Human Reproduction Update Advance Access originally published online on November 29, 2004
Human Reproduction Update 2005 11(1):33-41; doi:10.1093/humupd/dmh050
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Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization
Genetic and Molecular Research, Melbourne IVF, 320 Victoria Parade, East Melbourne 3002, Victoria, Australia
Email: lwilton{at}mivf.com.au
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Abstract |
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Abstract
IntroductionNumerical chromosome errors are known to be common in early human embryos and probably make a significant contribution to early pregnancy loss and implantation failure in IVF patients. Over recent years fluorescent in situ hybridization (FISH) has been used to document embryonic aneuploidies. Many IVF laboratories perform preimplantation genetic diagnosis (PGD) with FISH to select embryos that are free from some aneuploidies in an attempt to improve implantation, pregnancy and live birth rates in particular categories of IVF patients. The usefulness of FISH is limited because only a few chromosomes can be detected simultaneously in a single biopsied cell. Complete karyotyping at the single cell level can now be achieved by comparative genomic hybridization (CGH). CGH enables not only enumeration of all chromosomes but gives a more complete picture of the entire length of each chromosome and has demonstrated that chromosomal breakages and partial aneuploidies exist in embryos. CGH has provided invaluable information about the extent of mosaicism and aneuploidy of all chromosomes in early human conceptuses. CGH has been applied to clinical PGD and has resulted in the birth of healthy babies from embryos whose full karyotype was determined in the preimplantation phase.
Key words: aneuploidy / comparative genomic hybridization / fluorescent in situ hybridization / preimplantation genetic diagnosis
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Introduction |
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A significant proportion of pregnancy wastage is caused by numerical chromosome imbalance or aneuploidy. This is evidenced by the observation that >50% of first trimester spontaneous abortuses are aneuploid (Hassold et al., 1980
; Chandley, 1984; Jacobs, 1992). Some of the most commonly seen aneuploidies are trisomies of chromosomes 16, 21 and 22 (Hassold et al., 1980; Kalousek, 1987; Griffin et al., 1997; Stephenson et al., 2002). Trisomies of other chromosomes are rarely seen in clinically recognizable pregnancies, presumably because they are highly lethal and are lost much earlier in gestation. This is probably also true of monosomies which, except for monosomy X (Turner's syndrome), are not observed in spontaneous abortuses. |
Chromosome errors in human embryos |
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It is estimated that at 10 weeks gestation,
5% of human conceptions are aneuploid (Boue et al., 1985). Given the lethality of many aneuploidies it is not surprising that analysis of the chromosome constitution of early preimplantation embryos reveals a much higher rate of aneuploidy soon after conception. It is estimated that >50% of all embryos contain one or more aneuploid cells. Many of these aneuploidies are rarely, if ever, seen in later stage fetuses.
Although some information about aneuploidy in preimplantation embryos has come from studies using traditional analysis of metaphase chromosomes, this approach is technically difficult and inefficient when applied to single cells. The vast majority of single blastomere chromosome analyses have been done using fluorescent in situ hybridization (FISH). FISH utilizes chromosome-specific DNA probes labelled with different coloured fluorochromes that are hybridized to the target DNA that has been fixed to a microscope slide. Computerized imaging systems enable fluorescent probe signals to be identified and counted. FISH works extremely well on interphase nuclei, obviating the need to obtain interpretable metaphase chromosomes from blastomeres. The first application of FISH to single cells (Griffin et al., 1992) used probes for the X and Y chromosomes and was soon used to determine the gender of embryos from patients who carried X-linked recessive diseases (Griffin et al., 1993, 1994). Since this time, more fluorochromes have become available, meaning that more chromosomes can be simultaneously analysed. Additionally, FISH signals can be washed off the nucleus and another set of probes for different chromosomes applied. Commonly seven to nine chromosomes are analysed in individual cells (Figure 1). Re-probing nuclei a third time leads to a degeneration of target DNA and a decrease in the efficiency of hybridization.
