Reproductive Technology

Tissue-Specific Effects of In Vitro Fertilization Procedures on Genomic Cytosine Methylation Levels in Overgrown and Normal Sized Bovine Fetuses

Abstract
Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable bovine day 80 fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1-protocol employed 0.01 units/ml FSH and LH in oocyte maturation medium and 5% serum in embryo culture medium whereas the IVF2-protocol employed 0.2 units/ml FSH and no LH for oocyte maturation and 10% serum for embryo culture. Comparisons with in vivo fertilized controls (n = 14) indicated an apparently normal phenotype for IVF1-fetuses (n = 5), but IVF2-fetuses (n = 10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinical-chemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2-, but not in IVF1-fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1- and IVF2-fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1-fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2-fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF-protocol. Our data indicate that bovine IVF- procedures can affect fetal genomic 5mC levels in a protocol and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and correlated endocrine changes.

Key words: Embryo • Conceptus • Developmental biology • Growth factors • In vitro fertilization

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