Table 2.

Schematic representation of studies investigating the impact of different types and modalities of use of mineral oils on embryo culture conditions and laboratory or clinical outcomes

Oil overlay and osmolality (evaporation prevention)
	Methods	Results	Materials
Olds et al. (2015)	Real-time pH data was evaluated following scheduled benchtop incubator openings in a microdrop culture system using a real-time pH monitoring device: the SAFE (Sterile Automated Fluoroscopic Evaluation) 35 µL or 50 µL of oil were used to cover 100 µL of culture media in SAFE Sens sensor cups and pH continuously evaluated in a benchtop incubator	pH of culture media under 35 µL of oil overlay resulted higher compared to the control after repeated incubator opening. More stable pH was ensured by covering the media with 50 µL of oil	Continuous pH monitoring of SAFE Sens sensor cups
Swain et al. (2018)	25-μL drops of media covered with 3.5 mL of 1 of 4 types of mineral oil to compare differences in osmolality after prolonged culture	Different mineral oils used for microdrop oil overlay result in different rates of media evaporation and resulting osmolality increase. Density of mineral oil seems to be an important factor to consider in terms of media evaporation	35-mm culture dish
Swain et al. (2016)	Measure of osmolality in microdrops (25 µL) of culture medium, in dishes prepared daily for 7 days and cultured in humidified and non-humidified incubators	Uninterrupted culture for up to 7 days in a non-humidified incubator resulted in an increase in media osmolality over time, while osmolality of microdrops in a humidified incubator remained unchanged	35-mm culture dish
Del Gallego et al. (2018)	Embryos cultured in time-lapse incubator in humidified and dry conditions (83 and 93 patients, respectively)	Significantly higher blastocyst rate in embryos cultured in humidified conditions (74.5% vs. 69.2%). No statistical differences in terms of pregnancy and miscarriage rates	Time-lapse multi-well dish
Yumoto et al. (2018)	18 culture dishes containing six 50-µL microdrops of single-step medium. Dishes were divided into three groups: (A) light oil (10.8 mPa/s at 37 °C), (B) heavy oil (36.5 mPa/s at 37 °C), and (C) washed oil (10.4 mPa/s at 37 °C), and placed in a non-humidified benchtop incubator for 6 days	Results showed that the osmotic pressure of microdrops covered by light oil significantly increased on day 3 onward compared to that of microdrops covered by heavy oil	Dish size not reported
Yumoto et al. (2019)	Three single-step culture media were incubated for 5 or 6 days covered with four different mineral oils in non-humidified or humidified incubators to investigate the stability of osmolality in microdrops under different conditions. The absolute water content and water activity, dynamic viscosity, and density for each oil were also analyzed	Osmolality significantly increased in non-humidified benchtop incubators. The range of the increase was affected by microdrop volume and by the type of mineral oil used to cover the drops. In contrast, microdrop osmolality did not change during 5-day incubation in a humidified benchtop incubator. The oil that showed the highest osmolality increase was analyzed and reported the lowest water content and water activity	35-mm plastic culture dishes
Mestres et al. (2021)	Several variables compared to assess the factors can affect evaporation and osmolality including media composition and supplementation, volume of mineral oil, incubator humidification, and the type of dish and incubator used	Evaporation and consequently osmolality increased according to humidity levels inside the incubators, the volume of mineral oil, and the type of culture media Volume of oil is crucial to stabilize osmolality in dry incubators	Six experiments were conducted in which different dishes and time-lapse dishes were compared with different volume of oil and culture conditions
Mullen (2021)	Analysis of 9 different commercially available oils and their effect on osmolality rise, toward the evaluation of surface area-to-volume, oil density and viscosity, oil overlay thickness, upon 7 days of culture in non-humidified benchtop incubator	The surface area-to-volume, low density, and low thickness of oil overlay directly affect the rate of osmolality increase. Moreover, a mathematical model of these three variables was developed predicting the rate of change of osmolality	Three different dishes were tested under different culture conditions: 35-mm dishes 96-well dishes 4-well dishes
Mestres et al. (2021)	Comparison of 13 different commercially available oils, detailing their viscosity and density, proneness to peroxidation, capacity to minimize fluctuations of the culture conditions (temperature, pH, osmolality), and potential toxicity	pH fluctuations are better mitigated by high-viscosity oils and only two of them analyzed in the study are able to minimize pH arise after 30 min outside the incubator. Osmolality and evaporation rate vary in low viscous oils and one of them analyzed showed huge arise of osmolality after 7 days of embryo culture, demonstrated as embryo-toxic after MEA test was performed	Different oils were analyzed and tested