Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

C. Gutiérrez-Mateo1,4, J. Benet1, D. Wells2, P. Colls3, M.G. Bermúdez2, J.F. Sánchez-García1, J. Egozcue1, J. Navarro1 and S. Munné2,3

1 Departament de Biologia Cel.lular, Fisiologia i Immunologia, Unitat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain, 2 The Institute for Reproductive Medicine and Science, St Barnabas Medical Center, 94 Old Short Hills Road, Livingston, NJ 07039 and 3 Reprogenetics, 101 Old Short Hills Road, Suite 501, West Orange, NJ 07052, USA

4 To whom correspondence should be addressed at: Departament de Biologia Cel.lular, Fisiologia i Immunologia, Unitat de Biologia, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.; Email: joaquinia.navarro{at}uab.es


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH–FISH results were obtained in 42 1PB–MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy ({chi}2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.

Key words: aneuploidy/CGH/first polar body/FISH/oocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Early embryonic wastage caused by chromosome abnormalities is thought to be one of the main factors which contribute to the low fertility rate in humans (Bahçe et al., 1999Go). Some evidence suggests that there is a negative selection against some chromosome abnormalities during the first stages of embryonic development (Boué et al., 1985Go). This may explain the fact that the rate of aneuploidies in cleavage-stage embryos (Munné et al., 1995aGo; Márquez et al., 2000Go) is much higher than that found in spontaneous abortions and liveborns (Hassold and Hunt, 2001Go).

The study of oocytes may produce meaningful data, as most embryo aneuploidies as well as most first trimester aneuploidies were classified as originating in female meiosis I (Nicolaidis and Petersen, 1998Go; Hassold and Hunt, 2001Go). In female meiosis, the metaphase I–anaphase I checkpoint, which regulates the proper alignment of the chromosomes in the meiotic spindle, is not as strict as in spermatogenesis (Hunt et al., 1995Go; LeMarie-Adkins et al., 1997Go). Thus, when an error occurs in this alignment, male meiosis is arrested, while female meiosis continues and produces aneuploid oocytes. Moreover, a similar distribution of aneuploidy for all chromosomes was found in human sperm, whereas in oocytes and cleavage stage embryos, some chromosome groups are more prone to be involved in aneuploidy (Pellestor, 1991aGo; Munné et al., 2004Go).

Two mechanisms leading to aneuploidy in human oocytes have been commonly reported: non-disjunction of bivalents (Zenzes et al., 1992Go; Dailey et al., 1996Go) and premature separation of sister chromatids (pre-division, PSSC) (Angell et al., 1993Go; Angell 1997Go). Whereas some authors indicated the co-existence of both mechanisms (Dailey et al., 1996Go; Verlinsky et al., 1999Go; Sandalinas et al., 2002Go; Cupisti et al., 2003Go; Pujol et al., 2003Go), others have found only non-disjuction (Zenzes et al., 1992Go; Benkhalifa et al., 1996Go) or PCSS events (Angell, 1997Go).

The reported incidence of chromosomal abnormalities in human oocytes and polar bodies varies widely between published studies (from 4.7 to 47.5%, average 27.7%). To date, all of the techniques used for studying aneuploidy in oocytes have been based on the spreading of the chromosome material on to slides, followed by methods such as: banding techniques (Pellestor, 1991bGo; Pellestor et al., 2003Go), fluorescence in situ hybridization (FISH) for up to nine chromosomes (Dailey et al., 1996Go; Verlinsky et al., 1999Go; Mahmood et al., 2000Go; Cupisti et al., 2003Go; Pujol et al., 2003Go), spectral karyotyping (SKY) (Márquez et al., 1998Go; Sandalinas et al., 2002Go) or multicolour fluorescence in situ hybridization (m-FISH) (Clyde et al., 2003Go). The dependence on spreading of chromosomes has led to problems not only with overlapping chromosomes, chromosome morphology and artefactual loss of chromosomes during spreading, but also because of the difficulty of obtaining chromosome banding in metaphase II (MII) chromosomes to allow identification of specific chromosome aneuploidies. FISH studies have an extra limitation, as less than a half of the whole karyotype can be analysed because accuracy per probe is reduced when large numbers of probes are combined.

Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows the analysis of the full set of chromosomes in single cells (Voullaire et al., 1999Go; Wells et al., 1999Go). CGH, as a DNA-based method which does not involve cell fixation, may overcome these limitations by analysing the whole set of chromosomes and giving a more accurate and reliable evaluation of the aneuploidy rate (both hyperhaploidy and hypohaploidy).

In female meiosis I, a set of chromosomes, with two chromatids each, segregate to the first polar body (1PB) while the oocyte in MII retains the reciprocal chromosome complement. The object of this study was to examine chromosome abnormalities in 1PB and MII oocytes to determine which chromosomes are more frequently implicated in aneuploidy events, and to determine the main mechanisms producing aneuploidy in female meiosis I. For this purpose, the whole chromosome complement of the 1PB was analysed by CGH and the corresponding MII was analysed by FISH in order to identify the aneuploidy mechanisms and confirm the results.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Oocyte and polar body recovery
A total of 66 oocytes was donated by 46 patients aged 23–42 years (mean 34.9) who, due to different aetiologies, were included in the IVF programme of the Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center (West Orange, NJ, USA). These oocytes were obtained in accordance with guidelines set by the internal review board of that Center. Written informed consent was obtained from all patients and the project was approved by our institutional ethics committee. Two kinds of oocytes were used in this study: oocytes retrieved at metapahse I (MI) stage and matured in vitro and MII oocytes that failed to fertilize after IVF or ICSI. Only those immature eggs that have reached MII stage (displaying a 1PB) 12–24 h after retrieval and only those which showed no pronuclei and presented a 1PB 24 h after fertilization were used in this study. The oocytes were coded with a number (indicating the patient) plus one letter that indicates the oocyte number for each patient.

1PB isolation and oocyte spreading
The zona pellucida was removed using acid Tyrode's solution. Usually the 1PB disassociates after zona pellucida removal, unless there is a cytoplasm bridge between the 1PB and the oocyte. In that case, vigorous pipetting may help to release the 1PB. After that, the 1PBs were isolated and washed in three phosphate-buffered saline (PBS)/0.1% polyvinyl alcohol droplets. The 1PBs were transferred to individual PCR tubes and 1 µl of sodium dodecyl sulphate (17 µmol/l) and 2 µl of proteinase K (125 µg/ml) were added. The sample was overlaid with light mineral oil and the lysis was performed incubating at 37°C for 1 h followed by 10 min at 95°C to inactivate proteinase K. Oocytes were spread after zona pellucida removal using the technique of Tarkowski (1966)Go, with some modifications. Briefly, the oocytes were placed individually in hypotonic solution (0.075 mol/l KCl) for 5–7 min. The oocytes were transferred to a grease-free slide in a minimal volume of hypotonic and five drops of freshly prepared Carnoy fixative (3 methanol:1 acetic acid) were dropped onto the oocyte. The position of the oocyte was circled on the slide with a diamond pencil. Every slide was then examined under a phase-contrast microscope and the presence of metaphase spread was ascertained. Finally, the slides were dehydrated through an alcohol series (70, 85 and 100%, 2 min each) and were frozen at –20°C until they were hybridized.

