How PGT-mt works

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1. Identifying the pathogenic mtDNA variant in the family

The starting point is an established molecular diagnosis. Diagnostic investigation increasingly starts with genomic analysis of DNA from blood and/or urine, with muscle examined when needed (Mavraki et al., 2023). Planning PGT-mt requires the exact variant description, evidence of its pathogenicity, heteroplasmy levels in the carrier’s accessible tissues and the maternal family history. For a variant of uncertain significance, the decision to perform PGT-mt needs particular justification.

2. Counselling and assessment of reproductive options

Options include natural conception with prenatal diagnosis, PGT-mt, donor oocytes, mitochondrial donation (transfer of nuclear material, for example the oocyte spindle or zygote pronuclei) where it is permitted, and forgoing a genetic link (Smeets et al., 2023; Castelluccio et al., 2025; Steffann, 2025). In the Newcastle programme, the choice between PGT and mitochondrial donation was based on the expected probability of obtaining an embryo with low heteroplasmy (Hyslop et al., 2025; McFarland et al., 2025; Pickett et al., 2019). Mathematical modelling can estimate risks and chances of success before ovarian stimulation (Ji et al., 2023). The ethics of PGT-mt — residual risk, choice of an acceptable heteroplasmy level, informed consent — are discussed separately (Bredenoord et al., 2009).

3. Obtaining oocytes and embryos

An ART cycle comprises ovarian stimulation, follicle aspiration, fertilisation — in PGT programmes usually by ICSI — and embryo culture to the biopsy stage (Hyslop et al., 2025). Ovarian response to stimulation, outcomes and follow-up in carriers have been described in a French cohort (Mayeur et al., 2023). The number of embryos available for testing is the main practical constraint.

4. Embryo biopsy

Biopsy is performed at the cleavage stage (one blastomere) or at the blastocyst stage (several TE cells); the advantages and limitations of each are discussed in Chapter 4. With TE biopsy, embryos are often cryopreserved while results are awaited, then transferred after warming.

5. Sample preparation and WGA

A biopsy consisting of only a few cells contains an extremely small amount of starting DNA. Analysis can be performed directly on the lysate (targeted amplification of the variant region) or after whole-genome amplification, which provides material for several tests, including PGT-A. WGA can bias allele ratios and introduce background errors; linearity, limit of detection and noise after WGA are therefore validated separately (Spath et al., 2021; Xia et al., 2024; Hu et al., 2026).

6. Quantifying heteroplasmy

PCR with restriction analysis

the variant creates or destroys a restriction site; proportions are estimated from the digestion products. Used, for example, in a large series of embryos from France (Rubens et al., 2025).

Pyrosequencing

quantitative sequencing of a short region; has limitations at very low variant proportions (Mizuguchi et al., 2025; Hyslop et al., 2025).

ARMS-qPCR

allele-specific quantitative PCR (Mitalipov et al., 2014).

Droplet digital PCR (ddPCR)

quantification of variant and wild-type mtDNA from the numbers of positive and negative droplets, with statistical correction for droplet occupancy; used to assess residual mtDNA after nuclear transfer (Tytgat et al., 2021).

Targeted NGS

deep sequencing of an mtDNA region, compatible with PGT-A on the same sample (Spath et al., 2021; Tofilo et al., 2026).

Whole-genome sequencing

a single assay assessing PGT-M targets, aneuploidy and mtDNA (Janssen et al., 2024; Xia et al., 2024).

These are alternative approaches, not stages of one procedure. No method is “best” in itself: the choice is determined by validation in a specific laboratory for a specific variant and sample type (Mavraki et al., 2023).

7. Quality control, limit of detection and analytical accuracy

A laboratory should demonstrate linearity across the full 0–100% range using mixtures of known composition, establish the limits of detection and quantification, assess reproducibility with replicate measurements, and use positive and negative controls (Mavraki et al., 2023). For samples of a few cells, contamination control and allele drop-out matter. “Variant not detected” means that the assay did not detect the variant under the validated test conditions; it does not establish that the true heteroplasmy level is 0%.

8. Joint assessment with PGT-A

A low heteroplasmy level alone does not make an embryo suitable for transfer. In the study by Spath and colleagues, half of the embryos tested (16 of 33) were aneuploid (Spath et al., 2021); the aneuploidy rate rises with the woman’s age (Volodyaev et al., 2025). Simultaneous assessment of mtDNA and aneuploidy was evaluated for clinical use with NGS (Spath et al., 2021) and discussed as an important goal for clinical testing (Hanson & Seli, 2021). How the two criteria combine quantitatively is shown in the PGT-mt × PGT-A model section.

9. Interpretation and discussion of transfer

Embryos are ranked by mutant load; when no embryo without detectable variant is available, the embryo with the lowest available load is considered. A programme chooses and justifies its cut-off in advance: in Newcastle, for example, embryos with heteroplasmy below 30% in a single day-3 blastomere were preferred for transfer (Hyslop et al., 2025). The decision is made together with the family, taking residual risk into account (Chapter 6).

10. Prenatal diagnosis and postnatal follow-up

After transfer, discussion of confirmatory prenatal diagnosis is recommended (Rubens et al., 2025). Its interpretation depends on the pathogenic variant being tested, the tissue examined (chorionic villi, amniocytes) and gestational age. Chorionic villi are less reliable because of placental heterogeneity; amniocyte analysis is reliable across gestational ages (Steffann et al., 2021). Postnatal measurements should be reported with the tissue, the child’s age and the method — otherwise they cannot be compared with the PGT-mt result.

This description is not a clinical SOP. There are no universal biopsy parameters, laboratory conditions or selection criteria; each element of the pathway is defined by local validation and regulatory requirements.