Hum. Reprod. Advance Access originally published online on August 6, 2004
Human Reproduction 2004 19(10):2231-2237; doi:10.1093/humrep/deh391
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Nuclear chromosomal localization in human preimplantation embryos: correlation with aneuploidy and embryo morphology
1 Departments of Obstetrics and Gynecology and 2 Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA
3 To whom correspondence should be addressed at: Baylor College of Medicine, Department of Obstetrics and Gynecology, 6550 Fannin Street, Suite 885, Houston, TX 77030, USA. Email: bischoff{at}bcm.tmc.edu
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Abstract |
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BACKGROUND: Spatial organization of chromosomes is hypothesized to reflect transcriptional activity and regulatory protein function. Preimplantation genetic diagnosis allows assessment of the spatial relationship of chromosomes in human blastomeres. We thus examined the localization of chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres from 6–8-cell stage embryos, correlating localization to aneuploidy and embryo morphology. METHODS: Following fluorescence in situ hybridization to enumerate chromosomes 13, 16, 18, 21, 22, X and Y, signal positions were localized within one of four concentric shells. Statistical analysis compared chromosome localization between euploid and aneuploid blastomeres as well as morphologically normal and abnormal embryos. RESULTS: Of 98 embryos, 109 blastomeres were evaluated. Within chromosomally normal blastomeres, no difference in the location of all seven chromosomes (P
0.10) was observed. However, a significant difference was observed between the organization of chromosomes in euploid versus aneuploid blastomeres (P0.001). Localization of chromosomes 13, 18, 21 and 22 was significantly different when an abnormality involving that chromosome existed (P0.001, P0.01, P0.025 and P0.01 respectively). CONCLUSIONS: We report for the first time that localization of chromosomes is altered in chromosomally aneuploid but not in chromosomally normal nor morphologically abnormal euploid blastomeres.
Key words: aneuploidy/chromosomal nuclear localization/preimplantation genetic diagnosis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Organization of the cell nucleus plays a critical role in gene function. Spatial organization of chromosomes within the mammalian nucleus is believed to be conserved and to influence gene expression (Brown et al., 1997
; Francastle et al., 1999). In cultured mammalian cells, analysis of chromosome organization shows a relationship between gene density and nuclear location. Chromosomes with the highest gene density are preferentially displaced towards the nuclear interior, whereas euchromatin-rich (gene-poor) chromosomes are located towards the nuclear periphery (Croft et al., 1999; Boyle et al., 2001; Cremer et al., 2001). The prototypic example is the X chromosome undergoing X-inactivation, located on the periphery of the nuclear membrane (X-chromatin). Thus, we hypothesize that altered nuclear chromosomal localization may be indicative of altered cellular growth and division, leading to aneuploidy.
The nuclear organization of every human chromosome in diploid lymphoblasts and primary fibroblasts was studied by Boyle et al. (2001). As we would have predicted, this group found the most gene-rich chromosomes (HSA-1, 16, 17, 19 and 22) to be localized within the centre of the nucleus; gene-poor chromosomes (HSA-2, 3, 4, 7, 8, 11, 13 and 18) were concentrated towards the nuclear periphery. Chromosomes 5, 6, 10, 14, 15, 20 and 21 were not found to have a significant bias either to the peripheral or to central locations. Boyle et al. (2001) found no correlation between chromosome size and position within the nucleus, although gene density clearly contributed to the nuclear positioning of individual chromosomes. Thus, disruption of the nuclear organization of chromosomes should result in altered interaction between chromatin and the nuclear membrane with subsequent deregulation of gene expression. Alternatively, both nuclear location and gene expression could reflect perturbation of the same underlying event.
To distinguish between these two possibilities, we studied blastomeres derived from preimplantation embryos of subjects undergoing preimplantation genetic diagnosis (PGD). Using fluorescence in situ hybridization (FISH) chromosome-specific probes, we hypothesized that the nuclear organization of individual chromosomes may reflect or play a role in cell division and embryonic development. Thus, we predicted altered nuclear position in aneuploid and morphologically abnormal but not in morphologically normal (euploid)embryos.
