Cytogenetic study of spontaneous abortions using semi-direct analysis of chorionic villi samples detects the broadest spectrum of chromosome abnormalities

Conventional tissue culturing and karyotyping of spontaneous abortions has limitations such as culture failure, external contamination and selective growth of maternal cells. Molecular cytogenetic techniques such as FISH, QF-PCR, and CGH allow diagnosis on uncultured cells but are also limited as to the spectrum of cytogenetic abnormalities detected. We describe the cytogenetic findings in a series of 116 first trimester arrested pregnancies, obtained through chorionic villi sampling (CVS) and semi-direct analysis that avoids some of the long-culture pitfalls such as maternal contamination, and compare our results with those that would have been obtained theoretically using molecular cytogenetic techniques. Samples were obtained by transcervical CVS from women with a diagnosis of missed abortion, most of them referred for cytogenetic prenatal diagnosis. Cytogenetic analysis was performed using semi-direct technique. A karyotype was obtained in 103 cases. Eighty-two abnormal karyotypes were found (80%), including 12 triploidies, 10 monosomies, 61 trisomies, and 9 structural abnormalities; a double abnormality being present in 10 cases. Between 10% and 50% of our abnormal results would have been missed using the most common molecular cytogenetic techniques. Semi-direct analysis of CVS may still be considered as a comprehensive, reasonably rapid, cost-effective and reliable method for detecting the broadest spectrum of chromosome abnormalities in missed abortions.     

Response to Dr. Alter

Traditional cytogenetic analysis of material from spontaneous abortions often fails due to non-viability of the tissues in cell culture. The 45,X constitution is common in early pregnancy loss. Fluorescent in situ hybridization (FISH) was used to diagnose 45,X in uncultured blood smears obtained from 2 cases of spontaneous pregnancy loss with fetal hydrops. The ability of chromosome specific alpha satellite probes to detect aneuploidy in uncultured cells should greatly increase the successful detection of chromosomal abnormalities in early abortuses.     

High-throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low-resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation-Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy-number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty-two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost-effective, high-throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.     

未培养羊水荧光原位杂交快速检测60例胎儿常见染色体数目异常

目的 应用细菌人工染色体(bacterial artificial chromosome,BAC)克隆自行制备荧光探针,对胎儿常见染色体数目异常(13,18,21,X,Y)行快速产前诊断.方法 利用中国医学遗传学国家重点实验室BAC库中相应染色体特异位点克隆,自行制备荧光探针.经外周血淋巴细胞染色体杂交验证后,用于胎儿未培养羊水的快速荧光原位杂交fluorescence in situ hybridization,FISH)检测,已检测60例羊水标本.结果 所有探针特异性均为100%,杂交成功率为97.86%;FISH结果与常规核型分析一致,检出21三体2例,18三体1例.有两例涉及其它染色体的结构异常未能检出.结论 自制探针用于未培养羊水快速FISH,使用标本量少、快速、简便,可有效检出上述5种染色体的数目异常,但本法不能检出其他染色体的数目异常和结构异常,其应用仍有一定局限性.     

The use of fluorescent in-situ hybridization (FISH) for the analysis of in-vitro fertilization embryos: a diagnostic tool for the infertile couple

We use triple colour fluorescent in-situ hybridization (FISH)to sex human embryos for preimplantation diagnosis of X-linkeddisease, to analyse chromosome numbers in embryos donated forresearch purposes and as a diagnostic tool for patients undergoinginfertility treatment, especially in cases where abnormal embryodevelopment occurs. We have reported on the use of FISH in acase where all embryos showed accelerated cleavage. Here wereport on the use of triple colour FISH in a case where fiveout of seven oocytes were multi-nucleated when examined forpronuclei. The embryos were spread whole using HCI/Tween 20and triple colour FISH performed with probes for chromosomesX, Y and 1 in a 2 h procedure. Two embryos were normal for theprobes used, and three showed abnormalities, including one 4-cellembryo where all nuclei were X, X, X, Y, 1, 1, 1, 1. FISH indicatedthat fertilization had occurred, but that the majority of embryoswere abnormal confirming that such embryos should not be consideredfor transfer. In these cases, or where there is recurrent in-vitrofertilization failure or spontaneous abortions, embryos in futurecycles can be examined using FISH to ascertain the level ofchromosome abnormality which may aid future infertility treatment.     

Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes

BACKGROUND: Preimplantation Genetic Diagnosis (PGD) using FISH to analyze up to nine chromosomes to discard chromosomally abnormal embryos has resulted in an increase of pregnancy rates in certain groups of patients. However, the number of chromosomes that can be analyzed is a clear limitation. We evaluate the reliability of using comparative genomic hybridization (CGH) to detect the whole set of chromosomes, as an alternative to PGD using FISH. METHODS AND RESULTS: We have analysed by CGH both, first polar bodies (1PBs) and metaphase II (MII) oocytes from 30 oocytes donated by 24 women. The aneuploidy rate was 48%. Considering two maternal age groups, a higher number of chromosome abnormalities were detected in the older group of oocytes (23% versus 75%, P < 0.02). About 33% of the 1PB-MII oocyte doublets diagnosed as aneuploid by CGH would have been misdiagnosed as normal if FISH with nine chromosome probes had been used. CONCLUSION: We demonstrate the reliability of 1PB analysis by CGH, to detect almost any chromosome abnormality in oocytes as well as unbalanced segregations of maternal translocations in a time frame compatible with regular in vitro fertilization (IVF). The selection of euploid oocytes could help to increase implantation and pregnancy rates of patients undergoing IVF treatment.     

Features of chromosomal abnormalities in spontaneous abortion cell culture failures detected by interphase FISH analysis

Cytogenetic analysis of reproductive wastage is an important stage in understanding the genetic background of early embryogenesis. The results of conventional cytogenetic studies of spontaneous abortions depend on tissue culturing and are associated with a significant cell culture failure rate. We performed interphase dual-colour FISH analysis to detect chromosomal abnormalities in noncultured cells from two different tissues-cytotrophoblast and extraembryonic mesoderm-of 60 first-trimester spontaneous abortions from which cells had failed to grow in culture. An original algorithm was proposed to optimize the interphase karyotype screening with a panel of centromere-specific DNA probes for all human chromosomes. The overall rate of numerical chromosomal abnormalities in these cells was 53%. Both typical and rare forms of karyotype imbalance were found. The observation of six cases (19%) of monosomy 7, 15, 21 and 22 in mosaic form, with a predominant normal cell line, was the most unexpected finding. Cell lines with monosomies 21 and 22 were found both in cytotrophoblast and mesoderm, while cells with monosomy 7 and 15 were confined to the cytotrophoblast. The tissue-specific compartmentalization of cell lines with autosomal monosomies provides evidence that the aneuploidy of different human chromosomes may arise during different stages of intrauterine development. The effect of aneuploidy on selection may differ, however, depending on the specific chromosome. The abortions also revealed a high frequency of intratissue chromosomal mosaicism (94%), in comparison with that detected by conventional cytogenetic analysis (29%; P<0.001). Confined placental mosaicism was found in 25% of the embryos. The results of molecular cytogenetic analysis of these cell culture failures illustrate that the diversity and phenotypic effects of chromosomal abnormalities during the early stages of human development are underestimated.     

Assessment of molecular cytogenetic methods for the detection of chromosomal abnormalities

Some marker chromosomes and chromosome rearrangements are difficult to identify using G-bands by Giemsa staining after trypsin treatment (G-banding) alone. Molecular cytogenetic techniques, such as spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH), can help to detect chromosomal aberrations precisely. We analyzed the karyotypes in 6 cases of multiple congenital abnormalities and 1 case of spontaneous abortion (case 2). Three cases (cases 1, 6, and 7) had marker chromosomes, and 4 cases (cases 2-5) had chromosomal rearrangements. The karyotypes in cases 1, 2, and 3 were determined using FISH with probes based on the clinical findings and family histories. Spectral karyotyping (SKY) analysis in cases 4-7 showed that this method is useful and saves time. The combination of SKY and FISH analyses defined the range of the ring chromosome in case 7. We demonstrated that a combination of G-banding, FISH, and SKY can be applied effectively to the investigation of chromosomal rearrangement and to the detection of marker chromosome origins. We suggest the use of these methods for prenatal diagnosis, in which the inherent time limitations are particularly important.     

