Maternal weight, gut microbiota, and the association with early childhood behavior: the PREOBE follow-up study.

BACKGROUND AND AIM: Maternal overweight and breastfeeding seem to have a significant impact on the gut microbiota colonization process, which co-occurs simultaneously with brain development and the establishment of the "microbiota-gut-brain axis", which potentially may affect behavior later in life. This study aimed to examine the influence of maternal overweight, obesity and/or gestational diabetes on the offspring behavior at 3.5 years of age and its association with the gut microbiota already established at 18 months of life. METHODS: 156 children born to overweight (OV, n = 45), obese (OB, n = 40) and normoweight (NW, n = 71) pregnant women participating in the PREOBE study were included in the current analysis. Stool samples were collected at 18 months of life and gut microbiome was obtained by 16S rRNA gene sequencing. Behavioral problems were evaluated at 3.5 years by using the Child Behavior Checklist (CBCL). ANOVA, Chi-Square Test, ANCOVA, Spearman's correlation, logistic regression model and generalized linear model (GLM) were performed. RESULTS: At 3.5 years of age, Children born to OV/OB mothers showed higher scores in behavioral problems than those born to NW mothers. Additionally, offspring born to OB mothers who developed gestational diabetes mellitus (GDM) presented higher scores in attention/deficit hyperactivity and externalizing problems than those born to GDM OV/NW mothers. Fusicatenibacter abundance found at 18 months of age was associated to lower scores in total, internalizing and pervasive developmental problems, while an unidentified genus within Clostridiales and Flavonifractor families abundance showed a positive correlation with anxiety/depression and somatic complaints, respectively. On the other hand, children born to mothers with higher BMI who were breastfed presented elevated anxiety, internalizing problems, externalizing problems and total problems scores; likewise, their gut microbiota composition at 18 months of age showed positive correlation with behavioral problems at 3.5 years: Actinobacteria abundance and somatic complaints and between Fusobacteria abundance and withdrawn behavior and pervasive developmental problems. CONCLUSIONS: Our findings suggests that OV/OB and/or GDM during pregnancy is associated with higher behavioral problems scores in children at 3.5 years old. Additionally, associations between early life gut microbiota composition and later mental health in children was also found.

Infant Gut Microbiota Associated with Fine Motor Skills.

BACKGROUND: During early life, dynamic gut colonization and brain development co-occur with potential cross-talk mechanisms affecting behaviour. METHODS: We used 16S rRNA gene sequencing to examine the associations between gut microbiota and neurodevelopmental outcomes assessed by the Bayley Scales of Infant Development III in 71 full-term healthy infants at 18 months of age. We hypothesized that children would differ in gut microbial diversity, enterotypes obtained by Dirichlet multinomial mixture analysis and specific taxa based on their behavioural characteristics. RESULTS: In children dichotomized by behavioural trait performance in above- and below-median groups, weighted Unifrac b-diversity exhibited significant differences in fine motor (FM) activity. Dirichlet multinomial mixture modelling identified two enterotypes strongly associated with FM outcomes. When controlling for maternal pre-gestational BMI and breastfeeding for up to 3 months, the examination of signature taxa in FM groups showed that Turicibacter and Parabacteroides were highly abundant in the below-median FM group, while Collinsella, Coprococcus, Enterococcus, Fusobacterium, Holdemanella, Propionibacterium, Roseburia, Veillonella, an unassigned genus within Veillonellaceae and, interestingly, probiotic Bifidobacterium and Lactobacillus were more abundant in the above-median FM group. CONCLUSIONS: Our results suggest an association between enterotypes and specific genera with FM activity and may represent an opportunity for probiotic interventions relevant to treatment for motor disorders.

The Nutritional Profile of Food Advertising for School-Aged Children via Television: A Longitudinal Approach.

