RRBS used in the current study has advantages such as the ability of conducting focused mapping on CpG-rich exon or promoter regions at a single base level, which is suitable for comprehensive analysis. Theoretically, analysis can be performed with as little as 1–10 ng of specimen; however, for other methods, gDNA of minimum volumes in μ grams is needed till date. Thus, to analyze methylation in early embryos, a large volume of specimens was needed. Therefore, RRBS is suitable for the analysis of methylation in early embryos with small amounts of gDNA [6, 7].
A limitation is that RRBS is a semi-comprehensive analysis rather than a comprehensive analysis of methylation such as post-bisulfite adaptor-tagging. However, the results of the present experiment focusing on CpG-rich sites are consistent with those of the comprehensive analyses.
A certain blastocyst sample volume is required for the RRBS protocol that is appropriate for analysis. If a large proportion of the blastocysts are in a methylated state that is largely deviated from others, this may affect the mean value obtained from analysis. Various reports indicate that the conditions of the culture medium affect the epigenetics of pre-implanted embryos, as observed in large offspring syndrome and Beckwith–Wiedemann syndrome [12, 13]. Furthermore, depending on the amino acids and electrolyte composition of the culture medium, the blastocyst development rate remarkably changes [14, 15]. In embryos that cease to grow or die, there are underlying problems of aneuploidy, metabolic requirements and ammonium production [16]; however, it is likely that the difference in the in vitro environment induced epigenetic changes, which consequently may have resulted in embryonic death [17].
In the history of culture media, Harrison successfully developed a medium in 1907. Since then, progress has been made with various approaches and improvements. In the 1950s, Whitten was the first to successfully culture eight-cell mouse embryos to the blastocyst stage [18]. Thereafter, the basic approaches to the most suitable culture medium have been the “let the embryo choose” principle and the “back to nature” principle [19]. The KSOM medium is a common culture medium based on the “let the embryo choose” principle. From the perspective of energy requirements switching from pyruvic acid in the early stage to glucose in the late stage of pre-implanted embryonic development [20], the KSOM medium contains well-balanced nutrients and the necessary buffer constituents.
When following the “back to nature” principle, another reason for selecting KSOM is that the in vivo environment changes with embryonic growth. It is believed that the medium should be close to the in vivo environment; thus, the concept of sequential media has gained popularity. However, continual changes in the culture medium increases stress on the embryo, and because the growth factors secreted by the embryo becomes diluted in the new medium, there are advantages and disadvantages to both a single medium and sequential media, and the question remains controversial [21]. In this study, we compared changes in methylation caused by the addition of amino acids and used KSOM as the basic medium.
It has been reported that amino acids show minimal impact in terms of blastocyst formation. However, with increasing concentration of amino acids, the probability of hatching increases, the number of inner cell masses increases and the formation of the extracellular matrix becomes closer to normal [16, 22]. Alternatively, it has been reported that EAAs reduce the blastocyst maturation and development rates, to which NEAAs show a compensatory effect. It has been reported that even with administration of NEAAs alone, good blastocyst outcomes are achieved [16, 23]. Conversely, Whitten and Conaghan developed embryos till the blastocyst stage in human and mice culture media without amino acids, and they reported that the 20 essential and NEAAs were not absolutely essential for growth [24]. Many other reports can also be found that question the most suitable constituents of culture media [25]. Ammonium is a product of amino acid metabolism; however, it is reported to be harmful for pre-implanted embryos [16]. The use of multiple culture media simulating the in vivo environment as much as possible has been found to improve embryo development [26]. As per the “let the embryo choose” principle, the most suitable medium for the embryo is experimentally determined [19], and as per the “back to nature” principle [27], the culture medium closely reproduces the in vivo environment. The question as to which of these is most suitable for the embryo remains controversial [28].
The reports mentioned above describe results limited to specific gene loci, imprinted genes and DMR; thus, it is possible that methylation is disrupted at other genes that were not analyzed. Amino acids may exert subtle effects (not to a degree of exerting lethal genome-wide changes). To elucidate this, we added amino acids with different constituents to the KSOM culture. Although there was no major difference in the blastocyst development rate according to the presence or absence of added essential and NEAAs, the development rate was lower in culture media containing both essential and NEAAs. This suggested that both types of amino acids may be important for the initial stage of embryonic development.
The current study is the first report that comprehensively analyzed changes in methylation according to different conditions of culture media in blastocysts.
In this study, we found that (1) uniform mapping was performed by RRBS; (2) there were many regions that maintained hypermethylation, particularly with NEAAs; (3) there was little overall effect of demethylation from reprogramming in the 5′UTR and promoter regions; and (4) specific changes were observed in imprinted genes, such as Nnat and Nespas.
Overall, results of our analysis revealed that CpG mapping was uniformly performed in all culture media, and there was no difference according to mapping between each culture medium, which shows that good data were obtained. This was also supported by the fact that CGI accumulation was mostly in CpG-rich promoter and exon regions (Figs. 1 and 2).
When focusing on each culture medium, as shown in Fig. 4, many sites were found that maintained methylation in medium with NEAAs compared with other culture media. This suggested that when compared with EAAs, NEAAs protected genes from demethylation caused by reprogramming.
In terms of protecting methylation disturbance, Fig. 5 shows that there was little change in methylation in the promoter and 5′UTR regions. This could suggest the presence of a mechanism to protect changes in methylation in regions that were important for translation from external factors (in the present study, to prevent stimulation, i.e., changes in amino acids).
Focusing on individual genes, we observed methylation changes in genes that have been reported to be defective in human imprinting disorders, such as the imprinted genes: Nnat, Nespas, Snrpn, Snurf, H19, and Mest.
As noted by Adam et al., changes in methylation of CpG in pre-implantation embryos are strongly affected by the culture medium constituents [29], and this also affects RNA expression such as in metabolism-related genes [23]. In the current experiment, we also found methylation changes in Acaca and Slc3a1 genes associated with metabolism.
In particular, methylation in the promoter region of Slc3a1 increased to hypermethylation only with NEAAs. We observed methylation disturbances in many of the same genes that have been previously reported to be prone to methylation disturbance owing to the external environment. In the present study, it was suggested that the addition of NEAAs induced a mechanism by which methylation was particularly preserved from external stimuli.
