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Preimplantation Genetic Diagnosis
Author: Peter Braude
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"� 2002 Nature Publishing Group Preimplantation genetic diagnosis (PGD) is a clinical diagnostic procedure that has evolved from the sub- stantial advances in assisted reproductive technology that have occurred since the first birth resulting from in vitro fertilisation (IVF) nearly 25 years ago. PGD was originally developed as an alternative to prenatal diagnosis to reduce the transmission of severe genetic disease for fertile couples with a REPRODUCTIVE RISK 1 .In PGD, cellular material from oocytes or early human embryos that have been cultured in vitro (FIG. 1) is tested for a specific genetic abnormality. After diag- nosis, only the unaffected embryos are selected for transfer to the uterus. In contrast to this specific and limited application, the same technology has recently been used more frequently to improve IVF success for infertile couples by screening embryos for com- mon or age-related ANEUPLOIDIES (aneuploidy screen- ing, PGD-AS). The first successful clinical application of PGD for genetic disease involved the use of PCR to amplify a spe- cific repeat on the Y chromosome to sex embryos in the presence of X-linked genetic conditions 1 ? in this case, adrenoleukodystrophy (ALD) and X-linked mental retardation. Substantial groundwork for the clinical application of PGD to various conditions (see below for further discussion) was undertaken in the late 1980s and, in 1992, the first live birth was reported following PGD for cystic fibrosis (CF) 2 . Preimplantation testing of embryos is not new 3 .In 1968, Gardner and Edwards were able to sex rabbit embryos using a sex-specific chromatin pattern in BLASTOCYST biopsies, before their transfer to the uterus 4 . Preimplantation testing of embryos is also used rou- tinely in animal husbandry to produce animals of the preferred sex 5 . However, the clinical application of this type of technology, in an attempt to prevent transmis- sion of genetic disease in humans, is still evolving. Measuring cytoplasmic enzyme activity in individual embryonic cells was first investigated, as a method of PGD, for clinical conditions characterized by an absence or a reduction of specific enzyme activity. Among such conditions are severe combined immunodeficiency dis- order 6,7 (SCID; adenosine deaminase deficiency), Lesch?Nyhan syndrome (LNS; hypoxanthinephospho- ribosyl transferase deficiency) and Tay?Sachs disease 8 (TSD; hexosaminidase deficiency). However, this method turned out to be of limited use when it became clear that it was difficult to distinguish maternally inher- ited enzyme activity that was present in the oocyte, from the embryo?s own enzyme activity. In the mid 1980s, the advent of PCR provided a far superior method for genetic testing, making it possible to carry out a diag- nostic test on highly concentrated and relatively pure amplified PCR fragments that spanned the appropriate genetic mutation 9 . The ability to extract DNA and genetically characterize single sperm and diploid cells PREIMPLANTATION GENETIC DIAGNOSIS Peter Braude, Susan Pickering, Frances Flinter and Caroline Mackie Ogilvie Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction. REPRODUCTIVE RISK The risk of establishing a pregnancy in which a fetus miscarries or has a phenotypic abnormality as a consequence of the familial genetic condition. ANEUPLOIDY The presence of extra copies, or fewer copies, of some chromosomes. BLASTOCYST A preimplantation embryo that contains a fluid-filled cavity called a blastocoel. NATURE REVIEWS | GENETICS VOLUME 3 | DECEMBER 2002 | 941 Centre for Preimplantation Genetic Diagnosis, Thomas Guy House, Guy?s Hospital, London SE1 9RT, UK. Correspondence to P.B. e-mail: obgyn@kcl.ac.uk doi:10.1038/nrg953 REVIEWS � 2002 Nature Publishing Group AMNIOCENTESIS A procedure in which a small sample of amniotic fluid is drawn out of the uterus through a needle that is inserted into the abdomen. The fluid is then analysed to detect genetic abnormalities in the fetus or to determine the sex of the fetus. CHORIONIC VILLUS SAMPLING (CUS). Sampling of the placental tissue of the conceptus for laboratory analysis. TRIMESTER One of the ~12-week stages into which pregnancy is divided for clinical purposes. FLUORESCENCE ACTIVATED CELL SORTING (FACS). A method whereby dissociated and individual living cells are sorted, in a liquid stream, according to the intensity of fluorescence that they emit as they pass through a laser beam. 942 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS until they are investigated after obstetric problems or the birth of an affected child. Couples in which one partner carries a dominant mutation will usually be aware of their risk, and might wish to avoid transmitting the disease to their offspring. PGD can be applied to three groups of genetic disor- der. The first category encompases single-gene disorders. These can be either autosomal dominant, autosomal recessive or X-linked recessive (BOX 1) ? in which the spe- cific mutation that is associated with the disease is known and can be amplified using PCR, or in which embryos that are likely to be unaffected can be identified using genetic linkage. The second category includes X-linked disorders in which the specific gene defect might not be known or where there is considerable genetic heterogeneity (for example, Duchenne muscular dystrophy; DMD), but the disorder can be avoided by sex selection. Chromosomal rearrangements, such as reciprocal or Robertsonian translocations fall into the third category (see below for further discussion). Before the development of PGD, limited options were available to couples with a reproductive risk. Fertile couples, or infertile couples who are following assisted conception treatment (such as IVF), might opt for some form of prenatal diagnosis of their condition once a pregnancy is established, either by AMNIOCENTESIS or CHORIONIC VILLUS SAMPLING (CVS) with the option of terminating an affected pregnancy. This decision is not taken lightly, as termination, especially late in the sec- ond TRIMESTER, can have substantial psychological and even physical morbidity. Some couples will not con- template termination because of religious or personal principles, whereas others, after a succession of termi- nations, might feel unable to accept further abnormal pregnancies. Other couples might consider the use of gametes from a donor who is not a carrier of the disorder. In most countries, sperm donation is more easily available than egg donation owing to the difficulty in recruiting egg donors and to the rigours of ovarian stimulation and egg retrieval (see below for details). For X-linked disorders, there is the possibility of sorting spermatozoa before insemination or in vitro fertilization. Although several methods have been reported 13 ,only FLUORESCENCE ACTIVATED CELL SORTING (FACS) produces a significant enrichment of the desired type of spermatozoa 14 .If donor gametes are unavailable or unacceptable for moral or religious reasons, adoption might be an alter- native. Other couples opt to remain childless rather than risk having an affected child, or passing on their genetic condition or carrier status. Preimplantation diagnosis provides an alternative way forward, not only for couples who have such reproductive risks, but also for couples who are unable to establish a viable pregnancy because of miscarriage caused by chromosome rearrangements. It is essential that any couple contemplating PGD receives genetic counselling to ensure that they have a good under- standing of the nature of the genetic disorder that could affect their child, and of the implications of its pattern of inheritance. They should also be fully provided a powerful impetus to pursue this technology clinically 10 . In addition, in situ hybridization techniques, based either on autoradiography 11 or on fluorescent markers 12 , facilitated PGD from single interphase nuclei. This review describes the current status of PGD. We have included relevant technical aspects to facilitate an understanding of both the clinical and laboratory prac- tice of PGD. We also discuss aneuploidy screening and the ethical dilemmas that might arise from the current practice of PGD, and anticipate some future develop- ments in clinical practice and technology. Reproductive risk and options Couples in which both partners carry the same autoso- mal-recessive gene disorder, in which the female carries an X-linked disorder or in which one partner carries a balanced chromosome rearrangement have a reproduc- tive risk (BOX 1). Embryos that are affected by a single- gene disorder are often viable, but the children