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Pre-implantation genetic diagnosis (PGD) is a combination of procedures that apply the latest scientific breakthroughs in order to evaluate the genetics of an embryo before placing the embryo in the womb. The scientists at Fertility Center and Applied Genetics of Florida, Inc. have been performing PGD since June of 2000. Our first live birth was reported in 2001. Since then, FC & AG of Florida, Inc. has been at the forefront of PGD in the Southeastern United States. In order to perform PGD, patients must undergo in vitro fertilization and embryo culture. On the third day of embryo culture in the IVF laboratory, a microscopic opening is made in the outer "shell" of the dividing embryo. This outer shell is called the zona pellucida. It is composed of complex sugar molecules and helps to hold the growing embryo together. Earlier in the process, the zona pellucida is also important in normal fertilization. By the third day of growth, a robust human embryo is usually made up of 5 to 10 individual cells called blastomeres.
A single cell or blastomere is obtained and a pertinent genetic evaluation is carried out on the single cell. The opening that is made in the zona pellucida is an extension of a very common procedure called assisted hatching. The embryo is placed back into the incubator in our laboratory. The blastomere is prepared and sent to a reference laboratory. Movie The pertinent genetic evaluation involves the analysis of chromosomes or the analysis of abnormalities in genes or segments of DNA. Here is a little background on this: Background Chromosomes are structures found in the center or nucleus of cells. A human has 46 chromosomes (23 pairs). Each of us received 23 chromosomes from our mother and 23 chromosomes from our father. Chromosomes are made of very long strands of DNA. Regions of the DNA strands in chromosomes are organized into definite structural entities called genes. Particular genes contain the code for particular protein molecules that direct or carry out all the millions of functions of our bodies. Having an extra portion of a chromosome or a missing portion of a chromosome is called aneuploidy. This can result in failure of implantation of the embryo, pregnancy loss, and other conditions such as infertility and Down's syndrome. PGD testing is more commonly offered to patients undergoing in-vitro fertilization (IVF) who are older than 35 years. These patients are at increased risk of miscarriage or birth defects. PGD may reduce these risks. PGD with an aneuploidy screen can assist the IVF team to select embryos more likely to result in a normal pregnancy. PGD for aneuploidy is also offered to patients who have a history of recurrent pregnancy loss, recurrent IVF failure, and patients with very prolonged unexplained infertility.
Analysis The biopsied cells are analyzed using a technique called fluorescence in-situ hybridization or FISH. This technique uses probes that are small pieces of DNA that are a match for the chromosomes we want to analyze. These probes are of different colors. The probes are applied to the biopsied cell and attach to the chromosomes. Under the microscope, the number of chromosomes can be seen in the nucleus of the cell. In this way, the chromosome pairs are counted. This analysis is accomplished in one day. Single-gene defects or DNA sequence abnormalities are analyzed using a different technique than that used for aneuploidy or translocations. This analysis requires the use of a technique called PCR (polymerase chain reaction). PCR amplifies the amount of DNA found in a single cell so that DNA and/or gene sequences can be determined. This requires previous knowledge of the specific DNA or gene abnormality. Patients will be required to submit blood samples so that the PCR lab can analyze the particular abnormalities found in their particular case.
The risk of the fixation for chromosomal or aneuploidy evaluation After embryo biopsy, the biopsied cell is glued (fixed) to a glass slide and an acid solution is used to remove all but the nucleus of the cell. The nucleus contains the chromosomes to be analyzed. After this process the cell that has been fixed is no longer viable; it cannot be returned to the live embryo from where it was obtained. Not all cells can be analyzed after fixation. A fraction of cells will not be analyzable due to unexpected degeneration of the nucleus either after fixation or during the processes involved in the FISH analysis that follows. It is estimated that 4-5% of cells fixed are not analyzable due to absence or degeneration of the nucleus and that 2-3% of embryos have biopsied cells that are fixed in a way that can prevent FISH analysis. Embryos without analysis results can still be replaced, but all the possible advantages of PGD will not apply.
