A 2025 study re-analyzed embryos that tested as mosaic by NGS-based PGT-A using SNP karyotyping and found that one-third of those “mosaics” were actually uniformly aneuploid.
The DNA analysis step of PGT-A can be performed using different techniques, including next-generation sequencing (NGS) and SNP karyotyping. NGS measures the overall copy number of chromosomes, while SNP karyotyping analyzes specific genetic markers on chromosomes to determine the parental origin of each chromosome. NGS-based PGT is probably the most common type of PGT-A performed these days.
Mosaic embryos can be identified using NGS when it shows that there’s an intermediate number of chromosomes (explained more below). There’s some evidence that NGS might not be fully accurate in determining mosaicism.
This post is a summary of a study by Popa et al. (2025), in which these two different PGT-A techniques were used to confirm mosaic results from a single trophectoderm biopsy. First, standard PGT-A with NGS was used to flag potential mosaics, and then SNP analysis was applied to confirm whether the abnormality was uniform (meiotic error) or present in only a subset of cells (mitotic error).
To be clear, this study tested only a single biopsy with NGS and then retested that same biopsy using SNP. It isn’t evaluating the accuracy of PGT-A itself, but is comparing NGS and SNP for the DNA analysis step of PGT-A. It’s not comparing multiple biopsy samples.
It’s sufficient to understand that this study is confirming mosaic results, but for those who want a bit more detail…What’s a meiotic error and mitotic error?
A meiotic error occurs during the formation of the egg or sperm and affects every cell of the resulting embryo, while a mitotic error happens after fertilization, leading to a mix of normal and abnormal cells (mosaicism). You can see the difference below, with two examples of a mitotic error that occur during the two-cell or four-cell stage.
It’s important to highlight that a meiotic errors leads to an abnormality in every cell, ie. the embryo is completely aneuploid. When applying PGT-A on these embryos, the results should clearly indicate that every cell is affected and is abnormal. However, some studies have found that, when using NGS, these uniform meiotic errors can sometimes be misinterpreted as mosaicism (typically caused by mitotic errors).
🔗 Original studies are referenced in this post or within the linked Remembryo posts.
💡 Reminder: Terms underlined with a dotted black line are linked to glossary entries. Clicking these does not count toward your paywall limit.
A closer look at NGS and SNP karyotyping
In this study, researchers used NGS first to flag embryos with mosaicism, and then used SNP karyotyping to confirm it. Let’s look at the difference in these techniques.
Next-generation sequencing (NGS)…
- is essentially a brute-force approach that counts how many copies of each chromosome, and the smaller pieces that make up these chromosomes, are present in the cells of the biopsy sample.
- It detects mosaicism when there’s an intermediate copy number — so if there’s 2.5 copies of chromosome 1, this suggests that half of the cells have 2 copies and the other half have 3 copies (ie. it’s 50% mosaic for trisomy 1).
- It doesn’t distinguish whether those copies come from the mother or the father.
In contrast, SNP karyotyping…
- analyzes specific genetic markers called “SNPs” or single-nucleotide polymorphisms that run along each chromosome.
- Each chromosome can have hundreds of thousands of these markers, and they’re specific to an individual. A SNP karyotype can reveal which chromosomes in the embryo come from which parent.
- An embryo normally inherits one set of chromosomes from each parent in equal ratios. When the ratios deviate from this balance, it indicates mosaicism resulting from a mitotic error, where only some cells carry a chromosomal abnormality. Conversely, if a chromosomal abnormality is uniformly present in all cells from the biopsy, it suggests a meiotic error that affected the entire embryo.
A third of mosaics by PGT-A with NGS were fully aneuploid
This study included 311 PGT-A cycles and identified 177 mosaic embryos after NGS-based PGT-A, with 141 of the mosaics analyzed further with SNP karyotyping.