might subsequently suffer from significant physical abnormal- ities or developmental delay. Those who inherit unbal- anced chromosome rearrangements might be similarly affected or miscarried. In the absence of a strong family history, such couples might not be aware of their risk Transfer only unaffected embryos to the patient Biopsied cell Affected Affected Affected Figure 1 | Principle of preimplantation genetic diagnosis. A single cell (or cells) is removed from each embryo of an in vitro-developing cohort, on which a diagnostic genetic test is carried out. Up to three of the embryos that are unaffected are transferred to the patient in the hope of establishing a pregnancy. Box 1 | Genetic risk Autosomal-dominant disorders, such as Huntington disease, Marfan syndrome and myotonic dystrophy affect anyone who inherits one copy of the mutant allele. Phenotypes of such disorders can be quite variable, and any child of an affected individual has ~50% chance of inheriting the condition. This figure is important in calculating the overall chance of success during a preimplantation genetic diagnosis (PGD) cycle, as 50% of embryos that are successfully fertilized and tested will not be suitable for implantation. So, the odds of achieving a successful pregnancy are lower than for couples who undergo PGD for an autosomal-recessive condition. Autosomal-recessive disorders, such as cystic fibrosis, spinal muscular atrophy and sickle cell disease only affect individuals who inherit two mutant alleles, one from each parent. Carriers (heterozygotes), who have just one copy of the altered gene, are usually asymptomatic, but if both parents are carriers, then each pregnancy has a 25% chance of being homozygous and is therefore affected. Three out of four tested embryos, on average, should be suitable for implantation. X-linked recessive disorders affect males who inherit a mutant allele on their single X chromosome, whereas female carriers, who have two X chromosomes, are usually phenotypically normal. � 2002 Nature Publishing Group REVIEWS informed about all alternative reproductive options. Couples in which one partner carries a lethal or debili- tating progressive genetic disorder, such as Huntington disease (HD), need to consider the welfare and arrange- ments for the care of any child who is born following PGD. This is a statutory requirement of the UK Human Fertilisation and Embryology Authority (HFEA), which issues licences for all new conditions that are considered for PGD 15 . However, as there is still a possi- bility of misdiagnosis using PGD (discussed in more detail below), couples should be encouraged to con- sider prenatal diagnosis to confirm preimplantation diagnosis. Couples who choose PGD need to be highly motivated, as the process is complicated, expensive and, in some cases, associated with a lower chance of having a healthy baby than conceiving conventionally (dis- cussed in more detail below). Clinical procedures and embryology Stimulation and oocyte retrieval. The increasingly sophisticated technology available in the assisted reproduction clinic is harnessed to provide oocytes or embryos for genetic testing in PGD. Controlled stimu- lation of the ovaries with exogenous GONADOTROPHINS leads to the recruitment of many FOLLICLES, and the process can be monitored by pelvic ULTRASONOGRAPHY 16 . When the number and size of the developing follicles is deemed appropriate, oocyte maturation is hormon- ally triggered. Between 34 and 38 hours later, the oocytes are collected by transvaginal ultrasound- guided aspiration of the follicular fluid. The oocytes are transferred to suitable culture medium and are either inseminated and left overnight to fertilize (IVF), or fertilized by intracytoplasmic sperm injection (ICSI), whereby single spermatozoa are injected directly into mature oocytes 17 (see ICSI in Online links box). ICSI is required for patients with reduced sperm quality (low numbers, poor motility or abnormal sperm morphology) or where IVF is not likely to occur successfully, for example, because of previous poor success in fertilization. ICSI is also recommended in all cases in which PCR is required for PGD, as the pres- ence of supernumerary sperm, buried in the ZONA PELLU- CIDA after IVF, might lead to a contamination of PCR reactions with paternal DNA and, therefore, to a possi- ble misdiagnosis. In some PGD centres, ICSI is used routinely to avoid the unexpected failure of fertiliza- tion 18,19 . The day after oocyte retrieval, embryos are examined for the presence of two PRONUCLEI that indi- cate normal fertilization (FIG. 2a). These embryos are separated from the failed or abnormally fertilized oocytes and are returned to culture for further devel- opment. A biopsy sample for genetic testing can then be obtained at various stages of development. Polar body biopsy. A mature oocyte is characterized by the presence of a first POLAR BODY that contains a com- plement of 23 BIVALENT maternal chromosomes. This discrete structure can be removed and used for genetic testing or for aneuploidy screening of the oocyte before fertilization 20,21 . On fertilization, a second polar a c e g b d f h Figure 2 | Early human preimplantation development in vitro. a | The first cleavage takes place ~22?30 hours post-fertilization (a post-fertilization, pronuclear stage embryo is shown), and the embryo divides at ~18 hour intervals thereafter. b?d | Individual BLASTOMERES can be seen clearly until the 8-cell stage, at which point they begin to flatten on each other in a process known as ?compaction?. e | Tight junctions form around the 16?32-cell stage of development, and the embryo becomes a tight ball of cells known as a ?morula? (f). g | The embryo continues to divide, now more quickly, and fluid begins to accumulate inside the embryo forming the blastocoelic cavity, which subsequently expands, giving rise to an expanded blastocyst. h | At about six days of development, the blastocyst hatches from the zona pellucida to begin implantation. The blastocyst is composed of two different cellular types, the outer trophectoderm, which is destined to give rise to extra- embryonic tissues, and a small compact ball of cells on the inside, the inner cell mass, which protrudes into the blastocoel and will give rise to the fetus 114 . NATURE REVIEWS | GENETICS VOLUME 3 | DECEMBER 2002 | 943 � 2002 Nature Publishing Group 944 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS cellular apposition becomes too great to separate indi- vidual cells. Biopsy at the two- or four-cell stage (FIG. 2b,c) involves removal of a large proportion of the cellular mass of the embryo, with detrimental effects on further developmental potential 25,26 .However,at the 8?12-cell stage (FIG. 2d,e), 3 days after oocyte retrieval, blastomeres retain TOTIPOTENTIALITY, and the embryo can be biopsied successfully even when com- pact 27 . Biopsy at this stage is, at present, the preferred option for many PGD centres. The biopsy of compact embryos is facilitated by a short pre-incubation in cal- cium- and magnesium- free medium, which reduces cellular apposition (FIG. 3b). The decision as to whether one or two cells should be removed is controversial. Removing two cells reduces the cellular mass of the embryo and, therefore, might reduce its developmental capacity. The accuracy of the diagnosis, however, is likely to be enhanced if embryos are replaced only when the results from both cells are concordant 28,29 . As the likelihood of pregnancy is, in part, dependent on the quality and number of embryos replaced, discordant results between cells could also reduce the number of embryos deemed suitable for replacement 28 , and might decrease preg- nancy rates. After genetic diagnosis, suitable embryos are usually transferred to the uterus on day four or day five (the blastocyst stage) of development 30 . The effi- cacy and safety of cleavage stage biopsy were first shown in studies using mouse embryos 25 , and this technique has since been used in many clinical proce- dures around the world 19,27 . Blastocyst biopsy. A major problem with polar body and/or cleavage stage biopsy is the paucity of material that is available, which might lead to an inaccurate and unreliable genetic diagnosis (see below for further dis- cussion). Biopsy of the embryo at the blastocyst stage obviates many of these problems as the embryo can con- tain up to 300 cells ? depending on the exact stage of development ? so more cells can be removed without apparent detrimental effect (FIG. 2g,h). In addition, because blastocyst biopsy involves the preferential removal of the more accessible TROPHECTODERM cells, the inner cell mass that is destined to become the fetus proper is unlikely to be damaged 31 ,thereby reducing pos- sible