Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist's view

Human Reproduction Update, Vol.8, No.1 pp. 11±20, 2002 Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist's view Kare...
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Human Reproduction Update, Vol.8, No.1 pp. 11±20, 2002

Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist's view Karen Sermon Centre for Medical Genetics, Dutch-speaking Brussels Free University, Laarbeeklaan 101, 1090 Brussels, Belgium To whom correspondence should be addressed. E-mail: [email protected]

The ®rst clinically applied preimplantation genetic diagnosis (PGD) was reported more than a decade ago and since then PGD has known an exponential growth. This ®rst report described the use of PCR to sex embryos from couples at risk for X-linked diseases. Not surprisingly, in the ®rst years, the development of PCR-based tests led to PGD for well-known monogenic diseases such as cystic ®brosis and thalassaemia. When ¯uorescent in-situ hybridization (FISH) was introduced it quickly replaced PCR-based methods, which had led to misdiagnoses, for sexing of embryos. FISH was also quickly introduced for aneuploidy screening, which has as its main aim the improvement of IVF results in patients with poor reproductive outcome, and later for PGD in translocation carriers. In this review, PGD for patients with a pre-existing genetic risk will be discussed, i.e. the monogenic diseases and the translocations, as well as different biopsy methods and promising new developments. Key words: embryo biopsy/PGD for chromosomal translocations/preimplantation genetic diagnosis/sexing of embryos/single-cell PCR

TABLE OF CONTENTS Introduction Biopsy methods: Polar body biopsy Cleavage-stage biopsy Blastocyst biopsy The use of FISH in sexing for X-linked diseases and PGD for translocation carriers The use of PCR for monogenic diseases Outcome of PGD: pregnancy rates and outcome of pregnancies and children born Future developments: Cryopreservation of biopsied embryos Comparative genomic hybridization PGD for monogenic diseases using molecular beacons Conclusions References

Introduction The aim of this review is to discuss the current practices in preimplantation genetic diagnosis (PGD) for patients with a preexisting genetic risk from a technical, as opposed to a clinical, point of view. These current concepts will be situated in a historical perspective to clarify how and why the most recent techniques came into use. PGD is a very early form of prenatal diagnosis. Oocytes or embryos obtained in vitro through assisted Ó European Society of Human Reproduction and Embryology

reproductive techniques are biopsied (polar bodies for the oocytes, blastomeres for the embryos) and the biopsied cells are used for genetic diagnosis. Only embryos shown to be free of the disease under consideration are subsequently replaced (Liebaers et al., 1998). Two major methods are currently used for diagnostic purposes in PGD: PCR, which allows for the exponential ampli®cation of a short DNA fragment is widely used for diagnosis in monogenic diseases, while ¯uorescent in-situ hybridization (FISH), which allows the visualization of chromosomal regions, is used in sexing, aneuploidy screening and in PGD for translocations (Lissens and Sermon, 1997). In PGD for aneuploidy screening (PGD±AS), embryos are screened for chromosomal abnormalities in patients with impaired fertility, e.g. older patients, patients with several failed IVF cycles, or patients with recurrent miscarriages. As the main aim of PGD±AS is to improve IVF results in these patients, in contrast to the other types of PGD where patients have a pre-existing genetic risk, PGD±AS will not be discussed in this paper. The ®rst clinical application of PGD was reported more than a decade ago and described sexing using PCR for sex-linked diseases (Handyside et al., 1990). Since then, PGD has known a steady growth and new applications and methodology are introduced regularly. Not surprisingly, as the ®rst method available for the analysis of single cells was PCR, the ®rst reports dealt with monogenic diseases, such as cystic ®brosis (Coutelle et al., 1989) and defects in the b-globin gene (Holding and Monk, 1989; Pickering et al., 1992). Later, radioactively


K.Sermon labelled probes used in in-situ hybridization were replaced by ¯uorescently labelled probes and then adapted to be used on single interphase nuclei (Grif®n et al., 1992). Fluorescent in-situ hybridization (FISH) was initially used for gender determination, but as it became more and more apparent that a large proportion of human preimplantation embryos showed aneuploidy and chromosomal mosaicism (Delhanty et al., 1993; Harper et al., 1994), FISH was also used for aneuploidy screening (Munne et al., 1995). Before the advent of telomere probes and their commercial availability (Scriven et al, 1998; Munne et al., 2000), unbalanced translocations in embryos had to be detected using speci®c probes developed often for one translocation occurring in only one family (Conn et al., 1999; Weier et al., 1999; Iwarsson et al., 2000). Other methods to detect translocations are chromosome painting of the ®rst polar body and fusion of polar bodies or blastomeres with mouse metaphase II oocyes or zygotes followed by induction of pronucleus formation and metaphase karyotyping (Verlinsky and Evsikov, 1999; Munne et al., 2000). Newer FISHrelated methods are primed in-situ labelling or PRINS (a type of PCR applied to spread nuclei) (Pellestor et al., 1996) and comparative genomic hybridization or CGH (Voullaire et al., 2000; Wells and Delhanty, 2000; Wilton et al., 2001a,b), comparing the hybridization of two differentially labelled genomic DNA samples on normal metaphase spreads. Although after PGD often no supernumerary healthy embryos are available, cryopreservation has been attempted in the minority of cases where healthy embryos were still available after transfer. Compared with the cryopreservation of non-biopsied embryos, the outcome of cryopreservation of biopsied embryos has been very bleak (Joris et al., 1999; Magli et al., 1999; Lee and MunneÂ, 2000). However, ongoing research to adapt current freezing protocols to the special needs of embryos with a breached zona pellucida has recently led to higher success rates (Lalic et al., 2001; Wilton et al., 2001b). The main aim of PGD is eventually to help couples to have healthy children, not only avoiding the genetic disease for which they are at risk, but also avoiding the possible consequences of embryo manipulation. The accuracy and safety of PGD and its in¯uence on the outcome of pregnancies and babies born after PGD will be discussed (ESHRE PGD Consortium Steering Committee, 1999, 2000; Strom et al., 2000).

