Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes.
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.
Peters
Dominika D
DD
Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
Lepikhov
Konstantin
K
Rodenacker
Karsten
K
Marschall
Susan
S
Boersma
Auke
A
Hutzler
Peter
P
Scherb
Hagen
H
Walter
Jörn
J
de Angelis
Martin Hrabé
MH
D000818:Animals; D019175:DNA Methylation; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D042282:Microdissection; D011247:Pregnancy; D015044:Zona Pellucida / Q000378:metabolism*; D015053:Zygote / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*