1 Departament de Biologia Cel·lular, Fisiologia i Immunologia, Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain, 2 Institute of Human Genetics and Anthropology, 07740 Jena, Germany, 3 The Institute for Reproductive Medicine and Science, St Barnabas Medical Center, 94 Old Short Hills Road, Livingston, NJ 07039, USA and 4 Reprogenetics, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052, USA
5 To whom correspondence should be addressed. E-mail: cristina.gutierrez{at}uab.es; joaquima.navarro{at}uab.es
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Abstract |
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Key words: aneuploidy/cenM-FISH/CGH/first polar body/oocyte
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Introduction |
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In recent years, several reports have used various fluorescence in situ hybridization (FISH) strategies for the analysis of human oocytes (Anahory et al., 2003; Cupisti et al., 2003
; Eckel et al., 2003
; Pujol et al., 2003
) basically those excluded from IVF cycles for being immature or those which remained unfertilized after insemination (Pellestor et al., 2005
). In these studies, one or two rounds of fluorescence in situ hybridization (FISH) are performed, but only about one-third of the chromosomes per cell can be analysed due to technical problems (Liu et al., 1998
).
A full chromosome count may provide meaningful data, since the analysis of only the chromosomes most frequently involved in aneuploidy is likely to underestimate the real incidence of aneuploidy in oocytes. Recent studies have reported that any chromosome can be involved in aneuploidy (Wilton, 2005) and that
30% of the chromosome abnormalities in oocytes and blastomeres would have gone unnoticed if FISH for nine chromosome-specific probes had been used (Wilton et al., 2003
; Gutiérrez-Mateo et al., 2004a
,b
).
To date, the analysis of the full set of chromosomes of the oocyte has been performed using four different techniques: conventional karyotyping (Wall et al., 1996; Angell, 1997
; Pellestor et al., 2002
), spectral karyotyping (SKY) (Márquez et al., 1998
; Sandalinas et al., 2002
), multiplex fluorescence in situ hybridization (M-FISH) (Clyde et al., 2001
, 2003
) and comparative genomic hybridization (CGH) (Gutiérrez-Mateo et al., 2004a
,b
). Whereas conventional karyotyping, SKY and M-FISH are techniques that strongly depend on the spreading on metaphase chromosomes on slides, CGH is a DNA-based method which does not require cell fixation.
CenM-FISH is a 24-colour FISH technique that uses centromere-specific probes labelled with different combinations of five fluorochromes. This technique allows for the simultaneous identification of all chromosomes, excluding chromosomes 13 and 21, which are not differentiated (Nietzel et al., 2001). It has been applied in constitutional and cancer cytogenetics for the identification of small, supernumerary marker chromosomes (sSMC) with nearly no euchromatin, which are difficult to classify using other techniques such as M-FISH, SKY or CGH (Henegariu et al., 2001
; Nietzel et al., 2001
). It has also been applied for karyotyping synaptonemal complexes in spermatocytes from controls and infertile males (Oliver-Bonet et al., 2003
; Sun et al., 2004
; Oliver-Bonet et al., 2005
).
The main aim of this work was to investigate the efficiency, advantages and limitations of cenM-FISH as a new technique for basic studies on human oocytes. The validation of the technique was performed by the reanalysis of the oocytes with conventional FISH and by CGH analysis of the corresponding first polar bodies (1PB). While CGH enables the detection of chromosome or chromatid errors of any chromosome, FISH is useful to distinguish between these sorts of errors (chromosome or chromatid). The frequency of aneuploidy, the implication of two mechanisms of maternal aneuploidy and the benefits of the analysis of the whole set of chromosomes are also discussed.
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Materials and methods |
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1PB isolation and oocyte spreading
The zona pellucida was removed using Tyrodes acid. Isolation and lysis of the 1PB were performed as previously described (Wells et al., 2002). Oocytes were spread after zona pellucida removal using a modification of a previously described method (Tarkowski, 1966
). The slides were frozen at 20°C until they were hybridized.
CGH analysis of 1PB
All of the procedure was performed as previously described (Gutiérrez-Mateo et al., 2004b). Briefly, DNA from the isolated 1PB and single buccal cells from a normal female (reference sample in the CGH experiment) were amplified using degenerate oligonucleotide-primed PCR (DOP-PCR). Whole genome amplification products were fluorescently labelled by Nick Translation (Vysis, Downers Grove, USA). 1PB DNA (test) was labelled with Spectrum ReddUTP (Vysis), whereas reference DNA was labelled with Spectrum GreendUTP (Vysis). Labelled reference and test DNA were co-hybridized to normal male (46, XY) metaphase spreads (Vysis) in a moist chamber at 37°C for 4072 h. Metaphase preparations were examined using an Olympus BX 60 epifluorescence microscope. An average of 10 metaphases per hybridization was captured and analysed using SmartCapture software and Vysis Quips CGH software, both supplied by Vysis. The average red:green fluorescent ratio for each chromosome was determined by the CGH software. 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 of material in the test sample respectively. Telomeric, centromeric and heterochromatic regions were excluded from the analysis due to being non-informative.
