Background: Embryo transfer and in vitro embryo culture result in an increased relative weight of the heart in singleton, but not in twin fetal sheep. However, IGF2 gene expression is increased in the fetal heart after in vitro embryo culture and transfer in both singletons and twins. IGF2 is known to activate PI3K signalling, which can cause cardiac hypertrophy.

Hypothesis: We hypothesized that there would be a differential effect of transfer and/or culture of the embryo on key molecules in the IGF/P13K signalling pathway in the heart of singletons and twins.

Methods: Embryos were collected 24h after artificial insemination of superovulated donor ewes, and were either transferred to an intermediate ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) for six days before transfer to recipient ewes. Naturally mated (NM) ewes were used as controls. At 144/145d gestation, fetal hearts were collected and abundance of signalling proteins was measured.

Results: There was increased abundance of Akt, 4EBP1 and Phosphorylated 4EBP1 (P<0.05) in hearts from singleton fetuses only while EIF4E was upregulated in the heart of both singletons and twins in ET, IVC and IVCHS compared to NM. However, there was a strong correlation between EIF4E and relative heart weight only in singletons (P<0.005). There was a decrease in ribosomal protein S6 (P<0.01) and phosphorylated ribosomal protein S6 (P<0.01) in all the treatment groups in both singletons and twins compared to NM.

Conclusions: This study suggests that the increase in heart weight in the fetus after embryo transfer or in vitro embryo culture may be due to increased translational efficiency as a result of enhanced IGF/PI3K signalling in the heart of singleton fetuses. The decrease in the protein abundance of ribosomal protein S6 and its phosphorylated form suggest its regulation by a different pathway.