Biopsy methods Polar body biopsy

Polar bodies may be considered as useless by-products of meiosis and can therefore probably be safely removed without further intervention or possible damage to the embryo proper. They contain the genetic complement of the oocyte and are consequently used for the indirect determination of the genetic status of the oocyte. The ®rst reports on polar body biopsy (PBB) concerned ®rst PBB only, which meant they were a true preconception diagnosis (Verlinsky et al., 1992). However, after a misdiagnosis occurred because of non-detected allele drop-out (ADO), the Chicago group, who are the most active proponents of PBB, switched to consecutive biopsy of the ®rst and second polar body, in order to have more complete control over such phenomena as cross-over and ADO (Verlinsky et al., 1996;


Strom et al., 1997). The main disadvantage of PBB is that the paternal contribution to the embryo cannot be analysed, ruling out PBB for autosomal dominant diseases and translocations transmitted through the father, and that sexing is impossible with PBB. Technically, PBB is quite straightforward. As oocytes may be damaged by the use of acidic Tyrode's solution, the most common method for zona pellucida (ZP) breaching is to form a slit in the ZP using ®ne needles. A more elaborate method is to apply two perpendicular slits to allow a ¯ap in the ZP to be raised for the introduction of a biopsy pipette (Cieslak et al., 1999). An alternative to mechanical ZP breaching is to use the laser, which will be discussed in the next paragraph. Cleavage-stage biopsy

Cleavage-stage biopsy is the most widely used technique and is also used at our centre. Early experiments on murine and human embryos had established that the 8-cell stage was most suitable for biopsy (Nijs and Van Steirteghem, 1987; Wilton et al., 1989; TarõÂn et al., 1992; Liu et al., 1993). Hardy et al. were the ®rst to demonstrate the feasibility of blastomere biopsy at the 8-cell stage in the human, showing the absence of a detrimental effect on further in-vitro development of biopsied embryos (Hardy et al., 1990). Several methods exist to retrieve one or two blastomeres from the embryo. In a ®rst step, a hole has to be made in the ZP to allow access to the blastomeres. This can be done by mechanical means, as explained above for PBB, but the most widely used method is the application of a thin stream of acidic Tyrode's to dissolve the ZP. In this way, a funnel-shaped hole is made. This method requires a certain amount of skill and led in our hands to a non-negligible amount of cell lysis during biopsy. The introduction of laser technology allowed a more precise and above all quicker and easier way of ZP hatching (Joris et al., 2000). The group of Dexeus Institute of Barcelona (Boada et al., 1998) mainly introduced the use of a 1.48 mm diode laser, which allows for a good control of the place and width of the application and leaves a well-delineated hole in the ZP. We have been using ZP hatching with the laser since June 1999 with good results (Joris et al., 2000). The most striking improvements when compared with acidic Tyrode's drilling is that the biopsy procedure takes only 50% of the time and that signi®cantly less blastomeres are lysed during biopsy [648/681 (95.2%) intact blastomeres using acidic Tyrode's versus 762/775 (98.3%) with laser]. The implantation and ongoing pregnancy rates were similar in both groups. Once the hole in the ZP is made, there are several possibilities for the removal of one or two cells. In the extrusion method, the hole in the ZP is placed at 12 o'clock and a gentle pressure is applied at 3 o'clock to squeeze out the blastomeres (Staessen et al., 1996). In our hands, this method often led to the lysis of the blastomere and was quickly replaced by the aspiration method (Ao et al., 1996). Here, a biopsy pipette is introduced through the hole and the blastomeres are gently aspirated partly inside the pipette and then removed from the embryo by retrieving the pipette. A last method that is mentioned for completeness is the ¯ow displacement method, whereby blastomeres are expelled through one hole in the ZP by a ¯ow introduced through a second hole in the ZP. There is no mention in the literature of the clinical use of this method.

