Association of Serum Choline and Its Metabolites with Infant’s Growth and Neurodevelopment from Birth to 12 Months

Association of Serum Choline and Its Metabolites with Infant’s Growth and Neurodevelopment from Birth to 12 Months | 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 Research Article Association of Serum Choline and Its Metabolites with Infant’s Growth and Neurodevelopment from Birth to 12 Months Xuying Tan, Jiaxin Zhuang, Yanfei Xing, Suyi Qiu, Liyi Guo, Cuizhen Gao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7058350/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 : Choline is an essential nutrient that plays crucial roles in cell structure maintenance, neurotransmission, and betaine synthesis. Trimethylamine N-oxide (TMAO) is biosynthesized from choline through metabolic processes mediated by gut microbiota and the liver. However, the relationships among serum choline, its metabolites, and early neurodevelopmental and growth remain unclear. Methods : This retrospective study enrolled 109 outpatients aged 0–12 months who underwent neurodevelopmental assessments using the China Developmental Scale for Children at the Guangzhou Women and Children’s Medical Center from January 2018 to September 2019. Residual blood specimens obtained post-clinical testing were collected for subsequent analysis. To mitigate temporal bias, both neurodevelopmental assessment and blood sampling were conducted within a 30-day window for each participant. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was employed to measure the serum concentrations of choline, betaine, and TMAO. Anthropometric parameters, including weight, length, and head circumference, were recorded at birth and 1, 3, 6, 8, and 12 months of age. Results : Serum choline levels were significantly associated with enhanced neurodevelopment in 6- to 12-month-old infants. Conversely, serum betaine concentrations exhibited a negative correlation with the full-scale developmental quotient and language quotient in infants aged 91 to 180 days. Serum TMAO showed no significant associations with most indices of infant neurodevelopment. Additionally, no statistically significant correlations were observed between serum choline or its metabolites and infant growth parameters from 0 to 12 months. Conclusions : Our study identified specific associations between choline concentration and neurodevelopment in 6- to 12-month-old infants, suggesting that choline status may be a pivotal determinant of neurodevelopment during early infancy. choline betaine trimethylamine N-oxide neurodevelopment growth Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Background Choline is an essential nutrient for normal cellular function, growth, and neurodevelopment in fetal and newborn infants[ 1 , 2 ]. Choline is the precursor of phosphatidylcholine, which are major components of cellular membranes. As for the function of choline in neurodevelopment, choline serves as a component of sphingomyelin, which is a constituent of the myelin sheath of nerve axons and facilitates efficient transmission of nerve signals[ 3 ]. The neurotransmitter, acetylcholine, is produced from free choline in cholinergic neurons. In addition, choline acts as a methyl donor by oxidized to betaine, which functions to donate methyl groups to homocysteine, producing the essential amino acid and methionine[ 1 , 3 ]. Trimethylamine N -oxide (TMAO) is produced when dietary choline is converted to trimethylamine by the gut microbiota and then oxidized to TMAO in the liver[ 4 ]. Choline that converted to betaine or TMAO is irreversible. Evidence from human studies revealed dietary choline supplementation during pregnancy and lactation have found improved cognitive, motor and language development before 24 months of age[ 5 – 7 ]. Additionally, increased maternal choline concentration also associated with improved memory, attention and processing speed in children[ 8 ]. Serum choline concentrations is highest at birth and remain elevated with a slow and gradual decrease during the first 2 years of life, which may be related to significant choline requirements for phospholipid synthesis in growing brain and other organs[ 9 ]. Therefore, choline and its metabolites serve as critical roles in the early growth and neurodevelopment[ 10 , 11 ]. TMAO has been implicated in the aging related cognitive function decline, neuronal senescence and synaptic damage in the brain[ 12 , 13 ]. Molecular investigations have shown that TMAO activates astrocytes and microglia and triggers a cascade of inflammatory responses in the brain[ 14 , 15 ]. However, the relation between TMAO levels and neurodevelopment in children is still unknown. Only a limited number of studies have examined relations between serum choline metabolite levels and neurodevelopment and growth in early childhood[ 16 ]. To date, no study has examined the impact of the infant’s, rather than the mother’s, choline metabolites levels on neurodevelopment. We hypothesized that higher choline and betaine concentrations are related to a higher neurodevelopment quotient and linear growth, while TMAO showed negative correlations in infants of 0–12 months. Therefore, we conducted a historical observing study to estimate the relation between choline status and the neurodevelopment and growth in infants of 0–12 months. Firstly, we aimed to determine the concentration of serum choline, betaine and TMAO after birth during 0-365 postnatal days and investigate the relation between choline status with age. Secondly, we aimed to examined the relationship between choline status and neurodevelopment and growth in infants of 0–12 months. 2. Subjects and methods 2.1 Study population This study was designed as an observational, descriptive, and retrospective study. We retrospectively reviewed the cases of outpatients who admitted to Guangzhou Women and Children’s Medical Center for the neurodevelopmental assessments between January 2018 and September 2019. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki. The study was reviewed and approved by the ethics committee of the Guangzhou Women and Children’s Medical Center (Number: [2021]027A01). Inclusion criteria were as follows: (Ⅰ) Term infant aged between 0 and 12 months; (Ⅱ) Patients who had completed the neurodevelopmental assessment scale; (Ⅲ) Patients with residual blood specimens after clinical analysis; (Ⅳ) Patients underwent neurodevelopmental assessment and blood sampling within 30 days to minimize temporal bias. The exclusion criteria were as follows: (Ⅰ) Patients with chromosomal or genetic abnormalities; (Ⅱ) Encephalodysplasia; (Ⅲ) Patients with neurological diseases or injuries. Participant screening process: of 46,344 patients screened, 45,634 were aged 0 to 12 months (Fig. 1 ). For the analysis, 44,720 cases without residual blood specimens were excluded, leaving 914 infants with preserved blood samples stored in the study biorepository. Among these, 153 infants underwent blood sampling and neurodevelopmental assessments within 30 days. After excluding 1 case with chromosomal abnormalities and 2 cases with brain injuries, 150 eligible infants were identified. Finally, 109 term infants (aged 0 to 12 months) with both neurodevelopmental test results and stored blood specimens were included in this study. 2.2 Serum choline, betaine and TMAO measurements Unsued residual blood specimens after clinical testing were collected for retrospective research and stored at -80°C within 24 hours after to maintain sample integrity. Residual blood specimens after clinical testing were obtained with informed consent and used in accordance with institutional review board guidelines. Serum choline, betaine and TMAO were measured using high performance liquid chromatography-tandem MS/MS (HPLC-MS/MS) (Agilent 6400 Series Triple Quad LCMS; CA, USA), with multi-reaction monitoring (MRM) functions[ 17 ]. 60 µL of either serum sample or standards was mixed with 100 µL of acetonitrile containing 10 µM of internal standards d9-TMAO (Toronto Research Chemicals Inc., Toronto, Canada) and d9-choline (Sigma-Aldrich, St. Louis, USA). The mixture was vortexed and then centrifuged at 13,000 × g for 10 minutes to precipitate proteins. The supernatant was injected into a normal-phase silica gel column (2.1 mm × 100 mm, 5 µm particle size). The column was eluted isostatically with 30% A solution (15 mM ammonium formate aqueous solution; pH 3.0) and 70% B solution (acetonitrile) at a 0.2 mL/min flow rate. For free choline, betaine, and TMAO, the within-day coefficients of variation (CVs) are 0.79%, 1.53%, and 1.62%, respectively. The between-day CVs for these compounds are 2.71%, 2.68%, and 6.53%, respectively. 2.3 Demographic characteristics Birth outcome data, such as gestational age, gender, birth weight, birth length and delivery mode were obtained from medical records of the hospital. Gestational age was based on last menstrual period and was confirmed by ultrasound scanning performed at ≤ 20 wk gestation. If the gestational age estimated by sonographic differed by > 2 wk from the last menstrual period, the sonographic dating was used[ 18 ]. 2.4 Neurodevelopmental assessment The infant’s neurodevelopment was evaluated by the China Developmental Scale for Children aged 0–6 years, which is an indigenous and diagnostic development assessment tool with Chinese norms. The China Developmental Scale for Children was developed by the Capital Institute of Pediatrics of China since the early 1980s. The scale includes 261 items on five areas, namely, gross motor, fine motor, language, adaptive behavior and personal-social. The five areas were consistent with the relevant subscales in Gesell and showed adequate reliability in children with typical development[ 19 ]. The full scale developmental quotient (DQ) or a subscale quotient less than 70 pointes indicates a developmental delay; a quotient between 70 and 79 points is slightly below the threshold for developmental delay, and a quotient greater than or equal to 80 points indicates no developmental delay[ 19 ]. The neurodevelopmental assessment was performed when the infant in well awake status by extensively trained and certified pediatric physiotherapists who had no knowledge of the results of the infants’ serum analyses. 2.5 Anthropometric measurements The anthropometric measurements of weight (in kilograms), length (in centimeters), and head circumference (in centimeters) were conducted by two trained and experienced nurses at the infant’s birth and at 1, 3, 6, 8 and 12 months of age. The infant’s weight was measured to the nearest 0.01 kg using a calibrated digital scale (SECA 335, SECA GmbH, Hamburg, Germany) without clothes and diapers. Without clothing and shoes, the length was measured to the nearest 0.1cm using a calibrated rigid height board (SECA 335, SECA GmbH, Hamburg, Germany). Head circumference was measured to the nearest 0.1cm using a non-elastic plastic tape measure. All measurements were repeated twice. The errors between each measurement should be less than 0.5 cm for body length, less than 0.01 kg for weight and less than 0.2cm for head circumference. The mean values were taken as the final values. 