Abstract
BackgroundAssisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences.ResultsOf the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth.ConclusionBy examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.
Highlights
Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock
Changes to Deoxyribonucleic acid (DNA) methylation was region specific; the embryonic disc showing almost no alterations compared to a significant number of differences in trophectoderm tissues
Methylation differences at the PLAGL1 locus were apparent by pyrosequencing; this is congruent with observations in the literature demonstrating the influence of ART on DNA methylation at imprinted loci
Summary
Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. Evidence that ARTs are not completely benign exists from analyses of bovine ART-derived embryos, which exhibit differences at morphological, physiological, transcriptional, chromosomal and metabolic levels compared to their in vivo-derived counterparts [7] Epigenetic mechanisms such as chromatin remodelling, histone modification and DNA methylation are fundamental to successful gametogenesis and are required for normal embryonic progression [2, 8]. Analysis of blastocysts [24], mid-gestation placentas [25] and full term liver and brain tissue [26], derived from superovulated females, indicated altered DNA methylation and/or gene expression at candidate imprinted DMRs. Most recently, findings from an investigation using a mouse model suggest that individual ART procedures cumulatively increase placental morphological abnormalities and epigenetic perturbations [27]. A recent investigation by Saenz-de-Juano et al demonstrated that embryos developed using an in vitro follicular culture (IFC) method inflicted no additional epigenetic alterations at a small number of imprinted genes (Snrpn, H19 and Mest) compared with conventional ovulation induction, suggesting that IFC is a suitable, patient-friendly alternative to ovarian stimulation [28]
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