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Materials and methods |
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Subjects
After institutional review board approval and written informed consent, couples (n=17) underwent IVF and ICSI followed by PGD. Table I summarizes information pertaining to each case: indication for PGD, maternal age, number of blastomeres analysed and total number of hybridization signals recorded. Given our goal of focusing on the role of nuclear localization among PGD embryos independent of indication, we evaluated cases of multiple failed IVF (n=7), recurrent pregnancy loss (n=2), structural chromosome abnormality (n=5: two reciprocal translocations, one robertsonian translocation, two Y-chromosome deletions), one mosaicism (45X/46,XX), one X-linked disorder for sexing (chordideraemia), one prior trisomy 13 live-born.
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Embryo morphology
At 72 h post fertilization, blastomere number, shape, size and fragmentation were recorded (Table II). Embryos were graded 1 to 5. In general, grades 1, 2 or 3 were considered morphologically abnormal; embryos graded 4 or 5 were considered morphologically normal.
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Blastomere biopsy
Oocytes were aspirated after controlled ovarian stimulation, first with GnRH agonist down-regulation and then with hMG stimulation. The third morning following fertilization by ICSI, the 6–8-cell stage embryos were biopsied between 08:00 and 12:00 h. From each embryo one or two blastomeres were aspirated using suction and a Humagen® biopsy needle. Blastomeres were washed in phosphate-buffered saline (PBS) for 2 min and transferred to a poly-L-lysine-coated slide as described by Coonen et al. (1994)
. Cytoplasm was cleared following two sequential incubations in 50–100 µl spreading solution (0.01 N HCl, 0.1% Tween-20). Nuclei were then washed in PBS, dehydrated through sequentially more concentrated ethanol (70, 90 and 100%) incubations, air-dried and processed for FISH. Embryo biopsy and blastomere fixation was performed by the same individual for all cases in an environmentally controlled room, minimizing variability due to factors such as operator, humidity and room temperature.
FISH–PGD probes
Direct-labelled chromosome-specific probes (Vysis, Inc.) were used in analysis of all blastomeres. Our FISH strategy involved two sequential hybridizations. In the first hybridization, probes specific to chromosomes 13 (13q14 locus-specific, LSI 13; SpectrumGreen-labelled), 18 (18p11.1-q11.1 alpha satellite centromere, CEP18; 1:1 equal mixture of SpectrumOrange:SpectrumAqua-labelled), 21 (21q22.13-q22.2 locus-specific, LSI 12; SpectrumOrange-labelled), X (Xp11.1-q11.1 alpha satellite centromere, CEPX; 1:1 equal mixture SpectrumOrange:SpectrumGreen-labelled) and Y (Yq12 satellite III sequence-specific, CEPY; SpectrumAqua-labelled) were used simultaneously to identify blastomeres/embryos with the correct number of FISH signals corresponding to each chromosome. Blastomeres having two signals for each of the autosomes (13, 18 and 21) and either two signals for the X chromosome alone (XX) or one signal for both the X and Y chromosomes (XY) were classified as euploid (normal) and, hence, subjected to a second hybridization using probes for chromosomes 16 (16q11.2 satellite II centromere, CEP16; SpectrumOrange labelled), 22 (22q11.2 locus-specific, LSI 22; SpectrumGreen-labelled) and chromosome 18 again. The second hybridization for chromosome 18 was perfomed as a positive hybridization control (alpha satellite centromere, CEP18; SpectrumAqua-labelled).
For the two reciprocal translocation cases (nos. 11 and 12), only blastomeres (n=4 total) identified as balanced/normal for the rearrangement were subsequently assessed for chromosomes 13, 18, 21, X and Y. Of the 13 blastomeres/embryos examined in the 21:21 robertsonian translocation case (no. 10), only one was found to be balanced/normal; all 13 were assessed for chromosomes 13, 18, 21, X and Y.