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescencein situ hybridization

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequences as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.     

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by comparative genomic hybridization (CGH), while the corresponding metaphase II (MII) oocyte was analysed by fluorescence in situ hybridization (FISH) to confirm the results. RESULTS: Matched CGH-FISH results were obtained in 42 1PB-MII doublets, of which 37 (88.1%) showed reciprocal results. The aneuploidy rate was 57.1%. Two-thirds of the aneuploidy events were chromatid abnormalities. Interestingly, the chromosomes more frequently involved in aneuploidy were chromosomes 1, 4 and 22 followed by chromosome 16. In general, small chromosomes (those equal to or smaller in size than chromosome 13) were more prone to aneuploidy (chi2-test, P=0.07); 25% of the aneuploid doublets would have been misdiagnosed as normal using FISH with probes for nine-chromosomes. CONCLUSIONS: The combination of two different techniques, CGH and FISH, for the study of 1PB and MII allowed the identification and confirmation of any numerical chromosome abnormality, as well as helping to determine the mechanisms involved in the genesis of maternal aneuploidy.     

Down syndrome with pure partial trisomy 21q22 due to a paternal insertion (4;21) uncovered by uncultured amniotic fluid interphase FISH

OBJECTIVE: To emphasize the usefulness and reliability of fluorescence in situ hybridization (FISH) on uncultured amniotic fluid cells in the prenatal diagnosis of common chromosomal aneuploidies. METHODS: FISH analyses utilizing centromeric, locus-specific or whole chromosome paint DNA probes specific for chromosomes X, Y, 13, 18, 21, and 4 were performed on uncultured amniotic fluid cells or the peripheral blood specimen from the father. Routine chromosome analysis was carried out as well. RESULTS: A prenatal case with partial trisomy 21 due to a paternal cryptic insertion (4;21) was ascertained by a rapid overnight FISH on uncultured amniotic fluid cells. The fetus was delivered at term and had classical features of Down syndrome. CONCLUSION: Our results stress the importance of FISH on uncultured amniotic fluid cells to supplement routine cytogenetics, especially in cases with abnormal ultrasound findings.     

用荧光原位杂交技术快速诊断100例早期自然流产胚胎染色体数目异常

目的探讨荧光原位杂交(FISH)技术用于诊断绒毛间期细胞染色体数目异常的临床应用价值。方法采用FISH技术对我院100例50-84天的流产绒毛进行7条染色体(13、16、18、21、22、X和Y)的快速检测。同时,将绒毛接种、培养,进行常规细胞染色体核型分析,作为FISH检测结果的对照。结果被检测的100例样本中,用FISH检测,均获得诊断结果,检测成功率为100%,而常规细胞染色体核型分析,则只有91例获得诊断结果,检测成功率为91%。FISH检测结果与常规细胞染色体核型分析结果均相符合。结论应用FISH技术检测未培养绒毛间期细胞染色体数目异常,具有快速,简便,使用样本量少等优势,具有一定的临床应用价值。     

Towards unlimited colors for fluorescence in-situ hybridization (FISH)

We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei.     

Screening for specific chromosome involvement in hematological malignancies using a set of seven chromosome painting probes. An alternative approach for chromosome analysis using standard FISH instrumentation

We report the application of multi-color fluorescence in situ hydribidization (FISH) for bone marrow metaphase cell analysis of hematological malignancies using a sub-set of the human karyotype for chromosome painting. A combination of chromosome probes labeled with three haptens enabled the construction of a "painting probe" which detects seven different chromosomes. The probe was used to screen three chronic myeloid leukemia (CML) derived cell lines and ten CML patient bone marrow samples for aberrations, additional to the Ph rearrangement, that are associated with the onset of blast crisis of CML. This approach was shown to identify karyotype changes commonly seen by conventional karyotyping, and in addition revealed chromosome changes unresolved or undetected by conventional cytogenetic analysis. The seven-color painting probe provides a useful, fast, and reliable complementary tool for chromosome analysis, especially in cases with poor chromosome morphology. This is a simple approach, since the probes can be displayed in a standard red/green/blue format accessible to standard fluorescence microscopes and image-processing software. The proposed approach using panels of locus-specific probes as well as chromosome paints will be useful in all diagnostic routine environments where analysis is directed towards screening for genetic rearrangements and/or specific patterns of chromosome involvement with diagnostic/prognostic value.     

