Profile of chromosomal aberrations in different gestational age spontaneous abortions detected by comparative genomic hybridization

doi:10.1016/j.ejogrb.2006.04.037

European Journal of Obstetrics & Gynecology and Reproductive Biology

Volume 131, Issue 2, April 2007, Pages 127-131

https://doi.org/10.1016/j.ejogrb.2006.04.037 Get rights and content

Abstract

Objective

To determine the frequency and type of chromosomal aberrations in different gestational age spontaneous abortions.

Study design

In the study, 106 spontaneous abortions (SAs) were studied by comparative genomic hybridization.

Results

The frequency of detected chromosomal aberrations was 37.7%. Numerical chromosomal aberrations were disclosed in 82.5% of the aberrant cases, while structural chromosomal aberrations-in 17.5%. Highest frequency of aberrations was detected in the blighted ovum specimens (62.5%) compared to missed and second trimester SAs (respectively, 36.0% and 34.8%). With regard to structural aberrations, the difference in the frequencies between blighted ovum specimens and second trimester SAs nearly reached statistical significance (p = 0.0847). However, due to the low number of blighted ovum specimens analyzed (n = 8), this result should be interpreted with due caution. The most frequently affected chromosomal arms were Xp and Xq (13.7% of aberrant chromosomal arms, each), followed by 16p (8.4%), 16q (8.4%), 15q (4.2%) and 19q (4.2%).

Conclusions

We describe for the first time a profile of chromosomal aberrations in SAs from different gestational ages, detected by CGH. Our results showed highest frequency of chromosome aberrations in blighted ovum specimens compared to other types of spontaneous abortions, higher rate of structural aberrations than reported before (17.5%) and some aberrations that so far were not found by CGH.

Introduction

It has been estimated that 10–15% of all clinically recognized pregnancies terminate with spontaneous abortions (SAs). Cytogenetic studies have revealed that fetal chromosome abnormalities account for about 50% of first trimester and near 30% of second trimester pregnancy losses. Most of these abnormalities are numerical chromosomal aberrations (86%) and a low percentage are structural chromosomal aberrations (6%) or other, including chromosome mosaicism (8%) [1]. Chromosome abnormalities are of meiotic or mitotic origin. The most common cause of aneuploidy is non-disjunction during meiosis. The resulting gamete either lacks a chromosome or has two copies of it, producing a monosomic or trisomic zygote, respectively. When non-disjunction occurs after fertilization (mitotic non-disjunction), one expects to see mosaicism. Karyotypes, obtained from oocytes indicate that 20–25% of these cells have missing or extra chromosomes, while the frequency of aneuploidy in the sperm cells is 3–4% [2], [3].

According to the observations of Sandalinas et al. the cells with haploidy, polyploidy (5n), monosomy (other than X or 21), multiple trisomy (in most but not all instances) and severe structural chromosome rearrangements would not survive cell cycle control beyond the eight-cell stage: lethal stage. Cells with single and some with multiple trisomy, monosomy 21, sex chromosomal aneuploidy, less severe structural chromosome rearrangements, tetraploidy and triploidy were assumed to survive cell cycle control and keep their ability of cell division: viable cells [4]. Qualitative or (semi)quantitative models for cause and effects of chromosomal abnormalities during pregnancy are scare. The “audit of trisomy 16 in man” [5] is an example of a detailed quantitative model confined to one specific chromosome (chromosome 16).

The presence of chromosomal abnormality in miscarriages explains the reason for the pregnancy loss [6]. Traditionally, the detection of chromosomal aberration in fetal tissues is performed by conventional cytogenetic methods. These methods rely on obtaining viable and dividing cells, tissue culturing, and harvesting metaphase chromosomes for analysis. Limitations may be due to macerated or fragmented samples from pregnancy loss, external contamination, culture failure and selective growth of maternal cells in long-term cultures of chorionic stroma [7].

Comparative genomic hybridization (CGH) is a molecular cytogenetic technique, which can detect aneuploidies-numerical and unbalanced structural chromosomal abnormalities. The CGH technique involves the simultaneous hybridization of genomic test and reference DNA, each labelled with a different fluorescent dye, to normal target metaphase chromosomes. By comparing the relative intensities of the two fluorophores along the length of each target chromosome, variations in DNA copy number between the test and reference genomes can be detected. Unlike conventional cytogenetic analysis, CGH analysis does not depend on dividing cells and can be employed anytime DNA is available. In the last years the CGH-array analysis has proven to become a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number [8], [9].