CGH analysis of 1PBs
DNA from the isolated 1PBs was amplified using degenerate oligonucleotide-primed PCR (DOP–PCR) as previously described (Wells et al., 2002Go), with some modifications. In brief, each PCR tube contained 1 x Buffer, 2 µmol/l DOP primer (CCGACTCGAGNNNNNNATGTGG), 0.2 mmol/l dNTP and 2.5 U of SuperTaq Plus polymerase (Ambion, USA) in a final volume of 50 µl. The sample was spun and heated to 94°C for 4.5 min; eight cycles of 95°C for 30 s, 30°C for 1.5 min and 72°C for 3 min; 40 cycles of 95°C for 30 s, 56°C for 1 min and 72°C for 3 min with a final extension step of 72°C for 8 min. The PCR program was carried out in a 9700 PE thermocycler (Applied Biosystems, USA). Isolated and lysed single buccal cells from a normal female were also amplified and used as reference sample in the CGH experiment. Whole genome amplification products were fluorescently labelled by Nick Translation (Vysis, USA) according to the manufacturer's instructions. 1PB DNA (test) was labelled with Spectrum Red–dUTP (Vysis), whereas reference DNA was labelled with Spectrum Green–dUTP (Vysis). Labelled reference and test DNA were mixed and ethanol-precipitated with 10 µg of Cot-1-DNA. The pellet was dried and redissolved in 10 µl of hybridization mixture [50% formamide, 2 x standard saline citrate (SSC), 10% dextran sulphate, pH 7]. Normal male (46,XY) metaphase spreads (Vysis) were dehydrated through an alcohol series (70, 85 and 100% for 2 min each) and air-dried. The slides were then denatured in 70% formamide, 2 x SSC at 73°C for 5 min and taken through a cold alcohol series and air-dried. The probes were denatured at 73°C for 10 min and were applied to the slide; a coverslip was placed on top and sealed with rubber cement. Hybridization was performed in a moist chamber at 37°C for 40–72 h. After hybridization, the slides were washed at high stringency in 0.4 x SSC/0.3% NP-40 at 73°C for 2 min, 2 x SSC/0.1% NP-40 for 2 min and dehydrated through an alcohol series and air-dried. Finally, the slides were mounted in Vectashield (Vector Laboratories, UK) containing 4',6-diamidino-2-phenylindole (DAPI). 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 (Digital Scientific Cambridge, UK) 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 for being non-informative.

FISH analysis of MII oocytes
The probes used in this study were centromeric, locus-specific or telomeric probes (all provided by Vysis; detailed in Table I) and were used in sequential panels: panels A, B and C. Panel A contained probes for chromosomes 13, 16, 18, 21 and 22 (MultiVysionPB, Vysis). Panel B contained a mixture of 1, 15, 17 and X probes. Panel C included two chromosome probes, one chromosome which worked as an internal control (usually for the X chromosome), plus a second probe that varies depending on the aneuploidy found by CGH in the sibling 1PB (see Table II). At least two rounds of FISH (panels A and B, testing nine chromosomes) were applied to each oocyte, while in some oocytes (those whose corresponding 1PB showed one or more aneuploidies involving chromosomes not included in panel A or B) a third panel was also applied. In general, telomeric and locus-specific probes were applied in the first round of hybridization, so the order of application of the probe sets was variable. Before the FISH procedure, the slides were postfixed at room temperature in 50 mmol/l MgCl2/PBS for 5 min, 3% formaldehyde/50 mmol/l MgCl2/PBS for 9 min and PBS for 5 min. The preparations were then dehydrated, air-dried, stained with DAPI and captured using a fluorescence microscope. DAPI was removed and the slides were dehydrated and air-dried. The first probe set was applied to each slide and covered with a 5 x 5 mm coverslip. The slides were then sealed with rubber cement and placed for 5 min on a hot-plate at 75°C. The probes were allowed to hybridize overnight at 37°C in a dark moist chamber. Post-hybridization washes were performed according to the manufacturer's instructions. Slides were mounted with DAPI or, in the case of the MultiVisionPB probe, in Vectashield antifade. The slides were examined using a fluorescence microscope with filters for the fluorochromes used. Capture and analysis was performed using SmartCapture software 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. The second and the third rounds of FISH were performed as described above.