FISH: denaturation and hybridization
Following our previously described protocol (McKenzie et al., 2003), probes were mixed, denatured for 5 min at 70°C, applied to coverslips and then mounted onto blastomeres. Slides with probe mixtures were simultaneously denatured for 5 min using a 76°C hot plate and then transferred to a humidity chamber for 6–9 h. Slides were ‘post-washed’ in 0.4 x sodium salt citrate at 72°C and counterstained with DAPI (4',6-diamidino-2-phenylindole II; Vysis, Inc.).
To re-probe a blastomere nucleus, slides were placed in 0.4 xstandard saline citrate for 5 min at 70°C to remove probes from first hybridization. Slides were then dehydrated in serially increasing concentrations of ethanol (70, 90 and 100%), and air-dried for the second round of hybridization. Probe mixture was denatured separately at 70°C for 5 min and then sealed onto slides followed by simultaneous denaturation on a 76°C hot plate for 5 min. Following a 6–8 h hybridization, slides were post-washed as described above.
Microscope analysis
FISH signals were visualized using a Zeiss Axioskop microscope equipped with multi-bandpass filters that allow simultaneous visualization of different colors. A digital imaging system (Applied Imaging, USA) with a cooled charged couple device (gray-scale) was used for image capture and data collection.
Localization of hybridization signals
Eight concentric maps with four quadrants each were created using computer graphics. Shapes and sizes of these maps reflect the various nuclear morphologies expected following the biopsy and fixation procedure. Given that all blastomere FISH experiments were image-captured, each grid map was copied onto transparency sheets that were then placed directly on the computer monitor. For each blastomere nucleus, a concentric map that best matched the nuclear border was chosen and used to map each hybridization signal. Each of the four quadrants was designated to be equally distant from each other, representing the most central area (Q1) and radiating out toward to the nuclear periphery (Q4) (Figure 1). If a signal was equally present on the grid line between two quadrants, a value of one-half signal was assigned to both quadrants. Location of each hybridization signal was recorded for both euploid (normal) and aneuploid cells.
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Statistical analysis
2-Analysis (
=0.05) was performed to test euploid (normal) versus aneuploid embryos for significant differences in the mean proportion of hybridization signals at a given localization (Q1, Q2, Q3 and Q4). This analysis was performed for each of the individual autosomes as well as for all chromosomes combined. |
Results |
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Correlation of chromosome location to chromosome number
Overall, 1009 hybridization signals were analysed in 109 blastomeres derived from 98 embryos. Among the 30 euploid embryos, 33 blastomeres were analysed and 336 hybridization signals were localized to one of the four concentric quadrants as shown in Table III. In euploid embryos, no significant difference was observed in the localization of hybridization signals for any of the seven chromosomes (
2=26.07, P0.10; df = 18) (Figure 2a). Even though some signals were locus specific (chromosomes 13, 21, 22) and others were centromeric (chromosomes 16, 18, X and Y), a few general trends were observed. Chromosomes 13, 18, 21 and X tended to be more central (quadrants Q2 and Q3) whereas chromosomes 16, 22 and Y tended to favour more peripheral locations (predominantly quadrants Q3 and Q4).
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In the 68 embryos identified as abnormal, 12 were structurally unbalanced and 56 were aneuploid. Among aneuploid blastomeres, a significant difference was observed, not only of the single aneuploid chromosome, but for all chromosomes (
2=36.81, P0.001; df = 3). When the blastomere was aneuploid, 82% of signals localized towards the peripheral region of the nucleus (44.9% in Q3; 37% in Q4; Table IV). In euploid blastomeres, 68.5% of signals were within the more peripheral region (Q3 and Q4; Table IV) (Figure 2b).
