 Pre-implantation genetic diagnosis (PGD) or Pre-implantation genetic screening (PGS) 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/PGS 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/PGS in the Southeastern United States. We are happy to announce the continuation of our progress with another first for Florida and the Southeastern United States. We are happy to report our first pregnancies with 24 chromosome “gene security network” microarray technology in October of 2009.
Fertility Center and Applied Genetics of Florida, Inc. and Dr. Pabon have one of the broadest experiences in Pre-implantation Genetics in the Southeastern United States. Our laboratory team is second-to-none. During the first few years of the program, outside consultants from IVF labs, L.L.C. were brought to Sarasota to collaborate on each and every PGD/PGS case. We’ve perfected our skill set to the highest level possible and our current Lab-Team is made up of two senior embryologists, with more than ten years experience in these procedures.
The Process:
In order to perform PGD/PGS, 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.
By the third day of growth, a robust human embryo is usually made up of 5 to 10 individual cells called blastomeres.
Three-day-old embryos
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.
Pictures here
Embryo biopsy process
Chromosome Basics - A Bit of Background Information
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 coding regions 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 congenital abnormalities such as Down's syndrome (Down’s syndrome results when the embryo and subsequent child has extra genetic information in the form of an extra chromosome 21.
A normal blood chromosome analysis
An abnormal blood chromosome analysis showing an extra chromosome 21
PGD/PGS testing is more commonly offered to patients undergoing in-vitro fertilization (IVF) who are older than 34 years. These patients are at increased risk of miscarriage or birth defects due to aneuploidy. PGD may reduce these risks. PGD/PGS with an aneuploidy screen can assist the IVF team to select embryos more likely to result in a normal pregnancy. PGD/PGS 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.
Table of aneuploidy risk as related to maternal age
Pre-implantation genetic analysis
The biopsied cells are analyzed using different techniques. Here we include a very brief description of each clinically available technique:
Fluorescent In-situ hybridation (FISH)
FISH is a technique that uses fluorescent probes that adhere to unique and very stable regions of the chromosomes that we want to analyze. These probes are of different colors and are applied to the biopsied cell (blastomere) and attach to the chromosomes. Under a fluorescent microscope, these fluorescent color probes can be seen in the nucleus of the cell. In this way, the chromosome pairs are counted. FISH technique requires very careful processing of the slides on which the blastomere is fixed (adhered). Unfortunately, only a few chromosomes can be studied at the time. This requires repeated washing of the cell and subsequent application of chromosome probes. This technique has a limitation when analyzing a single cell or blastomere. Accuracy or reliability of the results begins to suffer after 10 pairs of chromosomes. Therefore, most labs and clinicians stop at this number and choose to look at the chromosomes that are most pertinent in causing clinically recognized problems.
Microarray Techonology
Recently, the popularity of FISH has decreased due to a decrease in the price of “DNA microarray” technology that allows the analysis of all 23 pairs of chromosomes with no decrease in reliability. Microarray technology is becoming more common with several reference labs providing this service. It is our hope that market forces will continue to drive the price of this technology down. It is our expectation that the cost of FISH will decrease as the reference labs see much of their business go to microarray laboratories.
Polymerase Chain Reaction (PCR)
Single-gene defects or DNA sequence abnormalities are analyzed using different techniques 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 using FISH
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/PGS will not apply.
The Risk of the Preparation of Cells for PCR Analysis
As previously mentioned, single gene defects or DNA sequence rearrangements are analyzed through a technique called PCR. The technique 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.
Mosaic embryos are still a problem
Currently, most embryo biopsies involve the biopsy of one or two cells from an embryo composed of 5 to 10 cells. Multicellular embryos can be made of different cell types. Most research has shown that any single embryo has about a 7% chance of being mosaic. This means that if we sample a cell, that cell may not represent the part of the embryo that will become the fetus and baby. This means that the results are not absolute or “guaranteed.” Nothing is absolute or guaranteed in medicine. Fortunately, our clinic has never had an error regarding a diagnosis as it applies to a baby’s PGD/PGS.
Due to mosaicism in embryos, we always tell our patients to have all the required obstetrical testing without regard for what was tested prior to the pregnancy.
Possible Benefits of PGD/PGS
Aneuploid embryos can be indistinguishable in appearance and development from chromosomally normal ones. The PGD/PGS 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/PGS.
PGD/PGS 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/PGS increase when more embryos are available for analysis. If there are fewer than six embryos, there may not be any increase in the pregnancy rate. Nonetheless, even with few embryos, the information gained from PGD/PGS 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/PGS. The list of known single-gene defects for which we have specific probes grows each week.
Visit this page for an incomplete list of conditions that can be tested for with PGD.
Risks and limitations of PGD/PGS
Fertility Center & Applied Genetics 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 embryo biopsies is not without risk of injury to the 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/PGS 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/PGS 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 eight to ten 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. The new 23 chromosome pair microarray technology certainly provides much more information. Even with microarrays, there is no guarantee that results will be available on all the embryos. Even microarray technology does not give results regarding all the billions of genes that make up a human.
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/PGS 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/PGS or even promise that there will be benefits for any individual case.
PGD/PGS 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 the biopsied embryos is acceptably low. Numerous animal and human studies show that the removal of one or two cells from an embryo does not affect the normal development of the fetus and baby. This procedure is relatively new so the possible negative effects, if any, are unknown.
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