Molecular Human Reproduction, Vol. 9, No. 10, 577-585,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting
Submitted on March 14, 2003; accepted on May 20, 2003
1 Service de Génétique Moléculaire et Chromosomique, C.H.U. Arnaud de Villeneuve, 34295 Montpellier, Cedex 5, 2 CNRS UPR 1142, Institut de Génétique Humaine, 141 rue de la Cardonille, 34396 Montpellier Cedex 5 and 3 Laboratoire de Biologie de la Reproduction, Clinique St Roch, Montpellier, France
4 To whom correspondence should be addressed. e-mail: franck.pellestor{at}igh.cnrs.fr
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Abstract |
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The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non- disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.
Key words: aneuploidy/double-labelling/FISH/oocyte/polar body
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chromosomal abnormalities account for the majority of pre- and post-implantation human embryo lethality. Many of these abnormalities result from de-novo segregation errors during female meiosis (Hassold and Hunt, 2001). Consequently, the chromosomal analysis of mature oocytes is of great interest in order to investigate both the occurrence and the aetiology of these abnormalities. The chromosomal constitution of human oocytes was first studied by conventional karyotyping, but the difficulties of obtaining good quality metaphase spreads and of performing accurate chromosome identification have strongly limited the viability of this approach (Pellestor, 1991; Jacobs, 1992). The approximate nature of these studies was pointed out by Angell’s observation that premature separation of homologous chromatids through anaphase I might be a significant mechanism for human aneuploidy (Angell, 1991, 1997). The emergence of this particular mechanism of malsegregation introduced an important new parameter in the investigation of meiotic non-disjunction, but also suggested that the mis-scoring of single chromatids might have significantly biased the results of cytogenetic studies of human oocytes. Only a few recent reports based on large samples of karyotyped oocytes have provided reliable data on the abnormality rate and the underlying mechanisms of aneuploidy occurrence (Nakaoka et al., 1998; Pellestor et al., 2002).
In recent years, molecular cytogenetic techniques, such as fluorescence in-situ hybridization (FISH) and primed in-situ labelling (PRINS), have been adapted on human gametes (Pellestor et al., 1996; Egozcue et al., 1997). Due to its relative simplicity and the commercial availability of numerous DNA probes, FISH has become the standard technique for aneuploidy assessment in human gametes. In human oocytes, FISH appears to be a significant improvement over karyotyping because it overcames the difficulty of chromosome spreading and sometimes allows the parallel analysis of the first polar body.
Several FISH studies have already been published using simple or sequential multi-FISH (Table I). All these studies have confirmed the co-existence of whole chromosome non-disjunction and chromatid separation as mechanisms of maternal aneuploidy. However, some of these studies have displayed unbelievably high rates of aneuploidy (from 37 to 44%) in comparison with the recent and reliable karyotyping data (Nakaoka et al., 1998; Pellestor et al., 2002). This discrepancy may result from some technical aspects of the FISH procedure used. Indeed, all these FISH studies have only used one type of labelling per chromosome, i.e. centromeric probes or locus-specific probes. Because of the inherent risk of artefactual, failed or overlapped in-situ hybridization and the relatively low quality of some scored metaphases, such a simple approach can lead to interpretation mistakes. To overcome this potential source of error, we have conducted a sequential FISH study of human oocytes and polar bodies, using a combination of centromeric labelling and whole chromosome painting for chromosomes 9, 13, 16, 18, 21 and X. By allowing the accurate identification of both chromosomes and chromatids, this combined approach provides better reliability for FISH investigation of human oocytes. The data obtained from 104 oocytes are presented here.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Oocyte recovery and processing
Oocytes were obtained from consenting women undergoing IVF or ICSI protocols at the IVF clinic of Montpellier. All women involved in this study were counselled about the procedure and gave consent for their unfertilized oocytes to be donated for this analysis. The use of human in-vitro unfertilized oocytes was approved by the local ethics committee. Stimulation of multifollicular development was induced by successive administration of GnRH agonist and hMG or recombinant FSH. Follicle development was monitored by transvaginal ultrasound. About 36 h after hCG injection, the follicles were aspirated under transvaginal ultrasound guidance. The oocytes used in the present study, were those that had failed to fertilize when examined 16–18 h after being either incubated with sperm (IVF) or micro-injected with a spermatozoon (ICSI). These oocytes were maintained in culture for a further 24 h. After this additional culture, oocytes were observed in detail for the presence of pronuclei and polar bodies, using a phase contrast microscope. Only those that exhibited intact or fragmented first polar bodies (PB) and showed no evidence of fertilization were assigned to this study and then prepared for chromosome analysis. Oocytes with a degenerate aspect were also excluded from the study.