ethical concerns. Blastocyst biopsy normally takes place on day five or six after fertilization and involves making a hole in the zona pellucida before the removal of cells. The cells are biopsied either by gentle teasing using needles or by induced herniation of a trophecto- dermal vesicle 31, which can then be separated by physical means using needles or by a laser 32 . Blastocyst biopsy has been used successfully in mice, with a high survival rate of embryos, and live pups after re-implantation 33 . So far, it has not been extensively used in humans because of the difficulty in culturing embryos to the blastocyst stage. However, the development of sequential media that have been specifically designed for the long-term culture of embryos 34 , and the recent report of a human live birth after blastocyst biopsy 35 might encourage the increased use of this promising technique. body, containing a complement of 23 maternal chro- matids, is extruded from the oocyte and can also be tested to provide further confirmation (FIG. 3a). Polar body biopsy has the advantage that it samples extra- embryonic material and is therefore less likely to affect detrimentally subsequent embryonic development, and it might be considered ethically preferable by some. However, as it can only provide information about the maternal genotype, it cannot be used in cases of paternally derived disorders. In addition, where PREDIVISION OF CHROMATIDS or undetected recombi- nation between markers has taken place, a reliable diagnosis might not always be possible 22,23 . Cleavage stage biopsy. Individual cells of the cleaving embryo are distinct and discernible until around the 8?16-cell stage (day 3) when the embryo begins to undergo the process of compaction (FIG. 2f).From the 16-cell stage, TIGHT JUNCTIONS begin to form 24 and GONADOTROPHINS Hormones that are produced by the pituitary gland, which act on the gonads to control endocrine functions. Examples include follicle stimulating hormone and luteinizing hormone. FOLLICLES Structures in the ovary in which primary oocytes develop into mature oocytes before ovulation. ULTRASONOGRAPHY A technique in which sound waves are bounced off tissues and the echoes are converted into a picture (a sonogram). ZONA PELLUCIDA The glycoprotein coat that surrounds the oocytes and the early embryos of mammals. PRONUCLEUS The haploid nucleus of an egg or sperm. BLASTOMERE A cell that results from embryonic cleavage. POLAR BODY A small haploid cell that is produced during oogenesis and that does not develop into a functional ovum. BIVALENT A chromosome that has undergone replication. The two identical sister chromatids remain joined at the centromere. PREDIVISION OF CHROMATIDS The abnormal separation of chromatids during meiosis I (normally, sister chromatids separate during meiosis II) usually gives rise to gametes with a genetic imbalance. TIGHT JUNCTION A connection between individual cells in an epithelium that forms a diffusion barrier between the two surfaces of an epithelium. TOTIPOTENTIALITY The capacity of an undifferentiated cell to develop into any type of cell. TROPHECTODERM The outer layer of the blastocyst- stage embryo. a b Figure 3 | Polar body and cleavage stage biopsies. a | Polar body biopsy. Around 14?20 hours after normal fertilization, the zona pellucida of the zygote is breached by partial zona dissection using a microneedle and then a small aspiration capillary is introduced under the zona and the first and second polar bodies removed by gentle suction (reproduced with permission from Reproductive Genetics Institute, Chicago). b | Cleavage stage biopsy. Cleavage stage embryos are taken ~72 hours post-fertilization and held stationary on a glass micropipette by gentle suction. The zona pellucida is breached either by laser beam or by a jet of acidified Tyrodes solution. A sampling pipette is introduced into the embryo and a single nucleated blastomere is removed by suction. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | DECEMBER 2002 | 945 REVIEWS several problems with the technique were encountered subsequently. First, stringent precautions ? such as using gown, mask, gloves, filtered air, separate specific laboratory and equipment ? were required to avoid contamination with extraneous DNA of non-embry- onic origin 44 , amplification of which could lead to mis- diagnosis. Second, it soon became clear that in some cases, the target sequence failed to amplify although it was shown by other methods that it was present in the sample. Indeed, failure of amplification of the Y-chro- mosome-specific sequence in a male embryo led to the first report of PGD misdiagnosis 45,46 . Modifications were introduced in an attempt to improve the technique, including the development of a two-step NESTED PCR procedure that considerably improved sensitivity and specificity 47 . However, results in several independent laboratories indicated that, despite rigorous optimization of the procedure, sequences still occasionally failed to amplify when a sin- gle or a few cells were used as a source of DNA. Also, often only one allele at a heterozygous locus would amplify successfully, leading to the false assumption that the sample was homozygous 44,48,49 . So, for single-gene disorders, depending on which particular allele failed to amplify, heterozygous embryos could potentially be genotyped as either homozygous affected, in which case they were lost from the cohort of available embryos, or as homozygous normal and, therefore, as suitable for replacement 48,49 .Although this approach is acceptable in autosomal-recessive disorders, in which there is no abnormal phenotype in heterozygous carriers, in auto- somal-dominant disorders or in embryo sexing, the problems of undetected ALLELE DROP-OUT (ADO) or amplification failure make the misdiagnosis too likely for the single locus diagnosis to be acceptable in routine clinical use. To overcome these problems, Findlay and colleagues applied the relatively new technique of fluorescent PCR to single-cell genetic analysis. PCR amplification with fluorescently tagged primers was shown to be highly sensitive (~1,000-fold more sensitive than in the con- ventional analysis systems), reliable and accurate, and fewer PCR cycles were required, thereby reducing the time taken to reach diagnosis 50,51 . In addition, if different fluorescent tags are used, or different sized amplicons are designed, several different sequences can be ampli- fied simultaneously from an individual cell (multiplex PCR). The simultaneous amplification of two or more fragments, one containing the mutation that is associ- ated with the disorder and one or more containing polymorphic markers that are closely linked to that mutation, identifies which parental allele the embryo has inherited and indicates cases where ADO is likely to have taken place 41,48,52 . This approach substantially decreases the possibility of misdiagnosis 28 and provides the added assurance of a partial ?fingerprint? of the embryo, confirming that the amplified fragment is of embryonic origin 44,53 ; in addition, because only a single round of PCR is required, the overall time taken by the procedure is substantially reduced. Multiplex PCR also affords the opportunity to develop a generic diagnostic Cryopreservation after biopsy. Cryopreservation of sur- plus embryos with good morphology and that have reg- ular cleavage is now routine in IVF/ICSI procedures 36 . However, cryopreservation of surplus embryos after biopsy is more difficult as the zona pellucida has been breached. Usual protocols for cryopreservation were applied to embryos at the pronucleate, cleavage or blas- tocyst stages of development and depend on the slow diffusion of the cryoprotectant through an intact zona. These protocols might be suboptimal when used on biopsied embryos, and the initial attempts at cryop- reservation after biopsy resulted in a reduced survival rate compared with non-biopsied embryos at the same stage of development 37 . Recently, however, pregnancies have been reported after polar body biopsy 38 and after the cryopreservation of cleavage-stage biopsied embryos, which had been frozen using a modified pro- tocol 39 . In addition, Lalic et al. have reported excellent survival rates of biopsied cleavage-stage embryos that were allowed to develop to the blastocyst stage before freezing and an impressive implantation rate of 25% after embryo transfer 40 . These reports indicate that it will soon be possible to cryopreserve surplus biopsied embryos routinely after genetic diagnosis. Applications of PGD Single-gene disorders. Many genetic disorders can now be diagnosed using DNA from single cells 19,41,42 . However, having only one or two cells for analysis imposes several limitations on the genetic