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Volume 53 (3): 255-260, 2005
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
Preimplantation Genetic Diagnosis—An Overview
Guy's & St Thomas' Centre for PGD, Cytogenetics Department (CMO,PNS), and Assisted Conception Unit (PRB), Guy's & St Thomas' Hospital Trust, London, UK
Correspondence to: Caroline Mackie Ogilvie, Cytogenetics Department, 5th Floor, Guy's Tower, St Thomas St, London SE1 9RT, UK. E-mail: caroline.ogilvie{at}genetics.kcl.ac.uk
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Summary |
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 Top Summary Literature Cited |
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Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye. (J Histochem Cytochem 53:255–260, 2005)
Key Words: preimplantation genetic • diagnosis • aneuploidy screening • sex selection • embryo biopsy • assisted conception • chromosome rearrangements
COUPLES WITH GENETIC disorders including recessive or dominant single gene defects, sex-linked conditions, or chromosome rearrangements face a reproductive risk: affected pregnancies may result in miscarriage or in the birth of a child with significant phenotypic abnormality, sometimes resulting in early death. Such couples have a number of reproductive choices. They may (a) opt for prenatal diagnosis followed by pregnancy termination in the case of an affected fetus, (b) choose gamete donation or adoption, or (c) decide to remain childless. In the last 10 years, another possibility has become available—preimplantation genetic diagnosis (PGD). PGD aims to significantly reduce a couple's risk of transmitting a genetic disorder while at the same time provides a realistic chance for the birth of a healthy child. Since the early 1990s, this technology has expanded in scope and applications and is now an established reproductive option, offered worldwide at specialist centers.
PGD uses standard assisted reproduction technologies, including controlled ovarian stimulation, oocyte retrieval, in vitro fertilization/intracytoplasmic sperm injection (ICSI), and in vitro embryo culture (Pickering et al. 2003a
). The fertilized egg undergoes reductive cell division (Figure 1) and reaches the eight-cell stage around 3 days postfertilization. Morula formation is on day 4, and the embryo reaches the blastocyst stage on day 5 when the inner cell mass, a discrete clump of cells destined to become the fetus, is clearly differentiated from the trophectoderm, destined to form the extra-embryonic tissues.
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PGD requires the biopsy of material from either the oocyte and/or the developing embryo. The biopsied material is tested for the genetic condition, and unaffected embryos (usually no more than two) are transferred to the uterus. First and second polar bodies may be biopsied and the results used to infer the genetic status of the oocyte (Verlinsky et al. 1990
). This technique is labor intensive and cannot be used for conditions where the male partner carries the genetic disorder but has the advantage of removing only material that is non-functional, thus being minimally invasive and damaging. The most widely used approach is to biopsy single blastomeres from embryos on day 3 (De Vos and Van Steirteghem 2001), when 5 to 10 cells should be available. Embryos are first transferred to calcium-free medium to disassemble tight junctions and then stabilized by suction onto a holding pipette. The zona pellucida is breached by laser, acidic solution, or mechanical abrasion, and a single cell is removed by biopsy pipette (Figure 2). Material may also be taken from the embryo at blastocyst stage following breach of the zona pellucida. The trophectoderm tends to herniate, allowing cells to be excised for testing (Dokras et al. 1990). This ensures a larger amount of material for testing than either polar body or blastomere biopsy and does not impinge on the inner cell mass. The disadvantages of this approach are that culture conditions may not allow all embryos to progress to the blastocyst stage in vitro. In addition, blastocysts must be transferred to the uterus before hatching on day 6; the time available for diagnosis is thus very limited.
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For genetic testing, material from these biopsy procedures may be tested by polymerase chain reaction (PCR) amplification of specific sequences. Amplified fragments can be analyzed according to the specific requirements of the test; procedures such as restriction digestion, sequencing, and analysis of fragment length polymorphisms have been used (Sermon 2002
; Thornhill and Snow 2002). The introduction of fluorescence multiplex PCR (Findlay et al. 1996), now widely used, allows the incorporation of linked markers and ameliorates some of the single-cell PCR-associated problems such as allele drop-out and contamination. Alternatively, fluorescence in situ hybridization (FISH) protocols may be applied to genetic material (metaphase chromosomes from polar bodies or nuclei from blastomeres) spread onto microscope slides. Dehydration is followed by application of targeted probe mixes. Probes may be directly labeled with fluorochromes or indirectly labeled with reporter molecules, detected using fluorescent antibodies. The copy number of the targeted regions is ascertained by fluorescence microscopy. Problems associated with FISH testing include difficulties in obtaining the required probes with necessary fluorochrome/reporter molecule labeling, split signals/signal overlap, and probe target polymorphisms. This latter problem makes it advisable to test both reproductive partners with the required probes to identify any such polymorphisms, especially where the probes may have had limited validation in the clinical arena.
Whole genome amplification followed by comparative genomic hybridization (CGH) detects imbalance across the genome and has been used in clinical PGD cycles with blastomere biopsy, with successful outcomes (Voullaire et al. 1999; Wells and Delhanty 2000). The technology is time consuming, and biopsied 3-day embryos need to be frozen pending the results of the CGH, with associated compromise of embryo quality (Wilton et al. 2001; Munne and Wells 2003). CGH application to polar body analysis may be promising for the future (Wells et al. 2002).
