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.

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The developmental potential of early embryos is mainly dictated by the quality of the oocyte. Here, we explore the utility of the maternal spindle transfer (MST) technique as a reproductive approach to enhance oocyte developmental competence. Our proof-of-concept experiments show that replacement of the entire cytoplasm of oocytes from a sensitive mouse strain overcomes massive embryo developmental arrest characteristic of non-manipulated oocytes. Genetic analysis confirmed minimal carryover of mtDNA following MST. Resulting mice showed low heteroplasmy levels in multiple organs at adult age, normal histology and fertility. Mice were followed for five generations (F5), revealing that heteroplasmy was reduced in F2 mice and was undetectable in the subsequent generations. This pre-clinical model demonstrates the high efficiency and potential of the MST technique, not only to prevent the transmission of mtDNA mutations, but also as a new potential treatment for patients with certain forms of infertility refractory to current clinical strategies.

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Objective: To study the effect of zona opening (ZO) and 2 zona thinning (ZT) techniques on the hatching process of mouse embryos using a last-generation laser system and time-lapse microscopy (TLM). Design: Prospective randomized study. Setting: Private research center. Animals: A total of 267 F1 hybrid (B6/CBA) mice embryos were included. Intervention(s): Morulae were randomly selected and the zona pellucida (ZP) manipulated using a laser system according to 4 experimental groups: control (ZP intact, n ¼ 59), ZO (25 mm hole, n ¼ 70), ZT25 (25% perimeter thinned, n ¼ 71), and ZT35 (35% perimeter thinned, n ¼ 67). Embryo development was monitored by TLM until day 6. Main Outcome Measure(s): Time to first breach the ZP, hatching time, time to complete hatching, multiple breaching, multiple hatching, loss of cells, hole size, and embryo quality were analyzed. Result(s): No significant differences in the proportion of completely hatched embryos were found among groups. However, the time (average hours AE SD) to complete hatching was significantly delayed in the control group compared with all laser-treated groups: 118.3 AE 9.5 hours in the ZT25 group, 116.6 AE 8.7 hours in the ZT35 group, and 120.4 AE 9.9 hours in the ZO group. The applied laser techniques did not interfere with the quality of the blastocysts at day 5/6 of culture. Conclusion(s): ZO, ZT25, and ZT35 embryos hatched significantly earlier than the zona intact group without increasing the multiple hatching rates, suggesting an improvement of the hatching process. This study found that the pattern of the hatching process after ZT and ZO differs. (Fertil Steril Sci Ò 2021;2:43-9. Ó2020 by American Society for Reproductive Medicine.