Role of multicolor fluorescence in situ hybridization (FISH) in simultaneous detection of probe sets for chromosome 18, X and Y in uncultured amniotic fluid cells.

Major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. The purpose of this study was to evaluate the role of multicolor fluorescence in situ hybridization in simultaneous detection of probe sets for chromosome 18, X, and Y in uncultured amniotic fluid cells as a safer alternative method for aneuploidy detection prenatally. Fifty amniotic fluid samples were analyzed by FISH and standard cytogenetics. Mean time to obtain results was three days for fluorescence in situ hybridization and 20 days for karyotype. Fluorescence in situ hybridization was informative in 43 samples (86%), and within this group, two aneuploidies were correctly identified. This evaluation demonstrates that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory.     

应用荧光原位杂交技术进行早期难免流产胚胎绒毛细胞染色体检测的研究

目的探讨荧光原位杂交（FISH）技术用于早期难免流产绒毛间期细胞染色体非整倍体异常的临床应用价值。方法采用FISH技术对20例我院住院难免流产病人的流产绒毛进行5条染色体（21、13、18、X和Y）的快速检测。结果被检测的20例样本中,用FISH检测,均获得诊断结果,检测成功率为100%,FISH检测结果中有8例异常,异常率为40%,其中XO是难免流产中胚胎中最多的异常。结论应用FISH技术检测未培养绒毛间期细胞染色体数目异常,具有快速,简便,使用样本量少等优势,具有一定的临床应用价值。     

Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia

Multicolor spectral karyotyping allows simultaneous visualization of all human chromosomes and screening for chromosomal rearrangements without a priori knowledge of any abnormalities involved. Based on this potentially increased sensitivity, we investigated, in a preliminary manner, whether spectral karyotyping could detect cytogenetic aberrations in karyotypically normal leukemia. The test population was comprised of 28 cryopreserved, cytogenetically normal acute myeloid leukemia (AML) samples from patients registered to a randomized trial for previously untreated AML (SWOG 9031). Two normal and 12 samples with known cytogenetic aberrations were used to validate and establish the diagnostic accuracy of the spectral karyotyping assay and instrumentation in a clinical setting. Enumeration and region-specific DNA fluorescence in situ hybridization (FISH) probes verified discrepant results. In the validation data set, spectral karyotyping refined complex karyotypic rearrangements in six cases and defined the chromosomal origin of a "jumping" homogeneously staining region; however, the technology was less sensitive in the detection of subtelomeric rearrangements and double minute chromosomes. In the test population, spectral karyotyping identified previously undetected cytogenetic aberrations in two cases (7%) of karyotypically normal AML: a cryptic 11q23 translocation in 20/20 cells and a minor monosomy 7 clone in 3/21 cells (FISH, 10.5%). Both of these abnormalities are considered to confer a poor prognosis when based on classical cytogenetic prognostic criteria. As an adjunct to classical cytogenetics and standard FISH analyses, the additive resolution of spectral karyotyping, in particular, with chromosome paints spiked with subtelomeric and/or locus-specific probes, may allow significant gains to be made in diagnostic accuracy and recognition of genotype/phenotype prognostic relationships, and in defining underlying biologic mechanisms in cancer. Genes Chromosomes Cancer 28:318-328, 2000.     

