A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model

Study question: What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? Summary answer: The reliability ofPGDto determine the level of mitochondrialDNA(mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. What is known already: PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. Study design, size and duration: We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n ¼ 33), between first PBs, second PBs and zygotes (n ¼ 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n ¼ 10), 4- (n ¼ 10) and 8-cell embryos (n ¼ 11). Levels of mtDNA heteroplasmy in second PBs (n ¼ 20), single blastomeres from 8-cell embryos (n ¼ 20), TE (n ¼ 20) and blastocysts (n ¼ 20) were also compared. Participants/materials, setting,methods: Heteroplasmic mice (BALB/cOlaHsd), containingmtDNAmixtures ofBALB/cByJ and NZB/OlaHsd,were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryowas cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followedby restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples. Main results and the role of chance: Modest correlations and wide prediction interval [PI at 95% confidence interval (CI)] were observed in the level ofmtDNAheteroplasmy between first PBs and their corresponding MII oocytes (r2 ¼ 0.56; PI ¼ 45.96%) and zygotes (r2 ¼ 0.69; PI ¼ 37.07%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r2 ¼ 0.65; PI ¼ 39.69%), single blastomeres (r2 ¼ 0.42; PI ¼ 48.04%), TE (r2 ¼ 0.26; PI ¼ 54.79%) and whole blastocysts (r2 ¼ 0.40; PI ¼ 57.48%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r2 ¼ 0.92; PI ¼ 11.73%, r2 ¼ 0.86; PI ¼ 18.85% and r2 ¼ 0.85; PI ¼ 21.42%, respectively), and also between TE and whole blastocysts (r2 ¼ 0.90; PI ¼ 23.58%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r2 ¼ 0.81; PI ¼ 28.15%) and blastocysts (r2 ¼ 0.76; PI ¼ 36.43%). Limitations, reasons for caution: These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD. Wider implications of the findings: To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, and question the reliability of PGD using different stages of embryonic development. Study funding/competing interest(s): This work was funded by a doctoral grant provided by the Special Research Fund (BOF) to J.N. (grant number, 01D05611) and a postdoctoral grant provided by Research Foundation-Flanders (FWO) to M.V. (grant number, 1248413N). P.S. is holder of a fundamental clinical research mandate by FWO-Vlaanderen (Flemish fund for scientific research). Trial registration number: Not applicable