Early cleaving embryos result in blastocysts with increased aspartate and glucose consumption, which exhibit different metabolic gene expression that persists in placental and fetal tissues.

OBJECTIVES: Using time-lapse microscopy, previous research has shown that IVF mouse embryos that cleave earlier at the first division ('fast') develop into blastocysts with increased glucose consumption and lower likelihood of post-implantation loss as compared to slower cleaving embryos ('slow'). Further, metabolomics analysis employing LC-MS conducted on groups of 'fast' blastocysts revealed that more aspartate was consumed. With the worldwide adoption of single blastocyst transfer as the standard of care, the need for quantifiable biomarkers of viability, such as metabolism of specific nutrients, would greatly assist in embryo selection for transfer. METHODS: Here we describe the development of a targeted enzymatic assay to quantitate aspartate uptake of single blastocysts. RESULTS: Results demonstrate that the rates of aspartate and glucose consumption were significantly higher in individual 'fast' blastocysts. Blastocysts, together with placental and fetal liver tissue collected following transfer, were analysed for the expression of genes involved in aspartate and carbohydrate metabolism. In 'fast' blastocysts, expressions of B3gnt5, Slc2a1, Slc2a3, Got1 and Pkm2 were found to be significantly higher. In placental tissue derived from 'fast' blastocysts, expression of Slc2a1, Got1 and Pkm2 were significantly higher, while levels of Got1 and Pkm2 were lower in fetal liver tissue compared to tissue from 'slow' blastocysts. CONCLUSIONS: Importantly, this study shows that genes regulating aspartate and glucose metabolism were increased in blastocysts that have higher viability, with differences maintained in resultant placentae and fetuses. Consequently, the analysis of aspartate uptake in combination with glucose represents biomarkers of development and may improve embryo selection efficacy and pregnancy rates.

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

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 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). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the University of Melbourne. The authors have no conflicts of interest to declare.