Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray

Abstract

Objective: To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts.

Design: A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays.

Setting: University research laboratory.

Patient(s): Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom.

Intervention(s): None.

Main outcome measure(s): Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos.

Result(s): Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs.

Conclusion(s): The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts.

Keywords: CpG island; Preimplantation; blastocyst; epigenetic; methylation.

Supplemental Figure 1

Images of the human preimplantation embryos used in the MCAM study, indicating the number of methylated CGIs in each embryo. CGI = CpG island; MCAM = methylated CpG island amplification coupled with microarray.

Supplemental Figure 2

Analysis of the CGIs that were methylated in ≥4 embryos, to identify whether the methylated embryonic CGIs overlap with gene promoters, enhancers, or intragenic regions. The analysis of promoter, enhancer, and intragenic overlaps were performed independently; therefore, in some cases, the same CGI might overlap enhancer/promoter and intragenic regions, and therefore may have been counted more than once. CGI = CpG island.

Supplemental Figure 3

Principal component analysis and hierarchical clustering show that sample 4 was the most divergent, relative to the other 4 samples. PC = principal component.

Supplemental Figure 4

Cluster dendrogram of methylation pattern with bootstrap resampling.

Figure 1

Distribution of CpG islands that are methylated in blastocysts across the human genome, as generated by MCAM. Red bars represent CGIs methylated in all 5 embryos; green bars represent CGIs methylated in 4 of 5 embryos. CGI = CpG island; CpG = C phosphate G; MCAM = methylated CpG island amplification coupled with microarray.

Figure 2

(A–H) Examples of loci with CpG islands that are methylated in all embryos (5 of 5), as obtained from the University of California, Santa Cruz browser. Black horizontal bars represent methylation coverage across a defined CGI (green) for each embryo. Overlapping loci (RefSeq genes) are blue. (I–L) Examples of loci with CpG islands that are methylated in 4 of the 5 tested embryos. (M–P) Examples of loci with multiple contiguous methylated CpG islands in most embryos. For these panels, the corresponding methylation data, as obtained from the ENCODE data for the human cells lines GM12878, HI-hESC (human embryonic stem cell), K562, HeLa-S3, and HepG2 are shown.