Medium osmolality changes during continuous culture are affected not only by the humidification level but also by medium composition

The increasing use of continuous culture to the blastocyst stage and use of a non-humidified environment led to a debate on the risk of increasing osmolality with potential negative impact on developmental potential. Historical data pointing to an osmolality limit of 300 mOsm utilized simple media (e.g. no amino acids). Today, more complex media are used for embryo culture. The aim of this investigation was to determine the impact of osmolality changes in relation to the starting osmolality in a humidified as well as non-humidified environment and whether medium components protect against osmolality changes. Medium droplets of 20 µL were placed in a 60 mm dish under 9.5 mL oil in a humidified or non-humidified environment during 5 days. Osmolality was measured on day 0 and day 5 using 50 µL medium for each measurement. Triplicate measurements were performed. Medium with starting osmolality of around 260, 280, 300,310 and 320 mOsm were prepared by adding NaCl. A simple medium (SG-1) and modern culture medium (Gx-TL) were compared. In a second series, the impact of amino acids and hyaluronan on osmolality changes was evaluated. In a humidified environment, osmolality increase ranged between 4 and 6 mOsm for both media and all starting osmolarities. In a non-humidified environment, the osmolality increase was between between 21 and 45 mOsm using SG-1 while it was between 19 and 23 mOsm for Gx-TL. For SG-1, osmolality increases were more pronounced with increasing starting osmolality while for Gx-TL osmolality increases were similar, independent of the starting osmolality. With a starting osmolality of around 260 mOsm, the increase in a simple medium over 5 days was 21 mOsm in a non-humidified environment. Addition of hyaluronan or amino acids reduced the osmolality increase to 18 mOsm for each component. In this test, the osmolality increase in Gx-TL containing both amino acids and hyaluronan was only 16 mOsm. Modern culture media containing amino acids and hyaluronan reduce osmolality changes during continuous culture in a non-humidified environment, with increasing protection as osmolality increases.