Compared to the Sanger sequencing, for example, where it was possible to analyze only a small portion, the modern methods of sequencing (NGS) allow to read the larger part of the genome. The main advantages of this sequencing are shown in the preconception and preimplantation screening, but also the possibilities of prenatal, postnatal and population screenings have increased.

The NGS PGS process step by step

  1. DNA extraction.
  2. Creation of libraries.
  3. Sequencing.
  4. Evaluation of the existing problems.

There is a opportunity to study the genome completely, however it’s an expensive and not always necessary procedure. Targeted research of the eхome or a part of it is used more often. An Exome has 22,000 genes, a clinical exome - 4800 and a part of an exome (partial exome) - 500-100 genes, that may be of interest, while looking for a particular disease.

At the first stage, the material is prepared, then goes DNA splicing, and it’s analysis afterwards.

Preconception screening

For the elimination of hereditary diseases in a child, first of all- it is necessary to be aware of the personal risk of one’s own genome. Considering that each one of us is a carrier of 2-3 mutations, everyone has a risk of having a sick child.

Each disease can be caused by different mutations in a single gene: a section falling out, its duplication, etc. (If we draw an analogy with a book, the error may be in the disappearance of a sentence or of an entire chapter, as well as excessive letters or chapters). And all of these variations need to be found in order to accurately establish the diagnosis. Also it is important to understand, that not every mutation may lead to a clinical manifestation of a disease.

Monogenic diseases and NGS

Cause 20% of the child mortality, 18% (10) of pediatric hospitalizations, and is a tragedy of individual families. Therefore, the introduction of NGS, will help avoid the birth of such sick children.

If we draw an analogy with the Down’s syndrome, the introduction of screening, made it possible to sharply reduce the number of children, born with this diagnosis.

Preconception screening was proposed in the 1960s, but only really started in 2013, with the appearance of sequencing and in 2014-2016 once the procedures became cheaper, the amount of research grew.

The results of one of the researches

In 2015, a retrospective study of more than 2,500 genetic samples (from donors and patients, who during the programs use donor cells, as well as their own cells) was carried out in Spain It found that 5% of the couples, had pathogenetic changes of high-risk on 6 diseases (2% of them were X- linked diseases), 25% had the risk of possible carriage, because of the X-linked changes. Only 7% of the patients did not carry any mutations.

In this research a "Wide" panel was used, which has a number of disadvantages.

  • It includes 623 diseases - 549 genes (OMIM).
  • It makes difficult to interpret the variations, identification of clinically significant or not, the frequency, the dispersion in the database, there is an unknown significance of the variations (just cystic fibrosis alone, has more than a 1,000 significant mutations).
  • Very complex validation (48 samples) from thousands of variations.
  • Pretest and post-test counseling is made difficult, due to the large number of diseases and the unknown significance of individual mutations.
  • Spends a lot of time and money.
  • A shallow depth of research considering the wide range.

Who can be recommended to do preconception screening?

  • Egg cell and sperm donors. The creation of a Cryobank with a low carrier status of genetic diseases.
  • Testing of prospective parents during ART.
  • Testing of prospective parents in the population.
  • Testing of the population (when the procedures are cheaper).

NextGen 21

Developed by NGS clinic, X-linked disease, autosomal dominant and autosomal recessive are included. There is a total of 21 diseases in the panel. The presence of 21 genes simplifies the interpretation of the variations, makes the validation feasible, and the pre- and post-test counseling possible. Deep screening is carried out. Therefore the research panel includes diseases that met the following criteria.

  • High population frequency.
  • A well-known genetic etiology.
  • High penetrance.
  • Well-known pattern of inheritance.
  • The severity of the manifestations.
  • Later phenotypic expression.
  • Guidelines.

The composition of the panel (diseases): cystic fibrosis, phenylketonuria, spinal-muscular atrophy, myotonic dystrophy type I, DMD, Hattington disease, hemophilia A, beta-thalassemia, sickle cell disease, familial adenomatous polyposis I and type II mucopolysaccharidosis type 1, autosomal dominant polycystic kidney disease type I and II, Charcot-Marie-Tooth disease, von Hippel-Lindau disease, Wilson's disease, Gaucher's disease, Tay-Sachs disease, hereditary breast and ovarian cancer type I and II.

