Optical genome mapping enhances cytogenetic analysis in recurrent miscarriage: confirmation of a suspected (1;10) chromosomal translocation

Abstract

Background

Optical genome mapping (OGM) is a next-generation cytogenetic technique that may be beneficial for detecting subtle structural chromosomal alterations that can go unnoticed with conventional studies in couples with recurrent pregnancy loss.

Case presentation

We report the case of a couple referred to our assisted reproduction unit due to a history of recurrent pregnancy loss. Initially, conventional cytogenetic studies were performed to identify a possible genetic cause. To this end, the karyotypes of both members of the couple were determined. The fetal tissue from the third miscarriage was analyzed using comparative genome hybridization (CGH) array. Subsequently, the cytogenetic analysis of the couple was extended with the OGM technique. Basic infertility studies revealed normal results, and the karyotypes of both partners were initially reported as normal with respect to structural abnormalities. Following the third miscarriage, an array CGH analysis of the abortive tissue detected a deletion-duplication on chromosomes 1 and 10, respectively. Moreover, OGM revealed a balanced translocation between chromosomes 1 and 10 in the male which had not been detected through conventional karyotyping. A retrospective review of the karyotype by an expert cytogeneticist identified an apparent translocation that had previously gone unrecognized.

Conclusions

Structural chromosomal abnormalities may be underestimated in couples experiencing multiple miscarriages because they are not always accurately recognized by conventional cytogenetic techniques. OGM offers a valuable complement to these traditional methods by identifying chromosomal alterations that may have been overlooked by karyotyping, precisely characterizing the nature of the structural rearrangements. While OGM cannot currently replace karyotyping due to limitations such as the inability to detect certain translocations (e.g., Robertsonian translocations), it can enhance diagnostic accuracy and provide additional insights into the genetic causes of repeated pregnancy loss. Therefore, OGM may serve as a useful supplementary tool for improving diagnosis and management in affected couples.

Background

Recurrent pregnancy loss (RPL) is defined as the spontaneous loss of two or more pregnancies from conception until the 24th week of gestation [1]. It affects 2–4% of couples of reproductive age [2] and causes significant emotional distress, emphasizing the importance of identifying underlying causes to develop strategies to prevent recurrence.

Chromosomal abnormalities are among the most significant genetic cause of RPL, with balanced translocations being the most prevalent, affecting approximately 5% of cases. Reciprocal translocations have a population prevalence of 1 in 500 individuals. Currently, conventional cytogenetic techniques such as karyotyping and comparative genome hybridization (CGH) array are widely used to detect chromosomal abnormalities in couples with RPL. However, these methods fail to identify the underlying cause in approximately 50% of cases [1].

G-banded karyotyping has long been the cornerstone of chromosomal analysis in clinical practice. However, the resolution of these techniques is inherently limited; they typically detect alterations in the range of 5–10 Mb and may fail to discern cryptic rearrangements, particularly when breakpoints occur near subtelomeric or pericentromeric regions [2].

Furthermore, array CGH has improved diagnostic yield by allowing the detection of copy number variations (CNVs) at a relatively high resolution. Nevertheless, as a method based on relative DNA dosage, array CGH is unable to detect balanced rearrangements that do not involve a net gain or loss of genetic material. This limitation is particularly problematic in cases of RPL where a balanced translocation in one parent may lead to unbalanced gametes and subsequent embryonic lethality, even though the parental karyotype appears normal [3].

In recent years, optical genome mapping (OGM) has emerged as a transformative technology in the field of cytogenetics. Unlike conventional methods, OGM utilizes ultrahigh-molecular-weight DNA molecules that are fluorescently labelled and placed on a chip, a physical support that is inserted into an analysis device. Within this device, each linear DNA molecule moves through nanoscopic-sized channels or nanochannels, while a sensor records the positions of the fluorescently labeled probes. This approach enables genome-wide mapping with unprecedented resolution (often down to several hundred base pairs), thereby allowing the detection of a wide spectrum of structural variants, including balanced translocations, inversions, insertions, and deletions, without requiring prior hypotheses about affected regions, unlike targeted methods such as fluorescence in situ hybridization (FISH) [4–6].

