Preimplantation genetic diagnosis: current status and new developments

Transcription

1 Human Reproduction vol.12 no.8 pp , 1997 Preimplantation genetic diagnosis: current status and new developments Willy Lissens 1 and Karen Sermon However, owing to the risk of misdiagnosis (one male pregnancy had to be terminated; Hardy and Handyside, 1992), Centre for Medical Genetics, University Hospital and Medical School of the Dutch-speaking Brussels Free University, sexing by PCR has now largely been abandoned in favour of Laarbeeklaan 101, 1090 Brussels, Belgium the fluorescent in-situ hybridization (FISH) technique (Griffin 1 To whom correspondence should be addressed et al., 1994; Munné et al., 1994). FISH has an advantage over PCR for sexing in that it detects the X and the Y chromosomes Preimplantation genetic diagnosis (PGD) is a very early simultaneously, as a result of which not only the sex of the form of prenatal diagnosis aimed at eliminating embryos embryo can be determined but also whether there is aneuploidy carrying serious genetic diseases before implantation. To of the sex chromosomes (Harper et al., 1995). this end, two major technologies are in use: the polymerase Soon, other applications of PCR were developed, such as chain reaction (PCR) for monogenic deseases and fluores- for cystic fibrosis, a disease determined by a single gene cent in-situ hybridization (FISH) for chromosomal aberra- inherited in an autosomal recessive manner (Handyside et al., tions. In this review, a number of problems arising from 1992; Liu et al., 1994). A new problem soon emerged in the the use of these technologies, as well as their possible form of allelic drop-out (ADO), i.e. the non-amplification of solutions and new developments, are discussed. Concerning one or both alleles in a heterozygous single cell. This phenom- PCR, the phenomenon of allelic drop-out, as well as enon probably even led to misdiagnoses in PGD for cystic methods to reduce this problem, such as fluorescent PCR, fibrosis (Grifo et al., 1994; Harper and Handyside, 1994; are described. The advantages and disadvantages of sperm Verlinsky, 1996): in the reported cases, both parents were separation by flow cytometry as an adjunct to sex deter- carriers of different mutations, complicating accurate diagnosis mination for the avoidance of X-linked disease are discus- even more. ADO has since been well studied and a number sed. The application of FISH for aneuploidy detection is of improvements have been proposed. These will be discussed commented upon and the advances in cell recycling, in in this review. One of these improvements is fluorescent PCR, which PCR and FISH are combined, are analysed. Finally, where PCR fragments are not detected on ethidium bromidediseases for which PGD is currently possible are sum- stained gels, as is done routinely, but are labelled with a marized. fluorophore so that they can be detected when excited by the Key words: FISH/preimplantation genetic diagnosis/single- correct laser (Hattori et al., 1992). This fairly new technology cell PCR will also be discussed below. Another topic of this review is the selection of X-bearing spermatozoa which has been used as an adjunct to PGD in avoiding X-linked diseases. The FISH technique has also been refined: up to five different Introduction chromosomes can now be detected in a single cell simultaneously, The idea of preimplantation genetics is not new: in 1967 allowing screening for the commonest aneuploidies Edwards and Gardner succeeded in sexing rabbit embryos (e.g. trisomies 13, 18 and 21) (Munné et al., 1994). Moreover, at the blastocyst stage, after which they were successfully this multicolour FISH may be used to diagnose unbalanced transferred (Edwards and Gardner, 1967). In that article, the transmission of translocations in embryos from parents carrying authors predicted the use of similar technology in humans to a balanced translocation. Finally, the two techniques, FISH avoid genetic disease. However, we had to wait until the and PCR, meet in so-called cell recycling, where they are beginning of the 1990s for a useful technology to emerge that carried out consecutively on the same cell so as to be able to would allow the detection of mutations in single cells: the glean as much information as possible from one or two cells polymerase chain reaction (PCR) (Saiki et al., 1985). PCR (Thornhill and Monk, 