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FISH studies of normally developing, good quality preimplantation embryos have shown that 30–65% have an aneuploidy in at least one cell (Benadiva et al., 1996
; Delhanty et al., 1997; Iwarsson et al., 1999). When several blastomeres in embryos are analysed chromosomal mosaicism is observed. Mosaicism is where either some cells are diploid and others are aneuploid or where different cells have different aneuploidies. The proportion of embryos that show mosaicism ranges from 25 to >60% (Delhanty et al., 1993, 1997; Munné et al., 1994, 1997; Benadiva et al., 1996).
Most of the early embryos that have been analysed for aneuploidy have come from infertile patients from assisted reproduction programmes as access to embryos from fertile people is extremely limited. It is difficult to know whether the high frequency of aneuploidy reflects the underlying infertility experienced by the couples whose gametes produced these embryos or whether this is a true picture of the chromosomal errors that exist in early human conceptuses. Some fertile patients, who are carriers of single gene defect diseases such as cystic fibrosis or thalassaemia, access IVF to have preimplantation genetic diagnosis (PGD) for the disease that they carry. Embryos that were not transferred because they were affected with the disease have been analysed for chromosomes X, Y and 1 (Delhanty et al., 1997). These embryos had a high frequency of aneuploidy and particularly exhibited chromosomal mosaicism, demonstrating that chromosomal errors exist in embryos from fertile patients. It must be remembered that these embryos were produced by routine IVF procedures and the impact of hormonal stimulation and/or embryo culture on the existence of aneuploidy in embryos cannot be determined. The only way to truly determine the frequency of aneuploidy in early human conceptuses would be to study a reasonable number of in vivo-fertilized embryos from fertile women. Clearly this is not possible.
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PGD of aneuploidy |
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There is no doubt that in recent years the in vitro growth and development of human embryos has been improved by the use of novel culture media, and this translates to higher implantation and clinical pregnancy rates when these embryos are transferred to the uterus. Despite this, the implantation rate of human embryos in most IVF programmes is relatively low compared to other species and is
25–30% for all embryos. If one considers only the highest quality embryos with the best growth and morphology, then still about half of them fail to implant. There is no doubt that the high frequency of chromosomal aneuploidy observed in human embryos makes a significant contribution to implantation failure and early embryo demise.
There is some evidence that some infertile patients are predisposed to chromosome errors in their embryos (Zenzes and Casper, 1992). In an attempt to improve the prognosis for pregnancy after IVF treatment many centres perform PGD using FISH to identify chromosome errors in embryos. This is usually applied to three types of patients: (i) those with recurrent implantation failure (RIF), that is at least three failed IVF cycles or 
10 embryos transferred without pregnancy; (ii) advanced maternal age (AMA), the definition of which differs slightly between centres but is usually aged >37 years; and (iii) women who have suffered recurrent miscarriage. Most commonly FISH for five to nine chromosomes is applied to single cells biopsied from the embryos of these patients. The impact of PGD on these different patient groups has recently been reviewed (Wilton, 2002). There have been too few prospective randomized trials but those studies that have included some sort of control group have demonstrated that PGD for a number of key chromosomes increases the implantation rate (Gianaroli et al., 1999) and ongoing pregnancy rate (Gianaroli et al., 1999; Munné et al., 1999) in patients with advanced maternal age. In a recent study, Munné et al. (2003) have shown that, in a matched group of women aged >35 years, PGD increased the implantation rate and that this improvement was even greater in women who had had less than two previous failed IVF cycles or produced eight or more zygotes in the study cycle.
The benefit of PGD and aneuploidy testing in women with implantation failure is less clear. One controlled study showed no benefit of PGD for RIF patients (Gianaroli et al., 1999) although others have found reasonably good pregnancy rates in both AMA and RIF patients (Kahraman et al., 2000). In a group of older women, Munné et al. (2003) found that those with two or more previous failed IVF cycles did not benefit from PGD for aneuploidy. The ESHRE PGD consortium (ESHRE PGD Consortium Steering Committee, 2002), which collates data from many IVF and PGD centres throughout the world, has reported a clinical pregnancy rate per embryo transfer of 36% for AMA patients but only 11% for RIF patients.