2.6 Statistical methods Statistical analyses were performed using SPSS 20.0 software (SPSS, Chicago, Illinois, USA) and R 4.3.1 software (R Foundation for Statistical Computing, Vienna, Austria). Continuous variables were expressed as the mean ± standard deviations (SDs), and categorical variables were expressed as numbers and percentages (%). The included individuals were divided into three groups based on the age of blood collection: 0–90 days, 91–180 days and 181–365 days. Wilcoxon’s signed rank tests were used to compare choline, betaine and TMAO levels among different age windows in infants. Spearman's rank correlation coefficients were employed to evaluate the association between choline and its related metabolites with age, as well as with neurodevelopmental outcomes. After natural log transformation of serum choline, betaine and TMAO levels among three groups, one-way analysis of variance (ANOVA) with linear tests for trend were used to compare the differences between groups. Bonferroni post-hoc tests were used for multiple comparisons[ 20 ]. Associations of choline and its related metabolites with each of the five neurodevelopmental endpoints in different age windows were evaluated with separate multiple linear regression analyses, adjusted for gestational age, birth weight and gender. A two-sided significance level of 0.05 was used to evaluate the above statistical tests. In order to select the best model for the weight, length and head circumference parameters at each measurement point (at birth and 1, 3, 6, 8, and 12 months), linear, quadratic, and cubic growth curve models were constructed. The optimal model for growth curve construction was selected based on the lowest values of Akaike Information Criterion, corrected Akaike Information Criterion, and Bayesian Information Criterion[ 21 ]. Subsequently, linear mixed-effect regression models were used to evaluate associations between anthropometric parameters and serum concentrations of choline and its metabolites, while adjusting for covariates including age, gender, and gestational age. These models accommodated repeated measures and accounted for within-subject correlations by incorporating random intercepts, random slopes, and an unstructured covariance matrix. Multicollinearity was assessed using the variance inflation factor, with values < 5 indicating no significant collinearity[ 22 ]. Within each model, a two-sided P -value less than 0.05 was considered statistically significant. 3. Results 3.1. Characteristics of the study population A total of 109 patients who met these inclusion criteria were enrolled for the analysis. Characteristics were described across three age groups categorized by the age at blood sample collection. The gestational age of infants demonstrated significant difference among three groups ( P = 0.03). Specifically, the 181–365 days group had a gestational age of 39.27 ± 1.25 weeks, compared to 38.44 ± 1.24 weeks in the 0–90 days group and 38.90 ± 1.07 weeks in the 91–180 days group. No significant differences were observed among the three groups in birth weight, birth length, or delivery mode. The 0–90 days group consisted of 14 males (56%) and 11 females (44%), with a mean age of 52.28 ± 20.52 days at blood collection. The 91–180 days group included 25 males (55.56%) and 20 females (44.44%), with a mean age of 137.51 ± 23.39 days at the time of blood collection. The 181–365 days group comprised 19 males (51.4%) and 18 females (48.6%), with a mean age of 231.03 ± 51.07 days at the point of blood collection. Table 1 Characteristics of the study population ( n = 109) 0–90 days ( n = 24) 91–180 days ( n = 48) 181–365 days ( n = 37) P Gestational age 38.44 ± 1.24 38.90 ± 1.07 39.27 ± 1.25 0.03 Birth weight (g) 3057.08 ± 387.46 3012.67 ± 314.40 3036.49 ± 441.11 0.89 Birth length (cm) 49.25 ± 1.62 48.76 ± 1.61 47.70 ± 8.31 0.46 Male [n (%)] 14 (56%) 25(55.56%) 19 (51.4%) 0.91 Delivery mode [n (%)] 0.48 Vaginal delivery 13 (52%) 27 (60.0%) 23 (62.16%) 0.26 Abdominal delivery 12 (48%) 15 (33.3%) 12 (32.43%) Obstetric forceps 0 (0%) 3 (6.67%) 2 (5.41%) Age at blood collection (days) 52.28 ± 20.52 137.51 ± 23.39 231.03 ± 51.07 NA 3.2. Correlations of Serum Choline, Betaine, and TMAO Levels with Age Spearman correlation revealed no significant associations between serum choline, betaine levels and age (Fig. 1 A-B). Serum TMAO levels and TMAO-to-choline ratios exhibited significant positive correlations with age ( r = 0.353, P = 0.001, Fig. 2 C; r = 0.357, P = 0.001, Fig. 2 E). Serum levels of choline, betaine, and TMAO across different age groups are presented in Fig. 3 . Neither serum choline nor betaine levels, nor the betaine-to-choline ratio, showed statistically significant differences among age groups. By contrast, serum TMAO levels and the TMAO-to-choline ratio exhibited significant intergroup variations and were positively correlated with age ( P − diff = 0.04, P - trend = 0.02; P − diff = 0.03, P - trend = 0.01, respectively). The median serum TMAO level in 181–365-day-old infants was 6.94-fold higher than that in 0–90-day-old infants (1.11 µM vs 0.16 µM, P = 0.04). The median TMAO:choline ratio in the 181–365-day-old group was 7.5-fold higher than that in the 0–90-day-old group (0.03 vs 0.004 µM, P = 0.04). 3.3. Associations of Serum Choline, Betaine, and TMAO Levels with Neurodevelopment Spearman correlation analyses were conducted to examine associations between serum choline and its metabolite levels and neurodevelopment (Table 2 ). Serum choline levels showed positive correlations with both the full-scale developmental quotient (DQ) and fine motor quotient ( r = 0.22, P = 0.02 for both). No significant correlations were observed for serum betaine or the betaine/choline ratio with any neurodevelopmental measures. By contrast, serum TMAO levels exhibited negative correlations with the full-scale DQ ( r = − 0.22, P = 0.04) and gross motor quotient ( r = − 0.25, P = 0.02). We further investigated associations between choline and related metabolites and neurodevelopmental outcomes across three age cohorts. Results of multiple linear regression analyses adjusted for gestational age, birth weight, and gender are shown in Fig. 4 . In infants aged 0 to 90 days, no statistically significant associations were observed between choline, betaine, or TMAO and any neurodevelopmental outcomes. In infants aged 91 to 180 days, a 1-unit increase in z-score of ln-transformed serum choline levels was significantly associated with: a 5.27-point increase in full-scale DQ (95% CI: [0.43, 10.10], P = 0.03), an 8.56-point increase in the fine motor quotient (95% CI: [2.04, 15.07], P = 0.01), and an 8.45-point increase in the personal-social quotient (95% CI: [1.42, 15.47], P = 0.02). Conversely, serum betaine levels demonstrated significant negative associations with full-scale DQ and the language quotient. Specifically, each 1-unit increase in z-score of ln-transformed serum betaine was associated with a 4.68-point reduction in full-scale DQ (95% CI: [-9.23, -1.14], P = 0.04) and a 6.76-point decrease in the language quotient (95% CI: [-11.83, -1.69], P = 0.01). In 181-365-day-old infants, elevated ln-transformed serum choline levels were significantly associated with improvements in full-scale DQ, adaptive behavior, and language quotient. Specifically, each 1-unit increase in the z-score of ln-choline was associated with a 3.59-point increase in full-scale DQ (95% CI: [0.37, 6.81], P = 0.03). Similarly, ln-transformed serum betaine levels exhibited positive associations with personal-social quotient, with a 6.96-point increase observed per 1-unit z-score elevation (95% CI: [0.93, 12.99], P = 0.03). By contrast, serum TMAO levels were negatively associated with the gross motor quotient, with each 1-standard deviation increase in TMAO corresponding to a 7.38-point decrease in the gross motor quotient (95% CI: [-13.62, -1.15], P = 0.02). Table 2 Correlations of Serum Choline and Its Metabolites with Neurodevelopment Choline Betaine TMAO Betaine/Choline TMAO/Choline r P r P r P r P r P Full Scale DQ 0.22 0.02 -0.02 0.86 -0.18 0.11 -0.18 0.06 -0.22 0.04 Gross motor 0.001 0.99 -0.08 0.39 -0.26 0.02 -0.09 0.37 -0.25 0.02 Fine motor 0.22 0.02 0.07 0.51 -0.17 0.13 -0.1 0.3 -0.21 0.05 Adaptive Behavior 0.14 0.14 0.001 0.99 0.08 0.47 -0.12 0.21 0.03 0.8 Language 0.12 0.22 -0.1 0.99 -0.09 0.39 -0.17 0.86 -0.12 0.28 Personal-social 0.17 0.08 0.09 0.34 -0.06 0.61 -0.02 0.86 -0.11 0.31 3.4. No Correlations between Choline and Its Metabolite Levels and Infant Growth Linear mixed regression models were used to examine the associations between serum choline and its metabolites (betaine, TMAO) and infant growth indicators (weight, length and head circumference). After adjusting for age, gender, and gestational age, linear mixed model results showed no significant associations between choline, betaine, or TMAO levels and infant growth (length, weight, head circumference) during 0–12 months (Table 3 ). Notably, birth weight was positively correlated with all growth parameters ( P < 0.05). Gestational age and the infant’s age at blood collection were not significantly associated with any growth outcomes (Supplementary Table S1 –3). Table 3 Correlations of Serum Choline and Its Metabolites with Infant Weight, Length, and Head Circumference Serum choline and its metabolites Length Weight Head circumference β (95% CI) P β (95% CI) P β (95% CI) P Ln betaine 0.02 (-0.01, 0.06) 0.22 0.04 (-0.02, 0.09) 0.20 0.03 (-0.05, 0.11) 0.32 Ln choline 0.02 (-0.01, 0.06) 0.19 0.02 (-0.03, 0.07) 0.45 0.06 (-0.01, 0.13) 0.10 Ln TMAO -0.02 (-0.07, 0.02) 0.31 -0.02 (-0.08, 0.04) 0.52 0.04 (-0.05, 0.13) 0.44 Linear mixed regression models were employed and adjusted for age, gender, gestational age. 4. Discussion This study demonstrates that elevated serum choline levels are associated with improved neurodevelopment in 6–12-month-old infants. By contrast, serum betaine was negatively associated with the full-scale DQ and language quotient in infants aged 91–180 days. Serum TMAO was not associated with most measures of infant’s neurodevelopment. Moreover, no statistically significant associations were detected between serum choline or its metabolites and the growth parameters of infants from 0 to 12 months. Serum TMAO levels were negative correlated with age, while neither serum choline nor betaine concentrations exhibited age - related correlations.To our knowledge, this is the first research to explore the associations of serum choline, betaine, and TMAO with growth and neurodevelopment of infants from birth to 12 months. Choline is required for the synthesis of phosphatidylcholines, which are the major component of cell membranes, are essential for the growth and development[ 3 ]. Choline is also an essential component of sphingomyelin. Additionally, choline is present in the nervous system, which it serves for the synthesis of acetylcholine. An impressive array of animal studies has shown that prenatal choline supplementation enhances the cognitive ability in offspring, probably related to the improve in structure and function of hippocampal pyramidal cells[ 23 , 24 ]. Conversely, choline deficiency in late pregnancy showed hippocampal apoptosis and impaired memory offspring rats[ 25 ]. However, results from observational studies on choline and neurodevelopment were variable. A prospective study included 404 mother-child pairs found no correlation of physiologic free and total choline concentrations during