The effect of pronuclear transfer on human preimplantation development

Abstract

Mutations in maternally inherited mitochondrial DNA (mtDNA) can cause a range of complex diseases for which there are currently no curative treatments. Using IVF based techniques involving nuclear genome transplantation, it may be possible enable women who carry mtDNA mutations to have a genetically related child without the risk of transmitting disease. The central aim of this project is to perform preclinical studies testing the safety and efficiency of pronuclear transfer (PNT). Surprisingly, the PNT technique developed using abnormally fertilised zygotes was detrimental to survival of normally fertilised zygotes. We tested the possibility that this might be due to the relatively accelerated development of normally fertilised zygotes allowing insufficient time for recovery following transplantation of the pronuclei. Switching the timing of PNT to shortly after pronuclei appearance (ePNT) rather than shortly before disappearance resulted in increased survival. Further modification of the enucleation and embryo culture media resulted in improved blastocyst quality. As part of the optimisation process, I tested the effect and reversibility of drugs that are used to inhibit the cytoskeleton of oocytes and zygotes in preparation for manipulations. Comparison of two compounds, which directly inhibit actin polymerisation, revealed marked differences in the reversibility. However, latrunculin B, which is rapidly reversed, has a detrimental effect on blastocyst development compared with latrunculin A, which is more potent and less readily reversible. Finally, I analysed single-cell RNA-sequencing data to determine whether gene expression in human blastocysts is altered by ePNT. This work was done in collaboration with Dr Kathy Niakan at the Francis Crick Institute. The findings indicate no detectable differences in global or lineage-associated gene expression between control and good quality ePNT blastocysts. Analysis of mitochondrial gene expression revealed high variability in the level of expression both within and between blastocysts. However, this variability was observed in ePNT and control blastocysts, and there was no detectable difference between them. In conclusion, this study has tested PNT in normally fertilised human zygotes for the first time; results indicate no detectable harmful effects of the ePNT procedure. We therefore conclude that it is likely to give rise to normal pregnancies.