Non-Invasive Metabolic Live Cell Imaging of Early Embryo Development Using Novel Adapted Confocal Microscopy in Ageing

Abstract

Background: Developing non-invasive methods that are reliable to assess embryo quality has been a significant aim for assisted reproductive technologies. Changes in metabolic activity could lead to abnormal embryo development and implantation potential which could be potentially predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging of nicotinamide adenine dinucleotide (NADH) has been well studied in cell lines, however, evidence for its use in embryo development is limited. Measurements of NADH during advanced-reproductive-age could be a useful tool as the auto-fluorescence of NADH is a straightforward representation of mitochondrial function and cellular redox status. Aim: To determine potential differences in NADH levels associated in early embryo development from young and old mice using novel adapted confocal microscopy. Methods: Oocytes from two groups of 20 super-ovulated female mice (Young: 5-8 weeks old, n=10; Old: 45-52 weeks old, n=10) were inseminated through in-vitro fertilisation (IVF). Inseminated oocytes (Young:93; Old:64) were assessed for fertilisation and embryo development to the blastocyst stage. Early embryos (Young:44; Old:34) from both young and old groups were assessed for NADH levels during embryo development every 2 and 3 hours respectively in separated experiments, using arbitrary units of auto-fluorescence (AU). Results: IVF experiments showed no significant differences in fertilisation rates between age groups but with significant differences in blastocyst formation (Young: 90.9%; Old: 66.7%; p[Formula: see text]0.05). Embryos from young and old females showed significantly different NADH levels during embryo development (Young: 29,889 ±864AU; Old: 25,972 ±260AU; p[Formula: see text]0.0001). Zygotes that reached the blastocyst stage in old females had significantly decreased NADH levels compared to young females (Young: 31,508 ±853AU, Old: 26,417 ±186AU; p=0.0002). Conclusion: Our study indicates different levels of NADH activity during early embryo development associated to ageing and blastocyst formation. Non-invasive measurements of NADH could be applied to determine embryo metabolic activity associated to ageing using simple technology.