The prevalence of childhood obesity continues to increase. Screen time, one of the most documented reasons for the obesogenic environment, enhances childhood obesity, since advertisements for unhealthy food products are still broadcast on channels for children. This is presently one of the main challenges for the government in Spain, since the current laws and obligations are not updated. This study aims to analyze food advertising aimed at children on Spanish television in 2013 and 2018 on children's and general channels to test the effect of laws and obligations over time. In total, we viewed 512 h of the most viewed channels, two children's and two general channels, during the week and on weekends during specific periods of 2013 and 2018. Food advertising was categorized as core, non-core, and other food advertisement (CFA, NCFA, and OFA, respectively) according to the nutritional profile. A total of 2935 adverts were analyzed, 1263 in 2013 and 1672 in 2018. A higher proportion of NCFAs were broadcast on children's channels than in prior years, rising from 52.2% to 69.8% (p < 0.001). Nowadays, the risk of watching NCFAs on children's channels compared to general channels turns out to be higher (Odds ratio > 2.5; p < 0.001), due to exposure to adverts for high-sugar and high-fat foods such as cakes, muffins, cookies, and fried and frozen meals rich in fat. In conclusion, the trends of nutritional profiles in food advertising on television are worsening over time, since the prevalence of NCFAs was higher in 2018 than in 2013. Currently, CFAs are not mainly broadcast on children's channels, confirming high-risk exposure to non-core food advertising by watching them. Thus, food advertising laws and obligations should be adapted to increase compliance.

Association study of rs1801282 PPARG gene polymorphism and immune cells and cytokine levels in a Spanish pregnant women cohort and their offspring.

BACKGROUND: Peroxisome proliferator activated receptor gamma (PPARG) belongs to the nuclear receptor superfamily functioning as transcription factors to regulate cellular differentiation, development and metabolism. Moreover, it has been implicated in the regulation of lipid metabolism, as well as the maturation of monocytes/macrophages and the control of inflammatory reactions. The aim of this study was to evaluate the relationship between the Pro12Ala (rs1808212) PPARG gene polymorphism on immune molecular and cellular components in mothers and their offspring participating in the PREOBE study. METHODS: DNA from maternal venous blood samples at 24, 34 and 40 gestational weeks, plus cord blood samples was extracted. Pro12Ala PPARG polymorphism genotyping was performed, and immune system markers were analyzed by flow cytometry. RESULTS: Study findings revealed no effect of rs1808212 PPARG genotypes on innate immune parameters in mothers and their offspring; however, CD4 + /CD8 + ratio were decreased at 24 and 34 weeks in pregnant women carrying the CG (Pro12Ala) rs1808212 polymorphism, (p = 0,012 and p = 0,030; respectively). Only CD19 levels in peripheral blood were significantly higher at delivery in pregnant women carrying the CC (Pro12Pro) genotype (p ≤ 0.001). Moreover, there were statistically significant differences in leukocytes and neutrophils maternal levels at 34 weeks of gestation, being lower in carriers of Pro12Ala genotype (p = 0.028 and p = 0.031, respectively). CONCLUSIONS: Results suggest that Pro12Ala PPARG polymorphism may have an effect on some cell and immune parameters in pregnant women during pregnancy and at time of delivery. However, newborn innate immune system does not seems to be influenced by PPARG Pro12Ala polymorphism in cord blood.

Effects of Maternal Fish Oil and/or 5-Methyl-Tetrahydrofolate Supplementation during Pregnancy on Offspring Brain Resting-State at 10 Years Old: A Follow-Up Study from the NUHEAL Randomized Controlled Trial.