Therefore, OGM is a next-generation cytogenetic technique that complements conventional methods and offers enhanced detection of balanced translocations and other structural variants. These advantages make OGM a valuable tool for diagnosing recurrent miscarriages when a specific cause remains unidentified or when conventional techniques do not identify subtle chromosomal alterations due to interpretation challenges.

Case presentation

We present the case of a couple referred to the assisted reproduction unit due to RPL. The initial infertility evaluations, including conventional karyotyping of blood samples from both partners, did not reveal any significant chromosomal abnormalities. However, a retrospective and more detailed review of the karyotype identified an apparent reciprocal translocation.

Array CGH analysis of the abortive tissue revealed a 22.6 Mb deletion and a 10.5 Mb duplication affecting chromosome 1 (Chr1:629,044–23,266,981) and chromosome 10 (Chr10:123,057,977–133,563,090), respectively.

The OGM technique was subsequently used to establish a definitive diagnosis, confirming a balanced translocation between chromosomes 1 and 10 in the male partner. This finding provides insights into the cause of recurrent miscarriages and facilitates the planning of appropriate and personalized reproductive management.

OGM represents a significant advancement in understanding the genetic causes of RPL. Its ability to detect complex structural variants and balanced translocations with high resolution complements conventional cytogenetic techniques, reinforcing its utility as a diagnostic tool in couples with unexplained miscarriages.

Methods

Patients

A couple under follow-up in an assisted human reproduction program due to primary infertility. They have a history of three spontaneous miscarriages, all occurring before the 12th week of gestation, without available ultrasound study data. After ruling out other possible causes (gynecological, hematological, autoimmune, infectious, etc.), genetic studies were performed on the couple after the second miscarriage and on the fetal sample after the third miscarriage.

Genetic study results

Structural variants were described using the International System for Human Cytogenomic Nomenclature (ISCN 2024) [7].

Karyotyping

As the initial cytogenetic evaluation, conventional karyotyping via the G-banding method (400–550 bands resolution) was performed on both partners after two miscarriages at an external reference center.

Comparative genome hybridization (CGH) array

Analysis of the tissue from a third miscarriage was conducted via array CGH, which involves hybridization of the submitted sample with same-sex reference DNA (Agilent) on a KaryoNIM® 180 K platform (NIMGenetics®) for the detection of genetic alterations associated with prenatal and postnatal pathologies. For the bioinformatic analysis, the genomic construct GRCh38 and the ADM-2 statistical algorithm (window size: 0.5 Mb, threshold A = 6) were used, accepting alterations involving ≥ 5 consecutive probes.

The resolution of the analysis, in the case of prenatal samples, is approximately 250 kb for syndromes included in the prenatal design and 2 Mb for the rest of the genome. For postnatal samples, the resolution is approximately 15 kb for the 140 autism-related genes included in the design (75 kb for other regions of neuropediatric interest) and 100 kb for the rest of the genome.

Optical genome mapping

OGM was performed on both members of the studied couple after obtaining normal karyotypes and detecting the deletion-duplication in the abortive tissue.

In the first phase of the technique, high molecular weight DNA (> 250 kb) is extracted using the Bionano-Prep-SP-G2-BMA/PB-DNA-Isolation kit, following the protocol instructions. Long-chain DNA is labeled with a fluorophore by the action of the DLE-1 enzyme, which recognizes the specific 6-nucleotide sequence (CTTAAG) repeated throughout the genome (Bionano Prep Direct Label and Stain (DLS) Kit). The labeled DNA molecules are loaded onto a chip, linearized, and scanned using the Saphyr system (Bionano). The scanning process is digitized, generating an image with a labeling pattern that is compared to the human reference map, allowing visualization and characterization of structural variations and copy number variations present in the studied DNA.

Once the scanning process of the labeled long-chain DNA molecules is completed, minimum quality parameters of the technique are reviewed: Average N50 (> 150 kb): ≥ 230 kb; Label Density/100 kb: 14–17; Average Map Rate: ≥ 70%; Effective Coverage (> 150 kb): ≥ 340x.

The results analysis is performed using the Bionano Access software (via the de Novo Assembly pipeline) with the GRCh38 reference genome version. During the analysis, confidence filters recommended by the manufacturer (Bionano) are applied, and benign or polymorphic structural variants are filtered out.

