P-228 Reliability of blastomere versus trophectoderm biopsy in preimplantation genetic testing for mitochondrial DNA disorders

Abstract

Abstract Study question Can a single blastomere or trophectoderm (TE) biopsy accurately reflect the heteroplasmy levels of the whole embryo for mitochondrial DNA (mtDNA) disorders? Summary answer Heteroplasmy levels of single blastomere and TE are comparable to those of the rest of the embryo, including the inner cell mass (ICM). What is known already Oocytes of women carrying mtDNA mutations can display varying mutation loads – percentage of mutated mtDNA copies. Preimplantation genetic testing (PGT) aims at reducing the risk of having affected children by screening for embryos with minimal mtDNA heteroplasmy. The technique involves either a blastomere biopsy on cleavage stage embryos or TE biopsy from blastocysts, followed by genetic analysis to select embryos suitable for transfer. However, it remains unclear whether there is a uniform segregation of heteroplasmic mtDNA during the early stages of embryogenesis, and therefore whether the mutation load from blastomere or TE biopsies are representative of the whole embryo. Study design, size, duration We investigated the suitability of PGT for mtDNA disorders by comparing the mutation load between a single blastomere recovered at the eight-cell stage, TE cells and rest of the same blastocyst (n = 15) in a mouse model heteroplasmic for the m.5024C>T mutation. We also compared the levels of heteroplasmy between TE and ICM of heteroplasmic human blastocysts (n = 5) and between blastomeres of arrested cleavage stage embryos (n = 4) donated by two mtDNA disease patients. Participants/materials, setting, methods To obtain heteroplasmic mouse embryos, we mated female mice carrying a mitochondrial tRNAAla mutation (m.5024C>T) with wild-type males. Whereas in human experiments, we analysed preimplantation embryos from two different carriers of the m.3243A>G mutation, associated with MELAS syndrome, who showed 16% and 32% mutation load in their peripheral blood. After embryo biopsy, NGS was performed in three independent technical replicates and profiles were compared between samples from the same embryo to assess heteroplasmy levels. Main results and the role of chance Analysis of heteroplasmic mouse embryos showed a strong correlation in mtDNA heteroplasmy between blastomere and the corresponding embryo (r2 = 0.76), and between TE and the rest of the blastocyst (r2 = 0.94) (n = 15). Moreover, a strong correlation was observed between the biopsied blastomere and TE from the same embryo (r2 = 0.90). Heteroplasmy levels in the embryos analysed ranged from 55.3% to 88.3%, and the mean overall mutation load was 73.7 ± 7.9%. Development of biopsied embryos was comparable to those of non-manipulated mutant (n = 10) and wild-type embryos (n = 10) (blastocyst rate = 85.7%). For human blastocysts (n = 5), statistical analysis showed a strong correlation in heteroplasmy levels between ICM and TE portions (r2 = 0.98). Moreover, a modest correlation was observed among blastomeres of cleavage stage human embryos (r2 = 0.60) (n = 24 from 4 embryos). The mutation load in the embryos analysed ranged from 10.3% to 57.4%, and the mean overall heteroplasmy was 42.8 ± 12.2% in cleavage stage embryos and 38.6 ± 17.3% in blastocysts. Overall, concordance in heteroplasmy levels between single blastomeres, TE portions and corresponding blastocyst was established in human and mouse embryos. Limitations, reasons for caution These results should be further validated by increasing the sample size and by the inclusion of different mtDNA mutations. Furthermore, current data are suggestive, but not definitive, to guarantee that the mtDNA mutation load in the embryo will remain constant throughout life. Wider implications of the findings Taken together, results suggest that TE biopsy may be better than blastomere biopsy for mtDNA disorders. Moreover, biopsy at the blastocyst stage is likely less harmful for embryo development. Following, we will also investigate how heteroplasmic mtDNA segregates at the peri-implantation stages after the resumption of mtDNA replication. Trial registration number Not applicable