Delineation of Partial Chromosomal Abnormalities in Early Pregnancy Losses
Categoria dell'articolo: ORIGINAL ARTICLE
Pubblicato online: 06 mar 2025
Pagine: 23 - 32
DOI: https://doi.org/10.2478/bjmg-2024-0014
Parole chiave
© 2024 Bozhinovski Gj et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Pregnancy loss (PL), encompassing spontaneous abortion or miscarriage, refers to the premature termination of a pregnancy before fetal viability, typically before the 20th gestational week. Early pregnancy loss (EPL) denotes losses occurring within the first trimester (<12 weeks) [1]. Approximately 15% of couples with confirmed pregnancies experience EPL, with recurrent PL (RPL) affect around 2% of them [2]. The etiology of PL is complex, involving a confluence of maternal and fetal factors. Maternal factors include endocrine disturbances, uterine anomalies, implantation issues, and infections [3]. In the event of fetal cause of EPL, chromosomal abnormalities account for roughly half of EPL cases, whereas the underlying cause remains elusive in the other half. Chromosomal trisomies, are the most prevalent fetal chromosomal aberrations, constituting up to 56% of abnormal EPLs, with trisomy 16 being the most common [4, 5]. Triploidy and monosomy, each affecting about 15% of abnormal conceptuses, follow in frequency. Rarer chromosomal anomalies comprise a smaller proportion of these cases. Lately, with the development of the molecular genetic technologies, especially with the widespread application of the whole exome sequencing (WES), embryonic and fetal monogenic conditions, were also reported in EPLs. Genes linked to fetal death, such as
Partial chromosomal aberrations, involving gain or loss of chromosomal content, represent a significant factor contributing to EPL. Our previous research identified these aberrations in 8% of abnormal EPLs, demonstrating heterogeneity across different chromosomes [4].
Quantitative fluorescent polymerase chain reaction (QF-PCR) and multiplex ligation probe amplification (MLPA) have been established as valuable screening tools for chromosomal abnormalities in the context of EPL. These techniques have enabled detailed analysis of chromosomal aberrations, including partial chromosome abnormalities, revealing their diverse nature and impact on pregnancy outcome [4, 12–14]. Consequently, while these techniques provide a valuable first step in the diagnostic process, they may not yield a definitive genetic diagnosis in all cases since they cannot detect all abnormalities, such as interstitial chromosomal abnormalities or determine the size and gene content of the detected partial chromosomal abnormalities. Understanding these abnormalities is crucial for improving diagnostic accuracy, genetic counseling, and potentially developing preventive strategies for recurrent pregnancy loss. Given the profound implications of partial chromosomal imbalances for fetal development and pregnancy outcome, a comprehensive and in-depth characterization of these aberrations is imperative.
In this study, we employed array comparative genomic hybridization (aCGH) to delineate the genomic architecture of previously detected partial chromosomal imbalances, including their size, location, and gene content. This granular level of analysis is expected to provide deeper insights into the pathogenic mechanisms associated with these aberrations and contribute to a more comprehensive understanding of the complex fetal etiology of early pregnancy loss.
In a prior investigation, partial chromosomal abnormalities were identified in 16 out of 900 analyzed EPL samples [4]. More recently, an additional four samples exhibiting partial chromosomal abnormalities were detected and incorporated into the current analysis, bringing the total number of EPL samples included in this study to 20. These samples consisted of single chromosomal abnormalities in half, while the other half exhibited double chromosomal abnormalities.
All EPL samples referred to the Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, at the Macedonian Academy of Sciences and Arts, Skopje for analysis of chromosomal aneuploidies were selected by a collaborative team of gynecologists and pathologists. Standard histopathological analysis was performed on all EPLs, as described previously [14, 15]. To ensure ethical compliance, written informed consent was obtained from all study participants, and the research protocol received approval from the institutional ethics committee (Macedonian Academy of Sciences and Arts, 09-1047/6 from 04.05.2016).
All EPL samples were processed using standard phenol-chloroform or automated extraction methods to obtain genomic DNA for further analysis. To initially exclude maternal contamination and to screen for common aneuploidies, we employed quantitative fluorescent polymerase chain reaction (QF-PCR) with a panel of short tandem repeat (STR) markers targeting chromosomes 13, 18, 21, and the sex chromosomes [16]. This method allowed for rapid detection of trisomies and monosomies involving chromosomes 13, 18, 21 and X, as well as triploidies. To complement QF-PCR, multiplex ligation-dependent probe amplification (MLPA), using P036-Subtelomere Mix 1 and P070-Subtelomere Mix 2B probemixes, was implemented to identify chromosomal imbalances on all chromosomes.
