FISH (Fluorescent in situ Hybridization)

As mentioned before, genetic material for analysis is limited to one or two structures (polar bodies, one or two blastomeres). Furthermore, polar body biopsy is not applicable if the genetic disease(s) in question is (are) transmitted by the man. Direct chromosomal analysis is performed exclusively in the first or second polar bodies because, in more precocious cleavage stages (6 to 8 cells) it is rare to obtain metaphase chromosomes. FISH technique, developed only in the last decade, allows the determination of the specific chromosome number, by counting specific signs in the interphase nuclei. This technique is highly specific and has high accuracy. It can diagnose aneuploidy and balanced translocations.

PCR (Polymerase Chain Reaction) For the detection of single gene diseases

The PCR technique revolutioned DNA analysis. The procedure involves repeated DNA amplifications in order to obtain na adequate number of copies for analysis. The use of this technique allows us now to detect mutations in a single gene, in polar bodies or blastomeres. Amplified DNA can be submitted to mutation analysis by various methods, and method selection is determined by the characteristics of the mutation to be investigated.

Although the list of diseases possible to be investigated by PCR-PGD is growing fast, there still are many problems inherent to the technique. DNA contamination control is crucial. Even very small quantities of a strange contaminating DNA have adverse effects on the method, since this material would be amplified alongside with the original template of the biopsied material. Techniques necessary to produce a reliable and “clean” result must be rigorously maintained, na isolated place is pre-requisite to establish a PCR laboratory.

Other problems involve the possibility of preferential allele amplification, or even worse, allele dropout (ADO). Recent attempts to reduce ADOs have had promising results. Nevertheless, it remains obscure if complete ADO elimination is possible.

Cystic Fibrosis and Sickle Cell Anemia are the two most commonly identified diseases by PCR in the PGD cases. After analysis and PCR amplification, heteroduplex formation and restriction enzyme digestion can be performed to to obtain complementary information. Furthermore, whole genome amplification has the potential to allow genetic analysis of multiple loci.

FUTURE PERSPECTIVES

PGD procedure involves new and challenging clinical techniques and, for this reason, it is probable that it remains in research phase for many years. On the other hand, in a near future, PGD will become more important and more widely used, for the following reasons:

1. Demand for PGD is constantly growing. Some forms of male infertility that may be due to genetic disease (like congenital bilateral obstruction of the vas deferens) are now routinely treated by ICSI. The risk of transmitting genetic disease has become bigger, from the moment that normal fertilization barriers have been overcome by technology. The use of PGD will identify these diseases and prevent their transmission. Besides, the Human Genome Project is finished. So, about 25,000 human genes have been identified and sequenced, and their pathogenic relation are being clarified. This knowledge will provide basic information for future PGD application.

2. PGD technique refinement will improve biopsy efficiency and, therefore, will enhance sensitivity and accuracy for FISH and PCR, minimizing false positive and false negative results. With the laser technology, the opening in the zona pellucida will be a simple operation and the risk for oocyte and blastomere damage will also be reduced. Furthermore, FISH and PCR instruments are getting better, allowing us quicker and more accurate diagnoses. We can anticipate that, in the future, one only blastomere will be identified to exclude potential contamination.

3. Improvment in assisted reproduction technologies, particularly in understanding gametogenesis and embryogenesis will bring direct and indirect effect to PGD. The optimization of pre-embryo culture media, for instance, will allow more days for the pre-embryo to be kept in culture and, thus, more time for na accurate diagnosis. Besides, routines may be created to biopsy the trophoectoderm of growing blastocysts. Better survival rates after cryopreservation of biopsied embryos will also permit longer time to complçete the relatively complicated procedures involved in PGD.

4. Finally, and most important, new DNA technologies, new imaging systems and new “chips” will open unfathomable possibilities to locate, with simple precision, nucleotide variation in the whole of the human genome, thus allowing precise prediction of the pre-embryo genetic content. All these predictable developments will have profound impact in PGD and the latter, by its turn, in us, our children and our society as a whole. So, one of this century’s dilemmas will involve ethic barriers that will be risen considering the growing use of PGD.

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