15 Preimplantation Genetic Diagnosis Mandy G. Katz-Jaffe Colorado Center for Reproductive Medicine, Englewood, Colorado, U.S.A. INTRODUCTION In 1990, preimplantation genetic diagnosis (PGD) was introduced as an experimental procedure to genetically screen human embryos during an in vitro fertilization (IVF) cycle (1,2). More than a decade later, PGD has become an established clinical procedure in assisted reproductive technolo- gies with over 6500 PGD cycles performed worldwide, resulting in the birth of well over 1000 healthy babies and a pregnancy rate per transfer of approximately 24% (3). The safety of PGD is reflected in these comparable pregnancy rates with conventional IVF, as well as the equivalent incidence of birth abnormalities in the general population (4). PGD was initially per- formed for preexisting Mendelian-inherited monogenic disorders including X-linked disorde rs involving sex selection (1), cystic fibrosis (5), and Tay- Sachs disease (6). With the development of interphase single-cell fluorescent in situ hybridization (FISH) in the early 1990s, PGD has expanded to offer screening for chromosomal disorders including aneuploidy detection for clinically significant chromosomes (7,8) and translocations (9,10). PGD involves the molecular analysis of genetic material derived from oocytes or embryos during an IVF cycle. Only embryos identified as free of the indicated genetic disorder or chromosomal error are selected for transfer to the woman’s uterus. Consequently, an established pregnancy is expected to be unaffected with respect to the indicated genetic testing. 313 SOURCE OF GENETIC MATERIAL There are three different sources of genetic material potentially available for PGD: polar bodies from the initial conception, blastomeres from early cleaving embryos, and trophectoderm cells from the later stage blastocyst. A biopsy is performed to remove these cells for subsequent genetic analysis (11). Several procedures have been developed to create a hole in the zona pellucida including mechanically by conventional partial zona dissection (12), chemically using acid Tyrodes solution (13), or with the use of non- contact laser technology (14,15). The biopsy of these cells is predominantly performed under an inverted microscope with contrast optics using glass holding, needle, or suction micropipettes, and a set of micromanipulators attached to a pneumatic- or hydraulic-based system (11). The biopsy of polar bodies extruded by the oocyte or blastomeres from cleava ge-stage embryos is considered safe on the basis of implantation and pregnancy rates reported in the literature that are comparable with conventional IVF (16) . Polar body biopsy can be performed preconceptionally to remove the first polar body or post-fertilization to remove either only the second polar body or both polar bodies simultaneously (Fig. 1) (17,18). Polar bodies are naturally extruded from the oocyte with no further role in the development of the future embryo. They each have a set of chromosomes that are comp- lementary to those present in the oocyte. The first polar body is formed during meiosis (M) I of oogenesis and has a set of bivalent chromosomes, whereas the second polar body is formed during MII, after fertilization, and contains a haploid set of chromosomes. Both polar bodies give comp- lementary diagnostic readouts and by deduction infer the genetic status of the oocyte. The main advantages of polar body biopsy include the Figure 1 Polar body biopsy. 314 Katz-Jaffe extra-embryonic nature of the polar bodies and the additional amount of time available for gene tic analysis prior to embryo transfer. However, the major disadvantage of this technique is that the sex and paternal genotype are not available for the analysis, thus precluding polar body biopsy for the analysis of paternal mutations, gender determination, and chromosomal abnormalities arising from paternal meiosis. In addition, polar bodies undergo fragmentation rendering them often difficult to biopsy which can potentially lead to misdiagnosis if the embryologist is unable to retrieve all the polar body fragments. Currently, the preferred stage for obtaining cells for genetic diagnosis is a blastomere biopsy of the cleavage-stage embryo performed at the 6–10-cell stage on day three post-fertilization (Fig. 2) (16). This allows for the retrieval of a blastomere containing both the maternal and paternal gen- omes. As these blastomeres are totipotent, the biopsy of 1–2 cells from the developing embryo does not seem to drastically reduce either the mass or contribution to the fetus, thereby not affecting the future viability of the embryo (19,20). Reports of ongoing comparable clini cal pregnancy and implantation rates with conventional IVF confirm these earlier studies (4). The blastomere biopsy procedure can be performed by either gentle aspir- ation (Fig. 2) or by the nudge of the flow of biopsy media. Even if the embryo has begun compaction with gap junctions forming between the blastomeres, a blastomere biopsy can be performed after a short pre-incubation in calcium–magnesium-free media to reduce the cellular apposition. The major disadvantage of blastomere