Table 2.
Overview of the methodology and statistical analysis
| Reference | Sample tissue | Individual sites, regional or global level | Method | Epigenetic outcome | Statistical tests | Statistical thresholds | Model covariates† | Cell count correction | Authors’ inference about exposure–outcome associations* | |
|---|---|---|---|---|---|---|---|---|---|---|
| Epigenome-wide association studies | Yeung et al. (2020) [30] | Cord blood | Site-by-site | Infinium MethylationEPIC BeadChip (Illumina) | Subset quantile normalized β values | Linear mixed-effects models | FDR cut-off < 0.05 | 6 | Reference data for cord blood [101] | Association |
| Addo et al. (2019) [48,100] | Placenta | Site-by-site | Infinium MethylationEPIC BeadChip (Illumina) | M values for statistical procedures, β values for data presentation | Robust linear regression models | FDR cut-off < 0.05 (2 CpGs reached Bonferroni significance at p < 6.3 × 10−8) |
14 (and PCA) | Houseman [102] | Association | |
| Cardenas et al. (2019) [29] | Cord blood | Site-by-site and regional | Infinium Human-Methylation450 BeadChip (Illumina) | M values for statistical procedures, β values for data presentation | Robust linear regression models | FDR cut-off < 0.05 (16 CpGs reached Bonferroni significance at P < 1.34 × 10−7 in Project Viva, 1 site of which was confirmed in the replication cohort) |
10 (and PCA) | Houseman [102] | Association | |
| Gervin et al. (2017) [35] | Cord blood | Site-by-site and regional | Infinium Human-Methylation450 BeadChip (Illumina) | M values for statistical procedures, β values for data presentation | Linear regression models | FDR cut-off < 0.05 | 6 (and SVA) | Houseman [102] and reference data for cord blood [103] | Association | |
| Emes et al. (2013) [46] | Cord blood | Site-by-site and global (LINE-1) | Infinium Human-Methylation27 BeadChip (Illumina) | β values (log2-transformed upon t testing) | Hierarchical clustering of the β values of all sites, then t-tests of the mean scores across the identified clusters to infer differentially methylated sites | FDR cut-off < 0.05 | 0 | No | Association | |
| Smith et al. (2012) [47] | Cord blood and placenta | Site-by-site and global (average measure across all investigated CpGs) | Infinium Human-Methylation27 BeadChip (Illumina) | M values for statistical procedures, β values for data presentation | Linear mixed effects models | FDR cut-off < 0.05 Follow-up in placental tissue: one-sided p-values |
4 | No | Association | |
| Schroeder et al. (2012) [43] | Cord blood | Site-by-site | Infinium Human-Methylation27 BeadChip (Illumina) | M values for statistical procedures, β values for data presentation | Linear mixed effects models | FDR cut-off < 0.05 | 4 | No | Association | |
| Combined epigenome-wide and candidate gene studies | Gurnot et al. (2015) [31] | Cord blood |
Genome-wide Site-by-site Gene-specific CYP2E1 both individual sites (16 CpGs) and global methylation |
Genome-wide Infinium Human-Methylation27 BeadChip array (Illumina) Gene-specific PyroMark Q96 ID pyrosequencer (Qiagen) |
Genome-wide β values Gene-specific Methylation percentage |
Genome-wide Non-parametric Wilcoxon tests and linear regression models Gene-specific Linear regression models |
FDR cut-off < 0.05 | 0 | No | The authors propose an epigenetic mediation mechanism based on three observations, suggesting that the DNAm of CYP2E1 is an epigenetic mechanism to protect the unborn child from the adverse effects of SRIs |
| Non et al. (2014) [32] | Cord blood |
Genome-wide Site-by-site and regional Gene-specific Site-by-site; 10 candidate genes |
Genome-wide Infinium Human-Methylation450 BeadChip (Illumina) Gene-specific Pyromark Q24 Pyrosequencer (Qiagen) |
Genome-wide β values Gene-specific Methylation percentage |
Multivariate robust standard error regression models |
Genome-wide FDR cut-off < 0.1 Gene-specific Bonferroni correction accounting for the number of probes tested in each gene |
4 | No; the authors do not expect shifts in cell populations to influence much, as neither of the identified genes are important in inflammation or immune system functioning | Association | |
| Candidate gene studies | Galbally et al. (2020) [37] | Placenta and buccal cells (24–72 hours after birth) | Site-by-site; NR3C1 and NR3C2 (13 placental CpGs and 11 buccal CpGs for both genes) | SEQUENOM MassARRAY EpiTYPER platform | Mean methylation percentage of triplicate samples | Univariate ANOVAs | FDR cut-off < 0.25 | 0 | No, but state this as a limitation | Hypothesize that the DNAm of placental NR3C2 CpG 24 mediates the indirect effect of maternal depression on cortisol reactivity at 12 months. In this model, antidepressants may modify the effect of depression on DNAm. To strengthen the hypothesis, the authors performed a mediation analysis based on Hayes et al. [104]. From this analysis, the authors concluded that CpG 24 methylation in the placental NR3C2 reduces the association between maternal depression and infant cortisol reactivity |