pregnancy or in cord blood with children’s intelligence at 5 years old[ 18 ]. Another study presented that higher maternal choline intake was associated with modestly higher child visual memory score at age 7 years[ 26 ]. As in the clinical trial studies, women supplemented with phosphatidylcholine (that delivers 750 mg/d) during pregnancy and early lactation, showed no advantage in improvement of brain development in their infants[ 27 ]. Supplementation of choline during pregnancy in woman who exposed to infection[ 28 ] or alcohol[ 29 ] showed protective effect of choline in the brain development of their infants. Based on the above studies, the protective effect of gestational choline supplementation in the development of infant brain appeared in women exposed to a low-choline diet or other detrimental factors during pregnancy. As the brain continues to develop rapidly in the first 2 years after birth, choline status is likely to have an impact on neurodevelopment[ 30 ]. There is limited research conducted on choline nutrition after birth and neurodevelopment outcomes, particularly in infants and toddlers, and previous studies have generally yielded positive or null results. An observational study conducted in low income country including children aged 6–15 months and those with milk intake less than 236 ml/day, found no strong or consistent associations between plasma choline and growth and development[ 31 ]. Another study showed higher plasma betaine concentrations linked to better visual-motor skills, but not choline in healthy toddlers aged 2 years who did not meet the recommended dietary choline intake[ 32 ]. Clinical trial studies found provision of supplementary foods containing choline, along with other nutrients, led to improve in working memory and locomotor in young children in South Africa[ 33 , 34 ]. Additionally, higher choline and higher docosahexaenoic acid in breast milk was related to better recognition memory in 6-month-old infants[ 35 ]. Of note, choline alone may not provide benefit if other proteinogenic amino acids and fatty acids are not sufficient in the diet. Besides, early postnatal choline supplementation in children with diseases of the nervous system such as fetal alcohol spectrum disorders, revealed improved in memory function[ 36 ]. We found consistent and mostly positive associations of serum choline levels with developmental outcomes in infants aged 0–12 months. In the current study, the results from Spearman relation analyses indicated positive associations between serum choline levels and both full scale DQ and fine motor quotients in infants aged 0–12 months. However, no correlations were observed with betaine. After adjusting for age, birth weight and gender, positive associations between serum choline and neurodevelopment were revealed in infants aged 6–12 months. It is plausible that during the initial six - month period after birth, the choline reserves in infants are mainly derived from the choline stored during fetal development and the postnatal choline ingested from dairy products. Once complementary foods are introduced at six months of age, differences in the choline status among different infants gradually become evident. Consequently, the positive correlations between choline and neurodevelopment also manifested between 6–12 months of age. Betaine serves as a major methyl donor for homocysteine in the synthesis of methionine[ 37 ]. Our findings revealed that serum betaine concentrations were negatively correlated with the full scale DQ and language quotients in infants aged 91–180 days. We hypothesize that owing to the irreversible conversion of choline to betaine, the quantity of free choline available for the synthesis of acetylcholine and structural phospholipids, such as phosphatidylcholine and sphingomyelin, has been decreased. Choline levels showed higher in this setting than in previous reports[ 9 ]. The inconsistent results among the different studies may be due to differences in choline reserves during fetal development, feeding patterns, sociodemographic factors, and genetic polymorphisms in choline metabolizing enzymes that are unique to the study population. First, plasma choline concentration declined over the first year after birth[ 9 ], comparing choline status across studies with different age groups poses a challenge. Second, the amount of choline in breast milk varies widely and is affected by the diet of the nursing mother[ 38 ]. Besides, the complementary feeding period to induce animal source foods, which are rich in choline, may be particularly important to ensure adequate choline intake and impact on the choline status in infants[ 39 ]. Third, genetics may also play a role, given the choline metabolism is regulated by phenylethanolamine methyl transferase (PEMT) and influenced by the polymorphisms in gene PEMT[ 40 ]. Choline may be irreversibly catabolized by intestinal flora to produce TMAO[ 4 ], the main source of TMAO in the human body. Although TMAO is associated with the metabolic diseases[ 41 ] and impaired of cognitive function in adults[ 42 , 43 ], its effects in young children are unclear. TMAO can cross the blood-brain barrier and are involved in brain development, neurogenesis and behavior[ 44 ]. A cross-sectional study of 328 children aged 3–8 years revealed an association between elevated plasma TMAO concentrations and the severity of autism spectrum disorder and its symptoms, indicating that TMAO could be used as a biomarker of autism[ 45 ]. Animal studies have shown that TMAO induced cognitive impairment through aggravating oxidative damage[ 46 ], promoting neuroinflammation and impairment of mTOR signaling[ 47 ] and synaptic plasticity[ 48 ]. We discovered that in infants ranging from 0 to 12 months old, postnatal trimethylamine N-oxide (TMAO) levels exhibited a gradual increase with advancing age. However, in the present study, associations of TMAO with neurodevelopment were null except higher TMAO was associated with lower gross motor quotients in infants aged 6–12 months. Specific to the relationship between choline and growth, previous study showed that low serum choline, rather than serum betaine or TMAO concentration was correlated with stunting in young children aged 12–59 months living in low-income countries[ 16 ]. Another prospective cohort study found no associations between either the maternal blood choline and its metabolites or postnatal choline intakes and anthropometric measurements at 2 years[ 49 ]. An egg intervention trial in children ages 6 to 9 months showed the egg intervention increased the plasma concentrations of choline and betaine[ 50 ], with the improved in linear growth and reducing stunting[ 51 ]. The growth of bones requires the synthesis of phosphatidylcholines by osteoblasts and the endochondral plate[ 52 ]. In the animal study, the deficient of choline kinase β gene in mice have reduced bone mass and decreased limb length[ 16 ]. However, in the present study, general liner mixed model showed no significant correlation between choline, betaine and the growth parameters. There may be factors such as differences in the overall diet and multiple other nutrients and bioactive factors converging to influence linear growth during infancy. Some limitations are present in this study. First, the determination of serum choline and related compounds relied on remnants of clinically indicated samples. Previous study have reported that the concentration of choline remain stability at 4 ℃ with 24 h and the remaining serum specimens after clinical laboratory test were collected within 24 h[ 53 ]. Therefore, the measurement with remaining serum specimens is likely to have limited impact on the infant’s choline status. Second, due to the retrospective nature of this study, the blood sampling was not always available exactly on the day prior to the neurodevelopmental assessment. However, to make the correlation analyses possible, the blood samples we included were within 30 days matched with neurodevelopmental evaluation. 5. Conclusion In summary, we found few significant associations between the concentration of choline and neurodevelopment outcomes in 6–12-month-old infants. Our results suggest that choline status may be an important factor in neurodevelopment. Controlled clinical trials are warranted to determine whether increasing dietary choline intake or choline supplementation can improve linear growth and neurodevelopment during the first year of life. Abbreviations TMAO Trimethylamine N-oxide HPLC-MS/MS High-performance liquid chromatography tandem mass spectrometry MRM Multi-reaction monitoring DQ Developmental quotient ANOVA One-way analysis of variance IQR Inter - quartile range PEMT Phenylethanolamine methyl transferase Declarations Ethics approval and consent to participate The study was reviewed and approved by the ethics committee of the Guangzhou Women and Children’s Medical Center (Number: [2021]027A01). Consent for publication No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. Competing interests The authors declare no competing interests. Funding This work was supported by the funding “Nutrition and Care of Maternal &Child Research Fund Project” of Biostime Institute of Nutrition & Care (Grant/Award Number: 2020BINCMCF047) and Student Innovation Ability Enhancement Program of Guangzhou Medical University (Grant/Award Number: 02-408-240603131100) Author Contribution Conceptualization, X.T. and J.Z.; Methodology, Y.X.;; Formal Analysis, S.Q. and G.L.; Investigation, L.G. and Z.L.; Writing – Original Draft Preparation, X.T.; Writing – Review & Editing, C.G.; Visualization, Y.H.; Supervision, Y.H.; Project Administration, Y.H.; Acknowledgement Thank Clinical Biological Resource Bank of Guangzhou women and children’s medical center for providing the clinical samples. Availability of data and materials All data analyzed in the current research are available from the corresponding author on logical request. References Zeisel SH, da Costa KA. Choline: an essential nutrient for public health. Nutr Rev. 2009;67(11):615–23. 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Maternal choline supplementation during the third trimester of pregnancy improves infant information processing speed: a randomized, double-blind, controlled feeding study. FASEB journal: official publication Federation Am Soc Experimental Biology. 2018;32(4):2172–80. Caroline Signore PM, Ueland J, Troendle, Mills JL. Choline concentrations in human maternal and cord blood and intelligence at 5 y of age. Am J Clin Nutr. 2008;87:896–902. Ilcol YO, Ozbek R, Hamurtekin E, Ulus IH. Choline status in newborns, infants, children, breast-feeding women, breast-fed infants and human breast milk. J Nutr Biochem. 2005;16(8):489–99. Zeisel SH. The fetal origins of memory: the role of dietary choline in optimal brain development. J Pediatr. 2006;149(5 Suppl):S131–6. Blusztajn JK, Mellott TJ. Choline nutrition programs brain development via DNA and histone methylation. Cent Nerv Syst Agents Med Chem. 2012;12(2):82–94. Brunt VE, LaRocca TJ, Bazzoni AE, Sapinsley ZJ, Miyamoto-Ditmon J, Gioscia-Ryan RA, et al. The gut microbiome-derived metabolite trimethylamine N-oxide modulates neuroinflammation and cognitive function with aging. Geroscience. 