O-078 Mosaic embryos – are they accurately detected by PGT-A?
Authors: Popa T , Whitney E , Kanani M , Xanthopoulou L , Bakosi E , Davis C , O'Neill H , Ottolini C 
Published: June 2023

Study question

Following quantitative NGS for PGT-A, do current thresholds for mosaicism calling give a true reflection of the presumed mitotic origin of mosaic errors?

Summary answer

A large proportion of embryo mosaicism diagnosed following quantitative NGS-based PGT-A does not correlate with a true and expected mitotic origin, showing instead meiotic signatures.

What is known already

Mosaicism is a true biological phenomenon that can affect ongoing embryo development. However, the reproductive potential of mosaic embryos in assisted reproduction varies greatly across publications and is not fully understood. While the transfer of mosaic embryos could result in healthy live births, embryos containing meiotic errors almost never result in healthy live births. Since the clinical implications of meiotic and mitotic errors are very different, it is critical to accurately differentiate between the two. However, diagnosing mosaicism using current NGS-based PGT-A is not always accurate due to technical and biological limitations which may misclassify mosaic embryos.

Study design, size, duration

Follow-up analysis was carried out on 159 embryos previously categorised as mosaic by NGS-based PGT-A. To identify the origin of mosaic calls, SNP genotyping and karyomapping were performed on the same amplified DNA used for the clinical NGS analysis. Trophectoderm biopsy samples were obtained from embryos on days 5,6 or 7 of development from a single IVF centre between 2018-2022. For all PGT-A tested embryos, the reported mosaicism rate at the IVF centre was 12%.

Participants/materials, setting, methods

Samples were analysed using PGTai from CooperSurgical with a 30-80% CNV mosaicism range. Patients with mosaic embryos consented to karyomapping follow-up using sibling embryo reference DNA. The origin of mosaic events was defined by SNP inheritance patterns for the affected chromosome. Meiotic errors were defined by the presence of both haplotypes from one parent for trisomy, and the absence of a parental haplotype for monosomy. Mitotic errors were inferred following a result of biparental inheritance.

Main results and the role of chance

A total of 154 diploid embryos identified as mosaic by NGS-based PGT-A were re-analysed by SNP-genotyping. Of these, 30.5% (n = 47) displayed at least one error of meiotic origin following SNP genotyping.

Among the 154 embryos, 189 mosaic chromosomal abnormalities were identified. After SNP-genotyping, 30.7% of chromosomal abnormalities (n = 58) showed evidence of meiotic signatures: 52 whole-chromosome (24 gains, 28 losses), and 6 segmental errors (5 gains and 1 loss). Of 73 segmental errors detected in total, only 8.2% of them (n = 6) were identified to be of meiotic origin.

Following PGT-A, 7.8% of embryos (n = 12/154) displayed ≥3 chromosomal abnormalities in the mosaic range. Of these embryos, 58.3% (n = 7/12) displayed one or more meiotic aneuploidies. Overall, embryos showing multiple mosaic events were significantly more likely to have a least one error of meiotic origin than embryos containing one abnormality (McNemar’s test p-value= 1.171e-14).

Among mosaic calls above 50% CNV, 56.7% (n = 55/97) showed evidence of meiotic origin. In contrast, but importantly, in the low-moderate mosaicism range &lt;50% CNV, 3.3% of errors (3/92) also displayed meiotic signatures, including two whole chromosome errors (chr21 gain at 32% CNV and chr13 loss at 46% CNV), and one segmental error (dup(6)(pter-p12.1) at 33% CNV).

Limitations, reasons for caution

Since karyomapping relies on recombination events to identify meiotic trisomy, the true origin of intermediate copy-number changes cannot always be identified. Therefore, errors without recombination may be missed, potentially underestimating meiotic gains, especially segmentals. The absence of a parental haplotype cannot entirely rule-out a mitotic error from a biopsy sample.

Wider implications of the findings

Quantitative NGS methodologies are unable to accurately distinguish the origin of mosaic errors in PGT-A samples. Since the clinical impact of meiotic and mitotic errors differs greatly, this limitation could explain mosaic embryos’ reduced viability. Reliable identification of meiotic aneuploidies in presumed mosaic embryos is likely to improve clinical outcomes.

Trial registration number

not applicable