Aneuploidy 12 in a Robertsonian (13;14) carrier: Case report

C. Gutiérrez-Mateo1,4, L. Gadea2, J. Benet1, D. Wells3, S. Munné2,3 and J. Navarro1

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


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In translocation carriers, the presence of aneuploidy for the chromosomes unrelated to the rearrangement may lead to an additional risk of abnormal pregnancy or implantation failure. Consequently, it may be important to analyse not only the chromosomes involved in the rearrangement but also the rest of chromosomes. We combined spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to karyotype one unfertilized oocyte and its first polar body (1PB) from a Robertsonian translocation carrier t(13;14) aged 29 years who was undergoing IVF and preimplantation genetic diagnosis (PGD) for translocations and aneuploidy screening. Two out of four embryos were aneuploid, as a result of an adjacent segregation. The unfertilized oocyte had a normal/ balanced constitution of the chromosomes involved in the reorganization. However, this 1PB–metaphase II doublet was aneuploid for chromosome 12, the oocyte being hyperhaploid (24, X, +12) and its 1PB hypohaploid (22, X, –12). The application of CGH for the study of Robertsonian translocations of maternal origin will be useful to study imbalances of the chromosomes involved in the rearrangement, as well as alterations in the copy number of any other chromosome. The combination of PGD for translocations with aneuploidy screening could help to reduce the replacement of chromosomally abnormal embryos.

Key words: CGH/first polar body/oocyte/SKY/translocation


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Robertsonian translocations are the most common structural chromosome abnormality found in humans and consist of a fusion of the short arms of two acrocentric chromosomes. The trivalent formed during meiosis I can produce either normal/balanced gametes (alternate segregation) or unbalanced gametes (adjacent segregation). Although most female translocation carriers are fertile, they have a high risk of recurrent spontaneous abortions and produce chromosomally abnormal offspring.

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, {alpha}-satellite or subtelomeric) (Escudero et al., 2000Go), while first polar body (1PB) analysis has focused on enumerator probes or chromosome painting probes (Durban et al., 2001Go). PGD of translocations using FISH has achieved a significant reduction of spontaneous abortions (Munné et al., 1998aGo) and helps to reduce the risk of delivering trisomic offspring (Munné et al., 1998bGo). 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., 2000Go; Durban et al., 2001Go; Munné, 2002Go; Pujol et al., 2003bGo). 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., 2002Go; Pujol et al., 2003aGo). 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., 1998Go; Voullaire et al., 1999Go; Wells et al., 1999Go).

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.


    Case report
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The patient was a woman aged 29 years with primary infertility due to a Robertsonian translocation [45, XX, der(13;14)(q10;q10)]. She had five previous first trimester miscarriages; four were spontaneous and one was therapeutic. The patient underwent IVF treatment at the Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center (West Orange, New Jersey, USA). Oocyte retrieval was performed by transvaginal follicular puncture. Eight out of 15 oocytes were at metaphase II (MII) stage and five were fertilized after IVF. Four out of five resulting embryos were biopsied on day 3 for PGD of the translocation. Additionally, one unfertilized oocyte was donated for research. The oocyte and embryos were analysed in accordance with guidelines set by the internal review board of that centre. Written informed consent was obtained and the project was approved by the institutional ethics committee.


    Materials and methods
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Single polar body isolation and oocyte spreading
The oocyte presented a single polar body (1PB) and no pronuclei 24 h after IVF. The zona pellucida was removed using acid Tyrode's. Isolation and lysis of the 1PB was performed as previously described (Gutierrez-Mateo et al., 2004Go). The oocyte was spread after zona pellucida removal using the technique of Tarkowski (1966)Go. The position of the oocyte was circled using a diamond pencil and the slide was frozen at –20 °C until it was hybridized.

CGH analysis of the 1PB
The whole procedure was performed as previously described (Gutierrez-Mateo et al., 2004Go) 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., 1998Go) 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., 1998Go). 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., 1996Go). 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.