Current concepts in PGD When using acidic Tyrode's to drill the ZP, two pipettes are needed, one for the application of acidic Tyrode's and one for the biopsy itself. Unless three holders are used for the micromanipulation, changing the pipettes in between biopsies can be tedious and time consuming. This is why Inzunza et al. introduced an elegant method of single-needle blastomere biopsy (Inzunza et al., 1998). One larger pipette is used to drill the ZP with acidic Tyrode's, after which the acid solution is removed by aspiration including part of the medium, so that the medium surrounding the embryo as well as the medium in the pipette reaches a neutral pH again. This is then followed by the blastomere biopsy using the same pipette. After the embryo has reached the 8-cell stage it will start to compact. This phenomenon is even more marked since the introduction of sequential media and can complicate the removal of blastomeres. This is why several centres now incubate compacted embryos for a short time in Ca++- and Mg++-free medium before biopsy after reports in the literature indicated the safety of the method (Dumoulin et al., 1998). Blastocyst biopsy

The last moment in the embryo's development at which it can be biopsied is the blastocyst stage. Early on, blastocyst biopsy knew its proponents because the obvious advantage is that more cells are available for analysis (Varawalla et al., 1991) and that the embryo proper is not touched as the trophoblast cells are biopsied. The drawbacks, however, were that very few embryos in vitro make it to the blastocyst stage and that very little time is left for analysis. Moreover, it was known that trophoblast cells are often multinucleated or even in syncitium. The ®rst drawback may be partly overcome by the signi®cant improvement of media speci®c for blastocyst culture (Gardner et al., 2000), although the bene®t of these culture media is not yet clear. Apparently, the other disadvantages of blastocyst biopsy have led to abandonment of the method, as no recent publication mentions it.

The use of FISH in sexing for X-linked diseases and PGD for translocation carriers Usually, the mothers of boys affected with recessive X-linked disease (such as Duchenne's muscular dystrophy and haemophilia A and B) are healthy carriers and have a risk of 50% of having an affected boy at each pregnancy. As daughters from carrier mothers are not at risk to develop the disease, sexing of fetuses and selective termination of male fetuses has been used for the prevention of these diseases. Unfortunately, the karyotyping used in prenatal diagnosis is inef®cient at the single cell level, and the development of FISH which allowed enumeration of a limited number of chromosomes (e.g. X and Y chromosomes), opened new avenues for PGD. In-situ hybridization was ®rst developed using radioactively labelled probes, but became much more amenable to the fast and ef®cient analysis of several chromosomes simultaneously once the probes were labelled with ¯uorochromes of different colours (Grif®n et al., 1992). Brie¯y, the method consists of the following steps: the biopsied blastomere is spread on a glass slide using either a modi®ed Tarkowski method (Munne et al., 1995) or the HCl/Tween 20 method (Coonen et al., 1994), followed by a ®xation step. Fluorescently labelled probes that recognise speci®c sequences of

speci®c chromosomes are then applied on top of the spread cell and allowed to hybridize to the chromosomes. After a few washes to remove the excess of probe, the result can be analysed through a ¯uorescence microscope using ®lters for different colours. Alternatively, the probes can be labelled with a hapten, which is then indirectly detected with ¯uorescently labelled antibodies. Since the directly labelled probes are currently widely commercially available and give results faster, most centres will prefer these. Although several sophisticated PCR methods for sexing were published in the wake of a misdiagnosis caused by a nonampli®cation (Grifo et al., 1992; Hardy and Handyside, 1992; Levinson et al., 1992; Chong et al., 1993; Liu et al., 1994a), it soon became clear that FISH had several important advantages over PCR for sexing of embryos. Firstly, because the X and Y chromosome were detected, aneuploidies in these chromosomes could be clearly detected, e.g. an XO karyotype would not be picked up by PCR, but would be clearly seen after FISH. In the ®rst case, the embryo would be transferred and could lead not only to a baby with Turner's syndrome, but also, as sexing is usually done for X-linked disease, to a girl with the genetic disease under consideration (Harper et al., 1994; Staessen et al., 1999). When after some time more ¯uorochromes became available (®ve different colours can be used in one round, and after removing the probes a second round of hybridization for even more chromosomes can be performed), sexing could be combined with aneuploidy screening. Since many PGD patients are >36 years, this is an advantage. Secondly, FISH techniques are not ridden with problems such as contamination and allelic drop out (ADO), which make single-cell PCR a technically demanding technique. The main disadvantage of sexing for Xlinked disease is that half of the male embryos are healthy and will not be replaced, and that the replacement of carrier girls cannot be avoided. This, together with advances in molecular biology, has led to the fact that, if the mutation in a given family is known or can be analysed through linked markers, the couples who wish to undergo PGD will rather opt for a speci®c DNA diagnosis rather than simple sexing. This trend is very clear at our centre, where the relative number of cycles for sexing has been steadily declining (C.Staessen, personal communication). Reciprocal chromosome translocations are characterized by the exchange of fragments between chromosomes, while in Robertsonian translocations a whole chromosome is translocated to another one through centromeric fusion. Carriers of balanced translocations are usually phenotypically normal because no genetic material is missing or in excess. However, the unbalanced offspring of carriers can be abnormal, leading to births of children with multiple congenital anomalies (e.g. Down's syndrome), or more frequently carriers of translocations suffer from secondary infertility because of recurrent miscarriages. The risk for either normal or balanced offspring, unbalanced offspring or recurrent miscarriage can be approximately estimated according to the chromosomes involved and the size of the exchanged fragments (Scriven et al., 1998). The group of Verlinsky described a method to obtain metaphases from blastomeres by fusion of the biopsied blastomere with failed fertilized metaphase II human oocytes or human zygotes (Evsikov and Verlinsky, 1999), or later with mouse zygotes (Evsikov al., 2000). The resulting heterokaryons either spontaneously went into mitosis or were induced to cleave with ocadaic acid, after which they were ®xed and the metaphase