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Table I. Probes used in this study

 

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Table II. Fluorescent in situ hybridization (FISH) analysis of oocytes

 
Scoring criteria
Sperm chromosomes that had prematurely condensed were easily distinguished from the oocyte complement as they appear much more extended. Sperm chromosomes were excluded from the analysis

The probe sets used in this study were tested on spreads of normal male leukocytes (metaphase and interphase nuclei) using the conditions previously described. Only the probe for chromosome 4 showed cross-hybridization to non-target chromosomes. However, these signals had much lower fluorescence intensity and they were easily distinguished from the specific, more intense signals targeting chromosome 4.

Probes complementary to centromeric regions usually gave one large signal, or alternatively, two smaller paired signals corresponding to the two sister chromatids. For the subtelomeric or locus-specific (LSI) probes (for chromosome 13, 14, 19 and 21), which target telomeric regions or regions of the long arms of these chromosomes, two paired signals were expected.

The presence of additional signals was always considered as confirmed aneuploidies, while missing signals were only considered when the result was confirmed by CGH in the complementary 1PB.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 66 oocytes was obtained. Fifty-four 1PBs obtained from 36 patients were successfully analysed by CGH (81.8%) as 10 1PBs failed to amplify and two 1PBs gave no CGH result because the hybridization intensity of the test DNA was too weak. In order to confirm the CGH results in 1PBs, their complementary MIIs were fixed. Forty-five out of 54 MII showed an adequate metaphase spreading after fixation (83.3%), and FISH results for at least four chromosomes were obtained in 42 oocytes (77.7%) (Table II). Both CGH and FISH results were obtained in these 42 1PB–MII pairs donated by 33 patients (mean 35.8 years old, range 26–42), while 12 1PB–MII doublets had only the 1PB analysed by CGH (data not shown). In most of the cases, one (81.8%) or two (12.1%) oocytes were analysed by patient, but we also analysed three oocytes in one patient and a maximum of four oocytes from one other patient. Sixteen out of 42 oocytes were matured in vitro and 26 failed to fertilize (13 after IVF and 13 after ICSI).

Only six out of 42 MII had FISH results for fewer than seven chromosomes, whereas 36 MII had FISH results for seven to 11 chromosomes. A third round of FISH was necessary in 12 MII. The successive denaturation and washing steps deteriorate the chromosomes, which appeared hairy. However, FISH results were obtained in all 12 MII.

Out of 42 1PBs analysed by CGH, 23 presented results consistent with aneuploidy (Table III). In addition, one extra chromatid for chromosome 18 was found by FISH in MII 8a while CGH analysis of its 1PB indicated a normal profile for all the chromosomes. According to this, we counted 24 aneuploid doublets, instead of 23. Therefore, the aneuploidy rate was 57.1%. Most aneuploid 1PBs had one (14/24), or two (6/24) chromosomes implicated in aneuploidy, but we also found four 1PBs with extensive aneuploidy involving three or more different chromosomes (Figure 1). Twelve out of 14 single aneuploidies, five out of six double aneuploidies and three out of four extensive aneuploidies were confirmed by FISH analysing the MII complement.


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Table III. Summary of CGH and FISH data from aneuploid pairs 1PB–MII

 


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Figure 1. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis from doublet 23a (Table IV) showing reciprocal results between the two sibling cells, first polar body (1PB) and metaphase II (MII). Arrows indicate signals for each chromatid. (A) CGH results from the 1PB showing extensive aneuploidy involving chromosomes 6, 15, 17 and 22. (B) FISH results from the MII testing chromosomes included in panel A. An extra chromatid for chromosome 22 was found. (C) FISH results from panel B showing a missing chromatid for chromosomes 15 and 17. (D) FISH results from panel C testing chromosomes 6 and X. X chromosome worked as an internal control for non-hybridization.

 
A total of 42 aneuploidy events involving 18 different chromosomes was found by either CGH, FISH or both CGH and FISH (Table IV). Out of 39 aneuploidies found by CGH in 1PBs, 36 were confirmed by FISH (92.3%). In addition, three hyperhaploidies (for chromosomes 15, 18 and X) were found by FISH while the 1PBs showed a normal CGH profile for these chromosomes. FISH analysis revealed that less than one-third of these aneuplodies (11 + 1=12 out of 39, 30.8%) were whole chromosome alterations (non-disjunction of bivalent chromosomes), whereas 27 (25 + 2) out of 39 (69.2%) were chromatid abnormalities (pre-division of sister chromatids prior to anaphase I) (Figure 2).


