bioRxiv2024
STUDY QUESTIONWhat is the effect of different oxygen (O2) levels on the transcriptomic profile of bovine embryos during the in vitro culture? SUMMARY ANSWEREmbryos grown in hypoxia (6% O2) from zygotes until the blastocyst stage had the highest blastocyst formation rate, whereas normoxia (20% O2) delayed transcriptomic reprogramming and embryonic genome activation, and induced changes in energy metabolism gene expression. WHAT IS KNOWN ALREADYMammalian preimplantation embryo development is a complex sequence of events where, within a week, the zygote is reprogrammed to totipotency and subsequently diverges to embryonic and extraembryonic cell lineages for post-implantation embryo development. This period of development is sensitive to oxygen levels that can affect various cellular processes. STUDY DESIGN, SIZE, DURATIONIn 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, METHODSAs the readout for varied O2 effects, we performed RNA sequencing using 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 condition, adding ultrahypoxia for blastocyst stage embryos as the third condition. MAIN RESULTS AND THE ROLE OF CHANCEWe found that the initial cleavage rate was not affected by O2 levels but there was a clear difference in blastocyst formation rate. In hypoxia, 36% of embryos reached blastocyst stage while in normoxia the blastocyst formation rate was 13%. In final ultrahypoxia condition only 4.6% of embryos reached blastocyst stage. Transcriptomic profiles showed that normoxic conditions slowed down oocyte transcript degradation and embryonic genome activation. Key metabolic enzyme genes were also altered between hypoxic and normoxic conditions at the blastocyst stage. Both hypoxic and ultrahypoxic conditions induced energy production by upregulating genes involved in glycolysis and lipid metabolism typical to in vivo embryos. In contrast, normoxic conditions failed to upregulate glycolysis genes and only depended on primitive oxidative phosphorylation metabolism. We conclude that constant hypoxia culture of in vitro embryos provided the highest blastocyst formation rate and appropriate energy metabolism. Normoxia altered the energy metabolism and decreased the blastocyst formation rate. Even though ultrahypoxia at blastocyst stage resulted in a drop of blastocyst formation, the transcriptional profile of surviving embryos was normal. LARGE SCALE DATAThe raw data (BCL files) are available at Zenodo: XXXXX FASTQ files generated are available in the EMBLs European Bioinformatics Institute (EMBL-EBI)-BioStudies with accession number X-XXXX. LIMITATIONS, REASONS FOR CAUTIONThe 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 human should be taken with caution. WIDER IMPLICATIONS OF THE FINDINGSThis study supports previous literature on hypoxic culture conditions being the most suitable for in vitro embryo culture. In addition, we provide new insights on why embryos grown in normoxia do not have the same success rate as embryos grown in hypoxia. We did not observe any benefits of lowering the oxygen levels to 2%, calling for caution of switching to this culture system. STUDY FUNDING/COMPETING INTEREST(S)This project has received funding from the European Unions Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 813707.; Work in the JK laboratory is supported by Jane and Aatos Erkko Foundation, Sigrid Juselius Foundation, Liv och Halsa (Finland), Swedish Brain Foundation and Swedish Research Council. The study was also supported by the Estonian Research Council (grant no. PRG1076) and the Horizon Europe NESTOR project (grant no. 101120075). What this means for patients?During assisted reproduction treatment (ART) oocytes are fertilized in vitro by sperm to form an embryo. The embryos are then exposed to environmental effects during the 5 days of culture before implantation. Embryo culture takes place inside the incubator, where different oxygen levels can be used. In this study, we compared 3 different oxygen concentrations for embryo culture: atmospheric or normoxia (20%), physiologic or hypoxia (6%) and combined low and ultra-low concentration with initial culture at 6% oxygen until day 3 followed by 2% (ultrahypoxia) until day 5 of culture. We conducted this study because many ART clinics use different oxygen concentrations for embryo culture, and we aimed to provide insight on how these different concentrations impact embryo development by monitoring gene expression. As using human embryos for this type of experiments is not commonly approved, we used bovine embryos whose early development resembles that of humans. We found that culturing embryos for 5 days at constant low oxygen concentration (hypoxia) gives the highest number of embryos that reach the blastocyst stage (day 5) and have expected gene expression that provides the physiological path of embryo development. Culturing embryos at atmospheric oxygen concentration showed that only 13% of fertilised eggs reached the blastocyst stage at day 5, while culturing at the final ultra-low oxygen concentration had the lowest percentage of matured embryos, and we did not observe any benefits that would justify the use of such a culture system. We suggest that in vitro culturing of embryos at constant 5-6% oxygen gives the best results.