Detection of aneuploidy in spontaneous abortions using the comparative hybridization method

Comparative genomic hybridization (CGH) technique was used to examine a set of ten spontaneous abortions whose cell cultures were characterized by the lack of proliferation in vitro, and thereby, were not available for the analysis by means of routine cytogenetic methods. Five abortions (50%) had aneuploidy of autosomes, including trisomy 10, 14, 18, and 21, and monosomy 22. The latter variant of unbalanced chromosomal abnormalities is rarely detected in spontaneous abortions by use of conventional cytogenetic methods. The results were validated by using fluorescent in situ hybridization (FISH) analysis with centromere-specific DNA probes. Embryos with trisomy 10 and monosomy 22 displayed mosaicism with the frequencies of abnormal cell clones constituting 68 and 33% respectively. The advantages and limitations of the applying of CGH technique for detection of genomic abnormalities in both nonmosaic and mosaic forms are discussed.     

Spectrum of Cytogenomic Abnormalities Revealed by Array Comparative Genomic Hybridization on Products of Conception Culture Failure and Normal Karyotype Samples

Approximately 30% of pregnancies after implantation end up in spontaneous abortions, and 50% of them are caused by chromosomal abnormalities. However, the spectrum of genomic copy number variants(CNVs) in products of conception(POC) and the underlying genedosage-sensitive mechanisms causing spontaneous abortions remain largely unknown. In this study, array comparative genomic hybridization(a CGH) analysis was performed as a salvage procedure for 128 POC culture failure(POC-CF) samples and as a supplemental procedure for106 POC normal karyotype(POC-NK) samples. Chromosomal abnormalities were detected in 10% of POC-CF and pathogenic CNVs were detected in 3.9% of POC-CF and 5.7% of POC-NK samples. Compiled results from this study and relevant case series through a literature review demonstrated an abnormality detection rate(ADR) of 35% for chromosomal abnormalities in POC-CF samples, 3.7% for pathogenic CNVs in POC-CF samples, and 4.6% for pathogenic CNVs in POC-NK samples. Ingenuity Pathway Analysis(IPA) was performed on the genes from pathogenic CNVs found in POC samples. The denoted primary gene networks suggested that apoptosis and cell proliferation pathways are involved in miscarriage. In summary, a similar spectrum of cytogenomic abnormalities was observed in POC culture success and POC-CF samples. A threshold effect correlating the number of dosage-sensitive genes in a chromosome with the observed frequency of autosomal trisomy is proposed. A rationalized approach using firstly fluorescence in situ hybridization(FISH) testing with probes of chromosomes X/Y/18, 13/21, and 15/16/22 for common aneuploidies and polyploidies and secondly a CGH for other cytogenomic abnormalities is recommended for POC-CF samples.     

Comparative genomic hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions

More than 50% of spontaneous abortions (SAs) have abnormal chromosomes; the most common abnormalities are trisomy, sex chromosome monosomy, and polyploidy. Conventional cytogenetic analysis of SAs depends on tissue culturing and is associated with a significant tissue culture failure rate and contamination by maternally derived cells. Comparative genomic hybridization (CGH), in combination with flow cytometry (FCM), can detect numerical and unbalanced structural chromosomal abnormalities associated with SAs while avoiding the technical problems associated with tissue culture. Routine cytogenetic and CGH analysis was performed independently on tissue from 301 SAs. Samples shown to be chromosomally balanced by CGH were analyzed by FCM to determine ploidy. Of 253 samples successfully analyzed by both approaches, there was an absolute correlation of results in 235 (92.8%). Of the 18 cases with discrepancies between cytogenetic and CGH/FCM results, an explanation could be found in 17. Twelve samples produced a 46,XX karyotype by cytogenetics, whereas CGH/FCM demonstrated aneuploidy/polyploidy or a male genome, indicating maternal contamination of the tissue cultures. In two cases, where tetraploidy was demonstrated by cytogenetics and diploidy by FCM, tissue culture artifact is implied. In three cases, CGH demonstrated an aneuploidy, and cytogenetics demonstrated hypertriploidy. In one unexplainable case, aneuploidy demonstrated by CGH could not be detected by repeat CGH analysis, conventional cytogenetic, or FISH analysis. These results demonstrate that CGH supplemented with FCM can readily identify chromosomal abnormalities associated with SAs and, by avoiding maternal contamination and tissue culture artifacts, can do so with a lower failure rate and more accuracy than conventional cytogenetic analysis.     

