Application of CNV-seq combined with chromosome karyotyping in prenatal diagnosis of pregnant women with high-risk NIPT results

Non-invasive prenatal testing (NIPT), which utilizes fetal cell-free DNA (cffDNA) in maternal peripheral blood to assess the risk of fetal chromosomal abnormalities, has been widely used for prenatal screening. Chromosome karyotyping and low-depth whole-genome copy number variation sequencing (CNV-seq) are commonly used techniques in prenatal diagnosis, each with distinct advantages and limitations. This study aimed to investigate the application value of CNV-seq and chromosome karyotyping in the prenatal diagnosis of pregnant women with high-risk NIPT results. In this study, 73 pregnant women with high-risk NIPT results were enrolled. The women underwent amniocentesis during the second trimester to obtain amniotic fluid for CNV-seq and karyotyping. The detection rates of fetal chromosomal abnormalities between the two methods were compared in high-risk NIPT pregnancies, and the difference was analyzed using the McNemar test. Among 73 pregnant women with high-risk NIPT results, 48 fetal chromosomal abnormalities were detected by karyotyping and CNV-seq combined. Concordant positive results were obtained for 35 cases, which were predominantly common aneuploidies. In addition, CNV-seq alone identified 12 patients with microdeletions or microduplications, all of which were classified as variants of uncertain significance (VUS). Conventional karyotyping alone detected one case of low-level sex chromosome mosaicism. Moreover, we report the first case of a fetus prenatally diagnosed with sex chromosome mosaicism (47,XXY/46,XX/46,XY), identified using chromosome karyotyping combined with CNV-seq following a high-risk NIPT result for sex chromosomes. CNV-seq and chromosomal karyotyping each have advantages and limitations in the prenatal diagnosis of pregnant women with high-risk NIPT results. While CNV-seq is more effective for detecting microdeletions and microduplications, karyotyping is superior in identifying low-level chromosomal mosaicism and specific karyotype compositions in complex mosaic cases. Notably, we report the first prenatal case of the rare 47,XXY/46,XX/46,XY mosaicism, highlighting the complementary benefit of combined cytogenetic and molecular approaches for detecting complex chromosomal aberrations. Overall, the combined application of CNV-seq and karyotyping is recommended for prenatal diagnoses in high‑risk NIPT pregnancies. This study suggests that NIPT is valuable for screening fetal sex chromosome abnormalities and may providing useful support early prenatal counseling and pregnancy management.