Non-invasive preimplantation genetic testing for putative mosaic blastocysts: a pilot study

STUDY QUESTION: What is the potential of applying non-invasive preimplantation genetic testing (niPGT) for chromosome abnormalities in blastocysts reported with a mosaic trophectoderm (TE) biopsy? SUMMARY ANSWER: niPGT of cell-free DNA in blastocyst culture medium exhibited a good diagnostic performance in putative mosaic blastocysts. WHAT IS KNOWN ALREADY: Advances in niPGT have demonstrated the potential reliability of cell-free DNA as a resource for genetic assessment, but information on mosaic embryos is scarce because the mosaicism may interfere with niPGT. In addition, the high incidence of mosaicism reported in the context of PGT and the viability of mosaic blastocysts raise questions about whether mosaicism really exists. STUDY DESIGN, SIZE, DURATION: The study was performed between May 2020 and July 2020. First, clinical data collected by a single-center over a 6-year period on PGT for chromosome aneuploidies (PGT-A) or chromosomal structural rearrangements (PGT-SR) were analyzed. After confirming the reliability of niPGT, 41 blastocysts classified as mosaics by trophectoderm (TE) biopsy were recultured. The chromosomal copy number of the blastocyst embryo (BE, the gold standard), TE re-biopsy, and corresponding cell-free DNA in the culture medium was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data on patients enrolled for PGT at a single center from 2014 to 2019 were collected and the cycles with available putative mosaic blastocysts were evaluated. To verify the diagnostic validity of niPGT, eight aneuploid blastocysts were thawed and re-cultured for 14-18h. The concordance of the niPGT diagnosis results and the whole blastocyst testing results was analyzed. Forty-one blastocysts reported as mosaics from 22 patients were included and re-cultured for 14-18 h. The genetic material of the BE, TE re-biopsy, and corresponding cell-free DNA in the culture medium was amplified using multiple annealing and looping-based amplification cycles. The karyotype data from niPGT and TE re-biopsy were compared with that from the whole blastocyst, and the efficiency of niPGT was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Data on 3738 blastocysts from 785 PGT-A or PGT-SR cycles of 677 patients were collected. According to the TE biopsy report, of the 3662 (98%) successfully amplified samples, 24 (0.6%) yielded no results, 849 (212%) were euploid, 2245 (61.3%) were aneuploid, and 544 (14.9%) were mosaic. Sixty patients without euploid blastocysts opted for a single mosaic blastocyst transfer, and 30 (50%) of them obtained a clinical pregnancy. With the BE chromosome copy number as the gold standard, niPGT and TE re-biopsy showed reliable detection ability and diagnostic efficiency in eight putative aneuploid blastocysts. Of the 41 putative mosaic blastocysts re-cultured and re-tested, 35 (85.4%) showed euploid BE results. All but two of the blastocysts previously diagnosed with segmental chromosomal mosaic were actually euploid. In addition, all blastocysts previously classified as low degree (20-50%) mosaics were identified as euploid by BE PGT, whereas four of the six putative high degree (50-80%) mosaic blastocysts showed chromosomal abnormalities. The raw concordance rates of spent culture medium (SCM) and TE re-biopsies compared with BE were 74.4% and 82%, respectively, in terms of overall ploidy and 96.2% and 97.6%, respectively, per single chromosome when considering all degree mosaic results as true positives. However, when we set a mosaicism identification threshold of 50%, the concordance rates of SCM and TE re-biopsies compared with BE were 87.2% and 85% at the overall ploidy level and 98.8% and 98.3% at the chromosomal level, respectively. At the full ploidy level, the sensitivity and false negative rates for niPGT were 100% and 0, respectively. After adjustment of the threshold for mosaicism, the specificity of niPGT increased from 69.7% to 84.8% in terms of overall ploidy and from 96.1% to 98.9% at the chromosomal level. LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study is the small sample size, which decreases the strength of our conclusions. If possible, identifying the clinical outcome of niPGT on reassessed mosaic blastocysts would be further progress in this field. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first to explore the practicability of niPGT in diagnostic reassessment of putative mosaicism. The present study provides a novel opportunity for patients with only mosaic blastocysts and no euploid blastocysts, regardless of the technical or biological basis of mosaicism. Employing niPGT after 14-18 h of re-culturing might be a superior option for the best use of blastocysts because of its minimally invasive nature.