2021;43(1):377–94. Praveenraj SS, Sonali S, Anand N, Tousif HA, Vichitra C, Kalyan M, et al. The Role of a Gut Microbial-Derived Metabolite, Trimethylamine N-Oxide (TMAO), in Neurological Disorders. Mol Neurobiol. 2022;59(11):6684–700. Vogt NM, Romano KA, Darst BF, Engelman CD, Johnson SC, Carlsson CM, et al. The gut microbiota-derived metabolite trimethylamine N-oxide is elevated in Alzheimer's disease. Alzheimers Res Ther. 2018;10(1):124. Gao Q, Wang Y, Wang X, Fu S, Zhang X, Wang R-T, et al. Decreased levels of circulating trimethylamine N-oxide alleviate cognitive and pathological deterioration in transgenic mice: a potential therapeutic approach for Alzheimer’s disease. Aging. 2019;11(19):8642–63. Semba RD, Zhang P, Gonzalez-Freire M, Moaddel R, Trehan I, Maleta KM, et al. The association of serum choline with linear growth failure in young children from rural Malawi. Am J Clin Nutr. 2016;104(1):191–7. Holm PI, Ueland PM, Kvalheim G, Lien EA. Determination of choline, betaine, and dimethylglycine in plasma by a high-throughput method based on normal-phase chromatography-tandem mass spectrometry. Clin Chem. 2003;49(2):286–94. Caroline Signore PM, Ueland J, Troendle, Mills JL. Choline concentrations in human maternal and cord blood and intelligence at 5 y of age. Am J Clin Nutr. 2008;87(4):896–902. Jin Chunhua L, Lili RZ, Yue Z. The revision and according validity research of China developmental scale for China. Chin J Child Health Care. 2014;22:1242–6. McHugh ML. Multiple comparison analysis testing in ANOVA. Biochem Med (Zagreb). 2011;21(3):203–9. Johnson W, Balakrishna N, Griffiths PL. Modeling physical growth using mixed effects models. Am J Phys Anthropol. 2013;150(1):58–67. Rogerson P. Statistical Methods for Geography. London: SAGE Publications, Ltd; 2001. Available from: https://methods.sagepub.com/book/mono/statistical-methods-for-geography/toc JK MTWCMWB. Prenatal choline supplementation advances hippocampal development and enhances MAPK and CREB activation. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2004;18(3):545–7. Li Q, Guo-Ross S, Lewis DV, Turner D, White AM, Wilson WA, et al. Dietary prenatal choline supplementation alters postnatal hippocampal structure and function. J Neurophysiol. 2004;91(4):1545–55. Zeisel SH, Niculescu MD. Perinatal choline influences brain structure and function. Nutr Rev. 2006;64(4):197–203. Boeke CE, Gillman MW, Hughes MD, Rifas-Shiman SL, Villamor E, Oken E. Choline intake during pregnancy and child cognition at age 7 years. Am J Epidemiol. 2013;177(12). Cheatham CL, Goldman BD, Fischer LM, da Costa KA, Reznick JS, Zeisel SH. Phosphatidylcholine supplementation in pregnant women consuming moderate-choline diets does not enhance infant cognitive function: a randomized, double-blind, placebo-controlled trial. Am J Clin Nutr. 2012;96(6):1465–72. Freedman R, Hunter SK, Law AJ, Wagner BD, D'Alessandro A, Christians U, et al. Higher Gestational Choline Levels in Maternal Infection Are Protective for Infant Brain Development. J Pediatr. 2019;208:198–e2062. Jacobson SW, Carter RC, Molteno CD, Stanton ME, Herbert JS, Lindinger NM, et al. Efficacy of Maternal Choline Supplementation During Pregnancy in Mitigating Adverse Effects of Prenatal Alcohol Exposure on Growth and Cognitive Function: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Alcohol Clin Exp Res. 2018;42(7):1327–41. Obeid R, Derbyshire E, Schön C. Association between Maternal Choline, Fetal Brain Development, and Child Neurocognition: Systematic Review and Meta-Analysis of Human Studies. Adv Nutr. 2022;13(6):2445–57. Bragg MG, Prado EL, Caswell BL, Arnold CD, George M, Oakes LM et al. The association between plasma choline, growth and neurodevelopment among Malawian children aged 6–15 months enroled in an egg intervention trial. Matern Child Nutr. 2022;19(2). Wiedeman AM, Chau CMY, Grunau RE, McCarthy D, Yurko-Mauro K, Dyer RA, et al. Plasma Betaine Is Positively Associated with Developmental Outcomes in Healthy Toddlers at Age 2 Years Who Are Not Meeting the Recommended Adequate Intake for Dietary Choline. Randomized Controlled Trial. 2018;148(8):1309–14. Smuts CM, Matsungo TM, Malan L, Kruger HS, Rothman M, Kvalsvig JD, et al. Effect of small-quantity lipid-based nutrient supplements on growth, psychomotor development, iron status, and morbidity among 6- to 12-mo-old infants in South Africa: a randomized controlled trial. Am J Clin Nutr. 2019;109(1):55–68. Roberts SB, Franceschini MA, Silver RE, Taylor SF, de Sa AB, Có R et al. Effects of food supplementation on cognitive function, cerebral blood flow, and nutritional status in young children at risk of undernutrition: randomized controlled trial. BMJ. 2020. Cheatham C, Sheppard K. Synergistic Effects of Human Milk Nutrients in the Support of Infant Recognition Memory: An Observational Study. Nutrients. 2015;7(11):9079–95. Wozniak JR, Fuglestad AJ, Eckerle JK, Fink BA, Hoecker HL, Boys CJ, et al. Choline supplementation in children with fetal alcohol spectrum disorders: a randomized, double-blind, placebo-controlled trial. Am J Clin Nutr. 2015;102(5):1113–25. Ueland PM, Holm PI, Hustad S. Betaine: a key modulator of one-carbon metabolism and homocysteine status. Clin Chem Lab Med. 2005;43(10):1069–75. Holmes-McNary MQ, Cheng WL, Mar MH, Fussell S, Zeisel SH. Choline and choline esters in human and rat milk and in infant formulas. Am J Clin Nutr. 1996;64(4):572–6. Dietary. patterns, food groups, and nutrients as predictors of plasma choline and betaine in middle-aged and elderly men and women. American Journal of Clinial Nutrition. 2008. Wu C-H, Chang T-Y, Chen Y-C, Huang R-FS. PEMT rs7946 Polymorphism and Sex Modify the Effect of Adequate Dietary Choline Intake on the Risk of Hepatic Steatosis in Older Patients with Metabolic Disorders. Nutrients. 2023;15(14). Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013;368(17):1575–84. Xu N, Wan J, Wang C, Liu J, Qian C, Tan H. Increased Serum Trimethylamine N-Oxide Level in Type 2 Diabetic Patients with Mild Cognitive Impairment. Diabetes Metab Syndr Obes. 2022;15:2197–205. Zhu C, Li G, Lv Z, Li J, Wang X, Kang J, et al. Association of plasma trimethylamine-N-oxide levels with post-stroke cognitive impairment: a 1-year longitudinal study. Neurol Sci. 2020;41(1):57–63. Mudimela S, Vishwanath NK, Pillai A, Morales R, Marrelli SP, Barichello T, et al. Clinical significance and potential role of trimethylamine N-oxide in neurological and neuropsychiatric disorders. Drug Discovery Today. 2022;27(11):103334. Quan L, Yi J, Zhao Y, Zhang F, Shi XT, Feng Z, et al. Plasma trimethylamine N-oxide, a gut microbe-generated phosphatidylcholine metabolite, is associated with autism spectrum disorders. Neurotoxicology. 2020;76:93–8. Zhao L, Zhang C, Cao G, Dong X, Li D, Jiang L. Higher Circulating Trimethylamine N-oxide Sensitizes Sevoflurane-Induced Cognitive Dysfunction in Aged Rats Probably by Downregulating Hippocampal Methionine Sulfoxide Reductase A. Neurochem Res. 2019;44(11):2506–16. Zhou S, Liu J, Sun Y, Xu P, Liu JL, Sun S, et al. Dietary choline metabolite TMAO impairs cognitive function and induces hippocampal synaptic plasticity declining through the mTOR/P70S6K/4EBP1 pathway. Food & function; 2023. Govindarajulu M, Pinky PD, Steinke I, Bloemer J, Ramesh S, Kariharan T, et al. Gut Metabolite TMAO Induces Synaptic Plasticity Deficits by Promoting Endoplasmic Reticulum Stress. Front Mol Neurosci. 2020;13:138. Kadam Ii, Dalloul M, Hausser J, Vaday D, Gilboa E, Wang L, et al. Role of one-carbon nutrient intake and diabetes during pregnancy in children's growth and neurodevelopment: A 2-year follow-up study of a prospective cohort. Clin Nutr. 2024;43(6):1216–23. Iannotti LL, Lutter CK, Waters WF, Gallegos Riofrío CA, Malo C, Reinhart G, et al. Eggs early in complementary feeding increase choline pathway biomarkers and DHA: a randomized controlled trial in Ecuador. Am J Clin Nutr. 2017;106(6):1482–9. Iannotti LLLC, Stewart CP, Gallegos Riofrío CA, Malo C, Reinhart G, Palacios A, Karp C, Chapnick M, Cox K, Waters WF. Eggs in early complementary feeding and child growth: A randomized controlled trial. Pediatrics. 2017;140. Li Z, Wu G, Sher RB, Khavandgar Z, Hermansson M, Cox GA, et al. Choline kinase beta is required for normal endochondral bone formation. Biochimica et Biophysica Acta (BBA) -. Gen Subj. 2014;1840(7):2112–22. Ocque AJ, Stubbs JR, Nolin TD. Development and validation of a simple UHPLC-MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. J Pharm Biomed Anal. 2015;109:128–35. Additional Declarations No competing interests reported. Supplementary Files Supplementary2025620.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. 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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-7058350","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488805041,"identity":"112f6660-e20c-4199-a1ff-14cb4b2cce5f","order_by":0,"name":"Xuying Tan","email":"","orcid":"","institution":"Guangzhou Women and Children's Medical Center, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuying","middleName":"","lastName":"Tan","suffix":""},{"id":488805042,"identity":"ffe3e449-f346-4a52-a307-4a77c216b89a","order_by":1,"name":"Jiaxin Zhuang","email":"","orcid":"","institution":"Guangzhou Medical 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14:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7058350/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7058350/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87346837,"identity":"7609adff-e31c-4667-a98a-ad85ff3e2d0b","added_by":"auto","created_at":"2025-07-23 02:32:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66742,"visible":true,"origin":"","legend":"\u003cp\u003eFlow diagram for study sample selection.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7058350/v1/3f56a18f25f0740146900347.png"},{"id":87346838,"identity":"42ebef37-1fb0-4fce-b4d1-cf56d068b6d0","added_by":"auto","created_at":"2025-07-23 02:32:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103263,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplots of serum choline, betaine, and TMAO concentrations, along with betaine-to-choline and TMAO-to-choline ratios, by age in 109 infants, with Spearman correlation coefficients indicated.\u003c/p\u003e","description":"","filename":"floatimage21.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7058350/v1/7b92292a5110af0ead8cab80.jpeg"},{"id":87346846,"identity":"58319989-5f64-4c1b-bb8f-25c5a020ce4b","added_by":"auto","created_at":"2025-07-23 02:32:34","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81775,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot illustrating serum choline, betaine, and TMAO Levels. The box represents the inter - quartile range (IQR), with the median manifested as a horizontal line inside the box. The whiskers extend up to 1.5 times the IQR. Outliers are shown as separate circles beyond the whiskers.\u003c/p\u003e\n\u003cp\u003eSingle asterisk (*) indicates statistical significance at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \"ns\" signifies non-significance (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7058350/v1/d05ef559a5a131e6ee20533c.jpeg"},{"id":87346844,"identity":"47b84b38-1375-402e-9635-998e522d6e54","added_by":"auto","created_at":"2025-07-23 02:32:34","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":564098,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot of associations between choline, betaine, and TMAO and neurodevelopmental outcomes, adjusted for birth weight, sex, and blood collection age.