Mitochondrial DNA and maternal inheritance

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Genome organisation

The human mtDNA reference sequence (the revised Cambridge Reference Sequence, rCRS) is 16,569 bp long (Anderson et al., 1981; Andrews et al., 1999). The genes are tightly packed with few or no intergenic bases; some stop codons are not encoded in the DNA and are completed after transcription by polyadenylation (Anderson et al., 1981). The main non-coding region (about 1 kb, including the D-loop) carries the elements that control replication and transcription (Chinnery et al., 2000). Its hypervariable segments, such as HV2, are polymorphic and serve as neutral markers in studies of mtDNA segregation in embryos (Rubens et al., 2025).

The 13 mtDNA-encoded proteins are subunits of respiratory-chain complexes I, III, IV and V; complex II is entirely nuclear-encoded (Gorman et al., 2016). Variants are named by their position in the reference sequence: m.3243A>G means that adenine is replaced by guanine at position 3243. The variants that appear in the Atlas sections lie in tRNA genes (MT-TL1: m.3243A>G and m.3250T>C; MT-TK: m.8344A>G) and in protein-coding genes (MT-ATP6: m.8993T>G/C; MT-ND1: m.3697G>A; MT-ND3: m.10191T>C and m.10197G>A; MT-ND6: m.14487T>C).

mtDNA copy number: a quantity, not a constant

mtDNA copy number differs between tissues and individuals and is linked to variation in the nuclear genome: a population-scale analysis identified nuclear loci associated with both copy number and heteroplasmy (Gupta et al., 2023). Floros and colleagues counted a mean of 1.22×10⁶ mtDNA molecules in oocytes from three healthy donors, and about 1,000-fold fewer in primordial germ cells of human embryos at Carnegie stage CS12 (median 1,425, mean 1,446 copies per cell); the number of mitochondria barely changed, corresponding to about five molecules per organelle (Floros et al., 2018). A similar germline reduction in copy number has been described in mice, cattle and zebrafish, so it is evolutionarily conserved (Xie et al., 2026). In the early embryo, mtDNA replication is largely suppressed until the blastocyst stage, so the existing molecules are shared among an increasing number of cells (Rubens et al., 2025; Facucho-Oliveira & St John, 2009).

Copy number and heteroplasmy are different quantities. Copy number describes how much mtDNA there is; heteroplasmy describes the relative proportions of mtDNA variants.

Maternal inheritance

Strictly maternal transmission is the rule in humans and most animals. Luo and colleagues reported inheritance from both parents in three families (Luo et al., 2018). The interpretation of such observations remains under discussion, including the role of nuclear copies of mtDNA fragments (NUMTs); mechanisms preventing paternal transmission remain under investigation (Xie et al., 2026). Maternal transmission means that mtDNA does not recombine with a paternal copy and is passed on as a single haplotype; new variants arise through replication errors, damage to single-stranded mtDNA and oxidative processes (Iliushchenko et al., 2025). Such errors are either corrected by repair mechanisms or propagated through generations (Stewart & Chinnery, 2015). mtDNA accumulates substitutions 10–20 times faster than nuclear genes (Xie et al., 2026; Bi et al., 2023), yet the female germline appears to be protected against their accumulation: in oocytes, unlike blood and saliva, the frequency of new mutations did not rise with age (Arbeithuber et al., 2025). Germline selection is discussed in Chapter 3.

Heteroplasmy: definitions that matter in practice

Heteroplasmy level

The proportion of molecules carrying the variant among all mtDNA molecules at that position in the analysed sample. It is an average over all cells in the sample: in a multicellular biopsy, cells with different levels are pooled.

Homoplasmy

All mtDNA copies have the same sequence at the position under consideration.

Cell-to-cell mosaicism

Differences in level between cells of the same tissue. Even in day-3 embryos, individual blastomeres may carry variants not detected in neighbouring cells (Mertens et al., 2022). However, the study concerned natural variants in donor embryos without a known pathogenic mutation, mostly at low levels. For pathogenic variants in carriers, blastomeres of one embryo generally have similar levels (chapter 4) (Mertens et al., 2022; Monnot et al., 2011).

Low-level heteroplasmy

Rare variants below the detection limit of conventional methods are widespread, including in oocytes of healthy women (Bi et al., 2023).

Genetic features of mitochondrial disease

• Type of variant. Pathogenic mtDNA variants are often heteroplasmic, although homoplasmy also occurs. PGT-mt is applicable to heteroplasmy: if all oocytes carry the same proportion of the variant, there is no embryo with a lower heteroplasmy level to select, and other reproductive options are considered (Smeets et al., 2023; Castelluccio et al., 2025).
• Origin. The probability of disease recurring in subsequent children depends directly on the origin of the mutation: if the variant arose de novo, the risk for future siblings is low, although its presence in other maternal oocytes cannot be completely excluded. If inherited from the mother, the recurrence risk is substantial but unpredictable for an individual pregnancy because of the bottleneck (chapter 3) (Smeets et al., 2023). Therefore, before PGT-mt is discussed, the mother’s tissues — and, where possible, those of other maternal relatives — should be studied (Mavraki et al., 2023).
• Incomplete penetrance and variable expressivity. The same variant causes different syndromes in different carriers — from isolated symptoms to severe multisystem disease; severity depends on the mutant load in specific tissues; for m.8993T>G, for example, the clinical picture differs at different heteroplasmy levels (Gorman et al., 2016; Burr & Chinnery, 2024; Xie et al., 2026).
• Nuclear genes. Mitochondrial diseases caused by nuclear genes follow Mendelian inheritance; PGT-M, not PGT-mt, is used for them (Smeets et al., 2023).

PGT-mt applies when a specific pathogenic mtDNA variant has been identified in a heteroplasmic state in the woman considering pregnancy, preferably in several tissues.