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Table IV. Aneuploidy events found in first polar bodies and oocytes by CGH and/or FISH

 


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Figure 2. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) results from four doublets 1PB–MII (first polar body and metaphase II) with aneuploidy for chromosome 1. Arrows indicate orange signals for each chromatid 1. Additionally, green signals correspond to chromosome 15, aqua signals to chromosome 17 and yellow signals to X chromosome. CGH analysis allows the detection of full chromosome (non-disjunction) or chromatid abnormalities (pre-division) but our data also indicate that it is difficult to differentiate between 0:2/1:2 or 4:2/3:2 CGH ratios. (A) CGH pattern for chromosome 1 and FISH analysis on doublet 12a. CGH profile deviated to the right, indicating gain. Absence of FISH signals for chromosome 1 in the corresponding MII. (B) Results from doublet 9a. CGH analysis indicates gain of chromosome 1. Only one single chromatid was found in the MII. (C) CGH and FISH results on doublet 26a. CGH profile deviated to the left, indicating loss. FISH analysis revealed non-disjunction of bivalents during meiosis I. (D) Results from doublet 11a indicating loss of chromosome 1 in the 1PB. FISH results of the corresponding MII showed an extra chromatid for that chromosome.

 
The chromosomes most frequently involved in aneuploidy were chromosomes 1, 4 and 22 (five aneuploid events each, 11.9%) followed by chromosome 16 (four events, 9.5%). No aneuploidy affecting chromosomes 2, 5, 11, 12 and 20 were found. Data were analysed using the {chi}2-test. Small chromosomes (chromosomes 13–22) were more prone to aneuploidy ({chi}2 test, P=0.07), mostly those chromosomes included in group E.

Age-related aneuploidy has also been analysed. Seven out of 13 analysed 1PBs from five women <35 years old (mean 30.6, range 26–34) gave results consistant with aneuploidy, all of them confirmed by FISH. Additionally, 17 out of 29 oocytes from women aged ≥35 years (mean 38.1, range 35–42) were aneuploid. The difference between the aneuploidy rates in these two age-related groups was not statistically significant.

When comparing the aneuploidy rates and the mechanisms involved in aneuploidy (non-disjunction and pre-division) of the different types of oocytes analysed in this study, no significant difference was found between in vitro matured oocytes and unfertilized oocytes. In addition, there was no significant difference either when comparing failed fertilized ICSI oocytes with failed fertilized IVF oocytes (data analysed using Fisher's exact and {chi}2-test).

Out of the 42 matched 1PB–MII pairs where both CGH and FISH results were obtained, 37 showed complementary results (88.1%). Five 1PBs were found to give results that were not entirely complementary between the CGH interpretations of the 1PBs and the FISH results of the MIIs (Table III). Some doublets were in partial agreement as CGH of 1PBs 16a and 19a indicated imbalance affecting several chromosomes, but not all of them were confirmed by FISH results in MIIs. Other doublets were not in agreement at all. An extra chromatid for chromosome 18 was found by FISH in MII 8a but the 1PB analysis by CGH revealed a normal karyotype. On the other hand, CGH analysis of 1PB 10b revealed a gain affecting chromosome 16 which was not confirmed by FISH. Finally, doublet 32a was classified as normal despite a missing chromosome 1 detected by FISH in the MII. Its 1PB not only did not display the reciprocal gain, but showed a normal CGH profile for all the chromosomes, indicating artefactual loss of chromosome 1 during spreading of the MII.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The aim of this study was to determine the mechanisms that produce aneuploidy in oocytes as well as establishing which chromosomes are more frequently involved in aneuploidy. To achieve this, unfertilized and in vitro matured oocytes and their complementary 1PB were analysed. The main cause of failed fertilization after ICSI is a defective oocyte activation (Schmiady et al., 1996Go). Because ~70% of unfertilized ICSI oocytes have, in fact, a swollen sperm head within them (Flaherty et al., 1995Go), if we had analysed the oocyte by CGH, a sperm nuclei might have been amplified and given us false results. Therefore, the whole chromosome complement of the 1PB was analysed by CGH, whereas the oocytes were analysed by FISH in order to confirm the aneuploidies found in 1PBs.

