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Obstetrics & Gynecology 2000;96:281-286
© 2000 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Comparative Genomic Hybridization for Cytogenetic Evaluation of Stillbirth

G. C. M. L. CHRISTIAENS, MD, PhD, J. VISSERS, P. J. PODDIGHE, PhD and J. M. DE PATER, MSc

From the Division of Obstetrics, Neonatology and Gynaecology and the Department of Medical Genetics, University Medical Center, Utrecht, The Netherlands.

Address reprint requests to: G. C. M. L. Christiaens, MD, PhD University Medical Center Utrecht Wilhelmina Children’s Hospital Division Obstetrics, Neonatology and Gynaecology, KE.04.123.1 P. O. Box 85090 3508 AB Utrecht The Netherlands E-mail: l.christiaens{at}dog.azu.nl


   Abstract
Top
Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To ascertain the feasibility and reliability of comparative genomic hybridization for cytogenetic evaluation of macerated stillbirths.

Materials: We examined ten stillborn fetuses above 15 weeks’ gestation whose karyotypes were unknown because of tissue culture failure. Sixteen fetuses that were successfully karyotyped using prenatal or postnatal tissues were also examined as controls, including five pregnancy terminations with autosomal aneuploidy, one with sex chromosome aneuploidy, one with a chromosomal deletion; five macerated fetuses with normal karyotypes, three with autosomal aneuploidy, and one with sex chromosome aneuploidy and discrepancy between chorionic villi and fetus.

Results: All comparative genomic hybridization analyses in fresh and macerated tissues were successful except for one. All normal karyotypes and aneuploidies were confirmed. Comparative genomic hybridization failed in one fetus with a deletion of the short arm of chromosome 18. In the stillborn fetuses without known karyotypes, one aberrant profile was found; however, the results were not confirmed with interphase fluorescence in situ hybridization. In one fetus triploidy was diagnosed with DNA flow cytometry.

Conclusion: Comparative genomic hybridization is a valuable backup technique for aneuploidy screening in tissues from macerated stillborn fetuses when tissue culture fails. Gains or losses can subsequently be confirmed by fluorescence in situ hybridization, using DNA probes that focus on specific loci of a chromosome.

Five to sixteen percent of unselected stillborn fetuses have chromosomal aberrations, a percentage that is higher in macerated stillbirths and higher at younger gestational ages. Most aberrations are aneuploidies, but newly discovered, familial translocations and other structural chromosomal aberrations are found also.1–5 Seven to ten percent of cytogenetic aberrations in stillborn fetuses are not recognized specifically at postmortem examination or autopsy.5,6 Macerated stillbirth is a diagnostic challenge because dysmorphic features and even anomalies can be concealed by maceration and because of frequent (more than half) failure of in vitro tissue growth, which is essential for classic karyotyping.1,2,5,7 Other techniques for detecting chromosomal anomalies in stillborn fetuses are needed. Cytogenetic technologies such as fluoresence in situ hybridization, primed in situ hybridization, and comparative genomic hybridization recently have been introduced in maternal-fetal medicine.8 Fluorescence in situ hybridization and primed in situ hybridization target specific regions or loci of chromosomes, whereas comparative genomic hybridization studies gains or losses of all chromosomes at once. Primed in situ hybridization combines techniques of fluorescence in situ hybridization and polymerase chain reaction (PCR) by amplifying signal on the glass slide. It has been used for rapid prenatal diagnoses in cases of high probability9,10 and for rapid identification of markers.

Comparative genomic hybridization is a molecular-cytogenetic method developed and validated by Kallioniemi et al11,12 for genome-scale screening for chromosomal aberrations of solid tumors, which, like tissues from stillborn fetuses, lack sufficient high-quality metaphases and lack information about which chromosomes to look at specifically. With comparative genomic hybridization one can scan a genome for differences from a normal reference genome, without mitotic activity. It is based on a one-step hybridization between normal metaphase and a (1:1) mixture of normal reference DNA and DNA from the tissue to be analyzed (test DNA), each labeled with a different fluorochrome. Chromosome aneuploidies or structural anomalies in the test DNA are quantitated in a digital system that measures the ratio of both fluorochromes along the test metaphase. Comparative genomic hybridization singles out the chromosome involved so that targeted techniques can be applied for later confirmation. A limitation of the technique is that it detects only copy number changes relative to the average in the entire specimen and hence does not detect triploidy, a chromosomal aberration often resulting in stillbirth.13 It also gives no information on the actual chromosome architecture; eg, it does not discern Down syndrome based on three separate chromosomes 21 and Down syndrome caused by unbalanced translocation. Therefore, an autosomal aneuploidy of acrocentric chromosomes (13 and 21, but not 18) in a stillborn fetus will always have to be followed up with parental karyotyping. Comparative genomic hybridization also does not detect alterations that do not change copy numbers, such as balanced chromosome rearrangements, which might have phenotypic consequences if they occur de novo in fetuses. It also only detects changes in a substantial percentage of the cells. Contamination and mosaicism prevent reliable detection of aneuploidy. Comparative genomic hybridization tolerates 30–50% dilution of the aberrant DNA by normal DNA.12

