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Profiling placental DNA methylation associated with maternal SSRI treatment during pregnancy

ABSTRACT: Background: Selective serotonin reuptake inhibitors (SSRIs), used to treat prenatal maternal depression, have been associated with neurobehavioral disturbances in exposed neonates, though the underlying molecular mechanisms remain poorly understood. In utero exposure to SSRIs may have an effect on DNA methylation (DNAme) in the human placenta, which is an epigenetic mark that is established during development and is associated with gene expression. Methods: Chorionic villus samples from 64 human placentas were profiled with the Illumina MethylationEPIC BeadChip and clinical assessments of maternal mood were collected at multiple time points during pregnancy. Case distribution was 20 SSRI-exposed cases and 44 SSRI non-exposed cases. Maternal depression using a mean maternal Hamilton Depression score >8 to indicate symptomatic depressed mood (“maternally-depressed”) further classified cases into SSRI-exposed, maternally-depressed (n=14); SSRI-exposed, not maternally-depressed (n=6); SSRI non-exposed, maternally-depressed (n=20); and SSRI non-exposed, not maternally-depressed (n=24). To provide a replication cohort, Illumina 450K DNAme profiles were obtained from 34 additional cases from an independent cohort (n=17 SSRI-exposed, n=17 SSRI non-exposed). Results: No CpGs were differentially methylated at FDR < 0.05 comparing SSRI-exposed to non-exposed placentas, while adjusting for mean maternal Hamilton Depression score, nor comparing SSRI-exposed maternally-depressed to SSRI-non-exposed maternally-depressed cases. At a relaxed threshold of FDR < 0.25, five CpGs were differentially methylated (|Δβ| > 0.03) by SSRI exposure status. Four of these CpGs were covered by the 450K array and were examined in the replication cohort, but none replicated. Amongst SSRI non-exposed cases, No CpGs were differentially methylated (FDR < 0.25) comparing maternally depressed to not depressed cases. In sex-stratified analyses for SSRI-exposed versus non-exposed cases (females n=31; males n=33), three additional CpGs in females, but none in males, were differentially methylated at the relaxed FDR < 0.25 cut-off. Conclusions: We did not observe large-scale alterations of DNAme in placentas exposed to maternal SSRI treatment compared to placentas with no SSRI exposure. We also found no evidence for altered DNAme in maternal depression-exposed versus depression non-exposed placentas. This novel work in a prospectively-recruited cohort with clinician-ascertained SSRI exposure and mood assessments would benefit from future replication.

ORGANISM(S): Homo sapiens

PROVIDER: GSE203396 | GEO | 2023/01/12

REPOSITORIES: GEO

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Project description:204 placentas from three cohorts were processed for DNAme arrays in Vancouver, Canada. The three cohorts consisted of: (i) V-NORM, a normative population of term pregnancies recruited at BC Women’s Hospital (BCWH) in Vancouver, Canada (n=35), as part of a study on Epigenetics in Pregnancy Complications (EPIC); ii) V-SSRI, a population of pregnant individuals recruited in Vancouver, Canada in the 20th week of gestation (n=64), with/without clinical depression, and with/without the use of selective serotonin reuptake inhibitors (SSRIs); and (iii) QF-2011, a population of term pregnancies that were exposed to sudden-onset stress during gestation due to catastrophic flooding in the Australian state of Queensland in early January 2011 (n=105). DNA samples from all three cohorts were run on Illumina Infinium MethylationEPIC arrays in Vancouver, BC, Canada. Processing included DNA purification after extraction using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA), bisulfite conversion using the EZ DNAme Kit (Zymo Research, Orange, CA, USA), and hybridization to and processing of the Illumina Infinium MethylationEPIC BeadChip arrays according to the manufacturer’s protocol (Illumina, San Diego, CA, USA). Samples from the three cohorts were randomized and run in 3 array batches across 44 eight-sample chips as illustrated in Supp Figure 5. In the case of the V-SSRI and QF-2011 cohorts, sample exposure groups (SSRI exposed/non-exposed and objective flood stress high/low) and infant sex were carefully distributed across array chips (1-44) and rows (1-8) to minimize potential batch effects. DNAme data from raw IDAT files were read into R v4.2.2(54) and annotated with the Illumina Infinium MethylationEPIC v1.0 B4 Manifest. Several data quality control checks were undertaken using the R packages minfi(55,56), wateRmelon(57,58), and ewastools(59). First, each sample was assessed at 17 Illumina control probes to evaluate bisulfite conversion efficiency and array run quality; all samples passed the manufacturer-recommended thresholds at the control probes. Next, average total (methylated + unmethylated) fluorescence intensity was assessed between samples, and between array batches. All samples had similar total fluorescence, though samples run on the EPIC array in Batch 3 had slightly higher average intensities than those in Batch 1 and 2. Sample sex was assessed with the ewastools package(59), using the mean total fluorescence intensity (methylated + unmethylated) of the X and Y chromosome probes, normalized to the per-sample mean autosomal total fluorescence intensity, and was confirmed to match the clinically-reported sex of the infant in all cases. Sample genetic identity was assessed using the 59 SNP (‘rs’) probes on the EPIC array with the call_genotypes() and enumerate_sample_donors() functions (ewastools)(59). Finally, DNAme beta value density plots of all samples were visually assessed to determine overall similarity of the beta value distributions between samples, with no outliers identified.