biopsy is the invasive nature of the procedure on the embryo itself along with the reduction in cell Figure 2 Blastomere biopsy. Preimplantation Genetic Diagnosis 315 number and the potential influence on further fetal development. There has been debate in the PGD field as to whether one or two cells should be biop- sied. Clearly, the removal of two cells further reduces the cellular mass of the cleavage-stage embryo and may result in a reduction in developmental potential (21). Only a handful of studies have comp ared the outcome of one versus two biopsied cells, concluding that there were no decreases in implantation rates (22) with potentially fewer misdiagnoses (23). Ongoing prospective studies are underway to further address this question. A human blastocyst, depending on the exact stage of development, can contain over 100 cells. Hence, the biopsy of 6–10 cells from the outer layer of trophectoderm is unlikely to have a detrimental effect on the blastocyst’s mass or on the developing fetus that originates from the inner cell mass (ICM) (24–26). Prior to biopsy, the position of the ICM is identified so that the hole in the zona can be created on the opposite side of the blastocyst, reducing possible developmental and ethical concerns (Fig. 3). The cells are removed either mechanically by mild teasing using needles (24,27) or after herniation of the trophectoderm allowing biopsy by laser (Fig. 3) (28,29). The major advantage of this procedure is the larger amount of material available for the genetic testing, thereby increasing the reliability and accuracy of the diagnosis. However, the time for the analysis is limited to no more than 24 hours, as the blastocyst needs to implant at this stage. There are also uncertainties surrounding the genetic make up of the trophec- toderm in relation to that of the ICM, the future-developing fetus. Due to these issues and the fact that some clinics prefer not to culture to the Figure 3 Trophectoderm biopsy. 316 Katz-Jaffe blastocyst stage, only a limited number of IVF clinics perform blastocyst biopsy routinely. However, these clinics have reported ongoing clinical preg- nancies and healthy babies (28,29). PGD FOR MONOGENIC DISORDERS The current experience of PGD for monogenic disorders exceeds more than 1500 cycles comprising over 50 different conditions and the birth of more than 300 unaffected children (3). PGD has been performed for autosomal- recessive (e.g., cystic fibrosis), autosomal-dominant (e.g., Huntington disease), and X-linked (e.g., Fragile X) inherited disorders (3). PGD has been established as an acceptable form of early prenatal diagnosis with the spec- trum of conditions expanding with patient demand. The main motives behind couples seeking PGD are objection to potential therapeutic abortion ($47%), genetic risk coupled with low fertility ($32%), and repeated thera- peutic abortions of identified affected fetuses ($26%) (30). In addition to conventional monogenic disorders, PGD is now requested for conditi ons such as late-onset predisposition disorders, blood group incompatibility, and human leukocyte antigen (HLA) matching (3,31). In routine genetic diagnostic procedures, a starting template of at least 10 ng of DNA is usually available. However, a single cell contains only 6 pg of DNA (32) and has only two copies of each target locus. Some of the requirements that need to be addressed in PGD for monogenic disorders are the difficulties in the amplification of single-cell templates and the estab- lishment of a procedure for high amplification efficiency and accuracy. A considerable amount of time and resources are required for the development of reliable and accurate single-cell diagnostic tests including the preliminary mutation workup. Careful experimental practices and suitable facilities including allocated equipment and vigilant quality control are essential (16). Specific and individual mutation-detection systems have been developed to capture and visualize the different DNA variants involved in monogenic disorders, including single base pair substitutions, deletions, insertions, duplications, and trinucleotide repeat expansions (3,4). Polymerase chain reaction (PCR) is a common technique performed in PGD for monogenic disorders. It is a rapid, highly sensitive, and specific molecular technique that is capable of amplifying single copies of DNA tem- plate into large numbers with high fidelity (33). In a PCR reaction, several DNA sequences or loci can be independently amplified at the same time using multiple primer pairs in a technique called multiplex PCR. Once the DNA from the single cell has been amplified, there are numerous detection methods available to visualize the PCR products for the presence or absence of the specific DNA mutation or variant. The choice of the technique is usually dependent on the nature of the specific DNA mutation or variant and includes restriction endonuclease digestion (34,35), single-strand Preimplantation Genetic Diagnosis 317 conformational polymorphism (36), denaturant gradient gel electrophoresis (37), heteroduplex analysis (38), single nucleotide primer extension (39), and analysis of DNA fragment