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Next-Gen Sequencing Gets a Fix on Disease

Article Author: Neil McKenna
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Recent years have seen rapid advances in the capacity of molecular biological techniques to simultaneously interrogate multiple targets on omics platforms.

While these techniques are well established in basic research, they have more recently gained a foothold in clinical diagnostics, with an increasing number of laboratory tests incorporating some type of high-throughput or global-scale molecular analysis. Of these techniques, the one that arguably has the greatest potential to revolutionize our approach to diagnosing disease and tailoring therapies is next-generation sequencing (NGS).

NGS, which refers to a constellation of techniques in which DNA or RNA fragments are sequenced in parallel, offers significant increases in speed, scalability, and resolution over traditional sequencing methodologies. Testament to the considerable interest in NGS is the increasing number of conferences exclusively devoted to the topic.

One such event, GTCBio’s “Next-Generation Sequencing” conference, was recently held in San Diego. Presenters showcased the application of NGS-based diagnostic platforms in a variety of clinical settings.

Preimplantation Genetic Screening

Varied genetic prenatal conditions impact pregnancy and fetal development. Accordingly, preimplantation genetic screening (PGS) has emerged as an important clinical tool for identifying chromosomal aberrations.

Traditional prenatal screens have a number of limitations. Some screens, such as amniocentesis or chorionic villi sampling, are invasive; others, such as ultrasound or biochemical screening, are less invasive but limited in their sensitivity and specificity.

“In contrast to these techniques, noninvasive PGS-based on NGS has both superior detection sensitivity and specificity for chromosomal abnormalities,” said Keith Jones, Ph.D., vp of development at Illumina. “The Illumina verifi® test detects greater than 99% of all true-positive cases and has a cumulative false-positive rate of <0 .2="" br="">
The verifi test uses sequence information from across the genome. This approach, Dr. Jones suggested, allows for the rapid adoption of additional tests that may find abnormalities not readily detected using traditional screening approaches. Such abnormalities include sex chromosome aneuploidy, microdeletions, trisomy 9, and trisomy 16.

Approximately 1.3 million in vitro fertilization (IVF) procedures are performed globally each year; however, only 25% of the procedures meet with success. The low success rate is usually attributed to complicating factors associated with advanced maternal age and chromosomal aneuploidy in the embryo.

“The aim of PGS in the IVF setting is to select chromosomally balanced embryos during the IVF process and ensure that only euploid embryos—those with a normal number of chromosomes—are implanted during IVF procedures,” explained Dr. Jones. He added that PGS has been shown to improve implantation success rates and reduce the number of high-risk pregnancies associated with multiple egg transfers.

In Illumina’s VeriSeq™ PGS platform, genomic DNA from a single cell is amplified and sequenced to provide a genome-wide view of the copy number state of the embryo. The protocol takes less than a day and allows multiplexing of up to 24 samples per sequencing run, translating to an increased likelihood of identifying a viable embryo and decreasing the time between biopsy and an answer. “The broad dynamic range derived from the sequencing data makes interpretation clear with a high degree of confidence,” Dr. Jones asserted.

Testing for Minimal Residual Disease

“The overarching theme in the NGS molecular diagnostics space is that robust clinical validation is a must,” said Martin Moorhead, Ph.D., vp of computational biology and software development at Sequenta. The company’s LymphoSIGHT platform is an NGS-based immune repertoire analytical solution that combines multiplex PCR assays and informatics algorithms to interrogate rearranged immunoglobulin and T cell receptor genes.

“Our PCR process targets the CDR3 region and the immediate surrounding sequence, yielding amplicons that are typically around 150 base pairs in length, which is ideal for NGS analysis,” Dr. Moorhead pointed out. In the assay, sequencing of rearranged B or T cell receptor gene amplicons from patient lymphocyte samples allows for an absolute quantification of the number of each clonotype—cells all sharing the same rearranged receptor sequence in the original sample.

“The first clinical product we developed using the LymphoSIGHT platform is the ClonoSIGHT test for measuring minimal residual disease (MRD) in patients with blood cancers, including diffuse large B cell lymphoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and mantle cell lymphoma,” stated Dr. Moorhead. MRD refers to cancer cells that may remain in the body of a person with lymphoid cancer after treatment, and is the leading cause of relapse in this condition.

Testing for MRD can help determine whether treatment has been successful, provide important information about patient prognosis, and help guide additional treatment decisions. At its essence, the ClonoSIGHT assay compares cancer cell DNA sequences generated using the LymphoSIGHT platform in a diagnostic sample with those in follow-up samples to determine the presence of residual cancer cells.

“ClonoSIGHT test results, which are generated in seven days using our CLIA-certified, CAP-accredited laboratory, are provided in a simple, actionable report,” added Dr. Moorhead. “[The report] shows a patient’s MRD status and level as well as MRD trends over time.”

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Data source: Stork Fertility Center 2013 data
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6 Ways to Boost Your Odds of IVF Success

Original Source:

by Laura Tedesco

As the rate of first-time moms over age 35 rises—it’s up to 15 percent in the U.S. now—so does the country’s reliance on in-vitro fertilization. In fact, what was once a last-resort procedure has become increasingly routine: In 2012, U.S. doctors performed 165,172 fertility procedures—more than 99 percent of which were IVF, according to the most recent data from the Society for Assisted Reproductive Technology, reflecting a 7 percent increase over 2011.