To further elucidate the genomic architecture of the observed partial chromosomal abnormalities, array comparative genomic hybridization (aCGH) analysis was conducted following Agilent Technologies’ SureTag Complete DNA Labeling Kit (Agilent Technologies, Santa Clara, CA, USA). Genomic DNA extracted from the analyzed samples, along with sex-matched control DNA, was enzymatically fragmented using the restriction enzymes AluI and RsaI to ensure uniform DNA fragment sizes. Subsequently, the fragmented DNA samples were fluorescently labeled: the experimental DNA was labeled with Cyanine 5 (Cy5), while the control DNA was labeled with Cyanine 3 (Cy3). After purification, the labeled experimental and control samples were combined and co-hybridized onto Agilent Technologies’ CGH Constitutional G3 4x180k microarrays. Following hybridization, data acquisition was performed using Agilent Technologies’ DNA microarray scanner with surescan technology, ensuring accurate capture of fluorescence signals. The resulting data were subsequently processed and analyzed using Agilent’s Genomic Workbench software platform. This analysis provided a comprehensive visualization of CNVs, with detailed genomic annotations based on the UCSC hg19 genome assembly, facilitating precise mapping and interpretation of the chromosomal imbalances observed in the study.
To gain insights into the genetic factors underlying early pregnancy loss, we conducted a comprehensive analysis of a total of 32 chromosomal abnormalities detected by aCGH in 20 EPLs with partial chromosomal abnormalities. We have established a criterion for identifying recurrent chromosomal regions: a chromosomal region, included in deletion or duplication, had to be observed in more than two EPLs. We determined the smallest overlapping region (SOR) for each recurrent chromosomal region. This approach enabled us to pinpoint the specific genes and pathways that are likely involved in the pathogenesis of early pregnancy loss.
To gain a deeper understanding of the biological functions and processes associated with the identified genes, we conducted a Gene Ontology (GO) enrichment analysis utilizing the Panther software [12]. This analysis compares the frequency of specific GO terms within a group of genes involved in SORs. Significantly enriched GO terms were visualized using a bar plot, thus providing a clear and intuitive way to interpret the results of the enrichment analysis and identify the biological functions and processes and that are most likely to be involved in EPL.
To investigate the genomic landscape of the previously detected partial chromosomal abnormalities in EPLs with QF-PCR and subtelomeric MLPA methods, we have performed array CGH analysis on a total of 20 samples.
All 30 previously detected chromosomal abnormalities among the 20 studied EPLs were confirmed and 2 additional chromosomal abnormalities previously not detected with QF-PCR and subtelomeric MLPA methods were revealed. These additional abnormalities were out of the ligation sites of the used subtelomeric MLPA probes, therefore they were not detected previously.
Among the 20 investigated samples, the total number of detected partial chromosomal abnormalities was 32, of which 18 (56% of all detected abnormalities) were deletions and 14 were duplications (44%). Nine samples had single partial chromosomal abnormality, 10 samples carried double abnormalities, while one sample carried three partial abnormalities. All the characterized abnormalities were on distal chromosomal sites, except one sample which had an additional aberration located proximally (7q22.1q34) of the expected single chromosomal abnormality on chromosome 7q36, previously not detected with the QF-PCR and subtelomeric MLPA methods. Also, another sample with deletion on 1p region and duplication on 1q region detected previously by MLPA, was found to carry an additional duplication involving the 1p36.32p36.31 chromosomal region. Schematic presentation of the detected chromosomal abnormalities among the affected chromosomes is shown on Figure 1. The sizes of the abnormalities ranged from 1.04 Mb to 150.21 Mb, with average size of 33.2 Mb. Detailed genomic locations of the detected partial chromosomal abnormalities is presented in Table 1 and the gene content of the detected abnormalities is described in Supplementary Table 1.
Figure 1. Schematic presentation of the detected chromosomal abnormalities among the affected chromosomes. (Red bars-deletions; blue bars-duplications), *chromosomal abnormalities detected together with other abnormality, see Table 1.