1996). A part of this review will be allows the amplification of a well-defined DNA sequence dedicated to these new developments. In the last part of the exponentially, ending up with a workable amount of DNA, review, we shall summarize for which genetic diseases PGD even when starting from just one single cell. The first clinical has already been used and for which genetic diseases a test is application of this technique for preimplantation genetic diagnosis being developed. (PGD) was described by Handyside et al. (1990): Y- chromosome-specific sequences were amplified using PCR to determine the sex of embryos obtained from couples at risk Allele drop-out: what is it and how can it be avoided? of X-linked diseases. Only female embryos were transferred Allele drop-out (ADO) was first described on a completely and several healthy girls were born following this procedure. theoretical basis by Navidi and Arnheim (1991). These authors 1756 European Society for Human Reproduction and Embryology

2 Preimplantation genetic diagnosis calculated what the effects of contamination and (partial) others: when using a denaturation temperature of 90 C, 21% failure of amplification would be on the diagnosis of autosomal of heterozygous lymphocytes show ADO, at 93 C the ADO recessive and dominant diseases and on X-linked genetic rate decreases to 13%, finally to reach 5% when a denaturation diseases. They concluded that (partial) failure, or ADO as it temperature of 96 C is used. We have also found that the is now usually called, would create a particular problem for denaturation temperature has a profound effect on the ADO the correct diagnosis of autosomal dominant diseases, where rate: for CF/ F508, we were able to decrease the ADO rate the affected allele would fail to amplify, and recessive diseases from 25% (five out of 20 cells) when using 94 C, to 10% involving two different mutations, when only one mutation (two out of 20 cells) when using 96 C (unpublished data). would be analysed and the affected allele would fail to amplify. Ray and Handyside (1996) emphasized the need for stringent It took until 1994 before the phenomenon of ADO was fully lysis conditions, as well as the need for efficient separation of recognized in a clinical setting, when Ray et al. (1994) found the native double-stranded DNA. As other causes of ADO, that 25% of the blastomeres heterozygous for the F508 they proposed a strand break or other DNA damage by, for mutation in the cystic fibrosis (CF) gene analysed during example, endogenous endonucleases. Apart from raising the clinical PGD revealed ADO. The phenomenon can be tracked denaturation temperature, a number of strategies for decreasing retrospectively in a number of publications. Levinson et al. the ADO rate have been proposed, or at least for avoiding (1992) described a multiplex PCR for the sexing of embryos misdiagnoses as a result of ADO. Findlay et al. (1995a) have in which they amplified the amelogenin gene on the X and Y presented a large body of evidence to show that fluorescence chromosomes as well as the repetitive DYZ1 sequence on the PCR, which is much more sensitive than conventional PCR, Y chromosome. From their table it can be calculated that they further decreases the ADO rate, because a number of ADO had an amplification efficiency of 92% and an ADO rate cases are in fact preferential amplifications which are classified of 21%. We have also encountered ADO in a blastomere as ADO by conventional PCR. Another possibility is to develop heterozygous for the F508 mutation in the CF gene although a PCR strategy in which ADO is detected, or alternatively, it was not recognized as such at that time (Liu et al., 1993). where ADO does not lead to misdiagnosis (Lissens et al., Since then, a number of authors have described ADO in 1996). An example of the first strategy is the simultaneous different PCR assays, amplifying a considerable number of amplification of two different mutations in a compound heterozygous different DNA sequences. The authors who encountered the embryo using only one primer pair in the first PCR lowest ADO rates are Strom et al. (1994): in a series of 168 (remember these are the cases where misdiagnoses were heterozygous cells (116 for CF/ F508 and 52 for Factor VIII) reported), as described by Ray et al. (1996b) for β-thalassaemia only one cell showed ADO (0.6%). Even with cell-recycling, major