diagnosis. There are also severe time constraints, as results must be available within 12?48 hours of embryo biopsy to allow the transfer of suitable embryos at an appropriate preimplantation stage. So, there is great emphasis on the development of increasingly rapid, but robust, diagnos- tic assays that are effective at the level of a single cell. This situation is in complete contrast with that often experienced in prenatal diagnosis (PND), in which rela- tively large quantities of pure genomic DNA can be extracted from biopsied tissue samples, which are made up of many hundreds of cells ? or primary cell cultures derived from them. This DNA can be used directly for a diagnosis that, if confirmation is required, can be repeated several times over a period of several days. As it is not possible to detect directly the presence of a specific mutation in the DNA from a single cell, all sin- gle-gene PGD assays that have been developed so far rely on PCR to amplify the relevant DNA sequence from the biopsy sample 2 . The sample is lysed to release the nuclear DNA solution and the region of interest is amplified using specific primers in ~35?60 cycles of PCR. Amplified fragments can then be analysed according to the requirements of the assay. Several techniques have been used, including restriction digestion, sequencing and analysis of FRAGMENT LENGTH POLYMORPHISMS 41 . Single-cell PCR was first applied clinically in the pio- neering work of Handyside and colleagues, to sex embryos that were at risk of X-linked disease 1 . The basis of the test was the successful amplification of a Y-chro- mosome-specific repeat sequence in blastomeres from male embryos only 43 . Despite this early clinical success, FRAGMENT LENGTH POLYMORPHISMS The individual variation in the length of a particular region of DNA (such as a dinucleotide repeat), which, if the DNA is cut with a restriction enzyme or amplified using PCR, gives rise to the generation of differently sized fragments. NESTED PCR A technique for improving the sensitivity and specificity of PCR by the sequential use of two sets of oligonucleotide primers in two rounds of PCR. The second pair (known as ?nested primers?) are located in the segment of DNA that is amplified by the first pair. ALLELE DROP-OUT (ADO). The failure to detect an allele in a sample or the failure to amplify an allele during PCR. � 2002 Nature Publishing Group 946 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS biopsy for genetic diagnosis (excluding screening and social sexing) showed an overall clinical pregnancy rate of 22.4% per embryo transfer (17.3% per oocyte retrieval procedure undertaken) 19 . Biopsy was success- ful in 97% of cases, and the diagnosis was obtained in 86% of successfully biopsied blastomeres 19 . For single- gene disorders, 575 cycles resulted in 119 pregnancies (21% per egg retrieval and 25% per embryo transfer procedure). Five misdiagnoses using PCR were reported, two of which were for embryo sexing using PCR ? a method that is now considered obsolete. strategy for a particular disease, which is independent of the mutation present. Linkage analysis of polymorphic markers that are closely linked to the disease locus allows the identification of embryos at high risk in a broad range of patients who might be carrying different mutations in the same gene 54 . Pregnancy rates after PGD for single-gene disor- ders vary with the type of disorder and its pattern of inheritance. The cumulative data from the 1,197 cycles received by the ESHRE PGD consortium dur- ing 1999?2001 data collection for all forms of embryo PENETRANCE The proportion of affected individuals among the carriers of a particular genotype. If all individuals with a disease genotype show the disease phenotype, then the disease is said to be ?completely penetrant?. Box 2 | ?Designer babies? and the ethics of PGD The ability to select an embryo after genetic testing sometimes raises accusations of choosing a child to order, as a commodity that has been designed simply to meet the needs and desires of the parents. This view ignores the fact that most couples make the difficult choice of undergoing preimplantation genetic diagnosis (PGD) as their only hope of a viable pregnancy and of having a healthy child. Real ethical dilemmas arise in a small number of unusual cases 94,95 .For example, a couple both of whom have the dominant condition of achondroplasia might request PGD to avoid the homozygous affected embryos, which are generally lethal in utero, but wish to select only heterozygous embryos (which would give rise to children with achondroplasia), rather than unaffected embryos, to fit in with their lifestyle. Consideration of a case such as this must give paramount importance to the welfare of the child, but this is not always easy. Despite the medical problems that are associated with this condition, would an unaffected child in an achondroplastic family suffer more than an affected child in such an environment? A similar dilemma might occur in inherited deafness where a non-hearing child might be preferred. In a recent high-profile case, a non-hearing child was deliberately conceived using donor insemination by a male with a substantial genetic history of deafness, to be deaf like its lesbian parents (REF. 96). In other genetic conditions, a couple might request not to replace carrier embryos to try and eliminate the disease from their family. For example, a couple in which the male partner suffers from haemophilia requested the selection and transfer of male embryos only, to avoid fathering carrier daughters 97 . In this case, there was no risk of a serious genetic disease in the following generation, only in his grandchildren. Some might view this as a departure from the purpose of PGD ? to prevent the birth of an affected child ? and as a move towards positive eugenics. The sex-linked condition incontinentia pigmenti (IP) is also problematic. IP is lethal to affected male fetuses, which inevitably miscarry. The PENETRANCE in female carriers is variable, and daughters might have a far more severe phenotype than their mothers. A possible strategy for PGD in this case might therefore involve the selection and transfer of male embryos only, as those that inherit the X chromosome with the IP mutation will not survive, and all the survivors will be free of the disease 98 . However, some might feel that deliberately transferring embryos with a 50% risk of carrying the mutation is not an appropriate use of PGD. The ability to sex embryos using preimplantation embryo biopsy and FISH is another area of PGD that fuels substantial debate and controversy 19,99?101 . When there is one child or more of one sex in a family, the wish for a child of the other sex (referred to as ?family balancing?) has been viewed sympathetically by some in the United States 102,103 but remains controversial 104, and many consider that this is not a legitimate use of PGD 105 . Particular concerns arise when selection in a population is predominantly for one sex, where only one child is allowed, or where male offspring are favoured over female offspring for cultural and economic reasons. PGD for male embryo selection is practised in some Middle Eastern and Asian countries, as an alternative to prenatal diagnosis and abortion on gender grounds alone 101,106,107 . In the United Kingdom, sex selection of embryos for non-medical reasons using PGD is forbidden by the HFEA. However a new public consultation exercise is in progress, which seeks views on this issue to see if the code of practice should be changed. PGD was taken into a new dimension by the highly publicised case of Adam Nash. This boy was born, having been selected by preimplantation HLA typing, so that he could become a donor of haematopoietic stem cells for his sister who suffered from Fanconi anaemia (FA) 108 . As only 3 in 16 embryos would be both unaffected and also be a full HLA match (three out of four unaffected for the recessive disorder, and one in four a full HLA match), substantial numbers of embryos are likely to be discarded in the search for an embryo that is suitable for transfer. With only ~25% chance of pregnancy following embryo transfer, several attempts at PGD might be required to achieve a match and a pregnancy. This case has been followed by the birth of a child selected by PGD for tissue type compatibility with a sibling who suffered from leukaemia. The attempt to save the life of a sibling by having another child who might provide a suitable tissue match has been practised for years but in a rather hit and miss fashion, as never before has there been the opportunity for precise diagnosis. In the leukaemia case, there was no genetic risk to the new baby, and the PGD was carried out solely for the purpose of tissue matching 109 . Statistically, only one in four embryos is likely to be a suitable match, with an expectation that ~75% of the created embryos might be unsuitable, and possibly discarded. Although the merits of saving a sibling can be rationalized and commended, this process has met with great controversy 110 . Issues of consent and protection of children?s autonomy become paramount in these cases and should form the focus for giving an approval in cases or in countries where more general methods for regulation of PGD are in force. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | DECEMBER 2002 | 947 REVIEWS of the discarded male embryos will be normal, a fact that gives rise to ethical criticism of this method. In addition, some embryos might be aneuploid, triploid, haploid and so on. Collecting sufficient oocytes 55 , and hence generating enough embryos to give a reasonable chance of having at least two embryos for transfer, might be difficult to achieve in women who do not respond well to ovarian stimulation. Enrichment of the sperm sample for X-bearing spermatozoa by FACS (see above) would be a step towards improving PGD success rates, especially in women who produce only a small number of eggs. Although determination of the embryo?s sex by PCR was one of the earliest achievements of PGD, the possi- bility of misdiagnosis and the advent of in situ hybridization techniques have encouraged the develop- ment of more robust and reliable assays 12,56 . Since then, most centres have used fluorescence in situ hybridiza- tion (FISH) for sex determination 19 (FIG. 4). This assay has the added advantage of detecting abnormalities of sex chromosome copy number, so avoiding the transfer of such embryos. Like all techniques, FISH is not without problems. Signals can go undetected arising from two signals of the same colour overlying each other (signal overlap) or the failure of hybridization, whereas extra signals can appear, arising from signal splitting or anomalous fluo- rescence 57,58 . However, sex selection that uses probe sets such as that shown in FIG. 4 ? typically, green (G) for the X-chromosome centromere, red (R) for the Y-chro- mosome centromere and blue (B) for chromosome 18 centromere ? is very robust. To misdiagnose a normal male embryo (RGBB) as a normal female embryo (GGBB), two errors must occur: a red signal must be lost, and an extra, anomalous green signal must be gen- erated. This provides an effective internal check, ensur- ing that the chance of transferring a normal male embryo in error is very low. In some cases, embryos might be mosaic, or chaotic ? in this case, cells that make up the embryo have different random chromo- some constitutions 59,60 , probably as a result of defective cell cycle surveillance mechanisms. The biopsied cell (or cells) might not then be representative of the whole embryo 61 . However, grossly mosaic or chaotic embryos are unlikely to be viable, making the theoretical chance of establishing a normal pregnancy with a fetus of the ?wrong? sex extremely low. Nevertheless, one FISH mis- diagnosis occurred among the 78 cycles of social sexing, as reported to the ESHRE consortium 19 . Chromosome translocations. Reciprocal translocation (FIG. 5a), an exchange of two terminal segments from different chromosomes, is the commonest form of chromosome abnormality, which occurs ~1 in every 500 live births. With few exceptions, each reciprocal translocation is effectively unique to the family or indi- vidual in which it occurs. Robertsonian translocation (FIG. 5b), the centric fusion of two ACROCENTRIC CHROMO- SOMES, is less common and occurs in only ~1 in 1,000 individuals. Carriers of balanced familial translocations are nearly always phenotypically normal, as there is no This rate is higher than would be acceptable for mole- cular PND following CVS or amniocentesis, and reflects the difficulty of reaching a certain diagnosis using only one or two cells. X-linked disorders. Any X-linked disease for which no specific single-cell PCR test is available, can be consid- ered appropriate for PGD sex selection, although par- ticular situations might cause ethical dilemmas (BOX 2). According to the most recent report of the ESHRE PGD Consortium, the highest number of referrals in this category was for Fragile X syndrome (despite the complication that carrier females can be clinically affected), followed by Duchenne or Becker muscular dystrophies and haemophilia. Only 4% of the referrals were turned down by the PGD centre on ethical grounds 19 . On average, half of the embryos in any sex selection cycle will be unsuitable for transfer on the grounds of sex alone. It should be appreciated that, on average, half ACROCENTRIC CHROMOSOME A chromosome with the centromere located at one end. a b Figure 4 | PGD of X-linked disorders using FISH. Two nuclei that have been hybridized with probes that are complementary to sequences on chromosomes X (green), Y (red) and 18 (blue). a | A nucleus from the blastomere of a normal female embryo has two green and two blue signals, whereas b | a nucleus from a normal male has one red, one green and two blue signals. � 2002 Nature Publishing Group 948 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS chromosome that is involved in the translocation 63 . FISH strategies for detecting reciprocal translocations initially involved probes that spanned 64 or flanked 65,66 translocation breakpoints. These strategies had the advantage that embryos that had a normal chromo- some complement could be discriminated from those that carried a balanced translocation, but were limited by the time required to develop specific probes for each translocation carrier. An alternative approach for determining the chro- mosomal rearrangement status of the oocyte relies on polar body biopsy that uses whole chromosome-specific painting probes, sometimes in combination with ?-SATELLITE repeat- and locus-specific probes 67 . First polar bodies that are biopsied shortly after oocyte retrieval contain highly condensed, metaphase chromosomes, and chromosome-specific paints can therefore be used to show the relative position of the regions that are net loss of genetic material. Translocations are usually diagnosed when a family member is found to be infer- tile, suffers from recurrent pregnancy loss or has phe- notypically abnormal offspring arising from the pro- duction of genetically unbalanced gametes (in which chromosomal material has been lost or gained as a result of the translocation). An ideal PGD test for patients with balanced translocations would discrimi- nate unambiguously between different meiotic out- comes 62 . If this is not possible, then the priority must be to increase the individual?s chance of a successful preg- nancy and of having phenotypically normal offspring. As for sex selection, FISH is the method of choice for diagnosing chromosome rearrangements. To detect Robertsonian translocations, chromosome enumerator probes are used to count the chromosomes in the inter- phase nuclei of biopsied cells ? these probes can be chosen to bind at any point on the long arm of each ?-SATELLITE DNA Repetitive DNA sequences arranged in tandem arrays that usually lie near the centromere. 13.3 13.1 13 12.3 12.2 12.1 11.2 11.1 11 12 13.1 13.2 13.3 14 15 21.1 21.2 21.3 22 23 24.1 24.2 24.21 24.33 24.32 13 12 11.2 11.1 11.1 11.2 12 21.1 21.2 21.3 22 23 24 25 13.3 13.2 13.2 13.1 12.3 12.2 12.1 11.2 11.1 11 12 13.1 13.2 13.3 14 15 21.1 21.2 21.3 22 23 24.1 24.2 24.31 24.33 24.32 q p a b c 12 13 12 11.2 11.1 11.1 11.2 12 13 21 22 23 24.1 24.2 24.3 31 32.1 32.2 32.3 q p 14 der(12) der(17)17 13 12 11.2 11.1 11.1 11.2 12 21.1 21.2 22 23 24 25 13.1 22.3 22.2 22.1 21 11.2 11.1 11.1 11.1 11.1 11.2 12 13 21 22 23 24.1 24.2 24.3 31 32.1 32.2 32.3 14 21 der(14,21) 13 12 11.2 11.1 11.1 11.2 21 22.1 22.2 22.3 der(14,21) Alternate Adjacent-2 Adjacent-1 3:1 Figure 5 | Chromosome translocations. a | Reciprocal translocation. An ideogram of a reciprocal translocation between chromosomes 12 and 17. b | Robertsonian translocation. An ideogram of a Robertsonian translocation between chromosomes 14 and 21. c | Meiotic segregation modes. A diagram of four possible modes of segregation that might occur during meiosis in the presence of a reciprocal translocation. The dashed lines in each panel indicate how the chromosomes will segregate to the daughter cells. In the top panel, one cell will receive the two normal chromosomes (top left and bottom right), whereas the other cell will receive two translocated chromosomes (bottom left and top right). In the two middle panels, each cell will receive one normal and one translocated chromosome, whereas in the bottom panel, one cell will receive a single chromosome and the other cell three