Nature or nurture or both? Potential use of both DNA copy number and epigenetics in assessing the human blastocyst

Selecting the best embryo for transfer improves pregnancy rates in in vitro fertilization (IVF). Currently, the best embryo is selected based on morphology and genetic euploid status. This approach yields a high pregnancy rate of 60%–70%. Conversely, 30%–40% of these embryo transfers fail. What else can we do to optimize live birth rates further and to understand embryo development?

Switching from genetics, which is hardwired into each cell (the “nature” of each cell), to considering the effects of nurture is an option. Nurture, in this case, will focus on epigenetics. Can we use information from epigenetics to learn more about embryo development and what may impact our ability to select the best embryo to further improve pregnancy rates and improve the health of offspring?

Epigenetic processes link our genetic code to the environment, thereby letting outside factors influence our genome. Epigenetics is the process of regulation of our genes and is comprised of DNA methylation, posttranslational histone modifications, or chromatin remodeling for chromatin accessibility and chromosome conformation leading to changes in transcription. Technology to assess most of these epigenetic changes in the single cell is emerging. The epigenetic signatures are altered by external modifiers, including age, diet, body mass index, environmental pollution, and stress. The modified epigenome is also phenotypically impactful, resulting in various pathologies, including infertility or aneuploidy among others. Hence, understanding the “nature” or DNA sequence and copy-number variants (CNV) and “nurture” as in methylome analysis of the human blastocyst is a critical step in assessing the impact of epigenetics of the embryo.

The particular epigenetic process of DNA methylation allows for gene expression and physiologic processes, such as imprinting, gene silencing, and chromosome inactivation (1). These epigenetic modifications essentially are chemical tags and can be present in the form of phosphate, methyl, and acetyl groups (1). DNA methylation survives cell division because of the activity of DNA maintenance methyltransferase-1 isoforms that are DNA maintenance methylases. Of significance to IVF, more stressful IVF media causes loss of imprinting (decreased DNA methylation) in the blastocyst and this persists into offspring in the form of several diseases, such as Beckwith-Wiedemann syndrome. Other mechanisms of epigenetics, such as histone modification, also may have immediate effects on stemness and line-age imbalance in the implanting embryo, but may not persist in future cell divisions. Embryologically, epigenetic regulation is important in the early stages of embryo development and its susceptibility to environmental factors can have an impact on the determination of the fate of the cellular differentiation (1). Thus, DNA methylation potentially can be used to identify various cell types from each other.

The study by Olcha et al. (2) analyzed two preimplantation genetic testing-aneuploidy (PGT-A) tested abnormal embryos with whole-genome bisulfate sequencing on 17 inner cell mass (ICM) and 12 trophectoderm (TE) cells to assess their CNVs and methylation profiles at the individual cell level. With regards to CNVs, of the nine cells that were amplified using whole-genome bisulfate sequencing, all except one cell was consistent with the original PGT-A CNV analysis for embryo A (2). In embryo B, 11 of the 13 cells that were amplified were consistent with the original PGT-A CNV assessment (2). With regards to methylation profiling, they found that the average CpG methylation frequency was higher in morphologically-identified ICM cells than TE cells (2). However, for non-CpG methylation, both groups of cells were similar in terms of their methylation frequency (2). This study was able to differentiate ICM from TE cells as well as concurrently determine the CNV and methylome of each of these cells with fairly good reliability before optimization of the techniques. Their findings on these individual cells also may provide more information on the genetic complexities of various embryos and potentially the origin of embryonic mosaicism (2).

Therefore, the technique described in the study by Olcha et al. (2) in the current issue demands attention. The study has assessed the methylation patterns of the ICM and TE cell lines and proven that the methylation frequency and cell-to-cell methylation variability are distinct between the two cell types. The ICM has a higher frequency and higher variability in methylation compared with the TE cells. Although this is a small pilot study that looked only at two aneuploid embryos, this still is significant and demands further inquiry. Future studies with larger sample numbers comprising euploid and aneuploid embryos assessing the methylation profile of the ICM and TE will provide answers with regard to the reproducibility of this technique. If this is, indeed, reproducible in euploid and aneuploid embryos and the ICM and TE cell lines could be distinctly delineated by methylation patterns, it will have an immense role in lessening the number of mosaic embryo results that leaves the reproductive endocrinologist in a quandary on a daily basis.

In addition, the specific technique used in this study, bisulfite sequencing, has important attributes to allow for the detection of DNA methylation (CpG units) in a single cell. Bisulfite sequencing converts cytosine to uracil, which is converted and read during next-generation sequencing (NGS) as thymidine. However, CpG cytosines remain and are sequenced as cytosines. Next-generation sequencing after bisulfite conversion throws the largest “net” in sequencing all CpG and cytosine methylations as well as all other forms of large- and small-scale chromosomal abnormalities, such as translocations, deletions, or repeats. There are now many single-cell epigenomic testing methods (1, 3) that should be used to further diminish the “gap” the investigators address here between poor embryos that have undetected changes in DNA epigenomic integrity. Preimplantation genetic testing-aneuploidy has moved from narrowest to broadest identification methods: from single gene hybridizations, such as fluorescence in situ hybridization and polymerase chain reaction to arrays of gene hybridizations, such as comparative genome hybridization to global NGS that identifies and counts all sequences. Next-generation sequencing here is used to detect genetic variations, such as CNV and some types of epigenetic variation as illuminated by bisulfite sequencing. However, epigenomic assays through NGS have several other assays applicable to single cells that complement and make more accurate methods used here to determine epigenomic changes in ICM and trophectoderm. The focus also should include other means to assay DNA methylation and its effects on access to DNA for transcription: Assay for transposase-accessible chromatin-seq directly measures sequence-specific openness for transcription; 5 hydroxy-methyl-cytosine sequencing techniques, because bisulfite sequence conversion misses 5 hydroxy-methyl-cytosines that are transcriptionally active but in an intermediate state before demethylation; and Hi-C, which is a type of chromosome conformation capture adapted to a single cell that measures chromatin conformation and openness (1). These complementary genomic techniques to those used in this article should be applied to a larger pilot study of human embryos before widespread use with elective single embryo transfer.