137 PYRUVATE UPTAKE DURING EARLY CLEAVAGE PREDICTS BLASTOCYST DEVELOPMENT

Abstract

The widely accepted picture of embryo metabolism has originated from work carried out in a few specialised laboratories due to the technical difficulty of carrying out metabolite assays on small volume samples. We have refined the noninvasive enzymatic determination of spent embryo culture medium for use in a standard fluorescence plate reader. Using this widely accessible, highly sensitive system, we have re-examined early bovine embryo metabolism. We have measured the consumption of pyruvate and glucose and formation of lactate by embryos in individual culture at all stages of preimplantation development. The consumption of pyruvate and glucose and production lactate by in vitro produced bovine embryos at the 2- to 4-cell, 8-cell, morula, and blastocyst stages was measured. Embryos were incubated individually for 24 h in 4-µL drops of medium alongside control drops. After 24 h of culture, the morphological status of each embryo was recorded and compared to the stage recorded at the beginning of individual culture. Analysis of the control and embryo samples of media was performed with a Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) using an adapted ultramicrofluorometric technique previously described (Leese and Barton, 1984). Uptake or production over the 24 h period was calculated and expressed as pmol/embryo/h. A further experiment focusing on pyruvate consumption by early embryos was carried out. On Day 2, embryos were cultured individually for pyruvate analysis. On Day 3, embryos were assigned into 3 groups (low, intermediate, and high) on the basis of their pyruvate depletion and then cultured until Day 8. Significant differences in metabolic profile (glucose, lactate, and pyruvate) were tested by Student’s t-test and one-way ANOVA followed by Fisher’s LSD test post hoc using Statview (SAS Institute Inc., Cary, NC, USA). There was no difference in glucose depletion and lactate production between embryos that progressed and those that did not at the cleavage stages. However, 2- to 4-cell-stage embryos that progressed morphologically depleted significantly more pyruvate from the medium than those that did not progress (6.19 ± 0.66 v. 3.80 ± 0.52 pmol/embryo/h, respectively; P < 0.05, n = 88). Embryos with an intermediate level (6.14 ± 0.27 pmol/embryo/h) of pyruvate uptake were associated with the highest number of cells on Day 2 (5.43 ± 0.16, n = 60) and on Day 3 (6.37 ± 0.20, n = 60) and were also associated with the highest rate of blastocyst development on Day 8 in comparison with the groups which included embryos with low and high level of pyruvate uptake (68.3% v. 13.3% and 25.0%, respectively; P < 0.05, n = 180). Embryos on Day 2 of development with an intermediate level of pyruvate uptake have between 2 and 3 times the probability of reaching the blastocyst stage in comparison with early embryos with high or low level of pyruvate uptake. Our data suggests that, at least for pyruvate consumption, there is a middle optimum range of depletion, which correlates with high viability.