This is an abbreviation of Jason Swain (2019) publication with emphasis on the oil used for medium overlay written by Ann-Helene Jakobsson, Vitrolife.
The first time I came into contact with the usage of oil for cell culture was when I started working at an IVF clinic back in 1989. Previously having cultured somatic cells using large volumes of media in flasks placed in large humid box incubators the practice of oil cover was new to me. I remember that I found it strange to use a petroleum product for such important and sensitive cells as oocytes and embryos. Soon after my initial attempts as an embryologist, the clinic received oil bottles which contained mold, from a company long forgotten. This unfortunate event made us stop using oil and instead culture embryos in larger volumes. We also used humidified incubators and transferred embryos on day 2, I guess that is why we got away with this culture method and still achieved pregnancies. Outcomes were far from optimal though.
A few years later we had learned more and understood the importance of using oil cover to prevent evaporation of media and returned to the use of oil. Still we found the use of the light oil messy since oil drops seemed to be everywhere, on incubator shelves, the floor and in the LAF hoods. We used all sorts of oil, I´m embarrassed to say. In the early 1990ties there was not so much to choose from, no mouse embryo tested oils were available and knowledge about the risks of using poor quality oil was limited. We tried to wash the oil with medium to reduce the risk of potential toxicity, I must add in our defense.
Later, when we extended embryo culture to the blastocyst stage we learned the hard way that all oils (and embryo culture dishes too for that matter) do not work and that some oils can be very detrimtal to embryo development. Today, embryo culture is not always performed in large box humid incubators and embryo culture to the blastocyst stage is becoming more and more frequent. This puts higher demands than ever before on the choice and use of oil in IVF laboratories.
Dr Jason Swain has written a review article entitled “Controversies in ART: considerations and risks for uninterrupted embryo culture” which was published in RBMOnline 2019, in which he addresses (among other topics) important issues when using oil in IVF. The following are excerpts from his article.
The most obvious reason to use oil is to prevent evaporation of the small volume media droplets. Evaporation results in an increase in medium osmolality which affects cell volume control and can impair embryo development if it increases to above 300mOsm/kg even though the presence of amino acids in the embryo culture media act as osmolytes. It is thus important to use a single-step medium with a somewhat lower starting osmolality to diminish the risk of increased osmolality to above 300mOsm/kg after 5-7 days of embryo culture, including time for medium equilibration.
Evaporation and increased osmolality as a consequence is a risk that has increased with modern embryo culture techniques using dry incubators. The longer the medium remains in the incubator the more evaporation occurs. Culturing embryos to the blastocyst stage in a single-step medium without medium renewal means that the medium may be in the incubator for up to 180 hours. Using an appropriate volume of oil to cover the medium is very important as shown by Carpenter et al (2018) who demonstrated that using 7 ml of instead of 3 ml of oil in the same size dish resulted in a decreased risk of evaporation. The size of the drop can also affect the rate of evaporation as described by Iwata et al. (2016) and Swain (2018).
Another important consideration is that media can evaporate also when covered under oil (Swain et al, 2016). Again, it must be emphasized that the amount of oil used to cover the medium drops is very important. Also, the type of oil used affects the evaporation rate according to Swain (2018). A heavier and more dense oil will reduce the evaporation compared to a lighter oil. Therefore, Swain concludes, the use of a heavier, denser oil many benefit evaporation for uninterrupted culture in a dry environment. Presence of lids on dishes or washing of oil in dry incubators does not affect the rate of media evaporation and resulting osmolality increase (Swain et al, 2016). The conclusion is that the water portion of culture media is likely not being absorbed into the oil and that evaporation through the oil is actually occurring.
An additional important aspect of evaporation is the fact that pH also increases (Swain, 2015). This increase is caused by the increased concentration of sodium bicarbonate when evaporation occurs. Since all other solute concentrations also will increase when media evaporates, the embryos are not exposed to the same components or concentrations as intended with the original composition of the medium.
Volatile organic compounds
Swain writes further that with prolonged exposure of a culture dish with oil overlay to the confines of the incubator during uninterrupted culture comes the potential for accumulation of harmful volatile organic compounds (VOCs). VOCs that are present in room air may also accumulate within the culture system, especially if using high oxygen culture. Therefore, particularly with extended, uninterrupted culture, precautions should be taken to ensure low VOC levels in the laboratory and in the incubator specifically (Mortimer et al. 2018).
