Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos

Abstract

Background: In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos. In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data.

Results: We show that: 1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium. 2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium. 3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16.

Conclusion: Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

Figure 1

Experimental group design. In the two first experimental groups (groups A and B), the fertilization and early embryo development were conducted in vivo. Fertilization was realized in vivo for group C and in vitro for group D. In these two groups (groups C and D), the early embryo development was obtained by culture in two different culture media: M16 and G1.2/G2.2. For in vivo fertilization groups (A, B and C), the mating performance was checked by the presence of a vaginal plug. The superovulation of females was induced in groups B, C and D.

Figure 2

Schematic representation of the H19 gene. The position of H19 ICR, number of CpG nucleotides analyzed for methylation status and position of primer set used for expression analysis are indicated. For methylation analysis of H19 ICR, two regions were studied: ICR^{CTCF 1–2} (574 bp) and ICR^{CTCF 3–4} (660 bp). The positions of the CpGs analyzed in each sequence are shown by lollipops (19 CpGs in ICR^{CTCF 1–2} and 23 CpGs in ICR^{CTCF 3–4}, respectively). The four CTCF binding sites are depicted as grey boxes within the ICR^{CTCF 1–2} and ICR^{CTCF 3–4}. For proximal part of H19 promoter (white box with PP), 8 CpGs were analyzed. For expression analysis, real time quantitative RT-PCR was performed using the Taqman technology with two primers (black arrows) chosen to encompass an intron (wedge indicates the exon-exon splice junctions) and a universal probe (white box).

Figure 3

Blastocyst maturity. At day 4, each blastocyst was observed and classified into four different categories according to the maturity characteristics (size of embryonic cavity and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χ² test, P < 0.05)

Figure 4

Embryo cleavage kinetics according to culture media and fertilization method. Daily observations for all cultured zygotes and color code classification according to the cell number are depicted. At day 0, two pronuclei were classically observed (2 PN) and defined the zygote stage. Zygotes obtained after in vitro fertilization (Figure 4A group D) or after in vivo fertilization (Figure 4B group C) were then cultured in M16 or G1.2/G2.2 medium. The results are expressed as percentage of total zygote (PN) number at day 0 for each experimental group and each culture condition. At each culture day, the number of atretic embryos was determined based upon the observation of necrosis signs.

Figure 5

Methylation analysis of individual blastocysts by bisulfite conversion followed by direct sequencing and cloning/sequencing. For each analyzed blastocyst, bisulfite mutagenesis was performed. After PCR amplification the methylation status of CpG positions was determined by direct sequencing (Figure 5A) and cloning/sequencing (Figure 5B). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure 5C).

Figure 6

Direct sequencing analysis of H19 ICR in individual blastocysts. Eight examples of sequences obtained by direct sequencing of bisulfite mutated genomic DNA of individual blastocysts without (Figure 6A) or with methylation defects (Figure 6B) are shown.

Figure 7

Methylation status of H19 ICR (A: ICR^{CTCF 1–2} region analysis and B: ICR^{CTCF 3–4} region analysis) for individual blastocysts determined by bisulfite/sequencing analysis. For each group, methylation status of individual blastocysts was analyzed by direct sequencing and is represented by the black and white lollipops. The CTCF binding sites are shaded in grey. Only examples are shown for clarity.

Figure 8

Direct sequencing analysis of proximal part of H19 promoter in individual blastocysts. Examples of sequences obtained by direct sequencing of bisulfite mutated genomic DNA of H19 promoter in individual blastocysts without (Figure 8A) or with methylation defects (Figure 8B) are shown.

Figure 9

Methylation status of ICR^{CTCF 1–2} according to the blastocyst maturity in group D. For each maturity stage, the results are expressed as a percentage of the blastocyst number.

Figure 10

H19 gene expression patterns in individual blastocysts according to experimental groups. H19 expression was determined by real time quantitative RT-PCR and calculated taking into account expression of a housekeeping gene (Sdha) and an internal standard sample. The results are presented as proportion of blastocysts with or without detectable H19 transcripts in Sdha expressing blastocysts (Figure 10A). The relative H19 RNA level is expressed according to the equation: H19 RNA quantity = 2 ^-ΔΔCt with ΔΔCt = (Ct(H19)-Ct(Sdha)_sample - Ct(H19)-Ct(Sdha)_standard). For each group, individual blastocyst expression is represented. The bars indicate the median expression value. The size of symbols is proportional to blastocyst number with the same H19 expression (Figure 10B).