The Race to Equilibrate

Abstract

Background: While it is well documented that culture in hypoxic conditions produces superior outcomes, the mechanics of establishing such conditions within the embryo’s liquid environment is less clear. It is not uncommon for laboratories, while ‘culturing’ in low-oxygen, to equilibrate dishes in CO2 only environments prior to use, meaning embryos are exposed to high oxygen tension in their media even after placing into the hypoxic environment, but for how long is so far unclear. Aim: To assess the equilibration rate of oxygen within embryo culture media when exposed to hypoxic conditions. Method: A handheld blood gas analyser (EPOC system, ‘BGEM’ cartridges) was used to determine the pO2 of G1+ culture media after exposure to a reduced oxygen environment. Results: Culture media in atmospheric oxygen (with or without the presence of CO2) showed mean pO2 values of 150mmHg. Media fully equilibrated over 24 hours in 5% oxygen fell to approximately 70mmHg. This was well above the theoretically expected value but shown to be consistent across multiple measurements including various dish and incubator types. Initial equilibration to hypoxic conditions occurred relatively rapidly, with dishes undergoing 50% of the required oxygen reduction in only 30 minutes. Within 1.5 hours of exposure 5% O2, media achieved 83% of the required change, with full equilibration reached at approximately 2.5 hours. Pre-equilibration of dishes in 6% CO2 did not appear to affect the oxygen diffusion rate compared with non-equilibrated media. Oxygen exhibited similarly rapid diffusion into culture media when dishes equilibrated at 5% O2 were exposed to atmospheric conditions, with pO2 increasing by 1.46mmHg per minute. Conclusion: The diffusion rate of oxygen appears to be significantly faster than the more widely studied CO2, however, oxygen tension in media still requires greater than 2 hours to reach optimum levels, requiring us to consider oxygen in our equilibration practices.