The incidence of chromosomal abnormalities in human oocytes and polar bodies varies widely between studies. Karyotyping data indicate that ~16% of oocytes are aneuploid (Zenzes et al., 1992Go; Nakaoka et al., 1998Go; Pellestor et al., 2003Go); the aneuploidy rate found using SKY or m-FISH varies from 16.7 to 39% (mean 31.3%) (Márquez et al., 1998Go; Sandalinas et al., 2002Go; Clyde et al., 2003Go). Data derived from FISH studies displayed even more variation, depending mainly on the number of chromosomes and the chromosomes that were analysed. While some researchers have found relatively low rates of aneuploidy (up to 9.6%) analysing two (chromosomes 18 and X), six (chromosomes 9, 13, 16, 18, 21 and X) or eight (1, 9, 12, 13, 16, 18, 21 and X) chromosomes (Dyban et al., 1996Go; Anahory et al., 2003Go; Cupisti et al., 2003Go), others have found a higher rate of chromosome abnormalities. In recent studies, the aneuploidy rate of MII oocytes analysing chromosomes 13, 16, 18, 21 and 22 (Kuliev et al., 2003Go) was 41.7%, analysing chromosomes 1, 7, 13, 18, 21 and X was 44% (Martini et al., 2000Go) and testing chromosomes 1, 13, 15, 16, 17, 18, 21, 22 and X (the same chromosomes tested in panels A and B) was 47.5% (Pujol et al., 2003Go). In the present study, a 57.1% aneuploidy rate was found (mean age 35.8 years). This high rate could be attributed to the higher number of chromosomes being analysed compared to other studies. Moreover, it is important to emphasize that all the techniques mentioned above (SKY, m-FISH, banding techniques and FISH) are based on the spreading of the chromosome material on slides, and some problems related to overlapping chromosomes, chromosome morphology, as well as artefactual loss of chromosomes during fixation have been found. To date, only 35% of the oocytes fixed gave a SKY result because of the overlapping chromosomes (Sandalinas et al., 2002Go) and 25.8% of the missing chromosomes or chromatids found in 1PB were considered artefactual using FISH (Pujol et al., 2003Go). The difficulty of distinguishing between real hypohaploidies or artefactual losses explains why some publications have considered only hyperhaploidies to evaluate the aneuploidy rate (Mahmood et al., 2000Go; Cupisti et al., 2003Go). In this study, a small excess of hyperhaploidies was found in the MII (22 hyperhaploidies versus 17 hypohaploidies), whereas the 1PB analysis showed more hypohaploidies. These findings are in agreement with other reports where more hypohaploidies were detected in 1PB (Kuliev et al., 2003Go; Pujol et al., 2003Go). However, when studying large series of cleavage stage embryos, more monosomies have been found (Munné et al., 2004Go). Therefore, this excess of monosomic embryos may not be produced in the oocyte but during the first mitotic divisions, and some mechanisms such as anaphase lag may be involved (Munné et al., 2004Go). In our hands, the combination of two techniques, CGH as a DNA-based method that allows the identification of any chromosome abnormality (both, hyperhaploidy and hypohaploidy) and FISH to confirm the aneuploidy events found in 1PB, provides a reliable and accurate estimation of the aneuploidy rate in oocytes.