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Limitations of FISH in PGD |
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Although FISH has been applied extensively throughout the world to identify chromosome abnormalities in embryos, its usefulness is limited primarily because only a small number of chromosomes can be identified in each embryo. This restriction is imposed by the limited number of distinct fluorochromes which are available to label the DNA probes. This can be partly overcome by using the technique of proportional labelling but this cannot delineate overlapping signals which are labelled with the same fluorochrome. Also, the more probes that are applied simultaneously the more likely it is that signals will overlap. Overlapping signals of different fluorochromes can be distinguished using computerized imaging systems which allow individual colour planes to be independently analysed but overlapping signals of the same colour cannot be differentiated and can lead to misdiagnosis. A common approach is to use FISH for five or six chromosomes simultaneously and then once these results have been analysed to wash the signals off and re-probe the nucleus for another two, three or four chromosomes. This cannot be done repeatedly as the DNA degenerates and signal efficiency is reduced when nuclei are re-probed for the third time (Liu et al., 1998
). Most laboratories look for chromosomes X, Y, 13, 16, 18, 21 and 22 because these are some of the most common aneuploidies seen in spontaneous abortuses. But recent evidence shows that aneuploidies of other chromosomes are also relatively common in embryos and so a choice must be made about which chromosomes to analyse when using FISH. |
Complete karyotyping of embryos |
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Ideally one would like to be able to identify every chromosome in single blastomeres for PGD. An obvious approach is to utilize traditional karyotyping techniques of analysing metaphase chromosomes. This has been achieved by a number of workers (Wilton, 1993
; Jamieson et al., 1994; Santalo et al., 1995; Clouston et al., 1997) but is too inefficient at the single cell level to be applicable to PGD. It is extremely difficult to obtain interpretable metaphase chromosomes when only one cell is available for analysis. For most of the cell cycle the DNA is in interphase. Exposure to mitotic inhibitors such as colchicine does increase the number of cells in metaphase but too often results in short, over-condensed chromosomes that are not informative when spread for karyotyping. Moreover, when only one cell is available for spreading, the chance that chromosome overlap prevents interpretation of the karyotype is high.
Other, more novel methods, to induce metaphase chromosome formation in blastomeres have been attempted. These include chromosome conversion where the blastomere is fused with an oocyte of another species (Verlinsky and Evsikov, 1999; Willadsen et al., 1999). This has produced chromosomes that can be karyotyped but in almost one-quarter of the fused cells no human DNA could be identified (Willadsen et al., 1999). The technique has not been used for complete karyotyping in clinical PGD although it has been applied clinically in conjunction with FISH for the analysis of embryos from translocation carriers (Willadsen et al., 1999; Verlinsky et al., 2002).
Spectral karyotyping techniques that utilize probes labelled with a combination of fluorochromes can also be used to enumerate chromosomes. This works very well in metaphase nuclei (Schröck et al., 1996) but can only identify 10 chromosomes in interphase cells (Fung et al., 2000) and has an efficiency of 50% on blastomeres (Fung et al., 2000; Weier et al., 2001).
To date, the most successful approach for complete karyotyping of single cells is comparative genomic hybridization (CGH). CGH was originally developed by cancer biologists who needed to analyse aneuploidy in small numbers of cells obtained from solid tumours (Kallionemi et al., 1992 and see review by Wells and Levy, 2003). CGH works by a comparison of the sample or test DNA with known normal or reference DNA (Figure 2). Test DNA and reference DNA are labelled with a green and red fluorochrome respectively and then simultaneously applied to a microscope slide that is covered with normal human male metaphase chromosomes. The labelled test and reference DNA are allowed to hybridize for 2–3 days and then computerized imaging systems are used to analyse the red and green fluorescence along the length of the metaphase chromosomes (Figure 2). A number of metaphase spreads are analysed and the fluorescence profiles for individual chromosomes are compiled (Figure 3). If, for example, the test DNA was normally diploid for chromosome 6 then there would be an equal amount of green and red fluorescence on all the copies of chromosome 6 on the microscope slide. The green:red fluorescence ratio for chromosome 6 would be 1:1 and the test DNA would be diagnosed as normal for that chromosome. If, however, the test cell was monosomic for chromosome 6 then there would be less green labelled chromosome 6 DNA in the mixture that was hybridized to the slide and all the copies of chromosome 6 on the slide would have a decreased ratio of green:red fluorescence. On the CGH profiles, this would be seen as a shift of the fluorescence ratio to <1 and the test DNA would be diagnosed as missing a copy of chromosome 6. This green:red fluorescence ratio can be determined for every chromosome, enabling a complete molecular karyotype to be generated.