Recent studies have shown that maternal supplementation with folate and long-chain polyunsaturated fatty acids (LC-PUFAs) during pregnancy may affect children's brain development. We aimed at examining the potential long-term effect of maternal supplementation with fish oil (FO) and/or 5-methyl-tetrahydrofolate (5-MTHF) on the brain functionality of offspring at the age of 9.5-10 years. The current study was conducted as a follow-up of the Spanish participants belonging to the Nutraceuticals for a Healthier Life (NUHEAL) project; 57 children were divided into groups according to mother's supplementation and assessed through functional magnetic resonance imaging (fMRI) scanning and neurodevelopment testing. Independent component analysis and double regression methods were implemented to investigate plausible associations. Children born to mothers supplemented with FO (FO and FO + 5-MTHF groups, n = 33) showed weaker functional connectivity in the default mode (DM) (angular gyrus), the sensorimotor (SM) (motor and somatosensory cortices) and the fronto-parietal (FP) (angular gyrus) networks compared to the No-FO group (placebo and 5-MTHF groups, n = 24) (PFWE < 0.05). Furthermore, no differences were found regarding the neuropsychological tests, except for a trend of better results in an object recall (memory) test. Considering the No-FO group, the aforementioned networks were associated negatively with attention and speed-processing functions. Mother's FO supplementation during pregnancy seems to be able to shape resting-state network functioning in their children at school age and appears to produce long-term effects on children´s cognitive processing.

Long-Chain Polyunsaturated Fatty Acids, Homocysteine at Birth and Fatty Acid Desaturase Gene Cluster Polymorphisms are Associated with Children's Processing Speed up to Age 9 Years.

Both pre- and early postnatal supplementation with docosahexaenoic acid (DHA), arachidonic acid (AA) and folate have been related to neural development, but their long-term effects on later neural function remain unclear. We evaluated the long-term effects of maternal prenatal supplementation with fish-oil (FO), 5-methyltetrahydrofolate (5-MTHF), placebo or FO + 5-MTHF, as well as the role of fatty acid desaturase (FADS) gene cluster polymorphisms, on their offspring's processing speed at later school age. This study was conducted in NUHEAL children at 7.5 (n = 143) and 9 years of age (n = 127). Processing speed tasks were assessed using Symbol Digit Modalities Test (SDMT), Children Color Trails Test (CCTT) and Stroop Color and Word Test (SCWT). Long-chain polyunsaturated fatty acids, folate and total homocysteine (tHcy) levels were determined at delivery from maternal and cord blood samples. FADS and methylenetetrahydrofolate reductase (MTHFR) 677 C > T genetic polymorphisms were analyzed. Mixed models (linear and logistic) were performed. There were significant differences in processing speed performance among children at different ages (p < 0.001). The type of prenatal supplementation had no effect on processing speed in children up to 9 years. Secondary exploratory analyses indicated that children born to mothers with higher AA/DHA ratio at delivery (p < 0.001) and heterozygotes for FADS1 rs174556 (p < 0.05) showed better performance in processing speed at 9 years. Negative associations between processing speed scores and maternal tHcy levels at delivery were found. Our findings suggest speed processing development in children up to 9 years could be related to maternal factors, including AA/DHA and tHcy levels, and their genetic background, mainly FADS polymorphism. These considerations support that maternal prenatal supplementation should be quantitatively adequate and individualized to obtain better brain development and mental performance in the offspring.

On the relationship between head circumference, brain size, prenatal long-chain PUFA/5-methyltetrahydrofolate supplementation and cognitive abilities during childhood.

Head circumference in infants has been reported to predict brain size, total grey matter volume (GMV) and neurocognitive development. However, it is unknown whether it has predictive value on regional and subcortical brain volumes. We aimed to explore the relationship between several head circumference measurements since birth and distributions of GMV and subcortical volumes at later childhood. We examined seventy-four, Caucasian, singleton, term-born infants born to mothers randomised to receive fish oil and/or 5-methyltetrahydrofolate or placebo prenatal supplementation. We assessed head circumference at birth and at 4 and 10 years of age and cognitive abilities at 7 years of age. We obtained brain MRI at 10 years of age, on which we performed voxel-based morphometry, cortical surface extraction and subcortical segmentation. Analyses were controlled for sex, age, height, weight, family status, laterality and total intracranial volume. Prenatal supplementation did not affect head circumference at any age, cognitive abilities or total brain volumes. Head circumference at 4 years presented the highest correlation with total GMV, white matter volume and brain surface area, and was also strongly associated with GMV of frontal, temporal and occipital areas, as well as with caudate nucleus, globus pallidus, putamen and thalamus volumes. As relationships between brain volumes in childhood and several outcomes extend into adulthood, we have found that ages between 0 and 4 years as the optimal time for brain growth; postnatal factors might have the most relevant impact on structural maturation of certain cortical areas and subcortical nuclei, independent of prenatal supplementation.