    Results
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of the analysis are given in Table I. Four embryos were biopsied and analysed by FISH. Two out of four cleavage stage embryos were aneuploid for one of the chromosomes implicated in the rearrangement, indicating an adjacent segregation of the trivalent. No aneuploidy involving chromosomes 16, 18, 21 and 22 was found. Two embryos were normal or balanced for the chromosomes analysed and therefore they were transferred to the mother. Unfortunately, no pregnancy was obtained.


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Table I.

 
CGH analysis of the first polar body (1PB) and SKY results of the MII oocyte are shown in Figure 1. The interpretation of the CGH profile from the 1PB revealed an abnormal karyotype 22, X, –12, as the CGH ratio for chromosome 12 showed a deviation below the threshold of 0.8, indicating loss. The reciprocal gain was found by SKY in the corresponding MII oocyte, confirming the CGH results and revealing that the oocyte had an extra chromosome 12 (24, X, +12). In this case, no aneuploidy affecting the chromosomes involved in the rearrangement (chromosomes 13 and 14) was found, indicating an alternate segregation. Moreover, the SKY analysis of the MII oocyte showed isolated chromosomes 13 and 14, with two chromatids each. Therefore, the translocated chromosome der(13;14) would have been segregated to the 1PB.



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Figure 1. Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) analysis of a metaphase II (MII) oocyte and its first polar body (1PB). (a) Inverted diamidino-2-phenylindole image to show the MII oocyte chromosomes spreading. (b) Composite spectra image after SKY. (c) 24-colour karyotype. An extra bivalent 12 is present within the MII oocyte. (d) CGH image of the 1PB. Chromosome 12 appears more green. (e) CGH profiles of chromosomes 12, 13 and 14. The CGH ratio of chromosome 12 is deviated to the left (<0.8) indicating loss. The CGH ratio of chromosomes 13 and 14 is close to 1.0 indicating a normal copy number of these chromosomes in the 1PB.

 

    Discussion
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this case report, of the four blastomeres and one unfertilized oocyte analysed, two of the blastomeres and the oocyte were aneuploid. Two aneuploidies were related to the chromosomes involved in the translocations and one was unrelated. It is important to note that there is only one previously published FISH study in which chromosome 12 has been assessed in oocytes (Cupisti et al., 2003Go). Consequently, aneuploidy of chromosome 12, such as detected in this case, is rarely observed in oocytes or cleavage-stage embryos, even in series which have used techniques such as CGH to analyse the copy number changes of every chromosome (Gutierrez-Mateo et al., 2004Go).

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., 2002Go; Pujol et al., 2003bGo), 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., 2004Go; Los et al., 2004Go).

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., 2001Go; Gianaroli et al., 2002Go; Pujol et al., 2003bGo) and others finding no evidence (Blanco et al., 1998Go; Oliver-Bonet et al., 2002Go). 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., 2000Go; Oliver-Bonet et al., 2002Go; Pujol et al., 2003bGo). 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., 2004Go). Considering that the frequency of aneuploidy in unfertilised oocytes can rise up to 47.5% (Pujol et al., 2003aGo), 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., 1998aGo). 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., 1998aGo). The difficulty in obtaining good 1PB spreads explains why <25% of polar bodies can be karyotyped by SKY (Márquez et al., 1998Go; Munné, 2002Go; Sandalinas et al., 2002Go).

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., 2004Go). 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., 2002Go), and recently the present authors have demonstrate their reliability to detect unbalanced segregations of a maternal translocation (Gutierrez-Mateo et al., 2004Go).

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Jorge Sánchez and Pere Colls for their collaboration in this work. This work was supported by Ministerio de Sanidad (FIS PI020168) and CIRIT (2001 SGR-00201). It has also been supported by Ministerio de Educación, Cultura y Deportes (Cristina Gutiérrez-Mateo has a Beca para la formación de Personal Universitario; FPU).


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 Introduction
 Case report
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 23, 2004; accepted on December 21, 2004.