A limited number of CGH analyzes have been performed on spontaneous abortions. In 1998, Daniely et al. published data on 27 SAs investigated by CGH, where chromosomal aberrations were found in 48% of the samples [10]. In the study of Lomax et al. including 253 SAs and Barett et al. including 368 SAs, chromosome defects were established in 53% of the cases [11], [12]. Highest rate of chromosomal changes (72%) was found in spontaneously aborted human specimens that had failed to grow in culture (72%) [13].

To further enrich the data we present the profile of chromosomal aberrations in miscarriages, detected by CGH in Bulgaria.

Section snippets

Sample collection

Samples included materials from 106 SAs (83 first- and 23 second trimester abortions) with history of advanced maternal age (20.4% of the women were 35 years old or higher), repeated pregnancy loss (0.9%), phenotypic abnormalities of the embryo or foetus (17.9%), infertility problems (1.9%), or retroplacental hematoma, detected by ultrasonography (5.7%). The remaining cases did not belong to any of the above-mentioned groups (53.2%).

The samples were obtained by residua abrasions from the

Results

The CGH analysis revealed 37.7% overall frequency of chromosomal aberrations (40 out of 106 cases), distributed as follows: 82.5% numerical aberrations (aneuploidy 47.5%, monosomy X 25.0% and hyperdiploidy 10.0%) and 17.6% structural aberrations (Table 1, Table 2).

With regard to the clinical appearance, highest frequency of aberrations was detected in the blighted ovum specimens (62.5%) compared to missed (36.0%) and second trimester SAs (34.8%) (Table 3).

Numerical aberrations were found in 50%

Comment

Detection of aneuploidies in SAs is especially important for reproductive counseling. The presence of an aneuploidy requires prenatal diagnosis in the future pregnancies, because of possible unbalanced segregations associated with advanced maternal age or balanced structural rearrangements.

Comparative genomic hybridization (CGH) represents an alternative molecular cytogenetic technique capable of detecting chromosomal imbalances by reverse fluorescence in situ hybridization. As the technique

Acknowledgements

This work was supported by grant of the Ministry of Education and Science—Upgrading of Research Infrastructure no. 05/01.08.2005.

