Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children1,2,3, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue4. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States5. Mitochondrial replacement therapies or techniques (MRT) circumventing mother–to–child mtDNA disease transmission involve replacement of oocyte maternal mtDNA6,7,8. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother’s oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer8, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor–to–maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.
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The authors acknowledge the OHSU Embryonic Stem Cell Research Oversight Committee and the Institutional Review Board for oversight and guidance. We thank all study participants for tissue donations and the Women’s Health Research Unit staff, University Fertility Consultants and the Reproductive Endocrinology and Infertility Division in the Department of Obstetrics and Gynecology, Oregon Health and Science University for support and procurement of human gametes. Studies were supported by the Leducq Foundation, OHSU institutional funds and Cincinnati Children’s Hospital Research Foundation. Work in the laboratory of J.C.I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, the Leona M. and Harry B. Helmsley Charitable Trust and the Moxie Foundation. A.P.L. was partially supported by a fellowship from the Hewitt Foundation. P.M.R. was partially supported by a fellowship from Fundación Alfonso Martín Escudero.
The authors declare no competing financial interests.
Reviewer Information Nature thanks E. Shoubridge and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Families with mitochondrial disease and women of reproductive age were recruited for MRT. All Leigh syndrome families had an existing, severely affected child. F, family; non-syn, non-synonymous; NA, not applicable. b, Family 5 was selected from an extensive MELAS syndrome pedigree. Clinical phenotypes of A3243G MELAS syndrome varied even with similar heteroplasmy levels. Asterisk indicates the first MELAS patient diagnosed clinically. Heteroplasmy in blood (upper panel) and urine (lower panel). Squares, males; circles, females. B, blood; U, urine. NT, not tested. Percentage values refer to heteroplasmy; y, years-old.
a, Age of oocyte donor(s) was similar between carrier and healthy groups. b, c, Ovarian reserve (b) and antral follicle count (c) were significantly lower in carriers than in healthy oocyte donors. d, e, Duration of controlled ovarian stimulation (d, COS) was longer in carriers and peak oestradiol (e, E2) on day of hCG trended to be lower in carriers. f, g, Total oocyte yield (f) and number of mature oocytes (g) were significantly lower in carriers than healthy oocyte donors. n = the number of controlled ovarian stimulation cycles. Data are represented as mean ± s.d. *P < 0.05. h, Baseline characteristics and cycle outcomes. Carriers had a lower anti-Müllerian hormone (AMH) levels. Lower peak oestradiol levels were measured in cED1 and cED2. Birth control method: combined oral contraceptive, medroxyprogesterone. i, Analysis of heteroplasmic mtDNA variants detected in MII oocytes. De novo indicates unique mutations found in individual oocytes; inherited indicates mutations shared with other oocytes, sibling children or egg donors. n = the number of mutations in individual oocytes. j, anti-Müllerian hormone levels, a measure of ovarian reserve, were not correlated with mtDNA copy number. n = the number of oocytes. NS, not significant. Source data files are available for a–g.
a, Whole mtDNA sequencing was performed to identify haplotypes of all egg donors and spindle transfer was performed to match various haplotypes. Cyto, cytoplast; Karyo, karyoplast. b, Normal fertilization (2 pronuclei) and abnormal fertilization (1 or 3 pronuclei); see arrows. Scale bars, 50 μm. c, Carrier spindle transfer showed higher abnormal fertilization than controls. One zygote from the control spindle transfer group with three pronuclei developed to a blastocyst and displayed a 69, XXY karyotype. d, The blastulation rate was similar between vitrified cytoplasm with fresh spindles and vitrified spindles with fresh cytoplasm (P > 0.05; t-test). e, Control spindle transfer embryo development as a function of donor mtDNA matching distances. The numbers on the top of bars are percentage of embryo development. n = the number of embryos; NS, not significant (P > 0.05; t-test).