SNP karyotyping revealed that 46 out of 141 mosaics, or 32.6%, had meiotic errors, meaning that 32.6% of embryos identified as mosaic by NGS were not truly mosaic but were uniformly aneuploid. In other words, NGS-based PGT-A only predicted 67.4% mosaics accurately.
This is the big result right here, but let’s also look more closely at how this breaks down in terms of high/low level mosaic.
From these 141 mosaic embryos, there were 191 chromosomal abnormalities that were mosaic after NGS-based PGT-A (ie. some had more than a single chromosomal abnormality). From these 191 mosaic abnormalities by NGS, 56 were fully aneuploid by SNP and 135 were confirmed mosaic by SNP.
So many of the high level mosaic embryos were actually aneuploid after SNP testing! The researchers performed a separate analysis and confirmed this — the higher the % mosaic by NGS, the higher the chance of it actually being fully aneuploid (by SNP analysis).
This was true only for whole chromosome mosaic embryos by NGS (there was a similar trend with segmentals, but it wasn’t statistically significant).
However, the researchers note that there was no reliable cutoff for % mosaic where mosaic by NGS results were aneuploid by SNP, as some high level mosaics were still mosaic by SNP (44 out of 135, if you look at the chart above).
This is also showing that about half of high level mosaics are truly mosaic (45.3%, or 44 out of 53+44=97).
Conclusions
This study found that a third of mosaics tested by NGS were fully aneuploid when tested by SNP, and were therefore “misclassified” by NGS, according to the authors. The rest of the mosaics were still mosaic.
This means that up to a third of “mosaic” embryos by NGS that are potentially transferred are actually aneuploid, and that the commonly used NGS-based PGT-A isn’t very accurate at identifying mosaic embryos.
Another analysis showed that a higher % mosaics were more likely to be aneuploid. About half of high level mosaics were actually aneuploid using the SNP test.
To be clear, this study tested only a single biopsy with NGS and then retested that same biopsy using SNP. It isn’t evaluating the accuracy of PGT-A itself, but is comparing NGS and SNP for the DNA sequencing step of PGT-A. They found that the commonly used NGS method alone wasn’t accurate in properly identifying mosaics, and a second method was needed to do this. It’s not comparing multiple biopsy samples.
So why is NGS less accurate? The authors state that it may be due to technical errors, or artifacts, during DNA processing/analysis that can create false signals that look like mosaicism, when in fact the result should be aneuploid.
Since they were able to properly identify mosaics and aneuploids when using both NGS and SNP, the authors call for the combination of these methods for PGT-A.
While this shows that NGS-based PGT-A isn’t as accurate as SNP, this doesn’t change the fact that a single biopsy may not be representative of the blastocyst! As discussed in my recent post The problems with PGT-A: A 2025 opinion, this is one of the several problems with PGT-A.
Additionally, although I didn’t discuss it, this study found five triploid embryos by SNP that were classified as mosaic by NGS. I’ve discussed the value of NGS and SNP in diagnosing polyploid embryos in another post.
Reference
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About Embryoman
Embryoman (Sean Lauber) is a former embryologist and the founder of Remembryo, an IVF research and fertility education website. After working in an IVF lab in the US, he returned to Canada and now focuses on making fertility research more accessible. He holds a Master’s in Immunology and launched Remembryo in 2018 to help patients and professionals make sense of IVF research. Sean shares weekly study updates on Facebook, Instagram, and Reddit regularly. He also answers questions on Reddit or in his private Facebook group.
Related posts:
Trophectoderm and ICM biopsies match depending on the abnormality
Does a PGT-A biopsy match the rest of the embryo?
Mosaicism much more common than previously thought, using more sensitive PGT-A technique
More sensitive PGT-A test shows that nearly all blastocysts, fetal tissues are mosaic
Mathematical model predicts that fully euploid embryos are rare
The problems with PGT-A: A 2025 opinion
High euploidy rates found after using PGT-A on whole embryos
PGDIS releases 2021 guidelines for mosaic embryo transfers