The research, led by a team at the Faculty of Medicine Ramathibodi Hospital, Mahidol University, in Bangkok, and published in the British Journal of Biomedical Science, set out to answer a deceptively simple question: does the timing of when you collect the embryo’s culture medium matter for the reliability of non-invasive preimplantation genetic testing, known as niPGT? The investigators hypothesized that extending embryo culture by just six hours, from 48 to 54 hours after the cleavage stage, might increase the amount of cell-free DNA released by the embryo into its surroundings, thereby improving the quality of the genetic signal that can be captured from the medium.

The biological logic behind this hypothesis is grounded in embryology. Aneuploid embryos, those carrying the wrong number of chromosomes, are known to develop more slowly than their euploid counterparts, with delays in key developmental milestones such as compaction and blastulation often exceeding six hours. Because cell-free DNA in the culture medium is thought to originate from apoptotic cells shed by the embryo during its normal cellular turnover, a modest extension of the culture period could, in theory, allow more time for this DNA to accumulate, particularly in embryos with delayed developmental kinetics. The researchers reasoned that capturing sufficient embryonic DNA would be especially important for those slow-developing embryos, which are more likely to be aneuploid and thus the very ones a screening test most needs to identify correctly.

To test this idea, the team enrolled 15 infertile women aged between 30 and 45 years who were undergoing IVF at their reproductive endocrinology and infertility unit between August 2020 and February 2021. From these patients, a total of 58 embryos were procured and analyzed. All embryos were created through intracytoplasmic sperm injection, a technique in which a single sperm is injected directly into a mature egg, and then cultured sequentially in droplets of specialized fertilization and cleavage media. On the third day after fertilization, when embryos had reached the cleavage stage, each was subjected to laser-assisted hatching, a procedure in which a small opening is made in the protective zona pellucida surrounding the embryo. This step is critical for non-invasive testing because it facilitates the release of cellular material into the culture medium as the embryo continues to develop.

Each patient’s embryos were assigned to one of two groups, with all embryos from a given patient biopsied at the same time point to avoid any overlap between conditions. One group had their trophectoderm biopsied and their spent culture media collected 48 hours after the cleavage stage, while the other group underwent the same procedures 54 hours after cleavage. The trophectoderm is the outer layer of cells that will eventually form the placenta, and biopsying it on day five of development is the current standard of care for preimplantation genetic testing for aneuploidy, or PGT-A. By comparing the genetic results obtained from the spent culture media against those from the trophectoderm biopsy, the researchers could assess how well the non-invasive approach matched the conventional invasive one.

On the laboratory side, the spent culture media samples were processed using a commercial non-invasive PGT kit that employs whole genome amplification, a technique that makes millions of copies of whatever DNA is present in the sample so that it can be sequenced. The amplified DNA was then subjected to ultra-low-pass whole genome sequencing, a cost-effective approach that reads only a small fraction of the genome but enough to determine whether the embryo has the correct number of each chromosome. The sequencing depth was approximately 0.01 times, generating between 400,000 and 500,000 reads per sample. Negative control samples, consisting of culture droplets that had never contained embryos but were cultured under identical conditions, were processed alongside the experimental samples to monitor for environmental contamination.

The results were revealing in several ways. First, the amount of cell-free DNA found in the spent culture media was remarkably consistent between the two time points, with median quantities of 27.8 nanograms per milliliter at 48 hours and 25.8 nanograms per milliliter at 54 hours, a difference that was not statistically significant. The success rate of DNA amplification was 93.1 percent at both time points, and the negative control samples yielded negligible DNA quantities of less than one nanogram per milliliter, confirming that the laboratory protocols were effective at preventing contamination. However, when the researchers compared the ploidy results from the spent culture media against those from the trophectoderm biopsy, the overall concordance rate was just 53.7 percent, meaning that the non-invasive test agreed with the conventional biopsy in only about half of the cases.

Breaking down the results further, the ploidy concordance rate was 48.2 percent in the 48-hour group and 59.3 percent in the 54-hour group, a difference that was not statistically significant. The sensitivity of the non-invasive test for identifying euploid embryos was 55.6 percent, while its specificity was 52.8 percent. The positive predictive value, which reflects the probability that an embryo called euploid by the non-invasive test truly is euploid, was just 37 percent. The negative predictive value, which reflects the probability that an embryo called aneuploid by the non-invasive test truly is aneuploid, was 70.4 percent. Interestingly, the 54-hour group showed a trend toward a higher negative predictive value, suggesting that sampling at a later time point might improve the ability to correctly exclude aneuploid embryos, though the wide confidence intervals in the data indicate that the subgroup analysis was statistically underpowered.