a, Energy metabolism was measured and compared between neural progenitor cells (NPCs) derived from MRT and control embryos carrying either donor or maternal mtDNA. OCR, oxygen consumption rate (representing oxidative phosphorylation); ECAR, extracellular acidification rate (representing glycolysis). NPCs from MRT ES cells with 6 and 49 SNP differences displayed comparable oxygen consumption rates to NPCs harbouring maternal or donor mtDNA except maximal respiration in the 49 SNP group. The oxygen consumption rate was reduced in NPCs with 33 SNPs. The oxygen consumption rate data were normalized by live cell DNA content. *P < 0.05 versus host mtDNA, **P < 0.05 versus donor mtDNA, ***P < 0.05 versus spindle transfer (t-test for 6 SNPs and one-way ANOVA for 33 and 49 SNPs, n = 16 technical replicates). Data are shown as mean ± s.e.m. NS, not significant. b, Mitochondrial respiratory chain enzyme activity in differentiated cells from spindle transfer ES cell lines. Mitochondrial complex I and IV activities in differentiated cells from spindle transfer ES cell lines carrying donor mtDNA with 49 SNP differences were comparable to controls with either donor or maternal mtDNA (P > 0.05). One-way ANOVA, n = 4 technical replicates. Source data file is available for b.
a, Summary of in vitro and in vivo differentiation of ES cells derived from spindle transfer or SCNT embryos carrying donor mtDNA. Similar to control IVF ES cells, all tested MRT ES cells produced teratoma tumours in vivo and formed neural progenitor cells (NPCs) and cardiomyocytes in vitro. NT, not tested. b, Histological analysis of differentiated tissues from MRT ES cells. The representative tissues were collected and used for mtDNA carry-over analysis and measurements of mitochondrial function. Scale bars, 1 mm (live image) and 50 μm (haematoxylin and eosin (H&E) and immunochemistry staining).
a, Aneuploidy rate in blastocysts determined by a comparative genome hybridization array was not significantly different in spindle transfer groups compared to controls. b, Karyotype abnormality rate in ES cells determined by G-banding analysis was also comparable among spindle transfer groups (P > 0.05). Normal, 46 XX or 46 XY. Number inside bars, the number of blastocysts or ES cell lines (abnormal/total). Simple χ2 tests.
a, Maternal mtDNA (X2c haplotype) heteroplasmy increased during extended in vitro culture and reached homoplasmy in pooled cultures or in individual colony subcultures (n = 1). See also Supplementary Table 4. b, Respiratory chain complex 1 (COM I) and complex 4 (COM IV) enzyme activities were measured in fibroblasts carrying various human mtDNA haplotypes used in this study. No significant differences were noted (P > 0.05). One-way ANOVA, n = 4 technical replicates. Source data file is available for b.
Sequence differences between egg donor haplotypes understudy by whole mtDNA sequencing (syn, synonymous; non-syn, nonsynonymous; frmshft, frameshift). (XLSX 35 kb)
mtDNA variants in oocytes and somatic tissues from mutant carriers and children and healthy oocyte donors (syn, synonymous; non-syn,non- synonymous; frmshft, frameshift). (XLSX 25 kb)
Maternal mtDNA analysis in ESCs derived from ST blastocysts (syn, synonymous; non-syn, non- synonymous; frmshft, frameshift). (XLSX 66 kb)
Analysis of maternal mtDNA heteroplasmy in ESCs derived from somatic cell nuclear transfer (SCNT) blastocysts (syn, synonymous; nonsyn, non- synonymous; frmshft, frameshift). (XLSX 47 kb)
Maternal mtDNA heteroplasmy changes in 18 ST-ES and 8NT-ES cell lines. Filtered reads were aligned to the human mitochondrial sequence reference NC_012920.1 followed by variant calling. Presented nucleotide positions indicate difference from the reference or between different human haplotypes. (XLSX 90 kb)
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Kang, E., Wu, J., Gutierrez, N. et al. Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations. Nature 540, 270–275 (2016). https://doi.org/10.1038/nature20592
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