Preimplantation Genetic Diagnosis (PGD)

What is Preimplantation Genetic Diagnosis?

Preimplantation Genetic Diagnosis (PGD) is presented as a very early form of diagnosis based on new Assisted Reproduction techniques, making genetic study of embryos possible before being transferred to the mother’s uterus.

Patients for whom PGGD is offered

Preimplantation genetic diagnosis is suitable for couples where one or both partners are carriers of monogenic diseases:

• Identification of sex in carriers of monogenic diseases related with sexual chromosomes, such as: Duchenne muscular dystrophy, Fragile X syndrome, Hydrocephaly, etc.)
• Identification of dominant autonomous monogenic diseases (Huntington’s chorea, Myotonic dystrophy, Marfan syndrome, etc.)
• Identification of recessive autonomous monogenic diseases (Cystic fibrosis, Tay-Sachs disease, Beta thalassemia, Fanconi anaemia, etc.

This genetic study also offers carriers of structural (Translocations, Inversions, etc.), and numerical (mosaics for Klinefleter syndrome, etc.) chromosomal anomalies.

Limitations of the PGD

• A PGD cycle should not be carried out without a minimum number of 10 oocytes.
• There is a percentage of embryos which do not endure the embryonic biopsy and which degenerate or stop their development.
• 80% of the biopsied embryos reach blastocyst.
• The PGD is not an alternative to prenatal diagnosis, but an option for maximum reduction of the likelihood of starting pregnancies with foetuses which carry genetic anomalies.

Undertaking a subsequent prenatal diagnosis is always recommended due to the high rate of mosaicism present in the embryos.

In conclusion, the genetic analysis of human embryos during the IVF cycle (PGD) is the combination of advances in embryology and molecular genetics. The results allow the identification of genetic anomalies at the embryonic level in stages prior to implantation, preventing the transfer of affected embryos.

The PGD technique can select chromosomically normal embryos before their transfer to the uterus.

Today, preimplantation genetic diagnosis has become a powerful tool for improvement of the effectiveness of in vitro fertilisation programmes.

What does the PGD process consist of?

1. Biopsy of blastomeres.

After an in vitro fertilisation process and through micromanipulation techniques, 1 or 2 blastomeres are extracted from the embryo which are normally on a third day in a stage of 6-8 cells. The embryonic biopsy is carried out in an inverted microscope with a micromanipulation system similar to that used for the ICSI technique. With a holding pipette, the embryo is held, and with an aspirating pipette a hole is made in the pellucid zone, immediately proceeding to aspirate 1 or 2 blastomeres on which appropriate analysis will be carried out. The elimination of these cells from the embryo does not affect its viability or capacity for survival, as the remaining cells are still totipotent and capable of forming a complete, perfectly normal individual.

2. Genetic studies.

A) Study of aneuploidies and structural alterations if the condition we wish to locate is due to a numerical or structural anomaly of the chromosomes (chromosomal translocations or inversions). To do so, the FISH (Fluorescent In Situ Hybridisation) technique is used, which only studies a few chromosomes, or CGH arrays (microarray-based comparative genomic hybridisation) which expands the study to all chromosomes. Today, massive sequencing techniques are being used, offering genetic analysis of all DNA. It is a more powerful, faster, more precise technique.

FISH allows dyeing of the centromeric area or specific regions of different chromosomes with various coloured fluorochromes. It is used today to simultaneously identify chromosomes 16, 22,13, 18, 21 and sexual chromosomes X and Y.

Full study of 24 chromosomes (CGH array). Through the use of new genetic analysis technologies, we have tools (arrays) which provide us with all genetic information of the embryo, numerical and/or structural alterations. A detailed sequencing of each chromosome of each embryo is carried out, allowing alterations to be detected in minute regions which may lead to genetic disorders.

CGH arrays consist of an analysis of the whole DNA of the patient, of all their chromosomes, comparing this with that of a healthy control sample. It allows the study of all chromosomes in their full scope. It is more precise than FISH in the detection of aneuploidies and structural alterations.

Through a comprehensive chromosomal screening (CCS), massive sequencing techniques of DNA allow the study of almost all DNA of the 46 chromosomes which make up our karyotype. It gives us precise information on the genetic health of the embryo and is more precise than arrays with CGH.

The study of aneuploidies or structural chromosomal alterations is mainly intended for:

• Women of advanced age
• Women who have had repeated miscarriages, with a suspected genetic cause.
• Presence of severe male factor.
• Repeated failure of IVF implantation.
• Alterations in chromosomes of parents.

One of the main applications of all these techniques of molecular biology within in vitro fertilisation is the determination of the sex of embryos. Currently, a combination of PCR and FISH techniques offer us practically 99% reliability in determining the sex of preimplantation embryos. In Spain, it is only possible to select the sex with these techniques when there is a high likelihood of transmission of a serious genetic disorder related with sex.

B) Study of Monogenic Diseases if the condition we wish to locate is due to one or several mutations and we wish to know whether the embryo is a carrier of any of these, the PCR (polymerase chain reaction) technique is used, which will allow us to amplify the DNA of the embryo and detect the presence or absence of the genetic mutation we are investigating in its genome.

PCR consists of analysing the mutation of the gene responsible for a hereditary condition in one or two cells obtained after the biopsy of the embryo. On the third day of the embryo’s development, when it has 6-8 cells, a hole is made from which one cell is extracted, on which the genetic study will be carried out.

After the embryo analysis process, and once we have checked and selected the healthy embryos, the embryo transfer is carried out as in a conventional IVF treatment. The results obtained with these techniques are similar to those obtained with classic IVF.

Applications

• Achondroplasia
• Alpha thalassemia
• Fanconi anaemia
• Spinocerebellar ataxias type 1, 2 and 3
• Spinal muscular atrophy
• Spinal and bulbar muscular atrophy
• Charcot-Marie-Tooth disease
• Facioscapulohumeral dystrophy
• Myotonic dystrophy
• Duchenne and Becker muscular dystrophy
• Amyotrophic lateral sclerosis
• Tuberous sclerosis
• Multiple extoses
• Cystic fibrosis
• Haemophilia A and B
• Huntington’s chorea
• Lynch syndrome
• Marfan syndrome
• Neurofibromatosis type I and II
• Osteogenesis imperfecta
• Autosomal dominant polycystic kidney disease
• Autosomal recessive polycystic kidney disease
• Tay-Sachs disease
• Fragile X
• Beta thalassemia