CenM-FISH of the oocytes
CenM-FISH probes are non-commercial centromere- or subcentromere-specific probes which were prepared as previously described (Nietzel et al., 2001). Briefly, each probe was labelled separately by DOP-PCR with Spectrum GreendUTP, Spectrum ReddUTP, Spectrum OrangedUTP, diethylaminocoumarine-dUTP and/or biotindUTP according to the labelling scheme shown in Table I. Labelled probes were mixed and ethanol-precipitated with 2 µg of Cot-1-DNA to avoid cross-hybridization of the different centromere-specific probes. The pellet was dried and stored at 20°C until it was used. The pellet was then resuspended in 10 µl of hybridization mixture [50% formamide, 2 x standard saline citrate (SSC), 10% dextran sulphate, pH 7], denatured for 5 min at 75°C and prehybridized for 10 min at 37°C.
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The cytoplasm was removed using pepsin (50 µg/ml) in HCl 10 mmol/l at 37°C. Prior to FISH, the slides were postfixed at room temperature in 50 mmol/l MgCl2/PBS for 5 min, 1% formaldehyde/50 mmol/l MgCl2/PBS for 2 min and PBS for 5 min. The preparations were dehydrated, air-dried, stained with DAPI and captured. DAPI was removed using 2 x SSC/0.05% Tween 20 solution for 5 min and then the slides were dehydrated and air-dried. The slides were denatured in 70% formamide, 2 x SSC at 70°C for 1 min and then taken through a cold alcohol series and air-dried. The cenM-FISH probe was applied to each slide and covered with a 5 x 5 mm coverslip. The slides were sealed with rubber cement and were allowed to hybridize overnight at 37°C in a dark moist chamber.
After hybridization, the slides were washed three times in 50% formamide, 2 x SSC at 45°C for 10 min, followed by 10 min in 2 x SSC at 45°C and finally 5 min in 4 x SSC/0.05% Tween 20 at 45°C. Biotin labelling detection was performed using a Cy5avidin system. Finally, the slides were mounted in Vectashield (Vector Laboratories, Peterborough, UK) containing DAPI. Metaphase preparations were examined using an Olympus BX 60 epifluorescence microscope equipped with a CCD camera. Capture and analysis was performed using SpectraVysion Software (Vysis). After capture, the slides were rinsed in distilled water before being dehydrated in an alcohol series.
FISH analysis of MII oocytes
Centromeric, locus-specific and/or telomeric probes (Vysis) were used to reanalyse the oocytes after cenM-FISH. The probes were hybridized in one or two rounds of FISH, as previously described (Gutiérrez-Mateo et al., 2004a). The slides were examined using a fluorescence microscope with filters for the fluorochromes used. Capture and analysis was performed using SmartCapture software (Digital Scientific Cambridge, UK) and IPLab (Scanalytics, Inc., Vysis, USA). After visualization of the first round, the slides were rinsed in distilled water before being dehydrated in an alcohol series.
Scoring criteria
The cenM-FISH probe was tested on spreads of normal male leukocytes. These centromeric probes usually gave one large signal or, alternatively, two smaller paired signals corresponding to the two sister chromatids. The presence of additional signals was always considered as confirmed aneuploidies, whereas missing signals were only considered when the result was confirmed by CGH in the corresponding 1PB.
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Results |
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The reciprocity rate between 1PB-CGH and oocyte-cenM-FISH was 93% (13/14), since one missing chromosome 4 was found by cenM-FISH in oocyte K whereas CGH analysis of its 1PB indicated a normal profile for all of the chromosomes. According to this, doublet K was classified as normal, since an artefactual loss of chromosome 4 during spreading of the oocyte may have happened.
Four out of 16 oocytes were aneuploid (25%) (Table II). Except for the loss of chromosome 4 in doublet K, all aneuploid events found by cenM-FISH were confirmed in a second or third round of FISH and by the CGH analysis of the corresponding 1PB. The four abnormal oocytes were donated by four patients. A 32 year old patient donated the 1PB-oocyte doublet D, which presented a pre-division of chromatids of chromosome 13. Doublet E (Figure 1) was donated by a 36 year old patient and had a double aneuploidy for chromosomes 1 and 8 due to a non-disjunction and a pre-division of chromatids respectively. 1PB-oocyte pair L had several aneuploidies involving chromosomes 10, 18 and 19 and it was obtained from another patient, aged 38 years, who suffered from ovarian dysfunction. Finally, a 42 year old patient donated doublet M, which had two pre-divisions of chromatids, one affecting chromosome 10 and the other affecting chromosome 21.
