Preimplantation diagnosis: a realistic option for assisted reproduction and genetic practice Anver Kuliev and Yury Verlinsky Purpose of review Introduction Preimplantation genetic diagnosis (PGD) allows genetically Introduced 14 years ago, preimplantation genetic diag- disadvantaged couples to reproduce, while avoiding the nosis (PGD) permits genetic testing before the transfer of birth of children with targeted genetic disorders. By embryos to the mother, which has distinct advantages in ensuring unaffected pregnancies, PGD circumvents the establishing pregnancies unaffected by tested disorders possible need and therefore risks of pregnancy termination. and without the potential need for pregnancy termina- This review will describe the current progress of PGD for tion. Approximately 7000 PGD cases have been per- Mendelian and chromosomal disorders and its impact on formed worldwide, which have resulted in the birth of reproductive medicine. more than 1000 healthy children [1]. PGD can be used Recent findings for the diagnosis of late-onset diseases with genetic Indications for PGD have expanded beyond those used in predisposition, preimplantation HLA typing, and other prenatal diagnosis, which has also resulted in improved non-traditional prenatal testing; thus, PGD complements access to HLA-compatible stem-cell transplantation for other methods used in prenatal diagnosis [2]. By permit- siblings through preimplantation HLA typing. More than ting selection of euploid embryos for transfer, PGD 1000 apparently healthy, unaffected children have been improves reproductive outcome, which has resulted in born after PGD, suggesting its accuracy, reliability and its extensive use in assisted reproduction practices [1, safety. PGD is currently the only hope for carriers of 2–5]. This review will describe the current progress of balanced translocations. It also appears to be of special PGD for Mendelian and chromosomal disorders and its value for avoiding age-related aneuploidies in in-vitro impact on reproductive medicine. fertilization patients who have a particularly poor prognosis for a successful pregnancy; the accumulated experience of thousands of PGD cycles strongly suggests that PGD can Approaches to preimplantation genetic improve clinical outcome for such patients. diagnosis Summary PGD involves genetic testing either of oocytes in the case PGD would particularly benefit poor prognosis in-vitro of maternally derived genetic abnormalities or of single fertilization patients and other at-risk couples by improving cells derived from preimplantation embryos. Normally reproductive outcomes and avoiding the birth of affected screened embryos are subsequently transferred to the offspring. patient, which ensures the establishment of an unaf- fected pregnancy [6]. These methods have not been Keywords shown to have detrimental effects on embryonic devel- expanding indications, improving reproductive outcome, opment, even after sequential polar body and embryo preimplantation genetic diagnosis (PGD), PGD accuracy, biopsy [7,8]. Blastocyst biopsy, which is becoming more preimplantation HLA typing common, may be used to confirm the diagnosis based on polar body or blastomere biopsy [9]. Each of these Curr Opin Obstet Gynecol 17:179–183. # 2005 Lippincott Williams & Wilkins. methods has advantages and disadvantages, and the choice depends on clinical circumstances. Embryo biopsy Reproductive Genetics Institute, Chicago, Illinois, USA is associated with reduced embryo cell number with a Correspondence to Anver Kuliev, 2825 North Halsted Street, Chicago, IL 60657, possible negative impact on viability. Despite this poten- USA tial drawback, embryo biopsy is the method of choice for Tel: +1 773 472 4900; fax: +1 773 871 5221; e-mail: [email protected] paternally derived dominant conditions, translocations, Current Opinion in Obstetrics and Gynecology 2005, 17:179–183 gender determination, and HLA typing. Abbreviations FISH fluorescent in-situ hybridization Oocyte testing, which is performed by analysis of the first HLA human leukocyte antigen and second polar bodies (PB1 and PB2), permits an PB1, PB2 first and second polar bodies PCR polymerase chain reaction assessment of the maternal genetic contribution to the PGD preimplantation genetic diagnosis subsequent embryo. This technique is the method of choice for maternally derived genetic abnormalities. # 2005 Lippincott Williams & Wilkins Although information on gender and paternal mutations 1040-872X is not provided, oocyte testing can be used for autosomal 179 180 Prenatal diagnosis recessive disorders and for maternally derived dominant fingerprinting, and detection of common aneuploidies. mutations and translocations. It can be particularly useful Real-time PCR, which reduces the