The application of more stringent parameters for mosaic classification in blastocysts-stage primplantation genetic testing for aneuploidies reduces false positive mosaic rates without comprising true detection

Study question: How do mosaic diagnostic thresholds setting affect the accuracy of Next Generation Sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A)? Summary answer: When single trophectoderm biopsy is tested, wide mosaicism thresholds (i.e.,20-80%) increase false positive calls compared to more stringent ones (i.e.,30-70%) without improving true detection rate. What is known already: Highly sensitive NGS-based technologies for PGT-A allows precise identification of intermediate chromosome copy number alterations potentially associated to chromosomal mosaicism in trophectoderm biopsies. Nevertheless, differences in technical validation procedures and in detection thresholds employed for diagnostic calls could lead to incorrect classification of normal and abnormal embryos into the mosaic category. Overcalling mosaicism in trophectoderm biopsies lowers PGT-A accuracy, ultimately affecting patients treatment outcome from both a clinical and a psychological standpoint. In this study, we evaluated diagnostic predictivity of different mosaicism classification criteria by employing blinded analysis of chromosome copy number values (CNV) in multifocal blastocyst biopsies. Study design, size, duration: The accuracy of different mosaicism diagnostic cut-offs was assessed comparing chromosomal CNV in intra-blastocysts multifocal biopsies. Enrolled embryos were donated for research between January and December 2019. The Institutional Review Board at the Near East University approved the study (project: YDUl20l9l70-849). Embryos showing euploid/ aneuploid mosaicism in their clinical trophectoderm (TE) biopsy (n=36) and euploid embryos (n=23) were disaggregated into 5 portions: the inner cell mass (ICM) and 4 TE biopsies. Overall, 295 specimens were analysed. Participants/materials, setting, methods: Fifty-nine donated blastocysts were warmed, allowed to re-expand and disaggregated in TE biopsies and ICM. PGT-A analysis was performed using Ion ReproSeq PGS kit and Ion S5 sequencer (ThermoFisher). Sequencing data were blindly analysed with Ion-Reporter software. Intra-blastocyst comparison of raw NGS data was performed employing different thresholds commonly used for mosaicism detection. CNV for each chromosome were reported as mosaic, according to 30-70% and 20-80% criteria. Categorical variables were compared using Fisher’s exact test. Main results and the role of chance: To minimize the impact of technical over biological variation, intermediate CNV were classified as confirmed mosaic according to the following criteria: 1) detection of the same mosaic alteration in at least 3 biopsies or in 1 additional biopsy over the 50% threshold, or 2) detection of a reciprocal mosaic pattern involving the same chromosome. If the same alteration was uniformly detected (>50%) in all biopsies, the embryo was classified as uniform aneuploid. When the high mosaicism threshold was considered (50-70%), the aneuploidy finding, mosaic or uniform, was confirmed in 82.5% of patterns (14/17; 95%CI=54.6-96.2). In particular, 35.3% of cases (6/17; 95%CI=3.0-16.8) were uniform aneuploid. For the low mosaicism category, 30-50%, putative mosaicism was confirmed in only 5.3% of the cases (3/57; 95%CI=1.10-14.62). When 20-50% threshold was applied, a significantly higher number of false mosaic alterations were observed, and the confirmation rate dropped to 1.8% (n=3/168;95%CI=0.37-5.13; P<0.001). In particular, the inclusion of very-low mosaicism (20-30%) results only added false positive results but no true mosaic case (66.1%;n=111/168;95%CI=58.38-73.19;P<0.001). In the euploid embryos group, none of the results obtained in the 39 cases of 20-30% and in the 30-50% range were confirmed. Limitations, reasons for caution: The study involved only blastocysts initially diagnosed as euploid or mosaic. Uniform aneuploid embryos were not evaluated at this stage. This approach involved the analysis of mosaicism thresholds at the embryo level and future studies will need to evaluate these criteria in relation to clinical predictive values following embryo transfers. Wider implications of the findings: Based on an embryo re-biopsy model, single TE biopsy results showing low mosaicism, particularly the very-low range (20%-30%) shouldn’t be considered as mosaicism diagnoses. The application of the lower stringency threshold for mosaic classification (i.e.,20%) leads to misclassification of embryos, increasing false positive calls and lowering accuracy of PGT-A analysis.