Article

Mammalian Genome

, Volume 20, Issue 9, pp 664-673

Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes

  • Dominika D. PetersAffiliated withInstitute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)
  • , Konstantin LepikhovAffiliated withDepartment of Natural Sciences – Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland
  • , Karsten RodenackerAffiliated withInstitute of Biomathematics and Biometry, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)
  • , Susan MarschallAffiliated withInstitute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)
  • , Auke BoersmaAffiliated withInstitute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)
  • , Peter HutzlerAffiliated withInstitute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)
  • , Hagen ScherbAffiliated withInstitute of Biomathematics and Biometry, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH)
  • , Jörn WalterAffiliated withDepartment of Natural Sciences – Technical Faculty III FR 8.3, Biological Sciences, Institute of Genetics/Epigenetics, University of Saarland
  • , Martin Hrabé de AngelisAffiliated withInstitute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH) Email author 

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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.