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The obvious advantage of CGH over FISH is that the copy number of all chromosomes can be determined. In addition, CGH provides a more detailed picture of the entire length of each chromosome, enabling the detection of imbalance of chromosomal segments. Although FISH is routinely used to determine ploidy of particular chromosomes, all it really demonstrates is that the target sequence of the probe (often located in the centromere) is present. It does not provide information about the remainder of the chromosome.
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Single cell CGH |
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Although CGH was originally developed to determine aneuploidy in small numbers of cells (Kallionemi et al., 1992
, 1993) there is insufficient DNA in a single cell for hybridization to target chromosomes as required for CGH. Multiplex PCR has been used successfully for a number of years to amplify several loci from single cells (Wells and Sherlock, 2001) but CGH requires non-specific amplification across the entire genome. A number of whole genome amplification techniques have been tested for their ability to evenly amplify random sequences from single cells (Wells et al., 1999). Two techniques, degenerate oligonucleotide primed PCR (DOP–PCR; Telenius et al., 1992) and primer extension preamplification (PEP; Zhang et al., 1992) gave more even amplification of the whole genome than tagged PCR and alu PCR (Wells et al., 1999). However, only DOP–PCR produced sufficient amplified product from single cells for use in CGH. In this first demonstration of single cell CGH, Wells et al. (1999) successfully identified trisomies of chromosomes 13, 14, 18 and 21 in individual amniocytes known to be trisomic for these chromosomes. In this study, four blastomeres from a single human embryo were also analysed by CGH. Three were reported to be monosomic for chromosome 1 and the fourth cell was diploid, demonstrating human embryo mosaicism previously identified by FISH studies (Wells et al., 1999).
Confirmation that DOP–PCR and CGH could be used to detect aneuploidy in single cells was also demonstrated by Voullaire et al. (1999) who analysed amniocytes trisomic for chromosomes 13, 18 or 21. Although the limits of detection of chromosomal amplifications seen in tumour cells using CGH is 4–5 Mb (Piper et al., 1995), detection of monosomy or trisomy is restricted to imbalances greater than 20 Mb (Griffin et al., 1998). The limit of detection of chromosome imbalance and partial aneuploidy using CGH on single cells is closer to 40 Mb (Voullaire et al., 1999). Detailed protocols for performing single cell CGH in the laboratory setting have recently been published (Wilton and Voullaire, 2004).
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Determination of aneuploidy using CGH on human blastomeres |
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The first extensive analysis of CGH on a series of human blastomeres was the analysis of >60 blastomeres from 12 normally fertilized, good quality embryos (Voullaire et al., 2000
). As would probably be expected given that every chromosome was assessed, CGH analysis found a lower proportion (25%) of diploid embryos than found using FISH. The high frequency of mosaicism was confirmed but, very interestingly, CGH demonstrated, for the first time, that partial aneuploidy existed in early human embryos (Voullaire et al., 2000). One of the 12 embryos had cells with post-zygotic breakage of chromosomes 2 and 10 so that the cells had loss and gain of fragments of these chromosomes. This is an important observation because these types of errors are not detectable by FISH, which only detects the target sequences of the FISH probes, and had not previously been known to exist in human embryos and they may offer another explanation for the failure of many human embryos to implant (Voullaire et al., 2000). In this study of only 12 embryos, abnormalities were found for a number of chromosomes that are not part of the standard FISH panel used in PGD for aneuploidy. These included chromosomes 1, 2, 4, 6, 7, 8, 9, 10, 11 and 12 (Voullaire et al., 2000). Interestingly the three embryos in the study whose growth and development were of such high quality that they would have been preferentially selected for transfer using routine embryology criteria were all abnormal, two having consistent aneuploidy in every cell and one being extensively mosaic (Voullaire et al., 2000).
