In China, congenital anomalies occur at a prevalence of approximately 5.6% (Xu et al., 2020). These developmental defects result from aberrant fetal morphogenesis in utero, potentially affecting the fetus’s anatomy, physiology, and cognitive development (Zhou et al., 2024). Consequences range from intrauterine fetal demise to congenital malformations and neurodevelopmental disorders. Chromosomal abnormalities, such as aneuploidy, triploidy, and large-scale deletions or duplications, are the primary etiological factors (Kagan et al., 2022). Moreover, submicroscopic chromosomal aberrations and copy number variations (CNVs) contribute significantly to the pathogenesis of congenital anomalies (Cao et al., 2023; Chen et al., 2023). Currently, pregnancy termination following prenatal diagnosis of fetal abnormalities remains the predominant management strategy (Graf et al., 2023).

Amniotic fluid karyotyping has long been regarded as the gold standard for diagnosing chromosomal abnormalities. For 5 decades, this technique has been widely utilized to identify conditions such as aneuploidy, polyploidy, balanced structural rearrangements, and large chromosomal segment abnormalities (Luo et al., 2023). However, this method has several limitations: it requires amniotic fluid cell culture, requires skilled technicians, is time-consuming, and lacks the resolution to detect copy number variations smaller than 5 Mb (Saldarriaga et al., 2015).

To address these limitations, genome-wide copy number variation sequencing (CNV-seq) based on low-depth whole-genome sequencing, combined with quantitative fluorescent PCR (QF-PCR) targeting short tandem repeats and chromosomal microarray analysis (CMA), has been recommended in China for prenatal diagnosis (Clinical et al., 2019; Prenatal et al., 2023). These advanced methodologies offers high-throughput capabilities, rapid turnaround times, and the ability to identify small CNVs(Chen et al., 2024; Qiao et al., 2022; Cai et al., 2023; Santirocco et al., 2021). Notably, CNV-seq demonstrates higher sensitivity, improved resolution, and greater robustness with suboptimal specimens compared to CMA (Cohen et al., 2011; Cohen et al., 2015; Ma et al., 2021). Nevertheless, these methods cannot detect balanced chromosomal translocations (Qiao et al., 2022). Therefore, integrating karyotyping with CNV-seq and QF-PCR may provide a more comprehensive assessment of chromosomal abnormalities.

In this study retrospectively analyzes data from 617 cases that underwent combined testing, aiming to evaluate the clinical utility of integrating karyotyping, CNV-seq, and QF-PCR in prenatal diagnosis.

Our integrated approach combining conventional karyotyping with CNV-seq and QF-PCR yielded a chromosomal abnormality detection rate of 12.5% (77/617), surpassing both isolated karyotyping (9.7% [60/617]) and CNV-seq/QF-PCR alone (8.3% [51/617]). This enhanced detection rate surpasses previously reported rates for CNV-seq as a standalone method and demonstrates comparable efficacy to the combined use of karyotyping and CNV-seq described in the literature (Wang et al., 2018; Zhang et al., 2022; Zhang et al., 2023; Ge et al., 2024).

Karyotyping remains the gold standard for chromosomal analysis, effectively detecting structural aberrations larger than 5–10 MB, aneuploidies, and balanced translocations. Our findings corroborate previous studies (Tang et al., 2024) demonstrating comparable efficacy between karyotyping and CNV-seq/QF-PCR in detecting major structural and numerical abnormalities, including trisomy 21 and 18. Notably, karyotyping identified chromosomal polymorphisms in 3.7% (23/617) of cases, two cases of balanced translocations, and one case of trisomy 20, underscoring its utility in detecting morphological chromosomal variants (Jing et al., 2021). While chromosomal polymorphisms and balanced translocations are not typically considered direct causative factors for congenital anomalies, emerging evidence suggests potential associations with subsequent infertility and oncological developments (Liehr, 2022; Pei et al., 2021; Ralapanawe et al., 2023; Verdoni et al., 2021). Thus, early identification of these variants provides valuable prognostic information for clinical surveillance and management strategies.

Our analysis detected CNVs in 5.3% (33/617) of cases, comprising 2.1% (13/617) pathogenic CNVs and 3.2% (20/617) variants of uncertain significance (VUS), consistent with published literature (Han et al., 2024; Xu et al., 2020; Zhang et al., 2022). These CNVs remained undetectable by conventional karyotyping. Notably, most CNV-carrying fetuses demonstrate no overt ultrasonographic abnormalities during gestation (Hilger et al., 2020; Jiang et al., 2024; Xue et al., 2021), underscoring the limitations of traditional prenatal screening methods and the value of incorporating molecular diagnostic techniques. However, the clinical interpretation of CNVs presents ongoing challenges. We observed two cases where fetuses carrying pathogenic CNVs (22q11.21 and 15q11.2 microduplications) exhibited normal phenotypes postnatally, with asymptomatic maternal carriers. These observations align with growing evidence of incomplete penetrance and variable expressivity in certain pathogenic CNVs(Hilger et al., 2020; Hu et al., 2017; Ramalingam et al., 2011), challenging the traditional paradigm of universal pregnancy termination for all pathogenic variants. Moreover, the detection of VUS poses significant counseling challenges, potentially inducing unwarranted anxiety and precipitous decision-making. In one instance, pregnancy termination was pursued due to family anxiety despite the absence of parental comparative analysis. This highlights the importance of exercising caution when CNVs are detected. Physicians and families should consider parental origin testing to inform decision-making and guide appropriate clinical management.

Although the mechanistic underpinnings and preferential genomic locations of CNVs remain not fully understood, our cohort showed a predominance of 22q11.21 microduplications (6/13) and 16p11.2 microduplications (2/13) among pathogenic CNVs. This distribution differs from previous reports by Zhang et al. (2023), who identified 15q11.2 microdeletions and 22q11.21 microduplications as the most prevalent pathogenic variants. VUS demonstrated no discernible pattern of occurrence. Intriguingly, we observed a preponderance of maternal inheritance for pathogenic CNVs, while VUS showed predominantly paternal inheritance. However, these findings warrant validation in larger cohorts.

Our data demonstrated that the detection rates of chromosomal abnormalities was was significantly higher in high-risk NIPT results (57.6%) compared to other risk categories, validating NIPT’s utility as a screening modality. Nevertheless, these findings reinforce that NIPT should not be employed as a diagnostic tool in isolation.

In conclusion, our findings validate the enhanced diagnostic yield of integrating karyotyping with CNV-seq and QF-PCR. However, the single-center, retrospective nature of this study necessitates further validation in larger, multi-center cohorts.