IS MTDNA CONTENT ASSESSMENT AN INDICATOR OF THE AMOUNT OF MITOCHONDRIA (TOTAL AND ACTIVE ONES) IN THE HUMAN BLASTOCYST?: A CONFOCAL MICROSCOPY APPROACH

to assess if mitochondrial DNA (mtDNA) content per trophectoderm (TE) cell (Mitoscore®) could serve as a predictor of total mitochondrial mass and activity in the human blastocyst. A prospective cohort study was performed in 51 vitrified aneuploid blastocysts with known Mitoscore® data. Blastocysts were warmed, maintained 6 hours under in vitro culture conditions and stained with mitochondrial dyes. Nonyl acridine orange was used for staining the total mitochondrial content, Mitotracker Deep Red was used for staining the active mitochondrial content and Dapi was used for staining the nucleus. Confocal microscopy was used to take three photos of the focal plane of each blastocyst. The analysis was performed with two different methods: Adjusting a fluorescence value for each mitochondrial dye and evaluating the differences in gains values required (N=17) between embryos and setting the same gain values for each dye and calculating the differences in fluorescence emitted between embryos using the image analysis software Image J (N=34). Data was statistically analysed by linear regression and analysis of variance ratios. We observed that there was no correlation (p>0.05) between the mtDNA content either with the number of mitochondria per cell or with the number of active mitochondria per cell using both methods of analysis (fixed fluorescence, N=17 and fixed gains values, N=34). Most importantly, when we analyzed the variance ratios (variance of a subsample divided by variance of the global population) of the total embryos fluorescence for each dye (mitochondrial mass variance ratio=0,99 and active mitochondria variance ratio=1,89) within a group of embryos with the same Mitoscore® (Mitoscore® variance ratio=0,0096) we observed differences in terms of Mitoscore®’ emitted fluorescence and vice versa (mitochondrial mass variance ratio =0,11 and Mitoscore® variance ratio = 0,79; active mitochondria variance ratio=0,007 and Mitoscore® variance ratio= 0,97). Confocal approach showed that no correlation could be established between Mitoscore® and total and active mitochondrial content in human blastocysts.