K.Sermon plate analysed using whole chromosome paints (a FISH method whereby probe mixtures cover the complete chromosomes). Although this method seems to be ef®cient in obtaining readable karyotypes (131 analysable metaphases from 164 blastomeres, 86%), except from one centre (Willadsen et al., 1999; using bovine oocytes), no other centre has attempted to use this method, probably because of the technical complexity. Polar body biopsy was used by the group of Munne et al.; ®rst polar bodies are still in metaphase when biopsied soon after their extrusion, and these metaphases can be analysed using chromosome painting (Munne et al., 2000). Although this method had a high ef®ciency (10 pregnancies from 22 cycles, 45%), it could not be used for male carriers. For Robertsonian translocation, simple enumeration of the chromosomes leads to a reliable diagnosis, but the situation is more complex for reciprocal translocations. The ®rst probes used for PGD of reciprocal translocations were probes isolated from cosmid, YAC or BAC libraries and spanning or ¯anking the breakpoint. These probes gave very accurate results because they allowed the distinction between normal and balanced genotypes, but were very time-consuming to isolate (Conn et al., 1999; Weier et al., 1999; Coonen et al., 2000; Iwarsson et al., 2000). Our group was the ®rst to describe the use of commercially available probes (here a diagnostic kit for DiGeorge syndrome) for the most common reciprocal translocation, t(11;22) (Van Assche et al., 1999). The publication and later commercial availability of subtelomeric probes (Scriven et al., 1998; Munne et al., 2000) opened completely new avenues for PGD for reciprocal translocations. A combination of two subtelomeric probes distal to the breakpoint with a different colour for the two chromosomes involved and a centromeric probe labelled with a third colour for one involved chromosome allows for the distinction between normal/balanced genotypes and unbalanced genotypes. Caution must be raised that recombination cannot be detected using this probe combination, but can be ignored because of the low frequency of this event. This method has very soon been adopted by most centres performing PGD for reciprocal translocations, including our own. PGD for other chromosomal rearrangements, such as pericentric inversion for chromosome 5 and DiGeorge syndrome, have also been successfully performed using in-house developed probes (Iwarsson et al., 1998a,b). A number of striking features have emerged from the analysis of embryos from translocation carriers. The ®rst is the high number of unbalanced embryos in carriers of reciprocal translocations. Munne et al. found 72/102 abnormal oocytes after polar body biopsy from this type of patient (Munne et al., 2000). Coonen et al. found 20/24 unbalanced embryos from two siblings (a man and a woman) carrying a t(3;11) (Coonen et al., 2000). Using the blastomere nucleus conversion technique, Evsikov et al. found 87/131 (66%) of the analysed embryos (19 from Robertsonian, 112 from reciprocal translocations) to be unbalanced (Evsikov et al., 2000). A possible difference as to the number of unbalanced gametes produced in males and females may exist, and may be different from the data available from the analysis of miscarriage material, prenatal diagnosis and live offspring, but currently numbers are too small to be conclusive. Several authors analyse sperm from male translocation carriers before embarking on PGD to be able to estimate the risks and counsel the patients as thoroughly as possible (Van Assche et al.,