Detailed genomic locations of the detected partial chromosomal abnormalities
| Sample | Chromosome band | Start-stop positions | Deletion/Duplication | Length (Mbp) | |
|---|---|---|---|---|---|
| 1 | Abp-24 | 3p26.3-p26.1 | 73914-7761159 | Deletion | 7.68 |
| 2 | Abp-118 | 6p22.3-q27 | 20695423-170911240 | Duplication | 150.21 |
| 3 | Abp-141 | 17p13.3-p13.2 | 59739-4424713 | Duplication | 4.36 |
| 18q21.2-q23 | 49010709-78010032 | Deletion | 28.99 | ||
| 4 | Abp-154 | 8p23.3-p23.1 | 176814-12686483 | Deletion | 12.51 |
| 5 | Abp-264 | Xp22.33 | 169064-2344976 | Duplication | 2.17 |
| 6 | Abp-359 | 11q24.2q25 | 127546842-134927114 | Deletion | 7.38 |
| 7 | Abp-498 | 9p24.3-p23 | 204193_9092170 | Deletion | 8.88 |
| 8 | Abp-565 | 8q24.13-q24.3 | 123993466-146294098 | Duplication | 22.3 |
| 13q14.3-q34 | 54627609-115089535 | Deletion | 60.46 | ||
| 9 | Abp-612 | 3q25.33-q29 | 160109099-197845254 | Duplication | 37.73 |
| 9p24.3-p22.3 | 204193-14453418 | Deletion | 14.25 | ||
| 10 | Abp-627 | 7q22.1-q34 | 101658846-140064713 | Deletion | 38.4 |
| 7q36.3 | 158074913-159118566 | Deletion | 1.04 | ||
| 11 | Abp-760 | 18q12.1-q23 | 25698937-78010032 | Deletion | 52.31 |
| 12 | Abp-805 | 1p36.33-p36.32 | 564424-2352253 | Deletion | 1.78 |
| 1p36.32p36.31 | 2394396-5710339 | Duplication | 3.31 | ||
| 1q21.1q44 | 142617943-249212668 | Duplication | 106.59 | ||
| 13 | Abp-815 | 1p36.33-p36.13 | 564424-17886109 | Deletion | 17.32 |
| 14 | Abp-840 | 13q13.3-q34 | 39536567-115093155 | Deletion | 75.55 |
| 16q21-q24.3 | 64826408-90148400 | Duplication | 25.32 | ||
| 15 | Abp-882 | 1p36.33-p36.11 | 564424-24570889 | Deletion | 24.01 |
| 11q11-q25 | 55361638-134904063 | Duplication | 79.54 | ||
| 16 | Abp-914 | 13q32.3-q34 | 101077865-115105297 | Duplication | 14.02 |
| 18q21.31-q23 | 55906486-78010032 | Deletion | 22.1 | ||
| 17 | Abp-941 | 9q21.11-q34.3 | 70984337-141087916 | Duplication | 70.1 |
| 18 | Abp-942 | 6q22.31-q27 | 122649716-170911237 | Duplication | 48.26 |
| 7p22.3-p13 | 45064-45032669 | Deletion | 44.98 | ||
| 19 | Abp-990 | 1p36.33-p35.2 | 746608-31284865 | Duplication | 30.53 |
| 18q21.2-q23 | 49862572-78010032 | Deletion | 28.14 | ||
| 20 | Abp-998 | 15q26.1-q26.3 | 93296596-102388476 | Deletion | 9.09 |
| 22q13.1-q13.33 | 37984158-51219009 | Duplication | 13.23 |
Chromosome 1 exhibited the highest frequency of abnormalities, with six of the detected alterations mapped to this chromosome. Chromosome 18 was the second most affected, harboring four abnormalities, followed by chromosome 13 with three abnormalities.
To identify significant CNV regions and to determine the molecular functions and biological processes of the genes contained in these regions who are associated with early pregnancy loss (EPL), we first focused on recurrent regions (>2) among the 32 detected partial chromosomal alterations determining the SOR regions.
With this approach, we identified a total of six recurrent events. The most common recurrent event was on the terminal part of the chromosome 1p, with SOR of 1.78 Mb in the 1p36.33-p36.32 regions, shared by five deletions/duplications Common recurrent events were found on the terminal part of chromosomes 18q (SOR of 22.1 Mb in the 18q21.31-q23 region) and chromosome 13q (SOR of 14.02 in the 13q32.3-q34 region). With two recurrent events were chromosomes 6q, 9p and 11q, with SOR of 48.26 Mb in the 6q22.31-q27 region, SOR of 8.88 Mb in the 9p24.3-p23 region and SOR of 7.38 Mb in the 11q24.2q25 region. Schematic presentation of the SOR regions is presented on Figure 2. The OMIM genes in every recurrent abnormality were identified and the respective diseases caused by those genes were paired. The most common disorders caused by the identified genes were multisystem syndromes, which may explain their pathogenicity in early human development. The OMIM genes and the associated disorders are shown in Supplementary Table 2.
The detected smallest overlapping regions (SORs), (Red bars-deletions; blue bars-duplications).
Gene Ontology (GO) enrichment analysis was conducted using Panther, and significantly enriched GO terms were visualized through bar plots (Figures 3 and 4). The analysis revealed that the genes are predominantly associated with molecular binding activities, enzymatic functions as catalytic molecules, or ATPase activity.