Comparative genomic hybridization in clinical cytogenetics.

We report the results of applying comparative genomic hybridization (CGH) in a cytogenetic service laboratory for (1) determination of the origin of extra and missing chromosomal material in intricate cases of unbalanced aberrations and (2) detection of common prenatal numerical chromosome aberrations. A total of 11 fetal samples were analyzed. Seven cases of complex unbalanced aberrations that could not be identified reliably by conventional cytogenetics were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome-specific probes. Four cases representing common prenatal numerical aberrations (trisomy 21, 18, and 13 and monosomy X) were also successfully diagnosed by CGH. We conclude that CGH is a powerful adjunct to traditional cytogenetic techniques that makes it possible to solve clinical cases of intricate unbalanced aberrations in a single hybridization. CGH may also be a useful adjunct to screen for euchromatic involvement in marker chromosomes. Further technical development may render CGH applicable for routine aberration screening.     

Molecular Cytogenetic Characteristics of Chromosome Imbalance in Spontaneous Human Abortion Cells with Low Proliferative Activity in Vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ovum and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ovum, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously aborted embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1–2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Molecular cytogenetic characteristics of chromosome imbalance in cells of spontaneous human abortion fetuses with low proliferative activity in vitro

Karyotyping of noncultivated cells of 60 first-trimester spontaneous abortions (blighted ova and missed abortions) was carried out using fluorescence in situ hybridization (FISH) with centromere-specific DNA probes for all chromosomes of the karyotype. Conventional cytogenetic study of these abortions was impossible because of cell culture failures. The algorithm is proposed for molecular cytogenetic FISH analysis of interphase karyotypes. Chromosome abnormalities were found in 32 fetuses (53.3%). In groups of missed abortions and blighted ova, the frequency of numerical chromosome abnormalities was 50 and 60%, respectively. Both the numerical chromosome abnormalities typical of spontaneous human abortions (autosomal trisomies, sex chromosome aneuploidy, and polyploidy) and a relatively rare type of genomic imbalance unidentifiable by standard cytogenetic analysis (autosomal monosomies 7, 15, 21, and 22 in mosaic state) were observed. The frequency of these type of chromosome abnormalities comprised 19% of all known karyotype abnormalities determined in spontaneously perished embryos. Note that the level of confined placental mosaicism in embryos with low cell proliferative activity was 25%, which is substantially higher than the corresponding parameter (1-2%) determined by prenatal diagnosis of chromosome abnormalities in developing embryos. The results of interphase FISH analysis of cells with low proliferative activity in vitro suggest that the pathology of early fetal development and missed abortion in humans are associated with a wider spectrum of chromosome abnormalities.     

Chromosomal disorders in women with spontaneous abortions

The results of examination of 120 women with spontaneous abortions are presented. The cytogenetic analysis showed that in karyotypes of the patients there are chromosome aberrations of different types: translocations (17.5%), monosomy (13.3%), trisomy (10%), mosaicism (5.8%) and other forms (4.1%). Phenotypic manifestation of chromosome anomalies with spontaneous miscarriages (abortions) is discussed in the work.     

Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages

Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations.     

Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage

Altogether, 750 cases of spontaneous abortion between the fifth and 25th week of gestation were analyzed cytogenetically by the direct-preparation method using chorionic villi. The majority of cases (68%) were derived from early abortions before the 12th week of gestation. The frequency of abnormal karyotypes was 50.1%; trisomy was predominant (62.1%), followed by triploidy (12.4%), monosomy X (10.5%), tetraploidy (9.2%), and structural chromosome anomalies (4.7%). Among trisomies, chromosomes 16 (21.8%), 22 (17.9%), and 21 (10.0%) were prevalent. The frequency of chromosomally abnormal abortions increased with maternal age but only because of an increase of trisomy. Polyploidy and monosomy X, however, decreased. Mean maternal age was significantly increased for trisomies 16, 21, and 22 and was highest for trisomies 18 and 20. The results obtained are within the range of variability reported earlier from tissue culture-type studies. A consistent feature during our study is the excess of females in chromosomally normal abortions (male:female sex ratio 0.71). According to the methodology applied, maternal cell contamination and undetected 46,XX molar samples cannot have influenced the sex ratio. However, a bias introduced by social status or maternal age cannot be excluded. With the more rapid and convenient direct preparation of chorionic villi, reliable cytogenetic data on causes of spontaneous abortions can be obtained.     

Double trisomy in spontaneous abortions

Cytogenetic data on products of conception from spontaneous abortions studied over a 10-year period have been reviewed for double trisomies. A total of 3034 spontaneous abortions were karyotyped between 1986 and 1997. Twenty-two cases with double trisomy, one case with triple trisomy, and a case with a trisomy and monosomy were found. The tissues studied were mostly sac, villi, or placenta. The gestational age ranged from 6 to 11 weeks and the mean age was 8.2 ± 1.7 (SD) weeks. The mean maternal age in years was 35.9 ± 5.3. Of the twenty-two cases, four were mosaics. All but two of the cases involved autosomal aneuploidies. The double trisomies included chromosomes 2, 4, 5, 7, 8, 12, 13, 14, 15, 16, 17, 18, 20, 21, and 22. The chromosomes that were trisomic in more than one double trisomy case were numbers 16 (8 cases), 8 (5 cases), 15 (4 cases), 2, 13, and 21 (3 cases each), and 5, 7, 14, 18, 20, 22, and X (2 cases). The triple trisomy involved chromosomes 18, 21, and X. The monosomy and trisomy case was a mosaic, with a monosomy 21 in all cells and some cells also with a trisomy 5. The double trisomies cited for the first time in this study were 4/13, 5/16, 8/14, 8/15, 14/21, 15/20, and 7/12. The pooled mean maternal age for double trisomy cases (34.1 ± 5.7 years) was higher than that for single trisomy cases (31 ± 6.1 years). The difference was statistically significant at P = < 0.001. The pooled mean gestational age of spontaneous abortions was lower for double trisomy (8.7 ± 2.2 weeks) than for reported single trisomy cases (10.1 ± 2.9 weeks). This difference is also statistically significant at P = < 0.001. The sex ratio among double trisomies was 15 females to 13 males. This difference was not statistically significant from the expected 1 : 1. Received: 27 June 1997 / Accepted: 4 September 1997     

Chromosomal mosaicism in spontaneous abortions: Analysis of 650 cases

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase MFISH and DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy. Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (∼ 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.     

Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.     

Reciprocal syndromes caused by deficiency duplication resulting from maternal t(10;18)(p12;q22) translocation

The detection of a familial translocation, t(10;18)(p12;q22), has made possible the observation in type and countertype of two related persons with opposite chromosomal imbalance: trisomy 18q22----18qter with monosomy 10p12----10pter in one of the two and monosomy 18q22----10pter in the other. In each case the abnormalities attributable to monosomy overrule those attributable to monosomy overrule those attributable to the associated trisomy.     

