|Vol. 8, No. 1 / February 2010
Preimplantation diagnosis for genetic disorders
PGD has made selection of unaffected embryos and improved reproductive outcomes a reality that promises to influence standards in ART.
Reproductive Genetics Institute, Chicago, Illinois
Preimplantation genetic diagnosis (PGD) enables couples at risk for having offspring with a genetic disorder to have a healthy child without having to undergo prenatal diagnosis and possible termination of pregnancy. Although introduced for the same indications as prenatal diagnosis, PGD is a fundamentally different aspect of assisted reproductive technology (ART): rather than prevent the birth of an affected child, it allows patients to establish only unaffected pregnancies.
1PGD has many benefits beyond avoiding heritable disease, including the potential to improve pregnancy outcomes by facilitating selection of only chromosomally normal embryos. It has been used where prenatal diagnosis would be inappropriate, eg, genetic predisposition for disease. Lastly, it is contributing to advances in stem cell therapy and research.
Dr Kuliev reports no relevant commercial or financial relationships.
†Dr Verlinsky died July 16, 2009.
Overview of PGD
Material for PGD may be obtained by biopsying (1) the oocyte, by removing the first and second polar bodies, which are naturally extruded during maturation and fertilization of the oocyte prior to embryo formation; (2) the blastomere at day 3 (
); or (3) the blastocyst at day 5.
The biopsied material from the oocyte and embryo is tested for single gene defects using polymerase chain reaction (PCR) analysis, or for chromosomal anomalies using fluorescence in situ hybridization (FISH) analysis. As the reliability of microarray technology improves, combined testing of causative genes, chromosomal errors, and health-related genetic variability predisposing to disease may become possible in an integrated analysis.
Experience shows that accurate diagnosis may require the use of 2 or 3 PGD methods, particularly in instances of multiple indications. For certain single indications, as well as for greater accuracy in avoiding embryos with numerical chromosomal errors, sequential testing of the egg and embryo may be more reliable than a single test.
Further improvement and simplification of the procedure is necessary to ensure that sampling does not affect embryo viability and that genetic testing detects all errors in a chromosome set that could decrease embryo survival. Sequential testing of both oocytes and embryos helps clinicians avoid the problem of mosaicism, which is the main limitation of testing at the cleavage stage. Blastocyst biopsy provides a sufficient number of sample cells for microarray analysis of all of the chromosomes.
Knowledge of mutation is a general requirement for clinicians undertaking PGD. Linkage analysis of DNA from family members or haplotyping is, however, a more universal approach that allows the clinician to track inheritance of a genetic defect without actually testing for the gene itself.
This technique may prove useful for patients known to be carriers of a genetic disorder when the specific sequence information is not available.
Biopsy procedures for PGD
Knowledge of mutation is required for clinicians undertaking PGD.
Improving prognosis for in vitro fertilization patients
Selection of embryos with the highest developmental potential based on morphologic parameters is limited at present. Approximately half of embryos obtained from patients with a poor in vitro fertilization (IVF) prognosis are chromosomally abnormal,
and therefore likely to be lost during implantation or postimplantation development. This phenomenon is well established by data showing the prevalence of chromosomal abnormalities at different stages of embryonic development. At birth, only 0.5% of babies have a chromosomal abnormality, but such abnormalities are found in at least 50% of oocytes and preimplantation embryos, of which only 20% survive implantation.
Testing of spontaneous abortions from IVF couples not undergoing PGD frequently reveals a chromosomal abnormality.
PGD allows clinicians to avoid transferring an abnormal embryo—provided sufficient numbers of embryos are available for testing—thereby potentially improving pregnancy outcomes in patients with a poor IVF prognosis.
Inherited genetic disease: PGD vs prenatal diagnosis
The first patients to be offered PGD were those at risk of having a child with a Mendelian disorder. The most frequent indications continue to be disorders with a simple pattern of genetic inheritance, such as cystic fibrosis, hemoglobin disorders, and dynamic mutations (eg, muscular dystrophy, fragile X–linked mental retardation).
The choice between the use of prenatal diagnosis and PGD mainly depends on the patient’s view of pregnancy termination. In the past, prenatal diagnosis was offered to patients only if the couple was willing to terminate a pregnancy in the event that a genetic abnormality was diagnosed. PGD makes it possible to avoid this difficult decision while achieving pregnancy with an embryo free of inherited disease.
Concerns regarding embryo biopsy led to the development of pre-embryonic genetic diagnosis, a procedure in which predictive genotyping occurs at an earlier stage than in traditional PGD, as testing occurs on polar bodies prior to pronuclear formation. Because this approach avoids embryo micromanipulation and discard, pre-embryonic genetic diagnosis is more ethically acceptable to some patients.
To date, pre-embryonic genetic diagnosis has been applied in cases of maternally derived gene defects and chromosomal abnormalities. In the future, it may also be used for paternally derived abnormalities.
Pre-embryonic genetic diagnosis allows predictive genotyping at an earlier stage.
PGD can also be used by couples in which one partner is affected. For example, in the case of a woman with a genetic abnormality, PGD may be used to screen the maternal contribution to the embryo and select only embryos for transfer from mutation-free eggs. Unfortunately, if the mother is affected by an autosomal recessive disorder, embryo biopsy is the only option, because all oocytes are affected and prediction is limited to testing for a paternal contribution in the embryo.
