O-269: Development of a sensitive assay to measure lactate production of preimplantation embryos

Abstract

Reliable indicators of embryo viability are rate of glucose/pyruvate consumption and lactate production. The ability to non-invasively quantify embryo metabolism within culture media is a valuable tool. Current non-invasive methods of measuring embryo metabolism are based on generation or utilization of reduced pyridine nucleotides, NADH and NADPH. This method requires excitation of NADH/NADPH with ultraviolet (UV) light which can damage embryo DNA thus requiring manual removal of media, nanoliter pipetting, and dilutions that can introduce quantitative errors. Long-term goals of these studies are to develop metabolic assays that can be performed in microfluidic embryo culture and analysis platforms that remove manual media manipulation and dilution, do not use UV light, and are “embryo-safe”. Study objectives were to optimize a system of measuring single embryo-produced lactate that would be compatible with microfluidic culture without use of UV light detection. Experimental study. Lactate assays were developed using Amplex UltraRed (AR), which is a colorless, stable and versatile fluorogenic substrate for horseradish peroxidase (HRP). In this assay, lactate oxidase (LOX) reacts with L-lactate to form pyruvate and H2O2. In the presence of HRP, H2O2 reacts with AR reagent in a 1:1 stoichiometry to generate a red-fluorescent oxidation product, resorfin. Amplex UltraRed lactate assays were optimized with 100 μM AR reagent, 0.2 U/ml HRP and 2U/ml LOX in three different reaction buffers (1X reaction buffer, glycine-hydrazine buffer, and 1X reaction buffer containing glycine and hydrazine) at pH 6.5, 7.5, and 8.5 over a span of 0-3 h. Standard curves of L-lactate in KSOM were established using 10-fold serial dilutions from 10nM to10mM. Lactate was measured in presence or absence of pyruvate and glucose to rule out interference from other substrate sources. A 3X3 cross-over design was used to compare assay sensitivity under varying concentrations of AR reagent (100, 10, and 1 μM) and HRP (0.2, 0.02, and 0.002 U/ml). Lactate was quantified with an excitation filter at 535 nm and an emission filter at 580 nm. Two-cell embryos from B6SJL-F2 mice were individually cultured in 10 μl KSOM w/o L-lactate for 3 h at 37°C in 5% CO2 in air. Embryo culture media was assayed at a 1:1 ratio with AR. Triplicate standard curves measuring 10-fold dilutions of lactate were performed each time embryos were assessed for lactate. The optimum buffer conditions for AR lactate assays was 1X reaction buffer at pH 7.5. There was no effect of glucose or pyruvate on AR lactate assay sensitivity. Reducing AR reagent to 10 μM and HRP to 0.002 U/ml greatly reduced background fluorescence and decreased the limit of detection to 600 nM lactate. Individual 2-cell embryo lactate production was measured at 44.9 ± 4.4 pmol/ 3h. Amplex UltraRed is a sensitive reagent that can be used to measure the production of lactate in the picomole range. These results provide a new method to non-invasively quantify preimplantation embryo metabolism in the absence of a UV light source. Future studies will focus on applying this technology to a microfluidic embryo culture and analysis system for real-time monitoring of embryo metabolism.