and Sermon et al. (1995a) for Tay Sachs disease. As a where PCR is carried out in suboptimal conditions because result of ADO, the embryo would appear to be homozygously FISH has to be performed on the same cells afterwards, they affected for either of both mutations, which is an impossible obtained an acceptable ADO rate of 12% (five cells out of 43) result. Another example is the multiplex PCR described by (Rechitsky et al., 1996). A number of causes of ADO have Verlinsky et al. (unpublished; presented at the 6th annual been put forward. Firstly, Harper et al. (1996) pointed out that meeting of the International Working Group on Preimplantation chromosomal mosaicism, and more specifically the presence Genetics, Rio de Janeiro, August 18, 1996), where the F508 of haploid cells in an otherwise diploid embryo, could lead to mutation and a nearby polymorphism in intron 6 of the CF misdiagnosis, especially in autosomal dominant diseases. The gene are amplified in multiplex and definite diagnosis is only way to reduce this type of misdiagnosis is to biopsy two ascribed only to cells showing concordant results for both loci. cells from the embryo. Secondly, a number of authors showed We have developed a single-cell PCR assay for Steinert s that the method used for lysing the cell before PCR has a disease, where only the healthy alleles of both parents are huge effect on the efficiency of the PCR, as well as on the amplified, rather than the expanded allele of the affected parent ADO rate. We (Sermon et al., 1995a) and others (Wu et al., (Sermon et al., 1997). Thus, only the presence of the healthy 1993; Gitlin et al., 1996; Kontogianni et al., 1996) showed allele of the affected parent would lead to a diagnosis of a that the alkaline lysis method as described by Cui et al. (1989) healthy embryo, whereas drop-out of this allele would lead to gave higher amplification efficiencies than did lysis in water the diagnosis of an affected embryo and to non-transfer of the followed by boiling, lysis in water followed by freeze thawing embryo concerned. and lysis in a complex buffer containing dithiotreitol (DTT) and proteinase K. Moreover, Wu et al. (1993) and Gitlin et al. (1996) revealed a dramatic difference in ADO rate between Fluorescent PCR: advantages and possible applications the different lysis protocols: a decrease from 19% (DTT and The technical intricacies of fluorescent PCR have been extensively proteinase K) to 10% (alkaline lysis) for CF/ F508 as shown described in Hattori et al. (1992). Briefly, PCR products by Wu et al. (1993), and a decrease from 89% (freeze are fluorescently labelled through the incorporation of fluorescently thawing) to 9% (alkaline lysis) for Tay Sachs disease as shown labelled primers. They are then detected after excitation by Gitlin et al. (1996). Thirdly, as demonstrated by a series by a laser during electrophoresis. The greatest advantage of of experiments by Ray and Handyside (1996) and Ray et al. this method is that it is much more sensitive than conventional (1996a) on blastomeres and lymphocytes, the denaturation PCR followed by staining of the PCR products with ethidium temperature at the start of the first PCR is primordial. The bromide. Only 35 PCR cycles are required to visualize PCR authors demonstrated this for the CF/ F508 mutation amongst products starting from single cells, as compared to

3 W.Lissens and K.Sermon cycles required for conventional single-cell PCR, thus obviating FISH-based procedures have been developed and used for sexing the need for nested PCR. Because of this, and because a prior in PGD, but as mentioned above FISH is now largely preferred to lysis of the cells is not necessary according to Findlay et al. PCR because of the additional advantage that sex-chromosome (1995a), the whole procedure is substantially simplified and aneuploidy can be detected. less sensitive to contamination. Moreover, the ADO rate is On a theoretical basis, half of the embryos produced after decreased dramatically by the use of fluorescent PCR: Findlay in-vitro fertilization (IVF) or intracytoplasmic sperm injection et al. (1995a) found an ADO rate for CF/ F508 of 4.3% using (ICSI) will be female. These female embryos can be used for a denaturing temperature of 94 C. This discrepancy might be transfer. The other half of the embryos will be male and these due to the fact that conventional PCR cannot