The selected oocytes were spread using the Tarkowski (1966) technique. Briefly, each oocyte was placed in hypotonic solution (1% sodium citrate) at room temperature for 6 min. The oocyte was then transferred to a grease-free slide and four drops of 16 µl of fresh fixative (3:1 methanol:acetic acid) were successively dropped onto the oocyte. The position of the oocyte was encircled with a glass marker. After fixation, each slide was examined under a light microscope (x10 and x40) in order to check for the presence of metaphase spread and a polar body. Their positions on the slide were noted. Slides with chromosomes scattered too widely were discarded. Finally, the selected slides were kept at room temperature for 1–3 days prior to the FISH procedure.
Sequential FISH procedure
In-situ labelling was sequentially performed on chromosomes 9, 13, 16, 18, 21 and X, using commercially available probes. The Vysis (USA) centromeric CEP probes directly labelled with Spectrum Green or Spectrum Orange, were used for the specific centromeric labelling of chromosomes 9, 16, 18 and X. Locus-specific probes LSI 13 and LSI 21 (Vysis) labelled with Spectrum Green and Spectrum Orange respectively were used on chromosomes 13 and 21. In combination with these probes, whole painting probes WCP (Vysis) labelled either with Spectrum Green or Spectrum Orange were used on each metaphase spread. For each analysed chromosome, the probe labels were chosen in order to complement the fluorescent green and red signals (Table II). All probes were used according to the manufacturer’s recommendations.
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Before the FISH procedure, fixed oocytes were denaturated in 70% formamide–2xstandard saline citrate (SSC), pH 7.0 at 73°C for 4 min, dehydrated in an ethanol series and air-dried. Three rounds of FISH, each combining the labelling of two chromosomes, were performed on each slide. Each hybridization mixture was prepared according to the manufacturer’s protocol, i.e. for a final volume of 5 µl:3 µl of buffer, 1 µl of distilled water and 0.5 µl of each probe. The mixture was denaturated at 73°C for 5 min just before being applied to the slide.
For the first round, 5 µl of hybridization mixture containing the first two combined centromeric probes (for instance CEP 9 and CEP 18) were deposited on the slide and sealed under a coverslip with rubber cement. The slide was then incubated at 37°C for 90 min in a humidified box. After hybridization, both the rubber cement and the coverslip were gently removed and the slide was washed twice in 4xSSC for 4 min. During this step, the mix of complementary painting probes (WCP 9 and WCP 18) was prepared and denaturated (same conditions). After draining the excess 4xSSC solution off the slide, the second mix was applied to the slide and sealed under a coverslip. The slide was incubated for 16–18 h at 37°C. Following hybridization, the coverslip was removed and the slide was washed for 2 min in 0.4xSSC at 72°C, and then in 2xSSC+0.05% Tween 20 at room temperature. The slide was counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), mounted in the antifading medium Vectashield (Vector Laboratories, USA), and examined using a Leica DMRB microscope (x40 and x100) equipped with single and triple band pass filters for DAPI, fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). After recording of the results of the first FISH round, the slide was washed twice in 4xSC + 0.05% Tween 20 for 2x15 min at room temperature in order to remove immersion oil, coverslip and antifade solution. The slide was briefly rinsed in phosphate-buffered saline (PBS) and distilled water before being dehydrated in an ethanol series and incubated for 2.5 min at 73°C in denaturation solution. This short denaturation step allowed removal of previous hybridized probes and to gently denaturate chromosomes. Then the second FISH round (for instance, targeting the chromosomes 16 and X) was performed as described for the first round. The procedure was similar for the third round, except that the LSI 13 and LSI 21 were directly combined with the corresponding 13 and 21 WCP and consequently hybridized for 16–18 h at room temperature.