Late-onset genetic disorders
Since the introduction of PGD, its use has expanded to testing for nearly 200 conditions,
including late-onset disorders with genetic predispositions. The latter disorders were never an indication for prenatal diagnosis—because these disorders may not clinically manifest during the lifetime, pregnancy termination cannot be justified. In this regard, PGD has a distinct advantage because only embryos that are free of a genetic predisposition are selected, obviating concerns about pregnancy termination.
At present, PGD is used to identify such cancer predispositions as ataxia-telangiectasia, familial adenomatous polyposis, familial colorectal cancer, breast cancer (BRCA1 and BRCA2), von Hippel-Lindau syndrome, retinoblastoma, neurofibromatosis types 1 and 2, Gorlin syndrome, tuberous sclerosis, and familial posterior fossa brain tumor.
PGD for disorders that have a genetic predisposition raises important ethical issues, such as the use of PGD for Alzheimer disease. Many at-risk couples carrying the genes predisposing to late-onset disorders would not otherwise forgo pregnancy without PGD. But PGD allows these patients to have unaffected offspring, rather than have to decide whether to prevent the birth of a child found through prenatal diagnosis to have inherited a predisposition to disease.
Identifying HLA-compatible embryos for stem cell therapy
Nondisease testing is another controversial PGD application, but it is likely to be more widely used in the future with the anticipated expansion of stem cell therapy, which requires an HLA-identical donor. Only PGD provides a realistic method of yielding an unaffected, HLA-compatible child, which may be a potential donor of stem cells for the affected sibling. The probability of identifying an embryo that is HLA-compatible with the affected sibling is only 1 of 4, which, together with the probability of 3 of 4 of detecting unaffected embryos in a couple at risk for the autosomal recessive disorder, constitutes the overall likelihood of a genetically normal HLA-compatible fetus of 3 in 16.
The application of PGD for the treatment of disorders in affected siblings is already a reality, resulting in successful stem cell therapy in children with congenital and acquired disorders for which there are no alternative radical treatments.
The first application of PGD for stem cell therapy was performed in Fanconi anemia (FA).
Embryos selected on the basis of being free of FA but HLA-compatible with the FA-affected sibling were transferred, resulting in birth of an unaffected child who became an HLA-compatible donor for the sibling who required bone marrow transplantation.
The success of hematopoietic reconstitution in the affected sibling spurred interest in applying PGD to stem cell therapy for sporadic bone marrow failures. This treatment approach was first used in a patient with Blackfan-Diamond anemia (BDA); as a result of stem cell therapy, this patient was no longer dependent on blood transfusions.
Preimplantation HLA typing will likely become the most attractive indication for PGD. In the majority of cases, HLA embryo selection is necessary for the treatment of a nongenetic sporadic or acquired condition, so preimplantation HLA typing is being performed without testing for a causative gene. In addition, patients of advanced age who request preimplantation HLA typing gain the added benefits of improved quality of transferred embryos and enhanced reproductive outcomes.
Preimplantation HLA typing has now been performed in hundreds of cases for conditions ranging from thalassemia, FA, Wiscott-Aldrich syndrome, X-linked adrenoleukodystrophy, X-linked hyper IgM syndrome, X-linked hypohidrotic ectodermal dysplasia with immune deficiency, incontinentia pigmenti, leukemias, and inherited and sporadic forms of BDA.
Lastly, PGD provides a unique opportunity for establishing disease-specific embryonic stem cell lines. A repository of human embryonic stem cell lines with genetic and chromosomal disorders has recently been established, representing a unique in vitro model for stem cell research on genetic and acquired disorders.
With the opportunity to investigate the primary disease mechanisms, it may be possible to one day develop treatments for genetic disorders.
Overcoming research limitations
Although some have not obtained the expected benefits of PGD on reproductive outcomes,
PGD is on the rise, having been used in approximately 40,000 cases to date.
Failure to observe improved reproductive outcomes with PGD may be due to inappropriate biopsy procedures, which affect the viability of the biopsied embryos, or inadequate genetic testing.
PGD has been used in approximately 40,000 cases.
In the absence of randomized controlled studies involving a sufficient number of cycles, comparison of reproductive outcomes in the same individual with and without PGD is useful in defining the clinical effects of PGD.
These reports demonstrate that implantation, spontaneous abortion, and take-home baby rates are significantly improved after PGD. In these studies, implantation improved 5-fold, spontaneous abortions were reduced more than 2-fold, and more than twice as many babies went home after PGD (
Reproductive outcomes before and after PGD for aneuploidies
PGD contributes to improved HLA-compatible stem cell transplantation.
It is not surprising that PGD is a growing aspect of ART in facilities that are equipped and staffed to perform the embryo/oocyte biopsy procedures and have the molecular genetic laboratory capabilities for genetic testing.
PGD may eventually become a routine procedure for IVF patients of advanced reproductive age who are more likely to have chromosomally abnormal embryos or for younger patients with a poor prognosis. By replacing the practice of selecting embryos for transfer based on morphologic criteria with selection of chromosomally normal embryos, PGD may improve the overall standards for ART. PGD allows patients at risk of having a child with a genetic disorder to select unaffected embryos. PGD contributes to improving HLA-compatible stem cell transplantation and has extraordinary potential for furthering development of the cellular therapy for genetic and acquired disorders.
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