Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage

• Published in: Human reproduction (Oxford) 2015-03, Vol.30 (3), p.543-552
• Main Authors: Lee, Y.S.L., Thouas, G.A., Gardner, D.K.
• Format: Article
• Language: English
• Subjects: Amino Acids - metabolism; Animals; Blastocyst - metabolism; Carbohydrate Metabolism; Culture Media - chemistry; Embryonic Development; Fertilization in Vitro; Glucose - metabolism; Kinetics; Mice; Mice, Inbred C57BL; Time Factors
• ISSN: 0268-1161; 1460-2350
• DOI: 10.1093/humrep/deu334

STUDY QUESTION What is the relationship between cleavage stage embryo kinetics, blastocyst metabolism and subsequent embryo viability? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, 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. MAIN RESULTS AND THE ROLE OF CHANCE 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 deve