Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia



This study used noninvasive, fluorescence lifetime imaging microscopy (FLIM)-based imaging of NADH and FAD to characterize the metabolic response of mouse embryos to short-term oxygen deprivation. We investigated the response to hypoxia at various preimplantation stages.


Mouse oocytes and embryos were exposed to transient hypoxia by dropping the oxygen concentration in media from 5–0% over the course of ~1.5 h, then 5% O2 was restored. During this time, FLIM-based metabolic imaging measurements of oocyte/embryo cohorts were taken every 3 minutes. Experiments were performed in triplicate for oocytes and embryos at the 1- to 8-cell, morula, and blastocyst stages. Maximum hypoxia response for each of eight measured quantitative FLIM parameters was taken from the time points immediately before oxygen restoration.


Metabolic profiles showed significant changes in response to hypoxia for all stages of embryo development. The response of the eight measured FLIM parameters to hypoxia was highly stage-dependent. Of the eight FLIM parameters measured, NADH and FAD intensity showed the most dramatic metabolic responses in early developmental stages. At later stages, however, other parameters, such as NADH fraction engaged and FAD lifetimes, showed greater changes. Metabolic parameter values generally returned to baseline with the restoration of 5% oxygen.


Quantitative FLIM-based metabolic imaging was highly sensitive to metabolic changes induced by hypoxia. Metabolic response profiles to oxygen deprivation were distinct at different stages, reflecting differences in metabolic plasticity as preimplantation embryos develop.

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  1. 1.

    Wales RG, Brinster RL. The uptake of hexoses by pre-implantation mouse embryos in vitro. J Reprod Fertil. 1968;15:415–22.

    CAS  Article  Google Scholar 

  2. 2.

    Brinster RL. Studies on the development of mouse embryos in vitro. II. The effect of energy source. J Exp Zool. 1965;158:59–68.

    CAS  Article  Google Scholar 

  3. 3.

    Brinster RL. Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J Reprod Fertil. 1965;10:227–40.

    CAS  Article  Google Scholar 

  4. 4.

    Biggers JD, Whittingham DG, Donahue RP. The pattern of energy metabolism in the mouse oöcyte and zygote. Proc Natl Acad Sci U S A. 1967;58:560–7.

    CAS  Article  Google Scholar 

  5. 5.

    Hewitson LC, Leese HJ. Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. J Exp Zool [Internet]. 1993;267:337–43 Available from:

    CAS  Article  Google Scholar 

  6. 6.

    Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil. 1984;72:9–13.

    CAS  Article  Google Scholar 

  7. 7.

    Gardner DK, Leese HJ. Non-invasive measurement of nutrient uptake by single cultured pre-implantation mouse embryos. Hum Reprod. Oxford University Press. 1986;1:25–7.

    CAS  Article  Google Scholar 

  8. 8.

    Chason RJ, Csokmay J, Segars JH, DeCherney AH, Armant DR. Environmental and epigenetic effects upon preimplantation embryo metabolism and development. Trends Endocrinol Metab. 2011;412–420.

  9. 9.

    Takahashi M. Oxidative stress and redox regulation on in vitro development of mammalian embryos. J Reprod Dev. 2012:1–9.

  10. 10.

    Van Blerkom J. Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod Biomed Online [Internet]. 2008;16:553–69 Available from:

    Article  Google Scholar 

  11. 11.

    Harvey AJ. Mitochondria in early development: Linking the microenvironment, metabolism and the epigenome. Reproduction BioScientifica Ltd. 2019:R159–79.

  12. 12.

    Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol Elsevier Ltd. 2009:346–53.

  13. 13.

    Magnusson C, Einarsson B, Nilsson BO. Oxygen consumption by the mouse blastocyst at activation for implantation. Acta Physiol Scand. 1986;127:215–21.

    CAS  Article  Google Scholar 

  14. 14.

    Wale PL, Gardner DK. Oxygen Regulates Amino Acid Turnover and Carbohydrate Uptake During the Preimplantation Period of Mouse Embryo Development1. Biol Reprod. Oxford University Press (OUP). 2012;87.

  15. 15.

    Swain JE. Controversies in ART: can the IVF laboratory influence preimplantation embryo aneuploidy? Reprod BioMed Online. Elsevier Ltd. 2019:599–607.

