1 Departament de Biologia Cel·lular, Fisiologia i Immunologia, Unitat de Biologia i Genètica Mèdica, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain, 2 Reprogenetics, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052, 3 Institute for Reproductive Medicine and Science, St Barnabas Medical Center, 94 Old Short Hills Road, Livingston, NJ 07039, USA
4 To whom correspondence should be addressed. Email: cristina.gutierrez{at}uab.es; Email: joaquima.navarro{at}uab.es
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Abstract |
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Key words: CGH/first polar body/oocyte/SKY/translocation
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Introduction |
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Several fluorescent in situ hybridization (FISH) approaches have been used for the preimplantation genetic diagnosis (PGD) of Robertsonian translocations in order to select gametes or embryos resulting from an alternate segregation. PGD based on blastomere analysis has involved the use of enumerator probes (locus-specific, -satellite or subtelomeric) (Escudero et al., 2000
), while first polar body (1PB) analysis has focused on enumerator probes or chromosome painting probes (Durban et al., 2001
). PGD of translocations using FISH has achieved a significant reduction of spontaneous abortions (Munné et al., 1998a
) and helps to reduce the risk of delivering trisomic offspring (Munné et al., 1998b
). However, it is important to emphasize that some Robertsonian translocation carriers fail to achieve a pregnancy after the replacement of embryos diagnosed as normal or balanced by PGD, and may even suffer from first trimester miscarriages (Escudero et al., 2000
; Durban et al., 2001
; Munné, 2002
; Pujol et al., 2003b
). This could be explained by the presence of chromosome abnormalities involving other chromosomes different from those implicated in the rearrangement, which are generally the only ones analysed by FISH.
Interchromosomal effect (ICE) is defined as a structural chromosome abnormality that affects the meiotic segregation of other chromosomes unrelated to the rearrangement. Recently, some authors have combined PGD for translocations with PGD for aneuploidy screening (PGD-AS) for the analysis of up to 10 selected chromosomes (Gianaroli et al., 2002; Pujol et al., 2003a
). These authors suggest that an ICE may be important in the case of Robertsonian translocations, as a higher rate of aneuploidy was found for the chromosomes not involved in the reorganization compared with controls or with reciprocal translocation carriers.
Comparative genomic hybridization (CGH) and spectral karyotyping (SKY) are two techniques, which, unlike FISH, allow the analysis of the whole chromosome complement in a single cell (Márquez et al., 1998; Voullaire et al., 1999
; Wells et al., 1999
).
We report on the use of CGH and SKY to detect imbalances not only of the chromosomes involved in a translocation, but also abnormalities of any other chromosomes in one MII oocyte and its 1PB. The use of CGH is desirable for the 1PB because karyotyping them by regular techniques or SKY is very inefficient.
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Case report |
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Materials and methods |
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CGH analysis of the 1PB
The whole procedure was performed as previously described (Gutierrez-Mateo et al., 2004) without modifications. Briefly, DNA from the isolated 1PB and a single buccal cell from a normal female (used as a reference sample in the CGH experiment) were amplified by DOP-PCR, labelled by nick translation and co-hybridized to normal male (46,XY) metaphase spreads (Vysis, USA) in a moist chamber at 37 °C for 48 h. Metaphase preparations were examined using an Olympus AX 70 epifluorescence microscope. Metaphases were captured and analysed using a Cytovision Ultra Workstation (Applied Imaging, UK). The average red:green fluorescent ratio for each chromosome was determined by the CGH software supplied by Applied Imaging. Deviations of the ratio <0.8 (the test DNA is under-represented) or >1.2 (the test DNA is over-represented) were scored as loss or gain respectively of material in the test sample. Telomeric, centromeric and heterochromatic regions were excluded from the analysis for being non-informative.
Spectral karyotyping analysis of the MII oocyte
SKY was performed using the SKY kit (Applied Spectral Imaging, Inc., USA) as previously described (Márquez et al., 1998) with some modifications. In brief, slides were denatured in 70% formamide/2 x standard saline citrate at 70 °C for 1 min and taken through a cold alcohol series and air-dried. Denatured SKYPaint probe was applied (1 µl), covered with a 6 x 6 mm coverslip and sealed with rubber cement. Hybridization took place at 37 °C in a dark, moist chamber for 48 h. Post-hybridization washes and indirect labelling detection was done as previously described without modifications (Márquez et al., 1998
). The fixed oocyte was examined using an Olympus BX 60 epifluorescence microscope equipped with a triple bandpass fluorescence filter set (SKY-1; Chroma Technology). SKYVision spectral karyotyping software (Applied Spectral Imaging) was used to analyse both the inverted DAPI image and the RGB image obtained from the excitation of the five fluorochromes present in the SKY probe mixture.