PCR technology is applicable to single gene disorders such as cystic fibrosis (Goossens et al. 2003) or spinal muscular atrophy (Daniels et al. 2001), where the familial mutations are known. Single-cell PCR tests for PGD have now been developed for over 30 different monogenic diseases (ESHRE 2002). The latest published data collection from the European Society for Human Reproduction and Embryology (ESHRE 2002) details a 21% pregnancy rate per oocyte retrieval and a 25% pregnancy rate per embryo transfer for monogenic diseases. Three misdiagnoses were reported. The availability of affected embryos following PGD cycles has allowed the possibility, with appropriate consent, for the creation of stem cell lines. These cell lines will provide an invaluable in vitro resource for studying the development and etiology of the phenotype arising from these single gene defects (Pickering et al. 2003b).
The first clinical application of PGD used PCR amplification of Y chromosome-specific sequences for the determination of embryo sex in a case of sex-linked disease (Handyside et al. 1990). Although initially successful, it became apparent that PCR amplification at the single cell level is prone to difficulties such as amplification failure, allele dropout, and contamination with extraneous DNA, and misdiagnoses resulted in the transfer of male embryos in two cycles. Sex determination is therefore now usually carried out using FISH technology.
FISH for single-cell sex determination uses repeat sequence probes, fluorescently labeled, specific for the centromere regions of the X and the Y chromosome, and usually uses a third probe for the centromere of an autosome. Signals specific for each chromosome can be visualized and counted in the fixed nucleus (Figure 3). This protocol has proved to be very robust (Kuo et al. 1998) and has a built-in "internal check," whereby two mistakes must occur for the misdiagnosis of a male embryo as female (failure to detect the Y-specific signal and detection of an extra X-specific signal). However, sex determination for sex-linked disease is not ideal, because 50% of male embryos will be unaffected by the disorder. As specific protocols for mutation detection are developed, perhaps using generic solutions such as minisequencing (Fiorentino et al. 2003), it is likely that the need for sex selection in this context will decline.
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In many centers, chromosome rearrangements (such as Robertsonian and reciprocal translocations and inversions) constitute a significant proportion of the PGD workload. One person in 500 is a phenotypically normal carrier of such a rearrangement, many of which are family specific. Carriers requesting PGD typically present with reduced fertility, recurrent miscarriage, or live birth of a child with genetic imbalance arising from the chromosome rearrangement. Early protocols for PGD developed specific breakpoint-spanning probes for each rearrangement (Munne et al. 1998a
); however, an approach using subtelomere-specific FISH probes in combination with centromeric probes obviates the need for specific probe development for each rearrangement (Scriven et al. 1998). For a reciprocal translocation, there are 32 possible outcomes of meiotic segregation; of these, the "normal" and "balanced carrier" embryo nuclei will show identical signal patterns (using the generic subtelomere/centromere approach), corresponding to their balanced genetic status, whereas the 30 abnormal outcomes will give different signal patterns. Depending on the probe combination chosen, with "internal checks", some will minimize the risk for misdiagnosis of abnormal embryos as normal/balanced (Figure 4). PGD has allowed the study of female meiotic segregation of reciprocal translocations (Mackie Ogilvie and Scriven 2002), previously unattainable due to the inaccessibility of female gametes.
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Success rates for PGD for chromosome rearrangements vary widely among centers. A review of data from three large centers reports a 29% pregnancy rate per oocyte retrieval and 38% per embryo transfer (Verlinsky 2001
). The Guy's and St Thomas' Centre for PGD has a success rate of 19% per oocyte retrieval and 29% per embryo transfer, whereas the ESHRE Consortium reports 16.8% per oocyte retrieval and 21.4% per embryo transfer. The ESHRE PGD Consortium results included one misdiagnosis for this application due to injudicious choice of probes.
PGD FISH protocols have been harnessed to address the problem of low IVF success rates for some groups of patients. These include older women (over 37 years), couples with recurrent implantation failure following IVF, and couples with recurrent miscarriages. FISH probes for the common viable abnormalities of chromosome copy number (trisomy for chromosomes 13, 18, and 21, and monosomy X), as well as for those abnormalities found in products of conception (trisomy for chromosomes 15, 16, and 22), have been used to test oocytes (Verlinsky et al. 1996) and cleavage-stage embryos (Munne et al. 1998b). There are few published controlled trials for the efficacy of aneuploidy screening, but those that exist indicate that PGD-AS increases the embryo implantation rate and reduces the miscarriage rate (Gianaroli et al. 2002; Munne et al. 2003). However, this remains a highly specialized procedure, potentially effective in experienced hands but of unknown benefit when applied across the board. The ESHRE PGD Consortium reports a 28% pregnancy rate per cycle started following PGD-AS for advanced maternal age, 28% for couples with recurrent miscarriage but only 7% for couples with recurrent IVF failure. Two affected pregnancies following PGD-AS have been reported to the ESHRE PGD Consortium. PGD-AS is now the most common application for PGD, accounting for 39% of PGD activity in 2001 compared with 23% for single gene disorders and 21% for chromosome rearrangements (ESHRE 2002). Use of these technologies is still to be balanced against the likelihood of a positive outcome without them, in appropriate randomized trials.
