PGD-CENTRE

By learning more and more about the human genome a growing number of diseases can be detected by PGD and thus more patients can be treated. On the other hand, the early PGD (involving the egg or the embryo at the 6-8 cell stage) has been limited by low number of cells (one or two cells). This can be eliminated by taking a sample on Day 5, when it is possible to test multiple cells. In this case, however, the currently applied techniques do not make the genetic test result available on Day 5, which is the optimal time for implanting the embryo.

Recently developed vitrification methods are much more efficient in freezing the embryo, and blastocyst survival after thawing is as much as over 90%. In this way, the embryo can be implanted in a later cycle, when the genetic test results are available.

Frozen embryo

Prenatal diagnostics helps to prevent the birth of children affected with genetic diseases to parents who belong to a high-risk group. Genetic tests are performed by removing a small sample of the placenta (chorionic villus sampling or CVS), or by amniocentesis. If an abnormality is found, he only option is to terminate the pregnancy.

Pre-implantation genetic diagnostics (PGD) is the earliest form of prenatal diagnostics as it involves the genetic testing of the pre-embryo before implantation, from the time of fertilization up to the blastocyst stage.

Thus, one of the advantages of PGD is that genetic abnormalities can be detected before pregnancy occurs, as only unaffected embryos are transferred to the uterus for implantation. According to the generally accepted position in the medical profession, PGD is recommended where couples are at risk of passing to the child so-called monogenically transmitted diseases, or there is an increased risk of numerical or structural chromosomal abnormalities.

Micromanipulator

A micromanipulator is used for the sophisticated intervention that involves the oocyte or the embryo. This device is capable of carrying out movements that the human hand is unable to execute. Micromanipulative techniques are used in the following areas:

• To assist fertilization: intracytoplasmic sperm injection (ICSI)
• Assisted hatching (AH): to improve implantation
• Sampling for pre-implantation genetic screening: polar body biopsy, blastomere biopsy, trophectoderm biopsy

Micromanipulation system

1. Motor-drive course manipulator
2. Hydraulic joystick for fine manipulation (oil hydraulic)
3. Laser module for interventions involving the zona pellucida and/or embryonic cells

PGD is carried out as part of in vitro fertilization (IVF or “test tube baby”) treatment only. After follicle stimulating hormone treatment several oocytes, or eggs, are retrieved from the woman. The oocytes are then fertilized in vitro, outside the body. The embryos developing from the zygotes are generally implanted in the uterus on day 3 or day 5 of development. In the course of the procedure it is possible to take samples for genetic testing. In the case of oocytes and zygotes (i.e. fertilized eggs) the test is aimed at identifying a genetic change in the so-called polar body.

• Polar body biopsy: In the case of oocytes and zygotes (i.e. fertilized eggs) the test is aimed at identifying a genetic change in the so-called polar body. The drawback of the procedure is that it provides information only about the mother’s genome, and also that only a single cell can be tested.
• Blastomere biopsy: A blastomere (i.e. a sample of two or three cells) of the three-day-old embryo is tested. Implantation will take place on day 5. The drawback of this method is that only one or two cells are analysed and thus the genetic opinion does not cover of all of the embryos, and also there is a relatively high rate (5-10 %) of false negative or false positive results.
• Trophectoderm biopsy: On day 5, at the blastocyst stage, a small portion of the so-called trophectoderm layer consisting of 20-50 cells is removed for testing. This is the most reliable procedure, so in future it will probably become the most widely used method. Its drawback is that the embryos must be frozen and implantation can take place only in the next cycle.

Oocyte in metaphase II of the meiotic process

Gametes are produced by meiosis. Meiosis takes place in two stages. Meiosis of the oocyte ready to be fertilized arrests at meiosis II, the second phase, and in a normal case it is only completed after fertilization as a result of activation triggered by the sperm.
ZP: zona pellucida (the outer layer of the oocyte)
OL: oolemma (cytoplasm membrane of the oocyte)
ST: polar body (the cell resulting from the first meiotic phase)

In the course of the procedure the oocyte or the embryo is placed in a special medium. A tiny incision or hole is made on the zona pellucida, generally by means of laser, and the cell or cells to be tested are retrieved through the hole with a micropipette, a very thin glass capillary.

Blastomere biopsy

On day 3 the pre-embryos are generally at the 5-10 cells stage. At this stage one or two cells (blastomere) are removed by means of a micromanipulator from the embryo for the purpose of pre-implantation genetic diagnostics.

After preparation the sample is taken to the genetic lab, where various molecular biochemical test methods are applied (FISH, PCR, CGH) depending on the purpose of the test and the disease to be detected.

PGD is recommended primarily in the following cases (Sermon K. et al, 2004):

• Monogenically transmitted diseases: In this category, currently only sex-linked diseases can be tested in Hungary by sexing. Screening for other monogenetic diseases by PGD is expected in the near future.
• Chromosomal disorders:
  • – In parents carrying a balanced translocation, generally detected as a result of tests related to spontaneous miscarriage, infertility, or when a previous conception resulted in an unbalanced translocation.
  • – Aneuploidy, or numerical chromosomal abnormalities that are generally detected by infertility tests.
• Pre-implantation genetic screening (PGS): Primary candidates include women of advanced maternal age or repeated IVF-ET failure. However, this procedure is currently at the stage of trial and therefore it is currently not supported in Hungary.
• HLA matching: This indication is currently not supported and cannot be administered in Hungary.

Intracytoplasmic sperm injection (ICSI)

ICSI is a micromanipulation in vitro fertilization technique, where a single sperm is injected directly in the cytoplasm of an egg. It is most commonly used where sperms are unable to fertilize the egg (due, for instance, to low sperm count, poor motility, etc.). When PGD is carried out ICSI is generally used for fertilization to avoid a false test result caused by the genome of residual sperm adhered to the zona pellucida.