PGD METHODS

Currently, the following methods are employed for genetic investigations:

FISH (fluorescence in situ hybridization) molecular cytogenetic method was developed to reveal certain DNA sequences of a chromosome and can be applied in case of both metaphase and interphase nuclei.

The method is based on the binding of a DNA probe labeled with a fluorescent dye to the DNA of a chromosome of the examined sample.

Fluorescence in situ hybridization (FISH)

FISH can be used for the detection of quantitative and qualitative chromosomal aberrations such as deletions (including microdeletions), translocations, reduplications and aneuploidies. In case of interphase chromosomes, FISH serves as a diagnostic method for trisomy 21, 18 and 13 or aberrations of sex chromosomes.

The method enables:

• Screening of preimplantation embryos for the most frequent aneuploidies of chromosomes 8, 9, 13, 14, 15, 16, 17, 18, 21, 22, X, Y;
  
• The determination of embryo genetic gender;
  
• Embryo PGD in carriers of chromosomal rearrangements.
  

The limitations of the method are:

• The method cannot identify 24 types of chromosomes (FISH diagnosis involves a limited number of probes allowing the detection only of the most common chromosomal anomalies);
  
• The immense dependence on the quality of fixation (signal overlap, a low efficacy of hybridization, disruptions in the blastomere fixation process, up to 6-10% of embryos without diagnosis);
  
• The method cannot rule out mosaicism;
  
• The diagnosis is performed for a specific pathology and cannot rule out an anomaly in the regions that are not examined;
  
• The method cannot tell a balanced embryo from a normal one (FISH analysis is used to identify embryos with unbalanced abnormalities. Balanced types (as in a parent with a translocation) cannot be identified using this method. Hence among embryos recommended for transfer there can be both normal embryos and embryos carrying a chromosomal rearrangement);
  
• The method cannot detect uniparental disomy;
  
• The method cannot be combined with other PGD types (for monogenic diseases, HLA).
  

At present, real-time PCR is actively employed for PGD.
The method utilizes the general principles of PCR consisting in a doubling (amplification) of a DNA region between primers with the help of a fragment of DNA polymerase. As a result, the number of fragments increases in a geometric progression. After 30-40 cycles, their number exceeds a few milliards, which enables their detection by means of various methods. The main difference is that the amount of the amplified DNA is measured in real time after each cycle of amplification, and amplicons are identifi
The method cannot identify 24 types of chromosomes (FISH diagnosis involves a limited number of probes allowing the detection only of the most common chromosomal anomalies);
The immense dependence on the quality of fixation (signal overlap, a low efficacy of hybridization, disruptions in the blastomere fixation process, up to 6-10% of embryos without diagnosis);
The method cannot rule out mosaicism;
The diagnosis is performed for a specific pathology and cannot rule out an anomaly in the regions that are not examined;
The method cannot tell a balanced embryo from a normal one (FISH analysis is used to identify embryos with unbalanced abnormalities. Balanced types (as in a parent with a translocation) cannot be identified using this method. Hence among embryos recommended for transfer there can be both normal embryos and embryos carrying a chromosomal rearrangement);
The method cannot detect uniparental disomy;
The method cannot be combined with other PGD types (for monogenic diseases, HLA).

This technique allows the determination of point mutations and of the quantitative content of DNA in a sample with a high degree of confidence. It is also used to determine the level of gene expression.

PGD for specific monogenic diseases is performed if there have been cases of such a disease in the familial history. First, parents are preliminarily tested for a mutant gene, whereafter the IVF procedure is carried out in combination with PGD so that to identify this very genetic defect in the embryos. This method enables a fast detection of the most common aneuploidies. The accuracy of the method is comparable to that of FISH.

The advantages are:

• The possibility of performing all types of PGD;
  
• The possibility of detecting the level of mosaicism of about 20-30%;
  
• The possibility of conducting PGS and PGD simultaneously;
  
• A relative cheapness and the possibility of simultaneous analysis of a much larger number of embryos;
  
• The speed at which investigations are conducted;
  
• The economy of time and effort.
  

