DNA methylation signatures in cord blood of ICSI children.

study questionDoes ICSI induce specific DNA methylation changes in the resulting offspring? SUMMARY ANSWER: Although several thousand analyzed CpG sites (throughout the genome) displayed significant between-group methylation differences, both ICSI and spontaneously conceived children varied within the normal range of methylation variation. WHAT IS KNOWN ALREADY: Children conceived by ART have increased risks for medical problems at birth and to the extent of present knowledge also in later life (i.e. impaired metabolic and cardiovascular functions). One plausible mechanism mediating these ART effects are epigenetic changes originating in the germ cells and/or early embryos and persisting during further development. STUDY DESIGN, SIZE, DURATION: We compared the cord blood methylomes and candidate gene methylation patterns of newborns conceived through ICSI or spontaneously. PARTICIPANTS/MATERIALS, SETTING,

methodsUmbilical cord bloods were obtained from healthy newborn singletons conceived spontaneously (53 samples), through ICSI (89) or IVF (34). Bisulfite-converted DNA samples of 48 ICSI and 46 control pregnancies were used for genome-wide analyses with Illumina's 450K methylation arrays. Candidate genes from the methylation screen were analyzed in all three groups by bisulfite pyrosequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Altogether, 4730 (0.11%) of 428 227 analyzed CpG sites exhibited significant between-group methylation differences, but all with small (β < 10%) or very small (β < 1%) effect size. ICSI children showed a significantly decreased DNA methylation age at birth, lagging approximately half a week behind the controls. ART-susceptible CpGs were enriched in CpG islands with low methylation values (0-20%) and in imprinting control regions (ICRs). Eighteen promoter regions (six in microRNA and SNORD RNA genes), four CpG islands (three in genes including one long non-coding RNA), and two ICRs contained multiple significant sites. Three differentially methylated regions were studied in more detail by bisulfite pyrosequencing. ATG4C and SNORD114-9 could be validated in an independent ICSI group, following adjustment for maternal age and other confounding factors. ATG4C was also significant in the IVF group. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The observed epigenetic effects are small and there are numerous potential confounding factors such as parental age and infertility. Although our study meets current standards for epigenetic screens, sample size is still two orders of magnitude below that of genome-wide association studies. WIDER IMPLICATIONS OF THE

findingsOur study suggests an impact of ICSI on the offspring's epigenome(s), which may contribute to phenotypic variation and disease susceptibility in ART children. Epigenetic regulation of gene expression by different classes of non-coding RNAs may be a key mechanism for developmental programming through ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a research grant (no. 692185) from the European Union (ERA of ART). There are no competing interests.