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A total of eight aneuploid events involving chromosomes 1, 8, 10, 13, 18, 19 and 21 were found, five of them were chromatid abnormalities and three were due to non-disjunction. Age-related aneuploidy was not analysed because the sample size was too small.
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Discussion |
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The incidence of chromosome abnormalities derived from FISH studies varies widely, basically depending on the number of chromosomes and the chromosomes that were analysed. Some studies have found relatively low rates of aneuploidy analysing six (chromosomes 9, 13, 16, 18, 21 and X, 9.5%) or seven chromosomes (1, 9, 13, 16, 18, 21 and X, 8%) (Anahory et al., 2003
; Cupisti et al., 2003
), whereas others have found up to 47.5% aneuploid oocytes analysing nine chromosomes (Pujol et al., 2003
). A high variation is also found even if we take into consideration only those studies which have evaluated the whole set of chromosomes to estimate the aneuploidy rate. Data obtained from conventional karyotyping reports indicate that
17% of the oocytes are aneuploid (Pellestor et al., 2005
), while the aneuploidy rate found using SKY or M-FISH varies from 16.7 to 39% (mean 31.3%) (Márquez et al., 1998
; Sandalinas et al., 2002
; Clyde et al., 2003
). The aneuploidy rate presently found is consistent with these studies, since four out of 16 doublets had errors (25%). However, the aneuploidy rate found recently by CGH is higher, as
52% of the 1PB-oocyte pairs were aneuploid (Gutiérrez-Mateo et al., 2004a
,b
). These variations could be attributed to patient-specific factors, differences in maternal age, source of oocytes, cohort size and study design (Clyde et al., 2003
). Some aspects such as the fixation technique (if used), the incidence of artefactual loss of chromosomes, the presence of overlapping chromosomes as well as the criteria to select the analysed oocytes should also be taken into account to explain these discrepancies. In our hands, the analysis of both 1PB and metaphase II oocytes provides the best internal control to make a reliable and accurate estimation of the aneuploidy rate in oocytes.
In this work, eight aneuploid events were found. Apart from an artefactual loss of chromosome 4 in doublet K, all chromosome abnormalities detected by cenM-FISH could be confirmed by FISH and CGH in the complementary 1PB. Five aneuploidies were due to premature separation of sister chromatids (pre-division), whereas three were due to non-disjunction of bivalent chromosomes. It is important to note that both mechanisms can operate in the same cell leading to aneuploid doublets with missing or extra chromosomes and chromatids (Table II, doublets E and L). This finding is consistent with recent studies which have also indicated the co-existence of two mechanisms of maternal aneuploidy, with chromatid anomalies being the most common (Verlinsky et al., 1999; Sandalinas et al., 2002
; Pellestor et al., 2003
; Gutiérrez-Mateo et al., 2004a
). Interestingly, in the case of doublet E (Figure 1), CGH analysis of the 1PB was sensitive enough to determine the mechanism leading to aneuploidy. In this 1PB, deviation to the left of the CGH ratio of chromosome 8 was close to the threshold of 0.8, which corresponded to a single chromatid loss, as indicated by the cenM-FISH analysis of the corresponding oocyte. On the other hand, the sharper deviation of the CGH ratio of chromosome 1 (<0.5) corresponded to the lack of chromosome 1 in the 1PB, as the cenM-FISH results of the complementary oocyte showed. Nevertheless, it is important to emphasize that as long as the deviation of the CGH profile was close to the threshold of 0.8, non-disjunction of bivalents or premature separation of sister chromatids cannot easily be distinguished using CGH, as recently reported by our group (Gutiérrez-Mateo et al., 2004a
). Other techniques, such as SKY, M-FISH or cenM-FISH, usually enable researchers to determine whether a chromosome abnormality is due to pre-division or to non-disjunction. However, it is important to emphasize that SKY and M-FISH, unlike cenM-FISH, use whole chromosome painting probes which commonly inflate the chromosomes and make the distinction of the mechanism of aneuploidy harder (Márquez et al., 1998
; Sandalinas et al., 2002
). Consequently, in some cases an additional conventional FISH analysis after SKY was necessary to investigate pre-division (Márquez et al., 1998
).
Even though age-related aneuploidy was not analysed because the sample size was too small, it is important to note that three out of the four aneuploid doublets came from women aged >35 years.