Chimerism and multiple numerical chromosome imbalances in a spontaneously aborted fetus

We report on a case of chimerism and multiple abnormalities of chromosomes 21, Xand Yin spontaneous abortion specimen. To the best our knowledge the present case is the first documented chimera in a spontaneously aborted fetus. The application of interphase fluorescence in situ hybridization (FISH) using chromosome enumeration and site-specific DNA probes showed trisomy X in 92 nuclei (23 %), tetrasomy X in 100 nuclei (25 %), pentasomy of chromosome X in 40 nuclei (10 %), XXY in 36 nuclei (9 %), XXXXXXYY in 12 nuclei (3 %), XXXXXYYYYY in 8 nuclei (2 %), trisomy 21 and female chromosome complement in 40 nuclei (10 %), normal female chromosome complement in 72 nuclei (18 %) out of 400 nuclei scored. Our experience indicates that the frequency of chimerism coupled with multiple chromosome abnormalities should be no less than 1 : 400 among spontaneous abortions. The difficulties of chimerism identification in fetal tissues are discussed.     

Uses and limitations of two molecular cytogenetic techniques for the study of arrested embryos obtained through assisted reproduction technology

We studied chromosomal abnormalities in arrested embryos produced by assisted reproductive technology with fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in order to determine the best technique for evaluating chromosomal aneusomies to be implemented in different situations. We examined individual blastomeres from arrested embryos by FISH and arrested whole embryos by CGH. All of the 10 FISH-analyzed embryos gave results, while only 7 of the 30 embryos analyzed by CGH were usable. Fifteen of the 17 embryos were chromosomally abnormal. CGH provided more accurate data for arrested embryos; however, FISH is the technique of choice for screening in preimplantation genetic diagnosis, because the results can be obtained within a day, while the embryos are still in culture.     

Cytogenetic analysis from DNA by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.     

Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human MII oocytes and first polar bodies

Single cell comparative genomic hybridization (CGH) was employed to extensively investigate 24 unfertilized or in vitromatured meiosis II oocytes and their corresponding first polar bodies (PBs), to determine how and whether all 23 chromosomes participate in female meiosis I errors and to accurately estimate the aneuploidy rate in the examined cells. Results were obtained for 15 oocytes and 16 PBs, representing 23 eggs (MII oocyte-PB complexes) donated from 15 patients (average age 32.2 years). Abnormalities were detected in ten eggs, giving an overall aneuploidy rate of 43.5%. In all, fourteen anomalies were scored, with the fertilized oocyte being at risk of monosomy in eight cases and at risk of trisomy in six; chromosomes of various sizes participated. CGH was able to give a comprehensive aneuploidy rate, as both absence of chromosomal material and the presence of extra copies were accurately scored. The aneuploidy mechanisms determined were: classical whole univalent non-disjunction; chromatid predivision prior to anaphase I, leading to metaphase II imbalance. There was also evidence of germinal mosaicism for a trisomic cell line. Three patients appeared to be predisposed to meiosis I errors, based on the presence of either multiple abnormalities in one or more of their examined cells, or of the same type of abnormality in all of their cells. Exclusion of these susceptible patients reduces the aneuploidy rate to 20%. Various hypotheses are put forward to explain these observations in order to stimulate research into the complex nature of female meiotic regulation.     

Q-banding of chromosomes in human spontaneous abortions

Chromosome studies of 242 spontaneous abortions were carried out by Q-banding technique. The abortuses were selected for study because they were phenotypically abnormal, had not progressed beyond 12 weeks development or were from women with repeated abortions. Chromosome anomalies were found in 126 (52%) of the abortuses. Of these, 71 (56%) were trisomies. Trisomies were found for all the autosomes except Nos. 1, 3, 5, 11, 17 and 18. Triploidy was the second commonest anomaly in this series, making up 26 (21%) of the total anomalies. About 70% of these had an XXY sex chromosome complement. Only 16 (13%) of the abortuses had X monosomy, a lower frequency than would be expected in an unselected study. Tetraploidy was found in 8 abortuses and the 5 remaining specimens had various anomalies. These included 3 translocations, a trisomy 21,X monosomy and a ring chromosome 13. Except for the greater frequency of XXY than XXX sex chromosomes in the triploids, there was no evidence of a distortion of the sex ratio, either among the trisomic or among the chromosomally normal abortuses.     

Detection of structural and numerical chromosome abnormalities in interphase cells using spectral imaging.

Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.