荧光原位杂交在产前诊断胎儿先心病22q11微缺失中的应用

目的应用荧光原位杂交（FISH）技术检测胎儿染色体22q11微缺失,以探讨该技术在胎儿先天性心脏病病因检测中的临床应用。方法对41例产前诊断有各类心脏畸形、且染色体核型分析结果未见明显异常的胎儿以及1例先证者进行FISH检测,检测探针位于22q11微缺失综合征微缺失关键区域22q11的TUPLE1基因,与22q末端ARSA基因。结果 41例胎儿FISH检测均成功,所有胎儿22q11两位点均未发现微缺失;一胎儿家庭的1名先证者经检测确证为22q11微缺失综合征患者。结论胎儿心脏畸形有多种病因,常规染色体检查仅能检出其中一部分染色体数目异常,对于各种微缺失综合征,仍然需要FISH、芯片等更高分辨率的技术手段应用,对相关可能的致病位点进行针对性检测或筛检,以提高病因检出率,防止心脏畸形患儿出生。     

Reflex fluorescent in situ hybridization testing for unsuccessful product of conception cultures: A retrospective analysis of 5555 samples attempted by conventional cytogenetics and fluorescent in situ hybridization

IntroductionThe use of chromosome analysis on products of conception from spontaneous abortions is recommended to identify a genetic etiology. However, 20% of products of conception cultures are unsuccessful due to microbial contamination or lack of viable dividing cells. Our laboratory implemented a reflex fluorescent in situ hybridization (FISH) assay to detect numeric chromosome abnormalities for unsuccessful cultures.Materials and MethodsAll products of conception samples were simultaneously processed for both chromosome analysis and FISH analysis. If the chromosome analysis was unsuccessful, interphase FISH was performed for chromosomes 13, 16, 18, 21, 22, X, and Y. To assess the performance of the FISH assay, a 3-year retrospective comparative analysis of the FISH results versus chromosome results was performed.ResultsOf 5555 total specimens, 4189 (75%) represented chorionic villi/fetal tissue and 1366 (25%) represented tissue of unidentified origin. Of the 1189 tissues of unidentified origin with chromosome or FISH results, 1096 (92%) were XX, indicating that the majority of these tissues are likely maternal in origin. Of the 3361 successful chromosome studies on the chorionic villi/fetal tissue specimens, 1734 (52%) samples had a chromosome abnormality. Of the 762 successful FISH studies on chorionic villi/fetal tissue specimens that were unsuccessful by chromosome studies, 181 (25%) had an abnormal result with the targeted FISH panel. Overall, the FISH panel detected approximately 70% of the chromosome abnormalities in products of conception detectable by karyotype. When the FISH panel results were combined with chromosome analysis for the 4189 chorionic villi/fetal tissue specimens, the overall abnormality rate is 47%.ConclusionsOur reflex FISH assay proved useful for the detection of common chromosome aneuploidies in products of conception samples that failed conventional chromosome analysis. Because of its limited view of the genome, cautious interpretation of FISH results is required for all samples, in particular, trisomy of an acrocentric chromosome, which may represent a Robertsonian translocation. An algorithmic approach to the genetic evaluation of products of conception specimens, with the potential for initial evaluation by a FISH panel, may be warranted.     

Detection of monosomy 7 by fluorescenceIn situ hybridization in acute nonlymphocytic leukemia and myelodysplastic syndrome

Summary Fluorescencein situ hybridization (FISH) with a chromosome 7 specific alpha satellite DNA probe was used to detect monosomy 7 in interphase and metaphase cells obtained from patients with myelodysplastic syndrome (MDS) and acute nonlymphocytic leukemia (ANLL). Chromosome analysis revealed monosomy 7, either alone or as part of a complex chromosome abnormality, in all cell samples. FISH analyses of 12 marrow samples and a blood sample using a chromosome 7 specific alpha satellite DNA probe revealed a single fluorescence spot in 80.5–97.5% of interphase cells indicating monosomy 7. In contrast, 83.5–92.0% of the same cells had two copies of chromosome 17 as two fluorescent spots were detected using a chromosome 17 specific alpha satellite DNA probe used as a positive control. The proportion of interphase cells with monosomy 7 did not correlated with the percentage of metaphase cells with monosomy 7 detected by conventional karyotyping or with the percentage of blast cells in the bone marrow.