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PGD/PGS Methods of Genetic Analysis

There are various screening techniques laboratories will use with preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). These methods include:

  • Fluorescent in situ hybridization (FISH) is an older technique used most often for PGS
  • Polymerase chain reaction (PCR) is used mostly for PGD, sometimes for PGS
  • Comparative genomic hybridization (CGH) is used for PGS
  • Single nucleotide polymorphism (SNP) analysis is used for PGS

For the last 20 years, PGD and PGS has been mostly performed on cleavage stage (Day 3) embryos after the biopsy of one to two cells, using PCR and FISH for the analysis. Today, there is a move toward blastocyst (Day 5) and polar body biopsy, and CGH and SNP arrays have been introduced clinically for PGS and PGD.

Fluorescent In Situ Hybridization (FISH)

FISH is used for the determination of sex for X-linked diseases, chromosomal abnormalities and aneuploidy screening. When FISH is used to evaluate the genetic make-up of an embryo, the embryos are grown to the Day 3 stage, and a single cell is removed from each embryo. The cells are then attached to a glass slide, packaged and sent to the fertility clinic's genetic testing laboratory partner for evaluation. The FISH technique involves testing fragments of DNA that are specific to each chromosome. Probes (small pieces of DNA that are a match for the chromosomes being analyzed) are placed on the slide with the cell from the embryo and will attach to the chromosome target.

FISH, however, cannot fully access all the chromosomes — a human cell contains 23 pairs of chromosomes, but FISH analysis allows accurate assessment of only 10 to 12 chromosomes in each biopsied cell. This means that many abnormal embryos, incapable of forming a successful pregnancy, remain undetected and may be transferred. Chromosomes that can be analyzed with FISH probes include X, Y, 1, 13, 16, 18, and 21. "The problem with FISH is that you can never analyze more than a selected number of chromosomes at a time," says Houston fertility doctor Randall C. Dunn with Fertility Specialists of Houston. "And the most that ever really came into practical use was 10 at a time."

Polymerase Chain Reaction (PCR)

PCR, sometimes called DNA amplification, is used for the diagnosis of single gene defects, including dominant and recessive disorders. It is a technique in which a particular DNA sequence is copied many times in order to facilitate its analysis. PCR rapidly multiplies a single DNA molecule into billions of molecules. PCR requires sufficient amounts of a pure, high-quality sample of DNA, which is sometimes difficult to obtain from a single cell.

"For PGD testing, we use PCR-based techniques to look at the gene of interest within the DNA sample," says Lauren Isley, Genetic Counselor with Genesis Genetic Institutes. "When building our probe, we identify what are known as polymorphic markers that are associated with the particular genetic change or mutation that runs in the family. If it is appropriate for the case, we also directly sequence the DNA to ensure that our lab can, indeed, visualize the mutation. This work will allow us to perform targeted testing in just 24 hours on the embryo samples."

Comparative Genomic Hybridization (CGH)

With CGH, the embryo nucleus is labeled with a fluorescent dye and a control cell is labeled using another color (ie, red or green). The ratio between the two colors is compared. If the chromosomal analysis shows an excess of red, the embryo nucleus contains an extra chromosome. If an excess of green is apparent, then the embryo nucleus is missing one of these chromosomes. CGH allows genetic specialists to examine all 23 chromosomes and provides a more detailed picture of the entire length of the chromosome, which may detect imbalance of chromosomal segments.

The CGH technique takes approximately 72 hours, which means embryos must be frozen to provide the time necessary to obtain a diagnosis. A newer advanced technique offered in some labs is called array CGH or microarray CGH (mCGH). This is an accelerated CGH protocol providing results in 24 hours for all chromosomes. "Our laboratory uses array CGH to test for chromosome abnormalities," Isley says. "We use data points from DNA within the embryo samples to screen for extra or missing chromosomes."

Using mCGH allows testing of all 23 pairs of chromosomes in an embryo.

Single Nucleotide Polymorphism (SNP) Analysis

SNP is another newer technique that can examine all 23 chromosome pairs. SNPs are single bases, the building blocks of DNA, which can be in a different sequence in different individuals. For SNP microarrays, the technical strategy is a little different than for CGH. Dr. Dunn prefers SNP because it can detect not only copy number changes, but it can detect inversions and translocations.

"Translocation is when, for example, there is a piece of chromosome 15 and a piece of chromosome 12 flipped on each other," Dr. Dunn explains. "Part of 15 is on 12, and part of 12 is on 15. The person themselves will be healthy. The No. 1 thing for them usually is they show up here having recurrent miscarriages, or they even had a baby that maybe made it toward term and then was found to be missing one of those little tail pieces."

There are several advantages to the newer screening techniques (array CGH and SNP), with some of the most important being that 23 chromosome pairs can be analyzed in the embryo, and arrays allow simultaneous testing of all chromosomes at once.


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