Molecular function profiling of recurrent genes in the studied regions.
An analysis of the biological processes associated with frequently occurring genes within the specified recurring regions.
In terms of biological processes, the recurrent genes identified in this study primarily encode proteins involved in essential functions such as biological regulation, cellular processes, and metabolic processes (Figure 4). Furthermore, several genes within the affected regions were linked to developmental processes, indicating that their dysfunction could contribute to early developmental abnormalities, potentially leading to pregnancy loss.
This study aimed to refine the characterization of previously identified partial chromosomal abnormalities in EPLs from early pregnancy losses (EPLs) by employing aCGH. Our findings significantly expand upon the initial QF-PCR and subtelomeric MLPA data, providing a detailed understanding of the genomic landscape in these cases. The identification of multiple abnormalities within individual samples, unidentified by the previous analyses underscores the complex genetic etiology of EPLs. For instance, in one sample we identified an additional abnormality located proximally to the expected single chromosomal abnormality on chromosome 7q36, which had not been previously detected using the QF-PCR and subtelomeric MLPA. In another sample, we discovered an additional duplication on the 1p36.32p36.31 chromosomal regions, despite the initial distal deletion and duplication on 1p and 1q regions respectively. Interstitial chromosomal abnormalities account for approximately 5–10% of chromosomal anomalies in early pregnancy losses (EPLs) [18, 19]. These abnormalities cannot be detected using methods such as the previously used QF-PCR and subtelomeric MLPA, underscoring the limitations of these techniques. This highlights the critical need for high-resolution approaches, such as aCGH in EPLs with no chromosomal abnormality detected by subtelomeric MLPA.
Our study’s findings are particularly noteworthy given the large size of the CNVs detected (averaging 33.2 Mb), suggesting that these genetic alterations may disrupt multiple genes and are essential for normal embryonic development. The frequent involvement of chromosomes 1, 18, and 13 in these abnormalities is in line with their established association with pregnancy loss [20, 21]. These chromosomes have previously been implicated in recurrent pregnancy loss (RPL), particularly in studies of aneuploidy and large chromosomal rearrangements [22]. Chromosomal copy number variations (CNVs) in key regions such as 1p36.33-p36.32, 9p24.3-p23, 11q24.2-q25, 13q32.3-q34, and 18q21.31-q23 have been strongly implicated in early pregnancy loss (EPL). These regions host genes critical for apoptosis, placental development, and cellular signaling. For example, 1p36.33-p36.32 harbors genes essential for apoptosis, while abnormalities in 11q24.2-q25 involve genes like
Around 1/3 of all genes in the recurrent regions are associated with multisystem syndromes, as shown by the OMIM genes enrichment analysis (Supplementary Table 2). Some of the genes are strongly associated with EPLs due to their critical roles in embryonic development and placental function. For instance,
It is essential to acknowledge the limitations of this study, including the retrospective design and relatively small sample size. These factors may restrict the generalizability of our findings. The majority of the detected chromosomal abnormalities could have arisen from parental balanced chromosomal translocations [37]. Unfortunately, this cannot be confirmed at the time, since we were unable to perform parental karyotypes. The probabilities of different reproductive outcomes for carrier individuals of reciprocal balanced translocations are commonly based on an estimate of the likelihood of a fetus to survive with chromosomal imbalances resulting from the adjacent-1 segregation, while conceptions with other unbalanced segregations may not be viable [38]. This can lead to a more precise risk assessment and proper genetic counselling for the next pregnancies of the couples.
The focus on CNVs also does not exclude the possibility of other genetic factors contributing to EPLs, such as single nucleotide variants (SNVs) or epigenetic changes that were not assessed in this study. Furthermore, while aCGH offers higher resolution than many traditional methods, it does not capture all types of genomic variation [39]. Future studies should aim to integrate CNV data with whole-genome sequencing and epigenetic profiling to develop a more comprehensive understanding of the genetic landscape of EPLs. Functional validation studies using in vitro and in vivo models are critical to elucidating how these genetic changes affect cellular and developmental processes [40–42]. This comprehensive research approach will be essential for understanding the underlying mechanisms of EPLs.
In conclusion, our study provides valuable insights into the partial chromosomal abnormalities associated with EPLs. The identification of recurrent CNV regions and their associated genes represents a significant step towards understanding the complex genetic etiology of this condition. Although, subtelomeric MLPA reliably detects telomeric partial chromosomal abnormalities in EPLs, aCGH is essential for detection and precise characterization of all CNVs, thus enhancing diagnostic and counseling strategies in affected couples.