Abstract
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.
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Acknowledgments
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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Peters, D.D., Lepikhov, K., Rodenacker, K. et al. Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes. Mamm Genome 20, 664–673 (2009). https://doi.org/10.1007/s00335-009-9227-0
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DOI: https://doi.org/10.1007/s00335-009-9227-0