Chromosomal microarray (CMA) is a high-resolution technique that can identify submicroscopic chromosomal aberrations or copy number variants (CNVs). It is used to detect CNVs as small as 50-100 Kb [1]. Thus, CMA provides approximately 100-fold higher resolution than conventional karyotyping. CNVs are common in the human genome [2,3], but some may cause various genetic disorders. CMA is recommended as a first-tier test in children with congenital structural anomalies, neurodevelopmental disorders, and autism spectrum disorders [4,5]. In prenatal settings, CMA analysis revealed clinically relevant CNVs in 6.0% of fetus with anomalies with a normal karyotype [6]. In a systematic review of prenatal CMA, additional relevant CNVs are identified in 3.1-7.9% of fetuses with structural anomalies [7]. Currently, both the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine (SMFM) recommend offering CMA in pregnancies with fetal structural anomalies [8].

As prenatal CMA is increasingly adopted in clinical practice, questions remain about the incidence of significant CNVs according to anomalous fetal organ systems. It is well known that fetuses with multiple anomalies are more likely to be associated with significant CNVs than those with isolated anomalies [9-11]. Many studies have reported that pathogenic CNVs (P CNVs) are frequently detected in prenatal ultrasound anomalies, particularly in cases of congenital heart diseases [12,13]. However, studies on the incidence of significant CNVs in specific organs have not been widely published [10].

In this study, we aimed to analyze the frequency of significant CNVs according to the involved organ system in fetuses with ultrasound-detected abnormalities.

1. Participants

This retrospective observational study included pregnant women who underwent a prenatal ultrasound and CMA analysis at CHA Gangnam Medical Center between April 2019 and July 2023. This study received approval from the ethics committee of CHA Gangnam Medical Center (IRB number: 2023-12-010). Clinical data were obtained from the electronic medical records of patients. The requirement to obtain informed consent was waived. The inclusion criteria were as follows: 1) women who underwent invasive tests (chorionic villous sampling, or amniocentesis); 2) women with abnormal findings on prenatal ultrasound; and 3) women who had both Giemsa banding (G-banding) karyotype and CMA performed, with normal karyotyping was identified.

2. Prenatal ultrasound

Abnormal ultrasound findings were classified as single organ system and multiple organ systems. The single organ system was categorized into increased nuchal translucency (INT), central nervous system (CNS), face and neck system, thorax system (heart and lung), gastrointestinal system, genitourinary (GU) system, musculoskeletal system, early-onset fetal growth restriction (FGR), oligohydramnios, and soft markers. INT was defined as nuchal translucency thickness ≥3.0 mm. Early-onset FGR was defined as an estimated fetal weight ≤10th percentile with onset before 32 weeks of gestation. Oligohydramnios was defined as the maximum vertical pocket <2 cm. Soft markers included choroid plexus cyst, ventriculomegaly, increased nuchal fold thickness, hypoplastic or short nasal bone, pyelectasis, short long bones, clinodactyly, and single umbilical artery. Soft markers were excluded from the classification of multiple organ systems.

3. Giemsa banded karyotyping and chromosomal microarray

An invasive test was performed, and cells were obtained from the chorionic villi or amniotic fluid. Cytogenetic analysis for fetal karyotyping was performed using the conventional G-banding analysis method.

CMA was performed using the CytoScan Optima assay platform (Thermo Fisher Scientific). The genomic DNA (250 ng) extracted from chorionic villi or amniotic fluid was digested by NspI and amplified using ligation-mediated polymerase chain reaction (PCR). The PCR product was purified, quantified, fragmented using DNase I, labeled with biotin, and hybridized overnight (16-18 hours) to a CytoScan Optima array. After hybridization, the sample was washed and stained with streptavidin using the GeneChip Fluidics Station 450 (Thermo Fisher Scientific). The array was scanned using the GeneChip Scanner 3000 (Thermo Fisher Scientific) to generate a CEL file. The CEL file was analyzed using Chromosome Analysis Suite (Thermo Fisher Scientific) and converted to a CYCHP file to visualize the status of genomic copy number and absence of heterozygosity. The CNVs called in the output file were classified according to the technical standards for CNV interpretation by American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). CNVs were classified into benign, likely benign, variants of uncertain significance (VUS), likely pathogenic (LP), and pathogenic (P). Among these, the P, LP, and VUS were reported as clinically significant variants.

4. Statistical analysis

Categorical and continuous variables with normal and non-normal distributions were expressed as N (%), mean±standard deviation (SD), respectively. The data analyses were performed using the Statistical Package for Social Sciences version 26.0 (IBM Corp.). A P<0.05 was considered statistically significant.

