Preimplantation Genetic Diagnosis or PGD is an exciting new technology that can detect genetic abnormalities such as disease-causing mutations and chromosomal abnormalities to prevent the conception of abnormal pregnancies or children. PGD has made the headlines recently in some controversial cases, including the use of PGD to prevent transmission of adult-onset diseases such as Alzheimer’s; and the use of PGD to help conceive an HLA-matched child to donate bone marrow for a sibling with Fanconi’s anemia. However, what is even more exciting than these headline stories is that PGD may also be a very effective treatment option for women who suffer recurrent first trimester pregnancy loss, for women with a history of a chromosomally abnormal pregnancy, and for women with age-related infertility. PGD can be used to detect aneuploidy in a very young embryo, before the embryo attaches to the mother’s uterus. Aneuploidy is a condition in which an embryo has an abnormal number of chromosomes. Aneuploidy is the cause of 50 to 70% of first trimester miscarriages, down’s syndrome and other birth defects. By preventing the transfer of abnormal embryos, the risk for miscarriage and aneuploidy can be significantly reduced. At this time, only a few centers have significant experience with this technique, but this technology is spreading rapidly and will likely become more routine within the next few years. This article reviews PGD and the Saint Barnabas experience with this technique.
How is PGD performed?
PGD requires the creation of embryos in the laboratory. Therefore, patients must undergo in vitro fertilization or IVF. This technique involves ovarian stimulation of multiple egg-containing follicles using injectable hormones called gonadotropins (Follistim, Gonal f, Pergonal, Repronex, etc). While the patient is under sedation or anesthesia, her eggs are retrieved through the vagina using a needle and ultrasound guidance to aspirate the eggs. The eggs are then inseminated with the husband’s sperm in the laboratory. Once fertilization occurs, embryos are allowed to grow in the laboratory for a total of 3 days after egg retrieval. After 3 days of culture, normal embryos will divide and should reach the 6 to 8 cell stage. At this stage of development, each cell has the potential to become an entire human being and removal of a single cell will not harm the embryo.
An embryo biopsy is performed by creating an opening in the zona pellucida, the “shell” around the embryo. A single cell from the embryo is removed through this opening using gentle suction and a micropipette (Figure 1). The procedure is performed using a special microscope with micromanipulators – special devices designed for delicate microscopic procedures. The cell is then fixed upon a slide and the embryos that have been biopsied are placed back into an incubator to await the results of the biopsy.
For the diagnosis of aneuploidy a technique called fluorescence in-situ hybridization (FISH) is used. This technique uses small pieces of DNA (probes) attached to fluorescent labels that bind to specific chromosomes in the cell. Once the probes are bound, the glow-in-the-dark signals are read under a fluorescent microscope, so that the number and type of chromosomes present in that cell can be determined (figure 2). The analysis takes approximately 1 day to accomplish. Embryos that are normal by the analysis are then transferred to the woman’s uterus on day 4 or 5 after the oocyte retrieval. Due to the limited amount of material being analyzed and the brief window of time in which to obtain a diagnosis, only 9 out of the 24 types of chromosomes can be analyzed. However, the chromosomes analyzed (13, 15, 16, 17, 18, 21, 22, X and Y) are those responsible for the majority of early pregnancy losses.
What are the results of PGD of aneuploidy?
PGD of aneuploidy can be used to reduce the chance of miscarriage. In a group of IVF patients at IRMS at Saint Barnabas, matched for maternal age, number of previous IVF cycles and response to fertility drugs, there was a significant 68% reduction in the rate of miscarriages in the group that underwent PGD compared to the group that did not (the control group). The control group experienced a 40% miscarriage rate while the PGD group experienced a 13% miscarriage rate.
Other benefits of PGD of aneuploidy: less Down’s syndrome, improved pregnancy rates and lower rates of high order multiple births.
The rate of Down’s syndrome and other abnormal pregnancies is also significantly reduced by PGD. In addition, in patients undergoing IVF, PGD for aneuploidy may improve the chance of conception by increasing embryo implantation rates. It is thought that PGD improves the process of selecting embryos for transfer, allowing embryologists to choose embryos most likely to result in a normal pregnancy. By improving embryo selection, and allowing fewer embryos to be transferred, PGD can also assist in reducing the frequency of high order multiple births after IVF.
What are the risks and limitations of PGD?