Reassuringly, subsequent reports of CGH analysis of blastomeres have found similar outcomes. In another 12 embryos investigated, an identical frequency of diploid embryos and very similar observations of mosaicism and post-zygotic chromosome breakage were observed (Wells and Delhanty, 2000). Trussler et al. (2004) analysed two or three cells from each of 40 embryos and observed that 57% of embryos were normal for all chromosomes. This frequency of normality is somewhat surprising given that FISH studies of less than half the chromosomes show that fewer embryos are normal. This may be because only about half of the total cells from each embryo were examined using CGH. It has been observed that some embryos may be normal for most cells and have an aneuploidy in only one or two cells (Voullaire et al., 2000, 2002). Some of the embryos that were diagnosed as normal may have had aneuploidies in the cells that were not examined. Instead of CGH, the remaining cells of the embryo were subjected to FISH for the chromosomes predicted to be aneuploid by CGH in an attempt to verify the CGH results (Trussler et al., 2004). The FISH result confirmed the CGH result on many occasions but often did not. This may have been because of CGH inaccuracy in some instances but was also very likely to be due to chromosomal mosaicism within the embryos. Chromosomal mosaicism in embryos means that FISH cannot be used to validate CGH outcomes on blastomeres.
A similar discrepancy between cells has been reported by Voullaire et al. (2002) who analysed the remaining cells of embryos that had been diagnosed as aneuploid after PGD using CGH (see later) demonstrating, again, that chromosomal mosaicism is very common in early human embryos. As well as embryos that contained cells with different abnormalities, many embryos were a mixture of normal and abnormal cells (Voullaire et al., 2002).
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The application of CGH to clinical PGD |
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The first successful clinical application of CGH in PGD was for a 38 year old woman who had suffered many years of primary infertility and then recurrent implantation failure in IVF including some embryos that had been diagnosed as normal for five chromosomes using FISH on biopsied blastomeres. CGH analysis of biopsied blastomeres found only one of five embryos to be normal for every chromosome. This predicted XX embryo was transferred and resulted in the birth of a healthy female infant (Wilton et al., 2001
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This patient was included in a series of 20 women with implantation failure who had PGD for aneuploidy testing using CGH (Wilton et al., 2003). Some of these women chose to have FISH analysis for chromosomes 13, 16, 18, 21 and 22 of blastomeres from half of their embryos and CGH on the remaining embryos. The fetal heart pregnancy rate and implantation rate was higher after transfer of embryos diagnosed as normal by CGH compared to FISH but the sample size was too small for this difference to be statistically significant (Wilton et al., 2003). Sixty per cent of the embryos analysed by CGH had at least one chromosome abnormality. Many biopsied blastomeres had multiple abnormalities and 11% had a partial aneuploidy. Interestingly at least one aneuploidy of every chromosome was observed in these blastomeres biopsied from just 126 early embryos. Many of the aneuploidies, including partial ones, had not previously been described in human embryos. This is of interest in comparison to the aneuploidies seen in miscarriage, some of which are extremely rare (for example trisomy 5 and trisomy 19) occurring in <1 in 1000 spontaneous abortuses (Jalal et al., 2004). This demonstrates that these aneuploidies do occur in embryos but are probably highly lethal and affected conceptuses never reach that stage of a clinically recognizable pregnancy. Clearly CGH is able to detect more chromosome errors than FISH and it was conservatively estimated in this study that FISH for five (13, 16, 18, 21, 22) or nine (XY, 13, 14, 15, 16, 18, 21, 22) chromosomes would have missed the aneuploidies in 38 and 25% of embryos respectively and diagnosed them as normal (Wilton et al., 2003).
