Video one:

Video two:

Biopsy is extremely crucial technique in IVF-PGS cycles. In Stork fertility center, for example, one in twenty to twenty-one biopsied samples may fail to be amplified. The videos showed one of the biopsy techniques — pipet shearing. In this kind of manipulation, both biopsied cell amount and quality are of the essences. 

The biopsied sample in video one resulted in amplification failure, and the cells seemed degenerated yet. In contrast, biopsied sample in video two resulted in successful amplification.

The traditional prenatal examinations include chorionic villus sampling (CVS) and amniocentesis. However, the induction would be required if either CVS or amniocentesis revealed significant chromosomal abnormalities, and it becomes psychological stress. Perimplantation Genetic Screening（PGS）is an advanced technology, combining IVF and cell molecular biology, taking the third day embryo (about 6-10 cells now) or the 5th day blastocyst cell for chromosome analysis. The widely used analysis platform is based on array comprehensive hybridization (aCGH), but the recent researches reported that the next-generation sequencing platform performs better resolution and sensitivity. In addition, the low-rate aneuploidy is displayed more clearly in the NGS than in the aCGH.

Stork Fertility Center:

1. Although PGS is not allowable in Japan so far, it does help the patients to increase the success rate. 

(http://taiwanivfsfc.blogspot.tw/2014/01/to-make-choosing-embryo-easier-array.html)

2. Do I need to do PGS?

The patients are recommended to take PGS test,

a. Advanced age

b. Recurrent miscarriage (more than two)

c. Male infertile factors

d. Repeated failures in the IVF programs

e. Reported chromosome abnormalities

3. New platform to PGS test:

Nest generation sequencing (NGS( is now applied in the PGS realm and could provide better accuracy. 

(http://taiwanivfsfc.blogspot.tw/2014/09/next-gen-sequencing-gets-fix-on-disease.html)

Two new techniques aim to improve the likelihood that implanting a single embryo will allow a patient to have a baby.

A. Screening Chromosomes
The technique furthest along, and already used in select clinics around the world, is called preimplantation genetic screening. In this procedure, cells are removed from the embryo on day five (or day six) to see if the normal amount of genetic material is present. An embryo should have 23 pairs of chromosomes; those with extra chromosomes or missing chromosomes are considered less viable.

"Chromosome abnormality is the main cause of miscarriage," says Richard Scott, clinical and scientific director at Reproductive Medicine Associates of New Jersey, a fertility clinic and the reproductive endocrinology and infertility division of Rutgers University's Robert Wood Johnson Medical School.

(The process is different from searching for a specific disease or genetic disorder that is likely to manifest later in life, which is known as a preimplantation genetic diagnosis.)

Chromosomal screening has shown promise in three small, randomized studies. A study co-authored by Dr. Scott, for instance, found that transferring a single embryo after chromosomal screening resulted in a similar rate of live births as transferring two untested embryos.

Still, some experts say more research is needed.
"A major study with an adequate number of patients is needed to definitively determine the degree of benefit," says Christos Coutifaris, chief of reproductive endocrinology at the Perelman School of Medicine at the University of Pennsylvania, adding that this is especially true given the price tag. The procedure costs about $4,000—a hefty fee on top of an already expensive process that often is paid for out-of-pocket. The average IVF cycle costs about $12,400, according to the Society for Assisted Reproductive Technology. B. Monitoring Cell Division

Time-lapse imaging, which costs about $1,500, is a less invasive method of evaluating embryos. It's already used in clinics around the world, but new types of time-lapse screening are just becoming available commercially. With time-lapse imaging, thousands of pictures are taken to record a fertilized egg cell dividing. Eggs dividing atypically are unlikely to survive.

"The embryo's fate can be determined very early in development," says Barry Behr, director of Stanford University Medical Center's IVF laboratory.

Dr. Behr co-wrote a study identifying three markers that determine if a four-cell embryo (on day two) is likely to reach blastocyst, a critical stage where it has divided into about 120 cells (on day five) and has a better chance of implanting in the uterus. The study created an algorithm to do the analysis automatically.

Auxogyn Inc., based in Menlo Park, Calif., and Unisense FertiliTech AS, based in Copenhagen, make devices that use time-lapse imaging. Both companies are coming to market with products that use algorithms to automate the evaluation process. Currently most clinicians analyze time-lapse imaging subjectively.

Time-lapse imaging may also work in tandem with chromosomal screening, since it provides information about an embryo's metabolisms unavailable through genetic testing. An initial study was promising.

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.”

Original resource: http://www.genomeweb.com/sequencing/indias-supratech-micropath-developing-ngs-based-tests-pgs-newborn-dx-cancer

NEW YORK (GenomeWeb) – Supratech Micropath, a reference and diagnostic lab in the state of Gujarat, India, is developing next-generation sequencing-based tests for preimplantation genetic screening and diagnosis, newborn diagnosis, and cancer.