Cultural effects on neurodevelopmental testing in children from six European countries: an analysis of NUTRIMENTHE Global Database.

Cultural background is an important variable influencing neuropsychological performance. Multinational projects usually involve gathering data from participants from different countries and/or different cultures. Little is known about the influence of culture on neuropsychological testing results in children and especially in European children. The objectives of this study were to compare neuropsychological performance of children from six European countries (Belgium, Germany, Italy, The Netherlands, Poland and Spain) using a comprehensive neuropsychological battery and to apply a statistical procedure to reduce the influence of country/cultural differences in neuropsychological performance. As expected, the results demonstrated differences in neuropsychological performance among children of the six countries involved. Cultural differences remained after adjusting for other confounders related to neuropsychological execution, such as sex, type of delivery, maternal age, gestational age and maternal educational level. Differences between countries disappeared and influence of culture was considerably reduced when standardised scores by country and sex were used. These results highlight the need for developing specific procedures to compare neuropsychological performance among children from different cultures to be used in multicentre studies.

Visual evoked potentials in offspring born to mothers with overweight, obesity and gestational diabetes.

BACKGROUND: Overweight, obesity, and gestational diabetes (GD) during pregnancy may negatively affect neurodevelopment in the offspring. However, the mechanisms are unclear and objective measures of neurodevelopment in infancy are scarce. We hypothesized that these maternal metabolic pathologies impair cortical visual evoked potentials (cVEPs), a proxy for visual and neuronal maturity. DESIGN: The PREOBE study included 331 pregnant women stratified into four groups; normal weight (controls), overweight, obesity, and GD (the latter including mothers with normal weight, overweight and obesity). In a subsample of the offspring at 3 months (n = 157) and at 18 months (n = 136), we assessed the latencies and amplitudes of the P100 wave from cVEPs and calculated visual acuity. RESULTS: At 3 months of age, visual acuity was significantly poorer in offspring born to GD mothers. At 18 months of age, there were no differences in visual acuity but infants born to GD mothers had significantly longer latencies of cVEPs when measured at 15', and 30' of arc. The group differences at 30' remained significant after confounder adjustment (mean [SD] 121.0 [16.0] vs. 112.6 [7.6] ms in controls, p = 0.007) and the most prolonged latencies were observed in offspring to GD mothers with concurrent overweight (128.9 [26.9] ms, p = 0.002) and obesity (118.5 [5.1] ms, p = 0.020). CONCLUSIONS: Infants born to mothers with GD, particularly those with concurrent overweight or obesity, have prolonged latencies of visual evoked potentials at 18 months of age, suggesting that this maternal metabolic profile have a long lasting, non-optimal, effect on infants´ brain development.

The impacts of maternal iron deficiency and being overweight during pregnancy on neurodevelopment of the offspring.

Both maternal Fe deficiency (ID) and being overweight or obese (Ow/Ob, BMI≥25 kg/m2) may negatively affect offspring brain development. However, the two risk factors correlate and their independent effects on infant neurodevelopment are unclear. PREOBE is a prospective observational study that included 331 pregnant Spanish women, of whom 166 had pre-gestational Ow/Ob. Fe status was analysed at 34 weeks and at delivery, and babies were assessed using Bayley III scales of neurodevelopment at 18 months. In confounder-adjusted analyses, maternal ID at 34 weeks was associated with lower composite motor scores at 18 months (mean 113·3 (sd 9·9) v. 117·1 (sd 9·2), P=0·039). Further, the offspring of mothers with ID at delivery had lower cognitive scores (114·0 (sd 9·7) v. 121·5 (sd 10·9), P=0·039) and lower receptive, expressive and composite (99·5 (sd 8·6) v. 107·6 (sd 8·3), P=0·004) language scores. The negative associations between maternal ID at delivery and Bayley scores remained even when adjusting for maternal Ow/Ob and gestational diabetes. Similarly, maternal Ow/Ob correlated with lower gross motor scores in the offspring (12·3 (sd 2·0) v. 13·0 (sd 2·1), P=0·037), a correlation that remained when adjusting for maternal ID. In conclusion, maternal ID and pre-gestational Ow/Ob are both negatively associated with Bayley scores at 18 months, but independently and on different subscales. These results should be taken into account when considering Fe supplementation for pregnant women.

