P–250 Mineral oil with high viscosity improves the stability of pH and osmolality in the human in vitro culture system
Authors: Mestres E , Matia-Algué Q , Villamar A , García-Jiménez M , Casals A , Acacio M , Calderón G , Costa-Borges N 
Published: August 2021

Abstract

Study question
Do commercial mineral oil brands differ in their capacity to stabilize the human embryo culture system, and is this related to the oil’s viscosity?

Summary answer
While the oils’ viscosity only had minor effects on temperature maintenance, it showed a direct correlation with the stability of pH and osmolality during culture.

What is known already
Mineral oil is a key component of the in vitro embryo culture system, which stabilizes temperature, pH and osmolality of the media during culture. Its use has been implemented worldwide for several decades and many manufacturers currently produce and commercialize oil intended for human embryo culture. Unfortunately, oil remains as one of the less characterized products in the IVF laboratory due to a lack of standardized nomenclature, production and testing. With differing physico-chemical properties, such as viscosity, oils produced by various manufacturers could behave differently to the same culture conditions and, thus, its use may need to be adjusted accordingly.

Study design, size, duration
Viscosity was quantified in three high-viscosity (H-V) and three low-viscosity (L-V) oils with a viscosity-meter. The required time for media’s pH to equilibrate using each oil was studied, as well as its subsequent stability outside the incubator for 30min. In-drop temperature was assessed during 15min when taking a dish outside the incubator, and again when putting it back. Additionally, each oil’s capacity to avoid media evaporation was studied with daily osmolality measurements during 7 days.

Participants/materials, setting, methods
pH equilibration was measured with a continuous pHmeter (Log&Guard, Vitrolife) in 4-well dishes prepared with 600µl of medium and 500µl of oil. For the other experiments, 35mm dishes with 4ml of oil and 20µl media droplets were used. pH stability was assessed after 0, 15 and 30min outside the incubator with a blood-gas-analyzer (epoc,SiemensHelthineers). A fine-gauge thermocouple was used to measure in-drop temperature loss/recovery. Daily osmolality readings were taken with a vapor pressure osmometer (Vapro5600,Wescor).

Main results and the role of chance
The selected oil samples had a viscosity of 115, 111, 52, 22, 18, and 12cP. The medium’s pH took approximately 12h to completely equilibrate under H-V oils, while it took less than 4h in L-V. Similarly, the rise in pH after 30min on a heated stage outside of the incubator with room atmosphere was 0.03, 0.04, 0.06, 0.13, 0.17, and 0.26, respectively.
Dishes were taken out of the incubator and placed on a heated surface. In the first five minutes, the in-drop temperature loss ranged between –0.22 and –0.13oC/min, with no significant differences observed between oil types. However, temperature plateaued at a significantly higher value in L-V oils (36.5oC), compared to H-V brands (36.25–36.1oC; p = 0.0005). By contrast, all samples followed a similar pattern when the dishes were returned to the benchtop incubator, with temperature taking around 7 minutes to completely recover.
Some media evaporated in all oil groups during the 7-day culture in a dry benchtop incubator. The linear regression performed to compare the evaporation rate between groups showed a statistically significant correlation between oil viscosity and the rate of evaporation (p &lt; 0.0001), with an osmolality rise ranging between +2.55mmol/kg/day in the most viscous oil and +6.29mmol/kg/day in the least viscous.

Limitations, reasons for caution
While the selected oils for this study represent a wide range of options in the market, future projects could widen this selection and include additional tests, such as optimized bioassays. Results may vary between centers, and thus each laboratory should test and optimize their culture system with their own settings.
Wider implications of the findings: Different oil brands have shown differing physico-chemical properties that have a direct effect on the culture system and the stability of several culture conditions. These results may be of major importance to adapt the settings and methodologies followed in each IVF laboratory according to the type of oil being used.

Trial registration number
Not applicable