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ABSTRACT
The Pre-Implantation Genetic Diagnosis is a prenatal diagnostic technique for the study of chromosomal and genetic alterations in the embryo before its relocation in the maternal uterus.
This technique, for the reason why is performed on the embryo before the start of a clinical pregnancy, turns out to be the earliest developed/the most precocious analysis of every prenatal diagnosis currently practicable (amniocentesis, chorionic villus sampling/CVS).
DGP’s targets are to verify that the embryo has not chromosomal anomalies and/or it is not affected by a particular genetic pathology (recessive, dominant or X- linked) present in the parents.
The first PDG clinical applications took place in England, in the end of the Eighties, in patients who were carriers of genetic diseases linked to chromosome X. The determination of the sex of the embryos of these patients allowed the selective transfer of those feminine (healthy and/or healthy carriers) with the intent of avoiding the implantation of a male embryo, whose disease risk was of the 50%.
This technique is practiced by taking a sample of one or two cells (blastomeres) from the embryo obtained in vitro or taking a polar body from the oocyte in its maturation phase/period in order to analyse the genetic material. As a matter of fact, during the ovogenesis, at the moment of the first meiotic division, it generates a new cell and a first polar body waste and, at the second division the second polar body is released too.
The first polar body contains a genetic structure which is specular to that present in the oocyte itself. The analysis of this little cell, which has not any biological role and degenerates after few hours, furnishes important information about the genetic status of the oocyte and can be considered as an alternative to the embryos biopsy, made to carry out the pre-implantation genetic diagnosis.
This technique finds its application in all those couples that are at risk to transmit a certain genetic disease to their child, indeed, it results useful to prevent the transmission of matrilineal genetic diseases (dominant or X-linked) or recessive in the case that the father is a carrier of the injured allele too.
The pre-implantation genetic diagnosis in all cases of monogenic disease (a disease caused by the mutation of a single gene) is made by a molecular analysis (PCR,
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Polymerase Chain Reaction) which allows to analyse specific sequences of DNA from the taken cells to determine, within reasonably short time, the embryos which have inherited the genetic mutation from the couple and/or from the single heterozygous partner.
The thesis has the goal to develop a PCDG protocol by researches on polar body to obtain a valid alternative to the pre-implantation diagnosis on the blastomeres, avoiding ethic/religious problems which this technique has always risen. Therefore this research is applicable also in the hypothesis that the prohibition of blastomeres analysis would be restored in Italy.
The genetic diagnosis on polar globules does not result without limits, indeed, is not possible to determine the formation of any mosaicism (cells taken from the same embryo that can present a different karyotype) and the presence of patrilineal transmission genetic diseases, except for the cases in which the disease is autosomal recessive transmittable.
For a reference laboratory for the prenatal diagnosis is fundamental to create a protocol for this kind of technique’s activation, which is at present handled only in few private centres with high costs.
This method has also a decisive and innovative impact for the prenatal diagnosis in order to avoid the recourse to therapeutic abortion, which is often devastating from a psychological point of view and also not always accepted because of personal beliefs as the best solution for a pregnancy.
In the laboratory where I worked we tried to recreate and to improve a minisequencing protocol presented in the article of F. Fiorentino et al (2003) in order to create a method which would allow to effect the pre-conception genetic diagnosis. We concentrated on the most widespread mutation in Italy and Europe, responsible for the cystic fibrosis, the ΔF508 of the gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator).