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7058350/v1/ac90ed328a192302366c765a.jpeg"},{"id":88931280,"identity":"cad5beda-8e0d-4e8e-b452-3210b5e23419","added_by":"auto","created_at":"2025-08-12 21:46:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1848681,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7058350/v1/a93941d2-a931-46af-a2d0-6fdb3e73571c.pdf"},{"id":87346839,"identity":"f24a33d4-b661-4e31-9225-e9339ba3e280","added_by":"auto","created_at":"2025-07-23 02:32:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":32983,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary2025620.docx","url":"https://assets-eu.researchsquare.com/files/rs-7058350/v1/a3018dd33295be8a77cceeb6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Association of Serum Choline and Its Metabolites with Infant’s Growth and Neurodevelopment from Birth to 12 Months","fulltext":[{"header":"1. Background","content":"\u003cp\u003eCholine is an essential nutrient for normal cellular function, growth, and neurodevelopment in fetal and newborn infants[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Choline is the precursor of phosphatidylcholine, which are major components of cellular membranes. As for the function of choline in neurodevelopment, choline serves as a component of sphingomyelin, which is a constituent of the myelin sheath of nerve axons and facilitates efficient transmission of nerve signals[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The neurotransmitter, acetylcholine, is produced from free choline in cholinergic neurons. In addition, choline acts as a methyl donor by oxidized to betaine, which functions to donate methyl groups to homocysteine, producing the essential amino acid and methionine[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Trimethylamine \u003cem\u003eN\u003c/em\u003e-oxide (TMAO) is produced when dietary choline is converted to trimethylamine by the gut microbiota and then oxidized to TMAO in the liver[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Choline that converted to betaine or TMAO is irreversible.\u003c/p\u003e\u003cp\u003eEvidence from human studies revealed dietary choline supplementation during pregnancy and lactation have found improved cognitive, motor and language development before 24 months of age[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, increased maternal choline concentration also associated with improved memory, attention and processing speed in children[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Serum choline concentrations is highest at birth and remain elevated with a slow and gradual decrease during the first 2 years of life, which may be related to significant choline requirements for phospholipid synthesis in growing brain and other organs[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, choline and its metabolites serve as critical roles in the early growth and neurodevelopment[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. TMAO has been implicated in the aging related cognitive function decline, neuronal senescence and synaptic damage in the brain[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Molecular investigations have shown that TMAO activates astrocytes and microglia and triggers a cascade of inflammatory responses in the brain[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the relation between TMAO levels and neurodevelopment in children is still unknown.\u003c/p\u003e\u003cp\u003eOnly a limited number of studies have examined relations between serum choline metabolite levels and neurodevelopment and growth in early childhood[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To date, no study has examined the impact of the infant\u0026rsquo;s, rather than the mother\u0026rsquo;s, choline metabolites levels on neurodevelopment. We hypothesized that higher choline and betaine concentrations are related to a higher neurodevelopment quotient and linear growth, while TMAO showed negative correlations in infants of 0\u0026ndash;12 months. Therefore, we conducted a historical observing study to estimate the relation between choline status and the neurodevelopment and growth in infants of 0\u0026ndash;12 months. Firstly, we aimed to determine the concentration of serum choline, betaine and TMAO after birth during 0-365 postnatal days and investigate the relation between choline status with age. Secondly, we aimed to examined the relationship between choline status and neurodevelopment and growth in infants of 0\u0026ndash;12 months.\u003c/p\u003e"},{"header":"2. Subjects and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Study population\u003c/h2\u003e\u003cp\u003eThis study was designed as an observational, descriptive, and retrospective study. We retrospectively reviewed the cases of outpatients who admitted to Guangzhou Women and Children\u0026rsquo;s Medical Center for the neurodevelopmental assessments between January 2018 and September 2019. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki. The study was reviewed and approved by the ethics committee of the Guangzhou Women and Children\u0026rsquo;s Medical Center (Number: [2021]027A01).\u003c/p\u003e\u003cp\u003eInclusion criteria were as follows: (Ⅰ) Term infant aged between 0 and 12 months; (Ⅱ) Patients who had completed the neurodevelopmental assessment scale; (Ⅲ) Patients with residual blood specimens after clinical analysis; (Ⅳ) Patients underwent neurodevelopmental assessment and blood sampling within 30 days to minimize temporal bias. The exclusion criteria were as follows: (Ⅰ) Patients with chromosomal or genetic abnormalities; (Ⅱ) Encephalodysplasia; (Ⅲ) Patients with neurological diseases or injuries.\u003c/p\u003e\u003cp\u003eParticipant screening process: of 46,344 patients screened, 45,634 were aged 0 to 12 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For the analysis, 44,720 cases without residual blood specimens were excluded, leaving 914 infants with preserved blood samples stored in the study biorepository. Among these, 153 infants underwent blood sampling and neurodevelopmental assessments within 30 days. After excluding 1 case with chromosomal abnormalities and 2 cases with brain injuries, 150 eligible infants were identified. Finally, 109 term infants (aged 0 to 12 months) with both neurodevelopmental test results and stored blood specimens were included in this study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Serum choline, betaine and TMAO measurements\u003c/h2\u003e\u003cp\u003eUnsued residual blood specimens after clinical testing were collected for retrospective research and stored at -80\u0026deg;C within 24 hours after to maintain sample integrity. Residual blood specimens after clinical testing were obtained with informed consent and used in accordance with institutional review board guidelines. Serum choline, betaine and TMAO were measured using high performance liquid chromatography-tandem MS/MS (HPLC-MS/MS) (Agilent 6400 Series Triple Quad LCMS; CA, USA), with multi-reaction monitoring (MRM) functions[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 60 \u0026micro;L of either serum sample or standards was mixed with 100 \u0026micro;L of acetonitrile containing 10 \u0026micro;M of internal standards d9-TMAO (Toronto Research Chemicals Inc., Toronto, Canada) and d9-choline (Sigma-Aldrich, St. Louis, USA). The mixture was vortexed and then centrifuged at 13,000 \u0026times; g for 10 minutes to precipitate proteins. The supernatant was injected into a normal-phase silica gel column (2.1 mm \u0026times; 100 mm, 5 \u0026micro;m particle size). The column was eluted isostatically with 30% A solution (15 mM ammonium formate aqueous solution; pH 3.0) and 70% B solution (acetonitrile) at a 0.2 mL/min flow rate. For free choline, betaine, and TMAO, the within-day coefficients of variation (CVs) are 0.79%, 1.53%, and 1.62%, respectively. The between-day CVs for these compounds are 2.71%, 2.68%, and 6.53%, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Demographic characteristics\u003c/h2\u003e\u003cp\u003eBirth outcome data, such as gestational age, gender, birth weight, birth length and delivery mode were obtained from medical records of the hospital. Gestational age was based on last menstrual period and was confirmed by ultrasound scanning performed at \u0026le;\u0026thinsp;20 wk gestation. If the gestational age estimated by sonographic differed by \u0026gt;\u0026thinsp;2 wk from the last menstrual period, the sonographic dating was used[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Neurodevelopmental assessment\u003c/h2\u003e\u003cp\u003eThe infant\u0026rsquo;s neurodevelopment was evaluated by the China Developmental Scale for Children aged 0\u0026ndash;6 years, which is an indigenous and diagnostic development assessment tool with Chinese norms. The China Developmental Scale for Children was developed by the Capital Institute of Pediatrics of China since the early 1980s. The scale includes 261 items on five areas, namely, gross motor, fine motor, language, adaptive behavior and personal-social. The five areas were consistent with the relevant subscales in Gesell and showed adequate reliability in children with typical development[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The full scale developmental quotient (DQ) or a subscale quotient less than 70 pointes indicates a developmental delay; a quotient between 70 and 79 points is slightly below the threshold for developmental delay, and a quotient greater than or equal to 80 points indicates no developmental delay[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The neurodevelopmental assessment was performed when the infant in well awake status by extensively trained and certified pediatric physiotherapists who had no knowledge of the results of the infants\u0026rsquo; serum analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Anthropometric measurements\u003c/h2\u003e\u003cp\u003eThe anthropometric measurements of weight (in kilograms), length (in centimeters), and head circumference (in centimeters) were conducted by two trained and experienced nurses at the infant\u0026rsquo;s birth and at 1, 3, 6, 8 and 12 months of age. The infant\u0026rsquo;s weight was measured to the nearest 0.01 kg using a calibrated digital scale (SECA 335, SECA GmbH, Hamburg, Germany) without clothes and diapers. Without clothing and shoes, the length was measured to the nearest 0.1cm using a calibrated rigid height board (SECA 335, SECA GmbH, Hamburg, Germany). Head circumference was measured to the nearest 0.1cm using a non-elastic plastic tape measure. All measurements were repeated twice. The errors between each measurement should be less than 0.5 cm for body length, less than 0.01 kg for weight and less than 0.2cm for head circumference. The mean values were taken as the final values.