Understanding High-Density versus Targeted Sequencing for PGS
The newest and most widespread method used for preimplantation genetic screening (PGS) available to practitioners and patients today, involves the use of Next Generation Sequencing (NGS) technology. I will admit that I was hesitant to endorse the new technology when it first became available because there were only a few published studies describing the application of this powerful genomic technology for PGS, and there was little consistency in the way the technology was applied in those studies. As a result, we spent the last full year doing our own, in-house validation and examining the clinical impact of this new technology relative to our past experience with PGS. Despite the similarity in the technological platform described as NGS, many differences exist in the actual test methods used by the different laboratories offering preimplantation genetic screening. As a reproductive healthcare provider, I believe it is important to understand the differences between the different tests described as NGS, and how those differences may impact your patients and your practice.
In recent years, microarray-based methods were the most common technology used for PGS. Depending on the type, these microarrays targeted 3000 to 300,000 different sites from the 24 chromosomes in each embryo. The microarray technology was far superior to the older methods used for PGS testing such as FISH or qPCR that evaluated fewer than 100 target sequences. All available clinical evidence supports the conclusion that the increased coverage made possible by the microarray-based tests lead to increased rates of detection of chromosomal abnormalities in embryos. In most cases, the increased rate of detection meant fewer apparently normal embryos were available for transfer, but the benefit was unmistakable as a corresponding increase in pregnancy rate and a reduction in miscarriage rate were also observed for each embryo.
One of the new NGS-based methods available for preimplantation genetic screening today uses a Targeted NGS approach. Although the exact details are not known, this type of targeted NGS typically uses a pre-defined set of DNA sequences (~22,000 on average), to interrogate all 24 chromosomes (Kinde et al, 2012). Although there is a lack of published data comparing the specificity and sensitivity of these methods to other NGS platforms or microarray analysis; many providers who use this type of Targeted NGS, report equivalent pregnancy rates relative to their past experience with microarray-based PGS. This may not be surprising since the microarray and targeted-NGS formats are similar in the design and number of DNA sequences interrogated per embryo. Essentially both assays are asking if “a specific, target-sequence is present or absent, and in what relative abundance?” Thus their experience with targeted-NGS is, “just fine” because it is not worse than their experience with microarray-based PGS. But that is no strong endorsement, nor is it an apples-to-apples comparison of NGS technologies (whole genome vs. targeted).
Alternatively, NGS tests like CombiPGS, use a so-called high density, whole-genome approach to examine up to 1,000,000 DNA sequences for each embryo. The sensitivity and specificity of this method are well documented (Fiorentino et al., 2014). Rather than searching for and quantifying a subset of pre-defined sequences from each chromosome, this type of NGS assay instead examines sequences uniformly distributed throughout the entire genome, and calculates their relative abundance. Many IVF centers, including my own, are experiencing improved clinical outcomes once again, with increased pregnancy rates and decreased miscarriage rates relative to microarray-based PGS. This suggests that high-density, whole-genome NGS assays, like CombiPGS are identifying abnormalities that were previously missed by the microarray-based assays, as expected.
If the argument in favor of targeted NGS is only that it is “as good as” array-based screening, then we have missed the true potential of NGS technology. Single cell genomic technologies will continue to advance, and thus the amount of data available for each embryo will continue to grow in the future. In my own practice, we want to ensure that we have access to all of the data that can be obtained to improve our patient’s outcomes. It’s that simple.