Most preimplantation genetic diagnosis (PGD) centres that perform aneuploidy screening (PGD-AS) in either cleavage stage embryos or polar bodies use probes for chromosomes most commonly involved in aneuploidy in live births (X, Y, 13, 18 and 21) (Munné and Weier, 1996Go; Verlinsky and Kuliev, 1997Go) but also for chromosomes involved in spontaneous abortions (14, 15, 16 and 22) (Munné et al., 1998aGo,bGo, 2003Go). In this study, panels A and B included probes for nine different chromosomes to cover the most frequent abnormalities detected in cleavage stage embryos and oocytes (chromosomes 1, 13, 15, 16, 17, 18, 21, 22 and X). However, these two panels revealed only 26 out of 42 aneuploidy events (61.9%). Thus, if we had not used CGH, 16 aneuploidies (38.1%) involving chromosomes 3, 4, 6, 7, 8, 9, 10, 14 and 19 would have been missed. It is important to note that oocytes or 1PB with extensive aneuploidy would be scored as abnormal even if FISH screening only detected one chromosome error (i.e. doublet 1PB–MII 2a, Table III). Despite this fact, 25% (6/24) of the 1PB classified as aneuploid would have been misdiagnosed as normal using FISH with probes for the nine chromosomes mentioned above. These results are consistent with a recent study in which 25% of the blastomeres diagnosed as aneuploid with CGH would have been incorrectly diagnosed as normal using FISH for nine chromosomes (Wilton et al., 2003Go). On the other hand, an 11.9% aneuploidy rate was found for chromosome 1 (Table IV). If we have chosen the current panel of nine chromosomes used by most PGD centres and therefore chromosome 1 had not been included in panel B, 10 out of 24 1PB (41.7%) classified as aneuploid by CGH would have been incorrectly diagnosed as euploid with FISH.

When comparing aneuploidy rates for different chromosomes in cleavage stage embryos with the incidence of these aneuploidies in spontaneous abortions (Simpson, 1990Go), some aneuploidies were found to survive less often than others (Munné et al., 2004Go). Bahçe et al. (1999)Go found trisomy 1 at a frequency of 16% in cleavage stage embryos, while to our knowledge there is only one reported case of trisomy 1 in first trimester conceptions (Hanna et al., 1997Go). Taken together, all these data suggest that aneuploidies of chromosomes not involved in trisomic offspring or spontaneous abortions, such as chromosomes 1, 4 and 17, may be common at conception but fail to implant or shortly die after implantation. Therefore, an ideal PGD-AS should analyse also these chromosomes as their involvement in aneuploidy may have an effect, not causing spontaneous abortions, but a decrease in implantation rates (Bahçe et al., 1999Go).

When studying age-related aneuploidy, no significant differences were found considering two maternal age groups (<35 and ≥35 years old), suggesting that the sample size was probably too small. In addition, it is worth noting that in a 27 year old woman (patient 34), we found three aneuploid 1PB from a total of four analysed 1PB. The inclusion of four oocytes from this patient, who may have a tendency to produce aneuploid oocytes despite her age, may bias our results, as previously suggested (Zenzes et al., 1992Go).

Previous reports have suggested that unbalanced pre-division and non-disjunction did not increase significantly with time in culture (Munné et al., 1995bGo; Boiso et al., 1997Go), whereas other authors suggest the contrary (Pickering et al., 1988Go; Dailey et al., 1996Go). In this study, no significant difference was found between in vitro matured oocytes and unfertilized oocytes concerning aneuploidy rates and aneuploidy mechanisms. On the other hand, no differences were found either when comparing unfertilized oocytes after ICSI or IVF, as previously reported (Edirisinghe et al., 1997Go).