The disadvantages are:

• the method can only identify specific chromosomal changes and cannot detect abnormalities beyond the region between primers;
  
• A lot of effort put into the preparatory stage;
  
• The absence of commercial sets.
  

The modern alternative to FISH is the method of microarray-based comparative genomic hybridization.
aCGH is a competitive in situ hybridization of two genomic DNA libraries: one obtained from the analysed tissue/single cells, the other one serving as a control library (from peripheral blood lymphocytes of a healthy person), derived in equal proportions and labeled with different fluorochromes on the membrane/microarray where thousands of locus-specific DNA fragments of a healthy person are located.

The use of microarrays has conditioned the high resolution, the objectivity and the high informative value of obtained results.

A microarray containing about 40000 samples

This method allows testing all 24 chromosomes (1-22, XY) simultaneously as well as analysing multiple genes and whole genomes in a single experiment. The risk of a false positive and a false negative result in case of CGH is determined to be no more than 1%.

The possibilities of aCGH:

• The identification of numerical chromosomal abnormalities (aneuploidies), including the mosaic ones;
  
• The detection of structural chromosomal aberrations:
  

• Unbalanced translocations;
  
• Microdeletions/microduplications;
  
• Unbalanced subtelomeric rearrangements;
  
• Marker chromosomes;
  
• The possibility of detecting dysomias.
  
  

The limitations of aCGH:

• The results of the investigation do not rule out the existence of balanced rearrangements:
  

• Reciprocal translocations;
  
• Inversions;
  
• Robertsonian translocations;
  

• They do not rule out unbalanced rearrangements beyond the resolution level:
  

• Deletions and duplications below the limit of sensitivity;
  
• Point mutations.
  
  

If rearrangements are detected at the limit of sensitivity of the method, FISH and/or PCR verifications are due.

• A limited possibility of detecting polyploidy and mosaicism (from 30%).
  

The effectiveness of the method depends on the quality of baseline material, the significance of deviations as well as on the disorders and special features of whole genome amplification (WGA).
New generation sequencing is a technique for DNA and RNA nucleic acid sequence identification aime at obtaining a formal description of its structure.
It turned out that the classic Sanger sequencing (the chain termination method) was not appropriate for a fast routine sequencing of human genomes for clinical purposes. Hence, the need in new whole-genome sequencing technologies arose.

NGS (New Generation Sequencing) employs the most modern methods of genetic information reading for embryo testing and gives new diagnostic possibilities. The method provides comprehensive information about the DNA of an embryo as regards diseases or genetic mutations.

The advantages are:

• The possibility of testing all 24 chromosomes simultaneously with unprecedented accuracy;
  
• An increased accuracy of mosaic aneuploidy diagnosis;
  
• The possibility of telling a balanced embryo from a normal one;
  
• The possibility of detecting uniparental disomy (the analysis of parental samples);
  
• The possibility of identifying haploids and triploids;
  
• The possibility of identifying micro and macro rearrangements (from 10 bp);
  
• The possibility of simultaneous diagnosis of monogenic diseases;
  
• In case of PGD NGS, an additional molecular code is assigned to each sample, thus ruling out the possibility of a mistake;
  
• The low price of expendables.
  
  

The limitations are:

• Tetraploids cannot be identified;
  
• The high cost of the equipment.
  
  

In the course of genome sequencing, we obtain information about the whole DNA in 23 chromosomes of human cells containing about three milliard nucleic acid pairs. This information includes sequences of all genes (20 thousand) as well as non-coding regions (that comprise the major part of the human genome and participate in gene regulation in particular). Thus a single genome investigation (sequencing) yields a massive array of information that will be used in clinical practice in combination with both already existing data and data that will be obtained by scientists in the course of scientific advance in the future.