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To evaluate if localization of an individual chromosome is influenced by whether that specific chromosome is euploid or aneuploid, aneuploid blastomeres were stratified based on the chromosome(s) involved. The 56 aneuploid embryos were characterized by chromosomal nullisomies (n=6), monosomies (n=25), trisomies (n=24) and mixed (n=5). In addition
5% (n=4) of the abnormal nuclei were classified as haploid, triploid or tetraploid. Chromosome localization was found to be more peripheral in both monosomic and trisomic blastomeres, when compared to diploid blastomeres. Among monosomic blastomeres, monosomy 18, 22 and 16 were most frequent. For chromosomes 18 and 22, the monosomic signal was always peripheral, localizing to Q3 or Q4. However, for trisomic nuclei, the parental origin of the extra chromosome may be important; thus, identifying the ‘extra’ chromosome is not feasible by FISH. Despite this, the overall localization of all three signals within trisomic nuclei was also peripheral within Q3 and Q4. Figure 3 shows the proportion of signals localized in central (Q1) to peripheral (Q4) regions in euploid compared to aneuploid blastomeres, for each of the five autosomes. Localization of chromosomes 13, 18, 21 and 22 was significantly different when an abnormality for that chromosome was observed (2=18.13, P0.001, df = 3; 12.61, P0.01, df = 3; 9.46, P0.025, df = 3; 13.08, P0.01, df = 3 respectively). When aneuploid, autosomes generally have a tendency to localize close to the periphery of the nucleus (Figure 3). This was not observed for chromosome 16 (2=0.85, P1.0, df = 3).
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In cases in which two blastomeres were analysed, localization of signals was consistent in both nuclei derived from the same embryo. In three of these nine embryos, the FISH results were normal in both blastomeres and 50–65% of the FISH signals localized to the periphery. In the remaining six embryos found to be aneuploid, nearly all of the signals were peripheral.
Localization in blastomeres with structurally normal versus unbalanced chromosomes
For the 21q:21q robertsonian translocation, FISH for chromosomes 13, 18, 21, X and Y was performed on 13 blastomeres. Among 12 blastomeres classified as unbalanced, we observed no significant difference in localization of any chromosome, compared with either chromosomally normal/balanced blastomeres derived from non-homologous robertsonian and reciprocal translocation cases (n=5) or from euploid cases (n=30) (2=5.8 to 2.7, P1.0, df = 2).
Correlation of chromosome localization to embryo morphology
Two of the 17 couples had no normal embryos and were therefore excluded from analysis. For each of the remaining 15, there was no difference (P=0.818) in localization of chromosomes when comparing morphologically normal embryos (grades 4 or 5) to morphologically abnormal embryos (grades 1, 2 or 3) (Figure 4).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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This is the first study in which relative nuclear localization of chromosomes in human blastomeres from 6–8-cell stage embryos has been compared between euploid and aneuploid embryos as well as between morphologically normal and abnormal embryos. We show that localization of chromosomes differs significantly in chromosomally normal versus aneuploid blastomeres. Chromosomes present in triplicate or monosomic were preferentially localized to the periphery. Our data also suggest that among normal blastomeres individual chromosomes each appear to prefer a specific region in the nucleus. In addition, there appears to be correlation between outcomes among embryos, suggesting that PGD patients are likely to repeat the same pattern of chromosomal abnormalities (Ferraretti et al., 2004
).
The localization of chromosomes did not differ when comparing morphologically normal versus abnormal embryos. This is consistent with studies showing that morphological appearance does not necessarily predict a healthy embryo, up to 40% of embryos with normal morphology being aneuploid (Munné et al., 1993, 1994).
Our results are based on extrapolation of data from the two-dimensional (2D) blastomere preparations. The pivotal assumption is that the relative organization of the nucleus is preserved. Indeed, Croft et al. (1999) compared location of hybridization signals for HSA-18 and -19 in human fibroblasts between confocal laser scanning microscopy and traditional 2D preparations: three-dimensionally (3D) preserved cells fixed with paraformaldehyde and not subjected to hypotonic swelling proved consistent with the orientations of 2D specimens. Similarly, data collected from hypotonically treated flattened nuclei in Drosophila were consistent with data collected from 3D preserved nuclei (Csink and Henikoff, 1998).