One inherent limitation of CGH is that it cannot detect whole ploidy errors, that is, it cannot distinguish for example haploid, triploid, tetraploid cells from diploid ones. However, many, if not most, of these aberrations are detected by embryologists at fertilization when they closely observe pronuclei and discard any zygotes that have one or three or more pronuclei. Additionally, it is possible that many of the tetraploid cells observed in early embryos represent a normal developmental process, perhaps underlying the later formation of the syncytial trophoblast.
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Cryopreservation of biopsied embryos |
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The greatest limitation of the approach taken by Wilton et al., 2001
, 2003) is that, because CGH is a relatively labour intensive procedure, it takes several days to obtain a result. This is longer than 3 day old embryos can be maintained in culture and so biopsied embryos had to be frozen until results were available and thawed and transferred in a subsequent cycle. Biopsied embryos have been shown to have a very poor survival rate after freezing and thawing using techniques that are successful for intact cleavage stage embryos (Joris et al., 1999; Magli et al., 1999; Ciotti et al., 2000). The most commonly used cryopreservation protocol for intact embryos used propanediol as a cryoprotectant. This has recently been modified so that there is an increased sucrose concentration in the freezing solutions which has been demonstrated by others to improve the survival of frozen–thawed oocytes (Fabbri et al., 2001). Also, human serum albumin was replaced with maternal serum (Jericho et al., 2003). This resulted in high rates of survival of both whole embryos and individual blastomeres that were not different from intact control embryos that had no breach in the zona pellucida (Jericho et al., 2003). This protocol was used to cryopreserve the biopsied embryos whose blastomeres were subjected to CGH in the only clinical application of CGH on embryos (Wilton et al., 2003).
The necessity to cryopreserve biopsied embryos while CGH results are obtained is not ideal and there is probably a loss of implantation potential of 30% (Edgar et al., 2000). However, as it has been argued (Wilton et al., 2003), in the clinical application of CGH most of the embryos would have been frozen irrespective of whether CGH was done or not. The loss of implantation potential only affects the two or perhaps three embryos that would have been transferred fresh had CGH not been done.
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CGH on polar bodies |
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One way of circumventing the need for embryo cryopreservation but still permitting complete karyotyping using CGH is to analyse the first polar bodies biopsied from oocytes prior to fertilization (Wells et al., 2002
). Polar body biopsy is done within hours of oocyte collection and so enables much more time to perform CGH. There has been a single case report of this approach where 1 out of 10 oocytes was predicted to be chromosomally normal based on the polar body CGH analysis. This fertilized and developed into a morphologically normal embryo which was transferred but no pregnancy resulted (Wells et al., 2002). Although CGH on first polar bodies avoids the need for embryo cryopreservation, it is limited because only maternal meiosis I errors can be detected. Maternal meiosis II (Kuliev et al., 2003) and paternal meiosis I errors that originate in sperm would not be detected, although the latter probably occur at low frequency (Martin and Rademaker, 1990; Martin et al., 1996; Downie et al., 1997). Chromosomal mosaicism in embryos, where either some cells are normal and others are aneuploid or where cells within an embryo have different aneuploidies, is very well documented (Harper et al., 1995; Delhanty et al., 1997; Munné et al., 1998a) and must occur during the early cleavage stages of the embryo (Malmgren et al., 2002). Clear examples of this phenomenon where there has been post-zygotic chromosome breakage have been reported (Voullaire et al., 2000). Analysis of the first polar body would not detect any of these errors. These limitations could be partly overcome by analysing first and second polar bodies from oocytes (Gutiérrez-Mateo et al., 2004).
A more extensive study to determine the reliability of CGH on first polar bodies found that, from 25 oocyte and first polar body pairs from women with a normal karyotype undergoing IVF, there was an aneuploidy frequency of 48% (Gutiérrez-Mateo et al., 2004). Interestingly, in 20% of cases the oocyte and matching polar body did not have complementary karyotypes, often because the CGH profiles were difficult to interpret and gave doubtful results (Gutiérrez-Mateo et al., 2004).
