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PREIMPLANTATION GENETIC DIAGNOSIS

PGD can be performed only on embryos in vitro (in a laboratory). That means this test is always performed in conjunction with an in vitro fertilization cycle.

In Vitro Fertilization (very brief summary)

Medication is given to stimulate the production of multiple eggs.

  • Egg retrieval is performed using an ultrasound guided needle.

  • Eggs are then subjected to intracytoplasmic sperm injection (ICSI), regardless of the quality of the husbands sperm, to avoid possible genetic contamination by other sperm  sticking to the egg coat. The injected eggs are placed in the incubator to allow fertilization and embryo growth to the 6-10 cell stage.

  • At this point, one or two cells will be biopsied from the embryo(s) and PGD will be performed.

  • Normal embryos are transferred to the mother's uterus on day 4-5 following egg retrieval.

  • Embryo Biopsy

    To enable screening of a human embryo before transfer to the uterus, it is possible to remove one or two cells from the 6-10 multi-celled embryo without compromising it, so that the genetic material in these cells can be analyzed. It must be noted that in routine genetic analysis there are usually hundreds of cells available for processing, however, with embryo biopsy only one or two cells are commonly available, and they must contain a nucleus to allow determination of the genetic status of that embryo. The biopsy method is relatively straightforward, but this does not mean that it is an easy procedure to undertake. The embryos are typically biopsied at the pre-implantation stage on day three of development. At this point, the embryo will be composed of between 4 and 12 cells that are still distinct from each other. On the third day, however, single cells can be individually removed without disrupting the adjacent cells in the embryo. However, at the latest on day 4 the embryo begins to compact, a process whereby the individual cells lose their clear outline and become more closely associated with the each other.

    Removing Cells

    At this time the embryo is still surrounded by a glygoprotein coat, the zona pellucida, and to remove any cells this coat must first be pierced. This can be done either using acidified culture medium that "dissolves" the zona pellucida locally, or more conveniently a hole can be made with a laser, allowing a glass micro-pipette to be pushed through and extract a cell. The hole that is drilled is usually made a little smaller than the cell itself, and this helps to maintain the integrity of the embryo within its coat during further development in the IVF lab. During manipulation on an inverted microscope the embryo is held in a warm culture medium that allows the cells to be removed with a minimum of trauma to the overall embryo. The removal of up to a quarter of an embryo is not known to be deleterious to its further development, as the embryo can compensate for the loss of some cells at this early stage of development All cells at this stage are still totipotent, meaning, that each is capable of developing into a complete embryo



    Embryo Biopsy (illustration)

    Analysis

    Once a single cell (a blastomere) is removed, it is either fixed on a glass slide for chromosomal analysis, or placed in a small tube of chemical buffer for single gene diagnosis. The cells are then analyzed using techniques called fluorescence in situ hybridization (FISH) or DNA analysis. During the genetic analysis, the embryos are usually grown to the fifth day of development at which time they may either be at the late morula or blastocyst stage. Those embryos found to be free of genetic abnormalities are then placed into the uterine cavity.

    Other Issues

    Misdiagnosis

    Misdiagnosis can occur due to mosaicism within the embryo. Some embryos may contain blastomeres (cells produced by the cleavage [division] of a fertilized egg) which are genetically normal and, within the same embryo, other blastomeres which are abnormal. This is called mosaicism. For this reason, a diagnosis may be incorrect. This may result in the transfer of an embryo carrying a chromosome abnormality or the failure to transfer a normal embryo.

    Experimental error can also account for a misdiagnosis. Improper cell fixation techniques, DNA denaturation errors, allelic drop-out or amplification of contaminated DNA can lead to a wrong diagnosis.

    A recent report of the European Society of Human Reproduction and Embryology (ESHRE) documented the PGD results from 25 consortium members from 1999 to 2001. There were 8 confirmed misdiagnoses from 451 PGD tested embryos; 1% (3/305) for chromosome analyses and 3.4% (5/146) for single gene disorders.

    Are there risks associated with PGD?

    The micromanipulation techniques used for blastomere biopsy are safe with little risk to the embryo. The risk of accidental damage to the embryo during biopsy is less than 1%. There is no risk to the embryo following chromosomal or single gene defect analysis because the analyzed cells are not put back into the embryo. There may be a slightly lower likelihood of implantation after embryo biopsy compared with an embryo not having been biopsied. Other risks may become apparent over time, but are far outweighed by the potential benefits for each couple.

    Procedures In The Embryo Fertilization Laboratory
    Who Takes Care of the Eggs, Sperm and Embryos in the IVF Laboratory?


    The embryologist is responsible for the culture, maintenance and protection of the patients, eggs, sperm and embryos. Having received specialized training and meeting requirements for certification for the reproductive technology laboratory, the embryologist administers the laboratory's operation including the maintenance and monitoring of the equipment; b) prepares for and participates in clinical procedures such as egg retrieval and embryo transfer; c) performs the assisted reproductive techniques to achieve fertilization and embryo development; d) documents and records all laboratory events pertinent to a patient's treatment cycle; and e) is an integral member of the multi-disciplinary treatment team.

    What is The Sequence of Events in the Laboratory for an IVF Cycle Involving PGD?

    When a patient initiates a treatment cycle, a specific plan is developed and established in the IVF Laboratory. Elements of the plan address the following: the fertilization procedure, how many eggs are expected; will the patient wish to freeze extra fertilized embryos.

    On the day before egg retrieval, the culture medium is prepared. Culture vessels which will hold eggs, and the test tubes in which the sperm are processed are labeled and placed in the incubator and dedicated workspaces, respectively. A patient laboratory chart is prepared to confirm her identity and the semen specimen used for preparation of the sperm for fertilization of her eggs, to provide a record of all eggs and embryos and to record the names of the embryologists and physician and the techniques and procedures performed by them, confirm the patient’s identity at embryo transfer, and to record the culture media used for the patient's cycle to fulfill quality assurance and control requirements.

    At egg retrieval, the patient's identity is confirmed, and her eggs placed in her labeled dishes. The corresponding semen specimen is accepted after the identification on the label of the specimen container is confirmed and recorded in the patient's chart. Motile sperm are isolated from the semen sample, by a "swim-up" procedure. According to the treatment plan, after confirmation of the identities of both eggs and sperm, the eggs are injected within two to eight hours of retrieval.

    After an incubation period of 15-18 hours, the eggs are examined to determine if fertilization has occurred. Fertilization is confirmed when two pronuclei (one each from the sperm and egg) are observed. In the next 24 hours, the onset of cell division is confirmed. The egg, with the union of genetic complements from each parent, will divide into two cells, and each can divide into two cells. In this way, the embryo expands in cell number and stage of development - the egg has become an embryo. On day 3 after egg retrieval, embryos can be selected for transfer. If there are extra fertilized eggs or embryos, these can be frozen and stored for potential use in a future cycle.

    The longest study of children born from in vitro fertilization and related treatments is reassuring in terms of intelligence scores and psychological health funded by the European Union this involved more than 1500 children from Britain, Belgium, Sweden, Denmark, and Greece tracked up to age 5. The researchers assessed the physical development and family relationships and intellectual psychological and social development.

    There were no differences from the norm in regard to birth weight and height, nor in intelligence, language skills and motor skills or in behavior and temperament.


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