u003e\n\u003cp\u003eIn summary, our report proposes for the first time that choline and its metabolites TMA and TMAO during pregnancy can not only predict fetal development during pregnancy, but can even extend to one year after birth. Especially the opposite association between TMAO and TMA in offspring development may be key to ensuring healthy fetal development, which suggests that it is more necessary to focus on the transformation of metabolites TMA and TMAO while paying attention to the adequacy of choline during pregnancy in women.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.L. and K.L. contributed to the statistical analysis, interpretation of data, and manuscript writing. Z.W., X.H., Y.X. and C.C. contributed to sample measurement and data management. All authors contributed to results interpretation. P.N.B. established CLIMB cohort. X.X., P.N.B. and R.S. reviewed and edited manuscript. X.X., H.Z. and H.Q. contributed to the conception, design of the study, and study supervision. X.X. and H.Z. are the guarantors of this work and, as such, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript. The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82071734, 82271715, and 82301929)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u003c/strong\u003e \u003cstrong\u003eavailability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data for our sample testing is available, since the individual patient data contain confidential information, it can be supplied only in an anonymised format to suitably qualified researchers who can make appropriate institutional commitments relating to data security and confidentiality. The data of participants in CLIMB cohort can be applied for from the creator PNB according to reasonable requirements.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAshorn P, Ashorn U, Muthiani Y, Aboubaker S, Askari S, Bahl R, Black RE, Dalmiya N, Duggan CP, Hofmeyr GJ, Kennedy SH, Klein N, Lawn JE, Shiffman J, Simon J, Temmerman M, UNICEF\u0026ndash;WHO Low Birthweight Estimates Group. Small vulnerable newborns-big potential for impact. Lancet. 2023, 401(10389):1692-1706.\u003c/li\u003e\n\u003cli\u003eLawn JE, Ohuma EO, Bradley E, Idueta LS, Hazel E, Okwaraji YB, Erchick DJ, Yargawa J, Katz J, Lee ACC, Diaz M, Salasibew M, Requejo J, Hayashi C, Moller AB, Borghi E, Black RE, Blencowe H, Lancet Small Vulnerable Newborn Steering Committee, WHO/UNICEF Preterm Birth Estimates Group, National Vulnerable Newborn Measurement Group, Subnational Vulnerable Newborn Measurement Group. 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Choline: Exploring the Growing Science on Its Benefits for Moms and Babies. Nutrients. 2019 Aug 7;11(8):1823. doi: 10.3390/nu11081823.[7] Subramaniam S, Fletcher C. Trimethylamine N-oxide: breathe new life. Br J Pharmacol. 2018, 175:1344\u0026ndash;1353.\u003c/li\u003e\n\u003cli\u003eSmith GC. Researching new methods of screening for adverse pregnancy outcome: lessons from pre-eclampsia. PLoS Med. 2012;9(7):e1001274.\u003c/li\u003e\n\u003cli\u003eYung HW, Hemberger M, Watson ED, Senner CE, Jones CP, Kaufman RJ, Charnock-Jones DS, Burton GJ. Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction. J Pathol. 2012, 228(4):554-64.\u003c/li\u003e\n\u003cli\u003eIwawaki T, Akai R, Yamanaka S, Kohno K. Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability. Proc Natl Acad Sci U S A. 2009, 106(39):16657-62. \u003c/li\u003e\n\u003cli\u003eMakhija L, Krishnan V, Rehman R, Chakraborty S, Maity S, Mabalirajan U, Chakraborty K, Ghosh B, Agrawal A. Chemical chaperones mitigate experimental asthma by attenuating endoplasmic reticulum stress. Am J Respir Cell Mol Biol. 2014, 50(5):923-931.\u003c/li\u003e\n\u003cli\u003eGong B, Zhang L, Pang C, Lam DS, Yam GH. Trimethylamine N-oxide alleviates the severe aggregation and ER stress caused by G98R alphaA-crystallin. Mol Vis. 2009, 15:2829-2840.\u003c/li\u003e\n\u003cli\u003eZhao Z, Xin F, Zhou D, Xue YQ, Liu XL, Yang RX, Pan Q, Fan JG. Trimethylamine N-oxide attenuates high-fat high-cholesterol diet-induced steatohepatitis by reducing hepatic cholesterol overload in rats. World J Gastroenterol. 2019, 25(20):2450-2462. \u003c/li\u003e\n\u003cli\u003eLin X, Zhang Y, He X, Chen Y, Chen N, Liu J, Wang M, Li Y, Yang H, Fan L, Hou Y, Li J, Wu C, Qi H, Zhang H, Xiao X. The Choline Metabolite TMAO Inhibits NETosis and Promotes Placental Development in GDM of Humans and Mice. Diabetes. 2021, 70(10):2250-2263.\u003c/li\u003e\n\u003cli\u003eGuest I, Varma DR. Selective growth inhibition of the male progeny of mice treated with trimethylamine during pregnancy. Can J Physiol Pharmacol. 1993, 71(2):185-7.\u003c/li\u003e\n\u003cli\u003eGuest I, Varma DR. Developmental toxicity of methylamines in mice. J Toxicol Environ Health. 1991, 32(3):319-30. \u003c/li\u003e\n\u003cli\u003eJalandra R, Makharia GK, Sharma M, Kumar A. Inflammatory and deleterious role of gut microbiota-derived trimethylamine on colon cells. Front Immunol. 2023, 13:1101429.\u003c/li\u003e\n\u003cli\u003eQiu Z, Li G, An T. In vitro toxic synergistic effects of exogenous pollutants-trimethylamine and its metabolites on human respiratory tract cells. Sci Total Environ. 2021, 783:146915. \u003c/li\u003e\n\u003cli\u003eRestini CBA, Fink GD,Watts SW. Vascular reactivity stimulated by TMA and TMAO: Are perivascular adipose tissue and endothelium involved? Pharmacol Res. 2021, 163: 105273.\u003c/li\u003e\n\u003cli\u003eOngun MC, Orgul G, Celik C, Bariskaner H. Contractile effect of trimethylamine and trimethylamine-n-oxide on isolated human umbilical arteries. J Obstet Gynaecol Res. 2023, 49: 1736-1742.