Non-Invasive PGT-A may be Superior to Standard PGT-A

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Non-invasive PGT-A may be superior to standard PGT-A

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

A prospective study was initiated to compare the accuracy of non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) and preimplantation genetic testing for aneuploidy (PGT-A) simultaneously.

Standard PGT-A involves biopsying the trophectoderm of an embryo, causing a loss of 5 to 10 cells with unknown consequences. In contrast, niPGT-A samples only the cell-free DNA contained within the spent embryo culture medium (solution) eliminating this concern.

After obtaining informed consent, 20 surplus embryos marked for disposal at the Akita University Hospital was collected for this study. Mean patient age was 35.6 ±3.2 years. Of the 20 embryos, 10 were originally frozen on day 5, and the other 10 on day 6. All embryos were then thawed, with 5 day old embryos cultured in blast medium for 24 hours, while 6 days embryos were similarly cultured for 3 hours only.

With all embryos now at day 6 of development, biopsy of the trophectoderm was carried out and the spent culture medium collected for analysis and comparison.

Culturing of embryos continued after biopsy, up to and including day 10 as per protocol described in other studies. Once embryos stopped developing, became detached or reached day 10, samples from the center of the embryo (or whole embryo) was collected for analysis and comparison with day 6 samples.

Preparation of sample was carried out using the SurePlex DNA Amplification System followed by next-generation sequencing (NGS) and data analysis using Bluefuse Multi software. A total of 60 samples was collected (PGT-A =20, niPGT-A = 20, Outgrowth = 20) and 55/60 analysed by NGS (19/20 PGT-A, 19/20 niPGT-A, 17/20 Outgrowth). The five that could not be analysed was a result of excessive noise in the data. No significant difference in DNA concentration was noted between day 5 and 6 cultured embryos, implying results should not be biased to either group.

Initial comparison of the embryos that continued developing up to day 10, and classification of euploid or abnormal (aneuploid / mosaic) by each respective technique, showed an accuracy rate of 77.8% (7/9) for PGT-A, 80% (8/10) niPGT-A and 70% (7/10) for Outgrowth samples.

Next analysis of autosomal concordance rate, to evaluate how accurate both PGT-A and niPGT-A reflects the chromosomal status of the whole embryo for polyploidy and mosaicism, showed a concordance rate of 43.8% (7/16) for PGT-A and 56.3% (9/16) for niPGT-A, compared to Outgrowth samples as the control group.

Concordance rate of sex chromosomes, for PGT-A and niPGT-A compared to Outgrowth samples was more accurate featuring identical scores, 87.5% (14/16). Interestingly analysing concordance rate vs embryo development of day 10 embryos also showed improved concordance with Outgrowth samples via niPGT-A (70%) compared to PGT-A (55.6%).

Finally based on these results, a range of statistical values was calculated to compare the accuracy of PGT-A and niPGT-A with respect to Outgrowth samples.

Positive predictive value87.5%88.9%
Negative predictive value75.0%100%
False positive rate14.3%12.5%
False negative rate22.2%0%

Overall niPGT-A displayed a higher diagnostic accuracy and 0% false negative rate. In addition to the non-invasive method of sampling, niPGT-A was superior to PGT-A according to all measurements performed in this study.


In this study, the accuracy of routine PGT-A was significantly lower than non-invasive PGT-A, with only 87.5% vs 100% sensitivity and 77.8% vs 87.5% specificity, explained by the biological differences that exist within the embryo, between the inner cell mass (ICM) and trophectoderm (TE).


  1. Small number of embryos
  2. Test method may not accurately represent embryo outcomes following implantation

Similar studies

Huang L, et al. (2019). Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy.

Popovic M, et al. (2019). Extended in vitro culture of human embryos demonstrates the complex nature of diagnosing chromosomal mosaicism from a single trophectoderm biopsy.

Capalbo A, et al. (2018). Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions.

Ho J R, et al. (2018). Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos.

Xu J, et al (2016). Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.


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