Cell-free DNA in spent culture medium effectively reflects the chromosomal status of embryos following culturing beyond implantation compared to trophectoderm biopsy

Abstract

This prospective study evaluated the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA in spent culture medium, as well as that of preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm (TE) biopsy after culturing beyond implantation. Twenty frozen blastocysts donated by 12 patients who underwent IVF at our institution were investigated. Of these, 10 were frozen on day 5 and 10 on day 6. Spent culture medium and TE cells were collected from each blastocyst after thawing, and the embryos were cultured in vitro for up to 10 days. The outgrowths after culturing beyond implantation were sampled and subjected to chromosome analysis using next-generation sequencing. Chromosomal concordance rate, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), false-positive rate (FPR), and false-negative rate (FNR) of niPGT-A and PGT-A against each outgrowth were analyzed. The concordance rate between the niPGT-A and outgrowth samples was 9/16 (56.3%), and the concordance rate between the PGT-A and outgrowth samples was 7/16 (43.8%). NiPGT-A exhibited 100% sensitivity, 87.5% specificity, 88.9% PPV, 100% NPV, 12.5% FPR, and 0% FNR. PGT-A exhibited 87.5% sensitivity, 77.8% specificity, 87.5% PPV, 75% NPV, 14.3% FPR, and 22.2% FNR. NiPGT-A may be more accurate than PGT-A in terms of ploidy diagnostic accuracy in outgrowths.

Consistency between chromosomal status analysis of biopsied human blastocyst trophectoderm cells and whole blastocyst cells

“…Therefore, the development of a new technology that could replace or complement the results of analysis performed using PGT-A, such as using cfDNA present in the culture supernatant, [14][15][16] is warranted.…”

“…Using the current PGT‐A method, it is difficult to accurately determine the actual chromosomal status of the whole embryo. Therefore, the development of a new technology that could replace or complement the results of analysis performed using PGT‐A, such as using cfDNA present in the culture supernatant, 14 , 15 , 16 is warranted.…”

“…Despite recent reports on PGT‐A using cell‐free DNA (cfDNA) from spent embryo culture medium, 14 , 15 , 16 the consistency between the actual blastocyst chromosomal status and the origins of cfDNA has been debatable. At present, PGT‐A implemented globally in clinical practice uses TE biopsy samples.…”

Embryonic Cell-free DNA in Spent Culture Medium: A Non-invasive Tool for Aneuploidy Screening of the Corresponding Embryos

“…As a result, we cannot identify mosaic embryos accurately because they show DNA heterogenicity, leading to a false genetic diagnosis (35)(36)(37)(38). However, mosaic embryos can give birth to healthy babies (39,40), but the dynamic implantation decreases compared to euploid embryos (41,42). In addition, researchers have found a discrepancy between the chromosomal states of cells in trophectoderm (TE) biopsies, so these cells do not necessarily represent the whole embryo (40,43,44).…”

“…Recent studies have shown detection of cf-DNA in biological fluids, blastocyst fluid, and spent culture medium of in vitro cultured embryos (39,40,(45)(46)(47)(48)(49)(50), opening new roads for the implementation of non-invasive procedures in assisted reproductive technology. The cf-DNA that can be detected by the embryonic developmental culture material, SCM, seems to be the best choice for non-invasive PGT (ni-PGT).…”