The risks of PGD include damage to the embryo during the biopsy procedure. Embryo damage is an “all or none” phenomena. If an embryo is damaged, it will stop growing. Embryos that continue to grow after the biopsy do not become abnormal as a result of the biopsy. The risk of embryo damage at our center is currently 0.9% but can vary widely depending upon the experience and skill of the person performing the biopsy. Embryo damage rates can be much higher in the hands of an inexperienced embryologist. If the embryo continues to grow after PGD, it will not sustain any injury and will not be at greater risk for miscarriage or for birth defects. In fact as stated above, if the results of the PGD are normal, these risks will be decreased.
As discussed earlier in this article, PGD of aneuploidy is currently limited to 9 out of the 24 types of chromosomes. An embryo that is deemed normal by PGD could have an abnormality in one of the other 15 types of chromosomes that were not analyzed by PGD. In addition, because the analysis is performed using FISH, the abnormalities that are detected are in chromosome number only. Other types of structural abnormalities, where the chromosome number may be correct, but the chromosome itself is abnormal, such as additions, deletions or translocations may not be detected as they would be in chorionic villus sampling (CVS) or amniocentesis. CVS and amniocentesis are tests that are performed on the fetus during early pregnancy.
Because hundreds of cells are analyzed, CVS and amniocentesis can detect conditions known as mosaicism, where some cells of the fetus are chromosomally normal and some cells are not. Since only a single cell is analyzed during PGD, mosaicism may lead to misdiagnosis. A mosaic embryo does not have the same chromosomal component for all cells, so that a single cell will not reflect the chromosomes of the entire embryo. Because of these limitations, the error rate for PGD for the chromosomes analyzed (including mosaics, false positive and false negative results) is approximately 7% at our center, while the error rate for CVS and amniocentesis is typically less than 1%. The error rate may vary between programs depending upon the experience of the personnel performing the PGD. Because of this difference in error rates, PGD cannot be considered a substitute for CVS or amniocentesis. The decision about whether or not to have CVS or amniocentesis should not be based upon whether or not PGD was performed. At this time, our center recommends that patients at high risk for abnormalities (such as patients who themselves have a chromosomal abnormality) have CVS or amniocentesis even if PGD has been performed. On the other hand, many couples who have experienced recurrent miscarriage would rather take the small risk of missing an abnormality because they did not have CVS or amniocentesis, rather than taking the small risk of losing a normal pregnancy due to complications from CVS or amniocentesis. Speaking with a genetics counselor who is familiar with PGD can be helpful in making this complex decision.
Which patients should have PGD?
Recurrent first trimester pregnancy loss
Recurrent pregnancy loss (defined as 3 or more consecutive miscarriages) affects approximately 1% of the population. However, after 2 consecutive miscarriages, it is reasonable to have a full evaluation and to consider treatment. The evaluation for recurrent miscarriage should rule out genetic, anatomic, endocrine, and immunologic causes for recurrent miscarriage. Many physicians will also rule out blood clotting disorders, although this testing remains controversial. The evaluation should be individualized, but typically includes history and physical exam, transvaginal pelvic sonogram, hysterosalpingogram or saline hysterosonogram, complete blood count, thyroid testing, antithyroid antibodies, prolactin, lupus anticoagulant, anticardiolipin and antiphosphostidylserine antibodies, karyotyping of both partners (blood chromosomal analysis) and possibly an endometrial biopsy and screening for blood clotting disorders.
Approximately 5 to 8% of couples with a history of recurrent pregnancy loss will have an abnormal karyotype or chromosomal abnormality, usually a balanced translocation. A balanced translocation means that all the normal chromosomal material is there in the right amounts (“balanced”), but it is arranged in an abnormal way (“translocated”) such that when the individual who carries the balanced translocation makes eggs or sperm, most of the eggs or sperm carry abnormal amounts of chromosomal material (are “unbalanced”). This can lead to infertility, miscarriage and birth defects depending upon the particular type of translocation. These couples can have an over 95% chance of miscarriage. PGD can be performed for couples with a balanced translocation, allowing them to implant only normal or chromosomally balanced embryos, thus dramatically reducing their risk of miscarriage (from over 95% to less than 10%). PGD for translocations is technically much more complicated than PGD of aneuploidy. Patients with a translocation should be seen by a genetics counselor to review their options. A referral for PGD at a center with experience performing this type of analysis can then be made if the couple desires it.