Scoring criteria
The chromatids of oocytes and polar bodies were easily distinguished because of the more compact and often degenerated aspect of polar body material. There was no confusion with sperm nuclei, which display sets of single chromatids, which are always very extended after sperm decondensation. The use of combined locus-specific or centromeric probes with whole painting probes led to the visualization of two complementary signals (red and green) for each chromatid. The distinct observation of these combined signals was the criterion for the identification of the targetted chromosomes or chromatids and the scoring of segregation abnormalities (whole chromosome non-disjunction, single chromatid or balanced pre-division of chromatids). Two homologous chromatids from one univalent were considered as separated when their centromeric signals were separated by more than the diameter of two centromeric signals. This configuration was classified as balanced pre-division. For chromosomes 13 and 21, because LSI probes label loci on the long chromosomal arms, and because oocyte chromosomes frequently display non-adjacent (floating) arms, the identification of separated chromatids was acheived by observing separation of the two painting signals. Oocytes with multiple chromatid separations were considered to be degenerating and were not included in the study.
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Results |
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A total of 234 unfertilized oocytes, obtained from 76 women, was processed for chromosomal analysis. Sixty-three cells were discarded because of their degenerative aspect. Of the 171 remaining oocytes, good metaphase spreading was obtained for 104 cells. The other 67 oocytes were considered unsuitable for analysis because they contained no chromosomal material or because they displayed excessive spreading or fragmentation of chromosomes. The overall spreading efficiency was therefore 60.8% (104/171). The 104 analysed oocytes came from 45 women, aged 21–42 years (mean age: 31.6 years). Of these 104 oocytes, 56 (53.8%) displayed an analysable first polar body.
According to the described protocol, several combinations of probes were applied on each metaphase spread. The specificity and efficiency of each probe were first tested on metaphase and interphase nuclei. The hybridization efficiency reached 100% for painting probes, 99% for centromeric probes and 97% for locus-specific probes. Three rounds of hybridization were successfully performed on 79 oocytes and 43 polar bodies. In all these cases, distinct labelling was obtained in each round of hybridization with both centromeric (or locus-specific) and whole chromosome painting probes, giving a hybridization efficiency of 75%. In other cases (25 oocytes and 13 polar bodies), it was only possible to perform two rounds of hybridization because of the decreasing quality of the metaphases through the successive hybridization and washing steps (deterioration of the metaphase, presence of debris covering some chromosomes).
The results of the FISH analysis are given in Table III, and examples of in-situ labelling are presented in Figures 1 and 2. A total of 84 (80.7%) oocytes displayed a normal pattern of FISH signal (Figure 1). When possible, these data were confirmed in the corresponding first polar body. Discordance between signals in metaphase II and the first polar body were found in two oocytes. In these two cases, a signal was absent in the polar body whereas the corresponding oocyte metaphase presented a normal signal. Due to the lower quality of the polar body material, this probably resulted from failure of hybridization. Two cases of chromosomal hyperhaploidy were observed for chromosomes 9 and 16 respectively (Figure 2A). One case of hypohaploidy for chromosome 16 was also noted and confirmed by the observation of two chromosomes 16 in the corresponding polar body. The presence of supernumerary single chromatids was observed in seven oocytes (6.7%) and confirmed in five cases by the analysis of polar bodies (Figure 2B, D). All the investigated chromosomes, except chromosome 13, were implicated in the occurrence of extra chromatids. In one case, an extra chromatid 21 was associated with the supernumerary chromosome 9 described above (Table III, case P25). In 12 oocytes (11.5%), the balanced separation of homologous chromatids was observed (Figure 2E, F). Although the implication of balanced chromatid separation in the formation of aneuploidy was controversial, we chose to distinguish these events and involve them in the description of the abnormal chromosomal constitution with regard to the defined scoring criteria (see Discussion). The distribution of the abnormalities per chromosome is given in Table IV. It should be noted that only chromosome 16 was involved in all types of abnormalities. On the other hand, chomosome 13 was only implicated in one case of balanced chromatid separation. Due to the relatively small size of our metaphase sample, the correlation between the abnormalities and the maternal age was not assessed.
