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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. Meanwhile, cell-free DNA in SCM reporting lacks uniform diagnostic interpretations. Considering that this test is meant to determine which embryos are relegated to be discarded, PGT-A with cell-free DNA in SCM should not be permitted to be applied in routine clinical settings for diagnosis purpose.
Given the challenges intrinsic to invasive biopsy procedures, there is increasing interest in developing noninvasive procedures. Clinically, sampling DNA from embryo spent culture media would be preferable over that from TE biopsy. Though SCM based PGT-A demonstrated some encouraging results through successfully amplified DNA and detected genomic sequences in the majority of PGT-A cases. Still, more research based on clinical outcomes is required to determine if SCM can be a reliable selection tool in PGT-A [14]. However, the SCM- based PGT-A outcomes are still subject to variation from the aspect of full concordance when compared to those obtained from whole embryos or biopsy specimens [15,16,17]. Nonetheless, this procedure opens a new avenue for quantitative and qualitative evaluation of chromosomal status. More importantly, SCM PGT-A would also likely increase accessibility to patients due to the nature of sequencing cell-free DNA in SCM with less invasive [14], simpler, safer, and lower PGT-A cost. We sought to evaluate whether this DNA in SCM can be liable source of information about the genetic status of the embryo.
One of the major challenges for cell-free DNA assessment is potential contamination by maternal DNA from granulosa cells [22]. Regarding this challenge, we focused on optimizing procedures to ensure that cell-free DNA derives only was from corresponding blastocyst. Through DNA amplification, WGA results will give well-identified chromosomal status of the corresponding blastocyst, opening the way for clinical use of this non-invasive technology.
An overview result of ploidy is shown in Table 1. For 75 blastocysts, informative results (successful DNA amplification with interpretable NGS results) were obtained from 100% TE (trophectoderm biopsy) and WB (zona-free whole blastocysts). One of 75 (1.33%) SCM specimens showed maternal contamination. Interpretable NGS results in the SCM group were 78.67% (59/75). These results were significantly lower than their corresponding TE and WB groups. Of the NGS results per sample, there were no significant differences observed in SCM, TE, or WB aneuploid rates based on their interpretable NGS results: SCM 91.53% (54/59); TE 86.67% (65/75); and WB, 81.33% (61/75).
In this study, the full chromosome concordances of SCM, TE and whole blastocyst contribute experimental evidence to the validation of PGT-A at the blastocyst stage. Due to intrinsically low quantity/abundance and poor integrity associated with DNA samples in SCM, it resulted in diagnosis biases. Subsequently, the results negatively contributed to our full concordance assessment so that the full concordance rate between TE and WB was greater than that of SCM and WB. Because of this bias, we conclude that SCM provides less accurate and reliable high-throughput methodology for PGT-A. Although cell-free DNA in SCM has the potential to represent a safe and simple strategy in PGT-A, it still requires further validation. This procedure works as expected, consistently achieves expected results, but is not comparable to currently used tests for TE biopsies. Therefore, it is a rule-in test, as opposed to a rule-out test [41] and could be used for optimizing noninvasive embryo prioritization. With further improvement, it remains a potential tool for noninvasive PGT-A. The diagnostic efficiency of cell-free DNA in SCM may ultimately require custom-tailored design methodology for sampling strategies, tailored blastocyst culture media components, sample storage to prevent the length of the DNA in SCM sample from further degradation, and new WGA techniques for the efficient amplification of degraded/short DNA fragment samples.
Like most submitted manuscripts, this manuscript underwent revisions after peer review. With submission of the revised manuscript for re-review, the corresponding author advised this journal that one of the original co-authors, a representative of the company that produced the assay used in the study to detect cell-free DNA in spent media, had withdrawn her co-authorship. This journal's policy of full transparency requires disclosure of this fact. 2b1af7f3a8