Evidence for High Frequency of Chromosomal Mosaicism in Spontaneous Abortions Revealed by Interphase FISH Analysis
Institute of Pediatrics and Children's Surgery, Russian Ministry of Health, Moscow, Russia (SGV,ADK); National Center of Mental Health, Russian Academy of Medical Sciences, Moscow, Russia (IYI,VVM,IVS,YBY); and Moscow Medical I.M. Sechenov's Academy, Moscow, Russia (EAK)
Correspondence to: Y.B. Yurov, National Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe sh.2, 119152, Moscow, Russia. E-mail: y_yurov{at}yahoo.com; i_yurov{at}mail.ru
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Summary |
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Key Words: mFISH spontaneous abortions chromosomal abnormality chromosomal mosaicism
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Introduction |
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Approximately 15% of all clinically recognized pregnancies are spontaneously aborted before 20 weeks of gestation. Chromosomal abnormalities are recognized as one of the main causes of spontaneous abortions. Previous studies have estimated the frequency of chromosomal abnormality in spontaneous abortions as 5070% (Boue et al. 1975; Ohno et al. 1991
; Jobanputra et al. 2002
; Stephenson et al. 2002
). For such studies, one of the most suitable molecular cytogenetic techniques is mFISH. The most common cause of spontaneous abortions is de novo numerical chromosome abnormalities, such as trisomy of autosomes 13, 14, 15, 16, 21, and 22; sex chromosome aneuploidy, and polyploidy (Jobanputra et al. 2002
; Stephenson et al. 2002
). Regular forms of chromosomal abnormalities in human fetuses are the result of meiotic errors leading to the appearance of chromosomally abnormal gametes. In contrast, mosaic forms are the result of postmeiotic errors. The frequency of chromosome mosaicism in spontaneous abortions during the prenatal period has not been evaluated, probably because of the absence of criteria for defining a sample as a mosaic. It was demonstrated that postmeiotic errors could lead to a high incidence of mosaic forms of chromosomal abnormalities (aneuploidy, haploidy, and polyploidy) in human preimplantation embryos in vitro (Bielanska et al. 2002
). Application of FISH with DNA probes to chromosomes X, Y, 2, 7, 13, 16, 18, 21, and 22 have indicated that
50% of human preimplantation embryos are chromosomal mosaics. If chromosomal mosaicism occurring in the preimplantation stage does not affect implantation, embryos with mild aneuploidy will be candidates for fetal or confined placental mosaicism (Bielanska et al. 2002
). However, the incidence of chromosomal mosaicism in spontaneous abortions has not previously been investigated by molecular cytogenetic methods (FISH). This problem is further complicated in the case of low-level chromosomal mosaicism occurring in spontaneous abortions. Therefore, it seems to be of great interest to carry out mFISH analysis in interphase nuclei of spontaneous abortions using a set of DNA probes containing the probes for the chromosomes mentioned above. In the present study, we have applied a set of original DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y and mFISH to assess the efficiency of the technique for the investigation of the chromosome complement in spontaneous abortions. Special attention was paid to assessment of chromosome mosaicism occurrence.
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Materials and Methods |
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mFISH
The set of original chromosome-specific DNA probes from the collection developed at the Cytogenetics Laboratory, National Center of Mental Health, Moscow, Russia was used in mFISH studies. DNA probes specific to chromosomes 1, 9, 13, and 21; 14 and 22; 15, 16, 18, X, and Y were selected for the study. Technical characteristics of DNA probes and their processing for FISH have been described previously in detail (Vorsanova et al. 1986; Yurov et al. 1987
,1996
,2002
; Soloviev et al. 1995
,1998
).
Labeled DNA probes were combined in the following order: (a) chromosome Yspecific probe (labeled by Cy3), chromosome Xspecific probe (labeled by fluorescein-FluorX), chromosome 1specific probe (labeled by biotin or AMCA); (b) chromosome 9specific probe (labeled by biotin) and chromosomes 13/21-specific probe (labeled by Cy3); (c) chromosome 16specific probe (labeled by biotin) and chromosomes 14/22specific probe (labeled by Cy3); and (d) chromosome 15specific probe (labeled by biotin) and chromosome 18specific probe (labeled by Cy3).