Maternal, fetal and perinatal alterations associated with obesity, overweight and gestational diabetes: an observational cohort study (PREOBE).

BACKGROUND: Maternal overweight, obesity, and gestational diabetes (GD) have been negatively associated with offspring development. Further knowledge regarding metabolic and nutritional alterations in these mother and their offspring are warranted. METHODS: In an observational cohort study we included 331 pregnant women from Granada, Spain. The mothers were categorized into four groups according to BMI and their GD status; overweight (n:56), obese (n:64), GD (n:79), and healthy normal weight controls (n:132). We assessed maternal growth and nutritional biomarkers at 24 weeks (n = 269), 34 weeks (n = 310) and at delivery (n = 310) and the perinatal characteristics including cord blood biomarkers. RESULTS: Obese and GD mothers had significantly lower weight gain during pregnancy and infant birth weight, waist circumference, and placental weight were higher in the obese group, including a significantly increased prevalence of macrosomia. Except for differences in markers of glucose metabolism (glucose, HbA1c, insulin and uric acid) we found at some measures that overweight and/or obese mothers had lower levels of transferrin saturation, hemoglobin, Vitamin B12 and folate and higher levels of C-reactive protein, erythrocyte sedimentation rate, ferritin, and cortisol. GD mothers had similar differences in hemoglobin and C-reactive protein but higher levels of folate. The latter was seen also in cord blood. CONCLUSIONS: We identified several metabolic alterations in overweight, obese and GD mothers compared to controls. Together with the observed differences in infant anthropometrics, these may be important biomarkers in future research regarding the programming of health and disease in children. TRIAL REGISTRATION: The trial was registered at clinicaltrials.gov, identifier ( NCT01634464 ).

Folate and long-chain polyunsaturated fatty acid supplementation during pregnancy has long-term effects on the attention system of 8.5-y-old offspring: a randomized controlled trial.

BACKGROUND: During fetal and perinatal periods, many nutrients, such as long-chain polyunsaturated fatty acids [contained in fish oil (FO)] and folate, are important in achieving normal brain development. Several studies have shown the benefits of early nutrition on children's neurocognitive development. However, the evidence with regard to the attention system is scarce. OBJECTIVES: The aim of this study was to analyze the long-term effects of FO, 5-methyltetrahydrofolate (5-MTHF), or FO+5-MTHF prenatal supplementation on attention networks. DESIGN: Participants were 136 children born to mothers from the NUHEAL (Nutraceuticals for a Healthy Life) project (randomly assigned to receive FO and/or 5-MTHF or placebo prenatal supplementation) who were recalled for a new examination 8.5 y later. The response conflict-resolution ability (using congruent and incongruent conditions)), alerting, and spatial orienting of attention were evaluated with behavioral measures (Attention Network Test), electroencephalography/event-related potentials (ERPs), and standardized low-resolution brain electromagnetic tomography (sLORETA). RESULTS: Children born to mothers supplemented with 5-MTHF alone solved the response conflict more quickly than did the placebo and the FO+5-MTHF groups (all P < 0.05). Differences between ERP amplitudes for the conflict conditions were also observed. sLORETA analysis showed higher activation of the right midcingulate cortex for the incongruent condition. In addition, a significant slowing down of response speed depending on the warning cue in the 5-MTHF and FO groups was observed. CONCLUSIONS: Folate supplementation during pregnancy, rather than FO or FO+5-MTHF supplementation, improves children's ability to solve response conflicts. This advantage seems to be based on the higher activation of the midcingulate cortex, indicating that early nutrition influences the functionality of specific brain areas involved in executive functions. This trial was registered at clinicaltrials.gov as NCT01180933.