Since comparative genomic hybridization was introduced, most studies have focused on oncogenetics, and only a few on other clinical applications.14–16 In this study we examined the effectiveness of the technique to detect or exclude chromosomal aberrations in stillborn fetuses, with the aim of introducing it as a backup technique in cases in which tissue culture and standard karyotyping fail.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
We studied tissues of two groups of fetuses of various gestational ages. The first group (n = 16) comprised fetuses whose karyotypes had been determined before or after birth, including seven cases of pregnancy termination for genetic reasons (fresh tissues) and nine stillborn fetuses (macerated tissues). The second group (n = 10) were stillborn fetuses without known karyotypes because of tissue culture failures. All material was taken with patients’ consent either to confirm antenatal diagnosis or for postnatal cytogenetic evaluation. The tissues to be used for comparative genomic hybridization were stored at -30C until further processing. Test DNA and reference (control) DNA, respectively, were isolated from each study sample and from peripheral blood of normal women according to standard protocols. We did not match test and reference DNA for sex phenotype.

Comparative genomic hybridization was done according to the methods of Kallioniemi et al11 with minor modifications. High-quality metaphase spreads (CGH-target slides, Vysis Inc., Downer’s Grove, IL) were used from karyotypically normal men. Test DNA and reference DNA (each 1 µg) were labeled with digoxygenin (Roche Ltd., Basel, Switzerland) and biotin (Gibco, Life Technologies Inc., Rockville, MD) respectively, precipitated in the presence of 200 µg Cot-1 DNA (highly repetitive DNA), and resuspended in 12 µL of hybridization mixture. After denaturation, the DNA mixture and metaphase spreads were hybridized for 72 hours at 37C. The hybridized test and reference DNA were viewed according to standard procedures using FITC (green) and Cy3 (red), respectively. By using fluorescence microscopy and digital image analysis, the ratio of green-to-red fluorescence was quantified, and changes in sequence copy number were established (Cytovision, Applied Imaging Corporation, New Castle, UK), resulting in a decreased green-to-red ratio (shift to the left) in cases of chromosomal loss or deletion in the test DNA and an increased green-to-red ratio (shift to the right) in cases of chromosomal gain. Threshold values for losses and gains were 0.75 and 1.25, respectively. We intended to analyze ten of each chromosome for which we needed up to ten meta-phases on average. Telomeric and centromeric regions, which contain highly polymorphic repetitive DNA sequences, are blocked to various extents by Cot-1-DNA. They might show false gains or losses, so were left out of the analysis.

One part of the samples was also processed for interphase fluoresence in situ hybridization.17 Plasmid probes pUC1.77,18 LSI 13/RB-1 (Vysis Inc., Downer’s Grove, IL), pH17H8,19 p2000a5,20 LSI-21 (Vysis), LSI-X (Vysis), and LSI-Y (Vysis) were used for demonstrating chromosomes 1, 13, 17 (centromere and q-telomere), 21, X, and Y, respectively. The DNA probes were hybridized to cell preparations. Demonstration of the biotinor digoxygenin-labeled DNA probes was done according to standard procedures using avidin-Cy3 and fluorescein (FITC)-conjugated sheep anti-digoxygenin, respectively. Counterstaining of the DNA was done with diamino-2-phenylindole (DAPI).