Results

The karyotypes of both members of the couple were initially reported as normal, with chromosomal formulas of 46,XX for female (Fig. 1A) and 46,XY for male (Fig. 1B).

Fig. 1.

A and B The karyotypes analysed for female (46,XX) (A) and male (46,XY) (B) were initially considered normal, but a more detailed review identified an apparent translocation in the male, involving chromosomes 1 and 10

In the abortive tissue sample, array CGH revealed a 22.6 Mb deletion in the 1p36.33-p36.12 region (Fig. 2C) and a 10.5 Mb duplication in the 10q26.13-q26.3 region (Fig. 2D), both terminal and heterozygous. The chromosomal formula (ISCN 2024) was as follows:

$$\text{arr}\left[\text{GRCh38}\right]\text{1p36}.\text{33p36}.12\left(629,044\_23,266,981\right)\times 1,10\text{q26}.\text{13q26}.3\left(123,057,977\_133,563,090\right)\times 3$$

Fig. 2.

A and B Analysis of fetal tissue via CGH array revealed a loss of 22.6 Mb on the short arm of chromosome 1 affecting region 1p36.33-p36.12 (A) and a gain of 10.5 Mb in the 10q26.13-q26.3 region of the long arm of chromosome 10 (B)

This deletion-duplication identified in the abortive tissue, suggested the possibility of an unbalanced structural rearrangement potentially inherited from one of the parents. Consequently, the study was expanded on the OGM technique. OGM revealed a balanced reciprocal translocation between chromosomes 1 and 10 in the male (Fig. 3), in a heterozygous state, with a Variant Allele Frequency (VAF) of 50%.

Fig. 3.

Circos plot of the male obtained via the Bionano Acces program via the OGM technique. The balanced reciprocal translocation t(1;10)(p36.12;q26.13) is represented as a pink line connecting chromosomes 1 and 10 in the Circos plot. From the outer circle to the inner circle of the Circos plot, the following are represented: 1. Chromosomes and their cytobands; 2. Masked areas (difficult to analyse because they are heterochromatic regions); 3. Area of structural variants; 4. Zone of Copy Number Variation (CNV); 5. Area of Loss of Heterozygosity (LOH) and 6. In the center of the plot, translocations are shown as lines connecting the genomic loci involved

The breakpoints were described in the following chromosomal formula: (ISCN 2024): ogm[GRCh38] t(1;10)(p36.12;q26.13)(23,269,706;123,048,129) [0.5]. Thus, OGM not only confirmed the translocation but also precisely characterized the nature of the rearrangement, demonstrating its value as an excellent complementary tool to conventional cytogenetic techniques.

In light of these results, a more detailed review of the males's karyotype was requested by another expert cytogeneticist, which revealed an apparent translocation with visible alterations in the terminal regions of chromosomes 1p and 10q that had not been initially detected.

Discussion and conclusions

Chromosomal aberrations are broadly categorized based on their size. Microscopic aberrations are those potentially visible under a microscope, typically with a net imbalance greater than 10 Mb, while submicroscopic aberrations are smaller than 10 Mb or involve cryptic unbalanced translocations or low-level mosaicism, which may go undetected in conventional karyotyping. However, it is important to note that some abnormalities exceeding 10 Mb may also be cryptic and not easily recognizable in the banded chromosome pattern. This limitation often arises when the derivative chromosome closely resembles the size and staining pattern of its normal homologs. In such cases, the minimum detection threshold of G-banding may be insufficient to identify these alterations, particularly in regions with similar staining patterns or when the structural change does not result in a substantial size difference between homologs.

These challenges highlight the subjectivity of G-banding techniques, where interpretation heavily depends on the observer, and underscore the need for cytogenetics laboratories to implement rigorous internal and external quality control programs. Discrepancies between laboratories in analyzing chromosomal aberrations larger than 10 Mb have been documented in external quality control studies, with analytical accuracy ranging from 80 to 99% [8–10]. The advent of next-generation cytogenetic techniques, such as OGM, has introduced a more objective and high-resolution approach to addressing these limitations.

