PREIMPLANTATION
GENETIC DIAGNOSIS (PGD)

By Barry Behr, PhD, HCLD & Victor Ivakhnenko, HCLD

Genetic errors arise from deletions or insertions of genetic material, abnormal numbers of whole chromosomes or genes, and even from misplacement of a single base in the DNA sequence. Genetic abnormalities can range from relatively harmless to severe: from vitamin deficiencies and food allergies to cancer, birth defects and infant mortality.

In recent years, significant advances in technology have enabled researchers to trace many disorders and diseases to their roots in the genetic code. Chromosome stretches, or even isolated genes, can now be used as markers to identify individuals at risk for certain illnesses. Additionally, the Human Genome Project, which aims to identify the chromosome location and DNA sequence of every human gene, is providing an everexpanding catalogue of potential genetic markers. The ability to recognize these genetic warning signs is rapidly becoming most effective tool for prevention, diagnosis and treatment of genetically based disorders.

An estimated 60 percent of all naturally occurring reproductive losses in pregnancies are associated with chromosomal abnormalities in the embryo. A normal embryo has 22 pairs of chromosomes called autosomes and 1 pair of sex chromosomes (XX or XY). Embryos that do not carry the normal pair of each chromosome are called aneuploids. Those that contain three copies of a particular chromosome (Trisomy) are the cause of some genetic disorders such as Down's syndrome (Trisomy 21). Other less common trisomies of chromosomes 13, 16, 18 and 22. Embryos that contain only one copy of a chromosome (Monosomy) are by and large nonviable.

Abnormal aneuploid embryos, either with monosomy (one missing) or trisomy (an extra one), are usually normal in appearance. It is not possible to distinguish these morphologically from other embryos. It is only through genetic analysis that they can be differentiated. Without such an analysis, many of these embryos are unknowingly transferred to patients. Depending on the specific abnormality, in IVF pregnancies, research has shown that chromosomal abnormalities such as aneuploidies (extra or missing chromosomes per cell) of the embryo increase either the risk of spontaneous miscarriage, the development of a genetically abnormal child or no pregnancy at all. Preimplantation genetic diagnosis (PGD) refers to the procedure involved in obtaining genetic diagnosis prior to embryo implantation (or embryo transfer). PGD is based on the ability of the human preimplantation eggs and embryos to continue their development into normal pregnancy after microsurgery (embryo or polar body biopsy), since cleavage stage cells of the embryo are pluripotent and removal of one or two cells at this time does not appear to affect further development of the embryo. PGD involves several obligatory steps: genetic counseling; reproductive counseling and treatment; in Vitro fertilization and genetic laboratory with DNA technologies such as fluorescence in-situ hybridization (FISH) for sex determination and screening for chromosomal abnormalities and polymerase chain reaction (PCR) for single gene diseases.

The technique entails in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), microsurgical removal of one or two blastomeres at the six- to eight- cell stage usually three days after fertilization, molecular (by PCR) in case of single gene diseases or molecular cytogenetic analysis (e.g. fluorescence in situ hybridization FISH) in case of chromosome abnormalities, studies of the biopsied cells, and uterine transfer of unaffected embryos. In cases of X-linked recessive diseases, sexing and selective transfer of female embryos can be performed.

An alternative source of material that has been used for PGD, when the disorder tested for is of maternal origin, is the polar body. A polar body is a small section of an egg and contains the complementary set of chromosomes present in the oocyte. Therefore, the genotype of the oocyte can be deducted by examining that in the polar body. The first polar body of an egg has been extruded prior to the egg retrieval and thus before fertilization. This polar body is not necessary for complete embryonic development and is available for analysis. A second polar body is extruded at the time of oocyte fertilization by a sperm. These polar bodies, removed using micromanipulation, can be a valuable source of genetic information. By using fluorescent-tagged genetic probes (Fluorescent in situ hybridization or FISH), we can examine them, thus allowing the chromosomal makeup of the oocyte to be inferred. Studies have shown that the majority of embryo aneuploids (85%) are due to the female oocyte. The remainder is of sperm origin.