\u003c/li\u003e\n\u003cli\u003eChhibber-Goel J, Gaur A, Singhal V, Parakh N, Bhargava B, Sharma A. The complex metabolism of trimethylamine in humans: endogenous and exogenous sources. Expert Rev Mol Med. 2016, 18: e8.\u003c/li\u003e\n\u003cli\u003eJoo EH, Kim YR, Kim N, Jung JE, Han SH, Cho HY. Effect of Endogenic and Exogenic Oxidative Stress Triggers on Adverse Pregnancy Outcomes: Preeclampsia, Fetal Growth Restriction, Gestational Diabetes Mellitus and Preterm Birth. Int J Mol Sci. 2021, 22(18):10122.\u003c/li\u003e\n\u003cli\u003eHoyles L, Pontifex MG, Rodriguez-Ramiro I, Anis-Alavi MA, Jelane KS, Snelling T, Solito E, Fonseca S, Carvalho AL, Carding SR, M\u0026uuml;ller M, Glen RC, Vauzour D, McArthur S. Regulation of blood-brain barrier integrity by microbiome-associated methylamines and cognition by trimethylamine N-oxide. Microbiome. 202, 9(1):235. \u003c/li\u003e\n\u003cli\u003eHuang S, Mo T, Norris T, Sun S, Zhang T, Han T, Rowan A, Xia Y, Zhang H, Qi H, Baker PN. The CLIMB (Complex Lipids In Mothers and Babies) study: protocol for a multicentre, three-group, parallel randomised controlled trial to investigate the effect of supplementation of complex lipids in pregnancy, on maternal ganglioside status and subsequent cognitive outcomes in the offspring. BMJ Open. 2017, 7(10):e016637.\u003c/li\u003e\n\u003cli\u003eChen YT, Zhang T, Chen C, Xia YY, Han TL, Chen XY, He XL, Xu G, Zou Z, Qi HB, Zhang H, Albert BB, Colombo J, Baker PN. Associations of early pregnancy BMI with adverse pregnancy outcomes and infant neurocognitive development. Sci Rep. 2021 Feb 15;11(1):3793.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 8 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3970231/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3970231/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe concept of Small Vulnerable Newborns (SVN), encompassing small for gestational age (SGA), low birthweight (LBW), and preterm birth (PTB) has recently proposed [1, 2]. Collectively, these births represent the main cause of neonatal mortality and specific interventions to address SVN have great potential to improve maternal and infant outcomes. Choline is an essential nutrient for many aspects of fetal development and metabolism, but despite this, upwards of 90% of pregnant women fail to achieve recommend adequate intake [3]. At present, the role of choline deficiency in pregnancy on SVN risk remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eHere, we measured choline and its metabolites, TMA an TMAO by liquid chromatography-mass spectrometry (LC-MS/MS) in first (11-14 weeks), second (22-28 weeks), and third (32-34 weeks) trimester plasma of 1231 pregnant women from the Complex Lipids in Mothers and Babies (CLIMB) cohort to investigate the relationship between choline TMA, TMAO in pregnancy and the risk of SVN. We also explored the relationship of these measures with later infant developmental outcomes of SVN and non-SVN births including physical and brain development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePregnant women with SVN births showed no differences in circulating choline throughout pregnancy but had significantly lower TMAO in the first two trimesters, and higher TMA in the third trimester, relative to non-SVN births. However, TMAO levels during pregnancy were positively correlated with the risk of SVN, while TMA levels were negatively correlated with the risk of LBW and PTB in SVN. Supporting findings included positive associationbetween TMAO during pregnancy and birth weight and birth length, while negative association between TMA and birth weight and birth length. In the receiver operating characteristic (ROC) curves, inclusion of choline and its metabolites TMA and TMAO during pregnancy in the models markedly increased the predictive performance for the model which contained traditional risk factors for SVN including age, body mass index, fasting blood glucose , one-hour postprandial blood glucose, two-hour postprandial blood glucose, and placental growth factor. Moreover, during pregnancy, the TMAO was not only positively correlated with fetal development, but also with the development of postnatal offspring at six months old and 1 year old, while TMA was the opposite.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe abnormal choline metabolism is potentially associated with the risk of SVN and the development of fetus and postnatal offspring, with particular attention paid to the opposite association between TMAO and TMA in offspring development, which may be an effective means of preventing SVN and ensuring healthy fetal development.\u003c/p\u003e","manuscriptTitle":"Choline and its Metabolites Trimethylamine and Trimethylamine N-oxide Potentially Predict the Risk of Small Vulnerable Newborn and the Development of 1-Year Infants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 17:10:44","doi":"10.21203/rs.3.rs-3970231/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6d4187b-d371-4b2b-a219-6b2cb1f02367","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29064229,"name":"Health sciences/Risk factors"},{"id":29064230,"name":"Health sciences/Biomarkers/Predictive markers"}],"tags":[],"updatedAt":"2024-03-19T20:53:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-05 17:10:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3970231","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3970231","identity":"rs-3970231","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}