Conventional cytogenetic techniques often underestimate the prevalence of structural chromosomal aberrations, particularly balanced reciprocal translocations with breakpoints distal to the centromere. These can result in derivative chromosomes that closely resemble normal homologs, as observed in our case. Despite the presence of a balanced reciprocal translocation between chromosomes 1 and 10 in the male partner, it was not initially detected by G-banded karyotyping. The finding was retrospectively visible upon expert re-evaluation, highlighting the importance of careful and specialized analysis in conventional cytogenetics. Additionally, array CGH is unable to detect balanced translocations. Before OGM became available, addressing such cases required complex strategies, including FISH techniques like chromosomal painting or subtelomeric fluorescent probes, depending on the suspected chromosomal involvement.

We acknowledge that incorporating FISH could have added an additional layer of validation; however, due to resource constraints and the consistency between the OGM and array CGH findings, we considered it sufficient to establish a definitive diagnosis in this context.

The high-resolution capability of OGM enables the detection of subtle balanced chromosomal rearrangements, such as the rare (1;10) translocation described herein. Similarly, other authors have demonstrated the utility of OGM in reclassifying cases of recurrent pregnancy loss (RPL) where traditional methods have failed. For instance, Rao et al. [11] identified cryptic balanced rearrangements that had gone undetected by both G-banded karyotyping and array CGH using OGM. Furthermore, Ren et al. [12] and Yin et al. [13] emphasized that incorporating OGM into diagnostic workflows improves detection rates and facilitates personalized reproductive management strategies, such as preimplantation genetic testing (PGT) and preconception genetic counseling. In the prenatal setting, Zhang et al. [14] demonstrated that OGM is also effective in detecting clinically relevant chromosomal aberrations not visible with conventional cytogenetic techniques.

Despite its advantages, OGM has inherent constraints that must be acknowledged. Structural variants involving centromeres, acrocentric short arms, telomeres, or generally repetitive and poorly covered regions (e.g., Robertsonian translocations or isodicentric chromosomes) are often undetectable due to sequence homology or the use of masking filters. Additionally, OGM is unable to detect single nucleotide variants (SNVs) and structural variants smaller than 500 base pairs. While OGM excels in detecting larger balanced and unbalanced structural variants with high resolution, it cannot identify triploidies or higher-order polyploidies, which are important in diagnostic contexts. Low-level mosaicism may also be missed, particularly when variants are detectable only by the CNV pipeline. Ultra-high molecular weight DNA is essential for analysis, and poor DNA integrity can lead to false negatives or incomplete data. The method's reliance on DNA quality and sample size limits its applicability [15].

Carriers of balanced chromosomal translocations are at risk of producing unbalanced gametes due to complex mechanisms during meiosis. Specifically, during meiotic prophase I, chromosomes involved in a translocation form a quadrivalent structure that may segregate abnormally. This abnormal segregation can result in gametes with duplications, deletions, or numerical imbalances. Such anomalies are associated with spontaneous abortions, genetic syndromes, and reduced embryonic viability, accounting for the reproductive difficulties observed in carriers of balanced translocations (3).

Despite its limitations, the implementation of OGM in routine clinical practice has the potential to benefit couples experiencing RPL. By providing high-resolution detection of subtle or previously unrecognized chromosomal rearrangements, OGM can complement existing diagnostic methods, enabling more personalized interventions. Additionally, its integration into workflows that include advanced techniques such as preimplantation genetic testing (PGT) may help reduce the risk of transmitting chromosomal imbalances in future pregnancies, thereby contributing to improved reproductive outcomes.

Abbreviations

OGM

Optical genome mapping

RPL

Recurrent pregnancy loss

CGH

Comparative genome hybridization

CNV

Copy number variation

FISH

Fluorescence in situ hybridization

ISCN

International System for Human Cytogenomic Nomenclature

Author contributions

MMA, MB and JRV wrote the main manuscript text; JAC, AP and FRC revised the manuscript; GA and BR prepared the figures and the tables; and AC designed the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by grants from the Andalusian Government and confounding by FEDER funds (B-CTS-410-UGR-20) and (Group CTS-143).

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The scientific ethics committee of CEIm provincial de Granada approved this case report.

Consent for publication

Patients referred to in this manuscript signed their consent for the publication of this article. It will be provided if necessary.

Competing interests

The authors declare no competing interests.