NIPT was routinely performed at our center between January 2022 and December 2024, during which a total of 10,025 pregnancies were screened. Among these, cases that screened positive for sex chromosome aneuploidies and subsequently underwent confirmatory invasive testing were reviewed. The three cases presented are those in which the final diagnostic work-up confirmed chromosomal structural abnormalities. This study was approved by the Institutional Review Board of CHA Gangnam Medical Center (IRB No. GCI 2025-07-006).

NIPT was performed using a massively parallel sequencing approach for whole-genome analysis. Maternal peripheral blood (10 mL) was collected in Cell-Free DNA BCT™ tubes (Streck, Omaha, NE, USA). Plasma was isolated by centrifugation at 1,200 × g for 10 min at 4 °C, followed by a second centrifugation at 16,000 × g for 10 min at 4 °C. Cell-free fetal DNA was extracted from 1 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Library preparation was conducted using the Ion Plus Fragment Library Kit (Thermo Fisher, Waltham, CA, USA). Sequencing was performed on the Ion S5™ XL System (Life Technologies, Singapore) at an average depth of 0.3×. A total of 12–14 samples were processed per Ion 540™ Chip (Thermo Fisher Scientific). Each sample generated >5 million raw reads, with uniquely mapped reads exceeding 65.0%. The assay screened for common autosomal trisomies (T21, T18, T13), chromosomal microdeletions/duplications, and SCAs.

Z-scores for each chromosome were derived from normalized chromosomal representation. Whole-chromosome aneuploidy was determined using Z-score analysis, with |Z| ≥ 3 defined as high risk and −3 < Z < 3 considered low risk.

To assess CGG repeat expansions in the FMR1 gene (OMIM #309550), genomic DNA (gDNA) from amniocytes or peripheral blood was amplified using the LabGscan™ FRAXA PCR Kit (Labgenomics, Gyeonggi-do, Republic of Korea), following the manufacturer’s instructions. The HUMARA assay, a PCR-based method, was used to distinguish maternal and paternal X chromosome alleles and assess methylation status. As previously described (Allen et al., 1992), 0.5–1 μg of gDNA was digested with the methylation-sensitive restriction enzyme HpaII, followed by PCR amplification. PCR products were analyzed on the ABI 3500Dx Genetic Analyzer (Applied Biosystems, CA, USA) using GeneMapper software (Applied Biosystems).

Southern blotting targeting the FMR1 locus was employed to clarify ambiguous PCR results, such as homozygous patterns or failed amplification due to large repeat expansions. DNA (7–10 μg) was digested with EcoRI and EagI (New England Biolabs, Ipswich, MA, USA) (Gold et al., 2000). A biotin-labeled probe (StB12.3: forward 5′-CGC​CAA​GAG​GGC​TTC​AGG​TCT​CCT-3′; reverse 5′-GAG​ACT​GTT​AAG​AAC​ATA​AAC​GCG​GG-3′) was used for hybridization. Signal detection was performed using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Rockford, USA).

Fragment-based molecular assays were performed to assess clinically relevant copy-number changes and sex-chromosome abnormalities. Multiplex Ligation–Dependent Probe Amplification (MLPA) using SALSA probemixes P245 Microdeletion Syndromes-1A, P070 Subtelomeres Mix-2B, and P185 Intersex (MRC Holland, Amsterdam, Netherlands), following the manufacturer’s instructions. PCR products were analyzed using the ABI 3500Dx Genetic Analyzer and GeneMarker® software v3.0.1 (SoftGenetics, LLC, State College, PA, USA). Devyser Compact kit (Devyser, Hägersten, Sweden) for the rapid detection of trisomies 21, 18, and 13, and SCAs were performed according to the manufacturer’s protocols. Y-chromosome microdeletion testing targeted the azoospermia factor (AZF) region using standard sequence-tagged sites (STS) markers. Devyser Compact kit and Y-chromosome microdeletion testing amplified products were analyzed by capillary electrophoresis on the ABI 3500Dx platform and GeneMapper software v6 (Applied Biosystems).

Karyotyping and CMA were performed on samples obtained via amniocentesis or chorionic villus sampling. Cells were cultured in BIO-AMF™ medium (Biological Industries, Cromwell, CT, USA), and metaphase spreads were prepared following standard G-banding protocols. A total of 20–30 metaphase cells were analyzed per sample. CMA was conducted using the Affymetrix CytoScan 750K array (Affymetrix, Santa Clara, CA, USA), comprising >750,000 oligonucleotide probes (including single-nucleotide polymorphism and non-polymorphic probes). Data were analyzed using the Chromosome Analysis Suite software (Affymetrix), based on the hg19 human genome assembly.

A 38-year-old woman (G2P1) who conceived via in vitro fertilization (IVF) underwent NIPT at 12+1 weeks’ gestation due to advanced maternal age. Routine first-trimester ultrasonography revealed no fetal abnormalities. Maternal anthropometric characteristics, including weight, height, and BMI, were unremarkable. NIPT indicated a high risk for monosomy X (fetal fraction [FF] = 6.44%; X chromosome Z-score = −3.13) (Figure 1A). Following genetic counseling, karyotyping and CMA were performed at 16+3 weeks of gestation. The fetal karyotype was 46,XX, but CMA detected a 3.6 Mb deletion in the Xq27.3–q28 region [arr (Xq27.3q28) (146,806,191_150,386,543)×1] and a 4.8 Mb duplication in the Xq28 region [arr (Xq28) (150,408,165_155,233,731)×3] (Figure 1B). The maternal CMA result was normal.