Once the evaluation is complete, many patients will not have an identifiable cause for their miscarriages and therefore, no obvious treatment options. Without any treatment, couples with this history have a 40 to 70% chance of a successful live birth, depending upon how many miscarriages they have had and whether they have any previous live births. Thus, doing nothing, with close follow up can be a reasonable option depending upon the couple’s preference. Some couples will feel more comfortable just having close follow up: very early, serial blood pregnancy testing; early, serial transvaginal sonograms; testing for estrogen and progesterone levels with hormone supplementation as needed. Many couples, once they know that their testing is normal will feel comfortable with this less aggressive approach. For couples that desire a more aggressive approach, IVF with PGD may be offered to significantly reduce the risk of first trimester loss due to aneuploidy. Ultimately, success rates will depend upon multiple factors including the experience and skill of the embryologist performing the biopsy, the experience and quality of the IVF laboratory and IVF program and individual patient characteristics such as maternal age and past medical history
Previous chromosomally abnormal child or pregnancy
For patients with a previous child or pregnancy with a chromosomal abnormality, PGD can reduce the risk of certain abnormalities in the patient’s next pregnancy. This may be an attractive alternative to post conception testing for patients as they may be able to avoid termination of an abnormal pregnancy.
Advanced maternal age
As a woman ages, her risk for both miscarriage and chromosomally abnormal or aneuploid pregnancy increases markedly, and is the major reason why IVF success rates decline with increasing age of the female partner. The chance of embryo implantation after IVF is inversely correlated with the risk for aneuploidy. The higher the risk of aneuploidy, the lower the chance for pregnancy (Figure 3). By improving the ability to select embryos more likely to implant by eliminating embryos with some of the most common chromosomal abnormalities, PGD can improve implantation rates and ultimately live birth rates. Since the morphologic appearance of the embryo (the way the embryo looks under the microscope) is not an accurate reflection of the chromosomal make-up of the embryo, as a women ages, the ability of the embryologist to select the embryos most likely to implant decreases and the proportion of aneuploid embryos increases. So in older women, the beauty of the embryo, its “quality” or “grade” becomes disconnected from its ability to implant in the uterus. Often, it is the high quality embryos that are chromosomally abnormal, while the lower quality embryos are the ones that test normal. Without PGD, these lower quality embryos may not be the ones selected for transfer, if there are excess embryos available for transfer. For women 35 years of age and older undergoing IVF, our center has demonstrated that PGD for aneuploidy significantly improves pregnancy rates, reduces miscarriage rates and reduces trisomies if 6 or more embryos of good quality are available for analysis. This will not hold true at all centers, since the PGD pregnancy rate is a result of the individual patient characteristics, the PGD analysis, the skill and experience of the person performing the embryo biopsy and the overall success rates of the IVF program.
PGD of aneuploidy is a new and exciting technology that is becoming available at more IVF centers around the country. In order to undergo PGD, patients must conceive via in vitro fertilization. PGD of aneuploidy is a limited chromosomal analysis of early stage embryos prior to implantation. PGD of aneuploidy is an option for women with a history of recurrent first trimester loss that is unexplained or due to a chromosomal abnormality. PGD can significantly reduce miscarriage risk in these women. PGD may also improve pregnancy rates in women undergoing IVF.
If a woman wants to consider PGD of aneuploidy, she should have a consultation with a genetics counselor for a more extensive discussion on the procedure and its limitations. The experience and skill of the person performing the biopsy, the experience and quality of the IVF laboratory and program and individual patient characteristics such as age and past medical history will all have an impact upon success rates. While PGD can have significant benefits, it is a limited genetic test and is not a substitute for CVS or amniocentesis.
Copyright Serena H. Chen, MD 2004
Figure 1: Embryo biopsy. This series of photographs taken through a powerful microscope illustrates the process of making an opening in the shell (zona pellucida) of a 3 day-old embryo using a dilute acid solution and then using a biopsy pipette to remove a single cell (blastomere) from the embryo for analysis.
Photos courtesy of IRMS at Saint Barnabas, PA and Dr Santiago Munne.
Figure 2: FISH – Fluorescence In Situ Hybridization on a single cell from a 3 day-old embryo. Two fluorescent signals are seen for chromosomes 13, 16, 18, 21 and 22, giving a normal diagnosis for these particular chromosomes.
Photos courtesy of IRMS at Saint Barnabas, PA and Dr Santiago Munne.
Figure 3: As a woman ages, the risk for aneuploidy increases and the chance for embryo implantation decreases. In our experience, PGD for aneuploidy in womenover 35 can increase implantation rates by improving embryo selection.
Figure courtesy of IRMS at Saint Barnabas, PA and Dr Santiago Munne.