distinguish ADO are unsuitable for transfer. Levinson et al. (1995) separated X- from extreme preferential amplification of one or both alleles. and Y-chromosome-bearing spermatozoa by flow cytometry and Another possibility would be that, since fewer PCR cycles are used the X-chromosome-enriched fraction of spermatozoa for required in fluorescent PCR and PCR is exponential, a difference IVF in a couple at risk of X-linked hydrocephalus. The sperm in amplification efficiency between two alleles would be separation procedure is based on the mean 2.8% difference in less obvious in the first cycles of PCR. Since we found a high DNA content of spermatozoa containing an X or a Y chromo- rate of ADO for our single-cell PCR for Steinert s disease some. In two applications the separation resulted in 80% and (21% in blastomeres; Sermon et al., 1997), we adapted this 89% X-chromosome-enriched sperm fractions (purity assessed assay for fluorescent PCR. A substantial decrease in ADO was by FISH) that produced seven out of nine and 15 out of 16 female noted: 21% (5/39) heterozygous lymphoblasts showed ADO embryos after IVF. A healthy girl was born. The yield of female when assayed with conventional PCR, whereas only 5% (1/19) embryos available for transfer can therefore be considerably lymphoblasts showed ADO with fluorescent PCR (unpublished improved. Moreover the number of male embryos is minimal, results). Similar results were obtained with blastomeres: 1/ reducing the ethical complications of discarding these embryos, 19 (5%) of the heterozygous cells showed ADO. Another half of which are unaffected. application of fluorescent PCR would be multiplex PCR, which The advantages and disadvantages of the technology of sex is notoriously difficult with conventional PCR. Findlay et al. preselection and its application in the human have recently been (1995b) described a simultaneous fingerprinting, diagnosis of discussed in detail (Edwards and Beard, 1995). The technology sex and mutation analysis for CF in single cells. Successful is still expensive and very specialized such that it precludes large amplification (i.e. amplification of all microsatellites and both scale use at present. Moreover, the use of two potential mutagenic sexing alleles) was obtained in 71% of the samples, while agents in the separation procedure, the dye Hoechst and partial amplification (i.e. amplification of some but not all ultraviolet light, is of some concern in the human (Ashwoodmicrosatellite fragments) occurred in 25% of the samples. Smith, 1994; Munné, 1994), although there is no evidence for Although these results are no different from the ones obtained obvious anomalies in many animals born after sperm separation. by Wu et al. (1993) for a conventional multiplex amplifying Improvement of the technology in terms of separation efficiency CF/ F508 and HLA DQA1 (72% of the samples showed may in the future make sperm separation by flow cytometry, in successful amplification as defined above), it must be taken combination with ICSI, the procedure of choice for the preven- into account that the microsatellite chosen by Findlay et al. tion of X-linked disease without the need to apply PGD further (1995b) is far more complex (six different loci per genome) to embryos generated with purified X-bearing spermatozoa. and thus much more subject to ADO. In conclusion, according to Findlay et al. (1996) the following advantages of this method can be enumerated: (i) sensitivity Cell recycling is increased 1000-fold, (ii) accuracy is high (to 1 2 bp), (iii) The concept of cell recycling was first introduced in 1994 accurate multiplex PCR, (iv) highly accurate and reliable at (Thornhill et al., 1994) with the aim of broadening the range single-cell level, (v) fewer PCR cycles are required, (vi) signals of possibilities in single-cell analysis for PGD. Cell recycling can be seen easily even if they are very weak, (vii) sizing of PCR involves the analysis of the same single cell, fixed to a glass products is highly accurate and (viii) the use of fluorescence slide, first by PCR and then by FISH. In contrast to conventional eliminates the need for hazardous radioisotopes. The only PGD, two independent analyses of the same cell have become drawback seems to be the high cost of the procedure: an possible. These combined analyses may serve independently to automated DNA sequencer, used for the electrophoresis