A total of 346 prenatal CMA were performed between April 2019 and July 2023, and there were 164 (47.4%) cases with abnormal ultrasound findings. Among these cases, chorionic villous sampling was performed in 95 cases (57.9%), and amniocentesis in 69 cases (42.1%). The gestational age at the time of the procedure ranged from 10.6 to 23.4 weeks, with a mean of 15.5 weeks (Table 1).

Among the 164 cases, 156 involved abnormalities in a single organ system, while 8 involved abnormal ultrasound findings in single or multiple organ systems respectively. Table 2 shows ultrasound-detected abnormal findings according to the organ system. INT was the most common abnormal finding, observed in 53.0% (87/164), followed by thoracic abnormalities at 8.5% (14/164), CNS abnormalities at 7.9% (13/164), and early-onset FGR at 7.9% (13/164). In 9 (5.5%) of 164 cases with a normal karyotype, significant CNVs were detected. These significant CNVs included one case with both P and VUS, three with P, one with LP, and four with VUS (Table 2). The frequency of significant CNVs was higher in cases involving multiple than in single organ systems (12.5% vs. 5.1%). The distribution of significant CNVs by single organ systems was as follows: GU 16.7% (1/6), face and neck 16.7% (1/6), cardiac 7.7% (1/13), early-onset FGR 7.7% (1/13), and INT 4.6% (4/87). Significant CNVs were detected in multiple organ systems, including CNS and facial anomalies.

Details of the LP or P CNVs are described in Table 3. All five cases with LP or P CNVs involved deletions, with the most frequent deletion occurring on chromosome 22q (three cases).

We confirmed that prenatal CMA provides an additional diagnostic yield of 5.5%. Significant CNVs were more frequently detected in multiple than isolated abnormal ultrasound findings. These findings are consistent with the previous studies [6-11].

According to the organ system, the diagnostic yield of prenatal CMA was highest for GU, and face and neck anomalies followed by cardiac anomalies. Donnelly et al. [9] reported that isolated renal and cardiac anomalies were associated with the greatest significant incremental yield provided by CMA (15.0%, P=0.036, and 10.6%, P=0.012, respectively). The high correlation with GU anomalies in our study is consistent with these findings, although the small sample size likely influenced the results of both studies. Most studies have reported that cardiac anomalies are the most common isolated anomaly associated with P CNVs [12,13]. Compared to theses studies, the different distribution in our study is likely due to the small sample size. Although our study is limited by a small sample size, its strength lies in the detailed analysis CNV distribution according to the specific organ systems. Also the study is retrospective in nature, which inherently comes with limitations such as selection bias and the inability to control for all potential confounding variables.

In our study, INT was the most common abnormal ultrasound finding but showed the lowest frequency of significant CNVs. It is well known that INT is associated with common aneuplidies, structural anomalies, as well as genetic syndromes [14,15]. In isolated INT, P CNVs have been reported a rate of 2.6% to 5.3% [11,16]. Similar to our results, these studies found that INT had a lower frequency of CNVs compared to other structural anomalies.

Previous studies either performed conventional karyotyping or confirmed normal results only with quantitative fluorescent PCR or fluorescent in situ hybridization, leading to mixed findings [11,16]. The large-scale prenatal CMA study analyzed CNVs smaller than 10 Mb, including those larger than submicroscopic CNVs [10]. Therefore, previous study results may include chromosomal abnormalities detectable by conventional karyotyping within the CNVs identified by CMA. We confirmed normal results for all cases using conventional karyotyping, and analyzed submicroscopic CNVs (smaller than 5 Mb) in other chromosomes as well as common aneuploidies. In our study, there was one case with a 6.2 Mb deletion that was not detected microscopically. This deletion, located at 22q13.3 at the terminal end of chromosome 22, is difficult to distinguish microscopically even if it is larger than 5 Mb.

Our study applied stricter criteria for analyzing submicroscopic CNVs compared to previous studies, confirming that prenatal CMA is a useful diagnostic tool in cases of ultrasound abnormalities. Additionally, we demonstrated that the frequency of significant CNVs varies according to the organ systems. We expect that collecting and analyzing more cases will aid in counseling for prenatal CMA when there are ultrasound abnormalities.

None.

No fundings to declare.

Conception and design: YJH, DHC. Acquisition of data: BG. Analysis and interpretation of data: KH, SHK. Drafting the article: NKY. Critical revision of the article: HJP, MYK. Final approval of the version to be published: all authors.