   Results
Top
Abstract
 Materials and Methods
 Results
Discussion
 References
 
Tissues from the 16 fetuses with known karyotypes were used to ascertain reliability of comparative genomic hybridization in fresh (n = 7) and macerated (n = 9) specimens at various gestational ages (Table 1Go). Gestational ages ranged from weeks. Comparative genomic hybridization results were obtained in all samples but one. Five fetuses had normal karyotypes (all female) and were correctly identified by comparative genomic hybridization, including their sexes (Figure 1Go). Ten fetuses had numerical chromosomal abnormalities, including: trisomy 21 (n = 6), trisomy 18 (n = 1), trisomy 13 (n = 1), and Turner syndrome (n = 2). Comparison of these results with standard karyotyping found that all ten were correctly identified, including sex, and including the two cases of Turner syndrome (Figure 2Go). One of the cases of Turner syndrome had a discrepancy between karyotypes in chorionic villi (46,X,del(Y)(q12)), placenta (45,X/46,XY), and fetus (45,X). Comparative genomic hybridization in fetal skin showed a loss at the X chromosome, confirming Turner syndrome. One fetus had a cytogenetic deletion of the short arm of chromosome 18. Although that specimen was a pregnancy termination with fresh tissue that was delivered to the laboratory within 12 hours, comparative genomic hybridization was unsuccessful.


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  		Table 1. Tissues With Known Karyotype
 


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  		Figure 1. Full comparative genomic hybridization profile of a dead female fetus with normal karyotype in cultured amniocytes (case 11). The fetus was severely macerated at birth. The lines in the profiles represent, from left to right, ratios of 0.5, 0.75, 1.0, 1.25, and 1.5. A ratio of 1 represents the normal diploid state.

 


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  		Figure 2. Partial karyotypes, partial comparative genomic hybridization profiles and metaphases, or interphase fluorescence in situ hybridization (top right) images of female fetuses with, respectively, trisomy 21 (case 11) and trisomy 18 (case 10), a male fetus with trisomy 13 (case 1), and a fetus with Turner syndrome (case 3). Chromosomes that are not shown had normal profiles, without significant deviation from a ratio of 1.0. Arrows in the comparative genomic hybridization metaphases indicate the aneuploid chromosomes.

 
In the second part of the study, ten stillborn fetuses with gestational ages between 15 and 41 weeks and unknown karyotypes caused by culture failure were examined (Table 2Go). Nine fetuses were macerated to various degrees, and the interval between fetal death and birth ranged from 8 hours to at least 8 days. All tissues were processed successfully for comparative genomic hybridization. Nine of ten samples had normal results. Postnatal evaluation of those nine fetuses led to the following tentative conclusions about causes of death: uteroplacental insufficiency in three, twin-to-twin transfusion syndrome in two, abruptio placentae in one, nonimmune fetal hydrops in one, maternal urosepsis in one, and triploidy diagnosed by DNA flow cytometry of the placenta in one. In one case an abnormal comparative genomic hybridization pattern was found, namely, gains of the short arm and the terminal part of the long arm of chromosome 17. However, interphase fluoresence in situ hybridization with the probes p17H8 centromere and p2000a5 (q-telomere)19,20 showed two signals, excluding (partial) trisomy 17 (Figure 3Go).


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  		Table 2. Tissues From Stillbirths Whose Karyotype Was Unknown
 


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  		Figure 3. Partial digital comparative genomic hybridization profile of a phenotypic female fetus (case 26) with unknown karyotype and abnormal gains at the terminal ends of the long and short arms of chromosome 17. Interphase fluorescence in situ hybridization with probes specific for these regions shows two signals indicating euploidy. The gains at chromosomes 13–16 in the comparative genomic hybridization profiles are at the heterochomatin-containing areas and result from highly polymorphic repeat-sequence-rich areas that cannot fully be blocked by the Cot-1-DNA.

 

   Discussion
Top
Abstract
 Materials and Methods
 Results
 Discussion
References
 
In liveborn infants, postnatal karyotyping usually succeeds. However, in stillborn fetuses there is an overall success rate of only 60%, and even less with increased time since death. In our study, eight of 18 (44%) macerated stillborn fetuses were karyotyped successfully. Until now only a few studies have described the usefulness of comparative genomic hybridization in perinatal medicine. Bryndorf et al15 studied 11 fetal samples. Four were prenatal cases with unidentifiable material that was elucidated with comparative genomic hybridization, two were stillborn fetuses, and five were genetic confirmations after pregnancy termination. Wang et al21 used the technique to identify a marker chromosome, and Wegner et al22 analyzed a structural anomaly in a newborn with congenital anomalies and previously normal results in chorionic villi. Levy et al16 used comparative genomic hybridization to resolve the identity of aberrant chromosomal material in 12 newborn cases. They validated all results with fluorescence in situ hybridization. Daniely et al23 examined 50 spontaneous abortions of unspecified gestational ages. Classic karyotyping was successful in only 23 of 50 samples; however, all 50 were processed successfully by comparative genomic hybridization. In the 23 samples karyotyped, five aneuploidies were found with both techniques. Comparative genomic hybridization detected three additional chromosomal anomalies in the other 27 samples. One aneuploidy (mosaic trisomy 16 in 20% of cells) was missed by comparative genomic hybridization. Their study did not contain cases of triploidy or Turner syndrome.