of the confirm the gender of an embryo, for instance, for sexing for X- fluorescent PCR products, costs ~ , while one gel, on linked disease or for PGD of monogenic disease by PCR and to which 36 samples can be analysed, costs ~ 35. detect chromosome aneuploidies by FISH (e.g. in women older than 35 years). In the first attempts at cell recycling, PCR and FISH efficienc- Sex determination for the avoidance of X-linked disease ies were ~70% each, resulting in about half the cells being Sex determination in PGD is an alternative to gene-specific PCR diagnosed by both procedures. Optimization of both PCR and analysis for avoiding X-linked recessive diseases. The obvious FISH conditions have considerably increased efficiency to ~65% advantage of sexing over gene-specific PCR diagnosis is that is (Thornhill and Monk, 1996) and 85% (Rechitsky et al., 1996) of simultaneously diagnosed cells. Unfortunately, the ADO rate in cell recycling seems to be consistently higher than in conven- tional single-cell PCR (Rechitsky et al., 1996). Nevertheless, can be used for all X-linked diseases without the need to adapt the technology to each individual disease. A disadvantage is that half of the discarded male embryos are healthy. Both PCR- and 1758

4 the application of cell recycling in PGD, in future, seems to be promising especially when its potential might be extended to the use of, for instance, primer extension preamplification (PEP) and fluorescent PCR. Fluorescent in-situ hybridization The major application of FISH in PGD has so far been for gender determination for the prevention of X-linked recessive disease. The field of application of FISH, however, might extend in the future to all cases of IVF involving older women. Indeed, it was and Handyside, 1995). recently demonstrated that in morphologically normal embryos obtained after IVF, aneuploidy significantly increases with Preimplantation genetic diagnosis was also developed at our centre, although we have not yet applied it clinically (Sermon et al., 1995a,b). Other reports described single-cell PCR for sickle-cell anaemia, Tay Sachs disease and ornithine transcarbamylase deficiency (Morsy et al., 1992), osteogenesis imperfecta (Lida et al., 1996), and cystic fibrosis using a commercially available kit (Scobie et al., 1996). At our own centre, a number of diseases are the object of research work, such as β-thalassaemia, Charcot Marie Tooth disease type 1A, Huntington s disease and Marfan syndrome. Potentially, many other diseases could be detected by PGD (Delhanty maternal age (Munné et al., 1995b). This led to the suggestion Conclusion that aneuploidy might be a major contributing factor to implanta- A number of developments have not been discussed in this tion failure in older women undergoing IVF and that selection review, e.g. blastocyst biopsy and primer extension preampliof normal embryos could increase their chance of becoming fication (PEP) (Sermon et al., 1996), essentially because no pregnant and having normal children. The use of first polar body major breakthroughs have been reported in the literature lately, analysis for aneuploidy detection has been proposed and applied although we know of colleagues who are working on both topics. as an alternative to blastomere analysis especially since most Blastocyst biopsy seems still to be limited by the difficulties aneuploidies originate from maternal meiosis I non-disjunctions encountered in obtaining blastocysts in vitro, while PEP, which (Munné et al., 1995a; Verlinsky et al., 1995, 1996). Polar body is a time-consuming technique, seems to have found a serious analysis in combination with blastomere biopsy would also allow competitor in fluorescent PCR. Indeed, the major advantage of screening for aneuploidy resulting from the male. Up to five PEP is that different genetic loci can be analysed starting from chromosomes can be detected by FISH at the single-cell level one cell, while fluorescent PCR could do just that in a much allowing screening for the most common aneuploidies for chro- shorter time span. While technology improves in sensitivity, the mosomes 13, 18, 21 and the sex chromosomes (Munné et al., scope and the possibilities of PGD will continue to broaden out 1994). However, the usefulness of FISH analysis in IVF of and more patients will be helped with this technique. What women of advanced age has still to be proven