  16. 16.

    Duranthon V, Watson AJ, Lonergan P. Preimplantation embryo programming: Transcription epigenetics, and culture environment. Reproduction. 2008;135:141–50.

    CAS  Article  Google Scholar 

  17. 17.

    Zeng F, Baldwin DA, Schultz RM. Transcript profiling during preimplantation mouse development. Dev Biol. 2004.

  18. 18.

    Renard JP, Philippon A, Menezo Y. In-vitro uptake of glucose by bovine blastocysts. J Reprod Fertil. 1980;58:161–4.

    CAS  Article  Google Scholar 

  19. 19.

    Leese HJ. Non-invasive methods for assessing embryos. Hum Reprod. Oxford University Press. 1987;2:435–8.

    CAS  Article  Google Scholar 

  20. 20.

    Gardner DK, Leese HJ. Assessment of embryo viability prior to transfer by the non-invasive measurement of glucose uptake. J Exp Zool. 1987;242:103–5.

    CAS  Article  Google Scholar 

  21. 21.

    Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod [Internet]. 1996;11:1975–8 Available from:

    CAS  Article  Google Scholar 

  22. 22.

    Gardner DK, Wale PL, Collins R, Lane M. Glucose consumption of single post-compaction human embryos is predictive of embryo sex and live birth outcome. Hum Reprod. Oxford University Press. 2011;26:1981–6.

    CAS  Article  Google Scholar 

  23. 23.

    Papkovsky DB, Dmitriev RI. Imaging of oxygen and hypoxia in cell and tissue samples. Cell Mol Life Sci [Internet]. 2018;75:2963–80 Available from:

    CAS  Article  Google Scholar 

  24. 24.

    Hink MA, Bisselin T, Visser AJWG. Imaging protein-protein interactions in living cells. Plant Mol Biol [Internet]. 2002;50:871–83 Available from:

    CAS  Article  Google Scholar 

  25. 25.

    Wang Y, Shyy JY, Chien S. Bioengineering in Cell and Tissue Research: Fluorescence Live-Cell Imaging: Principles and Applications in Mechanobiology. Proteins [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008;10:1–38. Available from:

  26. 26.

    Van Munster EB, Gadella TWJ. Fluorescence Lifetime Imaging Microscopy (FLIM). Adv Biochem Eng Biotechnol. 2005:143–75.

  27. 27.

    Sanchez T, Wang T, Pedro MV, Zhang M, Esencan E, Sakkas D, et al. Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes. Fertil Steril. 2018;110:1387–97.

    Article  Google Scholar 

  28. 28.

    Ma N, de Mochel NR, Pham PD, Yoo TY, Cho KWY, Digman MA. Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach. Sci Rep. Nature Publishing Group. 2019;9:1–13.

    Article  Google Scholar 

  29. 29.

    Ghukasyan V, Heikal A. Natural biomarkers for cellular metabolism: biology, techniques, and applications. 2014.

    Book  Google Scholar 

  30. 30.

    Berg S, Kutra D, Kroeger T, Straehle CN, Kausler BX, Haubold C, et al. ilastik: interactive machine learning for (bio) image analysis. Nat Methods. Nat Res Forum. 2019;16:1226–32.

    CAS  Google Scholar 

  31. 31.

    Sanchez T, Venturas M, Aghvami SA, Yang X, Fraden S, Sakkas D, et al. Combined non-invasive metabolic and spindle imaging as potential tools for embryo and oocyte assessment. Hum Reprod [Internet]. 2019;34:2349–61 Available from:

    Article  Google Scholar 

  32. 32.

    Houghton FD, Thompson JG, Kennedy CJ, Leese HJ. Oxygen consumption and energy metabolism of the early mouse embryo. Mol Reprod Dev [Internet]. 1996 [cited 2020 Apr 9];44:476–85. Available from:

  33. 33.

    Mills RM, Brinster RL. Oxygen consumption of preimplantation mouse embryos. Exp Cell Res. Academic Press. 1967;47:337–44.

    Article  Google Scholar 

  34. 34.

    Leese HJ. History of oocyte and embryo metabolism. Reprod Fertil Dev. CSIRO. 2015:567–71.

