The demand for assisted reproductive technologies (ART) to create embryos in vitro has increased, and now constitutes 1 in 20 births in Australia. However, the success rates of ART have remained stagnant over the past two decades. In vitro cultured embryos exhibit poorer development compared to their in vivo counterparts, with slower growth and fewer reaching the blastocyst stage. Changes to the composition of culture media to promote in vitro embryo growth include the addition of specific amino acids. Proline, a non-essential amino acid, improves embryo cleavage and blastocyst development when included in mouse embryo culture medium without other amino acids. However, mechanisms by which proline works are poorly understood.
Live-cell imaging of 2-cell and 4-cell mouse embryos cultured in medium containing proline revealed a reduction in mitochondrial activity and reactive oxygen species (ROS). This decrease in mitochondrial activity and ROS was prevented by culture in tetrahydro-2-furoic acid (THFA), an inhibitor of proline oxidase, suggesting that the beneficial effect of proline on embryo development is dependent on proline metabolism.
Glutathione (GSH) is a major regulator of ROS and its production is, in part, dependent on proline metabolism. Live-cell imaging and liquid chromatography-mass spectrometry confirmed that proline metabolism increased both GSH levels and the GSH:GSSG ratio throughout preimplantation embryo development. This, in turn, reduces oxidative stress in the embryo and improves development.
In conclusion, proline improves in vitro embryo development by reducing oxidative stress, likely through increased GSH production and decreased mitochondrial activity. These findings should inform the development of improved embryo culture media used for ART.