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Based on the present results, it is possible to estimate the overall rate of aneuploidy in oocytes by using the formula proposed by Dailey et al. (1996) which involves the different pathways of non-disjunction: 1/2(A) + 1/2(B – D) + C, where A is the frequency of balanced chromatid pre-division, B is the frequency of unbalanced pre-division, C is the frequency of chromosome non-disjunction and D is the frequency of metaphases with both chromosome non-disjunction and unbalanced chromatid pre-division (one case in our sample; Table III, P25). The calculation gives an overall estimate of aneuploidy of: 1/2(11.5) = 1/2(6.7 – 0.9) + 2.9 = 11.5%.
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Discussion |
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In this paper, we describe a new approach for in-situ chromosomal investigation of human oocytes, based on the combination of two types of DNA probes for labelling each targeted chromosome. This procedure guarantees efficient and reliable identification of both chromosomes and single chromatids, and consequently an accurate estimate of numerical abnormalities. All the previous conventional FISH studies of oocytes (Table I) have used a unique labelling procedure, essentially centromeric probes or locus-specific probes, leading to the in-situ visualization of only simple or double dots. This could be an evident source of artefact and miscoring. Using such a simple procedure, Dailey et al. (1996) have estimated a 10.6% error rate of FISH in human oocytes. The shortcoming of a simple labelling system is accentuated by the use of the sequential FISH procedure, where two or three rounds of double or triple FISH labelling were performed on the same chromosome preparation. This re-utilization procedure constitutes a simple and economic approach for scoring several chromosomes, since it requires no extra instrumentation or additional filter sets. However, the use of several cycles of FISH labelling and washing inevitably diminishes the integrity and the morphologic quality of the chromosome spreads (Epstein et al., 1995). In addition, because of the apparent efficiency of FISH labelling, the initial quality of the metaphase spreads scored is often poor (Dyban et al., 1996; Martini et al., 1997, 2000). Thus, some of the previous FISH studies have reported implausibly high rates of abnormalities (37–44%) (Dailey et al., 1996; Martini et al., 1997, 2000), which might be attributable to these technical limitations. The FISH data must be considered with caution because, as pointed out by Martini et al. (2000), the identification of two spots in an oocyte is not as reassuring as the clear identification of two chromosomes. However, in comparison with standard FISH procedures, the double-labelling system presented here allows a more precise distinction of chromosomal material, with the simultaneous in-situ visualization of chromosome arms and centromeres (or specific loci). In the same way, spectral karyotyping employing 24 chromosome-specific painting probes has appeared to be an ideal approach. However, preliminary results obtained on small oocyte samples have indicated that chromosome spreading was also an important limitation for spectral karyotyping (Marquez et al., 1998; Sandalinas et al., 2002). In addition, this procedure necessitates more time for chromosome identification and remains expensive (Bezrookove et al., 2000).
