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STUDY QUESTION What factors associated with embryo culture techniques contribute to the rate of medium osmolality change over time in an embryo culture incubator without added humidity? SUMMARY ANSWER The surface area-to-volume ratio of culture medium (surface area of the medium exposed to an oil overlay), as well as the density and height of the overlaying oil, all interact in a quantitative way to affect the osmolality rise over time. WHAT IS KNOWN ALREADY Factors such as medium volume, different oil types, and associated properties, individually, can affect osmolality change during non-humidified incubation. STUDY DESIGN, SIZE, DURATION Several experimental designs were used, including simple single-factor completely randomized designs, as well as a multi-factor response surface design. Randomization was performed at one or more levels for each experiment. Osmolality measurements were performed over 7 days, with up to 8 independent osmolality measurements performed per treatment group over that time. For the multi-factor study, 107 independent combinations of factor levels were assessed to develop the mathematical model. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was conducted in a research laboratory setting. Commercially available embryo culture medium and oil was used. A MINC incubator without water for humidification was used for the incubation. Osmolality was measured with a vapor pressure osmometer after calibration. Viscometry and density were conducted using a rheometer, and volumetric flasks with an analytical balance, respectively. Data analyses were conducted with several commercially available software programs. MAIN RESULTS AND THE ROLE OF CHANCE Preliminary experiments showed that the surface area-to-volume ratio of the culture medium, oil density, and oil thickness above the medium all contributed significantly (P < 0.05) to the rise in osmolality. A multi-factor experiment showed that a combination of these variables, in the form of a truncated cubic polynomial, was able to predict the rise in osmolality, with these three variables interacting in the model (P < 0.05). Repeatability, as measured by the response of identical treatments performed independently, was high, with osmolality values being ± 2 of the average in most instances. In the final mathematical model, the terms of the equation were significant predictors of the outcome, with all P-values being significant, and only one P-value > 0.0001. LIMITATIONS, REASONS FOR CAUTION Although the range of values for the variables were selected to encompass values that are expected to be encountered in usual embryo culture conditions, variables outside of the range used may not result in accurate model predictions. Although the use of a single incubator type and medium type is not expected to affect the conclusions, that remains an uncertainty. WIDER IMPLICATIONS OF THE FINDINGS Using this predictive model will help to determine if one should be cautious in using a specific system and will provide guidance on how a system may be modified to provide improved stability during embryo culture. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Cook Medical. The author is a Team Lead and Senior Scientist at Cook Medical. The author has no other conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.