RELIABILITY OF THE NEXT-GENERATION SEQUENCING (NGS) DIAGNOSIS OF MOSAICISM TO PREDICT THE CHROMOSOMAL CONSTITUTION OF THE INNER CELL MASS

Abstract

To investigate the reliability of next-generation sequencing (NGS) for diagnosing mosaicism and the predictive diagnostic value of trophectoderm (TE) biopsy for assessing the genetic status of the inner cell mass (ICM) in mosaicism. Prospective study. Sixty-eight human embryos diagnosed clinically as mosaicism were rebiopsied and dissected into three parts: TE, ICM and the remaining parts of embryos (a combination of TE and ICM). All the samples were examined and compared with original clinical diagnosis. Whole genome amplification using the Malbac DNA amplification system followed by NGS via the ThermoFisher DA8600 platform was performed. Statistical analysis was performed using chi square. Among the 68 blastocysts, the original mosaicism diagnosis was confirmed in at least one additional biopsy in 14 (20.6%) blastocysts; 12 (17.6%) displayed de novo abnormalities; and 42 (61.8%) showed a euploid profile in all segments. Among 59 rebiopsied ICM specimens, the results for 10 (16.9%) were consistent with the original trophectoderm (TE) biopsy; 6 (10.2%) displayed de novo abnormalities; and 43 (72.9%) were euploid. Stratification analysis showed that the type of mosaicism within the original biopsy diagnosis was associated with the concordance across the embryos. The concordance rate with the ICM results was highest for whole-chromosome mosaicism (41.2%), followed by complex mosaicism (20.0%), while that for segmental and mixed mosaicism was lowest (3.3% and 0%, respectively). However, it was not associated with the gain or loss of chromosomes (P=0.138). Closer examination of the level of mosaicism within the original biopsy diagnoses revealed that the concordance rate increased with increasing levels of mosaicism (P<0.001). In addition, the second TE biopsy for mosaicism showed a sensitivity of 68.8% and specificity of 92.9% for predicting abnormalities in the ICM portions. This novel study exposes false-positive errors in NGS-based PGT and the limitations of a single TE biopsy for predicting the cytogenetic constitution of the ICM and whole embryos. It also provides suggestions for the transfer of mosaic embryos, indicating that those with segmental mosaicism and a low rate of aneuploidy should be prioritized and that a second TE biopsy is an option for doctors and patients facing a dilemma.