DIFFERENCES IN METABOLIC SIGNATURES BETWEEN THE INNER CELL MASS AND THE TROPHECTODERM IN DISCARDED HUMAN BLASTOCYSTS

To determine whether non-invasive metabolic imaging can detect differences in metabolic profiles between the inner cell mass (ICM) and the trophectoderm (TE) of discarded human blastocysts. Prospective observational study. We used 192 morphologically normal human vitrified blastocysts from 126 patients (mean age 35.5 ±4.75 years) that were discarded and donated for research under an approved institutional review board protocol. These embryos were warmed, cultured for 2h and imaged, to analyze their metabolic signatures. Metabolic function was assessed using Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM enables non-invasive imaging of the autofluorescent endogenous molecules, NADH and FAD+, essential coenzymes for cellular respiration and glycolysis. FLIM yields quantitative information on metabolite concentrations from fluorescence intensity and on enzyme engagement from fluorescence lifetimes. A single measurement provides 9 metabolic parameters (4 for NADH, 4 for FAD and Redox Ratio) which was used to separately analyze the metabolic signatures of the ICM and TE from each embryo. Metabolic parameters from the ICM were compared to those of the TE using paired t-test analyses while a sub analysis of embryo day (5 or 6) was performed using multilevel models. Our data showed statistically significant variations in all metabolic parameters between the ICM and the TE from discarded human blastocysts. Both NADH and FAD+ intensities, that correlate with the concentration of these coenzymes, were significantly different between the ICM and the TE (p<0.0001). Furthermore, the lifetimes of these molecules and the fraction engaged with enzyme were significantly different between the ICM and the TE (p<0.0001). These findings are in line with previous evidence in mouse blastocysts showing that the ICM of mouse embryos have a relatively quiescent metabolism compared with that of the TE. Additionally, metabolic signatures from the ICM and the TE correlated with blastocyst day. When comparing day 5 and day 6 embryos, distinct metabolic signatures were visualized in the ICM (6/9 FLIM parameters, p<0.002) and also in the TE (6/9 FLIM parameters, p<0.001). Non-invasive metabolic imaging can detect significant metabolic variations between the ICM and TE of discarded human blastocysts, suggesting different metabolic demands specific to the ICM and TE. Further studies on human embryo samples are planned which will investigate possible correlations between metabolic signatures of both the ICM and the TE with embryo morphology and pregnancy outcomes.