Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing

Boucret, Lisa; Bris, Céline; Seegers, Valérie; Goudenège, David; Desquiret-Dumas, Valérie; Domin‐Bernhard, Mathilde; Ferré-L’Hôtellier, Véronique; Bouet, Pierre‐Emmanuel; Descamps, Philippe; Reynier, Pascal; Procaccio, Vincent; May‐Panloup, Pascale

doi:10.1093/humrep/dex268

Cited by 39 publications

(38 citation statements)

References 31 publications

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“…In this sense, our data are in line to that reported by two groups, which recently observed the presence of few mtDNA variants with relatively low pathogenicity in human oocytes (Boucret et al., 2017, Kang et al., 2016b). Also, the study of Kang et al.…”

Section: Discussionsupporting

confidence: 93%

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

et al. 2018

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SummaryIn this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation.

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Section: Discussionsupporting

confidence: 93%

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

et al. 2018

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“…According to this criterion we found heteroplasmic changes over the entire mitochondrial genome in 26.32% of the embryos. This result agrees with previous research (Boucret et al , 2017), which means that probably ~1% of the embryos will carry a pathogenic point mutation. This would account for 1:10 000 de novo cases of mtDNA disease (Jacobs et al , 2007).…”

Section: Discussionsupporting

confidence: 93%

Comprehensive mitochondrial DNA analysis and IVF outcome

Lledó

Ortíz

Morales

et al. 2018

Human Reproduction Open

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STUDY QUESTION Do mitochondrial DNA (mtDNA) copy number and heteroplasmy in human embryos affect the ongoing pregnancy rate? SUMMARY ANSWER Our study suggests that mtDNA copy number above a specific threshold is associated with the ongoing pregnancy rate. WHAT IS KNOWN ALREADY Mitochondria play a vital role in cell function. Recently, there has been increasing research on mtDNA as a biomarker of embryo implantation. Although reports showed that high levels of mtDNA in the blastocyst are associated with low implantation potential, other publications were unable to confirm this. Confounding factors may influence the mtDNA copy number in euploid embryos. On the other hand it has been speculated that both mtDNA heteroplasmy and copy number contribute to mitochondrial function. Next generation sequencing (NGS) allows us to study in depth mtDNA heteroplasmy and copy number simultaneously. STUDY DESIGN, SIZE, DURATION A prospective non-selection study was performed. We included 159 blastocyst biopsies from 142 couples who attended our clinic for preimplantation genetic testing for aneuploidies (PGT-A), from January 2017 to December 2017. All embryos were biopsied on Day 5 or Day 6. The aneuploid testing was performed by NGS. All blastocysts were diagnosed as euploid non-mosaic and were transferred. The mtDNA analysis was performed once the embryo diagnosis was known. PARTICIPANTS/MATERIALS, SETTING, METHODS Sequencing reads mapping to the mtDNA genome were extracted from indexed bam files to identify copy number and heteroplasmy. The relative measure of mtDNA copy number was calculated by dividing the mtDNA reads by the nuclear DNA value to normalize for technical variants and the number of cells collected at the biopsy. All the results were subjected to a mathematical correction factor according to the embryo genome. Heteroplasmy was assigned by MitoSeek. MAIN RESULTS AND THE ROLE OF CHANCE The mean average copy number and SD of mtDNA per genome was 0.0016 ± 0.0012. Regarding heteroplasmy, 40 embryos were heteroplasmy carriers (26.32%). MtDNA variants were detected in coding and non-coding regions and the highest number of variants in an embryo was eight. With respect to IVF outcome for mtDNA copy number analysis, we set a threshold of 0.003 for the following analysis. The vast majority of the embryos were below the threshold (142/159, 89.31%) and 17 embryos were classified as having higher mtDNA levels. We showed a reduction in ongoing pregnancy rate associated with elevated mtDNA copy number (42.96% versus 17.65%, P < 0.05). This result was independent of maternal age and day of the biopsy: these factors were included as confounding factors because mtDNA copy number was negatively correlated with female age (25 –30 y: 0.0017 ± 0.0011, 30 –35 y: 0.0012 ± 0.0007, 35 –40 y: 0.0016 ± 0.0009, over 40 y: 0.0024 + 0.001...

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“…The application of duplex sequencing to single oocytes and oocyte pools allowed us to analyze putative de novo mtDNA mutations directly in germ cells (i.e., without the need to observe them in the next generation) with a resolution sufficient to capture frequency increase over a period of only 9 mo. Previous studies in humans are contradictory and either suggested no increase in the number of mutations in single oocytes with ovarian aging [ 35 ] or an increase in the number of de novo germline mutations with maternal age at conception [ 26 , 36 ] (the latter studies were based on the analysis of new heteroplasmies in the offspring, thus providing only indirect measurements of germline mutation frequencies). Studies of this phenomenon in mice have been scarce.…”

Section: Discussionmentioning

confidence: 99%

Age-related accumulation of de novo mitochondrial mutations in mammalian oocytes and somatic tissues

et al. 2020

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Mutations create genetic variation for other evolutionary forces to operate on and cause numerous genetic diseases. Nevertheless, how de novo mutations arise remains poorly understood. Progress in the area is hindered by the fact that error rates of conventional sequencing technologies (1 in 100 or 1,000 base pairs) are several orders of magnitude higher than de novo mutation rates (1 in 10,000,000 or 100,000,000 base pairs per generation). Moreover, previous analyses of germline de novo mutations examined pedigrees (and not germ cells) and thus were likely affected by selection. Here, we applied highly accurate duplex sequencing to detect low-frequency, de novo mutations in mitochondrial DNA (mtDNA) directly from oocytes and from somatic tissues (brain and muscle) of 36 mice from two independent pedigrees. We found mtDNA mutation frequencies 2- to 3-fold higher in 10-month-old than in 1-month-old mice, demonstrating mutation accumulation during the period of only 9 mo. Mutation frequencies and patterns differed between germline and somatic tissues and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. Additionally, we discovered a more pronounced genetic drift of mitochondrial genetic variants in the germline of older versus younger mice, arguing for mtDNA turnover during oocyte meiotic arrest. Our study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing directly in oocytes, which provided unprecedented resolution and minimized selection effects present in pedigree studies. Moreover, our work provides important information about the origins and accumulation of mutations with aging/maturation and has implications for delayed reproduction in modern human societies. Furthermore, the duplex sequencing method we optimized for single cells opens avenues for investigating low-frequency mutations in other studies.

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Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing

Abstract: This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centers, INSERM and the CNRS. There are nocompeting interests.

Cited by 39 publications

References 31 publications

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

Comprehensive mitochondrial DNA analysis and IVF outcome

Age-related accumulation of de novo mitochondrial mutations in mammalian oocytes and somatic tissues

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