The risk of the preparation of cells for PCR analysis As mentioned above, single gene defects or DNA sequence rearrangements are analyzed through a technique called PCR. PCR is a technically challenging process that may not yield results about every embryo biopsied. In our experience, this occurs less than 5% of the time.
Possible benefits of PGD Aneuploid embryos can be indistinguishable in appearance and development from chromosomally normal ones. The PGD results can guide the selection of embryos for replacement or transfer into the mother. Most chromosomally abnormal embryos either do not implant or spontaneously abort shortly after implantation. Thus, if only normal embryos are replaced, each embryo will have a higher chance of implanting and reaching term. The probability of conceiving a healthy child is increased through PGD. PGD for aneuploidy has been reported to double implantation rates in several studies, to reduce the rate of pregnancy loss by half and to increase take-home baby rates. The benefits of PGD increase when more embryos are available for analysis. If there are fewer than six embryos, there may not be any increase in the implantation rate. Nonetheless, even with few embryos, the information gained from PGD can assist in the decisions involved in an IVF cycle. Patients with specific chromosomal rearrangements (like translocations) or specific gene or DNA defects can avoid passing this to their offspring through the application of PGD. The list of known single-gene defects for which we have specific probes grows each week. Here is an incomplete list of conditions that can be tested for with PGD: Structural chromosomal defects:
Monogenic (single gene) diseases:
Autosomal dominant diseases:
X-linked diseases:
Risks and limitations of PGD FC & AG of Florida, Inc. performs strict quality control in order to assess whether any procedures performed in our laboratory lead to compromised embryo growth or potential. In other words, performing PGD is not without risk of injury to the pre-embryo. Thus far, we estimate the risk of damage to any biopsied embryo as less than one percent. Embryos that have been biopsied in our laboratory have developmental rates comparable to age-matched and diagnosis-matched controls. That is, the biopsy process does not appear to hurt embryos in our laboratory. Nonetheless, patients must realize that the PGD process involves a micromanipulation that could injure a dividing embryo so that it may subsequently arrest or degenerate. An additional and very important limitation of PGD must be understood. In the case of the routine screening for large chromosomal defects, patients must know that current technology limits the Fluorescent in situ hybridization (FISH) analysis to the use of only six to eight chromosome pairs. Humans have 23 pairs of chromosomes. The chromosomes most frequently tested are those that have been shown scientifically to be more commonly involved in subfertility and pregnancy loss. These are the sex chromosomes (X and Y), and chromosomes 13, 15, 16, 18, 21, and 22. The FISH analysis will usually give a reading of most of these chromosomes. Sometimes there may be incomplete readings. In those cases, the available results are interpreted in the light of the appearance of the developing embryo. Dr. Pabon reviews all the data with the patients and a decision is made based not only on the FISH results, but also on the appearance of the embryos. When all the FISH results are normal for the chromosomes tested, patients must understand that there may be abnormalities in other chromosomes pairs that were not tested for. Early embryonic development is complex. It has been shown that human embryos can develop into an abnormal or disorganized fetus even in the presence of a completely normal complement of 23 pairs of chromosomes. Most of the time, these abnormal pregnancies abort spontaneously. Patients must understand that PGD may fail in individual cases because of unforeseen technical malfunctions; this can include the loss of any individual cell during shipment to the testing laboratory. It is not possible to guarantee pregnancy after PGD or even promise that there will be benefits for any individual case. PGD may require the removal one or two cells from the day 3 embryo. Two cells are removed when it is uncertain that the initial cell contained a normal nucleus or when the diagnosis of single gene defects or DNA rearrangements is difficult. Dr. Pabon and the staff of Fertility Center and Applied Genetics of Florida, Inc. believe that the risk of injury to embryos involved in microsurgery is acceptably low. Numerous animal and human studies show that the microsurgery of the embryo needed to remove cells does not affect the normal development of the baby. This procedure is relatively new so the possible negative effects, if any, are unknown. Click here for more information on Pre-implantation Genetic Diagnosis (PGD)
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