Preimplantation screening

Why is a complete procedure chromosome preimplantation screening necessary (PGS-СCS)?

Most embryos are aneuploid and die immediately or before 8 weeks of gestation (except 13, 18, 21, X, Y, 16). Aneuploidy is the most common cause of failure during IVF and the most common cause of miscarriages, embryos meanwhile can have segmental aneuploidy or be polyploid.

Feasibility of techniques PGS-CCS - why is the procedure necessary?

  • Increases the probability of a full-term pregnancy and live birth.
  • Possibility to transfer only one embryo and exclude twins.
  • Possibility to not store aneuploid blastocysts.
  • No transfer of the unnecessary (embryos that will give a sick child or an abortion).
  • Reduce abortions and risks of abnormalities in children.
  • Reduce the time before the birth of the child and the stress. This point is extremely important, because multiple interruptions of pregnancy at it’s early stages or the birth of sick children, increases the cost and the frequency of IVF attempts or, on the contrary, having lost all hope, the woman refuses any further attempts. It is also important to reduce the time before the birth of the child for patients with low ovarian reserve.
  • Reduce the cost of the birth of one child.
  • Prospects of the further development of screening.

History of PGS technology

In 1991-1995. FISH diagnostics appeared and began to take root, but in 2011 the results of a meta-analysis that were published showed that FISH diagnostics, didn’t just not increase the likelihood of having a healthy baby, but also reduced the implantation rate by 39% (the negative impact of a blastocyst biopsy). Today we understand that biopsy of the 3rd day should not be held due to mosaicism.

In 2010-2012. appeared methods qPCR, aCGH, SNP and the examinations of 24 chromosome started, which greatly improved the results of screening.

2013 was the emergence of NGS. Gradually once the cost of the technique became lower it began to be used more actively. Possibly in the future not only trophectoderm biopsy will be carried out, but also blastocentesis, the study of the media for cultivation, possibly not only autosomal diseases will be connected but also monogenic diseases, mitochondrial, as well as the study of telomere length, transcriptome and micro RNA.

Who can be recommended for ASG-CCS?

To date, a consensus hasn’t been reached. There are three points of view.

  1. To perform PGS treatment for older women who have entered into an IVF program, as they have very high risk of aneuploidy.
  2. To perform PGS process for all women, because at a young age there is still a risk of aneuploidy at 30%, and that is a reserve for increasing pregnancy rates.
  3. Do not do PGS procedure for anyone.

Data from various studies on the prospect of PGS in different groups

Among the young patients and the older women and among even older women, the transfer efficiency of the transfer of one tested embryo was almost identical to the efficiency of the transfer of two embryos that were not tested, but in the second group, respectively, the frequency of multiple pregnancies was significant.

In the group of women who have had to recourse to the help of surrogate mothers, the pregnancy rate after the transfer of the tested embryos was 68%, and when transferring an untested two embryos - 38% (here the rate of multiple pregnancies was - 35%, and the frequency of miscarriages- 22%).

In the donor programs the pregnancy rates while using the tested embryo was- 73%, against 46% (without-PGS).

The effectiveness of CCS to date (data of 4 RCTs and cohort 7):higher implant rate and live birth rate, lower frequency of multiple pregnancy and miscarriages.

NGS \ MPS screening for aneuploidy

Sequencing: NGS and screening (testing) for aneuploidy. Validation of the method in 2015.

How does NGS work: fragmentation and amplification of millions of small fragments. An analysis of the number of copies - mono, di, trisomy ...

The introduction of NGS into practice at NGC clinic (Next Generation Clinic) (Reproduction and Genetics)

  • Comprehension of the necessity of transition to NGS in 2013, the purchase of equipment.
  • Development of the rules for trophectoderm sequencing - the source of DNA contamination in 2015.
  • Validation of the method in the aneuploid biopsies aCGH (with the permission from the ethics committee abnormal embryos were used).
  • Verification of NGS on WGA aliquots at mismatch.
  • First clinical results of PGS on 24 chromosomes with NGS and in 2016 - 2-3 starts in a week - 70% of the programs.
  • Creating a screening panel for monogenic diseases of our population with the use of sequencing (NextGen21).