size (40,41). Fluorescent technology has further increased sensitivity of these detection methods resulting in the requirement of fewer PCR amplification cycles and greater reliability. Real-time PCR is a more recent molecular technique that allows the mutation amplification and subsequent fluorescent detection procedure to be carried out in the same tube. Fluorescently tagged probes directed to either the normal or mutant sequence allow detection of the rate of amplifi- cation product accumulation to be measured directly by associated computer software as the PCR reaction proceeds (42,43). Unfortunate ly, there has been a slow uptake of this technology in PGD labs due to the enormous costs involved in purchasing the specialized equipment and consu mables. Several misdiagnoses have been reported by clinics around the world due to the complexity and sensitivity of single-cell PCR analysis (16). It is of vital importance that PGD relies on a positive result from the biopsied cell, thereby reducing the possibility of the transfer of affected embryos. The three main sources of potential misdiagnosis in PGD include external DNA con- tamination, complete amplification failure, and allele drop out (ADO). External DNA contamination is a major problem due to the limited starting template and the large number of PCR amplification cycles required. Even at normal detectable levels, external DNA contamination may disguise or overwhelm a single cell and cause a misdiagnosis. The main laboratory contaminants include previously amplified PCR products accumulated in the laboratory and skin cells from the technician. The implementation of strict experimenta l practices and appropriate facilities will essentially mini- mize this risk including the following examples: a dedicated laminar flow hood with ultraviolet light to destroy any DNA by thymidine cross-linking, the isolation of all equipment for PGD use only, filtration and autoclaving of reagents, aliquots for storage, long sleeve lab gowns, caps and masks, and frequent glove changes (44). Pre-testing of all reagents and solutions prior to a clinical PGD case is essential to confirm the reliability of the test as well as for contamination prevention. Other potential sources of contaminants include the cumulus cells that surround the oocyte and excess sperm bound to the zona pellucida at the time of fertilization. These cells could accidentally be removed along with the polar body or blastomere during the biopsy pro- cedure. It is therefore recommended for PGD of monogenic disorders that all oocytes be completely striped of their cumulus complex and that intracyto- plasmic sperm injection is chosen as the method for fertilization. It is also possible to detect contamination by the simultaneous PCR amplification of highly polymorphic DNA markers (45). This is similar to DNA fingerprinting techniques whereby highly polymorphic DNA markers allow the distinction between maternal and paternal alleles (46). As poly- morphic DNA markers obey the laws of Mendelian inheritance, the 318 Katz-Jaffe embryos of any couple can only inherit a specific combination of alleles at any particular locus (47). Therefore, an incorrect combination of alleles or the presence of extra foreign alleles is an indication that contamination has occurred. It has become increasingly common for PCR-based single-cell tests to include the amplification of several polymorphic DNA markers alongside the mutation loci used for diagnosis. If intragenic markers are chosen, they are linked in 100% disequilibrium to the gene of intere st and are unlikely to be separated by recombination during meiosis. Hence, these polymorphic DNA markers ha ve a dual purpose to recognize potential extraneous DNA contamination and act as a positive control for amplifi- cation (34,38). Complete amplification failure of a mutat ion locus is observed in 5–10% of single cells. The source of the failure could be due to a number of factors, including failure to transfer the single cell into the PCR tube, an enucleate cell, degradation or loss of the target DNA sequence, and/or inefficient cell lysis (48,49). In contrast, ADO is defined as the amplification failure of either the maternal or paternal allele, giving the impression that a locus is homozygous. ADO has been observed to affect both parental alleles randomly. PGD involving dominant monogenic disorders is particularly vulnerable to misdiagnosis from ADO by the transfer of affected embryos that are incorrectly diagnosed as unaffected homozygous. One method to prevent misdiagnosis by ADO is the simultaneous amplification of intra- genic polymorphic DNA markers in combination with the gene of interest. These markers would be inheri ted alongside the mutation locus providing additional loci for the detection of affected embryos. It is improb able that all loci amplified during a multiplex PCR reaction would be affected by ADO (38,50). The factors that cause ADO are yet to be completely eluci- dated; however, differences in PCR thermal cycling conditions, fragm ent size, incomplete cell lysis, the degradation of target template sequence, freez- ing and thawing, and poor specificity of