NGS – next-generation sequencing

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NGS – next-generation sequencing 2016-11-10T12:49:25+00:00

NGS – next-generation sequencing

PGD NGS method (preimplantation diagnosis based on next-generation sequencing platform) uses the most up-to-date techniques of human genome sequencing (reading of genetic information) for testing embryos and opens new diagnostic possibilities.

It is used as a part of in vitro fertilization and provides comprehensive information concerning embryo’s DNA for diseases or genetic mutations. It provides physicians with a unique opportunity to help couples who are exposed to an increased risk of genetic abnormalities in the foetus. This is the first solution of this kind in the world.

To date, in case of appropriate indications it was possible to carry out preimplantation genetic diagnosis with the use of FISH or microarray (aCGH) techniques. Both are effective, but they have their limitations.

PGS-NGS 360°™ – Pre-implantation Genetic Diagnosis using Next Generation Sequencing:

  • Possibility of testing all 24 chromosomes simultaneously with unprecedented accuracy – at a level of 99.999% (Q50 by Phred Quality Score)!
  • Possibility of testing for a variety of monogenic diseases with known genetic background!
  • Credible result
    Within PGS-NGS 360°™ each sample is assigned an additional molecular code, eliminating the possibility of any error since the moment of collecting material from the embryo. In addition, the test credibility is enhanced by a direct connection of DNA reading with the obtained information. Owing to chips within semiconductor technology in the world of DNA molecule is directly reflected in the electronic signal.

  • Embryo safety – reducing the number of biopsies for the diagnosis
    Usually just one embryo biopsy is sufficient to obtain a reliable result. In the case of existing methods, occasionally, the biopsy and the test had to be repeated.

  • The possibility of combining the tests of chromosomes and single-gene diseases in a single test!
    Until now it was not possible to combine chromosomal aneuploidy and monogenic diseases tests. Today, due to NGS it is possible, just one embryo biopsy will suffice.

  • NGS method is considered to be referential for all the other techniques
    DNA sequencing is referred to as the reference method (model for others), mainly due to the direct nature of the genetic material reading. Other methods (FISH and microarrays) use markers and light as change markers and indirectly test the genetic material. For this reason, these methods are currently being abandoned for the use of NGS.

  • Lower costs of test
    The special design of the PGM apparatus allows for a significant reduction in the cost of tests in comparison with existing methods. Due to which, we increase the availability of PGD preimplantation genetic diagnosis for patients.

The main advantage of the new PGS-NGS 360°™ INVICTA technology is its extraordinary sensitivity and precision. Test design is tailored to the needs of a given patient.

PGD/ PGS-NGS diagnostics is applied within the in vitro fertilisation procedure. First a woman goes through the stage of hormonal stimulation; then from her ovaries stimulated to more intensive activity the material is collected from which eggs develop (the so-called ovum pick up, or OPU stage). After the collection, in the laboratory of embryology these cells are subjected to appropriate preparation and fertilised with the sperm of the patient’s partner. From these cells, embryos develop. After 5 days, blastocyst embryos are sampled for testing. The specimens of material are sent to the INVICTA Molecular Biology Lab where the material is prepared and analysed in a special device. Only the embryos without genetic abnormalities are transferred to the patient’s uterus, thus the couple gains higher chances of having a healthy child without predispositions to syndromes resulting from chromosome number abnormalities or mutations in single genes.

In INVICTA it is possible to collect material from 5- or 6-day old blastocyst embryos – which allows performing the analysis of trophectoderm cells. We are one of very few clinics in the world which provide their patients with such possibility.

INVICTA operates its own Molecular Biology Lab; therefore there is no need to send the material collected from embryos to another facility which performs such tests. This significantly reduces the time of waiting for results and increases the effectiveness of the whole procedure. Where the material is collected from blastocyst embryos for the purpose of testing for aneuploidies, embryos may need to be frozen.

Yes. Currently INVICTA is the only Clinic in the world which performs these tests on a routine basis.

Much depends on the individual situation of patients – in 99% of cases the test allows to determine whether the embryo is healthy or at risk of developing a genetic disease. Chances of giving birth to a healthy child depend on the number of healthy embryos, patient’s health condition and age, and on the course of pregnancy. PGD diagnostics is the only solution permitting the detection of DNA abnormalities at such an early stage of development. Transfer of a healthy embryo to the patient increases the efficacy of the IVF program.

A standard diagnosis covers all 24 chromosomes (22 chromosomes and 2 sex chromosomes– X and Y) and is performed for aneuploidies (chromosome number abnormalities), which in most cases result in the death of embryo. Others cause serious genetic defects, including Down, Turner, Edwards, Patau or Klinefelter syndromes. This method is also used in screening for mutations in single genes. The diagnostics has to be performed earlier in future parents in order to determine the risk in their case. A couple diagnosed with mutations threatening the foetus is referred for PGD testing adapted to this result.

In the case of couples at risk who decide to perform PGD diagnostics it is always recommended that they perform prenatal tests during pregnancy. It is worth remembering that these tests allow to exclude both genetic abnormalities and DNA-independent foetal development defects.

The existing PGD diagnostics used mainly two techniques – FISH (technology based on fluorescence microscopy and used in testing for major genetic defects) and microarrays (so-called aCGH used in chromosome testing). The latest test method is next generation sequencing (NGS) of DNA.