The application of social sex selection uses the same technology as that used in PGD for sex-linked disease and raises significant ethical issues. In some countries, "family balancing" is allowed, i.e., where there is at least one child of one sex, which can be "balanced" by social sex selection (Malpani and Modi 2002). Much debate has taken place over the rights and wrongs of the desire of some couples for a child of a particular sex (Pembrey 2002; Robertson 2003). Many, however, feel it would be unethical to subject a woman to the invasive and potentially damaging PGD protocols for such a purpose, because less invasive techniques such as sperm sorting may be acceptable. A telephone survey of adults in the US indicated majority approval for PGD for genetic disease, HLA matching (see below), and cancer predisposition but majority disapproval for social sex selection and "desirable characteristics" (Vastag 2004). Social sex selection accounted for 9% of the PGD activity reported to the ESHRE PGD Consortium.
The provision of "saviour siblings" by PGD has aroused fierce controversy, some seeing this as an unacceptable step along the road to "designer babies." PCR protocols are used to provide an HLA type for each embryo, with the intention of matching an embryo to the sick child in the family (Verlinsky et al. 2001; Verlinsky et al. 2004). This embryo is then transferred in the hope of establishing a pregnancy; hence, having stored cord blood stem cells and potentially bone marrow as material for treating the older sibling. In the UK, the Human Fertilisation and Embryology Authority (HFEA) has recently reversed its ruling that HLA typing can only be carried out where the embryos are also being tested to exclude transmission of the familial genetic defect causing the sibling's pathology (http://www.hfea.gov.uk).
Difficulties and dilemmas arise with all protocols used for PGD as these protocols are subject to error. This error may be reduced by biopsying two cells rather than one and only transferring an embryo where the two cells give a concordant normal result. Another strategy for reducing error is to increase the number of FISH probes or linked markers used in each test. These strategies may well reduce the false-negative rate and avoid affected pregnancies, but they also increase the false-positive rate, which excludes some normal embryos from transfer. Embryo attrition is a significant problem in PGD, where on average 50% of embryos may be abnormal due to the genetic condition in the family (e.g., in the case of dominant single gene disorders or reciprocal translocations). The pool of embryos available for transfer may thus be very limited, and the exclusion of normal embryos is likely to significantly affect the chances of establishing a pregnancy. PGD should thus be seen as a balance between reducing the risk of genetic abnormality and maintaining a good chance of establishing a pregnancy. Heroic efforts to reduce misdiagnosis risks to a minimum may result in a disappointing "take-home baby" rate (Ogilvie 2003).
Multiple pregnancies are associated with clinical problems for the babies and carry risks for the pregnant mother. In addition, multiple pregnancy following PGD increases the risk of misdiagnosis. The transfer of more than two embryos may result in an unacceptable multiple pregnancy rate, and guidelines limiting the number of embryos that can be transferred following IVF/PGD are in place in some countries (see HFEA Code of Practice 6: www.hfea.gov.uk).
These risks should all be discussed with a couple prior to their PGD cycle so that they can make an informed decision about whether to proceed with the treatment. Most PGD centers recommend prenatal diagnosis in the event of a successful pregnancy, but uptake of this option is usually low, probably due to reluctance to endanger a precious pregnancy.
For regulation and data collection, in the UK the HFEA oversees and licenses all procedures relating to embryo creation and manipulation. A license is required for each PGD center for each new condition to be tested, including a separate license for each unique chromosome rearrangement. In contrast, there is no federal regulation of PGD in the United States (Braude et al. 2002). In Germany, only procedures of direct benefit to the embryo are allowed, and PGD is therefore prohibited at any point following pronuclear fusion. PGD is banned in some other European countries, including Italy, where draconian legislation has recently been passed.
The ESHRE PGD Consortium collects and analyzes data from over 60 PGD centers worldwide and publishes these results regularly, providing a standard reference in the literature (ESHRE 2002). Several groups, including the ESHRE PGD Consortium, undertake follow-up of children born following PGD to track the level of phenotypic abnormality and to assess the safety of PGD. The recently formed PGD International Society (PGDIS) (http://www.rbmonline.com) is also monitoring activity that will include data from PGD centers in the US that do not contribute to the ESHRE PGD Consortium.
In summary, PGD for couples at risk of conceptions with serious genetic disorders is firmly established as a valid reproductive option for couples to consider following appropriate genetic counseling. The procedure entails a balance of risks between establishing a successful pregnancy and minimizing the risk of misdiagnosis. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while at the same time provoking interesting ethical debates and keeping PGD firmly in the public eye.
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Acknowledgments |
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We thank Dr Susan Pickering for the embryo photographs shown in Figures 1 and 2.
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Footnotes |
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Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich-Schiller-University, Jena, Germany, April 17–18, 2004.
Received for publication June 22, 2004; accepted November 29, 2004
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