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical methods\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using SPSS 20.0 software (SPSS, Chicago, Illinois, USA) and R 4.3.1 software (R Foundation for Statistical Computing, Vienna, Austria). Continuous variables were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs), and categorical variables were expressed as numbers and percentages (%). The included individuals were divided into three groups based on the age of blood collection: 0\u0026ndash;90 days, 91\u0026ndash;180 days and 181\u0026ndash;365 days. Wilcoxon\u0026rsquo;s signed rank tests were used to compare choline, betaine and TMAO levels among different age windows in infants. Spearman's rank correlation coefficients were employed to evaluate the association between choline and its related metabolites with age, as well as with neurodevelopmental outcomes. After natural log transformation of serum choline, betaine and TMAO levels among three groups, one-way analysis of variance (ANOVA) with linear tests for trend were used to compare the differences between groups. Bonferroni post-hoc tests were used for multiple comparisons[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Associations of choline and its related metabolites with each of the five neurodevelopmental endpoints in different age windows were evaluated with separate multiple linear regression analyses, adjusted for gestational age, birth weight and gender. A two-sided significance level of 0.05 was used to evaluate the above statistical tests.\u003c/p\u003e\u003cp\u003eIn order to select the best model for the weight, length and head circumference parameters at each measurement point (at birth and 1, 3, 6, 8, and 12 months), linear, quadratic, and cubic growth curve models were constructed. The optimal model for growth curve construction was selected based on the lowest values of Akaike Information Criterion, corrected Akaike Information Criterion, and Bayesian Information Criterion[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Subsequently, linear mixed-effect regression models were used to evaluate associations between anthropometric parameters and serum concentrations of choline and its metabolites, while adjusting for covariates including age, gender, and gestational age. These models accommodated repeated measures and accounted for within-subject correlations by incorporating random intercepts, random slopes, and an unstructured covariance matrix. Multicollinearity was assessed using the variance inflation factor, with values\u0026thinsp;\u0026lt;\u0026thinsp;5 indicating no significant collinearity[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Within each model, a two-sided \u003cem\u003eP\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characteristics of the study population\u003c/h2\u003e\u003cp\u003eA total of 109 patients who met these inclusion criteria were enrolled for the analysis. Characteristics were described across three age groups categorized by the age at blood sample collection. The gestational age of infants demonstrated significant difference among three groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03). Specifically, the 181\u0026ndash;365 days group had a gestational age of 39.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 weeks, compared to 38.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 weeks in the 0\u0026ndash;90 days group and 38.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 weeks in the 91\u0026ndash;180 days group. No significant differences were observed among the three groups in birth weight, birth length, or delivery mode. The 0\u0026ndash;90 days group consisted of 14 males (56%) and 11 females (44%), with a mean age of 52.28\u0026thinsp;\u0026plusmn;\u0026thinsp;20.52 days at blood collection. The 91\u0026ndash;180 days group included 25 males (55.56%) and 20 females (44.44%), with a mean age of 137.51\u0026thinsp;\u0026plusmn;\u0026thinsp;23.39 days at the time of blood collection. The 181\u0026ndash;365 days group comprised 19 males (51.4%) and 18 females (48.6%), with a mean age of 231.03\u0026thinsp;\u0026plusmn;\u0026thinsp;51.07 days at the point of blood collection.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCharacteristics of the study population (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;109)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u0026ndash;90 days\u003c/p\u003e\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e91\u0026ndash;180 days\u003c/p\u003e\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;48)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e181\u0026ndash;365 days\u003c/p\u003e\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;37)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGestational age\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBirth weight (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3057.08\u0026thinsp;\u0026plusmn;\u0026thinsp;387.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3012.67\u0026thinsp;\u0026plusmn;\u0026thinsp;314.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3036.49\u0026thinsp;\u0026plusmn;\u0026thinsp;441.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBirth length (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e49.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.70\u0026thinsp;\u0026plusmn;\u0026thinsp;8.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale [n (%)]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14 (56%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25(55.56%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19 (51.4%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDelivery mode [n (%)]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVaginal delivery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (52%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27 (60.0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23 (62.16%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbdominal delivery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12 (48%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15 (33.3%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12 (32.43%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eObstetric forceps\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 (0%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (6.67%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (5.41%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge at blood collection (days)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e52.28\u0026thinsp;\u0026plusmn;\u0026thinsp;20.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e137.51\u0026thinsp;\u0026plusmn;\u0026thinsp;23.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e231.03\u0026thinsp;\u0026plusmn;\u0026thinsp;51.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Correlations of Serum Choline, Betaine, and TMAO Levels with Age\u003c/h2\u003e\u003cp\u003eSpearman correlation revealed no significant associations between serum choline, betaine levels and age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Serum TMAO levels and TMAO-to-choline ratios exhibited significant positive correlations with age (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.353, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.357, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSerum levels of choline, betaine, and TMAO across different age groups are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Neither serum choline nor betaine levels, nor the betaine-to-choline ratio, showed statistically significant differences among age groups. By contrast, serum TMAO levels and the TMAO-to-choline ratio exhibited significant intergroup variations and were positively correlated with age (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u0026thinsp;diff\u003c/sub\u003e = 0.04, \u003cem\u003eP\u003c/em\u003e-\u003csub\u003etrend\u003c/sub\u003e = 0.02; \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u0026thinsp;diff\u003c/sub\u003e = 0.03, \u003cem\u003eP\u003c/em\u003e-\u003csub\u003etrend\u003c/sub\u003e = 0.01, respectively). The median serum TMAO level in 181\u0026ndash;365-day-old infants was 6.94-fold higher than that in 0\u0026ndash;90-day-old infants (1.11 \u0026micro;M \u003cem\u003evs\u003c/em\u003e 0.16 \u0026micro;M, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04). The median TMAO:choline ratio in the 181\u0026ndash;365-day-old group was 7.5-fold higher than that in the 0\u0026ndash;90-day-old group (0.03 \u003cem\u003evs\u003c/em\u003e 0.004 \u0026micro;M, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Associations of Serum Choline, Betaine, and TMAO Levels with Neurodevelopment\u003c/h2\u003e\u003cp\u003eSpearman correlation analyses were conducted to examine associations between serum choline and its metabolite levels and neurodevelopment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Serum choline levels showed positive correlations with both the full-scale developmental quotient (DQ) and fine motor quotient (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.22, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02 for both). No significant correlations were observed for serum betaine or the betaine/choline ratio with any neurodevelopmental measures. By contrast, serum TMAO levels exhibited negative correlations with the full-scale DQ (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.22, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) and gross motor quotient (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.25, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e\u003cp\u003eWe further investigated associations between choline and related metabolites and neurodevelopmental outcomes across three age cohorts. Results of multiple linear regression analyses adjusted for gestational age, birth weight, and gender are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In infants aged 0 to 90 days, no statistically significant associations were observed between choline, betaine, or TMAO and any neurodevelopmental outcomes. In infants aged 91 to 180 days, a 1-unit increase in z-score of ln-transformed serum choline levels was significantly associated with: a 5.27-point increase in full-scale DQ (95% CI: [0.43, 10.10], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03), an 8.56-point increase in the fine motor quotient (95% CI: [2.04, 15.07], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), and an 8.45-point increase in the personal-social quotient (95% CI: [1.42, 15.47], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02). Conversely, serum betaine levels demonstrated significant negative associations with full-scale DQ and the language quotient. Specifically, each 1-unit increase in z-score of ln-transformed serum betaine was associated with a 4.68-point reduction in full-scale DQ (95% CI: [-9.23, -1.14], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04) and a 6.76-point decrease in the language quotient (95% CI: [-11.83, -1.69], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01). In 181-365-day-old infants, elevated ln-transformed serum choline levels were significantly associated with improvements in full-scale DQ, adaptive behavior, and language quotient. Specifically, each 1-unit increase in the z-score of ln-choline was associated with a 3.59-point increase in full-scale DQ (95% CI: [0.37, 6.81], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03). Similarly, ln-transformed serum betaine levels exhibited positive associations with personal-social quotient, with a 6.96-point increase observed per 1-unit