Diagnóstico genético pré-implantacional

Cuzzi, Juliana FabriciaVulcani-Freitas, Tânia MariaMotta, Priscila Cristina RodriguesHassun Filho, Péricles Assad

Background: The ranks of patients seeking preimplantation genetic diagnosis (PGD) to identify embryos with monogenic disorders like cystic fibrosis or thalassemia are growing rapidly. Even so, the most common indication for preimplantation embryo testing remains the risk of chromosomal imbalance. In most cases, the PGD strategy employed for chromosomal testing involves biopsying a single cell (blastomere) from each embryo at the 6 to 10-cell stage, 3 days after fertilization. The cell is placed on a microscope slide, fixed, and then subjected to cytogenetic analysis. While the biopsied cell is being analyzed, the rest of the embryo is maintained in culture. Most infertility clinics prefer to transfer the embryos no later than day-5 post fertilization. Consequently, PGD methods need to be extremely rapid, providing a result in less than 48 hours. Review: Most chromosomal PGD protocols employ fluorescence in situ hybridization (FISH). This approach involves the hybridization of chromosome specific DNA probes, labeled with different colors, to nuclei spread on a microscope slide. The method is rapid, performs equally well whether applied to interphase nuclei, and permits enumeration of up to 10 chromosomes per cell. Initially, the PGD for aneuploidy was envisioned that most of the patients seeking PGD for aneuploidy would be those who carry a chromosomal rearrangement. Couples in which one of the partners carries a chromosomal rearrangement frequently experience miscarriage or bear children due to chromosome imbalance. However, in recent years the vast majority of patients requesting PGD for aneuploidy have in fact been chromosomally normal individuals undergoing IVF. Early methods employed five FISH probes for PGD, focusing on the chromosomes most often found to be abnormal in prenatal samples (13, 18, 21, X, and Y). Aneuploidies for these chromosomes are sometimes compatible with survival to term, leading to aneuploid syndromes. This strategy was successful in reducing the number of such syndromes, but a statistically significant improvement in embryo implantation could not be shown. Later PGD studies expanded the number of chromosomes assessed to eight. This was achieved by performing two sequential rounds of FISH analysis, assessing five chromosomes in the first round and three more in the second. The three new chromosomes added to the PGD screen (15, 16, and 22) are frequently found to be aneuploid in miscarriages. The eight-chromosome PGD protocol led to a doubling of embryo implantation rates in two separate studies and reduced the number of spontaneous abortions. Although this problem can be partially overcome by performing sequential FISH experiments on the same cell, the accuracy of the method declines with each additional hybridization. The good news is that there is a method related to FISH­comparative genomic hybridization (CGH)­ that can detect aneuploidy that affects any chromosome within a sample. In addition to offering comprehensive detection, CGH can be used on interphase cells. Conclusion: In summary, the use of FISH for the purpose of PGD has already improved IVF outcome for several groups of infertile patients, including women aged 37 and above, those with recurrent miscarriages, women with a previous comprehensive screening for aneuploidy will likely further increase the benefit of PGD to these patients and maybe even a broader range of patients. The widespread availability of CGH for PGD is nearly at hand, but while we await final refinements and validation, PGD strategies can still be improved by making the best use of current methods and reassessing the chromosomes selected for screening.

1. aneuploidy
2. FISH
3. PGD
4. CGH