| Galbally et al. (2018) [51] | Placenta | Site-by-site; OXTR (16 CpGs) | SEQUENOM MassARRAY EpiTYPER platform | Mean methylation percentage of triplicate samples | One-way ANOVAs Significant antidepressant–DNAm associations explored with scatter plots and multiple regression models |
p < 0.05 | 1 | No | Association | |
| McLaughlin et al. (2017) [34] | Buccal cells (24–72 hours after birth) | Regional; ABCB1, CYP2D6, and OPRM1 (mean of all CpG DNAm values within a gene to compare across samples) | Pyromark Q24 Pyrosequencer (Qiagen) | Methylation percentage | One-way ANOVAs | p < 0.05 | 0 | No | Association | |
| Mansell et al. (2016) [45] | Cord blood (mononuclear cells only) | Site-by-site; NR3C1 promoter (21 CpGs) | SEQUENOM MassARRAY EpiTYPER platform | Log-transformed methylation percentage (mean of triplicate arrays; log base not specified) for regression modelling; mean methylation with no transformation for data presentation | Student’s t-test, ANOVA or pairwise correlation tests (as appropriate) to investigate the associations between exposures, DNAm, and covariates. Multivariate linear regression models to investigate the association of maternal mental well-being and DNAm of NR3C1 |
Bonferroni corrected p < 0.00079 (accounting for three maternal well-being measures and 21 CpG units) Unadjusted p < 0.05 |
10 | Determine monocyte and lymphocyte frequencies by FACS, used this as a covariate in the model | Association | |
| Gartstein et al. (2016) [38] | Cord blood | Site-by-site; SLC6A4 promoter (10 CpGs) | PyroMark MD System (Biotage, Qiagen) | Methylation percentages | Identified the most important regions of methylation over the 10 CpGs of SLC6A4 by PCA and literature searches. Using these CpGs, performed hierarchical multiple regression models including interaction terms |
p < 0.05b | 2 | No | Association | |
| Ciesielski et al. (2015) [33] | Placenta | Site-by-site; 15 genes (in 27 CpGs) | Infinium Human-Methylation27 BeadChip array (Illumina) | Adjusteda β values | Logistic regression to determine if the methylation of CpG sites were associated with low birth weight. Fisher’s exact tests and follow-up exact binomial tests were applied to compare the significant CpGs from the regression across the included groups |
FDR cut-off < 0.05 | 1 | No | Findings suggest that decreased DNAm of one placental LEPR CpG may be part of a biological mechanism linking ongoing maternal psychiatric disease and poor foetal growth. The authors base this proposal on the observation of associations across their variables and state that they rule out other explanations of this apparent DNAm pattern. Notably, the authors also emphasize that the observed associations do not by default suggest causation and that additional validation and characterization studies are needed | |
| Soubry et al. (2011) [52] | Cord blood | Regional level; IGF2 DMR (3 CpGs) and H19 DMR (4 CpGs) | Pyromark MD System (Biotage, Qiagen) | Mean methylation percentage from triplicate assays |
t-tests between mean methylation over all samples of the DMRs and the covariates Multiple regression models |
p < 0.05b | 7 (and SVA) | Validated that IGF2 and H19 DMRs were equal across different cell fractions from cord blood [105] | Association | |
| Devlin et al. (2010) [44] | Cord blood | Site-by-site; SLC6A (10 CpGs) and BDNF (12 CpGs) | PyroMark MD System (Biotage, Qiagen) | Methylation percentage | MANCOVA models | p < 0.05 | 0 | No, but the authors recognize this limitation | Association | |
| Oberlander et al. (2008) [36] | Cord blood (mononuclear cells only) | Site-by-site; NR3C1 (13 CpGs) and global (LINE-1) | PyroMark MD System (Biotage, Qiagen) | Methylation percentage | Multiple regression models | p < 0.05 | 0 | No | Findings suggest an association between increased 3rd trimester maternal depressed mood and higher HPA stress responsiveness in the 3-month-old infant. This association may potentially be mediated epigenetically through DNAm of human NR3C1. However, the authors acknowledge that to infer any functional consequences of neonatal cord blood NR3C1 CpG3 DNAm, they will need more direct evidence, and any mediational relationship cannot be deduced from their study |
†Includes all covariates adjusted for in the statistical models. Most studies also employed other strategies to adjust for or assess suspected covariates; these are presented in the Supplementary Table S3.
*Full explanation of the authors’ reasoning behind a causal relationship is in Supplementary Table S5.
aWhat is meant by ‘adjusted’ is not specified in the article.
bAssumed p < 0.05 based on which results are considered significant, but the p was not stated clearly in the article.
(M)AN(C)OVA: (multivariate) analysis of (co)variance; CpG: 5ʹ–Cytosine–phosphate–guanine–3ʹ site; DMR: differentially methylated regions; DNAm: DNA methylation; FACS: fluorescence-activated cell sorting; FDR: false discovery rate; HPA: hypothalamic-pituitary-adrenal; PCA: principal component analysis; (S)SRI: (selective) serotonin reuptake inhibitor; SVA: surrogate variable analysis.