CGH analysis of blastomeres has detected aneuploidies of all chromosomes (Wilton, 2005). In this work, aneuploidies for chromosomes 1, 8, 10, 13, 18, 19 and 21 were found. As previously reported (Wilton et al., 2003
; Gutiérrez-Mateo et al., 2004a
,b
), if we had used FISH to analyse only the chromosomes included in current PGD panels for aneuploidy screening (commonly chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y), some of these aneuploidies would not have been detected. Despite that, some of these aneuploid doublets would have correctly been identified as abnormal because they had multiple aneuploidies, some of them affecting chromosomes included in the nine-chromosome-FISH panel (i.e. doublets L and M) (Abdelhadi et al., 2003
). Nevertheless, it is important to emphasize that 2530% of the aneuploid doublets (i.e. doublet E in the present study) would have been incorrectly diagnosed as normal using FISH for nine chromosomes (Gutiérrez-Mateo et al., 2004a
,b
). Therefore, the analysis of only some selected chromosomes may underestimate the real aneuploidy rate of human oocytes (Anahory et al., 2003
; Cupisti et al., 2003
).
One disadvantage of cenM-FISH, as compared with SKY or M-FISH, is that chromosomes 13 and 21 cannot be differentiated by their colour code because both chromosomes have almost identical -satellite sequences (Nietzel et al., 2001
). However, in oocytes the distinction between these two chromosomes can usually be made on the basis of their size. Additionally, cenM-FISH provides information only about the centromeric regions of the chromosomes but not about the remainder of the chromosomes. Consequently, it cannot detect specific structural rearrangements, which can be identified with CGH, SKY or M-FISH, which give information about the full length of the chromosomes.
It is worth noting that cenM-FISH is not applicable to the analysis of interphase cells (i.e. blastomeres) because of the high risk of overlapping signals (a normal blastomere will show 46 centromeric signals, one for every chromosome). Moreover, like SKY and M-FISH, this technique is not appropriate for preimplantation genetic diagnosis (PGD) by polar body analysis either. Since the 1PB is a very small cell whose fixation requires a high level of skill, only 25% of polar bodies give chromosome spreads suitable for being karyotyped after Tarkowski fixation, so the efficiency would be too low for its clinical application (Márquez et al., 1998
; Munné, 2002
; Sandalinas et al., 2002
; Gutierrez-Mateo et al., 2005
).
On the other hand, the particular morphology of the oocyte chromosomes and the difficulty of obtaining good chromosome banding have limited the data obtained from the conventional karyotyping reports on discarded oocytes, since specific chromosome aneuploidy is rarely identified and some of the chromosome counts that have been published might be biased by the erroneous classification of single chromatids as additional chromosomes (Pellestor et al., 2005). Considering that unlike SKY and M-FISH, cenM-FISH can be performed on a previously G-banded slide (Nietzel et al., 2001
), this technique would be appropriate to reanalyse the whole-chromosome complement of previously karyotyped R-banded oocyte chromosomes (Wall et al., 1996
; Pellestor et al., 2002
).
Additionally, some supernumerary marker chromosomes (sSMC) had been found in infertile couples who were referred for assisted reproduction techniques (Clementini et al., 2005). Some of these markers have barely any euchromatin and thus they cannot be clearly identified by either M-FISH or SKY, which use expensive, commercial whole-chromosome painting probes that do not cover the centromeric regions of human chromosomes (Liehr et al., 2004
). CenM-FISH would enable the study and characterization of such markers as well as the identification of any other numerical chromosome abnormalities in human oocytes, as previously reported in prenatal and postnatal samples (Nietzel et al., 2001
).
The combination of cenM-FISH with 1PB-CGH analysis permits an accurate estimation of the aneuploidy rate and provides a useful new technique for basic studies on discarded human oocytes, but also on available fresh oocytes. The further application of this novel technique may reveal the incidence of aneuploidy of chromosomes not extensively studied by FISH in other reports or in preimplantation genetic diagnosis (PGD). Moreover, some IVF patients could be prone to certain aneuploidies, which may not be included in routine PGD panels. The cenM-FISH analysis of discarded oocytes from these patients may elucidate which ones these aneuploidies are, so the PGD in a subsequent cycle could be focused on these problematic chromosomes. Finally, cenM-FISH may allow the investigation of any relationship between aneuploidy and the aetiology of infertility, maternal age or hormone levels.
In conclusion, we have reported the first application of cenM-FISH in human oocytes and our preliminary results prove that cenM-FISH is a reliable method to detect any numerical chromosome abnormality in MII oocytes in a single round of FISH, either due to non-disjunction of chromosomes or premature separation of sister chromatids. The use of seven fluorochromes, instead of five, will allow a maximum of two-fluorochrome combinations for each chromosome. This may achieve a reduction of the number of signals per channel, thus facilitating the identification of signals in certain overlapping situations and improving the karyotyping efficiency of this technique (Azofeifa et al., 2000). Our results also confirm the advantage of a full-chromosome analysis over the FISH analysis of only some selected chromosomes.
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Acknowledgements |
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Submitted on April 15, 2005; resubmitted on July 12, 2005; accepted on July 14, 2005.
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