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To study the genomics/genetic factors associated with recurrent spontaneous abortion (RSA), as ∼50% of RSA are unexplained. However, chromosome abnormalities have been reported to play major role in RSA. We performed whole genome array-CGH based genomic analysis of forty four Saudi RSA patients to identify potential molecular and chromosomal abnormalities. We identified a total of 845 alterations, usually not detected by classic cytogenetic methods, in different genomic regions using a cut off value of −0.25 and 0.25 for structural loss and gain, whereas −1.0 and 0.58 were used for single copy number deletion and duplication respectively. We identified frequent (present at least in 10% of patients) alterations including three macro-alteration at 8p23.1, 10q11.21-q11.22 and 15q11.2 as well as large numbers of micro-deletions/amplifications with affected genes including 22q11.23 (GSTT1), 3p22.2 (CTDSPL), 6p21.32 (HLA), and 8p22 (MSR1). Pathway analysis of genes located in detected CNVs regions revealed the allograft rejection signaling, IL-4 signaling, and autoimmune thyroid disease signaling as the most significant canonical pathways associated with RSA. Whole genome array CGH technique can be used to identify potential genes, biofunctions and chromosomal abnormalities associated with RSA which is supported by our findings of a number of novel CNVs/genes (22q11.23/GSTT1, 3p22.2/CTDSPL, 6p21.32/HLA, 8p22/MSR1, and 14q32.33/AKT1) and pathways in patients affected with RSA. To improve diagnosis and treatment of RSA, a comprehensive procedure is needed for identification and validation of causative genes.
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Chromosomal alterations are rare and usually incompatible with life. It has been described that 4.2% of the chromosomal abnormalities found in miscarriages involve 19q, most of them in trisomic state and only one case presenting monosomy (Azmanov et al., 2007). In fact, the chromosome 19, which is one of the smallest human chromosomes, is the third in number of genes, making its imbalance more damaging (Grover et al., 2004).
Rearrangements in chromosome 19 are rare. Among the 35 patients with partial 19q trisomy described, only six have a breakpoint defined by array. The 19q duplication results in a variable phenotype, including dysmorphisms, intellectual disability and seizure. In a female patient, although G-banding at 550 band-resolution was normal, multiplex ligation-dependent probe amplification (MLPA) technique and genomic array showed a 10.6 Mb terminal duplication of chromosome 19q13. Fluorescent in situ hybridization (FISH) revealed that the duplicated region was attached to the short arm of chromosome 21 and silver staining showed four small acrocentrics with nucleolar organization region (NOR) activity, suggesting that the breakpoint in chromosome 21 was at p13. This is the first de novo translocation between 19q13.33 and 21p13 described in liveborn. The chromosome 19 is known to be rich in coding and non-coding regions, and chromosomal rearrangements involving this chromosome are very harmful. Furthermore, the 19q13.33→qter region is dense in pseudogenes and microRNAs, which are potent regulators of gene expression. The trisomic level of this region may contribute to deregulation of global gene expression, and consequently, may lead to abnormal development on the carriers of these rearrangements.
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This rate is lower than the 30–40% suggested by previous studies [15–17]. This can be explained by the fact that we have not tested all the chromosomal abnormalities involved in SA, including aneuploidies of chromosomes 16 and 22 that are relatively common in products of SA [17]. Of the 10 cases with abnormality, six are the result of early pregnancy loss (before 12th week of gestation), which confirms the hypothesis that most cases with abnormal karyotype, especially those with trisomies, abort early during pregnancy [18].
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L’avortement spontané correspond à la perte du conceptus avant 22 semaines de gestation alors que le fœtus pèse moins de 500 g. Une étiologie génétique est retrouvée dans plus de deux tiers des cas, l’aneuploïdie autosomale provoquant à lui seule plus de 70 % des avortements spontanés. L’objectif de ce travail était d’étudier les causes chromosomiques les plus fréquentes de perte fœtale. À partir de 220 produits d’avortement et de mort fœtale in utero, nous avons analysé les noyaux interphasiques extraits du villus chorionique utilisant des kits FISH (AneuVysion™) et l’ADN extrait de tissus fœtaux utilisant des kits MLPA (SALSA P036, P070, P245). L’âge gestationnel était de 7 à 38 semaines d’aménorrhée. Les résultats de l’analyse par FISH de 151 échantillons montraient 10 anomalies chromosomiques : trisomie 21 (n = 4 dont 1 mosaïque) ; trisomie 18 (n = 1) ; trisomie 13 (n = 1) ; triploïdie (n = 3) ; monosomie X (Turner) (n = 1). Les résultats de l’analyse par MLPA de 69 autres échantillons montraient 2 autres monosomies X (Turner). Les analyses FISH et MLPA sont des méthodes sensibles faciles à utiliser et rapides à mettre en œuvre pour la détection d’aneuploïdies chromosomiques. L’étape de culture cellulaire du caryotypage n’étant pas nécessaire, elles représentent un alternatif très intéressant pour diagnostiquer des anomalies génomiques de produits d’avortement dont la mauvaise qualité peut compromettre la culture cellulaire.
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View full text

Profile of chromosomal aberrations in different gestational age spontaneous abortions detected by comparative genomic hybridization

Abstract

Objective

Study design

Results

Conclusions

Introduction

Section snippets

Sample collection

Results

Comment

Acknowledgements

Baillieres Best Pract Res Clin Obstet Gynaecol

Curr Opin Genet Dev

Fertil Steril

Am J Hum Genet

Am J Hum Genet

Trends Genet

Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations: a review

Hum Genet

Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage

Hum Reprod

An audit of trisomy 16 in man

Prenatal Diagn

Clinical significance of morphologic and genetic examination of spontaneously aborted embryos

Am J Reprod Immunol

Array comparative genomic hybridization profiling of first-trimester spontaneous abortions that fail to grow in vitro

Prenatal Diagn

Detection of chromosomal aberration in fetuses arising from recurrent spontaneous abortion by comparative genomic hybridization

Hum Reprod

Comparative genomic hybridization: a new tool for reproductive pathology

Arch Pathol Lab Med