STUDY QUESTION Which lab-related factors impact the culture system’s capacity to maintain a stable osmolality during human embryo culture? SUMMARY ANSWER Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture. WHAT IS KNOWN ALREADY Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes. STUDY DESIGN, SIZE, DURATION The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation. PARTICIPANTS/MATERIALS, SETTING, METHODS Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates. MAIN RESULTS AND THE ROLE OF CHANCE Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator’s characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture. LIMITATIONS, REASONS FOR CAUTION While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories. WIDER IMPLICATIONS OF THE FINDINGS Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables. STUDY FUNDING/COMPETING INTEREST(S) This study was privately funded. TRIAL REGISTRATION NUMBER N/A.

STUDY QUESTION Are there significant differences between the available commercial oil brands used for human IVF? SUMMARY ANSWER Important differences have been detected among the tested oil brands in their potential to stabilize culture conditions and, more importantly, in their direct effect on embryo development and viability. WHAT IS KNOWN ALREADY Mineral oil is a critical component of the human culture system due to its protective and stabilizing roles during in vitro embryo development. Many different oils are available on the market, with differences in their viscosity, density and overall quality. STUDY DESIGN, SIZE, DURATION Thirteen different commercial oil brands were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS Each oil was firstly analyzed to assess its viscosity, density, peroxide value and potential oxidation. Secondly, the capacity of each oil to reduce pH, osmolality and temperature fluctuations during embryo culture and manipulation was compared. Lastly, a sensitive mouse embryo assay (MEA) protocol, previously optimized to detect toxicity in oils samples, was used to compare the overall quality of the different brands in terms of embryo developmental rates up to the blastocyst stage. At the end of the MEAs, a triple labeling protocol was applied to analyze Oct4+ cells, apoptotic cells and total cell counts in the blastocysts obtained by fluorescence microscopy. MAIN RESULTS AND THE ROLE OF CHANCE Significant divergences were detected in the rise of osmolality and the equilibration and stability of pH between different oils, which could be correlated to their physico-chemical characteristics. In particular, oil samples with a higher viscosity tended to offer an additional protection against fluctuations in the culture conditions, however, the differences in temperature stability between oils were minor. Two out of the 13 oil samples, which were commercially available, were identified as embryo-toxic by applying the MEA protocol with increased sensitivity for toxicity detection. Additionally, substantial differences in the total number of cells and the number of cells in the inner cell mass of the obtained blastocysts were also detected between oil groups. LIMITATIONS, REASONS FOR CAUTION A single lot of oil was used for each brand and, thus, lot-to-lot variations in oil quality could not be determined. However, several bottles from the same oil were included to account for potential intra-lot variability. WIDER IMPLICATIONS OF THE FINDINGS Commercial oils differ in both their physical characteristics and their performance in maintaining the stability of the culture conditions during in vitro embryo culture. Oil selection is important for embryo culture success. Additionally, the detection of embryo-toxic oils which had already been released to the human IVF market showcases the importance of applying sensitive MEA protocols for a better detection of toxicity in this type of samples. STUDY FUNDING/COMPETING INTEREST(S) This study was privately funded. TRIAL REGISTRATION NUMBER N/A.

No abstract

No 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 < 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