since only limited molecular biologists in the field, including the present authors, data are available to date. really hope for, is a major breakthrough in IVF technology. Besides its use in sex selection and aneuploidy detection, Indeed, a pregnancy rate of 20% per cycle, as is achieved at our FISH is likely to become important in the future for the diagnosis centre, still seems low to our PGD patients, who generally have of unbalanced transmission of parental balanced translocations. a normal fertility and conceive naturally without problems. Although we are not aware of publications related to this subject, To date, fewer than 100 children have been born following we know from meeting presentations and personal contacts that PGD (Harper, 1996). The experimental nature of the procedure several groups involved in PGD are testing the possibilities for must be thoroughly discussed with the patients requesting PGD. this application which requires the isolation of locus-specific Although the risk of misdiagnosis, as discussed above, seems to probes for each couple. be relatively small, most centres offering PGD propose confirmation by prenatal diagnosis through chorionic villus sampling or amniocentesis. Also, the safety of the biopsy procedures Genetic diseases that can be diagnosed through PGD Harper (1996) listed the diseases for which PGD had been carried out before February 1995: cystic fibrosis, being the most frequent autosomal recessive disease in the Caucasian population, was carried out at most centres, fragile X diagnosis had been reported by the Genetics and IVF unit, Virginia, USA, Duchenne s muscular dystrophy was reported by our own centre (Liu et al., 1995), Tay Sachs disease was reported by the Jones Institute, Virginia, USA (Gibbons et al., 1995), haemophilia was diagnosed at Cecolfes, Bogota, Columbia and RhD blood typing was reported by Avner et al. (1996) at the Hadassah University, Tel Aviv, Israel. Since then, a number of reports have added to this list. PGD for fragile X was also developed by Dreesen et al. (1995, 1996) and has already been applied in a number of cycles (J.Dreesen, personal communication). Harton et al. (1996) reported the birth of a healthy baby after PGD for Marfan syndrome. Two pregnancies were obtained after PGD for Steinert s disease at our centre (Sermon et al., 1997). PGD for Tay Sachs disease (blastomere or polar body biopsy) should be assessed by followup studies of children born after PGD (Simpson and Liebaers, 1996). This could be organized, for example, through the Interna- tional Working Group on Preimplantation Genetics, or through the ESHRE Special Interest Group on Preimplantation Genetics. Acknowledgements The authors acknowledge the assistance of the clinical, scientific, nursing and technical staff of the Centres for Medical Genetics and for Reproductive Medicine. Mr Frank Winter of the Language Education Centre edited the manuscript. This work is supported by several grants from the Belgian Fund for Medical Research and the Research Council of the Vrije Universiteit Brussel. References Ashwood-Smith, M.J. (1994) Safety of human sperm selection by flow cytometry. Hum. Reprod., 9,

5 W.Lissens and K.Sermon Avner, R., Reubinoff, B.E., Simon, A. et al. (1996) Management of rhesus Lida, T., Suzumori, K., Ikuta, K. et al. (1996) Identification of a Gly 862 to isoimmunization by preimplantation genetic diagnosis. Mol. Hum. Reprod., Ser substitution in the type I collagen gene from a single spermatozoon. 2, Mol. Hum. Reprod., 2, Cui, X., Li, H., Goradia, T.M. et al. (1989) Single-sperm typing: determination Lissens, W., Sermon, K., Staessen, C. et al. (1996) Review: preimplantation of genetic distance between the G -γ-globin and parathyroid hormone loci diagnosis of inherited disease. J. Inher. Metab. Dis., 19, by using the polymerse chain reaction and allele-specific oligomers. Proc. Liu, J., Lissens, W., Devroey, P. et al. (1993) Polymerase chain reaction Natl. Acad. Sci. USA, 86, analysis of the cystic fibrosis F508 mutation in human blastomeres Delhanty, J.D.A. and Handyside, A.H. (1995) The origin of genetic defects following oocyte injection of a single sperm from a carrier. Prenat. Diagn., in the human and their detection in the preimplantation embryo. Hum. 13, Reprod. Update, 1, Liu, J., Lissens, W., Silber, S.J. et al. (1994) Birth after preimplantation Dreesen, J.C.F.M., Geraedts, J.P.M., Dumoulin, J.C.M. et