z-score elevation (95% CI: [0.93, 12.99], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03). By contrast, serum TMAO levels were negatively associated with the gross motor quotient, with each 1-standard deviation increase in TMAO corresponding to a 7.38-point decrease in the gross motor quotient (95% CI: [-13.62, -1.15], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelations of Serum Choline and Its Metabolites with Neurodevelopment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"17\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eCholine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eBetaine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003eTMAO\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eBetaine/Choline\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c17\" namest=\"c15\"\u003e\u003cp\u003eTMAO/Choline\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFull Scale DQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e-0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u003cb\u003e0.04\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGross motor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e-0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFine motor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e-0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdaptive Behavior\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLanguage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e-0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePersonal-social\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e-0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c17\" namest=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4. No Correlations between Choline and Its Metabolite Levels and Infant Growth\u003c/h2\u003e\u003cp\u003eLinear mixed regression models were used to examine the associations between serum choline and its metabolites (betaine, TMAO) and infant growth indicators (weight, length and head circumference). After adjusting for age, gender, and gestational age, linear mixed model results showed no significant associations between choline, betaine, or TMAO levels and infant growth (length, weight, head circumference) during 0\u0026ndash;12 months (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, birth weight was positively correlated with all growth parameters (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gestational age and the infant\u0026rsquo;s age at blood collection were not significantly associated with any growth outcomes (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;3).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelations of Serum Choline and Its Metabolites with Infant Weight, Length, and Head Circumference\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSerum choline and its metabolites\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eLength\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eWeight\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eHead circumference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eβ (95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eβ (95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eβ (95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLn betaine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.02 (-0.01, 0.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.04 (-0.02, 0.09)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.03 (-0.05, 0.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLn choline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.02 (-0.01, 0.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.02 (-0.03, 0.07)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.06 (-0.01, 0.13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLn TMAO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-0.02 (-0.07, 0.02)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.02 (-0.08, 0.04)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.04 (-0.05, 0.13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003eLinear mixed regression models were employed and adjusted for age, gender, gestational age.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrates that elevated serum choline levels are associated with improved neurodevelopment in 6\u0026ndash;12-month-old infants. By contrast, serum betaine was negatively associated with the full-scale DQ and language quotient in infants aged 91\u0026ndash;180 days. Serum TMAO was not associated with most measures of infant\u0026rsquo;s neurodevelopment. Moreover, no statistically significant associations were detected between serum choline or its metabolites and the growth parameters of infants from 0 to 12 months. Serum TMAO levels were negative correlated with age, while neither serum choline nor betaine concentrations exhibited age - related correlations.To our knowledge, this is the first research to explore the associations of serum choline, betaine, and TMAO with growth and neurodevelopment of infants from birth to 12 months.\u003c/p\u003e\u003cp\u003eCholine is required for the synthesis of phosphatidylcholines, which are the major component of cell membranes, are essential for the growth and development[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Choline is also an essential component of sphingomyelin. Additionally, choline is present in the nervous system, which it serves for the synthesis of acetylcholine. An impressive array of animal studies has shown that prenatal choline supplementation enhances the cognitive ability in offspring, probably related to the improve in structure and function of hippocampal pyramidal cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Conversely, choline deficiency in late pregnancy showed hippocampal apoptosis and impaired memory offspring rats[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, results from observational studies on choline and neurodevelopment were variable. A prospective study included 404 mother-child pairs found no correlation of physiologic free and total choline concentrations during pregnancy or in cord blood with children\u0026rsquo;s intelligence at 5 years old[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Another study presented that higher maternal choline intake was associated with modestly higher child visual memory score at age 7 years[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As in the clinical trial studies, women supplemented with phosphatidylcholine (that delivers 750 mg/d) during pregnancy and early lactation, showed no advantage in improvement of brain development in their infants[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Supplementation of choline during pregnancy in woman who exposed to infection[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] or alcohol[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] showed protective effect of choline in the brain development of their infants. Based on the above studies, the protective effect of gestational choline supplementation in the development of infant brain appeared in women exposed to a low-choline diet or other detrimental factors during pregnancy.\u003c/p\u003e\u003cp\u003eAs the brain continues to develop rapidly in the first 2 years after birth, choline status is likely to have an impact on neurodevelopment[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. There is limited research conducted on choline nutrition after birth and neurodevelopment outcomes, particularly in infants and toddlers, and previous studies have generally yielded positive or null results. An observational study conducted in low income country including children aged 6\u0026ndash;15 months and those with milk intake less than 236 ml/day, found no strong or consistent associations between plasma choline and growth and development[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Another study showed higher plasma betaine concentrations linked to better visual-motor skills, but not choline in healthy toddlers aged 2 years who did not meet the recommended dietary choline intake[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Clinical trial studies found provision of supplementary foods containing choline, along with other nutrients, led to improve in working memory and locomotor in young children in South Africa[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, higher choline and higher docosahexaenoic acid in breast milk was related to better recognition memory in 6-month-old infants[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Of note, choline alone may not provide benefit if other proteinogenic amino acids and fatty acids are not sufficient in the diet. Besides, early postnatal choline supplementation in children with diseases of the nervous system such as fetal alcohol spectrum disorders, revealed improved in memory function[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe found consistent and mostly positive associations of serum choline levels with developmental outcomes in infants aged 0\u0026ndash;12 months. In the current study, the results from Spearman relation analyses indicated positive associations between serum choline levels and both full scale DQ and fine motor quotients in infants aged 0\u0026ndash;12 months. However, no correlations were observed with betaine. After adjusting for age, birth weight and gender, positive associations between serum choline and neurodevelopment were revealed in infants aged 6\u0026ndash;12 months. It is plausible that during the initial six - month period after birth, the choline reserves in infants are mainly derived from the choline stored during fetal development and the postnatal choline ingested from dairy products. Once complementary foods are introduced at six months of age, differences in the choline status among different infants gradually become evident. Consequently, the positive correlations between choline and neurodevelopment also manifested between 6\u0026ndash;12 months of age.\u003c/p\u003e\u003cp\u003eBetaine serves as a major methyl donor for homocysteine in the synthesis of methionine[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our findings revealed that serum betaine concentrations were negatively correlated with the full scale DQ and language quotients in infants aged 91\u0026ndash;180 days. We hypothesize that owing to the irreversible conversion of choline to betaine, the quantity of free choline available for the synthesis of acetylcholine and structural phospholipids, such as phosphatidylcholine and sphingomyelin, has been decreased.