A pilot study to investigate the clinically predictive values of copy number variations detected by next-generation sequencing of cell-free deoxyribonucleic acid in spent culture media

Objective Analysis of cell free DNA in spent culture media has been identified as a non-invasive alternative to trophectoderm biopsy for PGT-A. However, the clinical value has not been well studied. We performed a pilot study with the primary interest of estimating the positive predictive value of copy number variations to predict nonviable or aneuploid embryos and investigate the false positive risk. Design Diagnostic/prognostic accuracy study. Subjects Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF centre. Intervention Embryo selection was performed according to conventional grading, blinded to non-invasive PGT-A results. After clinical outcomes were established, spent culture media samples were analyzed. Main outcome measures Prognostic accuracy of copy number variations according to niPGT-A results to predict nonviability or clinical aneuploidy. Results One-hundred twenty patients completed the study. Interpretations of NGS profiles were as follows: 7.5% (n=9) failed quality control; 62.5% (n=75) no copy number variations detected; 30% (n=36) abnormal copy number detected. Stratification of abnormal NGS profiles were as follows: 15% (n=18) whole chromosome; 15% (n=18) uncertain reproductive potential. Intermediate copy number variation was evident in 27.8% (n=5) of the whole chromosome abnormalities. Negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk for nonviability compared to no detected abnormalities (RR 2.21, 95% CI:1.66-2.94) (p<0.001). No other copy number variations were associated with significant differences in sustained implantation or relative risks for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant differences in MAPD between certain subcategories of CNV abnormalities were apparent. Conclusion Whole chromosome abnormalities were associated with high positive predictive value and significant relative risk for nonviability. Embryos associated with other copy number variations had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of non-invasive PGT-A. Analysis of cell free DNA in spent culture media has been identified as a non-invasive alternative to trophectoderm biopsy for PGT-A. However, the clinical value has not been well studied. We performed a pilot study with the primary interest of estimating the positive predictive value of copy number variations to predict nonviable or aneuploid embryos and investigate the false positive risk. Diagnostic/prognostic accuracy study. Patients aged 35 and younger with an indication for IVF-ICSI and elective single frozen embryo transfer at a single, private IVF centre. Embryo selection was performed according to conventional grading, blinded to non-invasive PGT-A results. After clinical outcomes were established, spent culture media samples were analyzed. Prognostic accuracy of copy number variations according to niPGT-A results to predict nonviability or clinical aneuploidy. One-hundred twenty patients completed the study. Interpretations of NGS profiles were as follows: 7.5% (n=9) failed quality control; 62.5% (n=75) no copy number variations detected; 30% (n=36) abnormal copy number detected. Stratification of abnormal NGS profiles were as follows: 15% (n=18) whole chromosome; 15% (n=18) uncertain reproductive potential. Intermediate copy number variation was evident in 27.8% (n=5) of the whole chromosome abnormalities. Negative predictive value for samples with no detected abnormality was 57.3% (43/75). Whole chromosome abnormality was associated with a positive predictive value of 94.4% (17/18), lower sustained implantation rate (5.6%, 1/18), and higher relative risk for nonviability compared to no detected abnormalities (RR 2.21, 95% CI:1.66-2.94) (p<0.001). No other copy number variations were associated with significant differences in sustained implantation or relative risks for nonviability. Unequal sex chromosome proportions suggested that maternal contamination was not uncommon. A secondary descriptive analysis of 705 supernumerary embryos revealed proportions of NGS profile interpretations similar to the transferred cohort. Significant differences in MAPD between certain subcategories of CNV abnormalities were apparent. Whole chromosome abnormalities were associated with high positive predictive value and significant relative risk for nonviability. Embryos associated with other copy number variations had sustained implantation rates similar to those with no abnormalities detected. Further studies are required to validate the clinical applicability of non-invasive PGT-A.