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

Elena Perevedentseva,Hsin-Hou Chang,Alexander Krivokharchenko,Artashes V Karmenyan,Chia-Liang Cheng

doi:10.1038/s41598-019-42958-5

Abstract

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.

Highlights

Investigation of early mammalian development using various methods has an aim to understand fundamental mechanisms underlying the process
The optimal embryo developmental ability was observed at the Raman measurements with laser power up to 3 mW in the focal spot, exposure time 40–55 sec, number of the spectra measured from one embryo 4–6 times
The laser power and exposure were varied and the embryo viability after the measurement was estimated via observation of further development to select the most achievable optimal conditions of the measurements

Summary

Introduction

Investigation of early mammalian development using various methods has an aim to understand fundamental mechanisms underlying the process. Morphological and metabolomics studies don’t affect the survival capacity of oocytes and embryos These methods don’t allow investigation of cytoplasmic organelles or nuclear components distribution, development and monitoring biochemical processes in situ; in addition, they are not suitable for quantitative evaluation. Previous spectroscopic studies such as FTIR22 and Raman[23,24] have been performed on mouse oocytes and early embryos, but these works were limited by their investigation on fixed rather than live ova. Methods of analyzing the dynamics of biochemical modifications on the example of cortical F-actin in oocyte and conformational changes in protein and glycoprotein secondary structure of the zona pellucida structure[28,29] were suggested for estimation of cryopreserved matured ovine oocytes in vitro; and the laser parameters used, e.g. laser power up to 50 mW and 25 mW at 532 nm excitation wavelength can definitely affect the live embryos[27,28]

Objectives

Methods

Results

Discussion

Conclusion