chromosomes. A fifth mode (4 : 0), in which all chromosomes segregate to one cell only, is not shown. � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | DECEMBER 2002 | 949 REVIEWS translocations will be incompatible with a viable preg- nancy, so probe combinations can be chosen to give an internal check (see above) on the abnormal products that are likely to be the most frequent. In this case, two mistakes would be needed to misdiagnose the product as normal or balanced. For those translocations that carry a significant risk of viable abnormal pregnancies, empiri- cal data indicate that only one segregation mode (FIG. 5c) per translocation will give rise to a viable pregnancy. This is probably because only one mode will give rise to a level of genetic imbalance that can be tolerated up to such a relatively advanced stage of fetal development 69 . Algorithms for analysis of reciprocal translocations have been published 70 and can be used to establish the most likely viable mode of any such translocation. Probe com- binations can then be chosen to give an internal check for products of that segregation mode. One potential pitfall of PGD for chromosomal translocations is that some FISH probes cross-hybridize with other loci in the genome, and in some cases this cross-hybridization might be patient-specific 71 . In addi- tion, clinically insignificant polymorphisms, in which the target sequence for one FISH probe is absent, might occur in some individuals, potentially leading to misdi- agnosis. FISH probes should therefore always be tested on blood samples from both partners before being used for PGD. As 50?70% of gametes, and hence of embryos, might carry unbalanced chromosomes arising from the translocation, embryo cohort size is even more impor- tant in this case than in sex selection PGD. For the same reasons, the embryo-transfer stage might not be reached due to the absence of normal or balanced embryos. Ideally, a clear-cut result should be obtained on each embryo to avoid excluding potentially normal embryos for technical reasons. PGD for chromosome translocations has resulted in the birth of normal babies 19,65,72,73 . Success rates vary between centres, but a recent review of data from three large centres (two in the United States and one in Italy) reports an overall pregnancy rate of 29% per oocyte retrieval, increasing to 38% when calculated per embryo transfer 42 . The percentage discrepancy is caused by a substantial number of embryos found to be unsuitable for transfer. Numerical chromosome abnormalities. For any one couple, the recurrence of the same autosomal trisomy (for example, trisomy 21) in pregnancy losses, termina- tions or live births is rare 74 . Although such recurrences might arise by chance, the possibility of mosaicism in the germ line of one partner can seldom be completely excluded. PGD has been used to test embryos for the copy number of the chromosome that was aneuploid in the previous pregnancies 75 and is usually considered appropriate ? especially for couples who have religious or ethical objections to pregnancy terminations. An ideal test for chromosome copy number would include two probes, each labelled in a different colour, both for the ?at risk? chromosome, combined with a third probe for a different chromosome, to control for ploidy. involved in the translocation. This approach cannot be used in biopsied blastomeres because the cells might not be in metaphase and therefore the chromosomes might not be sufficiently condensed. Polar bodies with an unbalanced chromosome complement imply an unbal- anced chromosome complement in the oocyte; polar bodies with a normal complement indicate an oocyte with a balanced chromosome rearrangement, which will give rise to phenotypically normal offspring. Once fertilized, normally progressing embryos are transferred to the uterus, assuming they were of sufficiently good morphology. In the absence of such embryos, those with the balanced translocation could be transferred to the patient. As this method uses polar bodies, it is of course only applicable to translocations in female carriers. In 1998, a more general strategy for testing biop- sied cells from cleavage-stage embryos was developed. It used probes that are specific for the subtelomeric regions of the translocated segments 62,68 . In this proce- dure, two differentially labelled probes that are spe- cific for the chromosome arms that are involved in the translocation can be combined with a centromeric probe (or any probe that maps proximal to the break- point on either chromosome). The test does not dis- criminate between non-carrier embryos and those that carry the balanced form of the translocation, both of which should give rise to phenotypically nor- mal offspring. For any chromosome rearrangement, genetically unbalanced gametes are likely to be produced during meiosis. For reciprocal translocations, the prevalence of these unbalanced gametes is estimated to be between 50% and 70% (REF. 69). It is likely that each unique translocation will give rise to different proportions of possible segregation products, of which there are 32, including those that result from errors at meiosis II (REF. 62). The abnormal gametes produced by some Figure 6 | Aneuploidy screening using FISH. Fluorescence in situ hybridization (FISH) of a single four-cell embryo. The embryo has been hybridized with probes to chromosomes 13, 16, 18, 21 and 22, each of which is labelled with a different fluor. Signal splitting and differences in signal size within and between blastomeres show the difficulty of interpretting FISH results. � 2002 Nature Publishing Group 950 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS Enhancing in vitro fertilization success. Preimplantation screening for chromosome aneuploidy is carried out at many centres throughout the world. It is the most common reason for embryo diagnosis, with more than 2,000 treatment cycles reported for this purpose 42 . However, some feel that it is important to delineate between PGD for inherited genetic diseases, and PGD for the detection of sporadic chromosomal abnormality, to enhance IVF success. For this reason, the latter procedure has been designated as PGD-AS (aneuploidy screening) by the ESHRE consortium 19 or PGS (preimplantation genetic screening) by the HFEA, but it has been included in the definition of PGD in the United States 78 . The couples who opt for this test are infertile and undergoing IVF/ICSI to overcome their infertility ? the success of which is heavily influenced by maternal age and the previous reproductive history of the couple 79 . Advanced maternal age or repeated IVF failure might indicate that the infertility is caused by the production of ane- uploid gametes, and the test is designed to identify embryos that have a normal chromosome comple- ment. In addition, women over 36 years of age, who are at increased risk of producing a child with Down syndrome, or with other age-related chromosomal abnormalities, and who have already opted for IVF/ICSI because of their infertility, might wish to have their embryos screened for these more common viable abnormalities rather than go through PND and possible abortion. Individual embryos are biop- sied, and biopsy samples are examined for numerical chromosomal abnormality using 5?14 FISH probes (FIG. 6). One multicentre study investigating the effi- cacy of PGD-AS showed a decrease in miscarriage rate from 25.7% per patient in the control group to 14.3% in the group undergoing PGD-AS testing 80 . This difference was not statistically significant, although the difference was significant when the mis- carriage rate was expressed as a percentage of fetal heartbeats detected 80 (24.2% miscarried in the con- trol group and 9.6% miscarried in the tested group). There was also a significant increase (from 10.5% to 16.1%) in the ongoing pregnancy and ?delivered baby? rate in the PGD-AS group compared with the control group. Three centres (two from the United States and one from Italy) contributed to this data, and although the matching of couples for compari- son was not consistent between the centres (for example, age, previous cycles and number of folli- cles), the trends were similar in all three centres 80 . One of the pitfalls of PGD-AS is that some normal embryos might be excluded from the cohort that is considered suitable for embryo transfer because of errors in the test, which, especially in older women who might have small embryo cohorts, could result in the failure to reach embryo transfer. The ESHRE consortium data on PGD-AS during the past 12 months in participating centres 19 showed a 28% preg- nancy rate in PGD-AS for advanced maternal age (>35 years), but only a 7% pregnancy rate in women with recurrent IVF failure (excluding couples with Other chromosome abnormalities. Couples in which one partner carries a chromosome abnormality might wish to avoid transmitting this abnormality to their offspring. This is particularly true in cases such as the deletion of 22q11 that leads to Velocardiofacial syndrome (VCSF) or DiGeorge syndrome (DGS) in which the phenotype is variable and unpredictable 76 . Even monozygotic twins that carry the same deletion might have discordant phe- notypes 77 . This deletion can be detected in biopsied blas- tomeres using FISH probes. However, 50% of embryos from such an affected individual are likely to carry a chro- mosome abnormality, and there is no internal check in this assay, therefore, only one mistake would be required to misdiagnose an abnormal embryo as normal. In these circumstances, most centres would consider it necessary to biopsy two cells from each embryo, and only transfer embryos with concordant results from both cells. NICK TRANSLATION A method for in vitro DNA labelling. Nicks are introduced into the DNA by an endonuclease and are subsequently repaired using labelled residues. Reference DNA Nick-translate with labelled substrate Pool labelled DNA In situ hybridize to normal chromosomes Test DNA Figure 7 | Comparative genomic hybridization. In this technique, reference and test DNA samples are fluorescently labelled in NICK-TRANSLATION reactions. After hybridization of labelled probe mixes to normal chromosome spreads, relative fluorescent intensity is detected by capture of fluorescence using a cooled charge-coupled device camera. Dedicated software is used to compare ratios of green to red fluorescence along each chromosome and, hence, to identify genome imbalance in the test DNA. The figure is based on images supplied by Vysis, Inc. (Downers Grove, Illinois, USA). � 2002 Nature Publishing Group NATURE REVIEWS | GENETICS VOLUME 3 | DECEMBER 2002 | 951 REVIEWS have been reasonably successful, but CGH now seems to be the method of choice for enumerating the whole chromosome set in blastomeres 88,89 . Although the chro- mosomes are not visualized directly, copy number of every chromosomal region of 20 Mb or more can be assessed using CGH 90 (FIG. 7). Whole-chromosome ane- uploidies and even small structural aberrations have been detected in blastomeres using this method, but the technique is limited to detecting relative imbalance and, therefore, changes in whole PLOIDY cannot be seen 88,89 . The technique requires two to three days for diagnosis and is therefore, at present, unsuitable for routine use on cleavage-stage embryos or blastocysts without a cryopreservation step between biopsy and transfer. Improvements in the protocols might shorten this time and allow the diagnosis and transfer of fresh material. Until now, several ongoing pregnancies and the birth of one healthy child have been reported using this technique 39,91 . DNA microarray analysis is a rapidly evolving method of molecular analysis that could find several potential uses in PGD 92,93 . Although it is primarily used for gene expression analysis, microarrays could be used in routine PGD in screens for mutations in any one gene, or screens of several genes for several mutations. Embryos could then potentially be tested for serious susceptibility traits loci, such as the breast cancer 1 (BRCA1) gene. Microarrays could also be useful in PGD of specific diseases that are severely genetically heterogeneous and for which there are few common mutations, such as Duchenne muscular dystrophy. Such an approach could provide a useful generic test- ing procedure that is applicable to all patients that carry this disease. Finally, microarrays could replace the metaphase spreads that are now used to assess chromosome imbalance during CGH. At present, tech- nical limitations, such as the paucity of material that is available for hybridization, sensitivity and reliability of the data, and the cost of producing appropriate microarrays are likely to hinder their application in PGD for some considerable time. Conclusions PGD is a sophisticated form of early prenatal diagnosis that is carried out in a few specialized centres. However, the rapid advances in molecular genetics are likely to stimulate further the use of PGD and to encourage a substantial change in the way that genetic conditions in the offspring of certain patients are prevented. It is becoming apparent that the main demand for embryo biopsy will come from infertile patients seeking to improve their chances of successful IVF treatment and to reduce the risk of conceiving a child with an age- related aneuploidy. Indeed, it is likely that a combina- tion of approaches will be made possible by the molecu- lar examination of the entire chromosome complement, at the same time testing for common genetic diseases, such as cystic fibrosis. The challenge will be to regulate the use of PGD technology (BOX 3) for medical purposes and to limit or prevent its use for eugenic selection. translocations), indicating that, in the latter group, factors other than aneuploid gametes might be likely to be the main cause of infertility. More than 1,000 cycles of PGD-AS have been carried out in one US centre 81 , but clear benefits of this technique in terms of live birth rate per initiated cycle have yet to be shown in any large-scale prospective controlled study. It is important that this technology is properly evalu- ated 78 . An expensive test of unproved or limited effi- cacy might be readily taken up by women who are desperate to establish a pregnancy (the so-called ?technological imperative?; REF. 82), when in some cases the test might reduce their chances of preg- nancy by excluding normal embryos from the cohort available for transfer. Future research into the inci- dence of individual chromosome aneuploidies in early embryos might provide the means to design a more specific test that uses FISH probes for the most common early abnormalities. Alternatively, other technologies, such as comparative genomic hybridization (CGH) or microarrays might be used in future to allow the screening of the whole genome for genetic imbalance 68 (see below). Future developments Comparative genomic hybridization. Although PGD- AS has been shown to improve implantation rate in some patient groups, at present, only a limited subset of chromosomes can be screened using traditional FISH protocols 80,83 . Ideally, cytogenetic tests would involve a full KARYOTYPE ANALYSIS on metaphase chromo- somes. Unfortunately, preparing METAPHASE SPREADS directly from embryonic blastomeres by traditional methods has proved difficult, with only a small pro- portion of the blastomeres in any one embryo giving interpretable results 84,85 . Alternative methods for karyotyping, including fus- ing polar bodies or blastomeres with enucleated human oocytes or with bovine oocytes to induce mitosis 86,87 KARYOTYPE ANALYSIS The ascertainment of chromosome constitution by the light microscopy analysis of stained metaphase chromosomes. METAPHASE SPREADS The result of a cytogenetic method in which dividing cells are artificially arrested at metaphase, when chromosomes are shortened and condensed. The fixed material from such preparations is dropped onto microscope slides, where the chromosomes from individual cells form clusters or spreads, which can be stained and analysed. PLOIDY The number of sets of chromosomes in a cell (n). Normal human somatic cells are diploid (2n), with 2 sets of 23 chromosomes. Box 3 | Regulation of preimplantation genetic diagnosis There are substantial differences in the control of preimplantation genetic diagnosis (PGD) worldwide 111 . These differences are linked to the prevailing attitudes to assisted conception, invasive procedures on human embryos and the eugenics of embryo selection. In the United Kingdom, PGD, like all reproductive technology that involves in vitro human embryo manipulation, is strictly regulated by the HFEA (see the section entitled ?Clinical procedures and embryology ?) under the terms of the Human Fertilisation and Embryology Act (1990). According to this Act, and the consequent HFEA Code of Practice 15 , embryos may only be used for PGD and for research to develop new diagnostic methods under licence from the HFEA. The HFEA provides reassurance to the public that PGD is being undertaken only for serious genetic diseases and not for social purposes (BOX 2). PGD has only recently been allowed in France 112 , and then limited to three centres, whereas it is not allowed in Argentina, Austria, Switzerland and Taiwan 113 . In Germany, only those procedures that are of direct benefit to the embryo can be undertaken 111 . As PGD might result in the destruction of affected embryos, PGD is now not allowed due to deep-seated fears arising from that country?s sad history of eugenics. Although, encouragingly, the future use of PGD is now being debated in the German Parliament, more restrictive legislation is being proposed in Italy. There is no federal regulation of PGD in the United States 42 . � 2002 Nature Publishing Group 952 | DECEMBER 2002 | VOLUME 3 www.nature.com/reviews/genetics REVIEWS 1. Handyside, A. H., Kontogianni, E. H., Hardy, K. & Winston, R. M. