DOI: 10.5061/dryad.c2fqz61bw
title: Multi-omic brain and behavioral correlates of cell-free fetal DNA methylation in macaque maternal obesity models (NMR datasets, maternal plasma and infant brain)

description: Maternal obesity during pregnancy is associated with neurodevelopmental
 disorder (NDD) risk. We utilized integrative multi-omics to examine
 maternal obesity effects on offspring neurodevelopment in rhesus macaques
 by comparison to lean controls and two interventions. Differentially
 methylated regions (DMRs) from longitudinal maternal blood-derived
 cell-free fetal DNA (cffDNA) significantly overlapped with DMRs from
 infant brain. The DMRs were enriched for neurodevelopmental functions,
 methylation-sensitive developmental transcription factor motifs, and human
 NDD DMRs identified from brain and placenta. Brain and cffDNA methylation
 levels from a large region overlapping mir-663 correlated with maternal
 obesity, metabolic and immune markers, and infant behavior. A DUX4
 hippocampal co-methylation network correlated with maternal obesity,
 infant behavior, infant hippocampal lipidomic and metabolomic profiles,
 and maternal blood measurements of DUX4 cffDNA methylation, cytokines, and
 metabolites. Ultimately, maternal obesity altered infant brain and
 behavior, and these differences were detectable in pregnancy through
 integrative analyses of cffDNA methylation with immune and metabolic
 factors. 
description: All animals were housed at the California National Primate
 Research Center (CNPRC). Adult pregnant female rhesus macaques (Macaca
 mulatta) with male fetuses were selected for this study. All dams ranged
 in age from 7 to 12 years and were selected for lean and obese groups
 based on their Body Condition Score (BCS). Obese females had a BCS of at
 least 3.5 (range 1-5) which correlates with 32% body fat, and lean animals
 had a BCS of 2-2.5. Animals had maintained a consistent BCS for at least
 one year prior to selection for the study and pre-study physicals
 confirmed that none of the selected females were diabetic. All animals
 were maintained with standard indoor housing conditions at CNPRC and fed
 nine “biscuits” of commercial chow (High Protein Primate Diet Jumbo;
 LabDiet; 5047) twice daily while pregnant, received biweekly fresh
 produce, daily forage mixture, and ad libitum water. The caloric
 restriction group had the amount of chow restricted to prevent weight gain
 during pregnancy and the Pravastatin group was given 1 mg/kg of body
 weight. However, all dams, regardless of group, were provided twelve
 biscuits twice daily during nursing of 4 months or older infants. Dams
 were relocated to a single housing room around gestational day 70. 
 Approximately two weeks later they were paired with a compatible cage mate
 during daytime hours and were separated prior to feeding times. Dams were
 allowed to deliver naturally (~165 day gestation length) and mother-infant
 pairs were raised indoors until offspring were 6 months old. However, five
 pregnant dams required Cesarian deliveries for post-date pregnancies (~175
 gestation days) or as recommended by veterinarians for health reasons, two
 in the obese group, one in the pravastatin group and two in the lean
 control group. In those scenarios, infants were successfully reared to 6
 months of age by foster dams using established CNPRC protocols.
 Mother-infant dyads were housed indoors with another compatible
 mother-infant dyad during daytime hours if possible; however, this was not
 possible for 1 lean control dyad, 3 obese dyads, and 1 pravastatin dyad.
 Infant brain samples (hippocampus, hypothalamus, and prefrontal cortex)
 were collected on postnatal day 180 after infants were anesthetized with
 ketamine and euthanized with 120 mg/kg pentobarbital. Upon collection,
 samples were immediately frozen and stored at -80 °C.
 Fasting blood was collected in lavender top (EDTA) tubes from
 mothers once during the 1st and 2nd trimesters and twice during the 3rd
 trimester after anesthetization with 5-30 mg/kg ketamine or 5-8 mg/kg
 telazol. Plasma samples were filtered using Amicon Ultra Centrifugal
 Filters (3k molecular weight cutoff, Millipore) to remove proteins and
 lipids. Metabolites were extracted from each of the brain tissues as
 previously described (*1). To 207 µL of either plasma filtrate or brain
 tissue extract, 23 µL of internal standard containing DSS-d6 was added and
 samples were placed in 3 mm Bruker nuclear magnetic resonance (NMR) tubes.
 Proton NMR spectra were acquired on each sample at 25 °C using the
 noesypr1d pulse sequence on a Bruker Avance 600 MHz NMR spectrometer
 (Bruker, Billerica, MA, USA). The concentrations of individual metabolites
 are determined based on the addition of an internal standard, and analyzed
 using Chenomx NMRSuite (version 8.1, Chenomx Inc) to annotated the
 metabolites based on Reference Library 10.
 (*1) Hasegawa, Y. et al. Optimization of a Method for the
 Simultaneous Extraction of Polar and Non-Polar  Oxylipin Metabolites, DNA,
 RNA, Small RNA, and Protein from a Single Small Tissue Sample. Methods
 Protoc. 3, (2020).