One area where the non-invasive approach performed notably better was in determining the sex of the embryo. The overall concordance rate for sex determination reached 79.6 percent, with the 54-hour group showing a higher rate of 85.1 percent compared to 74.1 percent in the 48-hour group. While this difference was not statistically significant, the trend suggests that extending the culture period may improve the reliability of sex chromosome identification. The researchers note that for couples at risk of passing on X-linked genetic disorders, non-invasive sex determination could hold genuine value for informed reproductive planning, and some IVF clinics have already begun adopting similar techniques for clinical use, resulting in successful pregnancies.

The study’s findings largely confirm what previous research has suggested about the limitations of early sampling for non-invasive genetic testing. Prior studies have reported concordance rates between spent culture media and trophectoderm biopsy ranging from 62.1 percent to 88.9 percent, with higher rates typically achieved when sampling occurs at later developmental stages. Recent work has demonstrated that day-six sampling yields higher quantities of cell-free DNA and superior concordance rates, and some groups have even extended culture to day seven with promising results. The current study’s finding that a six-hour extension from 48 to 54 hours made no meaningful difference suggests that the relationship between culture duration and DNA yield may not be linear, and that substantially later sampling may be necessary before non-invasive testing can achieve the accuracy required for clinical application.

The researchers acknowledge several limitations of their work. The study was conducted at a single center with a small sample size, which limits the generalizability of the findings. There was no randomization or strict matching regarding the exact developmental stage and morphological grading of embryos, and variation in embryo development kinetics could influence the quantity and quality of DNA released into the culture medium. Perhaps most importantly, maternal DNA contamination in the spent culture media was not quantitatively assessed using techniques such as short-tandem repeat analysis or methylation profiling. Cumulus cells, which surround the egg and are shed during fertilization, have been identified as a significant source of maternal DNA that can contaminate the culture medium and confound the genetic results. Future studies should incorporate robust maternal DNA quantification techniques to ensure the accuracy and reliability of non-invasive testing results.

Despite the disappointing ploidy results, the researchers remain optimistic about the broader potential of non-invasive preimplantation genetic testing. The approach demands less technical expertise than traditional trophectoderm biopsy, which requires a highly skilled embryologist and specialized micromanipulation equipment. It also carries lower costs and eliminates the need for direct embryo manipulation, which could circumvent potential religious and legal dilemmas in regions where embryo biopsy is prohibited. The technique could be particularly valuable for IVF laboratories that lack access to a skilled embryologist for biopsy procedures, and it could reduce embryo damage by eliminating the need for an additional biopsy when combined with preimplantation genetic testing for monogenic disorders.

Looking ahead, the researchers argue that a deeper exploration of non-invasive preimplantation genetic testing is imperative before it can be integrated into routine clinical practice. The question of whether the DNA found in culture media truly represents the authentic karyotype of the embryo remains unresolved, with theoretical speculation suggesting that the spent medium contains apoptotic cells from both the inner cell mass and the trophectoderm, arising from either abnormal cells or the embryo’s inherent cellular clearance process. Large multicenter prospective studies have demonstrated that cell-free DNA represents both the inner cell mass and the trophectoderm, with concordance rates of 78 to 90 percent when maternal contamination is tightly controlled. However, other studies have reported much lower concordance rates of 20 to 60 percent, often attributed to maternal contamination or variations in laboratory protocols. Until these technical challenges are resolved and standardized protocols are established across laboratories, the promise of non-invasive embryo screening remains just that, a promise waiting to be fulfilled.

Subject of Research: Application of non-invasive preimplantation genetic screening for aneuploidy through spent embryo culture media analysis at 48 and 54 hours after embryo cleavage

Article Title: Application of non-invasive preimplantation genetic screening for aneuploidy through spent embryo culture media analysis at 48 and 54 hours after embryo cleavage

Article References: Application of non-invasive preimplantation genetic screening for aneuploidy through spent embryo culture media analysis at 48 and 54 hours after embryo cleavage. (n.d.). https://doi.org/10.3389/bjbs.2026.15905

Image Credits: AI Generated

DOI: 10.3389/bjbs.2026.15905

Keywords: Application, non-invasive, preimplantation, genetic, screening, aneuploidy, spent, embryo, culture, media, analysis, hours