The laboratory is CAP accredited and certified by India's National Accreditation Board for Testing and Calibration Laboratories. It has been offering molecular testing five years ago using PCR, Sanger sequencing, FISH, and other technologies. This year it began offering NGS tests that it markets as research-use only, and is aiming to market them for clinical use in 2015.

Parth Shah, a research scientist at Supratech Micropath, told Clinical Sequencing News that the lab has developed preimplantation tests and cancer panels on Thermo Fisher's Ion Torrent PGM. The lab currently has one PGM machine and runs around 20 to 30 tests per month, he said.

Shah said that the lab decided to go with the PGM over an Illumina system because of the cost and also because an Illumina system would have had too much throughput for the lab's needs. "In India, people are much happier with small panels," he said. And with the higher throughput of the Illumina system, the lab would need many more samples in order to fill the machine, which would have made turnaround times prohibitively long. Additionally, Shah said that Thermo Fisher has more clinical support available in India.

Thus far, he said that the lab has experienced some issues in calling variants in homopolymeric regions, but added that chemistry improvements are helping to resolve those issues. In addition, he said that the lab can now readily identify those errors, so the overall sensitivity of the assays is still very high.

The first NGS test the lab developed was a preimplantation aneuploidy test, which uses low-coverage whole-genome sequencing of single cells to look for chromosomal aneuploidies in embryos before they are implanted, Shah said. The group plans to publish its method in a journal this year.

Currently, most insurance plans in India do not cover these tests, Shah said, so patients pay out of pocket. The PGS test cost varies depending on how many embryos are screened, but is around $250 per embryo when eight are screened. The lab returns information about chromosomal aneuploidies, but will not give sex chromosome information unless there is an abnormality.

Supratech is also developing preimplantation genetic diagnostic tests for blood disorders like hemophilia and beta thalassemia, as well as for glycogen storage disorders, urea cycle disorders, muscular dystrophy, neuromuscular disorders, and lysosomal disorders.

These panels include between 20 and 30 genes each, said Shah, and can be used for both preimplantaion or newborn diagnoses.

In the realm of cancer, Shah said that the lab is offering a comprehensive AmpliSeq panel of around 400 genes, a 50-gene hotspot AmpliSeq panel, and smaller panels of fewer than 10 genes for specific types of cancer like lung or colon.

For the comprehensive and hotspot panels, the lab started with the Ion AmpliSeq kits and then "customized them for our own purposes," Shah said, adding or subtracting genes as necessary. "Where we find that the AmpliSeq panels are not sufficient, we develop our own amplicons and make them a part of those panels in order to expand them," he added.

In addition, in order to cater to oncologists in India that are not familiar with broad genomic testing and want to just analyze a few genes, Supratech offers a targeted lung cancer panel and a targeted colon cancer panel that each have only a handful of genes — fewer than 10 — but are sequenced to 1,000-fold coverage. Prices for the cancer panels also vary, but Supratech charges around 20,000 rupees ($328) for the hotspot panel, Shah said.

Thus far, Shah said the majority of tests run have been for inherited diseases and PGS. For cancer, the benefits are less obvious, and the test results more difficult for the clinician to interpret. In addition, a Sanger-based EGFR test will cost around $120, Shah said, so although a 50-gene panel test is more efficient from a cost-per-gene standpoint, physicians are not as interested in those other genes.

However, Shah said, he expects that cancer test volumes will pick up as more data accrues around the actionability of cancer genes and as physicians become more comfortable with the data.

In the future, the lab plans to develop assays to detect pathogens from body fluids, Shah said. He added that scientists from Supratech are working to validate a protocol established by a group at the University of California, Los Angeles on pathogen detection. In addition, he said the lab is working to develop a cancer panel for fusion transcripts. Currently, the lab has several PCR-based assays for gene fusions in cancer, but would like to incorporate all of those into an NGS assay.

NGS (next generation sequencing)-based preimplantation genetic screening has become another powerful platform in IVF realm. 

Array based	Sequencing based
It provides   vastly improved resolution and detection capability and more consistent results compared to traditional karyotyping, the American College of Medical Genetics (ACMG) recommends microarrays as the first approach for postnatal cytogenomic screening.	NGS offers complimentary chromosome aberration testing that can add additional mutational and higher-resolution data beyond the CNV (copy number variation) and AOH (absence of heterozygosity) capabilities of microarrays. With higher levels of functional resolution, NGS is ideal for measuring chromosome inversions, balanced translocations, and disease-associated point mutations.

(reference: Illumina. inc)