Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes
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In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.
KeywordsZona Pellucida Inner Cell Mass Laser Microdissection Post Fertilization Paternal Genome
This work was supported by the European Commission under FP6, by EMMA inf (RII3-CT-2004-506455) to MHdA and by a grant from the National Genome Research Network, NGFN + (01GS0850), to MHdA. We thank Stefanie Dunst, Monika Beschorner, Andrea Bäßler, Alexander Huber, and Bernhard Rey for excellent technical assistance. We are grateful to John Favor for valuable comments on this manuscript.
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