1999; Coonen et al. 2000). Another striking feature is the high incidence of mosaic and chaotic embryos, which seems to be higher in reciprocal than in Robertsonian translocations (Conn et al., 1998; Van Assche et al., 1999; Iwarsson et al., 2000; Munne et al., 2000; Magli et al., 2001). Thus, infertility in translocation carriers may not only be caused by the presence of unbalanced gametes, but also because of the high incidence of aneuploidy involving other than the translocation chromosomes. This is also re¯ected in the pregnancy rates obtained in translocation carriers: the ESHRE PGD Consortium reported a pregnancy rate of 19% per embryo transfer and 15% per oocyte retrieval, mainly caused by the low number of embryos suitable for transfer after PGD (27%) (ESHRE PGD Consortium Steering Committee, 2000). Again, the number of embryos analysed are small and the results are only indicative of general trends, but further follow-up and research both on the topic of the number of unbalanced gametes as well as on the number of mosaic and chaotic embryos is mandatory. Finally, MeÂneÂzo et al. have proposed to culture embryos from translocation carriers in vitro until the blastocyst stage, with the aim of achieving a selection of only normal/balanced blastocysts, reasoning that unbalanced embryos are not viable and would thus be selected against (MeÂneÂzo et al., 1997, 2001). Evsikov et al. have, however, shown that an equal proportion of normal/ balanced (14/44, 32%) and unbalanced embryos (23/87, 26%) make it to the blastocyst stage (Evsikov et al., 2000). The fact that many couples where one partner is a translocation carrier experience recurrent miscarriages, and that some of these translocations may even lead to live offspring, are also arguments against MeÂneÂzo's hypothesis.

The use of PCR for monogenic diseases PCR is a method by which short stretches of DNA are ampli®ed exponentially. Technically, PCR consists of three steps which are usually repeated between 20±40 times. The ®rst part of a PCR cycle consists of denaturation of the two complementary DNA strands at high temperature (~94°C), followed by an annealing step at a lower temperature, to allow two short, single-stranded complementary pieces of DNA (called primers) to anneal to the template DNA on either side of the DNA sequence to be ampli®ed. In the third step (elongation), a heat-resistant DNA polymerase (usually Taq DNA polymerase) inserts dinucleotide building blocks, starting from the primers. At the end of the cycle, the number of DNA molecules has doubled and the next cycle can start. Very early on, it was realised that PCR offered an important means by which to analyse the genetic content of single cells (Jeffreys et al., 1988) so, unsurprisingly, the ®rst reports of preclinical and clinical PGD tests followed very quickly (Coutelle et al., 1989; Handyside et al., 1990). The ®rst reports on the ampli®cation of unique sequences were neither very ef®cient nor gave clear results, because Taq polymerase tends to incorporate more and more mistakes as more cycles are applied (necessary when starting from only 6 pg DNA present in a single cell) and aspeci®c priming leads to aspeci®c bands. This problem was very soon solved with the introduction of nested PCR (Holding and Monk, 1989). Here, a ®rst round of ampli®cation with an outer set of primers is performed, followed by a second round using a small

Current concepts in PGD amount of the ®rst round as a template and a set of inner primers (i.e. primers amplifying a PCR fragment inside the outer primers). As experience with single cell PCR grew, three main characteristic problems became apparent: the ®rst one was the problem of speci®city when a large number of cycles are needed. Besides the nested PCR described above, other measures such as careful design of primers, the use of better performing DNA polymerases (Sermon et al., 1998b, 1999b) and the use of more sensitive detection methods (e.g. ¯uorescent PCR) (De Vos et al., 1998, 2000; Findlay et al., 1998; Sermon et al., 1998a,b, 1999a,b; Van de Velde et al., 1999; Goossens et al., 2000) were introduced and have led to signi®cant improvements. The second recurrent problem is contamination, either from genomic DNA from the operator or the patient (e.g. cumulus cells or sperm sticking to the zona) or carry-over contamination from PCR products ampli®ed earlier. The ®rst type of contamination can be avoided by using ICSI when PCR will be used for diagnosis (Liebaers et al., 1998) and by working in a sterile manner; the second type requires special precautions such as separate pre- and post-PCR rooms and dedicated material (Lissens and Sermon, 1997). Contamination can be detected through the use of blanks and multiplex PCR (see below). The third problem, which has led to at least three reported misdiagnoses, is ADO. This is the non-ampli®cation of one of the two alleles in a single cell, which can only be detected or lead to problems in a heterozygous cell. A difference should be made between ADO and preferential ampli®cation (PA), where one allele is less ampli®ed than the other. If less ef®cient detection methods are used, often the difference between ADO and PA cannot be detected. However, more sensitive detection methods (e.g. ¯uorescent PCR) allow for the clear distinction of these two phenomena. Here too, several strategies either for avoiding or detecting ADO have been described, the most important at this moment being multiplex PCR with linked markers, sometimes in combination with ¯uorescent PCR (Findlay et al., 1998; Rechitstky et al., 1998). Although nested PCR followed by analysis on ethidiumbromide-stained polyacrylamide gel electrophoresis (PAGE) or agarose gels is still widely in use, multiplex PCR included, more and more investigators turn to the use of ¯uorescent PCR because of the higher resolution (a difference in fragment length of one base pair can be distinguished) and higher accuracy [ADO rates can drop from 24% in conventional PCR to 6.5% in ¯uorescent PCR for myotonic dystrophy (Sermon et al., 1998a)] (Moutou and Viville, 1999; Piyamangkol et al., 2001). For more details, the reader is referred to other authors (Lissens and Sermon, 1997; Wells and Sherlock, 1998), but multiplex PCR will be discussed in depth. Multiplex PCR, which is the simultaneous ampli®cation of two or more DNA sequences, is used extensively in genetic diagnosis for more frequent diseases such as cystic ®brosis (e.g. INNOLiPA CFTR17+Tn ampli®cation kit, Innogenetics, Ghent, Belgium) and Duchenne's muscular dystrophy (Ray et al., 2001). The advantages of using multiplex PCR at the single cell level are considerable. Firstly, contamination with DNA of cellular origin can be detected when using any informative marker in the genome. If, for example, a marker on chromosome 21 were to be used, on top of detecting contamination, trisomy 21 would also be detected providing the parents are fully informative, i.e. carry four, or two each, completely different alleles (Cram et al., 2001; Piyamongkol et al., 2001). Secondly, if linked markers are