Mineral oil degradation
It is not only media and protein degradation that must be considered for extended embryo culture, also degradation of oil, more specifically oxidation of oil, may negatively affect embryo development as described by Otsuki et al. (2007 and 2009). Simply opening a bottle of oil resulted in increased peroxidation after several weeks compared with unopened oil. Also, peroxidation will occur at elevated temperatures and with exposure to light, especially UV-light. Initial peroxide content vary according to Otsuki et al. (2009) and storage of oil at room temperature for 4-12 months tend to increase peroxidation levels compared to storage at 2-8°C. Although elevated temperature and extended time lines may not be a concern to IVF laboratories, data suggest that increased peroxide levels, sufficient enough to impair embryo development, may be a risk under laboratory conditions (Inoue, 2017). Therefore, the initial quality of the oil is likely important to ensure that damaging peroxides are not present before culture. The starting oil quality, including VOC content, peroxide levels and ionic composition which may differ between oils (Martinez et al, 2017) may be more important if using uninterrupted culture and thorough oil quality testing by manufacturers is prudent.
Swain ends his review by concluding that the field of IVF has undergone a shift to more laboratories using modern incubators, many of which are non-humidified. A shift towards using single step media and uninterrupted culture has also taken place. This simplifies the process for embryologists and may reduce treatment costs. This approach offers a potential to reduce harmful environmental stress factors and improve embryo quality and outcomes. However, optimized quality control and culture conditions are required to successfully implement uninterrupted culture. Without impeccable laboratory conditions the new culture condition could be imparting stress. Factors such as evaporation and associated osmolality and pH increases as well as oil peroxidation and VOC accumulation, could offset any advantage of reduced dish and embryo handling.
As so thoroughly described by Swain (2019) evaporation of media is an important factor to consider for optimal embryo culture using modern equipment and methods. Dripping of oil that I personally found inconvenient during my early days in the IVF laboratory may be reduced by the use of a heavier oil and that fact alone may be a reason to consider using a heavy oil rather than a lighter version. If a heavy oil also can reduce evaporation then even better.
Carpenter G, et al. (2018). The impact of dry incubation on osmolality of media in time lapse culture dishes. Hum. Reprod. 33: i61
Inoue F, (2017). Effect of antioxidant addition to oil for embryo culture. Fertil. Steril. 108: e60
Iwata K, et al. (2016). Unstable osmotic pressure in microdrops cultured under mineral oil in non-humidifed incubators. Fertil. Steril. 106: e355
Martinez C A, et al, (2017). Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development. Theriogenology 103: 17–23
Mortimer D, et al. (2018). Cairo consensus on the IVF laboratory environment and air quality: report of an expert meeting. Reprod. Biomed. Online 36: 658–674
Otsuki J, et al. (2007). Peroxidation of mineral oil used in droplet culture is detrimental to fertilization and embryo development. Fertil. Steril. 88: 741–743
Otsuki J, et al. (2009). Damage of embryo development caused by peroxidized mineral oil and its association with albumin in culture. Fertil. Steril. 91: 1745–1749
Swain J, et al. (2015). Real time assessment of pH stability within an IVF isolette. Fertil. Steril. 103: e37
Swain J, et al. (2016). Washing mineral oil used for microdrop overlay does not improve stability of media osmolality. Fertil. Steril. 106: e355
Swain J. (2018). Different mineral oils used for embryo culture microdrop overlay differentially impact media evaporation. Fertil. Steril. 109: e53
Swain J. (2019). Controversies in ART: considerations and risks for uninterrupted embryo culture. Reprod. Biomed. Online Vol 39, Issue 1.
Topics: Embryo culture & transfer
Written by Dr. Ann-Helene Jakobsson
Ann-Helene has a PhD in Genetics and started her career in IVF as Lab Manager at the private clinic Fertilitetscentrum in Sweden. Ann-Helene is a popular lecturer at Vitrolife workshops where she makes complicated matters easy to understand.