Abnormal<i>DLK1/MEG3</i>imprinting correlates with decreased HERV-K methylation after assisted reproduction and preimplantation genetic diagnosis

Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an important role in retrotransposon silencing and genomic imprinting during mammalian development. Assisted reproduction technologies (ARTs) may be associated with increased stress and risk of epigenetic changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and imprinted genes. Here, we investigated whether the methylation status of Human Endogenous Retroviruses (HERV)-K LTR retrotransposons and the imprinting signatures of the DLK1/MEG3. p57(KIP2) and IGF2/H19 gene loci are linked during early human embryogenesis by examining trophoblast samples from ART pregnancies and preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status. Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to controls and this was associated with altered HERV-K methylation. No differences were detected in p57(KIP2) and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using bioinformatics, we found that while the genome surrounding the p57(KIP2) and IGF2/H19 genes differentially methylated regions had low coverage in transposable element (TE) sequences, the respective one of DLK1/MEG3 was characterized by an almost 2-fold higher coverage. Moreover, our analyses revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that TEs may be affecting the establishment of genomic imprinting under stress conditions.