Maternal Obesity, Overweight and Gestational Diabetes Affect the Offspring Neurodevelopment at 6 and 18 Months of Age--A Follow Up from the PREOBE Cohort.

BACKGROUND: Brain development in fetal life and early infancy is critical to determine lifelong performance in various neuropsychological domains. Metabolic pathologies such as overweight, obesity, and gestational diabetes in pregnant women are prevalent and increasing risk factors that may adversely affect long-term brain development in their offspring. OBJECTIVE: The objective of this research was to investigate the influence of maternal metabolic pathologies on the neurodevelopment of the offspring at 6 and 18 months of life. DESIGN: This was a prospective case-control study of 331 mother- and child pairs from Granada, Spain. The mothers were included during pregnancy into four groups according to their pre-gestational body mass index and their gestational diabetes status; overweight (n:56), obese (n:64), gestational diabetic (n:79), and healthy normal weight controls (n:132). At 6 months and 18 months we assessed the children with the Bayley III scales of neurodevelopment. RESULTS: At 6 months (n=215), we found significant group differences in cognition composite language, and expressive language. Post hoc test revealed unexpectedly higher scores in the obese group compared to the normal weight group and a similar trend in overweight and diabetic group. The effects on language remained significant after adjusting for confounders with an adjusted odds ratio for a value above median in composite language score of 3.3 (95% CI: 1.1, 10.0; p=0.035) for children of obese mothers. At 18 month (n=197), the offspring born to obese mothers had lost five points in language composite scores and the previous differences in language and cognition was replaced by a suggestive trend of lower gross motor scores in the overweight, obese, and diabetic groups. CONCLUSIONS: Infants of obese mothers had a temporary accelerated development of cognition and language, followed by a rapid deceleration until 18 months of age, particularly of language scores. This novel observation prompts further confirmative studies to explore possible placental and neurodevelopmental mechanisms involved.

Maternal PPARG Pro12Ala polymorphism is associated with infant's neurodevelopmental outcomes at 18 months of age.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are ligand activated transcription factors with crucial functions in lipid homeostasis, glucose metabolism, anti-inflammatory processes, placental development, and are involved in cognitive functions and neurodegenerative diseases. Polymorphisms in PPAR genes are shown to influence the activity of these receptors. AIMS: 1) To examine the association of PPARG Pro12Ala polymorphism in pregnant women and their offspring on infant's neurodevelopmental outcomes during the first 18 months of life; 2) to determine the influence of Pro12Ala polymorphism on fatty acid concentrations in plasma phospholipids and placental tissue. STUDY DESIGN: 138 mother-infant pairs from the PREOBE observational study were genotyped for PPARG Pro12Ala. Plasma phospholipids and placental fatty acid concentrations were measured at delivery. Infants' neuropsychological assessment at 6 and 18 months of age was performed using Bayley III. RESULTS: The effect of Pro12Ala on infant's neurodevelopmental outcomes was detected at 18 months, but not at 6 months of age. 18 months old infants born to mothers with wild-type Pro12 genotype had better cognitive (OR=5.11, 95% CI: 1.379-18.96, p=0.015), language (OR=3.41, 95% CI: 1.35-11.24, p=0.044), and motor development scores (OR=4.77, 95% CI: 1.243-18.33, p=0.023) than the Ala allele carriers. Pro12Ala variants did not seem to affect fatty acids concentrations in blood nor in placenta at delivery. CONCLUSIONS: Infants born to mothers with Pro12 genotype have better neurodevelopmental outcomes at 18 months of age than Ala allele carriers, indicating a long-term transplacental action of PPARγ variants on foetal brain development.