Humidified incubators are widely used for culturing human embryos. However, the use of non-humidified benchtop incubators is becoming more common due to their smaller size and lower risk of fungal contamination. We previously reported that the osmotic pressure of culture media increased more than 20 mOsm/kg after continuous culture for five days in non-humidified incubators. Microdrops of culture medium are typically covered with mineral oil, which prevents changes in the medium's osmotic pressure, pH and temperature. Although several types of mineral oils are commercially available, it is unknown how the different types affect culture conditions. In this study, we examined how different mineral oil viscosities influenced the osmotic pressure of culture media in non-humidified incubators. Basic Clinical Study. We prepared 18 culture dishes containing six 50-μl microdrops of single-step medium (265 ± 10 mOsm/kg). Dishes were divided into three groups: A, light oil (10.8 mPa/sec at 37°C); B, heavy oil (36.5 mPa/sec at 37°C); and C, washed oil (10.4 mPa/sec at 37°C), and placed in a non-humidified benchtop incubator for six days. The osmotic pressure of one dish per group was measured daily using an osmometer. Student t-tests were used for statistical analyses. The osmotic values for each group are expressed as the mean ± S.D. In group A, the osmotic pressure increased from 268.4 ± 0.5 mOsm/kg (day 0) to 280.5 ± 0.5 mOsm/kg (day 3), and then 300.7 ± 2.4 mOsm/kg (day 6). The osmotic pressure in group B rose from 267.6 ± 0.5 mOsm/kg (day 0) to 275.6 ± 1.1 mOsm/kg (day 3), and then 286.8 ± 1.3 mOsm/kg (day 6). As with group B, the osmotic pressure in group C elevated from 267.6 ± 0.5 mOsm/kg (day 0) to 276.8 ± 0.8 mOsm/kg (day 3), and then 287.8 ± 0.4 mOsm/kg (day 6). These results showed that the osmotic pressure of microdrops covered by light oil significantly increased on day 3 onwards compared to that of microdrops covered by heavy oil (P < 0.01). Elevations in osmotic pressure are minimized by using heavy or washed mineral oils. Low-viscosity mineral oil significantly increased the osmotic pressure of culture medium after three days in a non-humidified incubator. However, our results also showed that the osmotic pressure of culture media will inevitably increase in non-humidified incubators regardless of the viscosity of the mineral oil. Therefore, depending on the types of mineral oil, culture media, culture period and incubators used, it may be possible for the osmotic pressure of culture media to exceed the optimal culture environment threshold for human embryos. Although in vitro culture is essential for ART programs, this system is unstable and insufficient compared to the in vivo situation. To more closely align the ART in vitro culture environment to that found in vivo, we must focus on optimizing the materials available to us.
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