Abnormal Ultrasonography Overcomes NIPT's Inherent Limitations: Revealing Two Cases of NIPT False Negatives Caused by Trisomy 21 Mosaicism and a Literature Review.

Background: Non-invasive prenatal testing (NIPT) is widely adopted for its high sensitivity in detecting chromosomal abnormalities, but its accuracy for placental and fetal mosaicism is limited due to reliance on placental DNA, leading to false results and missed diagnoses. Prenatal ultrasound may signal chromosomal risks via soft markers. This study investigates ultrasound's diagnostic value for mosaicism in NIPT false-negative cases. Materials and Method: A retrospective analysis was conducted on two unique cases of trisomy 21 diagnosed at our hospital's prenatal diagnosis center. Case One: At 16+2 weeks of gestation, the pregnant woman's NIPT result was negative. However, ultrasound later revealed fetal cardiac abnormalities. Amniocentesis was performed for fetal karyotype analysis and chromosome copy number variation sequencing (CNV-Seq) and whole-exome sequencing (WES) were conducted. Multiple samples from the umbilical cord and placenta, retained after induced labor, were collected for CNV-Seq detection. Concurrently, peripheral blood chromosome analysis, CNV-Seq, and WES were performed on the fetus's parents. Case 2: At 18 weeks and 5 days of gestation, the pregnant woman's NIPT returned negative results. However, the subsequent ultrasound examination revealed several soft markers, prompting the performance of amniocentesis. Fetal karyotyping and chromosomal copy number variation sequencing (CNV-Seq) were subsequently performed. The pregnant woman signed the informed consent form before the test. This study received review and approval from the Ethics Committee of Suining Central Hospital (No.: KYLLMC20240020). Results: In Case One, karyotype analysis of amniotic fluid cells revealed 46,X? psu idic(21)(q22.3), while all placental tests returned normal results. Only the umbilical cord exhibited trisomy 21 syndrome, due to inconsistent karyotypes between the fetus and placenta. In Case Two, the amniotic fluid karyotype was 47,XY,+21[14]/46,XY[50] (mosaicism ratio 22%), indicating low fetal mosaicism NIPT failed to detect both instances of placental and fetal mosaicism, because it relies on the concentration and proportion of fetal free DNA in maternal plasma. Conclusion: Non-invasive prenatal testing (NIPT) exhibits inherent limitations in detecting placental and fetal mosaicism, whereas ultrasound soft indicators function as a "gatekeeper" in prenatal screening, thereby providing clinicians with critical evidence for decision-making.