Localization of signals to the periphery of the nucleus in presumed chromosomal aneuploidy suggests either that cells are undergoing programmed cell death (apoptosis) or that key functions (i.e. transcription of certain regulatory genes) maintaining nuclear organization may be altered. During apoptosis, chromosomal fragmentation and relocalization to membrane periphery (in preparation for packaging into apoptotic bodies) occurs prior to characteristic membrane changes. Thus, chromosomes localizing to the periphery may presage embryonic death. Conversely, genes regulating nuclear organization may be altered. As a result, inefficient or failed organizational maintenance in these nuclei can give rise to abnormal growth and/or errors in cell division leading to embryonic loss. For example, coordination of mitosis is mediated by cell cycle checkpoints under genetic control. The spindle assembly checkpoint in particular is crucial for ensuring fidelity of chromosome segregation. Several well-established cell cycle genes, such as centromeric protein Nuf2, are conserved in yeast, nematodes and humans and are critical for proper chromosome segregation (Nabetani et al., 2001). Overexpression or down-regulation of human Cdc14a phosphatase directly causes aberrant chromosome positioning in daughter cells and can lead to genomic instability (Mailand et al., 2002).
Proper orientation of the embryonic axis may be pivotal for early embryo cleavage and may affect chromosome segregation. The embryonic axis appears to be dependent on proper polarization of both chromatin and nuclear precursor bodies (Van Blerkom et al., 1995; Edwards and Beard, 1997; Payne et al., 1997). Edwards and Beard (1997) have postulated that pronuclei rotate within the ooplasm to orientate their axis towards the second polar body in order to achieve proper orientation for subsequent cleavage. Disruption of this orientation may manifest in uneven cellular division or chromosomal segregation, leading to aneuploidy in the resultant embryo (Gianaroli et al., 2003).
A caveat in our studies is that results are based on a single probe for each chromosome. Given that the observed localization of each chromosome may vary based on the chromosomal region to which the probes hybridize, further evaluation using probes that span the length of each chromosome will be necessary to assess more precisely nuclear location of each chromosome.
A further pitfall is that we evaluated blastomeres from patients having infertility. Ideally, a control group of reproductively normal patients would be studied. However, these ‘normal’ individuals would not be expected to undergo PGD, thus, such embryos would be difficult to collect. Alternatively, analysis of blastomeres from subjects undergoing PGD for single gene disorders (presumed to be reproductively normal) may prove to be more suitable as controls. Case no. 16 (X-linked chorioderemia) is such a suitable control; however, only 16 blastomeres were available for assessment. A correlative limitation is that data were generated by pooling all infertility diagnoses together. A better design would be stratification by indication. Although the limited number of patients in this study precluded such analysis, preliminary evaluation showed no significant difference between the types of indications (data not shown).
That we found perturbations in nuclear localization in embryos from women undergoing PGD for a variety of indications (Table I) supports the biological plausibility of peripheral localization indicating cellular disturbance. Of special interest is our finding of this in women with both prior trisomy and repeated IVF failure. This bears on the phenomenon of recurrent aneuploidy, which appears responsible for repetitive clinical pregnancy losses in otherwise normal women. Recurrent aneuploidy is known to extend to preimplantation embryos. In nine couples with repeated abortions, Pellicer et al. (1999) found that 58.5% of the PGD embryos were aneuploid compared to 16.7% of PGD embryos from controls with normal reproductive histories. Our data also suggest that mechanisms regulating nuclear organization are altered in infertile couples. Further investigation is warranted to elucidate the underlying mechanism associated with altered localization of chromosomes in preimplantation embryos.
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Acknowledgements |
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We thank the Baylor Assisted Reproductive Technology team (Linda Allen, R.N., Cindy Wherry, L.V.N., Daneeka Hamilton, B.S., Aimee Huynh, B.S.) and genetic counsellors (Sallie McAdoo, M.S. and Audrey Burke M.S.) for clinical management and assistance in gathering patient information.
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References |
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Submitted on April 20, 2004; accepted on June 3, 2004.
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