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Preimplantation genetic diagnosis (PGD)

Preimplantation genetic diagnosis (PGD) is an alternative to traditional prenatal diagnosis such as chorionic villus sampling (CVS) and amniocentesis after artificial insemination and/or ICSI.  The procedure was first proposed to determine the sex of embryos in couples at risk of X-linked disease.  Today PGD is offered to detect numeric chromosome abnormalities of 5 chromosomes nl: 13, 18, 21, the X and Y chromosomes making use of the fluorescent in situ hybridization (FISH) procedure.  In special cases where a known familial balanced chromosome translocation is present PGD may also be offered if appropriate FISH probes are available. The success of the complete PGD procedure is dependent on various factors nl: fertilization, embryo quality, successful embryo section, single blastomere retrieval onto a microscope slide, fixation, and successful FISH procedure and result interpretation.  This whole procedure influences the number of healthy embryos available for transfer.

Embryo section is an extremely difficult mechanical procedure.  A single blastomere has to be sectioned off from the blastocyst.  To achieve this, a very finely drawn glass “knife” is used and the blastomere must then be drawn into a capillary tube.  The sectioned blastomere must then be deposited onto a microscope slide to be fixed for the FISH procedure.  For the FISH procedure to succeed the nuclear membrane and cytoplasm of the blastomere must be removed.  This is achieved by a drastic chemical dehydration and heat procedure.  The actual FISH hybridization can only now be started. DNA-probes are short DNA sequences unique to specific areas on specific chromosomes and labeled with appropriate color molecules.  To bind to the specific complementary DNA sequences in the nucleus of a blastomere an excess of probe is added to the slide on which the blastomere has hopefully adhered to.  By a number of chemical treatment and heat cycles, excess DNA probe is washed away.

Analysis can then be performed by exposing the treated blastomere to UV light under a microscope.  The hybridization DNA probes are observed as colour signals after the attached colour molecules are excited.  By counting the number of different colour signals the number of those specific chromosomes is indicated and the genetic health of the whole blastocyst can be reported.  This procedure is 100% accurate and reliable.

The following problems must always be considered: 

Although the utmost care is taken to retrieve a complete single blastomere from the blastocyst it can happen that the blastocyst can break and only part of the complete cell is retrieved.

It may also happen that the sectioned blastomere is not ejected from the collection capillary tube onto the microscope slide.  This process is carefully monitored through the micromanipulator microscope, but it can be very difficult to clearly distinguish the blastomere in the capillary or on the microscope slide.

Once the blastomere has been deposited on the microscope slide it must adhere completely to the surface.  In the minority of cases the blastomere may be lost in the FISH procedure due to the stringency of the procedures.

Utmost care is taken to achieve good hybridization results, however in some cases the removal of the nuclear membrane is unsuccessful and no results can be obtained.

Dispersed despiralized DNA in the nucleus can also lead to weak hybridization and ambiguous results.

Finally it must be kept in mind that the success of the implantation is dependent on the overall health of the blastocyst (embryo) replaced.

 Considerations and problems of preimplantation genetic diagnosis.

  • Patients should be counselled on IVF procedures, possible prenatal diagnosis (a chorionic villus sampling/CVS or amniocentesis) even if PGD/PGS is performed.
  • Even with a successful IVF and PGD procedure, pregnancy is not guaranteed after transfer, and a term or near-term delivery is also not guaranteed.
  • Removal of a single cell without breaking it or causing serious damage is technically difficult.  Damage to the embryo (projected to be 0.1%) may accidentally occur during removal of the cell.
  • Analysis of a single cell has limitations, and misdiagnosis resulting from mosaicism (when the embryo has cells with different compositions) may occur. For this reason, prenatal diagnosis with either a CVS or amniocentesis should be considered to confirm the condition of the fetus.
  • A relatively large number of eggs or embryos may be found to be abnormal, thus leaving only a few or no healthy embryos for transfer.
  • For aneuploidy (numeric) screening, not all chromosomal or genetic abnormalities can be diagnosed with PGD because only a restricted number of chromosomes can be examined at one time during the course of a single procedure.
  • Currently, only a specific examination of a single biopsied cell is available. A single cell cannot be screened for multiple genetic conditions.
  • Some chromosome disorders may not be amenable to PGD due to an unavailability of suitable FISH locus specific or area specific probes for a specific chromosome disorder (i.e. chromosome deletions, or translocations). In the case of deletions and / or balanced chromosome translocations couples must have a chromosome analysis specifically to confirm detailed chromosome breakpoints. The process of validating and confirming the correct choice of FISH probes for such a procedure can take several weeks.

Considerations and problems of preimplantation genetic screening

Although initially heralded as a method to identify and avoid aneuploidy in embryos of women at increased risk asa result of advanced maternal age, recent studies suggest that success might be limited.  Most considerations and challenges listed in the PGD section also apply to PGS.

  • Technical limitations: Currently, FISH offers evaluation of less than half of the 23 chromosomes; usually only 5 are analyzed for the most commonly occurring numeric chromosome abnormalities. It must be noted that studies demonstrate that up to 25% of aneuploid embryos are characterized as normal because the abnormal chromosomes were not analyzed.
  • No or inconclusive results:  Approximately 30% of cells removed for screening may yield no or inconclusive results as a result of difficult technical protocols.
  • Limitation of single cell analysis: If non- disjunction occurs during meiosis, then all the cells in an embryo are aneuploidic. However, if disjunction occurs at mitosis, then two or more cell lines may be present in the embryo. Thus, a mosaic embryo with normal and abnormal cells may be misdiagnosed with the present single cell biopsy technique.
  • "Self-correction": Self-correction refers to evidence that mosaic embryos are able to halt the proliferation of abnormal cells and that many embryos identified as aneuploid will survive and be re-identified as normal.
  • Benefits: Results for PGS for advanced maternal age are mixed. Couples with recurrent pregnancy loss and established balanced translocation may benefit from PGS.