The hightest rates of abnormalities were found for chromosomes 1, 4 and 22, followed by chromosome 16. Chromosomes 16 and 22 have also been frequently involved in aneuploidy in other studies of polar bodies, oocytes and embryos (Sandalinas et al., 2002Go; Kuliev et al., 2003Go; Pujol et al., 2003Go; Munné et al., 2004Go). Classifying the chromosomes as large (chromosomes 1–12 and X chromosome) or small (chromosomes 13–22), small chromosomes were more prone to aneuploidy ({chi}2-test, P=0.07). These findings are in agreement with other studies, where small chromosomes are involved more frequently in aneuploidy (Pellestor et al., 2002Go; Sandalinas et al., 2002Go; Cupisti et al., 2003Go). One possible explanation is that small chromosomes tend to form fewer chiasmata during meiosis I, and reduced levels of recombination could predispose to non-disjunction (Koehler et al., 1996Go; Hassold et al., 2000Go; Hassold and Hunt, 2001Go; Sun et al., 2004Go).

FISH analysis of MII oocytes allowed the confirmation of 92.3% of the aneuploidy events found in 1PBs by CGH. Moreover, FISH analysis detected unequivocally two mechanisms of aneuploidy, revealing that up to two-thirds of the aneuploidy events (69.2%) were due to a PSSC, while only one-third was due to chromosome non-disjunction. Previous reports have also indicated the co-existence of these two mechanisms of maternal aneuploidy, with chromatid anomalies being the most common (Verlinsky et al., 1999Go; Pellestor et al., 2002Go; Sandalinas et al., 2002Go). On the other hand, older women seem also to accumulate more mitochondrial DNA mutations, either in oocytes or in the surrounding follicular cells (Bartmann et al., 2004Go). These mutations result in a reduced mitochondrial function (Schon et al., 2000Go). Cohesin is the protein complex that holds replicated sister chromatids together until anaphase II. Considering that meiosis is an energy-dependent process and that ATP is also required for DNA binding by Cohesin (Uhlmann, 2004Go), a deficiency in oxidative energy metabolism could result in a higher incidence of PSSC in older women, as previously reported (Sandalinas et al., 2002Go).

In CGH, hypohaploidy affecting single chromatids (DNA test: DNA reference ratio 1:2) is easier to detect than hyperhaploidy (ratio 3:2) as in hypohaploidy there is a loss of 50% of the chromosomal material, while in hyperhaploidy there is only a gain of 33% (Gutierrez-Mateo et al., 2004Go). In the present study we demonstrate the ability of CGH to detect not only extra or missing full chromosomes, but also single chromatid abnormalities. However, our data also show similar deviations of the CGH profiles in either chromosome (ratios 4:2 or 0:2) or chromatid (ratios 3:2 or 1:2) abnormalities. Therefore, we can conclude that CGH is useful to determine the implication of any chromosome in aneuploidy but is not the most suitable technique to test whether the chromosome abnormality is derived from a non-disjunction of bivalents or alternalively from a premature separation of sister chromatids at meiosis I.

In this work, five out of the 42 (11.9%) matched 1PB–MII pairs where both CGH and FISH results were obtained showed no reciprocal results. Two kinds of non-reciprocity have been scored: (i) doublets 1PB–MII which presented one or two hyperhaploidies with no reciprocal loss of material in the sibling cell (1PB or MII); (ii) a doublet which presented absence of FISH signals for chromosome 1 in the MII, while its 1PB displayed a normal karyotype, suggesting artefactual loss of chromosome 1 during fixation of the MII. Some authors have also found extra chromosomes or chromatids with no reciprocal loss of material in the complementary cell, suggesting a third mechanism of maternal aneuploidy: the possible existence of a gonadal mosaicism with a trisomic germ line in some of these patients (Mahmood et al., 2000Go; Cupisti et al., 2003Go; Pujol et al., 2003Go).

In conclusion, the combination of two different techniques, CGH and FISH, for the study of 1PB and MII allows the identification and confirmation of any chromosome imbalance, as well as determining the mechanisms involved in the genesis of maternal aneuploidy. Our data also have implications for PGD of aneuploidy, as some chromosomes that were involved frequently in aneuploidy are not included in current panels for aneuploidy screening.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Ana Castellano for her collaboration on this work. This study 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).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on May 24, 2004; accepted on August 18, 2004.