Navigating Non-invasive Preimplantation Genetic Testing for Aneuploidy (niPGT-A) – Technical Challenges and Ethical Considerations

Dear Editor,

Non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) is a revolutionary technique that assesses the chromosomal health of in vitro fertilisation (IVF) embryos without requiring an invasive biopsy procedure.[1] This thus offers a cheaper, faster, less labour-intensive and more convenient alternative to conventional preimplantation genetic testing (PGT),[1] by analysing cell-free DNA (cfDNA) released by the embryo into its surrounding environment, primarily from spent blastocyst media (SBM) and blastocoel fluid (BF).[2,3] The presence of embryonic DNA in SBM and BF is hypothesised to originate from cells undergoing apoptosis and necrosis, as well as mechanical stress from blastocoel expansion.[1] The methylation pattern of cfDNA indicates its origin from both the inner cell mass and trophectoderm, thus addressing mosaicism and providing a more comprehensive representation of the embryo’s chromosomal status.[4]

As a non-invasive genetic test, niPGT-A has the potential to increase pregnancy rates, reduce conception time, decrease the number of transfers needed and lower miscarriage rates.[5] However, its implementation raises ethical issues, primarily due to concerns about accuracy, DNA source, lack of standardisation and its role in clinical practice. Accuracy and reliability issues are of utmost concern with niPGT-A. The general rate of concordance between niPGT-A and conventional PGT-A varies widely between studies, ranging from 30.4% to 99.7%.[6,7] Upon reviewing data from several studies, Rubio et al. concluded that the concordance rates mostly range between 50% and 80%, which is currently inadequate for clinical recommendations.[6] A distinction must be made between “general concordance” (euploid versus aneuploid) and “full concordance” (matching specific chromosomal results), with full concordance being more challenging to achieve. Moreover, niPGT-A is prone to false positive and false negative results due to intrinsic characteristics of the DNA source, biological mechanisms of DNA shedding and technical challenges related to cfDNA sample quality and contamination.[6,7] The largest multi-centre study to date, involving 1301 blastocyst-stage embryos, reported a 8.3% false negative rate and 12.4% false positive rate with niPGT-A,[8] together with a sensitivity (proportion of correctly identified aneuploid embryos) range of 76.5%–91.3% and a specificity (proportion of correctly identified euploid embryos) range of 64.7%–93.3%.[8] This is concerning because false positives can lead to the discarding of viable embryos, while false negatives can lead to the transfer of abnormal IVF embryos.

The ability of niPGT-A to detect chromosomal mosaicism through SBM or BF sampling remains questionable, due to the low quantity and poor integrity of fragmented cfDNA.[7] Current techniques struggle to differentiate true mosaicism from technical errors,[9] and the question of whether cfDNA accurately reflects the genetic composition of mosaic embryos remains unresolved, due to uncertainty surrounding the origin and representativeness of cfDNA released into the SBM or BF. Current hypotheses of cfDNA originating from apoptotic or necrotic cells during normal embryo development or from abnormal cells remain highly disputed.[1,9] Another major technical and ethical challenge with cfDNA is minimising contamination from extraneous DNA, which can include residual maternal cumulus cells, polar bodies, excess sperm, or contamination from the laboratory environment.[1] The suggested use of intracytoplasmic sperm injection to reduce paternal DNA contamination in niPGT-A is ethically controversial due to potential risks to offspring health. Therefore, a higher prevalence of DNA amplification failure in niPGT-A is expected compared to conventional PGT-A utilising trophectoderm biopsy.

The clinical utility and implementation of niPGT-A are also hindered by a lack of standardised guidelines for sample collection, processing and analysis across different laboratories.[1] This lack of standardisation raises ethical questions about the reliability and comparability of niPGT-A results. Not surprisingly, there is controversy over its role as a prioritisation tool or a viability marker for selecting IVF embryos for transfer. The debate is over whether clinicians should receive detailed chromosomal results or just a priority score for niPGT-A. To date, the niPGT-A platform is still in the research and development phase, and extensive validation through large-scale, multi-centre randomised clinical trials is required to establish its clinical utility, safety and cost-effectiveness.

Rigorous and comprehensive genetic counselling is crucial to ensure IVF patients are fully educated about the choices, risks and benefits of niPGT-A.[1] This includes managing expectations regarding potential discordance with conventional PGT-A results, the likelihood of re-biopsy and the implications of mosaicism. The cost-effectiveness of niPGT-A compared to conventional PGT-A remains unclear, especially if high false positive/negative results require additional testing. The core issue of contention pits the risks of embryo biopsy with conventional PGT-A versus the inaccuracy of niPGT-A. Artificial intelligence (AI) and machine learning models in IVF embryo selection, such as niPGT-A data, face ethical and technical challenges. AI algorithms for predicting embryo ploidy often lack transparency, leading to ethical concerns among IVF professionals.[10] These require large volumes of high-quality data for training and validation, and a lack of standardisation in imaging capture systems and embryo incubation protocols can introduce variability. AI currently has limited capacity to predict complex chromosomal statuses like mosaicism.

In conclusion, while niPGT-A offers significant non-invasive advantages for IVF embryo assessment, its implementation is currently hindered by critical ethical and technical challenges. Key concerns include ensuring diagnostic accuracy in the face of contamination and overcoming the lack of standardised protocols across different IVF laboratories. Extensive validation through large-scale trials is essential, and critical issues related to contamination, standardisation and accuracy must first be resolved before safe and effective widespread clinical implementation of niPGT-A can occur. Furthermore, implementation requires specialised laboratory infrastructure and trained personnel, creating barriers for many clinical facilities. Until such issues are resolved, niPGT-A remains a prioritisation tool rather than a definitive diagnosis.

Author contributions

A. H. B. C. and N. S. contributed to writing this letter.

Conflicts of interest

There are no conflicts of interest.

Data availability statement

Not applicable.

Funding Statement

Nil.