Pushing the Limits of Detection: Investigation of Cell-free DNA for Aneuploidy Screening in Embryos

Objective: To determine the accuracy of cell-free DNA (cfDNA) in spent embryo medium (SEM) for ploidy and sex detection at the cleavage and blastocyst stages. To determine if assisted hatching (AH) and morphologic grade influence cfDNA concentration and accuracy.

Design: Prospective cohort.

Setting: Academic fertility center.

Patient(s): Nine patients undergoing IVF; 41 donated two-pronuclei embryos and 20 embryos from patients undergoing preimplantation genetic testing for aneuploidy (PGT-A).

Interventions(s): In a donated embryo arm, SEM was collected on days 3 and 5, with one-half of the embryos undergoing AH before and one-half after. In a clinical arm, SEM was collected on day 5 before trophectoderm (TE) biopsy. Samples underwent PGT-A with the use of next-generation sequencing. cfDNA results were compared with corresponding whole embryos and TE biopsies.

Main Outcome Measure(s): Concordance rates, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for ploidy and sex detection with the use of cfDNA.

Result(s): Of 141 samples, cfDNA was amplified in 39% and 80.4% of days 3 and 5 SEM, respectively. Concordances for ploidy and sex, respectively, were 56.3% and 81.3% between day 3 cfDNA and whole embryos, and 65% and 70% between day 5 cfDNA and TE biopsies. Day 5 cfDNA sensitivity and specificity for aneuploidy were 0.8 and 0.61, respectively. PPV and NPV were 0.47 and 0.88, respectively. Timing of AH and morphology did not influence cfDNA concentration or accuracy.

Conclusion(s): cfDNA is detectable on days 3 and 5, but more accurate on day 5. Although our data suggest moderate concordance rates, PGT-A with the use of cfDNA must be further optimized before clinical implementation.