Table 3. Human studies linking maternal obesity with DNA methylation changes in the offspring.

Studies using global methylation techniques
Study	Population	Design	Country	Tissue	Method	Findings
Michels, et al PLoS One. 2011; 6: e25254.	Epigenetic Birth Cohort 319 newborns with 316 placentas	cohort	USA	Cord blood Placental tissue	Global methylation using a LINE-1 bisulfite pyrosequencing assay	No associations between maternal prepregnancy BMI and global methylation in either tissue.
Herbstman et al. PLoS One. 2013; 8: e72824	Northern Manhattan Mothers & Newborns Study of the Columbia Center for Children’s Environmental Health n = 279 newborns with n=165 at 3 year follow-up.	cohort	USA	Cord blood Peripheral blood at 3 years	Global DNA methylation using the Methylamp™ Global DNA Methylation Quantification Kit (Epigentek Group Inc, NY).	Pre-pregnancy BMI was negatively predictive of both cord and three-year DNA methylation.
Morales, et al BMC Res Notes. 2014; 7: 278.	Avon Longitudinal Study of Parents and Children (ALSPAC) Cohort; n=88 term newborns. Replication study population including 170 term newborns born	cohort	UK	Cord blood	GoldenGate Cancer Panel I Array (Illumina Inc, USA) Validation using A PyroMark MD Pyrosequencing System (Qiagen) in replication cohort	No associations between maternal pre-pregnancy BMI and differentially methylated DNA at any CpG site No association of maternal pre-pregnancy BMI using pyrosequencing in replication cohort
Liu, et al, Environ Mol Mutagen 2014 223–230.	Boston Birth Cohort 309 Black (African American and Haitian), term newborns	cohort	USA	Cord blood	Illumina HumanMethylation27BeadChip	The methylation levels of 20 CpG sites were associated with maternal BMI. One CpG site (ZCCHC10) remained statistically significant after correction for multiple comparisons.
Sharp et al, Int J Epidemiol 2015; 44:1288-304.	ARIES (subset of ALSPAC) n-1018	cohort	UK	Cord blood	HumanMethylation 450 K	Compared with neonatal offspring of normal weight mothers, 28 and 1621 CpG sites were differentially methylated in offspring of obese and underweight mothers. A positive association, where higher methylation is associated with a body mass index (BMI) outside the normal range, was seen at 78.6% of the sites associated with obesity
Guenard et al, J Obes. 2013; 2013: 492170.	50 siblings aged 2 years 8 months to 24 years 11 months, , born before and after maternal bariatric surgery (25 BMS and 25 AMS offspring)	case/control	Canada	Peripheral blood	Genome-wide methylation analysis using the Infinium HumanMethylation450 BeadChip	5698 differentially methylated genes between BMS and AMS offspring. Main differences in inflammatory and immune pathways
Studies using candidate gene approach
Study	Population	Design	Country	Tissue	Method	Findings
Gemma et al, Obesity 2009;17(5): 1032-9.	88 newborns: 57 newborns with appropriate weight for gestational age (AGA), 17 SGA, and 14 LGA	cohort	Argentina	Umbilical cord	PPARGC1A promoter After bisulphite treatment of umbilical cord genomic DNA, a real-time methylation-specific PCR was used to determine the promoter methylation status in selected CpGs	positive correlation between maternal BMI and PPARGC1A promoter methylation in umbilical cord (Pearson correlation coefficient r = 0.41, P = 0.0007)
Hoyo et al, Cancer Causes Control 2012;23:635-45	438 participants in Newborn Epigenetics STudy (NEST)	cohort	USA	Cord blood	Bisulphite sequencinc	Lower IGF2 DMR methylation was associated with elevated plasma IGF2 protein concentrations, an association that was stronger in infants born to obese women. Elevated IGF2 concentrations were associated with higher birth weight
Soubry et al, BMC Mewd 2013; 11:29	79 newborns from the NEST cohort	cohort	USA	Cord blood	Bisulphite sequencing	Increase in DNA methylation at the IGF2 and H19 DMRs among newborns from obese mothers
Soubry et al, Int J Obes 2015;39: 650-7.	92 newborns from the NEST cohort	cohort	USA	Cord blood	Bisulphite pyrosequencing	Obesity in mothers was associated with an increase in methylation at the PLAGL1 DMR β-coefficient +2.58 (s.e.=1.00; P=0.01) and a decrease at the MEG DMR (β-coefficient 3.42 (s.e.=1.69; P=0.04).
Burris et al, Epigenetics 2015; 10:913-21.	531 infants from Programming Research in Obesity, Growth Environment and Social Stress (PROGRESS) cohort	cohort	Mexico City	Cord blood	aryl-hydrocarbon receptor repressor (AHRR) DNA methylation by bisulphite sequencing	AHRR DNA methylation was positively associated with maternal BMI (P = 0.0009) AHRR DNA methylation was 2.1% higher in offspring of obese vs. normal weight mothers representing a third of the standard deviation differences in methylation