FISH studies were performed as described in detail previously (Soloviev et al. 1994,1995
; Yurov et al. 1996
). For dual- and three-color hybridization, DNA probes were mixed in equal proportions (3.55 µl each probe at a concentration of 5 ng/µl for each probe). Hybridization was usually performed at 42C for 4 hr or overnight. The slides were postwashed in 50% formamide, 2 x SSC at 45C three times for 2 min and rinsed in 0.12 x SSC for 5 min. Detection of biotin-labeled probes was performed by the use of one layer of fluorescein isothiocyanate (FITC)-avidin or AMCA-avidin (Vector Laboraties; Burlingame, CA). Slides were mounted in antifade solution (0.2% p-phenylenediamine in 80% glycerol, 20 mM TRIS-HCl, pH 8.0), and 50100 ng/ml DAPI (4',6-diamidino-2-phenylindole-2HCl).
Microscopy
For epifluorescence microscopy, a Leitz Orthoplan microscope equipped with a 100 W lamp was used with the following filter sets (Leica Mikroskopie und Systeme; Wetzlar, Germany): A (No. 513,596) for DAPI fluorescence; I3 (No. 513,719) or GR (No. 513,821) for FITC signals; GR (No. 513,821) for both fluorescein and cyanine signals; N2 (No. 513,609) for Cy3 signals. All images were observed with the Plan-Neofluotar 63x or 100x oil immersion objectives.
Analysis of FISH Signals
From 300 to 600 nuclei were scored for each sample for each probe. Only intact and undamaged nuclei free of cytoplasm were analyzed. Nuclei with low signal intensities, diffuse signals, or absence of signals on both homolog chromosomes were considered to be hybridization failures and were not scored. Two small focal (or paired) signals of the same color and the same intensity, separated by a distance of less than the area of one signal, were considered to be a split signal from one chromosome. Interphase nuclei with one large signal of the same color and increased intensity of fluorescence with the absence of a second hybridization signal in an interphase nucleus were considered to be overlapping (or over-position) of two signals and were not scored.
The samples were considered normal (disomic) if more than 95% of all nuclei demonstrated two signals for autosomes and the XX or XY signal pattern for sex chromosomes. Informative abnormal samples with regular forms of chromosomal imbalances were defined as those in which more than 95% of the nuclei demonstrated definite abnormal patterns of the signals (monosomy, trisomy, tetrasomy, XXY signal pattern, polyploidy). Informative mosaic samples were defined as those in which more than 5% of the nuclei had a reproducible abnormal pattern of signals (no less than 1530 abnormal cells per sample with scoring of 300600 nuclei) different from normal (disomic) autosomal or normal sex chromosomal signals (XX and XY).
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Results |
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Discussion |
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A significant challenge in studies of chromosomal mosaicism in material of spontaneous abortions using standard cytogenetic and molecular cytogenetic techniques is the possibility of maternal cell contamination. In fetuses with female chromosome complement, the presence of maternal cells cannot be ruled out. This fact probably explains the prevalence of female fetuses over male fetuses seen during karyotype analysis of spontaneous abortion specimens. Careful sampling of villous tissue can help somewhat in solving this problem. For example, in our study of 150 spontaneous abortion specimens, two had both male and female cells, probably indicating the presence of maternal cell contamination. These samples were excluded from analysis. The male-to-female ratio was 0.78 (65 samples with male and 83 with female chromosome complements), indicating the prevalence of fetuses with female chromosomal complement. The female fetus predominance could be explained by both maternal cell contamination and the increased rate of male fetus loss resulting from hemizygocity for lethal X-linked mutations known to have a high frequency. Many clinical laboratories report an excess of normal female over normal male karyotypes, and some studies show that maternal cell overgrow is not uncommon (Griffin et al. 1997; Bell et al. 1999
). Therefore, contamination by maternal cells during processing of samples for cytogenetic or molecular cytogenetic (FISH) analysis should not significantly affect the results. However, in cases of suspected maternal cell contamination, a "DNA fingerprinting" assay can avoid inaccuracy of cytogenetic analysis (Bell et al. 1999
). Interestingly, in our study, the frequencies of mosaic forms of chromosomal abnormalities in samples with male and female chromosomal complements were practically identical50% (16/32 chromosomally abnormal) for male fetuses and 47.4% (27/57 chromosomally abnormal) for female fetuses. Therefore, there is no prevalence of mosaic forms in female fetuses, and the high incidence of chromosomal mosaicism detected in our representative study of 148 spontaneous abortions is unlikely to be explained by maternal cell contamination.