al. (1995) RS46 diagnosis of the cystic fibrosis F508 mutation by polymerase chain reaction (DXS548) genotyping of reproductive cells: approaching preimplantation in human embryos resulting from intracytoplasmic sperm injection with testing of the fragile-x syndrome. Hum. Genet., 96, epididymal sperm. J. Am. Med. Assoc., 272, Dreesen, J.C.F.M., Dumoulin, J.C.M., Coonen, E. et al. (1996) A test system Liu, J., Lissens, W., Van Broeckhoven, C. et al. (1995) Normal pregnancy for preimplantation diagnosis of the fragile-x syndrome. Tijdschrift voor after preimplantation diagnosis of a dystrophin gene deletion. Prenat. Fertiliteitsonderzoek (special issue ESHRE Maastricht), 10, Diagn., 15, Edwards, R.G. and Beard, H.K. (1995) Sexing human spermatozoa to control Morsy, M., Takeuchi, K., Kaufmann, R. et al. (1992) Preclinical models for sex ratios at birth is now a reality. Hum. Reprod., 10, human pre-embryo biopsy and genetic diagnosis. II. Polymerase chain Edwards, R.G. and Gardner, R.L. (1967) Sexing of live rabbit blastocysts. reaction amplification of deoxyribonucleic acid from single lymphoblasts Nature, 214, and blastomeres with mutation detection. Fertil. Steril., 57, Findlay, I., Ray, P., Quirke, P. et al. (1995a) Allelic drop-out and preferential Munné, S. (1994) Flow cytometry separation of X and Y spermatozoa could amplification in single cells and human blastomeres: implications for be detrimental for human embryos. Hum. Reprod., 9, 758. preimplantation diagnosis of sex and cystic fibrosis. Mol. Hum. Reprod., 1, Munné, S., Lee, A., Rosenwaks, Z. et al. (1994) Chromosome mosaicism in see Hum. Reprod., 10, human embryos. Biol. Reprod., 51, Findlay, I., Urquhart, A., Quirke, P. et al. (1995b) Simultaneous DNA Munné, S., Dailey, T., Sultan, K.M. et al. (1995a) The use of first polar bodies fingerprinting, diagnosis of sex and single-gene defect status from single for preimplantation diagnosis of aneuploidy. Mol. Hum. Reprod., 1, see cells. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, Hum. Reprod., 10, Findlay, I., Quirke, P., Hall, J. et al. (1996) Fluorescent PCR: a new technique Munné, S., Sultan, K.M., Weier, H.-U. et al. (1995b) Assessment of numeric for PGD of sex and single-gene defects. J. Assist. Reprod. Genet., 13, abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human Gibbons, W.E., Gitlin, S.A., Lanzendorf, S.E. et al. (1995) Preimplantation embryos before transfer. Am. J. Obstet. Gynecol., 172, genetic diagnosis for Tay Sachs disease: successful pregnancy after pre- Navidi, W. and Arnheim, N. (1991) Using PCR in preimplantation genetic embryo biopsy and gene amplification by polymerase chain reaction. Fertil. diagnosis. Hum. Reprod., 6, Steril., 63, Ray, P.F. and Handyside, A.H. (1996) Increasing the denaturation temperature Gitlin, S.A., Lanzendorf, S.E. and Gibbons, W.E. (1996) Polymerase chain during the first cycles of amplification reduces allele dropout from single reaction amplification specificity: incidence of allele dropout using different cells for preimplantation diagnosis. Mol. Hum. Reprod., 2, DNA preparation methods for heterozygous single cells. J. Assist. Reprod. Ray, P., Winston, R.M.L. and Handyside, A.H. (1994) Single cell analysis for Genet., 13, diagnosis of cystic fibrosis and Lesch Nyhan syndrome in human embryos Griffin, D.K., Handyside, A.H., Harper, J.C. et al. (1994) Clinical experience before implantation. Miami Bio/Technology Short Reports: Proceedings of with preimplantation diagnosis of sex by dual fluorescent in situ the 1994 Miami Bio/Technology European Symposium, Advances in Gene hybridization. J. Assist. Reprod. Genet., 11, Technology: Molecular Biology and Human Genetic Disease, 5, 46. Grifo, J.A., Tang, Y.X., Munné, S. et al. (1994) Healthy deliveries from Ray, P.F., Winston, R.M.L. and Handyside, A.H. (1996a) Reduced allele biopsied human embryos. Hum. Reprod., 9, dropout in single-cell analysis for preimplantation genetic diagnosis of Handyside, A.H., Kontogianni, E.H., Hardy, K. et al. (1990) Pregnancies cystic fibrosis. J. Assist. Reprod. Genet., 13, from biopsied human preimplantation embryos sexed by Y-specific DNA Ray, P.F., Kaeda, J.S., Bingham, J. et al. (1996b) Preimplantation diagnosis amplification. Nature, 344, of β-thalassaemia major. Lancet, 347, Handyside, A.H., Lesko, J.G., Tarin, J.J. et al. (1992) Birth of a normal girl