Project description:Transfer of frozen-thawed embryos leads to sex-specific DNA hypermethylation in both human and mouse placentas. : In humans, frozen ET led to placentas with significant alterations in DNA methylation with most genes demonstrating hypermethylation compared to placentas from fresh ET (4402 CpGs; 1600 genes; p-value < 0.05 in two–tailed unpaired t tests, and a mean methylation difference > 0.05). When compared to control samples, both frozen and fresh samples showed significant differences in methylation.(Frozen vs Control: 5600 CpGs, 2775 genes; Fresh vs Control: 4096 CpGS; 1914 genes; p-value < 0.05, mean methylation difference > 0.05). Paired analysis showed similar trends, despite controlling for maternal environment. Sex specific analysis revealed that these changes were primarily driven by male placentas, as seen by a larger number of CpGs that were differentially methylated, and a larger proportion of genes with at least 2 differentially methylated CpG sites in male placentas as compared to female placentas. (Males: 15572 CpGs, 4399 genes; Females: 7441 CpGs, 3070 genes; p-value < 0.05, mean methylation difference > 0.05). In order to isolate the effects of vitrification we utilized the mouse model, controlling for the effect of the maternal hormonal milieu. In the first genome-wide profiling of DNA methylation in a mouse IVF model, we found that trends in DNA methylation differences paralleled those seen in human placentas. Using similar significance cutoffs as for human samples, and BumpHunter analysis that identifies differentially methylated regions to compensate for a lower number of samples, placentas derived from frozen embryos were predominantly hypermethylated compared to placentas after fresh ET (589 DMRs, 445 genes). Both frozen and fresh embryo transfer samples demonstrated perturbations compared to control samples (Frozen vs Control: 1787 DMRs, 1319 genes; Fresh vs Control: 1119 DMRs, 808 genes). Consistent with the sex-specific trends we observed in human placentas, differences between samples derived from frozen compared to fresh embryo transfer were driven by changes in male placentas (Males: 1069 DMRs, 798 genes; Females:14 DMRs, 11 genes) . When considering the effect of IVF as a whole, murine placentas were predominantly hypomethylated, replicating existing human genome-wide methylation data. As seen previously, IVF led to changes in placental weight, placental morphology and microvessel density in mice, but no persistent changes were seen after embryo vitrification alone. Sexually dimorphic epigenetic changes could indicate differential susceptibility of male and female embryos to IVF-associated perturbations. This observation highlights the importance of sex-specific evaluation of the incidence of adverse outcomes. Similarities between changes seen in mouse and human samples underscore the suitability of the mouse model in evaluating the effect of ART on the epigenetic landscape. This convergence is especially valuable, given limited access to human tissues and the ability to isolate specific interventions in the mouse model.

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Profiling placental DNA methylation associated with maternal SSRI treatment during pregnancy

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