Human Reproduction, Vol. 17, No. 5, 1166-1170,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Multiplex interphase FISH as a screen for common aneuploidies in spontaneous abortions
1 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India, 2 Genetics Laboratory, New York Presbyterian Hospital, Columbia-Presbyterian Center, New York, USA, 3 Research Foundation for Mental Hygiene, New York State Psychiatric Institute and Graduate School of Arts and Sciences, Columbia University, USA, 4 Epidemiology of Developmental Brain Disorders Department, New York State Psychiatric Institute, Gertrude H. Sergievsky Center and Department of Epidemiology at Mailman School of Public Health, Columbia University, New York, USA and 5 Departments of Genetics and Development, and Pediatrics, Columbia University, New York, USA
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Abstract |
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BACKGROUND: A multiplex fluorescence in-situ hybridization (FISH) strategy using chromosome-specific probes for eight chromosomes as an initial screen for chromosome abnormalities in uncultured tissues from spontaneous abortions was evaluated. METHODS: Fifty-seven prefetal spontaneous abortions were studied by karyotyping cultured cells and using FISH on uncultured cells. Two probe sets were used, identifying chromosomes 13, 15, 16, 18, 21, 22, X and Y. RESULTS: Abnormalities were detected in 53% of cases by karyotyping, and 54% of cases by FISH. FISH detected an abnormality in four of five cases where cultures failed, and in two cases where maternal cells apparently overgrew the culture. FISH missed four trisomies not identifiable with the probe sets, and one trisomy because one probe set was unscorable. FISH using these probes identified 83% of all abnormalities detected by karyotyping. CONCLUSIONS: FISH can detect abnormalities in a significant proportion of cases where the culture fails to grow or is contaminated by maternal cell growth. Multiplex FISH as an initial screen, followed by culture and karyotyping in cases where no abnormality is detected, would identify a higher proportion of chromosome abnormalities in spontaneous abortion specimens than karyotype analysis alone.
Key words: aneuploidy/karyotyping/multiplex FISH/spontaneous abortion
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Introduction |
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The karyotype of a spontaneously aborted conceptus provides valuable clinical information. For example, knowledge that a loss is chromosomally abnormal may obviate the need for extensive diagnostic work-up or treatment of couples who experience recurrent spontaneous abortions. Approximately 50% of early (<15 weeks) losses are chromosomally abnormal, whereas for later (15–24 weeks) losses the frequency is approximately 20% (Warburton, 2000
). Standard cytogenetic diagnosis of spontaneous abortions requires either short- or long-term culture of fetal tissue. This labour-intensive procedure can fail due either to lack of successful cell growth in culture or to overgrowth of maternal cells in the specimen. Success rates range from 60 to 90% [the standard set by the American College of Medical Genetics (Committee on Laboratory Practices, 1999) is 75%]. Most clinical laboratories report an excess of normal female over normal male karyotypes, and a number of studies have shown that maternal cell overgrowth is not uncommon (Griffin et al., 1997; Bell et al., 1999).
Fluorescence in-situ hybridization (FISH) permits rapid determination of aneuploidy in interphase cells. Several studies have compared the results of karyotype analysis and FISH to detect aneuploidy after amniocentesis (Klinger et al., 1992; Ward et al., 1993; Bryndorf et al., 1996; Eiben et al., 1998; Jalal et al., 1998) or chorionic villus sampling (Bryndorf et al., 1997). Multiplex FISH has also been used to detect aneuploidy in preimplantation embryos or gametes (Munne et al., 1998; Verlinsky et al., 1998; Harper and Wells, 1999; Ruangvutilert et al., 2000).
For spontaneous abortions, FISH on uncultured cells might provide an efficient screen for numerical chromosomal changes, so that culture of cells would be required only for specimens where FISH failed to reveal an abnormality. This report evaluates the accuracy and yield of a multiplex FISH strategy on prefetal spontaneous abortions (i.e. losses in which the conceptus did not achieve a developmental age of 9 weeks). Based on knowledge about the frequencies of specific trisomies in spontaneous abortions and the availability of commercial multiplex probe sets, it was elected to use FISH to identify aneuploidy for chromosomes 13, 15, 16, 18, 21, 22, X and Y. This battery can detect not only many common trisomies, but also the other most frequent chromosomal anomalies in spontaneous abortions, namely, monosomy X, triploidy and tetraploidy. It can also distinguish between a normal male karyotype and maternal cell growth.
The ability of a given set of FISH probes to detect chromosomal abnormalities among spontaneous abortions depends upon the proportion and distribution of abnormal karyotypes in the sample. These characteristics vary with the distributions of maternal age and developmental age of the conceptus. To predict the expected frequency of abnormalities that could be detected with our set of probes, a recent hospital-based sample of 100 karyotyped prefetal losses ascertained for a research study was examined (Table I). The 100 karyotypes derived from 106 prefetal singleton spontaneous abortions set up in tissue culture. Six yielded no karyotype, either because of microbial infection or because the tissue failed to grow in culture. In a sample comparable with that in Table I, the two probe sets used could identify an abnormality in 54% of specimens, and detect 79% (54/68) of all abnormalities. Furthermore, FISH on uncultured cells can: (i) reveal cytogenetic abnormalities in specimens misclassified as normal female because of maternal cell growth in the culture; and (ii) provide clinically useful information for specimens where culture was unsuccessful.
