Raman spectroscopy on live mouse early embryo while it continues to develop into blastocyst in vitro

Laser based spectroscopic methods can be versatile tools in investigating early stage mammalian embryo structure and biochemical processes in live oocytes and embryos. The limiting factor for using the laser methods in embryological studies is the effect of laser irradiation on the ova. The aim of this work is to explore the optimal parameters of the laser exposure in Raman spectroscopic measurements applicable for studying live early embryos in vitro without impacting their developmental capability. Raman spectra from different areas of mouse oocytes and 2-cells embryos were measured and analyzed. The laser power and exposure time were varied and further embryo development was evaluated to select optimal conditions of the measurements. This work demonstrates safe laser irradiation parameters can be selected, which allow acquisition of Raman spectra suitable for further analysis without affecting the early mouse embryo development in vitro up to morphologically normal blastocyst. The estimation of living embryo state is demonstrated via analysis and comparison of the spectra from fertilized embryo with the spectra from unfertilized oocytes or embryos subjected to UV laser irradiation. These results demonstrate the possibility of investigating preimplantation mammalian embryo development and estimating its state/quality. It will have potential in developing prognosis of mammalian embryos in assisted reproductive technologies.