FISH of the biopsied blastomeres
On day 3 of development, the embryos were biopsied and analysed, as previously described (Munné et al., 1996). Two probes were used for test the chromosomes involved in the translocation, centromeric probe for chromosome 13 and subtelomeric probe for chromosome 14. Additionally, aneuploidy screening was performed using four more probes (chromosomes 16, 18, 21 and 22) hybridized during a second round of FISH.
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Results |
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Discussion |
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The fertilization of this oocyte, as long as the abnormality was not rescued in the second meiotic division, would have originated a trisomic embryo that may have failed to implant or died after implantation, causing a spontaneous abortion. The current PGD approaches use probes only for the chromosomes involved in the translocation. Even in the case of combining PGD of translocations and PGD-AS for the detection of common aneuploidies (chromosomes 1, 13, 14, 15, 16, 17, 18, 21, 22 and X) (Gianaroli et al., 2002; Pujol et al., 2003b
), chromosome 12 would not have been analysed and the oocyte would have been misdiagnosed as normal. It is worth emphasizing that the presence of embryo mosaicism may also increase the risk of misdiagnosis in PGD-AS, when only one cell is biopsied and analysed (Baart et al., 2004
; Los et al., 2004
).
Controversial data have been reported when studying interchromosomal effects (ICE) in sperm, embryos or oocytes from translocation carriers, with some reports finding evidence of this phenomenon (Pellestor et al., 2001; Gianaroli et al., 2002
; Pujol et al., 2003b
) and others finding no evidence (Blanco et al., 1998
; Oliver-Bonet et al., 2002
). As suggested, the occurrence of ICE could be case specific and it may depend on the type of chromosomes involved in the reorganization and the fragments concerned (Estop et al., 2000
; Oliver-Bonet et al., 2002
; Pujol et al., 2003b
). All these studies have been performed using FISH for the analysis of up to 10 selected chromosomes; thus some imbalances involving other chromosomes would have gone unnoticed (Gutierrez-Mateo et al., 2004
). Considering that the frequency of aneuploidy in unfertilised oocytes can rise up to 47.5% (Pujol et al., 2003a
), the aneuploidy identified may not be representative of real ICE. Nevertheless, the transfer of abnormal embryos misdiagnosed as normal using FISH could lead to an additional risk of abnormal pregnancy or implantation failure in these patients. Therefore, it may be important to analyse not only the chromosomes involved in the rearrangement or the chromosomes most commonly involved in aneuploidy, but also any other chromosome.
In this case report, we have combined two techniques that provide data on the whole chromosome complement: SKY and CGH. SKY uses 24 chromosome-specific painting probes labelled with a combination of one to five different fluorochromes, so every chromosome can be differentiated from the others by their spectral colour. Considering that 1PB chromosomes are in metaphase up to 6 h after oocyte retrieval, PGD of translocations can be performed using SKY to karyotype the 1PB chromosomes. This approach would allow the segregation of the translocation to be determined as well as permitting simultaneous analysis of the whole set of chromosomes. Moreover, SKY permits normal gametes to be distinguished from balanced gametes and therefore, when enough normal embryos were available, those would be preferentially transferred to avoid the perpetuation of the translocation in the family (Munné et al., 1998a). However, SKY is a technique highly dependent on the fixation and spreading of the sample and the 1PB is a very small cell whose fixation requires a high level of skill. Consequently, if the spreading were insufficient, the chromosomes would be too close to be analysed. Conversely, if the 1PB was excessively scattered the risk of artifactual loss of chromosomes during fixation would increase (Munné et al., 1998a
). The difficulty in obtaining good 1PB spreads explains why <25% of polar bodies can be karyotyped by SKY (Márquez et al., 1998
; Munné, 2002
; Sandalinas et al., 2002
).
On the other hand, CGH cannot detect alterations that do not involve gain or loss of DNA and therefore is unable to differentiate between balanced and normal gametes. However, the advantage of CGH compared with SKY is that being a DNA-based method, cell fixation is not required and as much as 83% of the 1PBs can be analysed, as recently reported (Gutierrez-Mateo et al., 2004). Consequently, CGH would be a useful tool not only to detect unbalanced segregation of translocations, but also to make a reliable detection of aneuploidy (both hyperhaploidy and hypohaploidy) of any other chromosome. CGH has been applied clinically for the PGD-AS through 1PB analysis (Wells et al., 2002
), and recently the present authors have demonstrate their reliability to detect unbalanced segregations of a maternal translocation (Gutierrez-Mateo et al., 2004
).
In conclusion, we have demonstrated that SKY and CGH can be combined for the study of any chromosome abnormality present in oocytes and polar bodies. More data using these methods may contribute to reveal which specific rearrangements induce an ICE during female gametogenesis. The clinical application of CGH for PGD of maternal translocations by 1PB analysis could help to reduce the replacement of aneuploid embryos and reduce the incidence of spontaneous abortions in these patients.
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Acknowledgements |
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References |
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Submitted on September 23, 2004; accepted on December 21, 2004.