Our study showed that comparative genomic hybridization can be done successfully in tissues from fresh stillborn fetuses and stillborn fetuses macerated to various degrees. It was accurate for diagnosing or excluding the most common aneuploidies, including Turner syndrome, and it confirmed the karyotypes found prenatally or postnatally in 15 of 16 fetuses. Comparative genomic hybridization was unsuccessful in one case. Nine of 10 stillborn fetuses with unknown karyotypes had normal comparative genomic hybridization results. Many of the stillborn fetuses were late fetal deaths in which obstetric causes dominated. One case showed an abnormal pattern at chromosome 17, found to be normal by fluorescence in situ hybridization. One of the advantages of comparative genomic hybridization is its genome-wide screening. When aberrations are suspected, diagnoses can be confirmed or rejected by techniques using specific probes for regions of interest, provided that such probes are available, which is increasingly the case. A case found to be triploid by DNA flow cytometry was not detected by comparative genomic hybridization because the latter only detects copy number changes relative to average copy number in the entire specimen.

In our study, we did not match reference DNA to fetal phenotypes but used female reference DNA and male metaphases for all fetuses. A male metaphase allows male test DNA to hybridize. Combined with female reference DNA, that produces a shift to the right on the Y chromosome in case of a male test case (chromosomal gain in the test DNA compared with reference DNA). In female test cases there is no Y signal and a balanced red-green hybridization on the X chromosome resulting in a green-to-red ratio of approximately 1. In Turner syndrome, no Y signal and a shift to the left (chromosomal loss in test DNA) on the X chromosome is expected. Our strategy correctly identified the sexes of all fetuses and the two cases of Turner syndrome. Comparative genomic hybridization using paraffin-embedded materials previously has been unsuccessful; therefore, part of the fetal postmortem tissues must be frozen to allow comparative genomic hybridization if tissue culture fails. An average laboratory needs about 6 months to establish the technique,15 which was the case in our laboratory, but comparative genomic hybridization can then obviate time-consuming and expensive tissue culture.

Despite its limitations, comparative genomic hybridization is a valuable backup technique for the cytogenetic analysis of (macerated) stillborn fetuses when no antenatal specimens are available or in cases of failure of postnatal tissue growth. It helps to focus molecular techniques such as interphase fluorescence in situ hybridization to specific chromosomes. We do not recommend comparative genomic hybridization to replace classic karyotyping but we strongly recommend a protocol for the cytogenetic investigation of stillbirth that includes frozen storage of postmortem tissues to allow eventual comparative genomic hybridization (Figure 4Go).



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  		Figure 4. Protocol for the cytogenetic investigation of stillbirth. CGH = comparative genomic hybridization; FISH = fluorescence in situ hybridization.

 


   Footnotes
 
Supported by the Wim Schellekens Foundation.

PII S0029-7844(00)00879-6

Received November 3, 1999. Received in revised form January 14, 2000. Accepted January 20, 2000.


   References
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9. Pellestor F, Girardet A, Lefort G, Andreo B, Charlieu JP. Use of the primed in situ labelling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y. Hum Genet 1995;95:12–7.[Medline]

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13. Sanchez JM, Franzi L, Collia F, De Díaz SL, Panal M, Dubner M. Cytogenetic study of spontaneous abortions by transabdominal villus sampling and direct analysis of villi. Prenat Diagn 1999;19: 601–3.[Medline]

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16. Levy B, Dunn TM, Kaffe S, Kardon N, Hirschhorn K. Clinical applications of comparative genomic hybridization. Genet Med 1998;1:4–12.[Medline]

17. Raap AK, Arnoldus EPJ, Nederlof PM, Smit VTHBM, Cornelisse CJ, van der Ploeg M. Detection of numerical and structural chromosome aberrations in interphase nuclei by fluorescence in situ hybridisation. In: Elder HY, ed. Transactions of the Royal Microscope Society. MICRO 90, London: 1990:661–7.

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