primer pairs could possibly explain the variability in observed ADO rates (51). The development of new reliable single-cell strategies, often for only one specific monogenic disorder, requires a major investment in resources, staff, finances, and time. Obviously, more adaptable and univers al tech- niques are required in PG D that will allow a wider range of mutations to be concurrently investigated. One platform that may be able to achieve this goal is microarray technology. Specific sequences of DNA incorporating dif- ferent mutations would act as probes on a microarray slide or chip, allowing hybridization between these known DNA probes and test DNA amplified from the single biopsied cell. Initial development of a specific cystic fibrosis deltaF508 array highlighted the diagnostic capability of microarrays for PGD (52). However, for this technology to be offered clinically, several issues need to be addressed including the reliable amplification of the whole genome from a single biopsied cell, a reduction in the complexity and time Preimplantation Genetic Diagnosis 319 for data analysis, and more cost-effective microarray platforms comprising DNA probes for numerous common monogenic disorders. Whole genome amplification (WGA) is a technique aimed at maximiz- ing the amount of information that can be obtained from a single cell or limited template. WGA theoretically involves the non-specific amplification of the entire genome, thereby increasing the amount of template for sub- sequent PCR reactions and multiple genetic analyses (53,54). There are several types of WGA protocols that have been developed to amplify DNA from small numbers of cells including primer extension preamplification (55), degenerate oligonucleotide primer PCR (56), and multiple displacement amplification (MDA) (57). Recently, MDA has been incorporated in clinical PGD for cystic fibrosis and b-thalassaemia resulting in two pregnancies (58). Some of the drawbacks to WGA methods include higher incidences of ADO, inaccurate size fragments, and inconsistent amplification of the whole gen- ome (59,60). It is paramount that any WGA protocol incorporated in clinical PGD be reliable, accurate, and complete in the amplification of the entire human genome from a single cell. PGD is considered an early form of prenatal diagnosis allowing high- risk couples to establish pregnancies free of the indicated genetic disorder. This technology is viewed as a positive contribution to the field giving cou- ples early reassurance and avoidance of therapeutic abortion. Indications for PGD will con tinue to grow with patient demand and advancing technology. The use of PGD for non- medical indications, including HLA matching for siblings suffering lethal diseases such as leukemia and late- onset diseases such as cancer predisposition, are also likely to become more common. These non-medical indications have attracted media attention and passionate public debate concerning the ethics of ‘‘designer bab ies.’’ In con- trast, it has been argued that PGD for non-medical reasons highlights the love and commitment of couples to treat and prevent disease in their chil- dren and therefore should be viewed as an acceptable treatment. CHROMOSOMAL ANEUPLOIDY SCREENING Chromosomal analysis of human IVF embryos using single-cell inter- phase fluorescent in situ hybridization (FISH) was first developed to screen for embryo sex, allowing for the detection of the two sex chromosom es (7,61). Over time, the number of chromosomes for detection has increased signifi- cantly, allowing for screening of chromosomal aneu ploidy in up to 9–10 chromosomes (62,63). Studies have shown that fetal chromosomal abnor- malities are associated with human implantation failure and pregnancy loss (64,65). Therefore, chromosomal aneuploidy screening in PGD was introduced for IVF patients who are considered to be at increased risk of producing embryos with chromosomal abnormalities. These at-risk groups include advanced maternal age (> 36 year), repeated miscarriages (RM), poor 320 Katz-Jaffe IVF prognosis (>3 failed cycles), and couples who carry a chromosome rearrangement (such as translocations and inversions). Chromosomal aneu- ploidy screening of these patients’ embryos should identify euploid embryos for the indicated chromosomes leading to a greater chance of implantation and clinical pregnancy. PGD for aneuploidy screening now accounts for the majority of PGD cycles worldwide, estimated at close to 5000 cycles (3). The benefit of PGD for aneuploidy screening has been reported by several groups showing an increase in implantation rates and decrease in miscar- riage rates (31,62,63,66,67). Single-cell interphase FISH is a rapid, reliable, and efficient technique capable of detecting up to 9–10 chromosomes in two rounds of hybridiza- tion on a single nucleus (62,63). Currently, fluorescent DNA probes for chromosomes X, Y, 13, 14, 15, 16, 18, 21, and 22 are being used in PGD for aneuploidy screening as they are involved in more than 50% of all chromosomal abnormal miscarriages (65). In the cases of chromosomal translocations, probes distal to the sites of chromosome breakage are used in addition