\u003c/p\u003e\u003cp\u003eCholine levels showed higher in this setting than in previous reports[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The inconsistent results among the different studies may be due to differences in choline reserves during fetal development, feeding patterns, sociodemographic factors, and genetic polymorphisms in choline metabolizing enzymes that are unique to the study population. First, plasma choline concentration declined over the first year after birth[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], comparing choline status across studies with different age groups poses a challenge. Second, the amount of choline in breast milk varies widely and is affected by the diet of the nursing mother[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Besides, the complementary feeding period to induce animal source foods, which are rich in choline, may be particularly important to ensure adequate choline intake and impact on the choline status in infants[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Third, genetics may also play a role, given the choline metabolism is regulated by phenylethanolamine methyl transferase (PEMT) and influenced by the polymorphisms in gene PEMT[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCholine may be irreversibly catabolized by intestinal flora to produce TMAO[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the main source of TMAO in the human body. Although TMAO is associated with the metabolic diseases[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and impaired of cognitive function in adults[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], its effects in young children are unclear. TMAO can cross the blood-brain barrier and are involved in brain development, neurogenesis and behavior[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. A cross-sectional study of 328 children aged 3\u0026ndash;8 years revealed an association between elevated plasma TMAO concentrations and the severity of autism spectrum disorder and its symptoms, indicating that TMAO could be used as a biomarker of autism[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Animal studies have shown that TMAO induced cognitive impairment through aggravating oxidative damage[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], promoting neuroinflammation and impairment of mTOR signaling[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and synaptic plasticity[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We discovered that in infants ranging from 0 to 12 months old, postnatal trimethylamine N-oxide (TMAO) levels exhibited a gradual increase with advancing age. However, in the present study, associations of TMAO with neurodevelopment were null except higher TMAO was associated with lower gross motor quotients in infants aged 6\u0026ndash;12 months.\u003c/p\u003e\u003cp\u003eSpecific to the relationship between choline and growth, previous study showed that low serum choline, rather than serum betaine or TMAO concentration was correlated with stunting in young children aged 12\u0026ndash;59 months living in low-income countries[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another prospective cohort study found no associations between either the maternal blood choline and its metabolites or postnatal choline intakes and anthropometric measurements at 2 years[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. An egg intervention trial in children ages 6 to 9 months showed the egg intervention increased the plasma concentrations of choline and betaine[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], with the improved in linear growth and reducing stunting[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The growth of bones requires the synthesis of phosphatidylcholines by osteoblasts and the endochondral plate[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In the animal study, the deficient of choline kinase β gene in mice have reduced bone mass and decreased limb length[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, in the present study, general liner mixed model showed no significant correlation between choline, betaine and the growth parameters. There may be factors such as differences in the overall diet and multiple other nutrients and bioactive factors converging to influence linear growth during infancy.\u003c/p\u003e\u003cp\u003eSome limitations are present in this study. First, the determination of serum choline and related compounds relied on remnants of clinically indicated samples. Previous study have reported that the concentration of choline remain stability at 4 ℃ with 24 h and the remaining serum specimens after clinical laboratory test were collected within 24 h[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, the measurement with remaining serum specimens is likely to have limited impact on the infant\u0026rsquo;s choline status. Second, due to the retrospective nature of this study, the blood sampling was not always available exactly on the day prior to the neurodevelopmental assessment. However, to make the correlation analyses possible, the blood samples we included were within 30 days matched with neurodevelopmental evaluation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, we found few significant associations between the concentration of choline and neurodevelopment outcomes in 6\u0026ndash;12-month-old infants. Our results suggest that choline status may be an important factor in neurodevelopment. Controlled clinical trials are warranted to determine whether increasing dietary choline intake or choline supplementation can improve linear growth and neurodevelopment during the first year of life.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTMAO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Trimethylamine N-oxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPLC-MS/MS \u0026nbsp; High-performance liquid chromatography tandem mass spectrometry\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Multi-reaction monitoring\u003c/p\u003e\n\u003cp\u003eDQ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Developmental quotient\u003c/p\u003e\n\u003cp\u003eANOVA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;One-way analysis of variance\u003c/p\u003e\n\u003cp\u003eIQR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Inter - quartile range\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePEMT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phenylethanolamine methyl transferase\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was reviewed and approved by the ethics committee of the Guangzhou Women and Children\u0026rsquo;s Medical Center (Number: [2021]027A01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the funding \u0026ldquo;Nutrition and Care of Maternal \u0026amp;Child Research Fund Project\u0026rdquo; of Biostime Institute of Nutrition \u0026amp; Care (Grant/Award Number: 2020BINCMCF047) and Student Innovation Ability Enhancement Program of Guangzhou Medical University (Grant/Award Number: 02-408-240603131100)\u003c/p\u003e\n\u003ch3\u003eAuthor Contribution\u003c/h3\u003e\n\u003cp\u003eConceptualization, X.T. and J.Z.; Methodology, Y.X.;; Formal Analysis, S.Q. and G.L.; Investigation, L.G. and Z.L.; Writing \u0026ndash; Original Draft Preparation, X.T.; Writing \u0026ndash; Review \u0026amp; Editing, C.G.; Visualization, Y.H.; Supervision, Y.H.; Project Administration, Y.H.;\u003c/p\u003e\n\u003ch3\u003eAcknowledgement\u003c/h3\u003e\n\u003cp\u003eThank Clinical Biological Resource Bank of Guangzhou women and children\u0026rsquo;s medical center for providing the clinical samples.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and materials\u003c/h3\u003e\n\u003cp\u003eAll data analyzed in the current research are available from the corresponding author on logical request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZeisel SH, da Costa KA. 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Clin Nutr. 2024;43(6):1216\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIannotti LL, Lutter CK, Waters WF, Gallegos Riofr\u0026iacute;o CA, Malo C, Reinhart G, et al. Eggs early in complementary feeding increase choline pathway biomarkers and DHA: a randomized controlled trial in Ecuador. Am J Clin Nutr. 2017;106(6):1482\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIannotti LLLC, Stewart CP, Gallegos Riofr\u0026iacute;o CA, Malo C, Reinhart G, Palacios A, Karp C, Chapnick M, Cox K, Waters WF. Eggs in early complementary feeding and child growth: A randomized controlled trial. Pediatrics. 2017;140.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Wu G, Sher RB, Khavandgar Z, Hermansson M, Cox GA, et al. Choline kinase beta is required for normal endochondral bone formation. Biochimica et Biophysica Acta (BBA) -. Gen Subj. 2014;1840(7):2112\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOcque AJ, Stubbs JR, Nolin TD. Development and validation of a simple UHPLC-MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. J Pharm Biomed Anal. 2015;109:128\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"choline, betaine, trimethylamine N-oxide, neurodevelopment, growth","lastPublishedDoi":"10.21203/rs.3.rs-7058350/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7058350/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Choline is an essential nutrient that plays crucial roles in cell structure maintenance, neurotransmission, and betaine synthesis. Trimethylamine N-oxide (TMAO) is biosynthesized from choline through metabolic processes mediated by gut microbiota and the liver. However, the relationships among serum choline, its metabolites, and early neurodevelopmental and growth remain unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: This retrospective study enrolled 109 outpatients aged 0–12 months who underwent neurodevelopmental assessments using the China Developmental Scale for Children at the Guangzhou Women and Children’s Medical Center from January 2018 to September 2019. Residual blood specimens obtained post-clinical testing were collected for subsequent analysis. To mitigate temporal bias, both neurodevelopmental assessment and blood sampling were conducted within a 30-day window for each participant. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was employed to measure the serum concentrations of choline, betaine, and TMAO. Anthropometric parameters, including weight, length, and head circumference, were recorded at birth and 1, 3, 6, 8, and 12 months of age.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Serum choline levels were significantly associated with enhanced neurodevelopment in 6- to 12-month-old infants. Conversely, serum betaine concentrations exhibited a negative correlation with the full-scale developmental quotient and language quotient in infants aged 91 to 180 days. Serum TMAO showed no significant associations with most indices of infant neurodevelopment. Additionally, no statistically significant correlations were observed between serum choline or its metabolites and infant growth parameters from 0 to 12 months.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our study identified specific associations between choline concentration and neurodevelopment in 6- to 12-month-old infants, suggesting that choline status may be a pivotal determinant of neurodevelopment during early infancy.\u003c/p\u003e","manuscriptTitle":"Association of Serum Choline and Its Metabolites with Infant’s Growth and Neurodevelopment from Birth to 12 Months","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 02:32:29","doi":"10.21203/rs.3.rs-7058350/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":"e86c08b4-9170-4420-b285-45127c20cb49","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T21:38:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 02:32:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7058350","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7058350","identity":"rs-7058350","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}