The feasibility of non-invasive PGT in preventing pathogenic mitochondrial DNA disease transmission in mice:...

Is cell-free mitochondrial DNA (cf-mtDNA) in spent blastocyst medium (SBM) a suitable biomarker for non-invasive preimplantation genetic testing (niPGT) for mtDNA diseases, and what factors influence the accuracy of niPGT results?

Oocytes isolated from mouse models were fertilized and cultured to the blastocyst stage using a two-step sequential culture procedure. For each blastocyst, SBM and the corresponding embryo were collected, and trophectoderm biopsies were performed exclusively on high-quality blastocysts. These samples were subsequently analysed using real-time polymerase chain reaction, or subjected to mtDNA direct amplification for next-generation sequencing.

The amplification rate of the target mtDNA site in SBM was 100% in both mice and humans, whereas the amplification rate of the GAPDH gene within the human nuclear genome was 75%. Linear regression analysis revealed strong correlation of the trophectoderm biopsy group (r² = 0.96, prediction interval 6.87 ± 0.06%) and the cf-mtDNA group (r² = 0.84, prediction interval 14.29 ± 0.11%) with the remaining whole embryo. However, in SBM from low-quality embryos, the r²-value was lower (r² = 0.17, prediction interval 37.80 ± 0.60%) compared with SBM from high-quality embryos (r² = 0.84, prediction interval 14.29 ± 0.11%). Notably, the sequential culture procedure minimized contamination from cumulus cells, and SBM samples contaminated with fewer than 10 spermatozoa did not affect the niPGT results.

Based on the sequential culture procedure, niPGT may serve as an alternative method for preventing maternal-to-offspring transmission of mtDNA diseases. However, this conclusion was based on an animal model, and the findings require further validation in humans.