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344, 768?770 (1990). The first pregnancies that resulted from the transfer of embryos that had been genotyped as female were reported here. Embryos from couples who were at risk of transmitting two different X-linked disorders were subjected to biopsy and the cell removed was sexed by the PCR of a Y-chromosome specific repeat sequence. 2. Handyside, A. H., Lesko, J. G., Tarin, J. J., Winston, R. M. & Hughes, M. R. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N. Engl. J. Med. 327, 905?909 (1992). 3. Edwards, R. G. Diagnostic methods for human gametes and embryos. Hum. Reprod. 2, 415?420 (1987). 4. Gardner, R. L. & Edwards, R. G. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature 218, 346?349 (1968). 5. Johnson, L. Gender preselection in mammals: an overview. Dtsch Tierarztl Wochenschr. 103, 288?291 (1996). 6. Benson, C. & Monk, M. Microassay for adenosine deaminase, the enzyme lacking in some forms of immunodeficiency, in mouse preimplantation embryos. Hum. Reprod. 3, 1004?1009 (1988). 7. Monk, M., Handyside, A., Hardy, K. & Whittingham, D. Preimplantation diagnosis of deficiency of hypoxanthine phosphoribosyl transferase in a mouse model for Lesch?Nyhan syndrome. Lancet 2, 423?425 (1987). 8. Sermon, K. et al. ?-N-acetylhexosaminidase activity in human oocytes and preimplantation embryos. Hum. Reprod. 7, 1278?1280 (1992). 9. Saiki, R. K. et al. Enzymatic amplification of ?-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350?1354 (1985). 10. Li, H. H. et al. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 335, 414?417 (1988). The successful PCR amplification of DNA sequences from individual diploid cells and from human sperm not only enabled the analysis of DNA sequence variation at the single-cell level, but also opened up the possibility of applying this technology clinically in PGD, to identify the presence of genetic mutations in embryos from carrier patients. 11. West, J. D. et al. Sexing the human pre-embryo by DNA?DNA in-situ hybridisation. Lancet 1, 1345?1347 (1987). 12. Griffin, D. K., Handyside, A. H., Penketh, R. J., Winston, R. M. & Delhanty, J. D. Fluorescent in-situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes. Hum. Reprod. 6, 101?105 (1991). 13. Beernink, F. J., Dmowski, W. P. & Ericsson, R. J. Sex preselection through albumin separation of sperm. Fertil. Steril. 59, 382?386 (1993). 14. Vidal, F. et al. Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X- or Y- chromosome haplotypes: a blind trial assessed by double and triple colour fluorescent in-situ hybridization. Hum. Reprod. 13, 308?312 (1998). 15. HFEA. Code of Practice, 5th edn [online] <http:www.hfea.gov.uk/forclinics/archived/chair_letters/ 00005.htm> (1999). 16. Khalaf, Y., Taylor, A. & Braude, P. R. Low estradiol concentrations after five days of controlled ovarian hyperstimulation for IVF are associated with poor outcome. Fertil. Steril. 74, 63?66 (2000). 17. Palermo, G., Joris, H., Devroey, P. & Van Steirteghem, A. C. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17?18 (1992). 18. Vandervorst, M. et al. The Brussels? experience of more than 5 years of clinical preimplantation genetic diagnosis. Hum. Reprod. Update 6, 364?373 (2000). 19. ESHRE Preimplantation Genetic Diagnosis Consortium. Data collection III. Hum. 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Liu, J., Van de Abeel, E. & Van Steirteghem, A. The in vitro and in vivo developmental potential of frozen and non frozen biopsied 8-cell mouse embryos. Hum. Reprod. 8, 1481?1486 (1993). 26. Hardy, K., Martin, K. L., Leese, H. J., Winston, R. M. & Handyside, A. H. Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage. Hum. Reprod. 5, 708?714 (1990). Showed that the removal of 1?2 cells from an 8-cell human embryo did not significantly affect its chances of subsequent in vitro development to the blastocyst stage. After substantial investigations into the safety of biopsy procedures in mouse embryos, this study underlined the efficacy of such techniques and endorsed their clinical application in PGD. 27. De Vos, A. & Van Steirteghem, A. Aspects of biopsy procedures prior to preimplantation genetic diagnosis. Prenat. Diagn. 21, 767?780 (2001). 28. Lewis, C. M., Pinel, T., Whittaker, J. C. & Handyside, A. H. 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Assist. Reprod. Genet. 15, 276?280 (1998). 62. Scriven, P. N., Handyside, A. H. & Ogilvie, C. M. Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenat. Diagn. 18, 1437?1449 (1998). 63. Scriven, P. N., Flinter, F., Bickerstaff, H., Braude, P. & Mackie Ogilvie, C. Robertsonian translocations?reproductive risks and indications for preimplantation genetic diagnosis. Hum. Reprod. 16, 2267?2273 (2001). 64. Munne, S., Fung, J., Cassel, M. J., Marquez, C. & Weier, H. U. Preimplantation genetic analysis of translocations: case- specific probes for interphase cell analysis. Hum. Genet. 102, 663?674 (1998). 65. Munne, S., Scott, R., Sable, D. & Cohen, J. First pregnancies after preconception diagnosis of translocations of maternal origin. Fertil. Steril. 69, 675?681 (1998). 66. Conn, C. M., Harper, J. C., Winston, R. M. & Delhanty, J. D. 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Fertil. Steril. 75, 861?864 (2001). Provides a useful overview of the ethical issues that surround gender selection for non-medical reasons. 104. Gleicher, N. & Karande, V. Gender selection for non-medical reasons. Fertil. Stertil. 78, 460?462 (2002). 105. Robertson, J. Sex selection for gender variety by preimplantation genetic diagnosis. Fertil. Stertil. 78, 463 (2002). 106. Kilani, Z. & Hassan, L. Sex selection and preimplantation genetic diagnosis at The Farah Hospital Reprod. BioMed. Online 4, 68?70 (2002). <http:www.rbmonline.com> 107. Kumar, A. Does preimplantation genetic diagnosis for gender selection really offer a solution for family balancing? A response to the article by Malpani and Malpani. Reprod. BioMed. Online 4, 10?11 (2002). <http://www.rbmonline.com> 108. Verlinsky, Y., Rechitsky, S., Schoolcraft, W., Strom, C. & Kuliev, A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA 285, 3130?3133 (2001). Describes the application of PGD to preselect a potential donor for an affected sibling who required stem cell donation. An HLA-matched, unaffected child was born after four attempts of PGD, and cord blood was used to successfully treat the affected sibling. 109. Meek, J. Baby with selected gene born in Britain. Guardian 7 (London, 2002). 110. Boyle, R. J. & Savulescu, J. Ethics of using preimplantation genetic diagnosis to select a stem cell donor for an existing person. Br. Med. J. 323, 1240?1243 (2001). 111. Gunning, J. Regulating assisted reproduction technologies. Med. Law 20, 425?433 (2001). 112. Viville, S. Preimplantation genetic diagnosis, finally a reality in France. Gynecol. Obstet. Fertil. 28, 873?874 (2000). 113. Jones, H. W. & Cohen, J. IFFS surveillance 01. Fertil. Steril. 76, S24?S25 (2001). 114. Gardner, R. L. in Implantation of the Human Embryo (eds Edwards, R. G., Purdy, J. M. & Steptoe, P. C.) 155?178 (Academic, London, 1985). Acknowledgements The authors thank the other members of the Centre for Preimplantation Genetic Diagnosis for helpful and regular discus- sion about the issues raised in this review. Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ breast cancer 1 OMIM: http://www.ncbi.nlm.nih.gov/Omim/searchomim.html adrenoleukodystrophy | Becker muscular dystrophies | cystic fibrosis | DiGeorge syndrome | Down syndrome | Duchenne muscular dystrophy | Fanconi anaemia | Huntington disease | incontinentia pigmenti | Lesch?Nyhan syndrome | Marfan syndrome | myotonic dystrophy | severe combined immunodeficiency disorder | sickle cell disease | Tay?Sachs disease | Velocardiofacial syndrome | X-linked mental retardation FURTHER INFORMATION American Society for Reproductive Medicine (ASRM): http://www.asrm.com European Society of Human Reproduction and Embryology (ESHRE): http://www.eshre.com ICSI: http://www.ivfdirect.com/Alternatives/ICSI.htm PGD: http://www.ivfdirect.com/Alternatives/PGD.htm Reproductive Genetics Institute: http://www.reproductivegenetics.com UK Human Fertilisation and Embryology Authority: http://www.hfea.gov.uk Access to this interactive links box is free online. 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