Polar bodies are efficient donors for reconstruction of human embryos for potential mitochondrial replacement therapy

In mice, the first (1^st) and second (2^nd) polar bodies (PBs) can be used in place of the female genome of oocytes or zygotes and efficiently support the generation of embryonic stem cells (ESCs) and normal offspring^1,2,3. These results indicate that both 1^st and 2^nd PBs can be used as nuclear donors for mitochondrial replacement therapy (MRT)⁴, which is an effective approach for preventing the transmission of disease-causing mutant mitochondria from mother to children⁵. Most recently, human 1^st polar body (PB1) has been successfully used to replace the maternal genome to generate functional oocytes, demonstrated by the generation of blastocysts after fertilization⁶. However, whether human 2^nd polar body (PB2) can be used as the nuclear donor for embryo reconstruction remains unknown. In this study, we establish protocols for the reconstruction of human oocytes or zygotes using 1^st or 2^nd PBs as donors under conditions without cytoskeleton disruptors. Normal human ESCs can be efficiently generated from reconstructed embryos by PB transfer (PBT). Importantly, mitochondrial DNA (mtDNA) carryover remains at low levels during subsequent ESC culture and differentiation.

A total of 75 PB1T embryos were successfully generated and cultured in vitro for 5 or 6 days. 19 good-quality blastocysts (4BC or better by Gardner's criteria) were obtained and cryopreserved until further analyses (Supplementary information, Table S1A and Figure S1A). 17 of them were successfully thawed. To test the genome origin of PB1T blastocysts, DNA samples were extracted from trophoblast cells dissociated by trophectoderm (TE) biopsy of 16 embryos and used for short tandem repeat (STR) genotyping. As expected, all of them showed precise match with the corresponding PB1 donors, demonstrating a high success rate of generation of PB1T embryos by our strategy (Supplementary information, Table S1B and S1C). We next analyzed the karyotype of PB1T embryos using the same DNA samples for preimplantation genetic screening (PGS). The results showed that 10 blastocysts were euploid. However, the rest 6 embryos sustained aneuploidy (Supplementary information, Table S1C), consistent with previous observations of generation of aneuploid embryos by IVF centers^9,10 and recent observations in human PB1T experiments⁶. Moreover, droplet digital PCR (ddPCR)¹¹ showed that the 10 blastocysts with normal karyotype displayed an average mtDNA carryover level of 0.26% (0.07% - 0.50%) (Supplementary information, Figure S1B). Taken together, these results demonstrate that PB1 can be used as nuclear donors for reconstruction of human oocytes.

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