used, ADO in one locus can be detected by comparing it with the result in the second locus. This approach was pioneered by Rechitsky et al. in polar bodies (Rechitsky et al., 1998). These investigators combine up to ®ve loci in one PCR (e.g. one for the mutation and four more for linked or non-linked markers). They calculated that using a duplex for a mutation and one linked marker decreased the risk for misdiagnosis due to ADO by at least half; e.g. the ADO rate for exon 10 of the CFTR gene was 33.3%, while the linked marker in intron 6 of the CFTR gene showed 20.4% ADO. When used together, ADO occurred simultaneously in both loci in only 13.9% of the cases. A third important advantage of multiplex PCR is that linked markers can also be used for diagnosis. Dreesen et al. described such a strategy for cystic ®brosis, for which at this moment >900 mutations, most of which are private, are known (Dreesen et al., 2000). Instead of developing a new test for each patient with a new cystic ®brosis mutation, these authors developed a single cell PCR amplifying four highly informative linked markers ¯anking the cystic ®brosis gene. Once this test is developed, it can be used for virtually all couples at risk for cystic ®brosis, provided they are informative for at least two ¯anking markers. The fact that contamination, ADO and recombination can be detected is an added bonus. A similar strategy has recently been described for Fragile X syndrome (Apessos et al., 2001). Ray et al. and Hussey et al. have developed a multiplex PCR covering parts of the dystrophin gene that are mostly involved in disease-causing deletions (Hussey et al., 1999; Ray et al., 2001). This test can be used for >70% of the Duchenne muscular dystrophy deletion carriers, but can detect neither contamination nor ADO, and although ADO is not a problem in this case, contamination could potentially lead to the transfer of an affected embryo. Usually however, as is the case in our centre, attempts are made to combine a speci®c mutation with (un)linked markers (Piyamongkol et al., 2001). Strategies and tests developed for the more common genetic diseases will now be reviewed. Cystic ®brosis, being the most common autosomal recessive disease in the Northern European population, has been from the beginning the focus of attention of several groups involved in PGD. The ®rst reports all used nested PCR of a fragment encompassing the most common mutation in cystic ®brosis, DF508, followed by analysis through heteroduplex formation on PAGE gels (Handyside et al., 1992; Verlinsky et al., 1992; Grifo et al., 1994; Liu et al, 1994b). Considering the fact that all these authors used boiling in water as a lysis method and the high ADO rates expected in the types of PCR used, it is not surprising that a number of misdiagnoses occurred (Grifo et al., 1994; Verlinsky et al., 1996). These led to, on one side, higher caution in investigators (e.g. by not accepting couples where each partner carried a different mutation, at least until more accurate testing was available) and, on the other side, the development of tests to detect or avoid ADO. These included the development of sequential ®rst and second polar body analysis (Strom et al., 1997), improvement of the PCR itself by increasing the denaturation temperature (Ray and Handyside., 1996), using a more stringent lysis method (Wu et al., 1993; Thornhill et al. 2001) and the introduction of ¯uorescent PCR for mutations or linked markers (Moutou and Viville, 1999; Dreesen et al., 2000; Goossens et al., 2000; Eftedal et al., 2001), and ®nally the use of multiplex PCR (Rechitsky et al., 1998; Goossens et al., 2001).