To our knowledge, the criteria for defining a sample as mosaic using interphase FISH analysis of spontaneous abortion specimens has not been reported. In recent publications in this field using FISH, informative mosaic samples were defined as those in which more than 20% of nuclei had a variation in signal number from the majority or showed a signal pattern other than the normal disomic autosomal or normal sex chromosomal (XX and XY) signals (Jobanputra et al. 2002). It must be noted that usually 50 nuclei or 20 metaphase spreads scored for each probe after FISH. In our study, samples were considered normal (disomic) if more than 95% of all nuclei demonstrated two signals for autosomes and the XX or XY signal pattern for sex chromosomes. To increase the sensitivity and accuracy of FISH analysis, we scored large cell populations for each spontaneous abortion specimen suspected of having chromosomal mosaicism (more than 1530 chromosomally abnormal cells per sample with scoring of 300600 nuclei). The low level (5%) of the starting value for scoring of mosaicism and the scoring of large cell populations as well as application of chromosome-enumerating probes for many chromosomes in mFISH studies probably explain the high incidence of mosaicim in spontaneous abortions. It is clear that classical cytogenetic studies based on analysis of 2050 metaphase spreads are not very effective for assessing low-level mosaicism. Interphase FISH studies allow scoring of a large cell population with high accuracy. Different criteria for defining samples as mosaic may explain the differences in the frequency of detecting mosaicism in the present study as compared with previously published results. However, it is necessary to note that this problem in relation to frequencies of mosaicism in spontaneous abortion has not yet been investigated.
It will be very useful to perform studies of chromosomal variations, including chromosomal mosaicim, in a control population, for example, in material of induced abortions, such as that performed by Horiuchi et al. (1997). In this work, the authors demonstrated that the number of chromosomally abnormal cells in cases of spontaneous abortions with mosaicism is significantly higher than that in induced or spontaneous abortion specimens without abnormal cell populations. However, the use of "internal" controls in large-scale studies, as performed in our study of 148 spontaneous abortions, seems to be adequate. The highly reproducible and regular appearance of chromosomal aberrations in samples with confirmed chromosomal abnormalities, as well as the presence of chromosomally normal samples (serving as "internal" controls) detected in our study, indicates that the mosaicism seen in spontaneous abortions is not likely to be explained as a FISH artifact.
The origin of chromosomal mosaicism in spontaneous abortion could be explained by meiotic as well as mitotic errors. The meiotic origin of mosaic aneuploidy has been proposed to explain confined placental mosaicism resulting from a correction of a trisomic conception by reduction to disomy. The previously accepted frequency range of both confined and generalized types of mosaicism is estimated at 510% and higher (Kalousek et al. 1992; Wolstenholme, 1996
). Postfertilization somatic errors occurring in the first trimester of pregnancy are an alternative mechanism. Postzygotic mitotic nondisjunction causing mosaic forms of aneuploidy usually occur in a small proportion (12%) of live-born children with chromosomal syndromes. The high incidence of chromosomal mosaicism throughout human preimplantation development in vitro was demonstrated by mFISH with an overall frequency of
50% (Bielanska et al. 2002
). It is possible to propose that postzygotic errors leading to chromosomal mosaicism could persist throughout preimplantation development in vivo. In the present study, the frequency of chromosomal mosaicism in spontaneous abortions of 317 weeks gestation and abnormal chromosome complement is 44.8%. These data indicate the fetal mosaicism due to postzygotic mitotic errors to be rather frequent. Chromosomal mosaicism and regular forms of aneuploidy involving different human chromosomes may contribute equally to a higher rate of spontaneous fetal loss. Regular and probably mosaic forms of aneuploidy are the result of meiotic errors, leading to the appearance of chromosomally abnormal gametes. Additionally, mosaic forms are known to occur due to postmeiotic errors after fertilization. Therefore, one can propose the existence of genetic, epigenetic, and environmental factors that are currently unknown and that are likely to selectively disturb chromosome segregation during mitotic division in somatic cells during human early fetal development.
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Acknowledgments |
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Footnotes |
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Received for publication May 26, 2004; accepted September 2, 2004
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Literature Cited |
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