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The physiological pH for human gametes and embryos is generally thought to be between 7.2 and 7.4. In order to maintain this range of pH during culture, i.e. to create a similar environment to that of the human reproductive tract, we use CO₂ gas inside various types of incubators (this blog post by Markus Montag – “Considerations for embryos culture at high altitude”, further explains this).

In vitro, the pH is generally maintained between 7.2 and 7.4.  All Vitrolife media are formulated to meet this narrow pH specification range, supporting optimal metabolic conditions. Maintaining a defined and physiological pH is through the inclusion of certain chemical components in the media called “pH buffers”. These pH buffers act as a weak acid or base. As a result, solutions containing such buffers can resist a change in pH caused by environmental changes.

The most commonly used pH buffer in IVF culture media is bicarbonate, which is the same buffer that is present in our blood. Carbon dioxide (CO2) in the atmosphere surrounding the culture dish will dissolve and equilibrate in the medium. Dissolved CO2 increases the amount of carbonic acid in the medium, releasing protons and therefore decreasing pH in the medium (see equation below). If the CO2 level in an incubator remains constant, the pH of the medium can be maintained.  Vitrolife culture media require a CO2 concentration of 6.0% at sea level, but the CO2 concentration can be adjusted by the end user to reach the desired pH. This ensures there is enough CO2 inside the incubator surrounding the culture dish, and in turn the formation of carbonic acid and protons, to maintain the specified pH of the culture media.

CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+

When a procedure is performed in atmospheric conditions outside of the incubator, the media containing gametes and embryos are exposed to a much lower CO2 concentration (about 0.02%).  This dramatic drop in CO2 concentration will cause a change in the carbonic acid and proton content in the medium and an increase in the pH to a level above what is optimal for gametes and embryo development.  Using an oil overlay on the dish will somewhat delay the effect of this drop in CO2 concentration and subsequent pH increase.  Thus, it is recommended to use media containing a pH buffer other than bicarbonate when performing procedures under atmospheric conditions.

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