Single-embryo transcriptomic atlas of oxygen response reveals the critical role of HIF-1α in prompting embryonic zygotic genome activation
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Cited by 3 publications
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Oxygen level alters energy metabolism in bovine preimplantation embryos
STUDY QUESTION: What is the effect of different oxygen (O2) levels on the transcriptomic profile of bovine embryos during the in vitro culture? SUMMARY ANSWER: Embryos 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 ALREADY: Mammalian 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, 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 5end 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 CHANCE: We 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 DATA: The 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 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 human 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. 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).
Oxygen level alters energy metabolism in bovine preimplantation embryos
STUDY QUESTION: What is the effect of different oxygen (O2) levels on the transcriptomic profile of bovine embryos during the in vitro culture? SUMMARY ANSWER: Embryos 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 ALREADY: Mammalian 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, 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 5end 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 CHANCE: We 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 DATA: The 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 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 human 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. 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).
Molecular Markers in Embryo Non-Development: Analysis of Gene Expressions (Ki-67, hTERT, HIF-1α) in Spent Embryo Culture Medium
An embryo culture medium is a specialized set of ambient conditions, technological equipment, and nutrients that embryos require to grow properly. We aimed to investigate the Ki-67, hTERT, and HIF-1α gene expression differences between developing and non-developing embryos in spent embryo culture medium. Ki-67, hTERT, and HIF-1α gene expressions were determined from the spent embryo culture medium containing developing and non-developing embryos of 20 normoresponder patients admitted to the Bahçeci Umut IVF Center. An increase in hTERT gene expression (p < 0.05) and a decrease in HIF-1α gene expression (p < 0.001) were observed in mediums of developing compared to the non-developing embryos. No difference was observed in Ki-67 gene expression (p > 0.05). While there was a correlation between Ki-67 and HIF-1α genes in the non-growing group (r < 0.01); no correlation was observed in the developing group (r > 0.05). Both normoresponder groups will be similar in terms of proliferation rate. The low HIF-1α expression that observed high telomerase activity in embryo development maintains continuity and avoids mechanisms that result in cell death. A molecular study of the embryo development in patients with similar characteristics may help to understand the pathogenesis of the disease and establish a diagnosis and specific treatment.
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