Rechitsky, S., Freidine, M., Verlinsky, Y. et al. (1996) Allele dopout in after in vitro fertilization and preimplantation diagnostic testing for cystic sequential PCR and FISH analysis of single cells (cell recycling). J. Assist. fibrosis. N. Engl. J. Med., 327, Reprod. Genet., 13, Hardy, K. and Handyside, A.H. (1992) Biopsy of cleavage stage human Saiki, R.K., Scharf, S., Faloona, F. et al. (1985) Enzymatic amplification of embryos and diagnosis of single gene defects by DNA amplification. Arch. β-globin genomic sequences and restriction site analysis for diagnosis of Pathol. Lab. Med., 116, sickle cell anemia. Science, 230, Harper, J.C. (1996) Preimplantation diagnosis of inherited disease by embryo Scobie, G., Woodroffe, B., Fishel, S. et al. (1996) Identification of the five biopsy: an update of the world figures. J. Assist. Reprod. Genet., 13, most common cystic fibrosis mutations in single cells using a rapid and Harper, J.C. and Handyside, A.H. (1994) The current status of preimplantation specific differential amplification system. Mol. Hum. Reprod., 2, diagnosis. Curr. Obstet. Gynecol., 4, Sermon, K., Lissens, W., Nagy, Z.P. et al. (1995a) Simultaneous amplification Harper, J.C., Coonen, E., Handyside, A.H. et al. (1995) Mosaicism of of the two most frequent mutations of infantile Tay Sachs disease in single autosomes and sex chromosomes in morphologically normal, monospermic blastomeres. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, preimplantation human embryos. Prenat. Diagn., 15, Sermon, K., Lissens, W., Devroey, P. et al. (1995b) Amplification of exon 11 Harton, G.L., Tsipouras, P., Sisson, M.E. et al. (1996) Preimplantation genetic of the gene for the α-chain of β-n-acetylhexosaminidase in single human testing for Marfan syndrome. Mol. Hum. Reprod., 2, blastomeres. Fertil. Steril., 63, Hattori, M., Yoshioka, K. and Sakaki, Y. (1992) Highly-sensitive fluorescent Sermon, K., Lissens, W., Joris, H. et al. (1996) Adaptation of the primer DNA sequencing and its application for detection and mass-screening of extension preamplification (PEP) reaction for preimplantation diagnosis: point mutations. Electrophoresis, 13, single blastomere analysis using short PEP protocols. Mol. Hum. Reprod., Kontogianni, E.H., Griffin, D.K. and Handyside, A.H. (1996) Identifying the 2, sex of human preimplantation embryos in X-linked disease: amplification Sermon, K., Lissens, W., Joris, H. et al. (1997) Clinical application of efficiency of a Y-specific alphoid repeat from single blastomeres with two preimplantation diagnosis for myotonic dystrophy. Prenat. Diagn., in press. lysis protocols. J. Assist. Reprod. Genet., 13, Simpson, J.L. and Liebaers, I. (1996) Assessing congenital anomalies after Levinson, G., Fields, R.A., Harton, G. L. et al. (1992) Reliable gender preimplantation genetic diagnosis. J. Assist. Reprod. Genet., 13, screening for human preimplantation embryos, using multiple DNA target- Strom, C.M., Rechitsky, S., Wolf, G. et al. (1994) Reliability of polymerase sequences. Hum. Reprod., 7, chain reaction (PCR) analysis of single cells for preimplantation genetic Levinson, G., Keyvanfar, K., Wu, J.C. et al. (1995) DNA-based X-enriched diagnosis. J. Assist. Reprod. Genet., 11, sperm separation as an adjunct to preimplantation genetic testing for the Thornhill, A.R. and Monk, M. (1996) Cell recycling of a single human cell prevention of X-linked disease. Mol. Hum. Reprod., 1, see Hum. Reprod., for preimplantation diagnosis of X-linked disease and dual sex determination. 10, Mol. Hum. Reprod., 2,

6 Preimplantation genetic diagnosis Thornhill, A., Holding, C. and Monk, M. (1994) Recycling the single cell to detect specific chromosomes and to investigate specific gene sequences. Hum. Reprod., 9, Verlinsky, Y. (1996) Preimplantation genetic diagnosis (Editorial). J. Assist. Reprod. Genet., 13, Verlinsky, Y., Cieslak, J., Freidine, M. et al. (1995) Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, Verlinsky, Y., Cieslak, J., Freidine, M. et al. (1996) Polar body diagnosis of common aneuploidies by FISH. J. Assist. Reprod. Genet., 13, Wu, R., Cuppens, H., Buyse, I. et al. (1993) Co-amplification of the cystic fibrosis F508 mutation with the HLA DQA1 sequence in single cell PCR: implications for improved assessment of polar bodies and blastomeres in preimplantation diagnosis. Prenat. Diagn., 13, Received on February 24, 1997; accepted on May 30,