  • PGS-NGS is an extremely sensitive method. The analysis includes several hundred thousand readings for each chromosome. Methods like aCGH (microarrays) enabled analysing only several dozen points on a chromosome and required knowing the target sequence, thus providing significantly less information. Now the analysis of one DNA fragment can be performed several hundred times providing extremely reliable and accurate results.
  • DNA sequencing is the method of the so-called direct genetic material reading; therefore it is referential for all other methods. In other words, every other method should be confirmed by sequencing. Next-generation sequencing makes this difference even more prominent and globally becomes an unrivalled model in testing huge amounts of DNA at the same time.
  • Next-generation DNA sequencing (NGS) is referred to as the reference method (model for others), mainly due to the direct nature of the genetic material reading. All other methods (FISH and microarrays) use markers and light as change markers. They also require the use of advanced optics which is prone to failure and not always provides reliable results. For this reason, these methods are currently being abandoned for the use of NGS.
  • In analysing the number of chromosomes, each chromosome is counted several dozen hundred times which accounts for the unsurpassable reliability and precision of PGS-NGS; any imprecision in a single reading won’t affect the test results as it is the case with aCGH technique where each chromosome is counted at most a dozen or so times. NGS is also unrivalled as regards testing our genes. Each gene is read hundreds of times, thus minimising the risk of misreading as compared with other techniques where the information is read from 2 to less than 10 times.
  • In addition, the test credibility is enhanced by a direct connection of DNA reading with the obtained information. Owing to chips within semiconductor technology in the world of DNA molecule is directly reflected in the electronic signal. The situation is different with indirect techniques where information reading does not originate directly from DNA but from light phenomena and their recordings. Obviously this introduces additional source of error and reduces the test reliability.
  • Within PGS-NGS each sample is assigned an additional molecular code, eliminating the possibility of error since the moment of collecting material from the embryo. Preparation of a sample for testing takes place without moving it out of the vial in which it was placed at sampling. In other techniques a tested sample is indistinguishable from others, therefore a human error plays more significant role, in particular where there is large amount of sample manipulations in multiple-stage tests.
  • NGS in semiconductor technology permits reading the entire continuous DNA record, and not only selected places far from each other as it is the case in microarray technology. We can compare it to the popular game of “joint the dots” where instead of the full picture an outline appeared depending on the guessing person’s idea. NGS supplies us with a high-definition photo, while microarrays – only a dotted piece of paper.
  • It is possible to perform a comprehensive diagnosis of frozen embryos without the prior diagnosis of the genetic background. If a couple completed the IVF program and learned about its predisposition to genetic diseases only after that, it may ask for performing the testing of embryos deposited in the bank. This way the woman doesn’t have to go through the hormonal stimulation stage again, and the cost of the entire process is significantly lower.
  • It is recommended that future parents have their DNA tests before they start trying for pregnancy. Woman and man alike may be carrying genetic mutations, despite good health. The new method allows determining which of these abnormalities may impact the risk of defect in the child of a given couple. Based on the obtained information, if the risk of disease in foetus is high, the couple can choose to undergo in vitro fertilisation with preimplantation genetic diagnosis and to eliminate the risk. For certain people this is the only chance of having healthy prodigy.

Yes; preimplantation genetic diagnosis is performed only as part IVF procedure – the material for testing is collected during embryo biopsy before a woman actually becomes pregnant.

Prior to taking the decision on performing PGD testing, consultation with a specialist in gynaecology or genetics is necessary. The specialist will confirm whether there are indications for performing these tests in the case of a given couple.

No; only embryos with appropriate morphology are qualified to tests. They need to be of good quality in order to ensure that the material collected for testing is of diagnostic quality and that the remaining cells have good chances of further development. Embryos of poor quality are not qualified to biopsy or to further PGD testing. This is also compliant with the guidelines of the European Society of Human Reproduction and Embryology (ESHRE).

It is worth remembering that couples are qualified to PGD testing for specified reasons. The indications include risk of passing on genetic diseases to prodigy, age of future parents, recurrent miscarriages or abnormal karyotype of female and/or male partner. Sometimes a genetic defect impairs the development of the fertilised egg to such extent that the result is pre-clinical (asymptomatic) loss of pregnancy or clinical miscarriage. In case of every woman there is a statistical risk that in consequence of fertilisation an embryo or foetus with significant chromosomal defect will develop in her uterus. In the case of couples with the problem of recurrent miscarriages – even if their cause is known – PGS-NGS will increase the chances of achieving normal pregnancy and giving birth to a healthy child.

Every embryo has unique DNA code and is different. If there is a significant risk of foetal genetic defect, PGD should be performed in all embryos (i.e. also in subsequent cycles within IVF procedure). Only this allows identifying potential defects.

Even theoretically this possibility is very limited as it relates only to the pool of genes contributed by parents and not the entire range of a given physical feature. Moreover, in Poland PGD is performed not for the purpose of ensuring the specific appearance or gender of a child. This test aims solely at diagnosing embryos for genetic risks which may contribute to the development of genetic diseases. It is performed with regard to specified medical indications and not for anybody’s whim.