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Material and methods |
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Specimen preparation
In order to compare FISH and karyotype results, tissues were used from 57 consecutive spontaneous abortions derived from: (i) the same hospital-based series of spontaneous abortions used to generate Table I
(n = 37); and (ii) specimens submitted to a clinical cytogenetic laboratory (n = 20). The 37 specimens in the FISH study were excluded from the sample described in Table I. All tissues were examined grossly under a dissecting microscope, freed of maternal decidua, and washed clean of blood. About 4–5 mg of tissue was used for FISH analysis, and about 10–20 mg for culture. Tissue for FISH was prepared in one of two ways. At the beginning of the study, the sample of villi was placed in 5 ml of hypotonic (KCl) solution for 20 min, and fixed in 3:1 methanol:acetic acid for 15 min. After replacing the original fixative with fresh fixative, the tissue was suspended in a few drops of 60% acetic acid, and the dispersed single cells were dropped onto clean slides and placed on a slide warmer at 45°C for 5–10 min. Two slides were prepared for each sample. In the latter part of the study, slides for FISH analysis were prepared directly from the collagenase-digested tissue used to set up the cultures, after hypotonic treatment and 3:1 methanol:acetic acid fixation.
Tissue for culture was prepared by digestion with trypsin and collagenase and cultured according to standard procedures in replicate cultures.
FISH analysis
FISH was performed using two sets of multicolour probe mixtures (Table II). Probe mix 1, the MultiVysion PB probe panel (Vysis, Inc., Downers Grove, IL, USA), identified chromosomes 13, 16, 18, 21 and 22. Probe mix 2 identified chromosomes 15, X and Y. In each mixture the probes were direct-labelled with different fluorophores, which could be visualized with appropriate filter combinations.
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Each probe set was applied to one of the two slides from each case. Hybridization and post-washing conditions were those recommended by the manufacturer. Slides were counterstained with 5 µl of a very dilute 4,6-diamidino-2-phenylindole (DAPI)-antifade solution and observed under a Nikon fluorescent microscope equipped with a filter wheel and cubes appropriate for SpectrumGreen, SpectrumOrange/Red, SpectrumGold, SpectrumAqua and SpectrumBlue fluorophores.
Nuclear signals were scored using a x60 oil objective. Twenty nuclei were scored for each of the eight chromosomes, and the number displaying one, two, three or four hybridized signals was recorded. In normal diploid cells, two signals were observed for each chromosome in a mean of 88% of 100 scored cells with probe mix 1, and a mean of 95% with probe mix 2. Aneuploidy was diagnosed when >20% of the cells showed an abnormal number of signals. In order to resolve ambiguities when signals were weak, fluorescent images were captured with the Cytovision (Applied Imaging, Inc., Santa Clara, CA, USA) system. The chromosome 15 probe used was subsequently found to have a 5–10% chance of cross-hybridizing with chromosome 14. In two cases, the FISH suggested a trisomy 15 and the karyotype did not. In both cases the discrepancy was confirmed as being due to cross-hybridization by FISH with D15Z1 on the metaphase chromosomes.
Karyotype analysis
Following collagenase and trypsin dissociation of the specimen, two primary cultures from each of the tissue samples were established using standard methods. Cultures were harvested after approximately 1–2 weeks. Karyotyping was performed on G-banded chromosome preparations using Cytovision (Applied Imaging) software. Initially, 10 cells were examined; if no abnormal clone was identified, 10 more cells were analysed.
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Results |
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Karyotyping was successful for 52 of 57 (91.2%) cases. Five cultures either failed to grow or were microbially contaminated. Interphase FISH was successful in all 57 cases with probe mix 2, but could not be scored accurately for two cases with probe mix 1. For the 51 specimens where both methods were informative, results were identical in 43 (84.3%). Culturing detected abnormalities in 52.6% of specimens (57.7% of all specimens successfully karyotyped), and FISH in 54.4% (Table III
). Of the 30 abnormal results detected by karyotype analysis, FISH identified 25 (83.3%). In two cases FISH confirmed the karyotype diagnoses of tetraploidy, showing that the diagnosis was not due to a cultural artefact. FISH also successfully detected one rare anomaly, complete monosomy 21, and confirmed mosaicism in one case where cells with both triploidy and 47,XX,+22 were present, suggesting a twin pregnancy.
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In the five specimens whose cultured cells failed to yield a karyotype (Table IV
, cases 1–5), FISH identified an abnormality in four (three trisomies, one monosomy X). For the fifth specimen, FISH suggested a normal male karyotype but, because the tissue was not tested for all trisomies or rearrangements, this diagnosis was not definitive.
