Abstract
Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy samples is labour intensive, invasive, and subject to sampling bias. In this study, we report on the efficacy and factors affecting accuracy of a technique we pioneered for minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A). Our technique uses cell-free embryonic DNA (cfeDNA) in spent embryo culture medium (SEM) combined with blastocoel fluid (BF) to increase the amount of assayable cfeDNA. We compared miPGT-A results (n = 145 embryos) with standard PGT-A analysis of the corresponding trophectoderm biopsy. We found that accuracy of miPGT was not related to blastocyst morphological grade. The overall concordance rate per sample for euploidy/aneuploidy status between miPGT-A and TE biopsy samples was 88/90 (97.8%), and was not different between good 47/48 (97.9%) and moderate/low quality blastocysts 41/42 (97.9%) (p > 0.05). Importantly, we also discovered that for cfeDNA analysis, the SurePlex whole genome amplification (WGA) kit can be utilized without an additional cell lysis/extraction DNA step; this efficiency likely reduces the risk of maternal contamination. Regarding origin of embryonic cfeDNA, the average amount of miPGT-A WGA-DNA we obtained from blastocysts with different morphological grades, as well as the size miPGT-A WGA-DNA fragments, suggest that it is unlikely that apoptosis and necrosis are only mechanisms of DNA release from the inner cell mass (ICM) and TE into BF and SEM.
Highlights
Low birth weight, following invasive PGT compared with IVF without embryo biopsy, conclusive evidence regarding the long-term health of the offspring after embryo biopsy will take some time to obtain[5,13,14]
Non-invasive preimplantation genetic testing (NIPGT) or minimally invasive preimplantation genetic testing using the cell-free embryonic nuclear DNA (cfeDNA) of spent embryo culture medium (SEM) and/or blastocoel fluid (BF) has the potential to eliminate the need for embryo biopsy, thereby avoiding potential risks related to that invasive procedure[21,22,23]
NIPGT-A, which is based on sequencing of cfeDNA likely released from both TE and ICMcells[24,25], may better represent the entire embryo compared to TE biopsy alone[26,27,28]
Summary
Low birth weight, following invasive PGT compared with IVF without embryo biopsy, conclusive evidence regarding the long-term health of the offspring after embryo biopsy will take some time to obtain[5,13,14]. Attempts to use cfeDNA for non-invasive preimplantation aneuploidy testing bring to light several factors that could potentially affect the accuracy of this approach reducing concordance rates with TE biopsy results. These include maternal contamination by cumulus and corona cells[33], cfeDNA degradation, low amounts of cfeDNA, variable DNA amplification efficacy and yield[7,36,37], and short DNA fragments[17]. NIPGT-A, based on sequencing of cfeDNA likely released from both TE and ICM cells, may better represent the entire embryo compared to TE biopsy alone[30,37,38,39]. It should follow that lower quality blastocysts, which generally have higher degrees of apoptosis, would result in a higher quantity of cfeDNA release and more accurate results from aneuploidy testing
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