276 IN VITRO FERTILIZATION PROCEDURES AFFECT THE HEPATIC GENOMIC CYTOSINE METHYLATION LEVEL AND THE PHENOTYPE OF 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 different in vitro fertilization (IVF) protocols on hepatic genomic cytosine methylation levels and on fetal phenotype. Two groups of IVF-fetuses (IVF1, n = 5 and IVF2, n = 10) were compared with control fetuses generated by artificial insemination (AI, n = 14). Both IVF protocols were previously employed to generate live offspring but differed with respect to gonadotropins in the oocyte maturation medium (0.01 units/mL b-FSH and b-LH for IVF1 versus 0.2 units/mL o-FSH for IVF2) and serum concentrations in the embryo culture media (5% estrous cow serum in IVF1 versus 10% estrous cow serum in IVF2). Analysis of variance (General Linear Model Procedure, SPSS for Windows version 12.0; SPSS GmbH Software, Munich, Germany) showed that fetus group significantly affected fetus weight and length (P < 0.01), heart (P < 0.05) and liver (P < 0.01) weight, and 5-methylcytosine (5 mC) content of liver DNA (P < 0.001). Comparison of group means (t-test) showed that methylation levels in both groups of IVF fetuses differed significantly from AI controls. We observed hepatic DNA hypomethylation (-15.4% vs. AI control, P < 0.01) in IVF1 fetuses, and hypermethylation (+11.6% vs. AI control, P < 0.001) in IVF2 fetuses, but only IVF2 fetuses showed phenotypic abnormalities. The IVF2 fetuses were significantly heavier (18.6%, P < 0.01) and longer (4.3%, P < 0.05) than AI fetuses, with increased heart (21.8%, P < 0.05) and liver (25%, P < 0.001) weights, and thus displayed an overgrowth phenotype. A clinical-chemical screen of 18 plasma parameters failed to detect abnormalities in IVF1 fetuses but revealed significantly increased levels of insulin-like growth factor 1 (40.8%, P < 0.001) and creatinine (28.5%, P < 0.05) in IVF2 fetuses. Our data indicate that bovine IVF-procedures can induce protocol-specific and persistent changes in hepatic cytosine methylation level with or without obvious concomitant changes in fetal phenotype. This suggests that epigenetic change after bovine IVF could be more widespread than previously thought and highlights the value of epigenetic diagnostic screening.