By Kevin Davies
February 22, 2013 | A reproductive clinic in New Jersey has successfully used next-generation sequencing (NGS) to screen embryos conceived in otherwise routine in vitro fertilization (IVF) cases prior to implantation. The news was reported in a talk yesterday evening at the Advances in Genome Biology and Technology (AGBT) conference by Dagan Wells, a geneticist at the University of Oxford.
“This could become a game changer in terms of IVF and a better reproductive option for couples,” Wells told Bio-IT World in an interview before his presentation.
The application of NGS in reproductive genetics is reaching new highs. Several companies have launched non-invasive prenatal screening tests for chromosomal abnormalities such as trisomy 21; two groups reported progress in determining the full sequence a fetal genome non-invasively; and Stephen Kingsmore and colleagues reported the rapid 50-hour genome analysis of newborns in the intensive care unit.
Now NGS is beginning to make its mark in IVF, says Wells, who first started working on preimplantation genetic diagnosis (PGD) as a Ph.D. student at University College London 21 years ago. He currently runs a lab at the National Institute of Health Research Oxford Biomedical Research Centre, and partners with Reprogenetics, a leading IVF clinic in New Jersey where he previously worked and helped set up PGD (in which DNA from a cell plucked from an undifferentiated 3-day embryo is analyzed for the presence of specific gene mutations and help instruct the selection of healthy embryos for implantation). IVF clinics such as Reprogenetics and Genesis Genetics in Michigan currently offer PGD for hundreds of different genetic disorders.
But genetic screening is also performed on regular IVF embryos in cases where there are no known genetic risk factors. “More than half of all embryos produced during IVF—and probably naturally as well—are chromosomally abnormal,” Wells says. “If you do transfer those [abnormal] embryos, most commonly they don’t implant. And if they do implant, the patients usually miscarry in the first trimester… Since we have these extremely high levels of chromosomal abnormalities, wouldn’t it be a good idea to ensure we’re not transferring those that are most abnormal to the patients?”
An IVF cycle produces 6-8 embryos on average, but transferring more than one embryo increases the risk of multiple implants and concomitant medical risks. Even twin pregnancies carry elevated risks of preeclampsia, cerebral palsy, and other conditions.
“The best outcome in an IVF cycle is a single embryo transfer leading to a single ongoing pregnancy birth,” says Wells. But even embryos that are chromosomally abnormal “all look exactly the same... Even dramatic chromosomal abnormalities don’t have any discernible effect on embryo morphology.”
Chromosomal analysis of IVF embryos was initially performed by FISH (fluorescence in situ hybridization), but results were mixed. The reason in hindsight, says Wells, was that the technique “at best only looked at half the chromosomes. At early stages of development, any chromosome can be abnormal, but you wouldn’t see it later on because they can be so lethal.”
FISH was eventually supplanted by microarray CGH (comparative genomic hybrization), which involves extracting DNA from the single cell of a 3-day embryo, followed by whole-genome amplification (WGA) and the CGH step, which reveals the copy number for each chromosome. Wells credits the British company BlueGnome—recently acquired by Illumina—for dedicating a lot of R&D into producing what he calls “a robust system” for this technology.
Clinical trials have shown that microarray CGH significantly improves pregnancy rates, reduces miscarriages and the incidence of disorders such as Down syndrome. Some clinics are now introducing more rapid screening, analyzing the embryo 48 hours later, at Day 5 post fertilization.
But a downside of microarray CGH is cost. “BlueGnome has done a great job of pushing cost down,” Wells says, but the price has started to plateau. Even with discounts, Wells says it costs his lab about $150/embryo. As some patients produce as many as 20 embryos, this can add thousands of dollars to an already pricey IVF process.
“We need to look for newer technologies for high accuracy tests, to lower costs, and confer benefits to a much larger group of patients,” says Wells. The logical next step is NGS, particularly given the possibility of multiplexing and barcoding to reduce costs.
For several reasons, Wells selected the Ion Torrent PGM platform from Life Technologies. “We wanted something user friendly, we could imagine it being moved into a diagnostics environment, and not too fiddly,” he says. “We also needed something that was fast… Because the Ion Torrent system was the quickest available for this kind of sequencing, we decided to go down that route.”
Even as the DNA test is being conducted at Day 3, the embryo continues to develop. “Once it reaches five days, it really needs to be transferred to the uterus in order to implant,” says Wells. “Outside that window, you risk the embryo having developed too far or the uterus no longer being receptive.” Speed as well as accuracy is of the essence.
Furthermore, many IVF clinics lack their own genetics expertise so they must send the cell to a specialist reference lab for testing, which takes a day. (The new trend of analyzing the embryo DNA later, at Day 5, also narrows the window.)
Wells and Ion Torrent have been working together for about one year. He says he tries to treat the embryo DNA just like any other genomic DNA sample. “Our experience with single cells, particularly embryos, is the simpler you keep the procedure, the better the chances of a robust result.”
Wells says the PGM has “a very nice user-friendly interface with iPhone-like apps. We use one of those that assigns each DNA fragment to its chromosome of origin, so we count how many fragments come from each chromosome. That’s pretty reproducible for a normal sample.”
The amount of sequencing required to achieve a statistically significant result is fairly light—less than 1x coverage across the genome. “We just need a few thousand fragments from each chromosome, about 5-10,000 reads for a small chromosome. That becomes statistically robust,” says Wells.
The total number of reads required is about 150,000 for each test. “We’re more than happy if we get 5 percent coverage of the genome, that’s more than sufficient to get a chromosomal diagnosis,” says Wells.
As far as multiplexing goes, his team has analyzed the DNA from 32 embryos on a single chip, and it is feasible to push that number up to 100 embryos. Cost is “significantly cheaper than microarray approaches,”—he quotes $70/sample in his presentation—and Wells expects that advantage to continue given the downward trajectory of NGS costs.
But WGA does introduce some biases—the quantity of reads is not exactly proportional to the size of chromosome. “But those distortions tend to be repeatable. It’s not diagnostically a problem so long as you have a normal sample for reference,” says Wells.
After extensive preclinical validation, Wells and colleagues were confident enough to apply the NGS screening process clinically in a couple of cases at Reprogenetics. Two pregnancies are progressing well, says Wells, who performed the data analysis.
The use of the PGM platform in an IVF setting is interesting in that, as a Life Technologies spokesperson confirmed for Bio-IT World, “The Ion PGM is for research use only and not intended for use in diagnostic procedures.” However, the IVF cases at Reprogenetics were conducted under a clinical research protocol (not as a clinical service). Moreover, according to Reprogenetics’ founder and director Santiago Munné, CLIA (Clinical Laboratory Improvement Amendments) does not regulate clinical analysis of preimplantation embryos. He adds that the testing of chromosomes is considered “screening” rather than “diagnosis”; by definition, IVF testing does not involve diagnosis of the fetus in utero.
Although early days, Wells says the benefits of NGS may go further in diagnosing single-gene disorders and shedding additional light on factors mitigating successful pregnancies.
“It is still the case that even if we transfer a chromosomally normal, morphologically perfect embryo, we still can’t guarantee a pregnancy. NGS may give us insight into what else is going on,” he says. For example, there are extensive variations in the levels of mitochondrial DNA between embryos. Says Wells: “It would be hard to imagine that doesn’t have some clinical effect.”