And how many of those procedures actually lead to deliveries? Among women younger than 35, the data shows that 46 percent of the embryo transfers resulted in live births. That number drops to 38 percent for women ages 35 to 37, and 28 percent for women ages 38 to 40.

Although those stats may sound discouraging, consider this: Only 55 percent of “chromosomally normal” embryos successfully become a baby, “whether you make them at home in bed, or whether you make them in a lab,” Jamie Grifo, M.D., program director of the NYU Fertility Center, told Yahoo Health. “It’s mostly not under your control. It’s about the luck of getting a good embryo.”

But that doesn’t mean the only way to boost your chances of success is to cross your fingers and hope for the best. “There are things the patients can do themselves to actually improve the odds of IVF,” A. Musa Zamah, M.D., an infertility expert at the University of Illinois at Chicago, told Yahoo Health. “And I don’t think those things are always fully utilized.”

Kick stress to the curb

Don’t stress too much about de-stressing — just devote a few minutes every day to unwinding without totally checking out from your daily routine. “Just because you’re stressed doesn’t mean you can’t get pregnant,” Grifo said. “If that were true, nobody in New York would have a baby.” In fact, he said, eliminating every source of anxiety — say, by quitting your job — may just leave you without a distraction, tempting you to fret 24/7 about getting pregnant (which is, in and of itself, a serious stressor).

That said, mental tension definitely doesn’t do your fertility any favors, since being in chronic fight-or-flight mode can compromise the quality of your eggs, Grifo said. Plus, the stress hormone cortisol may interfere with implantation, while also blocking estrogen action in your endometrium, said Sarah Berga, M.D., the chair of obstetrics and gynecology at Wake Forest University.

Easing stress, on the other hand, may actually help speed things along: In a 2011 Fertility & Sterility study, women who watched a clown perform magic tricks for about 15 minutes after undergoing IVF were nearly three times more likely to become pregnant than the control group. So figure out what works for you — knitting, yoga, watching funny cat videos, whatever — and commit to doing it daily.

Resist the urge to over-exercise

Being fit equals higher fertility, so constantly hitting the gym is a step in the right direction, right? Not necessarily. Exercise is a form of physiological stress, which means too much of it could hurt your pregnancy odds, noted Grifo. “If you’re running 60 miles a week or training for a marathon, that’s not a good thing,” he said. “If you’re doing an hour of cardio, seven days a week, that’s not a good thing.”

You should be especially cautious about high-intensity workouts during the actual stimulation phase of IVF, when you’re receiving hormone injections, said Zamah. “You don’t want to risk the ovaries twisting,” he explained. So what’s a reasonable workout regimen? Grifo recommended 30 to 40 minutes of cardio three times a week, with yoga or light weightlifting on the days in between.

Load up on vitamin D

It’s not just folic acid you should be popping: Consider taking a dose of D, too. “Vitamin D has long been thought to be primarily important for bone health,” said Zamah. “But it can really affect all aspects of health, including reproduction.” In fact, a 2014 study in the journal Human Reproduction found that D-deficient women who underwent IVF had a 39 percent lower chance of pregnancy than ladies with normal levels of the vitamin. Although the reverse isn’t yet proven — that taking D boosts your pregnancy odds — supplementing with 400 to 800 IUs of vitamin D per day could be “potentially beneficial,” Zamah said.

Establish a healthy weight — before attempting IVF

Don’t wait until you’re ready to start trying for a child to worry about the number you see on the scale. “Doctors might be reluctant to bring this up with patients, but weight can impact the success of IVF,” Zamah said. Specifically, being overweight has been linked to poor IVF outcomes, including decreased pregnancy rate, lower live birth rate, and increased risk of miscarriage. The likely explanation? “Obesity appears to hurt the eggs and maybe the endometrium,” said Berga.

So before you start IVF, aim to get your weight into the “normal” BMI range, or as close to it as possible. “Even if you’re not fully in the ‘normal’ range, a 5 to 10 percent reduction in body weight can really be beneficial for IVF outcomes,” Berga told Yahoo Health. Just don’t go too far — being underweight can have a similarly negative effect on pregnancy success.

Consider a DHEA supplement

A third of all IVF centers across the globe now use DHEA, a hormonal supplement shown to boost pregnancy chances in women with a shortage of viable eggs, according to a recent study review in Reproductive Biology and Endocrinology.How does it work? “DHEA might help the ovary respond to the fertility medications,” particularly in patients with a low number of eggs, explained Zhama. The suggested dose: 25 milligrams, three times a day.

Get the guy involved

Your body carries the baby, but his sperm also plays a role in your IVF odds. “The male partner needs some attention, too,” said Berga. In addition to him limiting his stress and watching his weight, your partner (or donor) can also try a more targeted approach to boosting the health of his swimmers: by popping multivitamins, plus an antioxidant supplement. “Nobody has shown one antioxidant blend to be superior to another for sperm,” Zamah said. “But there are multiple studies showing at least a potential benefit on sperm parameters.”
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