P-237 Non-invasive metabolic live cell imaging of early embryo development using adapted confocal microscopy; a safety study
Authors: Horta F , Newman H , Vargas-ordaz E , Cadarso V , Nosrati R , Neild A , Vollenhoven B , Mercer S , Catt S 
Published: June 2022

Abstract

Study question
Is it safe to use metabolic imaging to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence during embryo development using adapted confocal microscopy?

Summary answer
Non-invasive metabolic imaging is safe as no differences were observed between controls and illuminated embryos in terms of embryo development, blastocyst formation and implantation potential.

What is known already
Developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproductive technologies. Changes in metabolic activity could lead to cell death or abnormal embryo development and low implantation potential. This could potentially be predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging in embryos has been investigated through complex methodologies, however, scientific evidence for its utility during embryo development using simple technology remains unexplored. Measurements of metabolic activity could be a useful tool as the auto-fluorescence of molecules such as NADH is a straightforward representation of mitochondrial function.

Study design, size, duration
Super-ovulated female mice (n = 30) were subjected to mating with 10 males. In-vivo produced embryos collected at the 2-cell stage were divided in control group (n = 151), sham control group (n = 151) and illuminated group (n = 152). Illuminated embryos were assessed for NADH levels during embryo development every 3 hours using arbitrary units of autofluorescence (AU). Produced blastocysts were assessed for total cell and inner-cell-mass (ICM) number (Oct4 immuno-staining) and implantation potential through outgrowth assays in separate experiments.

Participants/materials, setting, methods
F1 (CBA/C57Bl6) mouse strain was used. NADH auto-fluorescence levels were measured during embryo development using adapted confocal microcopy (Olympus FV1200). A confocal Z-stacking function was used to record 15 focal planes using a 20x/0.95NA air objective of entire embryos, opening the confocal pinhole system completely. Then, images were collected and analysed using FIJI software (version: 2.0.0-rc-69/1.52n;ImageJ). Blastocyst cell number, formation rates and outgrowth rates for 4 days post blastocyst formation were compared between study groups.

Main results and the role of chance
Embryo culture experiments showed no significant differences in blastocyst formation rates between study groups (Control: 71.7%; Sham: 64.9%; Illuminated 71.7%; p &gt; 0.05). Similarly, the total number of cells (Control: 82.9±5.6; Sham: 76.5±3.3; Illuminated: 77.1±4.2; ± Standard error of mean [SEM]) and ICM cells (Control: 10.8±1.3; Sham: 9.4±0.7; Illuminated: 11.9±0.8; ± SEM) did not differ between groups (p &gt; 0.05). Outgrowth assays presented similar outgrowth areas during day5 to day8 post-blastocyst development between study groups (p &gt; 0.05). Illuminated embryos presented significantly different NADH activity levels during embryo development, particularly between the 2-cell stage (987.1±36.2AU), morulae stage (1226±31.5AU) and blastocyst stage (649±42.9AU; ± SEM; p &lt; 0.05). Embryos that did not reach the blastocyst stage presented a significantly different NADH activity profile during embryo development compared to those that did(p &lt; 0.05). Additionally, abnormal embryos also presented significantly decreased NADH activity levels at the 2-cell stage (Normal: 987.1±36.2; abnormal: 726.9±121.7AU; p &lt; 0.05) to the morulae stage (Normal: 1226±31.5; Abnormal:893.3±189AU; p &lt; 0.05). Our study indicates that measuring NADH activity levels during early embryo development present no negative effects in embryo developmental rates, blastocyst formation and implantation potential. Thus, non-invasive measurements of NADH could be applied to determine embryo metabolic activity during embryo development using simple technology and imaging techniques.

Limitations, reasons for caution
The study was conducted using a mouse model focused in early embryo development and implantation potential. Thus, studies on live birth are required to fully assess safety to further validate potential wider applications. Validation in ageing models is also required to assess potential applications for embryo selection.

Wider implications of the findings
Non-invasive measurements of metabolic activity could be applied to determine embryo metabolic activity using simple and safe technology. Further applications could link the use of simple non-invasive metabolic imaging with the latest time-lapse technology and artificial intelligence applications.

Trial registration number
N/A