Large chromosomal abnormalities, such as extra or missing chromosomes (aneuploidies), gender determination and unbalanced chromosomal translocations resulting from a parental balanced translocation can be detected by a laboratory procedure called fluorescence in situ hybridization (FISH). For this technique, DNA probes are labeled with colored fluorescent tags that light up so one can see specific chromosomes or genes under a microscope. The reagents are optimized for use with imaging software for probesignal enumeration. This software allows the simultaneous analysis of up to 12 different target-specific fluorophores in a single cell. However, up until now only 9 chromosomes can be accurately assessed during one analysis using FISH with up to a 10% error rate.

In cases involving more subtle abnormalities, on the scale of single genes or even DNA bases or single gene diseases, highly specialized techniques such as PCR are required. Such methods rely on the fundamental principles of the genetic code, and specifically on the ability to generate a matching, or complementary segment of DNA. Structurally, DNA is composed of two single strands attached to each other to form a double helix. The bases of one strand always bind to the bases (A,T,G &C) of the other in a specific fashion: A pairs with T, and G with C. If one knows the sequence of the bases in one strand, one can deduce the complementary sequence of bases in the other strand. Based on a known sequence of DNA, a synthetic copy of the matching strand called a DNA probe is created, it will then bind, or hybridize to that specific gene within a chromosome. The mutation in the carrier parent(s) needs to be characterized before PGD is applied.

Both FISH and PCR procedures typically take 24-48 hours to complete. However, since diagnostic tests are performed on a single cell, the possibility of misdiagnosis has to be considered. There are limitations of the test procedures, e.g. allele dropout in PCR, either non- specific or inefficient hybridization in FISH. New techniques like comparative genomic hybridization (CGH) offer the possibility to analyze all 23 pairs of chromosomes simultaneously for aneuploidy, translocations and single gene defects. Unfortunately, this technology is not clinically useful due to the time it takes to generate the results. It currently takes 4-5 days for the results to be obtained using CGH. This requires the biopsied embryos to be cryopreserved after biopsy to allow time for the analysis. There is a single report in the literature that has accomplished this approach successfully. Another technique that is also emerging and that may have application to PGD is Gene Chip technology where literally thousands of DNA sequences may be analyzed simultaneously. This technique is a little further off on the horizon.

PGD was first employed in 1989 with subsequent birth of normal females to couples at risk of various X-linked recessive diseases. The number of genetic diseases potentially diagnosable by PGD is vast. Examples of such disorders that have been reported include: chromosomal translocations, Down syndrome, Turner syndrome, DiGeorge syndrome, alfa-1- antitrypsin deficiency, beta- thalassemia, Charcot-Marie_Tooth disease, cystic fibrosis, Fancony anemia, fragile X syndrome, hemophilia A, Huntington disease, Lesch- Nyhan disease, Marfan syndrome, myotonic dystrophy, sickle cell anemia, and Tay- Sachs disease.

To date, a few hundred normal births have been achieved. The overall pregnancy rate per embryo transfer is ~ 25 % and birth rate is ~ 15%. Selective implantation of embryos with normal chromosome compliments have also been shown to result in high pregnancy rates with decreased spontaneous miscarriage rates. At present, there are approximately fifty PGD centers worldwide.

The U.S. Food and Drug Administration consider the procedure experimental. However, Huntington Reproductive Center remains committed to keeping pace with the rapid advances in the fields of genetics and human reproduction and making them available to couples as soon as is practically possible.

In the past 10 years, HRC has become one of the largest providers of assisted reproductive treatments in the U.S. Both the physicians and staff of HRC are committed to maintaining the highest standard of care in reproductive medicine in terms of moral and ethical practices. Significant experience in infertility treatment and embryo culture, highly skilled medical and laboratory personnel make it possible to offer PGD technology to couples at risk of having a genetically abnormal fetus which can help them avoid the birth of an affected child or having to face the painful decision of a pregnancy termination.