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The initial choice of a chromosome 15 probe, D15Z1, had a significant degree of cross-hybridization with chromosome 14. Subsequently, a switch was made to D15Z4, which does not have this problem. For two cases with normal male karyotypes, the FISH was originally read as trisomy 15; FISH on metaphase chromosomes, however, showed that the third FISH signal represented cross-hybridization of the D15Z1 probe to chromosome 14. In the present analyses (and those in Table IV
), these cases were not included as examples of discrepancy between FISH and karyotype results, as the error would not have occurred using probe D15Z4. There were discrepancies between karyotype and FISH results for 11 specimens. Seven of these were clinically significant.
For five specimens (cases 6–10), FISH failed to detect a trisomy identified by karyotype analysis. Four of these cases were trisomy of a chromosome for which no probe was used. The fifth was a trisomy 16 in a case where FISH with probe set 1 was not scorable.
For two specimens (cases 11,12) karyotyped as normal female, FISH identified an abnormality (one trisomy and one monosomy X/normal mosaic). It is believed that the normal female karyotypes resulted from growth of maternal cells in the culture; that is, the FISH analysis was accurate.
The remaining four discrepancies (cases 13–16) were not clinically significant because both methods identified a similar chromosomal anomaly. In three specimens, multiple anomalies were diagnosed by karyotyping and a single anomaly by FISH. In the fourth specimen, mosaicism for 45,X and a cell line with an additional small marker chromosome was scored by FISH as mosaic X/XX; the marker chromosome presumably derived from an X chromosome.
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Discussion |
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The aim of this study was to determine the accuracy and efficiency of FISH as a preliminary screen for chromosomal abnormalities among spontaneous abortions. An analysis of the results suggests that the approach would prove clinically useful. For patient management, the most important information is whether a karyotype is normal or abnormal—an abnormal result precludes any need for further diagnostic work-up or treatment, and has a better prognosis for future pregnancies (Strobino et al., 1986
; Ogasawara et al., 2000).
From Table I, it was predicted that FISH using a probe set for chromosomes 13, 15, 16, 18, 21, 22, X and Y would identify 54% of the sample as abnormal, and would detect 79% of chromosome abnormalities. In the present study, FISH identified 54.4% (31/57) of the sample as abnormal and detected 83% of the abnormalities detected by karyotype analysis—rates which were very similar to those predicted. In the 50 cases where karyotyping and both FISH probe sets were informative, FISH failed to identify an abnormality in four cases and karyotyping in two. The inaccuracies of the FISH diagnoses were as expected, namely that FISH missed trisomies of chromosomes for which enumeration probes were not used. Inaccuracies of karyotyping were apparently due to maternal cell growth. Others (Lomax et al., 2000) found a similar rate of maternal cell contamination in their sample.
FISH identified a significant number of abnormal specimens not found by standard culture methods. Notably, FISH provided a diagnosis of chromosomal anomaly in four of five cases where the culture produced no result and in two specimens that karyotype analysis identified as normal female. FISH for chromosomes 13, 15, 16, 18, 21, 22, X and Y should identify about 80% of the chromosomal anomalies commonly found in spontaneous abortion specimens. The procedure is rapid, does not require large amounts of fetal tissue, and avoids the cost of feeding, monitoring and harvesting cell cultures and analysing the karyotype microscopically. Because FISH allows analysis of large numbers of interphase nuclei, it is also an efficient method of identifying mosaicism.
Recently, comparative genomic hybridization (CGH), either alone or in combination with flow cytometry, has provided an alternative approach for the detection of chromosomal abnormalities in spontaneous abortions (Daniely et al., 1998; Lomax et al., 2000). CGH detects aneuploidy of all chromosomes, including partial monosomies and trisomies. It does not, however, reliably detect changes in ploidy (since signal ratios are normalized over the whole chromosome complement), nor does it detect low-grade mosaicism or balanced structural anomalies. Since changes in ploidy and mosaicism are common among spontaneous abortions, all normal CGH results must be followed by flow cytometry or standard cytogenetic diagnosis. CGH appears to be more expensive and labour-intensive than the FISH procedure outlined here, and also requires specialized software that is not available in all laboratories. However, a cost–benefit comparison of FISH and CGH has not been carried out.
Based on results obtained in the present study, it is suggested that multiplex FISH on uncultured cells using probes for chromosomes 13, 15, 16, 18, 21, 22, X and Y would provide a useful strategy for screening spontaneous abortion specimens. Cultures could then be processed for karyotyping only when FISH detects no abnormality. Given this procedure, a cytogenetic abnormality would have been detected in 36 (31 by FISH alone plus five by karyotype) of 57 specimens (63%), which is a substantial increase over the 53% (30/57) detected from analysis of cultured tissue alone.
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Acknowledgements |
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We thank Dr Grace Jorgensen and her colleagues for their help in providing access to their patients. These studies were supported by a grant from the National Institutes on Aging (R01 AG 15386). V.J. is a Senior Research Fellow of the Council of Scientific and Industrial Research, India.
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Notes |
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6 To whom correspondence should be addressed. E-mail: cuh{at}cancercenter.columbia.edu
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References |
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Submitted on April 24, 2001; resubmitted on November 12, 2001; accepted on January 9, 2002.
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