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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Key words: aneuploidy/karyotyping/multiplex FISH/spontaneous abortion
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Introduction |
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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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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 510 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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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 510% 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 12 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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There were discrepancies between karyotype and FISH results for 11 specimens. Seven of these were clinically significant.
For five specimens (cases 610), 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 1316) 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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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 analysisrates 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 costbenefit 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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Notes |
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References |
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Bryndorf, T., Christensen, B., Vad, M., Parner, J., Carelli, M.P., Ward, B.E., Klinger, K.W., Bang, J. and Philip, J. (1996) Prenatal detection of chromosome aneuploidies in uncultured chorionic villus samples by FISH. Am. J. Hum. Genet., 59, 918926.[ISI][Medline]
Bryndorf, T., Christensen, B., Vad, M., Parner, J., Brocks, V. and Philip, J. (1997) Prenatal detection of chromosome aneuploidies by fluorescence in situ hybridization: experience with 2000 uncultured amniotic fluid samples in a prospective pre-clinical trial. Prenat. Diagn., 17, 333341.[ISI][Medline]
Committee on Laboratory Practices (1999) Standards and guidelines for clinical genetics laboratories. 2nd edn. The American College of Medical Genetics, Bethesda, MD.
Daniely, M., Aviram-Golding, A., Barakai, G. and Goldman, B. (1998) Detection of chromosomal aberrations in fetuses arising from recurrent spontaneous abortion by comparative genomic hybridization. Hum. Reprod., 13, 805809.[Abstract]
Eiben, B., Trawicki, W., Hammans, W., Goebel, R. and Epplen, J.T. (1998) A prospective comparative study on fluorescent in situ hybridization (FISH) of uncultured amniocytes and standard karyotype analysis. Prenat. Diagn., 18, 901906.[ISI][Medline]
Griffin, J.K., Millie, E.A., Redline, R.W., Hassold, T.J. and Zaragoza, M.V. (1997) Cytogenetic analysis of spontaneous abortions: comparison of techniques and assessment of the incidence of confined placental mosaicism. Am. J. Med. Genet., 72, 297301.[ISI][Medline]
Harper, C.J. and Wells, D. (1999) Recent advances and future developments in PGD. Prenat. Diagn., 19, 11931199.[ISI][Medline]
Jalal, S.M., Law, M.R., Carlson, R.O. and Dewald, G.W. (1998) Prenatal detection of aneuploidy by directly labeled multicolored probes and interphase fluorescence in situ hybridization. Mayo Clin. Proc., 73, 132137.[ISI][Medline]
Klinger, K., Landes, G., Shook, D., Harvey, R., Lopez, L., Locke, P., Lerner, T., Osathanondh, R., Leverone, B., Houseal, T. et al. (1992) Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). Am. J. Hum. Genet., 51, 5565.[ISI][Medline]
Lomax, B., Tang, S., Separovic, E. Phillips, D., Hillard, E., Thomson, T. and Kalousek, D.K. (2000) Comparative Genomic Hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions. Am. J. Hum. Genet., 66, 15161521.[ISI][Medline]
Munné, S., Magli, C., Bahce, M., Fung, J., Legator, M., Morrison, L., Cohert, J. and Gianaroli, L. (1998) Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and livebirths: XY, 13, 14, 15, 16, 18, 21, 22. Prenat. Diagn., 18, 14591466.[ISI][Medline]
Ogasawara, M., Aoki, K., Okada, S. and Suzumori, K. (2000) Embryonic karyotype of abortuses in relation to the number of previous miscarriages. Fertil. Steril., 73, 300304.[ISI][Medline]
Ruangvutilert, P., Delhanty, J.D., Rodeck, C.H. and Harper, J.C. (2000). Relative efficiency of FISH on metaphase and interphase nuclei from non-mosaic trisomic or triploid fibroblast cultures. Prenat. Diagn., 20, 159162.[ISI][Medline]
Strobino, B., Fox, H.E., Kline, J., Stein, Z., Susser, M. and Warburton, D. (1986) Characteristics of women with recurrent spontaneous abortions and women with favorable reproductive histories. Am. J. Public Health, 76, 986991.[Abstract]
Verlinsky, Y., Cieslak, J. and Ivakhnenko, V. (1998) Preimplantation diagnosis of common aneuploidies by the first- and second-polar body FISH analysis. J. Assist. Reprod. Genet., 15, 285289.[ISI][Medline]
Warburton, D. (2000) Cytogenetics of reproductive wastage: from conception to birth. In Mark, H.F.L. (ed.), Medical Cytogenetics. Marcel Dekker, New York, pp. 213246.
Ward, B.E., Gersen, S.L., Carelli, M.P., McGuire, N.M., Dackowski, W.R., Weinstein, M., Sandlin, C., Warren, R. and Klinger, K.W. (1993) Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4500 specimens. Am. J. Hum. Genet., 52, 854865.[ISI][Medline]
Submitted on April 24, 2001; resubmitted on November 12, 2001; accepted on January 9, 2002.