K.Sermon From the literature and our own experience, we foresee that multiplex PCR using ¯uorescent PCR will soon become, for cystic ®brosis as for other diseases, the golden standard. Haemoglobin diseases are the commonest monogenic disorders overall: sickle-cell anaemia is caused by an A>T mutation at codon 7 in the b-globin gene and is common in Africa, while bthalassaemia is caused by many different mutations in the bglobin gene and is prevalent in the countries around the Mediterranean Sea. The development of PGD for b-globin diseases runs nearly parallel with cystic ®brosis: the ®rst reports used nested PCR followed by detection on agarose gels (Pickering et al., 1992; Monk et al., 1993; Xu et al., 1999). Later, more sophisticated methods such as ®rst and second polar body analysis (Kuliev et al., 1999), strand conformation polymorphism (SSCP) (El-Hashemite et al., 1997), denaturing gradient gel electrophoresis (DGGE) (Kanavakis et al., 1999; Vrettou et al., 1999) and ¯uorescent PCR (De Rijcke et al., 2001) led to a more reliable and successful application of PGD. Less common, but still rather frequent autosomal recessive diseases for which PGD has been developed include Tay-Sachs disease, which is as common as cystic ®brosis in the Ashkenazi Jewish population (Gibbons et al., 1995; Sermon et al., 1995; Liu et al., 2000), spinal muscular atrophy, which is the most common autosomal recessive disease after cystic ®brosis in the Northern European population (Dreesen et al, 1998; Fallon et al., 1999; Moutou et al., 2001), congenital adrenal hyperplasia (1/10000 live births) (Van de Velde et al., 1999) and medium-chain acyl-CoA dehydrogenase (MCAD; 1/10000 live births) (Sermon et al., 2000). At our centre, we have a particular interest in the development of PGD for dynamic mutations. This group of diseases is characterized by triplet repeats in the disease-causing genes, which can display instability and expand, thus disturbing the activity of the gene. They can be inherited in an autosomal dominant fashion (e.g. Huntington's disease and myotonic dystrophy), in an autosomal recessive fashion (e.g. Friedreich's ataxia), and in X-linked recessive (Kennedy's disease) or dominant (Fragile X syndrome) fashion. As these triplet repeats are highly polymorphic, we have developed a generalized approach for PGD for these diseases. Using PCR, the DNA fragment around the triplets was ampli®ed, but only in the healthy alleles, since the large expanded alleles are refractive to PCR ampli®cation. Two exceptions are Huntington's disease (Sermon et al., 1998b), where disease causing alleles are usually ~200±250 base pairs long and Kennedy disease (Georgiou et al., 2001). In this type of PGD, the informativity of the patients has to be tested ®rst, i.e. the alleles of the healthy parent must differ from the healthy allele of the affected parent. Although contamination probably led to one misdiagnosis for myotonic dystrophy (Sermon et al., 1998b), ADO will not lead to misdiagnosis in this type of PGD. We have developed tests for myotonic dystrophy (Sermon et al., 1998a), Huntington's disease (Sermon et al., 1998b), Fragile X syndrome (Sermon et al., 1999a) and Kennedy disease (Georgiou et al., 2001). Other authors have also described PGD for myotonic dystrophy (Piyamongkol et al., 2001). Spinocerebellar ataxia type 7 is another genetic disease caused by a dynamic mutation for which we have developed a test and which has been applied clinically.


PGD for fairly common autosomal dominant diseases has also been developed: we and others have developed PGD for Marfan's syndrome (Harton et al., 1996; Blaszczyk et al., 1998; Sermon et al., 1999b), osteogenesis imperfecta (De Vos et al., 2000) and Charcot-Marie-Tooth disease type 1A (De Vos et al., 1998).

Outcome of PGD: pregnancy rates and outcome of pregnancies and children born The ESHRE PGD Consortium has had a main aim of collecting data on PGD cycles and their outcome, particularly the safety of the procedure as related to pregnancies and children born. In the last report (ESHRE PGD Consortium Steering Committee, 2000), 1318 cycles were reported and 141 of these (16%) have led to a pregnancy. Because pregnancies and their outcome were collected separately from the cycle data, the reported pregnancies did not coincide with the 141 cycles with a pregnancy. A total of 163 pregnancies were reported, of which 138 went on to the second trimester. The occurrence of four misdiagnoses was notable, all having occurred after PCR and the birth of 37 twins and one triplet out of 123 deliveries (31% multiplets). Complications, both before and after birth, were not different in type or number from those found in a comparable ICSI population. Other parameters, such as birthweight and length, were also similar to an ICSI population. Another large cohort of children born after PGD was reported by Strom et al. (Strom et al., 2000). A total of 109 infants were described and here too, the conclusions were that children born after PGD are very comparable with children born after ICSI, and that PGD is a safe method to avoid the birth of children with genetic defects.

Future developments Cryopreservation of biopsied embryos

Most PGD cycles are an elimination contest, with loss of embryos at each stage and not least due to diagnosis, there are not usually many embryos left for cryopreservation. In the cycles reported for the ESHRE PGD Consortium, only 360 of the 4323 embryos analysed were cryopreserved. This bleak outcome is worsened further by the fact that biopsied embryos have a low survival rate after cryopreservation. Magli et al. found only 38% survival versus 61% in unbiopsied embryos (Magli et al., 1999), whereas we found 29.5% intact blastomeres in biopsied embryos versus 56.3% in non-biopsied embryos (Joris et al., 1999). Lee et al. reported a pregnancy after polar body biopsy and cryopreservation, but these authors also admit that although cryopreservation is the only alternative for supernumerary healthy embryos, the chances of pregnancy remain limited (Lee et al., 2000). We have tried to avoid the cryopreservation of biopsied embryos by freezing embryos before biopsy in patients with >16 good quality embryos at risk for an autosomal recessive disease where 75% of the embryos are expected to be transferable. It is obvious that this strategy cannot be applied very often. At the 2001 ESHRE Annual Meeting in Lausanne, hope was rekindled by two Australian groups who showed a higher survival rate after freeze±thawing and subsequent pregnancies (Lalic et al., 2001; Wilton et al., 2001b). Lalic et al. reported that 16/24 (75%) of the thawed embryos survived, while 4/14 (25%) of the subsequently

