Although the optimal osmolality for in vitro human embryo development is unknown, it makes sense that the osmolality of commercially available culture media is broadly in line with the osmolarity of the in vivo embryo environment (i.e. 280-290 mOsm/ kg). For example, the osmolality of SAGE 1-Step and global total LP is specified as 257-273 mOsm/ kg and 260-270 mOsm/kg, respectively.
The fact that culture media are often supplied with a slightly lower-than-physiological pH introduces the very important fact that evaporation of water from media will cause its osmolality to increase. It is this simple evaporation of water that needs careful consideration in the modern IVF lab.
It is commonplace nowadays to mitigate the effects of evaporation by using oil overlay (in addition, of course, to the advantages of temperature stability and protection from infection). However, it is a common misbelief that evaporation will not take place when oil overlay is used. The effect of evaporation through the oil overlay and in particular, the effect of oil volume can clearly be seen in Figure 1, where extremely high osmolalities are observed when a smaller volume of oil-overlay is used1. Recommended oil overlayer is minimum 2 mm.
Furthermore, the data shown in Figure 1 were derived using a ‘large’ medium drop volume (200μl) leading to the second important consideration in terms of evaporation namely, that which might occur as small droplets of culture medium are being prepared.
This phenomenon has been elegantly demonstrated by Jason Swain2 and colleagues who investigated the effect of different ways of preparing microdrops (of different volumes) on their osmolality. Figure 2 shows, in particular, that preparing microdrops of small volume and preparing microdrops at 37°C rather than at room temperature can cause a significant increase in osmolality.