Researchers from Italy's Genoma Molecular Genetics Laboratory in Rome have published a validation of an Illumina sequencing method for preimplantation genetic diagnosis and screening that they said reflects closely the methodology behind the VeriSeq commercial PGS test Illumina plans to launch later this year.
The study, published online this month in the journal Fertility and Sterility, retrospectively compared the sequencing approach to established aneuploidy results from previous array-CGH testing of 68 embryos, as well as results in single cells from cultured amniotic fluids or products of conception.
According to the authors, the sequencing method, which involved paired-end sequencing on the Illumina HISeq2000 of amplification products created using BlueGnome technology, showed 100 percent sensitivity and specificity in identifying aneuploid embryos, a promising indication of its clinical validity and potential utility compared to current methods.
Francesco Fiorentino, the study's first author told Clinical Sequencing News in an email that his group decided to work with Illumina to validate the method because it believed sequencing-based PGS could reduce costs and enhance precision compared to current methods, which rely on various types of microarrays.
"We wanted to use Illumina's sequencing technology because of its reliability, wider experience in the NGS field compared to other technologies, [and] also because [these] NGS instruments were already available in [our] lab," Fiorentino wrote.
According to Fiorentino, "from [a] technical point of view," the protocol used in his team's study is "very similar" to the protocol behind the commercial sequencing PGS test, VeriSeq, that Illumina has said it plans to launch during the second quarter of this year.
Based on the results of the lab's completed validation work so far, Fiorentino said Genoma will "certainly adopt" VeriSeq after its launch.
"We are very keen to introduce this technology on [a] clinical basis," he wrote, adding that the lab would consider other NGS approaches to PGS "only after being thoroughly validated and after having demonstrated similar performances and characteristics."
Tristan Orpin, senior vice president of Illumina’s reproductive and genetic health business told CSN in an email that Genoma has been a customer "for a number of years and was selected to take part in the beta validation and early access of the PGS solution using NGS [while] a number of other groups around the world are also validating this application on Illumina sequencing platforms.”
Apart from the Genoma lab's efforts using Illumina sequencing, other groups are also exploring alternate sequencing strategies for NGS-based preimplantation screening.
Reproductive Medicine Associates of New Jersey announced earlier this year that it is currently enrolling patients in a clinical validation trial of a targeted NGS strategy using the Ion Torrent PGM and a set of sequence-specific primers for preimplantation genetic testing to aid in the selection of embryos for transfer as part of in vitro fertilization.
Dagan Wells from the University of Oxford's Nuffield department of obstetrics and Xiaoliang Sunney Xie from Harvard University have also reported on single-cell sequencing techniques to improve IVF success rates.
BGI has also been testing a method, published in PLoS One, for detecting copy number variants from single-cell, low-pass, whole-genome sequencing on couples undergoing IVF, and China's Berry Genomics offers a single-cell sequencing test for preimplantation genetic diagnosis called Chrosure.
According to Fiorentino, though interest is growing, no group has yet published an extensive validation of NGS-based PGS.
"We are hopeful that publication of our work in a full, peer-reviewed format will allow others to assess and reproduce our data and begin to assess the use of NGS for preimplantation genetic screening and perhaps other forms of preimplantation genetic testing methodologies," he wrote.
According to Fiorentino and Illumina, the Genoma group's published study represents the first section of a three-phase strategy to validate the use of the Illumina method for comprehensive aneuploidy screening as a preclinical step "toward its routine use in the diagnosis of chromosomal aneuploidy on embryos."
These first validation results showed that the approach was 100 percent consistent in its aneuploid embryo calls with array-CGH results using Illumina’s 24sure array, a current widely-used test originally developed by BlueGnome, which Illumina acquired in 2012.
The second phase of the three-part validation strategy has focused on clinical application of the NGS protocol in a prospective trial involving analysis of human embryos at blastocyst stage of development.
Beyond this, the team is also studying pregnancy rates following transfer of screened embryos to establish that the NGS method does as well or better than current array methods in terms of its ultimate outcome in allowing successful births.
"The only effective way to demonstrate its clinical usefulness is to perform a well designed and well executed [randomized controlled trial], showing actual improvements in clinical outcomes. The results of the above study will be critical when considering the use of this new technology in a clinical setting and will help to outline the potential for routine clinical use of NGS-based preimplantation embryo assessment," Fiorentino wrote.
Fiorentino told CSN in a later interview that the team's followup prospective study is already complete and the group is submitting it now for publication.
"The results overlapped well with the validation that has just been published," he said. "We compared [NGS again] to array CGH results using the BlueGnome 24sure array and we achieved a very high rate of pregnancy."
Moving forward, Fiorentino said Genoma is also working on a study using mixes of euploid and aneuploid cells to assess the accuracy and resolution limit of the Illumina method for detecting chromosomal mosaicism.
"Now that trophectoderm biopsy is becoming the preferred stage for PGS, mosaicism may represent an issue in the analysis and interpretation of the results after [chromosomal screening]. Although its significance for implantation and the developmental potential of embryos is still unclear, it is reasonable to assume that this phenomenon is likely to influence IVF success rates," he wrote.
Illumina declined to comment further on when it expects to launch VeriSeq this year.