To further clarify whether the FMR1 region within this deleted segment was specifically affected, we performed FMR1 TP-PCR and Southern blot analysis. TP-PCR fragment analysis indicated that both mother and fetus had homozygous normal alleles (29 CGG repeats). Southern blotting showed a normal female pattern in the mother (2.8 kb active and 5.2 kb inactive X chromosomes), while the fetus exhibited only the 2.8 kb band, indicating a single active X chromosome (Figure 1C). To assess whether the X chromosome harboring the FMR1 deletion was subject to inactivation, the HUMARA assay was performed. Following digestion with the methylation-sensitive enzyme HpaII, only the paternally derived 269 bp allele was amplified in the fetus, indicating extremely skewed X-chromosome inactivation (XCI) (Figure 1D). The 4.8 Mb duplication in Xq28, encompassing MECP2 (OMIM #300005), was confirmed by MLPA, providing concordant genomic support for the structural abnormality initially detected by CMA (Figure 1E). Paternal CMA was not performed. The Xq27.3–q28 deletion involved neurodevelopmentally relevant genes, including FMR1 and AFF2, which have been associated with X-linked intellectual disability and cognitive/behavioral impairment (Wu and Li, 2022; Tekendo-Ngongang et al., 2021). In addition, the 4.8 Mb Xq28 duplication encompassed MECP2, overexpression of which is linked to MECP2 duplication syndrome, characterized by severe neurodevelopmental deficits (Akaba and Takahashi, 2025). These potential impacts were discussed during genetic counseling, and the patient elected to terminate the pregnancy at 21 weeks of gestation.

A 33-year-old woman (G1P0) underwent NIPT at 12+0 weeks of gestation. NT measured 1.1 mm, with no ultrasonographic anomalies. NIPT indicated a high risk for monosomy X and a vanishing twin pattern (FF = 9.92% for the X chromosome, X chromosome Z-score = – 8.54; FF = 1.46% for the Y chromosome, Y chromosome Z-score = 5.43) (Figure 2A). At 16+0 weeks, QF-PCR, karyotyping, and CMA were performed. QF-PCR of amniocytes indicated an XY pattern, but the peak was absent at Y-specific markers ZFYX and DYS448 (Figure 2B). Karyotyping of cultured amniocytes revealed a mosaic karyotype: 46,X,?r(Y) (p11.32q11.223)[20]/45,X [9] (Figure 2C). CMA identified a 7.4 Mb duplication in Yp11.31–p11.2 region [arr (Yp11.31p11.2) (2,650,141_10,073,965)×1∼2] and a 15 Mb deletion in the Yq11.21–q11.23 region [arr (Yq11.21q11.23) (13,800,703_28,799,937)×0] (Figure 2D). Parental testing was not performed; thus, the origin of the ring chromosome remains unknown. Following genetic counseling, the patient opted for termination of pregnancy at 20+6 weeks.

A 35-year-old woman (G1P0) underwent NIPT at 11+6 weeks of gestation. NT measured 1.1 mm, with no sonographic anomalies. The patient was underweight (height: 150 cm; weight: 41 kg; BMI: 18.2). Apart from being underweight, the mother did not present with any neurological, endocrine, gynecologic, or dysmorphic features of clinical concern. NIPT revealed a high risk for XYY (FF = 34.20%, X chromosome Z-score = – 2.61; Y chromosome Z-score = 6.46) and a 12.97 Mb microdeletion in the Xp22.33–p22.2 region (Figure 3A). After counseling, karyotyping, and CMA were performed on both the fetus and the mother at 16+5 weeks. QF-PCR of maternal blood showed a heterozygous pattern at the STR markers T3, DXS1187, T1, XHPRT, and DXS2390, indicating a normal female genotype (Figure 3B). However, the presence of a peak at the Y-specific DYS448 marker (Yq11.223) prompted Y chromosome microdeletion analysis to confirm the presence of the Y chromosome. The analysis identified six STSs in the AZFb region, each showing detectable peaks (sY117, sY134, sY152, sY157, sY158, and sY1206) (Figure 3C). MLPA revealed single-copy loss of SHOX (rsaXp22.33/Yp11.3 [SHOX]×1) and triplication of VAMP7 (rsaXq28/Yq12 [VAMP7]×3) (Figure 3D).

Maternal karyotyping identified a translocation: 46,X,der(X)t (X; Y) (p22.31; q11.222). CMA revealed a 15.6 Mb deletion in the Xp22.33–p22.2 region [arr (Xp22.33p22.2) (168,546_15,766,553)×1] and an 8.2 Mb duplication in the Yq11.222–q11.23 region [arr (Yq11.222q11.23) (20,596,144_28,799,937)×1] (Figure 4A). These findings indicate that the estimated maternal chromosomal structure is 46,X,der(X)t (X; Y) (p22.2; q11.222) (Figure 4B). Invasive testing of the fetus confirmed a normal male genotype, and the pregnancy was continued.

Taken together, a concise summary of the NIPT results, invasive diagnostic findings, definitive diagnoses, and clinical relevance of the three cases is provided in Supplementary Table S1.