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Researchers Demonstrate Clinical Utility of NGS in Preimplantation Aneuploidy Screening

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NEW YORK (GenomeWeb) – Following the success of whole-genome sequencing to noninvasively screen for fetal aneuploidy, a similar technique for screening embryos before they are implanted into women undergoing in vitro fertilization is quickly working its way into the clinic.

In a study published this month in Journal of Medical Genetics, researchers from the University of Oxford, led by Dagan Wells, a senior fellow in the Nuffield Department of Obstetrics and Gynecology at the University of Oxford, validated their technique on 61 samples. They found that low-pass whole-genome sequencing of embryos can identify chromosomal aneuploidies in less than 15 hours with 100 percent sensitivity and specificity, and they used it in a clinical setting to select embryos to implant in two women undergoing IVF. Both women subsequently gave birth to healthy babies.

Wells, who is also a director at New Jersey-based preimplantation genetic diagnostic firm Reprogenetics, told Clinical Sequencing News that the firm is now in the midst of a clinical trial comparing the NGS technique to standard of care for women undergoing IVF to see whether using NGS to choose chromosomally normal embryos improves pregnancy success rates. He anticipated Reprogenetics would launch a clinical test in the US by the end of the year.

While a cost for such a test has not been established, he anticipated it could be around 25 percent cheaper than other methods for evaluating chromosomal aneuploidy, such as array-based methods, which can cost around $2,000.

Wells previously presented on the technique at the 2012 Advances of Genome Biology and Technology meeting in Marco Island, Fla.

His group is not the only one pursuing the application. Illumina recently launched its VeriSeq test on the MiSeq to look for chromosomal aneuploidies in embryos prior to implantation, and the Reproductive Medicine Associates of New Jersey has been enrolling patients in a clinical validation trial of a targeted NGS-based preimplantation test. In addition, researchers at BGI said last year that more than 20 healthy babies have been born following a pre-IVF single-cell sequencing screen for chromosomal aneuploidies and large copy number variants.

Currently, IVF has only about a 25 percent success rate, Wells told CSN. One of the top predictors of pregnancy failure is that embryos contain chromosomal abnormalities, and studies have shown that evaluating aneuploidy can improve IVF success rates. Those studies have mainly used array CGH, Wells said, so the next step is to show that next-gen sequencing, which could be less expensive than array-based tests, can also improve pregnancy success rates.

In the Journal of Medical Genetics study, Wells' team performed single-cell whole-genome sequencing on 61 samples using Thermo Fisher's Ion Torrent PGM. The team started with 100 ng of input DNA and used multiple displacement amplification to amplify whole genomes from each cell, then used Ion's 200 bp sequencing chemistry. The 61 embryos included samples from cell lines, samples from five infertile couples of advanced reproductive age that were tested in a blinded fashion with both the NGS technique and array CGH, and seven clinical samples from two women undergoing IVF.

The team also multiplexed 32 samples and concluded that given the "robust" results, "even higher levels of multiplexing are likely to be possible," which would help reduce the cost further.

The group was able to complete the total protocol in under 15 hours; however, they wrote that even faster testing will be possible using the Ion Isothermal Amplification Chemistry, "which streamlines the sequencing procedure, eliminating the need for emulsion PCR." Using this approach, they were able to obtain data within eight hours. A fast turnaround time is critical, Wells said, because clinicians typically have only 24 hours after biopsy to do the test, unless the embryo is frozen.

After the researchers confirmed the accuracy of the protocol, they used it clinically to choose chromosomally normal embryos from two women aged 35 and 37 years who were undergoing IVF. Five blastocyst-stage embryos suitable for biopsy were produced in one IVF cycle and two in the other. For both women, embryos that were chromosomally normal were implanted, and both women gave birth to healthy babies.

The study also revealed another intriguing aspect that could help improve IVF success rates, Wells said. The whole-genome sequencing method also sequenced the entire mitochondrial genome from single cells to between 20x and 60x coverage. The researchers found differences in the quantity of mtDNA, even among embryos derived from the same parents. In the most extreme case, there was a 97-fold difference in mtDNA content between two embryos from the same parents. On average, though, the embryo with the largest amount of mtDNA was three to six times the quantity of mtDNA from the embryo with the least.

The group also found that embryos with high levels of mtDNA had a greater risk for being chromosomally abnormal. Wells said that including mtDNA analysis in embryo selection could help pick a more viable embryo. "The underlying basis isn't understood, but the clinical effect is significant," he said. It could be that the mtDNA is not functional, or that the embryo is upregulating its metabolism to deal with a stress. Regardless of the reason, mtDNA "does seem to have some link with embryo viability."

Wells' team also demonstrated that a targeted sequencing approach to screen for single gene disorders is feasible. They evaluated cells from a cell line homozygous for a common cystic fibrosis mutation. Following whole-genome amplification, they PCR amplified a 95 bp fragment that encompassed the known mutation. They then performed the same low-pass whole-genome sequencing technique, such that the PCR amplified fragment was sequenced to an average of 30x. The approach enabled the researchers to evaluate both chromosome aneuploidy as well as the cystic fibrosis mutation, and they detected the mutation in all of the cells.

Wells said that the use of sequencing to diagnose single-gene disorders is still a ways off from routine use in the clinic because it would add significant cost and time to the procedure.

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