title: Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage.
abstract: OBJECTIVE
  What is the relationship between cleavage stage embryo kinetics, blastocyst metabolism and subsequent embryo viability?
  
  
  CONCLUSIONS
  Embryos cleaving faster at the first cleavage division resulted in blastocysts with a larger inner cell mass (ICM), higher glucose consumption, lower glycolytic rate, higher aspartate uptake, lower global amino acid turnover and higher percentage of developing fetuses on E13.5 when compared with blastocysts that developed from slower cleaving embryos.
  
  
  BACKGROUND
  Previous research has shown that morphokinetics, blastocyst carbohydrate metabolism and cleavage stage amino acid metabolism of the preimplantation embryo can be used independently as markers of its developmental competence and subsequent viability.
  
  
  METHODS
  Morphokinetics of in vitro fertilized mouse zygotes were observed using a time-lapse imaging system and they were identified as 'fast' or 'slow' cleaving embryos. Spent culture media from resultant blastocysts were analysed for carbohydrate and amino acid utilization. Blastocysts either had their ICM and trophectoderm (TE) cell number determined, were cultured further in an outgrowth assay or transferred to a recipient female to assess implantation and fetal development.
  
  
  METHODS
  Morphokinetics of in vitro fertilized C57BL/6xCBA (F1) zygotes individually cultured in 2 µl drops of G1/G2 media with HSA under Ovoil in 5% O2, 6% CO2 and 89% N2 were analysed using a time-lapse incubator. At 72 h post-insemination, blastocysts were separated into quartiles derived from timing of the first cleavage division. Blastocysts were cultured for a further 24 h and spent media samples, including controls containing no embryos, were frozen and subsequently analysed for amino acid utilization using liquid chromatography-mass spectrometry. These blastocysts were then analysed over a further 1.5 h period for carbohydrate utilization and subsequently stained to determine ICM and TE cells. To analyse implantation potential, fetal quality and viability, additional 'fast' and 'slow' blastocysts were cultured further in an outgrowth model or transferred to recipient females.
  
  
  RESULTS
  Embryos cleaving faster at the time of first cleavage (first quartile, designated 'fast') were on average 2.5 h ahead of slower embryos (fourth quartile, designated 'slow', 15.1 ± 0.1 versus 17.6 ± 0.1 h, P < 0.001). On Day 5 of culture, blastocysts developed from 'fast' embryos had a larger ICM number (17.4 ± 2.1 versus 7.4 ± 2.0, P < 0.01), a higher glucose consumption (21.2 ± 1.2 versus 14.3 ± 1.0 pmol/embryo/h, P < 0.001) and a lower glycolytic rate (expressed as the percentage of glucose converted to lactate) (49.6 ± 2.8 versus 59.7 ± 2.8%, P < 0.05) compared with 'slow' embryos. Further non-invasive metabolomic analysis revealed that 'fast' blastocysts consumed more aspartate (2.2 ± 0.1 versus 1.8 ± 0.1 pmol/embryo/h, P < 0.05) and produced little or no glutamate compared with 'slow' blastocysts (0.02 ± 0.07 consumed versus 0.32 ± 0.11 pmol/embryo/h produced, P < 0.05). Transfer of 'fast' blastocysts to pseudo-pregnant recipients resulted in higher fetal survival post-implantation compared with 'slow' blastocysts (69.6 versus 40.4%, P < 0.01).
  
  
  CONCLUSIONS
  The timing of the first cleavage division was used to classify blastocysts as 'fast' or 'slow' embryos; however, a combination of several developmental kinetic markers (cleavage time of 3- to 8-cell, duration between cleavage division times) may be used to more accurately determine an embryo as 'fast' or 'slow'. Only the fastest and slowest quartiles (those embryos with the fastest and slowest first cleavage division) were analysed in this study.
  
  
  CONCLUSIONS
  These findings show that kinetically different embryos develop into blastocysts with different metabolic profiles and viability. Work is now being undertaken to determine if using these viability markers in combination will increase embryo selection efficacy and further improve implantation and pregnancy rates.
  
  
  BACKGROUND
  The study was funded by the University of Melbourne. The authors have no conflicts of interest to declare.
authors.affiliation: School of BioSciences, University of Melbourne, Melbourne, VIC 3010, Australia.
authors.forename: Y S L
authors.initials: YS
authors.lastname: Lee
authors.affiliation: School of BioSciences, University of Melbourne, Melbourne, VIC 3010, Australia.
authors.forename: G A
authors.initials: GA
authors.lastname: Thouas
authors.affiliation: School of BioSciences, University of Melbourne, Melbourne, VIC 3010, Australia david.gardner@unimelb.edu.au.
authors.forename: D K
authors.initials: DK
authors.lastname: Gardner
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D003470:Culture Media / Q000737:chemistry; D047108:Embryonic Development; D005307:Fertilization in Vitro; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D013997:Time Factors
chemical_list: D000596:Amino Acids; D003470:Culture Media; D005947:Glucose
keywords: metabolism; selection; time-lapse; viability