Comparative Genomic Hybridization (CGH)

A significant proportion of unsuccessful pregnancy is caused by numerical chromosome imbalance or aneuploidy supported by the evidence that >50% of fif irst trimester abortuses are aneuploidy. FISH was introduced to reveal aneuploidy in embryos in order to improve the pregnancy success. However, whether PGD using FISH technique will improve pregnancy rate of IVF treatmentcycle is still controversial due to its limitation in the number of chromosome studied. This is limited by the number of distinct fluorochromes which are available to label the DNA probes.

The more probes applied simultaneously the more overlapping signal will occur. Though signal overlapping can be partly overcome by using computerized imaging, signal overlapping of the same fluorochrome cannot be delineated.

A common approach is to use FISH for 5-6 chromosomes simultaneously and then once these results have been analyzed, the signals are washed-off and re-probing the nucleus for other 2-4 chromosomes is done. This cannot be done repeatedly as DNA degenerates and signal effiiciency is reduced with each round of re-probing. Recent evidences have shown that aneuploidies are not limited to particular chromosomes but can occur to any chromosome which can result in pregnancy wastages or infertility so there is an interest in analyzing every chromosome in a single blastomere for PGD. Conventional karyotyping using G-banding technique for the analysis of metaphase chromosomes has been reported but is too ineffif icient at the single cell level to be applicable to PGD.

To date, the most successful approach for complete karyotyping of single cells is comparative genomic hybridization (CGH). CGH has originally been applied to the detection of chromosome copy number changes in tumors. CGH does not require the preparation of chromosomes from the sample and in a single experiment reveals the copy number of every chromosome segment larger than 5-10 Mb in size depending on technique used.

Originally, published CGH protocols require 0.2–1.0 μμg of sample DNA whereas a single cell contains only 6 pg. This is later overcome by using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to amplify the whole genome before the CGH analysis. CGH works by a comparison of the sample or test DNA with known normal or reference DNA. Test DNA and reference DNA are labeled with a green and red f lfluorochrome respectively and then simultaneously applied to either human male metaphase chromosomes or microarray slide (fiF igure 1).

The green:red flflf luorescence ratio would normally be 1:1 for each euploid chromosome. If the test cell is monoploidy for a particular chromosome/segment then there would be a decreased ratio of green:red flflf luorescence in that particular chromosome/segment. If the test cell is polyploidy for a particular chromosome/segment then there would be an increased ratio of green:red flf lfluorescence in that particular chromosome/segment. The advantage of CGH is that besides analyzing the copy number of all chromosomes, it can also reveal chromosome microdeletions and/or microduplications.

Microarray-based CGH provides a higher resolution than CGH onto metaphase chromosomes which is limited to about 5 Mb. In order to perform PGD-CGH, the embryos should be cultured to blastocyst stage as blastocyst biopsy provides higher number of DNA copies for whole genome amplifif ication compared to the biopsy of cleavage stage embryo. Moreover, the embryo freezing/thawing protocol must be effif icient as all embryos need to be frozen after the biopsy and be transferred in the subsequent frozen/thawed cycle as the PGD-CGH result is generally available after 2-3 weeks from the day of biopsy.

In conclusion, CGH offers a new paradigm of pre-implantation genetic diagnosis to allow clinicians to offer aneuploidy testing of all chromosomes in pre-implantation embryos with an expectation of improving IVF success.

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