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CGH – Comparative Genomic Hybridization

In the early 1990s, a method known as comparative genomic hybridization (CGH) was developed for the evaluation of chromosomal number in tumors. In 1996, this method was developed and applied to embryos for the first time. CGH allows for the evaluation of every chromosome in a cell. This has been termed comprehensive chromosome screening and it is being used in IVF cycles today.

 

How CGH for IVF works

To understand how CGH works, you first have to understand a little about DNA. The nucleus of a cell contains the genetic information in the form of DNA. DNA are long dual chains of individual chemicals called nucleotides. There are four nucleotides which are commonly known by their one letter abbreviations: A (adenine),C (cytosine),G (guanine), and T (thymine). The information in DNA is “coded” by the specific pattern of these nucleotides in the chain.

dna.jpgThe two chains of the DNA (called helices because of the way they twist around each other) are held together by chemical bonds between the nucleotides. The “A” attaches to the “T” and the “G” attaches to the “C”. Thus, one strand or chain of DNA is “complementary” to the other strand. See the example below.

 

 Strand 1 – ATTACGCATA

Strand 2 – TAATGCGTAT

 

Notice that on one strand of DNA, the “A” always has a “T” opposite. A “C” always has a “G” opposite and so on. When two strands are attached to each other, they are said to be hybridized. The process of separating the two strands from each other is called denaturation. The process of sticking them together is called annealing.

 

Lets take an easy example. Lets say we have a sample chromosome with two DNA strands.

1) Heat it up until the two strands separate

2) Next use a chemical called an enzyme to cut the strand into several pieces

 

3) Attach a chemical to the pieces that glows fluorescent green

Next we take DNA from a well characterized source that we know is normal. We put the DNA through the same process but instead of labeling it green, we label it red. Finally, we take a chromosome and isolate the strand that is complementary to the DNA strands we just labeled.

 

We then add the green “sample” DNA fragments and the red “reference” DNA fragments to the chromosome. The green “sample” DNA and the red “reference” DNA will anneal to their complementary areas on the DNA within the chromosome. The chromosome becomes coated with thousands of red and green DNA fragments (See picture below). If the chromosome is then inspected under the microscope there will be equal amounts of both green and red fixed to the chromosome.

 

 Chromosomes analysis by CGH

 

 Now lets take another example. Lets say we have a cell that has three copies of a chromosome we are interested in studying. We go through the same process as before but now there are three times more green DNA fragments than there are red DNA fragments. When all the fragments are added to the chromosome, it will appear to be more green than red.

 

In general, the ratio of green to red fluorescence along the length of each chromosome reveals the relative number of chromosome copies in the test sample compared with the reference. An excess of green fluorescence on a specific chromosome is indicative of more chromosomes, whereas an excess of red fluorescence is indicative of fewer chromosomes.

 

In this picture, most of the chromosomes have equal amounts of red and green color. Chromosomes 13 and 21 are more red in color indicating the sample had less of the DNA from those chromosomes. In fact, this was due to one copy of the chromosome pair being missing, a condition known as monosomy. The X chromosome is more green indicating twice as many chromosomes and the Y chromosome is red indicating it is missing. This tells us that the embryo is female.

 

Amplification

 The CGH method requires 1 mg of DNA whereas a single cell contains about one billionth of that amount. So in order to do CGH, our scientists must first increase the amount of DNA. This process is called amplification. The most widely used method for doing this is called the polymerase chain reaction (PCR).

 

Advantages and Disadvantages of CGH

The previous method for assessing the number of chromosomes in an embryo is known as FISH. FISH technology was able to analyze only 9 or ten chromosomes in a cell. Human beings have 23 pairs of chromosomes so FISH left a considerable portion of the chromosomes untested. CGH allows us to study every chromosome in a cell.

 

FISH required extensive preparation of the cells before analysis. CGH only requires extracting and amplifying the DNA.

 

The main disadvantage of CGH was the length of time it took to get a result. IVF programs transfer embryos to the uterus between three and five days after fertilization. In the past, this was not sufficient time to get results with CGH. This required that the embryos be frozen and transferred at a later time. This was a successful strategy and babies have been born as a result of this method. It never became very popular however, because the techniques for freezing embryos was not efficient and therefore some embryos would not survive the freeze thaw process. More recently, a new technique for freezing embryos known as vitrification allowed for much higher efficiency and therefore led to renewed interest in CGH.

 

Finally, there is one type of chromosome problem that is missed with CGH. This occurs when all the chromosomes in an embryo have an extra copy. So instead of having two copies of every chromosome, you can have three copies (called triploidy) or four copies (tetraploidy) etc. The “polyploidies” do occur in embryos and can be responsible for failed implantation of an embryo or miscarriages. Polyploidies do not result in live born babies with birth defects.

 

CGH however, remained a complicated technique for most laboratories to perform. This led to the development of a newer form of CGH called CGH microarray.

 

CGH Microarrays

With microarrays, the green and red DNA fragments are allowed to hybridize to a large number of specially prepared DNA “probes” affixed to a microscope slide rather than a chromosome. Each probe is specific to a different part of a chromosome and occupies a discrete “spot” on the slide.

Microarrays have an advantage over conventional CGH in that the evaluation of the green or red fluorescence is simple and easily automated with a computer and software. This dramatically reduces the time required for hybridization and analysis. Currently, IVF1 uses a system that allows us to get results in 24 hours! This enables to biopsy an embryo at any stage of development and still get results in time to do a fresh embryos transfer.

In the example below, the cell being analyzed has the correct number of chromosomes for every chromosome except number 10. There are three copies of chromosome 10 (a trisomy) instead of two.

 

Trisomy 10 on a CGH Microarray