Three types of numerical abnormalities have been identified in our oocyte samples, i.e. chromosome non-disjunction, extra single chromatid and balanced chromatid separation (Figure 3). As illustrated in Figure 2, the precise distinction of these abnormalities was possible thanks to the combination of centromeric (or locus-specific) probes and whole chromosome painting probes. These findings confirm that both conventional non-disjunction and premature chromatid separation contribute to the occurrence of aneuploidy in human oocytes. We observed that abnormalities due to chromatid pre-division were more frequent than chromosome non-disjunctions (seven extra chromatids versus three chromosome non-disjunctions). This is in agreement with data of some previous FISH studies (Martini et al., 2000; Sandalinas et al., 2002), but also with the results of the recent karyotyping studies performed on large oocyte samples (Nakaoka et al., 1998; Pellestor et al., 2002). On 1397 karyotyped human oocytes, Pellestor et al. (2002) found 5.9% of single chromatid malsegregation versus 3.5% of whole chromosome non-disjunction. It has been suggested that extra free chromatids could be the result of in-vitro oocyte ageing (Dailey et al., 1996; Mailhes et al., 1998) or oocyte degeneration (Lim and Tsakok, 1997). However, the recent study of Sandalinas et al. (2002) using spectral karyotyping on fresh, non-inseminated human oocytes has indicated that chromatid pre-division was also significantly observed in fresh oocytes. Thus, the premature separation of chromatid at meiosis I clearly constitutes a major mechanism for trisomy formation in the human zygote. The emergence of this new class of chromosomal abnormalities has provided new insight into the mechanisms of non-disjunction in female meiosis. Particular interest has been trained on factors implicated in the cohesion between sister chromatids through the cell cycle. The nuclear proteins, called cohesins, have been identified as essential elements for the stable association of homologue chromatid and proper segregation during meiosis. Molecular studies on various species (yeast, Drosophila, mammals) have indicated that chromatid cohesion is a conserved mechanism which ensures physical attachment between chromatids until anaphase I (Watanabe and Nurse, 1999; Waizenegger et al., 2000; Nasmyth, 2001). The cohesins oppose the splitting force mediated by microtubules on kinetochores (Nicklas, 1997) and are even required for the association of chiasmate homologues during meiosis (Bickel et al., 2002). The gradual degradation or loss of these proteins appears to be a causal mechanism of maternal age-related non-disjunction, and thus the premature degradation or lack of these proteins might be responsible for the premature sister chromatid separation observed in oocyte metaphases (Wolstenholme and Angell, 2000; Pellestor et al., 2003).
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The observation of balanced separated chromatids could also be considered to be the reflection of premature separation of chromatids at anaphase I. Nevertheless, this hypothesis has been questioned by some authors suggesting that balanced separation was rather an artefact related to the time in culture (Kamiguchi et al., 1993; Dailey et al., 1996). Although the possibility of a technical origin of balanced separated chromatids could not be ruled out, the observation of such balanced separations in fresh, non-inseminated human oocytes (Sandalinas et al., 2002) contrasts with the artefactual explanation, and indicates that this phenomenon could be taken into consideration in the analysis of aneuploidy occurrence. In the present study, we have adopted strict criteria for the scoring of separated chromatids, and the use of the double-labelling system has allowed an unambiguous visualization of separated chromatids, as illustrated in Figure 2E and F. Consequently, in agreement with Mahmood et al. (2000), we consider that oocytes with balanced but significantly separated chromatids are at an increased risk for aneuploidy through anaphase II, after fertilization.
Balanced chromatid separation occurred most frequently for chromosomes 16 and 18. Although not statistically significant in our sample, this finding confirms previous observations made in FISH studies (Mahmood et al., 2000) and karyotyping studies (Angell, 1997; Pellestor et al., 2002). The prevalence of the chromatid separation in chromosomes of group E might be consistent with the mechanism of loss of chromatid cohesion. It could be speculated that there is a particular feature in the conformation or in the centromeric DNA sequence of chromosomes 16 and 18 which favours the occurrence of premature separation. If the variation in size of centromeric DNA sequence is likely to affect the efficiency of chromatid cohesion, small alphoid DNA domains could not bind enough centromere-associated cohesins to durably maintain cohesion between homologous chromatids. In larger chromosomes, the presence of several chiasmata could prevent the occurrence of premature chromatid separation. This could provide a plausible explanation for the prevalence of aneuploidy for small chromosomes in aged oocytes.
In this study we estimated an overall rate of aneuploidy in oocytes of 11.5%. This value is consistent with the most recent estimates drawn from the karyotyping of large series of human oocytes (8.5% in Nakaoka et al., 1998; 10.8% in Pellestor et al., 2002). Similar results were also reported in a few FISH studies (Dailey et al., 1996; Honda et al., 2002), but other FISH reports have displayed unbelievably high rates of aneuploidy (Dyban et al., 1996; Martini et al., 1997, 2000), indicating that the interpretation of FISH results remains critical. An aneuploidy rate of 11.5% is in better agreement with data from spontaneous abortions and clinically recognized conceptions (Jacobs, 1992). Taken together, all these data provide solid insight into the incidence of aneuploidy and the mechanisms of non-disjunction in female meiosis.
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Acknowledgement |
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This study was supported by a European grant Copernicus 2 (Contract ICA2-CT-2000-10012, proposal ICA2-1999-20007).
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References |
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