Embryo development and biopsy

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mtDNA in the embryo before implantation

The embryo inherits its mtDNA pool from the oocyte. Until the blastocyst stage, mtDNA replication is suppressed, so with each cell division the existing mtDNA is distributed between daughter cells and the mtDNA content per cell falls approximately twofold (Rubens et al., 2025). The resumption of replication and the regulation of copy number as cell lineages differentiate are closely linked to pluripotency (Facucho-Oliveira & St John, 2009). If molecules were distributed very unevenly, an embryo would have a “bottleneck” of its own; this is why how representative the biopsy sample is has to be tested experimentally (Rubens et al., 2025).

Evidence from the cleavage stage

• Monnot and colleagues studied 70 single blastomeres from embryos of two m.3243A>G carriers: the maximum difference between blastomeres of one embryo was 6 pp, with an intraclass correlation coefficient of 0.994; the level measured in two day-3 blastomeres differed from that in the whole day-5 embryo by less than 7 pp in 13 of 16 embryos and by 10–15 pp in the other three (Monnot et al., 2011).
• Sallevelt and colleagues found, among 294 blastomeres from 73 embryos of 9 m.3243A>G carriers (15% cut-off), only one potentially false-negative result (0.34%) and no false positives; they recommended a single-blastomere protocol, noting that two-blastomere biopsy lowers the live birth rate (Sallevelt et al., 2017a).
• For m.14487T>C, Sallevelt and colleagues found differences of 19.4 and 30.6 pp between blastomeres in 2 of 19 embryos (Sallevelt et al., 2017b).
• Mertens and colleagues showed that, for neutral and rare mtDNA variants, individual day-3 blastomeres already differ (early mosaicism), while the proportion of potentially pathogenic variants decreases between days 3 and 5 (Mertens et al., 2022).
• Tajima and colleagues examined 3PN embryos from couples without a known variant: their method detected 4–22% m.8993T>G, with a spread of 2–11 percentage points between blastomeres, within the method error (−5 to +6 percentage points); these data cannot estimate biological heterogeneity (Tajima et al., 2007).

Evidence from the blastocyst stage

• Treff and colleagues: for m.3243A>G, the TE level predicted the ICM level (r² = 0.97) (Treff et al., 2012).
• Rubens and colleagues: 112 embryos with pathogenic variants and 28 controls; variants were distributed evenly between day-4 blastomeres and between parts of the blastocyst, and levels remained stable from cleavage to blastocyst; however, in 1 of 8 control embryos single TE cells differed substantially (Rubens et al., 2025).
• Tofilo and colleagues: six blastocysts from an m.14487T>C carrier with a bimodal distribution; after warming and division into parts, values in the remaining TE and ICM closely matched the original biopsy (Tofilo et al., 2026).
• Hu and colleagues: a blastocyst with 17.8% m.3250T>C in the biopsy was divided into 16 samples of 3–5 cells; values ranged from 14.7% to 21.9%, mean 18.2% — results from small cell samples were more variable, but their mean matched the biopsy result (Hu et al., 2026).
• Whole-genome approaches allow different forms of PGT, including mtDNA assessment, on one biopsy (Janssen et al., 2024).

Within-embryo variability in numbers

The Within individual embryos section brings together paired measurements in the TE biopsy, ICM and remaining TE, as well as blastomere pairs. The spread within an embryo is typically an order of magnitude smaller than between embryos. The practical consequence: a difference of a few percentage points does not change the decision for embryos with extreme values, but it can move an embryo across the chosen cut-off if the measured level lies close to it.

Limitations of embryo microdissection methods

Cellular material is isolated after thawing, generally from embryos unsuitable for clinical use. Residual cells, cell debris and material under the zona pellucida cannot be equated with the cell lineages that form foetal tissues; the ICM of a blastocyst is not yet the epiblast (Tofilo et al., 2026). Comparisons of TE with ICM and of TE with remaining TE should therefore be considered separately, and study samples remain small, with uneven representation of mtDNA variants.

Biopsy as an intervention

Biopsy is an invasive procedure, and its parameters (stage, number of cells, how the zona pellucida is opened, culture and cryopreservation) are defined and validated by each clinic and laboratory. There is no universal protocol for PGT-mt; published recommendations concern the minimum number of TE cells for a reliable estimate of heteroplasmy (Rubens et al., 2025) and the choice between one and two blastomeres (Sallevelt et al., 2017a).

This description of stages and biopsy is educational. It is not a laboratory protocol and does not define biopsy parameters.