Differences between NGS and aCGH

After various studies were conducted, it was shown that the NGS method is more accurate and selects embryos more clearly- it can detect mosaic embryos unlike aCGH. And when these methods were compared, including the cases with and without PGS, the following results were obtained.

  1. Clinical pregnancy: NGS - 71%, aCGH - 53%, non-PGS - 42.5%.
  2. Pregnancies after 12 weeks: NGS - 66,7%, aCGH - 50%, non-PGS - 34.7%.
  3. Abortion: NGS - 6,7%, aCGH - 5%, no PGS - 15.5%.
  4. Twins: NGS - 0% aCGH - 5%, no PGS - 29.3%.

Why switch from aCGH to NGS?

In 2015 in a research of J.Grifo, 50 abortions were analyzed after transfer of aCGH euploid embryos. 52% of them were abnormal (5% triploids, 47% mosaic). It was concluded that if NGS was used, 54% of the abortions could have been avoided, as well as a huge amount of negative results (among the “no pregnancies” 26% were identified as mosaics with the use of NGS, which aCGH did not find).

NGS selects the embryos more strictly and clearly.

The advantages of NGS are significant: a more precise definition of mosaicism, segmental aneuploidy, more attractive price, which is gradually reducing, and the technique has great possibilities. NGS is inferior to aCGH only in time spent and in complexity.

Mosaicism of NGS (by one chromosome)

Mosaic embryos can be transferred, but only in 2nd or 3rd place, and if only one mosaic embryo was obtained.

If mosaicism detected less than 20%, then most likely, we are talking about the errors of the equipment and a set of euploid cells. Such an embryo can be tranferred.

When mosaicism is 80-100%, most likely, we are talking about aneuploidy, monosomy and trisomy.

Not recommended for transfer: trisomy + 13, 18, 21, 16, 15, 2, 7, X, Y; monosomy X or Y, as well as segmental aneuploidy.

Can be transferred: trisomy +1, 2 - 6, 8 - 12, 17, 19, 20, 22; monosomy.

Outcomes of birth after IVF - existing problems

  • After IVF procedure, in singleton pregnancies the rate of preterm birth and low birth weight infants is higher than in spontaneous singleton pregnancies.
  • When transferring fresh embryos after IVF, the rate of preterm birth is higher than at cryo transfer. This suggests that the level of estrogen, which is formed during the stimulation is not optimal, for the formation of the chorion and placenta.
  • With IVF the rate of birth defects increases.
  • A review of 38 studies of somatic pathologies of children after IVF showed that there is a higher risk of non-specific infections, parasitic diseases, genitourinary diseases, epilepsy, and seizures (perhaps due to LBW) and asthma. Cancer risk is not confirmed, but the risk is higher by 3 times for specific diseases - retinoblastoma and leukemia. Perhaps these facts are associated with higher anxiety of the patients after IVF and a large number of diagnoses.
  • Complications after IVF: multiple pregnancy (frequency in Russia - 23%, but in the population should be no more than 5%); Ovarian hyperstimulation syndrome (a complication that should not be there at all!); bleeding; ectopic pregnancy.

IVF though CMI

IVF in the CMI system - it's not really an effective appointment, because often it is apponted to women who have no indications for the procedure or they are hopeless. This way in the clinics, the patient that are really in need of IVF will not be accepted (so as to not reduce the performance), and the frequency of multiple pregnancies is too high due to the pursuit of CNB rates, or because of lack of cryo programs.

PGS Efficiency

IVF - is not an ideal procedure and PGS can reduce the incidence of complications (multiple pregnancy, ovarian hyperstimulation syndrome), reduce the time till pregnancy and childbirth, exclude the transfer of aneuploid embryos, etc. But it is important to understand that PGS - is the testing of the blastocyst trophectoderm and not the treatment of aneuploidy.


Do you consider it necessary to introduce PGS into the CMI system for carriers of chromosomal abnormalities?

Yes, it would be a reasonable solution, because the procedure is expensive and not all couples can handle it financially.

What is the Panel Next 21 Cost? How to convey to the obstetricians the necessity for PGS?

The cost is about 29 000 rubles. The reluctance to use PGS has more to do with financial reasons, but gradually the procedure is being implemented into practice more often.