133 NONINVASIVE ANALYSIS OF EMBRYO METABOLITES USING NUCLEAR MAGNETIC RESONANCE

Abstract

Wide-spread use of IVF has significantly increased the number of multiple births (Janvier et al. 2011 J. Pediatr. 159, 409–413). A potential solution to this problem is to develop improved methods for embryo selection to permit single-embryo transfer. Identification of a noninvasive technique to assess embryo implantation potential in assisted reproduction would greatly increase success rates and lead to more efficient single-embryo transfer. The aim of this study was to assess whether there are metabolic differences among embryos produced by IVF and embryos obtained by parthenogenetic activation. Matured bovine cumulus-oocyte complexes were fertilized in vitro according to our standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Presumptive zygotes were placed in individual drops of 50 μL of SOF. Zygotes were incubated in a humidified mixture of 5% CO₂, 6% O₂, and 88% N₂ in air at 39°C. For the parthenogenetic group, the oocytes were activated by 5 μM ionomycin in M199 + 10% FCS for 5 min, and incubation in 2 mM 6-DMAP in M199 + 10% FCS for 4 h. After 48 h, the zygotes were placed into WOW culture and the drops collected in tubes. The embryos were scored for quality on the basis of morphological criteria. Samples of media (40 μL) were added to 660 μL of a stock solution prepared by dissolving 5.0 mg of sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate in 50 mL of deuterium oxide. The sodium 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate acted both as a chemical shift reference and as an internal standard for the purposes of quantitation. Samples were analysed on a Varian VNS-750 NB (750 MHz) spectrometer (Agilent Technologies, Santa Clara, CA, USA). Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SAS, version 9, 1999, SAS Institute Inc., Cary, NC, USA), where the independent variable was the sample (IVF or parthenogenetic embryos and control media without embryos). Tukey's post-hoc test was used to perform multiple comparisons. The P-level was set at 0.05. All data were expressed as quadratic means with standard error of the means. The results, reported in Table 1, show that there were no statistical differences between embryo metabolites with IVF or parthenogenetic activation when we evaluated lactate, formate, myo-inositol, and pyruvate. However, we can see that there are differences when we focused on acetate and citrate. Parthenogenetic embryos produced more citrate than IVF embryos. It is well known that the Krebs cycle produces one molecule of acetate for each molecule of citrate. The present results support that as well with the concentration of acetate being greater in parthenogenetic than in the IVF embryos. These results are a first step in identifying noninvasive, quantitative parameters that indicate which embryos may be the most viable before transfer.

Table 1.  Results (least squares means ± s.e.)