Validation of preimplantation genetic tests for aneuploidy (PGT-A) with DNA from spent culture media (SCM): concordance assessment and implication

Abstract

Background: Spent culture medium (SCM) as a source of DNA for preimplantation genetic tests aneuploidy (PGT-A) has been widely discussed. Methods: Seventy-five blastocysts donated for research provided a unique possibility in which multiple specimens, including trophectoderm (TE) biopsy, SCM, and paired corresponding whole blastocyst (WB) specimens from the same blastocyst source, could be utilized for the purpose of this preclinical validation.Results: To conduct a validation ploidy concordance assessment, we evaluated the full chromosomal concordance rates between SCM and WB (SCM-to-WB), and between TE and WB (TE-to-WB) as well as sensitivity, specificity and overall diagnostic accuracy. 78.67% (59/75) of NGS results in the SCM group are interpretable, which is significantly lower than their corresponding TE and WB groups. This discrepancy manifests itself in intrinsically low quantity and poor integrity DNA from SCM. Subsequently, remarkable differences in full concordance rates (including mosaicism, and segmental aneuploidies) are seen: 32.2% (SCM-to-WB, 19/59) and 69.33% (TE-to-WB, 52/75), (p < 0.001). In such cases, full concordance rates were 27.27% (15/55) in SCM-to-WB, and, 76% (57/75) in TE-to-WB (p < 0.001) Collectively, the NGS data from SCM also translated into lower sensitivities, Positive Predictive Value (PPV), Negative Predictive Value (NPV), overall diagnostic accuracies, and higher Negative Likelihood Ratio (NLR).Conclusions: Our study reveals that DNA is detectable in the majority of SCM samples. Individual chromosomal aberration, such as segmental aneuploidy and mosaicism, can be quantitatively and qualitatively measured. However, TE still provides a more accurate and reliable high-throughput methodology for PGT-A. While cell-free DNA in SCM has the potential to represent a useful strategy in reproductive medicine.

Embryo selection through non-invasive preimplantation genetic testing with cell-free DNA in spent culture media: a protocol for a multicentre, double-blind, randomised controlled trial

“…PGT-A by trophectoderm biopsy or whole embryo and SCM showed a consistency rate between 78.2% and 100% 18–23. However, other studies reported a consistency rate between 32.2% and 56.3% 24 25…”

Non-invasive preimplantation genetic testing for aneuploidies: an update

“…All 42 embryos provided informative results, but only 85.7% had ploidy concordance across sample types (66.7% full and 19.0% partial). Yin et al, 2021 worked with 75 thawed day-5/6 embryos, where TE biopsy had already been performed and that were cultured for 24 h. Although the informativity rate was 78.7%, the ploidy concordance rate was 89.8% (32.2% full and 57.6% partial). Ho et al, 2018, Huang et al, 2019, Shitara et al, 2021and Chen et al, 2021a, also compared some of the SBM, not only with TE, but also with whole embryos (33, 48, 16 and 256, respectively), obtaining high informativity rates for the SBM (97.6%, 92.3%, 95% and 96.6%, respectively) but disparate ploidy concordance rates: 45.5%, 93.7%, 93.8% and 78.1%, respectively (Table 2).…”

“…The benefit of such procedure has not been confirmed (Ho et al, 2018). Other manipulations that were performed include vitrification (Xu et al, 2016;Huang et al, 2019;Jiao et al, 2019;Zhang et al, 2019b;Chen et al, 2020;Yin et al, 2021;Shitara et al, 2021;Li et al, 2021), collapsing of the embryo and/or biopsy before media collection (Feichtinger et al, 2017;Kuznyetsov et al, 2018;Huang et al, 2019;Jiao et al, 2019;Zhang et al, 2019b;Chen et al, 2020;Li et al, 2021;Sialakouma et al, 2021). Moreover, there are publications where a completely non-invasive protocol is performed achieving ploidy concordance rates with TE biopsy as high as 84% (Rubio et al, 2019;.…”

Incidence, dynamics and recurrences of reverse cleavage in aneuploid, mosaic and euploid blastocysts, and its relationship with embryo quality

“…Among these research contents, the abnormal cleavage pattern of embryos is an important one. Studies have found that abnormal cleavage directly affects the genome of a single cell in human embryos, resulting in chromosome loss and mosaicism, thereby reducing embryonic developmental potential, resulting in embryonic developmental arrest and significantly reduced blastocyst formation rates [16][17][18].…”