  35. 35.

    Ng KYB, Mingels R, Morgan H, Macklon N, Cheong Y. In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in human conception: A systematic review. Hum Reprod Update. Oxford University Press. 2018;24:15–34.

    Article  Google Scholar 

  36. 36.

    Redel BK, Brown AN, Spate LD, Whitworth KM, Green JA, Prather RS. Glycolysis in preimplantation development is partially controlled by the Warburg Effect. Mol Reprod Dev. 2012;79:262–71.

    CAS  Article  Google Scholar 

  37. 37.

    Krisher RL, Prather RS. A role for the Warburg effect in preimplantation embryo development: Metabolic modification to support rapid cell proliferation. Mol Reprod Dev. 2012;79:311–20.

    CAS  Article  Google Scholar 

  38. 38.

    Bagheri D, Kazemi P, Sarmadi F, Shamsara M, Hashemi E, Daliri Joupari M, et al. Low oxygen tension promotes invasive ability and embryo implantation rate. Reprod Biol. Elsevier Sp. z o.o. 2018;18:295–300.

    Article  Google Scholar 

  39. 39.

    O’Fallon JV, Wright RW. Quantitative determination of the pentose phosphate pathway in preimplantation mouse embryos1. Biol Reprod. Oxford University Press (OUP). 1986:34, 58–64.

  40. 40.

    O’Fallon JV, Wright RW. Calculation of the pentose phosphate and Embden-Myerhoff pathways from a single incubation with [U-14C]- and [5-3H]glucose. Anal Biochem. 1987;162:33–8.

    Article  Google Scholar 

  41. 41.

    Gardner HG, Kaye PL. Characterization of glucose transport in preimplantation mouse embryos. Reprod Fertil Dev. 1995;7:41–50.

    CAS  Article  Google Scholar 

  42. 42.

    Comizzoli P, Urner F, Sakkas D, Renard JP. Up-regulation of glucose metabolism during male pronucleus formation determines the early onset of the S phase in bovine zygotes1. Biol Reprod. Oxford University Press (OUP). 2003;68:1934–40.

    CAS  Article  Google Scholar 

  43. 43.

    Sanchez T, Seidler EA, Gardner DK, Needleman D, Sakkas D. Will non-invasive methods surpass invasive for assessing gametes and embryos? Fertil Steril. Elsevier Inc. 2017:730–7.

  44. 44.

    Dumollard J, Carroll MR, Duchen K, Campbell K, Swann K. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol 2009;20(3):346–53.

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This study received support from the following sources. A grant from Vivere Health (E.A.S.). Harvard Catalyst, The Harvard Clinical and Translational Science Center (National Institutes of Health Award UL1 TR001102); National Science Foundation (DMR-0820484 and PFI-TT-1827309); National Institutes of Health (R01HD092550-01); National Science Foundation Postdoctoral Research Fellowship in Biology (1308878 T.S.); Becker and Hickl GmbH sponsored research with the loaning of equipment for FLIM.

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EAS primarily contributed to study conception and design and data acquisition, analysis, and interpretation; drafted and critically revised the manuscript; and approved the final version for publication. TS contributed to study conception and design and data acquisition, analysis, and interpretation; drafted and critically revised the manuscript; and approved the final version for publication. DS contributed to study conception and design and data interpretation; critically revised the manuscript; and approved the final version for publication. MV contributed to design and data interpretation, and approved the final version for publication. DJN contributed to study conception, design and data interpretation, critically revised the manuscript, and approved the final version for publication.

Corresponding author

Correspondence to Emily A. Seidler.

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Conflicts of interest/Competing interests

TS and DJN co-hold patent US20150346100A1 pending for metabolic imaging methods for assessment of oocytes and embryos and patent US20170039415A1 issued for nonlinear imaging systems and methods for assisted reproductive technologies.

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Seidler, E.A., Sanchez, T., Venturas, M. et al. Non-invasive imaging of mouse embryo metabolism in response to induced hypoxia. J Assist Reprod Genet 37, 1797–1805 (2020).

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  • Embryo metabolism
  • Hypoxia
  • Noninvasive assessment
  • Metabolic imaging
  • FLIM
  • Mitochondria