P-129 Oxygen levels alter energy metabolism in bovine preimplantation embryos

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Abstract Study question What is the effect of oxygen (O2) levels on the transcriptomic profile and embryonic genome activation of bovine embryos during the in vitro culture? Summary answer Normoxia (20% O2) delayed transcriptomic reprogramming and embryonic genome activation (EGA), and induced changes in energy metabolism gene expression. What is known already Mammalian preimplantation embryo development is a complex sequence of events where, within a week, terminally differentiated need to be reprogrammed to totipotency and subsequently diverge to embryonic and extraembryonic cell lineages for post-implantation embryo development. This period of development is highly sensitive to environmental changes such as oxygen levels (O2), pH, and temperature. Many IVF clinics still use normoxia for the embryo culture instead of hypoxia. In addition, a new hypothesis of sequential O2 hypoxia followed by ultrahypoxia has emerged. The molecular evidence about the O2 effect mostly comes from mice which are not developmentally similar to humans. Study design, size, duration In this study, we used triplicate bovine embryos as a model for human embryogenesis to compare the influence of O2 levels on preimplantation embryonic development by culturing embryos either in normoxic (20% O2) or physiological hypoxic (6% O2) conditions, or sequential hypoxia until 16-cell stage and then switching to ultrahypoxic culture (2% O2). Participants/materials, setting, methods As the readout for varied O2 effects, we performed RNA sequencing using the 5’ targeted STRT-N method on single embryos. We compared zygotes, 4-, 8-, 16-cell, and blastocyst stage embryos grown in either normoxic or hypoxic conditions, adding ultrahypoxia for blastocyst stage embryos as the third condition. Main results and the role of chance The effect of O2 was not observed at the cleavage rate, however the clear difference was seen at the blastocyst formation rate. The highest number of embryos that reached the blastocyst stage was in hypoxia condition (36%), in normoxia it was 13% and in ultrahypoxia, this number was only 4.6%. Transcriptomic profiling showed that normoxic conditions slowed down maternal transcript degradation and EGA. At the blastocyst stage, normoxia embryos did not have key energy metabolism genes for glycolysis upregulated, but rather only depended on oxidative phosphorylation metabolism. When we compared hypoxia and ultrahypoxia blastocysts, they had similar transcription profiles. Both conditions induced proper upregulation of the energy metabolism genes involved in glycolysis and lipid metabolism which are typical for in vivo embryos. We found constant hypoxia culture system provides the best blastocyst formation rate and induces appropriate energy metabolism needed for later stages of development. Normoxia alters the EGA, and energy metabolism and decreases blastocyst formation rate. Despite having a similar transcription profile as hypoxia, ultrahypoxia led to the lowest blastocyst formation rate making this condition sub-optimal for the in vitro culture of embryos. Limitations, reasons for caution The limitation of this study is the use of bovine as an animal model instead of human embryos. Due to this, the direct translation of the results to humans should be taken with caution. Wider implications of the findings This study supports previous literature on hypoxic culture conditions being the most suitable for in vitro embryo culture. We provide new insights on the reason normoxia embryos don't have the same success rate as hypoxia embryos. We didn't observe any benefits of lowering O2 to 2%. Trial registration number No

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