to centromeric and proximal probes (68). The technical dif- ficulties encountered in regards to selecting appropriate FISH probes and optimizing protocols for each couple’s specific chromosome rearrangement are considerably time-consuming and expensive. However, these couples are considered to be one of the most motivated groups of PGD for aneuploidy screening due to their history of RM and infertility. A clear advantage has been documented in more than 500 clinical cycles with a fourfold reduction in miscarriage rates and an increase of live births (69,70). Successful FISH involves annealing of the single-stranded fluorescent- labeled DNA probes to its complementary target sequence on a specific chromosome. The biggest limitation to this technique is the fact that only one or two cells are available for analysis. The error rate for single-cell inter- phase FISH has been recorded in several studies at frequencies betw een 5 and 15% (18,68,71). Numerous variables could be responsible including signal overlap, signal splitting, cross-hybridization of FISH probes, and the presence of chromosomal mosaicism (72) . A greater number of monoso- mies have been diagnosed by single-cell FISH than trisomies. This could be due to insufficient binding, loss of DNA, poor probe penetration, or an overlap of chromosome signals due to the poor spread of the nucleus during fixation (68). In an attempt to counteract the possibility of misdiagnosis, a FISH scoring system has been implemented (68) to reduce the incidence of false-positive and false-negative results. Nevertheless, several misdiag- noses have been recorded, where aneuploid embryos were misdiagnosed as normal, but on transfer resulted in aneuploid pregnancies that either spontaneously aborted or were detected after prenatal diagnosis (16). Interestingly, the data from FISH analysis of human IVF embryos have revealed a high incidence of chromosomal mosaicism, with over 30% containing a proportion of aneuploid cells (61,73,74). These high rates of Preimplantation Genetic Diagnosis 321 chromosomal mosaicism observed in human IVF embryos are a major con- cern in chromosomal aneuploidy screening, questioning the validity of the test, with the possibi lity of transferring affected mosaic embryos (75,76). Embryonic chromosomal mosai cism is the existence of two or more differ- ent chromosomal complements in a single embryo. Mitotic cell division errors post-fertilization, appear to be responsible for the observed chromo- somal mosaicism in early human preimplantation development (77, 78). The chances of detecting mosaicism would depend on the timing of the mitotic cell division error, e.g., a non-disjunction event during the second cleavage division would resul t in a 25% chance of biopsing an aneuploid blastomere. Studies have also revealed frequent mosaicism in both the trophe ctoderm and ICM of human blastocysts (79–81), with the significance at this stage of human embryonic development still to be clarified. The current under- standing of the normal dynamics and regulation of mitotic chromosomal segregation during early embryonic cleavage divisions is critically insuf- ficient. Hence, several clinics support the biopsy and analysis of two blastomeres for chromosomal aneuploidy screening in order to reduce the chance of misdiagnosis due to mosaicism (22,82). However, this approach cannot completely overcome the possibility of a misdiagnosis, nor does it address the problem of mosaicism in human IVF embryos. One approach to gaining a better understanding of chromosomal mosaicism during human preimplantation development is to determine the underlying mechanisms causing this phenomenon includi ng the origin and nature of the cell division errors (83). Such knowledge may translate into a revision of current clinical and lab procedures to produce higher numbers of non-mosaic embryos available for transfer, thereby potentially improving implantation rates. Current single-cell interphase FISH methods are limited to the analysis of less than half of the human chromosomal complement. It is hypothesized that the development of a technique that can analyze all 23 pairs of human chromosomes will allow for the selection of entirely euploid embryos for transfer, furth er improving pregnancy rates and decreasing miscarriage rates for indicated couples. There are several alternatives that are currently being investigated including metaphase nuclear conversion. Customarily, cytogenetic techniques are performed on cells that are in meta- phase, when the nuclear membrane has broken down and the chromosomes are condensed allowing for the identification of each individual chromo- some. However, blastomeres are typically observed to be in interphase, when chromosomes are in an unrecognizable state. Metaphase nuclear con- version is a technique that fuses blastomeres or second polar bodies with enucleated or intact oocytes (mouse, bovine, or human) allowing for the metaphase visualization of all 23 pairs of chromosomes to identify both chromosomal aneuploidy and rearrangements. 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Comparison of the validity of preimplantation genetic diagnosis