#161 : Validation and Concordance of Non-Invasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A) Using Cell Free DNA from Spent Culture Media (SCM) with Gold Standard Trophectoderm Biopsy

Abstract

Background and Aims: Recent studies suggests that cell free(cf)-DNA is secreted in the spent culture medium (SCM) during embryo development, this can be used as non-invasive approach for PGTA. We aimed to validate Non-invasive Preimplantation Genetic Testing for aneuploidies (niPGT-A) using cf-DNA from SCM Methods: Prospective cohort study, tertiary centre from January-August 2021-2022. A total of 23 aneuploid/high mosaic embryos were donated by couples who had Trophectoderm (TE) biopsy for aneuploidy testing. All aneuploid blastocyst (WB) that were donated for research were thawed and transferred to RNAase and DNAase free PCR tubes. DNA extraction and amplification are carried out using Sureplex DNA amplification system. Libraries prepared using VeriSeqTM PGS Library Prep kit. A total of 24 libraries were pooled, denatured subjected to Next Gen Sequencing (NGS) using MiSeq system. Copy Number Variation (CNV) visualization and analysis for each sample was carried out using BlueFuse Multi Software. Results: NGS results from SCM and TE were respectively used against same positive control to find out accuracy with corresponding WB. NGS profiles generated from aneuploid WB, were manually analysed for CNV. Quality control(qc) was performed using Illumina qc requirements, only samples satisfying were used for analysis. Out of 23 embryos, 14 were aneuploid and 9 were high mosaic based on TE biopsy. Out of 14 aneuploid blastocysts on TE biopsy, 9 (64%) showed corresponding aneuploidy (full chromosomal concordance) on WB NGS profile. The rest 5 showed aneuploidy or mosaicism of a different chromosome on WB NGS profile. Only 5 out of 23 (21.7%) had full chromosome concordance between SCM and WB. The ploidy concordance between WB vs TE and WB vs SCM was 100% and 69.5% (16/23), which was statistically significant (p[Formula: see text]0.05). The decreased ploidy concordance with SCM could be due to suspected maternal contamination. Conclusions: Aneuploidy testing using Cf DNA in SCM is a promising technique but needs more research.