allele drop-out rate by in the diagnosis of X-linked diseases, while avoiding the almost half compared to conventional or fluorescent PCR 50% discard of healthy male fetuses associated with [6], can further improve diagnostic accuracy and be of gender-based determinations by blastomere evaluation particular value in cases with an insufficient number of [10]. Over 90% of chromosomal abnormalities originate informative markers. from maternal meiosis; thus, the polar body approach has particular relevance for PGD of aneuploidies related to Aneuploidy testing, at least for the chromosome with the maternal age. The high rate of mosaicism at the cleavage target gene, may be of special value in PGD for single- stage is a major limiting factor in blastomere-based PGD, gene disorders in women with advanced reproductive which, of course, does not impact on oocyte testing. age, because of the associated high aneuploidy rates in oocytes and embryos, and high prevalence of mosaicism Each of the PGD approaches, which are often comple- at the cleavage stage [3,4,19]. Without such information, mentary, should be available to ensure an accurate diag- the lack of a mutant allele due to chromosomal mono- nosis. As will be described below, the current standard of somy in the biopsied blastomere or oocyte cannot be PGD may require testing for a causative gene and HLA excluded; thus, the single-cell biopsy should ideally be typing along with aneuploidy testing, which in some tested simultaneously for the causative gene and specific cases cannot be done without a sequential polar body chromosome number. This can be achieved by adding and embryo biopsy. Further testing by blastocyst biopsy primers for chromosome-specific microsatellite markers may be necessary for confirmation of the polar body or to the multiplex PCR reaction designed for each genetic blastomere diagnosis. disorder. Accuracy of preimplantation genetic The accuracy of PGD for chromosomal aneuploidies can diagnosis also be improved. Biopsied single blastomeres may not The accuracy and reliability of PGD are key issues, represent the actual karyotype of the embryo, because up because of the limitations of single-cell DNA analysis. to half of the cleavage stage embryos have been shown to Critical to the design of any PGD strategy are methods to have chromosomal mosaicism [3,4,19]. Misdiagnosis due detect and avoid misdiagnosis. A key contributor to to mosaicism can be avoided by a fluorescent in-situ misdiagnosis is preferential amplification, known as hybridization (FISH) analysis in two or three steps, with allele-specific amplification failure (allele drop out), so initial testing for maternally derived aneuploidies (which special protocols are required to ensure the highest contribute at least 90% of all aneuploidies) by PB1 and possible allele drop-out detection rate [6,11–15]. Allele PB2 analysis, and subsequent removal and testing of drop-out rates in single-cell polymerase chain reaction single blastomeres to exclude paternally derived abnorm- (PCR) analysis have been shown to be around 10–20%, alities. Depending on the test results, a diagnosis may be depending on the type of cell tested [16], which would established from a blastocyst biopsy on day 5. lead to misdiagnosis, especially in compound heterozy- gous embryos [17,18]. Expanding indications for preimplantation genetic diagnosis Simultaneous detection of the mutant gene with up to Initially, indications for PGD, which were similar to those three highly polymorphic markers that are closely linked for prenatal diagnosis, generally included at-risk couples to the gene being tested effectively guards against mis- (25–50% chance of a transmitted genetic disorder) that diagnosis due to preferential amplification [6,11]. The could not accept pregnancy termination for an affected embryos should be transferred only if the polymorphic fetus. Recently, the indications for PGD have been sites and the mutation tests agree. Frequently utilized for extended to include conditions with low-penetrance, selecting mutation-free embryos in dominant conditions late-onset disorders with genetic predisposition, and and normal or heterozygous embryos in autosomal reces- HLA typing with or without testing for the causative gene. sive disorders, PGD should also include the detection of PGD has now been applied to more than 100 conditions, normal genes. Only multiplex amplification allows detec- with the most frequent indications still being cystic fibrosis tion of allele drop out, which provides critical protection and hemoglobin disorders. The choice between prenatal against the transfer of affected embryos. diagnosis and PGD mainly depends on the patient’s views on pregnancy termination, which are, of course, strongly Diagnostic errors