Mitochondrial DNA (mtDNA) diseases are maternally transmitted to offspring 1. It has been proposed to prevent mtDNA disease inheritance through mitochondrial replacement therapy (MRT), using nonpathogenic mitochondria from donor oocytes to replace defective maternal mitochondria. Three strategies, including maternal spindle transfer (MST), pronuclear transfer (PNT) and polar body transfer (PBT), have been applied in human embryos2,3,4,5. All these techniques require the use of cytochalasin B (CB) or nocodazole, the cytoskeleton disruptors, to assist the isolation of the chromosome-spindle complex or pronucleus. However, the safety of using these cytoskeleton disruptors in clinical applications of human embryo-related manipulation has not been rigorously evaluated. In this study, we show a novel technique, in which female pre-pronucleus (PPN) can be isolated shortly after fertilization in a condition without cytoskeleton disruptors and then be successfully used for MRT, leading to the generation of human embryonic stem cell (ESC) lines with mtDNA carryover at very low levels.

To test the feasibility of PPNT in human zygotes, we used immature MI oocytes donated by patients who were taking in vitro fertilization (IVF) treatments. MI oocytes were kept in in vitro maturation (IVM) medium for 18-24 h. Matured oocytes with the distinct first polar body (PB1) were collected and used for intracytoplasmic sperm injection (ICSI). PB1 was then removed. 3.5-4 hours post ICSI, zygotes with extruding PB2 were collected for isolation of PPN (Figure 1A). The PPN and PB2 complex could be easily isolated from the zygote in medium without CB using a pipette with a diameter of 20 μm (Figure 1B). PPN and PB2 complexes were kept in culture medium of human embryos for 2-3 h; PPN was then separated from PB2 spontaneously (Figure 1C). PPN was briefly exposed in Sendai virus extract and injected into the perivitelline space of a donor zygote whose PB1, PB2 and PPN have been removed through the same method. Membrane fusion between PPN and the donor zygote was usually observed within 20-30 min, resulting in reconstructed embryos.

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