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Diagnosis of chromosomal translocations using CGH |
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Another exciting potential application of CGH for PGD is the diagnosis of chromosomal imbalance in the embryos of reciprocal and Robertsonian translocation carriers. Currently, these imbalances are diagnosed using FISH with DNA probes specifically designed to account for all possible segregants of the chromosomes. The most common approach is to use probes that are either side of the breakpoints on the chromosomes (Munné et al., 1998b
; Pierce et al., 1998; Scriven et al., 1998; Delhanty and Conn, 2001); however, this often requires the use of telomeric probes. Telomeric probes are often quite small and it can be very challenging to develop FISH protocols that work reliably and reproducibly at the single cell level. In the majority of cases, particularly for reciprocal translocation carriers, a unique test must be developed for each couple. This can be very laborious, time-consuming and expensive.
The use of CGH to diagnose chromosomal imbalance would provide a much more generic solution which should be applicable to all translocations. Hence, PGD of embryos from translocation carriers could become an ‘off the shelf’ test. Individual blastomeres from embryos from translocation carriers have been karyotyped using CGH (Malmgren et al., 2002). These embryos had already been diagnosed by FISH as being unbalanced for the translocation. In two-thirds of the embryos the CGH result was in complete agreement with the FISH result and in 20% of the embryos there was agreement in at least one cell (Malmgren et al., 2002). The resolution of single cell CGH is reported to be 10–40 Mb (Voullaire et al., 1999; Malmgren et al., 2002) so CGH will only be able to diagnose translocations that are larger than this. Also, CGH is known to be unreliable in the telomeric regions (Kallionemi et al., 1994) so it would be difficult to detect small translocations with breakpoints close to the telomeres. Nevertheless, there are many patients who have larger translocations and CGH may prove to be a suitable approach to diagnose imbalances in their embryos.
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CGH using microarrays |
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The analysis of CGH results using metaphase chromosomes as the template, as described above, is time-consuming and labour intensive. CGH can also be performed using microarrays as the target DNA. These microarrays are made up of DNA sequences specific to human chromosomes spotted onto a platform, usually a glass slide. The principle is the same as metaphase chromosome CGH in that differentially labelled reference and test DNA are hybridized to the slide and differences in the fluorescence ratio are indicative of changes in DNA copy number. One key potential advantage for PGD applications is that array CGH takes less time, primarily because <24 h is required for the hybridization. Array CGH has been used to detect aneuploidy (Pinkel et al., 1998
; Pollack et al., 1999; Snijders et al., 2001). Recently, array CGH has been used to detect several aneuploidies including trisomy 21 and sex chromosome complement in single fibroblasts with 100% accuracy (Wells et al., 2004). These microarrays consisted of DNA clones derived from the telomeres of human chromosomes and therefore may be applicable to the diagnosis of chromosomal imbalance caused by translocations as well as partial aneuploidy of chromosome segments which have been shown to exist in embryos (Voullaire et al., 2000; Wells and Delhanty, 2000). Array CGH has also been used to detect trisomy of chromosomes 13, 15 and in single cells after DOP–PCR amplification (Hu et al., 2004). In this study, rather than each DNA sequence being spotted separately on the slide, a number of different sites on each chromosome were spotted on top of each other. This approach is less discriminating than metaphase CGH as partial aneuploidy and imbalance of chromosome segments would not be detected. Moreover, the array sometimes gave incorrect results for chromosomes 2, 4, 9, 11, 17 and 22 (Hu et al., 2004) and more refinement will be required before it is suitable for clinical application. |
Concluding comments |
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CGH on blastomeres has provided new insights into the extent of chromosome abnormalities in preimplantation embryos. It has clearly demonstrated that aneuploidies of all chromosomes exist and that chromosomal breakages are also relatively common during the first few cleavage divisions. All CGH studies on blastomeres have identified chromosome errors that would not have been detected if the cells had been analysed by FISH, confirming the benefit of complete karyotyping for PGD. However, CGH remains technically challenging and, in its current form, is likely to be performed in only a few laboratories that have appropriate skill and expertise in molecular biology. Research continues into ways to simplify CGH, in particular to shorten the time required to obtain results. Novel approaches such as the use of microarrays instead of metaphase chromosomes and multiplex fluorescent PCR on whole genome amplified DNA also hold great promise.
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