Embryo culturing conditions and the development of non-invasive preimplantation genetic testing for aneuploidy detection (NI-PGT-A)

Abstract

The absence of standardised culturing conditions or molecular testing methodologies, including whole genome amplification (WGA) used for non-invasive preimplantation genetic testing of spent embryo culture media for aneuploidy (NI-PGT-A) may explain the variable rates of concordance reported between the spent embryo culture media and embryo biopsy results to-date. Culture conditions impact the accumulation of embryonic and contaminating DNA in spent embryo culture media and optimisation of either the culturing conditions, molecular testing methodologies, or both, should yield the highest level of concordant results for NI-PGT-A. This study examined rates of ploidy concordance between spent embryo culture media and embryo biopsies to evaluate the impact of culturing conditions on NI-PGT-A results. Spent embryo culture media was collected from single embryo culture droplets following biopsy of the embryo for PGT-A then stored at -20°C, with ethics approval. Equivalent volumes of spent embryo culture media samples from 10μl-60μl culture droplets, from either continuous (n=4 labs) or two-step cultures (n=4 labs) were whole genome amplified using DOPlify® kit reagents (PerkinElmer). WGA DNA yield was assessed by gel electrophoresis and high sensitivity quantification using a Qubit® instrument (Thermo Fisher® Scientific). Next generation sequencing libraries were generated according to the PG-Seq™ kit 48 sample protocol and sequencing was performed on a MiSeq® instrument (Illumina®). Data was bioinformatically aligned to hg19, and WGA DNA yield, NGS metrics, and whole chromosome aneuploidy concordance with the PGT-A result for the embryo biopsy were determined. Whole genome amplification using the DOPlify® kit reagents resulted in the amplification of 78-100% of spent embryo culture media samples (WGA failure rate 0-22%). Ploidy concordance with the embryo biopsy ranged from 29-75% for autosomal chromosomes and 47-94% for sex chromosomes using a single-step culturing system (n=4), compared with concordance rates of 67-90% and 50-97% respectively when media was changed during the 5-6 day culture (n=4). DNA yield was not affected by embryo culture media droplet volume, or continuous or two-step culture. Sex chromosome concordance varied between individual labs, suggesting that embryological processes are important in NI-PGT-A testing. Further statistical analysis during the ongoing larger scale collaborative study will determine quality control parameters for acceptance of NI-PGT-A results. Successful NI-PGT-A using spent embryo culture media will possibly require specific culturing conditions and/or specialised molecular methodologies for accurate and representative amplification and testing of the embryonic DNA. In a step toward this, we identified that renewing culture media during IVF improves overall concordance rates between the embryo biopsy and spent embryo culture media for NI-PGT-A.