Current concepts in PGD transferred embryos implanted (Lalic et al., 2001). The success of these groups seems to be in the gentler and more progressive removal of sucrose from the freezing medium. Further research on larger numbers is needed to con®rm these initially encouraging results. Comparative genomic hybridization

Comparative genomic hybridization (CGH) was developed as a method to analyse the chromosome complement of solid tumours, which very often display highly rearranged and complex karyotypes. As it allowed karyotyping of cells in interphase, it was later adapted at the single cell stage (Voullaire et al., 2000; Wells and Delhanty, 2000; Wilton et al., 2001a). In CGH, a single cell to be analysed undergoes degenerate oligonucleotide primed (DOP) PCR reaction (a type of whole genome ampli®cation) during which the PCR fragments are labelled with a ¯uorochrome (e.g. green). A control cell undergoes the same treatment, but with a different ¯uorochrome (e.g. red). These two labelled PCR fragment mixtures are then hybridized together to a normal metaphase spread, after which the relative green/red ratio is measured using a CCD camera and image analysis with special software. Excess genetic material in the tested cell would light up greener, and loss of genetic material would light up redder. CGH at the single cell level would especially be useful in aneuploidy screening and in translocation cases, although as with subtelomeric probes, CGH does not allow for the discrimination between normal and balanced karyotypes. CGH, in contrast to multicolour FISH, allows for the analysis of the complete chromosome set of the embryo, and has already yielded surprising results. Aneuploidies involving chromosomes that are never found in miscarriage or other prenatal material can be found and every chromosome was found to be involved in aneuploidies. Some aneuploidies would not have been detected using multicolour FISH. At this moment, CGH still requires ~72 h of analysis, which is why embryos need to be cryopreserved after biopsy when CGH is used in a clinical setting (Wilton et al., 2001b). In the future, the metaphase to which the DOP±PCR fragments are hybridized could be replaced by microarrays containing sequences covering the whole genome, and would thus allow for a quicker analysis, obviating the need for cryopreservation. PGD for monogenic diseases using molecular beacons

Molecular beacons are short oligonucleotides which carry a ¯uorochrome on one end and a quencher on the other. As these molecular beacons form hairpin loops, in a normal state the ¯uorochrome is quenched and does not emit light. However, when the molecular beacon hybridizes to a complementary DNA sequence, the hairpin loop is opened and the ¯uorochrome is released from the quencher. The beacons have been incorporated into PCR reactions, where they hybridize to the PCR product during PCR cycling, and thus yield real-time results as to the amount of PCR product ampli®ed. The technology has been reported for sexing by ampli®cation of a Y-sequence, but in my opinion this can not replace FISH for sexing, which is much simpler and gives more information on the embryo's ploidy status (Pierce et al., 2000). Other applications could be the diagnosis of single nucleotide changes or other mutations involving small fragments, but here a method should be found to couple this type of analysis to the analysis of linked markers of the microsatellite

type (Wangh et al., 2001). Finally, the most important application of molecular beacons may be for mitochondrial diseases. Brenner et al. have already shown the use of molecular beacons to quantify heteroplasmy in mitochondria in peripheral blood (Brenner et al., 2001).

Conclusions PGD is now a well-established alternative to prenatal diagnosis. Two main methods for obtaining material from the preimplantation embryo are basically in use: the ®rst and second polar body biopsy, and blastomere biopsy at the cleavage stage. Depending on the genetic risk of the parents, FISH is then used for the diagnosis of chromosomal abberrations and PCR for the detection of monogenic disease. In chromosomal translocations, the introduction of subtelomeric probes has led to their wide-spread diagnostic use on interphase nuclei. For monogenic diseases, multiplex PCR, with or without detection by ¯uorescent PCR, is becoming the golden standard. Finally, most practitioners in the PGD ®eld are aware that close follow-up of children born after PGD is mandatory to ensure that the ®nal aim of PGD, i.e. to allow couples at risk to have healthy children, is met.

Acknowledgements I am indebted to the staff of the PGD team for their daily commitment to make PGD a workable alternative for our patients, to Profs Liebaers, Van Steirteghem and Devroey for their continuing support, and to the Fund for Scienti®c Research Flanders (FWO Vlaanderen) for post-doctoral support. The PGD team is supported by grants from the Fund for Scienti®c Research Flanders (FWO Vlaanderen), the Research Council (OZR) of the Dutchspeaking Brussels Free University, the Forton Fund and the Concerted Action of the Brussels Free University.

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Received on August 1, 2001; accepted on October 31, 2001

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