Methylome-wide analysis of IVF neonates that underwent embryo culture in different media revealed no significant differences

Abstract

A growing number of children born are conceived through in vitro fertilisation (IVF), which has been linked to an increased risk of adverse perinatal outcomes, as well as altered growth profiles and cardiometabolic differences in the resultant individuals. Some of these outcomes have also been shown to be influenced by the use of different IVF culture media and this effect is hypothesised to be mediated epigenetically, e.g. through the methylome. As such, we profiled the umbilical cord blood methylome of IVF neonates that underwent preimplantation embryo development in two different IVF culture media (G5 or HTF), using the Infinium Human Methylation EPIC BeadChip. We found no significant methylation differences between the two groups in terms of: (i) systematic differences at CpG sites or regions, (ii) imprinted sites/genes or birth weight-associated sites, (iii) stochastic differences presenting as DNA methylation outliers or differentially variable sites, and (iv) epigenetic gestational age acceleration.

Fig. 1. Genome-wide DNA methylation analysis of IVF neonates that underwent embryo culture in different media revealed no significant differences.

a Schematic overview showing sample collection from IVF neonates from the G5 versus HTF RCT as well as the inclusion of naturally conceived neonates, genome-wide DNA methylation data generation, data processing and analyses included in this study. b PCA of all CpG sites passing our QC criteria in data from UCB samples of IVF neonates that underwent embryo culture in G5 (gold) or HTF (blue) medium. c Density plot showing the distribution of beta values from all sites and samples within each group (G5 = gold, HTF = blue).

Fig. 2. Preprocessing of umbilical cord blood (UCB) methylome data.

a Scatter plot showing the projection of UCB samples (n = 105) into the PC space generated using reference data for sex prediction. The shape of the dots represents the recorded sex of the participants (circles = female, triangle = male), while the colour shows the predicted sex based on results from sEST (blue = male, pink = female, grey = not specified). b Violin plot showing the predicted cellular composition of the UCB samples, split by culture medium group (gold and blue represent G5 and HTF medium respectively). The violin plots are overlaid with boxplots where the horizontal lines represent the 25th percentile, median and 75th percentile respectively while the whiskers extend to the farthest data points that are no more than 1.5 times the IQR from the upper or lower quartile. c Heatmap showing associations between the principal components and biological/technical aspects of the samples. The colour gradient represents the −log10 of the p values. P values < 0.05 are shown. Significance of the correlation between age, maternal age, CD8-T cells, CD4T cells, NK cells, B cells, monocytes, granulocytes and nRBCs and the 8 PCs was tested using a permutation test with 10,000 permutations. The associations of the PCs with variables creating 2 groups (sample plate, sex, culture medium, pregnancy complication) and those creating three or more groups (Sentrix ID, Sentrix position) were tested using two-sided Wilcoxon rank tests and Kruskal–Wallis one-way analysis of variance respectively.

Fig. 3. Analysis of systematic methylation differences between G5 and HTF neonates: differentially methylated positions and regions.

Volcano plots showing differential methylation between G5 and HTF neonates where the grey dots represent all individual CpG sites (a, b) or multiple CpG sites aggregated into genomic regions, namely genes (c), promoters (d), CpG islands (e). Imprinted genes (c) and sites within them (a) are highlighted in purple while CpG sites associated with birth weight are shown in green (b). No significantly differentially methylation positions or regions (FDR adjusted p value < 0.1) were identified when comparing the two culture medium groups.

Fig. 4. Methylation outliers.

The main panel shows the number of hypomethylation (x-axis) and hypermethylation (y-axis) outliers per UCB sample (G5 = gold, HTF = blue). Distribution summaries, in the form of a density plot and boxplot, are shown for hypomethylation outliers and hypermethylation outliers in the top and right side panels respectively. Lines of the boxplot represent the 25th percentile, median and 75th percentile respectively while the whiskers extend to the farthest data point that is no more than 1.5 times the IQR from the upper or lower quartile. The axes are shown on a log10 scale. The groups were not found to be significantly different (p value > 0.1).

Fig. 5. Epigenetic gestational age acceleration.

Raincloud plot showing the GAA of each UCB sample in each culture medium group. Points represent individual samples of the G5 (gold) and HTF (blue) groups. Above a density plot and boxplot is shown. Horizontal lines of the boxplot represent the 25th percentile, median and 75th percentile respectively while the whiskers extend to the farthest data point that is no more than 1.5 times the IQR from the upper or lower quartile. GAA is represented in weeks. The groups were not found to be significantly different (p value > 0.1).