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Effect of culturing human embryos in group on the composition of spent culture media

In assisted reproductive technologies the number of human embryos maintained together per culture drop will affect the embryo development and embryo fitness for implantation and live birth. In the present work, it was evaluated how the number of embryos per culture drop affects the molecular fingerprint of spent culture media used till blastocyst stage (achieved at day 5 or 6), and consequently indirectly the embryo metabolism. It was considered embryos cultured in sequential media, with media changing at day 3, and maintained individually (n=19) or in groups of 2 (n=6) or 3 (n=21) embryos per culture drop. It was observed by principal component analysis of the second derivative infrared spectra that media at day 3 presented a different fingerprint than media at day 5 or 6, independently of the number of embryos maintained together. This highlight the differences between the two media and the high metabolic changes that occurs with the embryo metabolism till the blastocyst development stage. It was also identified specific ratios between spectral bands, either at day 3 either at day 5/6, that discriminate at a significance of 10%, cultures of individual embryos from embryos maintained in groups, and embryos maintained in groups of 3 in relation to embryos maintained in groups of 2. These results highlight the high impact of the number of embryos maintained in the same culture drop, in the culture media composition and consequently on the embryo metabolism. It was possible to identify molecular signatures specific to the culture time and number of embryos per culture drop, pointing to the high sensitivity and specificity of infrared spectroscopy to capture indirectly the embryo metabolism by non-invasive analysis of the spent culture media.