Background

Trisomy 21 (T21, also known as Down syndrome) is one of the most prevalent chromosomal abnormalities worldwide, occurring in approximately 1:700 live births [1]. Non-invasive prenatal testing (NIPT) has rapidly transformed the global prenatal screening landscape for common fetal chromosome aneuploidies because of its high sensitivity and specificity [2, 3]. NIPT evaluates the fetal cell-free DNA (cffDNA) fraction circulating in maternal blood, which can be detected at a gestational age (GA) as early as 9 weeks [4]. NIPT has been applied to screen high-risk patients for fetal aneuploidy as part of antenatal care and has increasingly been utilized in clinical practice.

Compared to other screening modalities, NIPT has unparalleled sensitivity and specificity for trisomy 21 [5, 6]. Over 99% of cases can be detected using NIPT, and the false-positive (FP) rate is less than 0.1% [7]. The cffDNA in maternal plasma originates from apoptotic cytotrophoblasts [8]; thus, in most pregnancies, the genetic components are identical between the placenta and fetal tissues. However, due to confined placental mosaicism, NIPT results may not always be representative of the true fetal karyotype, and both false-negative (FN) and FP results may occur [9,10,11,12]. Placental mosaicism [10], fetal chromosomal rearrangements, vanishing twin or co-twin demise [13], familial chromosomal abnormalities, and malignancy are common causes of FP NIPT results [14].

In contrast, among many clinical follow-up cases evaluated, FN NIPT results involving fetal aneuploidies have been rarely found [15, 16]. The presence of low cffDNA content and placental mosaicism has been associated with some FN findings, while others remain unexplained [17]. The effects of the aforementioned factors on FN NIPT results are unclear. Notably, there is a high possibility that FNs are clinically misdiagnosed, and the causes of FN NIPT results should be investigated. Clinical geneticists should recognize these FN results, and patients should be informed about discordant findings between NIPT and subsequent cytogenetic analyses.

This study reports two cases of fetal T21 associated with placental mosaicism that resulted in one FN NIPT result.

Case presentation

Case 1

A 23-year-old healthy primagravida woman with a single fetus was referred to the First Affiliated Hospital of Gannan Medical University. A serological screening at 12 weeks combined with a nuchal translucency measurement (2.1 mm) suggested a critical risk for fetal T21 of 1 in 529. A NIPT examination at another hospital yielded a negative result at 15 weeks (fetal DNA fraction: 15.67%, chromosome 21 Z scores: − 0.201; Table 1). However, the patient was referred to our hospital at 27GA for routine ultrasonography, which showed that the fetus exhibited right-sided pleural effusion (Fig. 1A). Subsequently, the pregnant woman was referred to a hospital in the city of Guangzhou for further evaluation. The ultrasound scans showed bilateral pleural effusions and nasal dysplasia at 28GA. At 29 weeks, trisomy 21 of the fetus was identified via Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) and Chromosomal Microarray Analysis (CMA) by cordocentesis. The patient was transferred back to our hospital and underwent elective termination at 30GA after genetic counseling and communicating with family members. After gaining the consent from the patient, we retrieved the amniotic fluid, maternal peripheral blood, six placental biopsies (three from the fetal side and three from the maternal side), and umbilical cord tissue at termination and examined these samples in detail to understand the biological basis of the discrepancy.

Table 1 NIPT results for cases 1 and 2
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Fig. 1
figure 1

The ultrasound examination image. A Ultrasound examination result at 27 weeks. B Ultrasound examination result at 22 weeks

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As shown in Table 1, positive Z-scores were not detectable for chromosome 21 among five NIPT tests performed at different laboratories. Although the third-party data before labor induction was found to be greater than 3 (Table 1), the fetal concentration at that time was very high. Placental mosaicism may explain the false negative NIPT result, and no obvious problems were found in the clinical NIPT detection process. In addition, copy number variation using next-generation sequencing (CNV-seq) analysis results suggested that the degree of mosaicism of trisomy 21 varied greatly among different placental tissue sites; in particular, the proportion of mosaicism of trisomy 21 in maternal-side placental tissues was less than 30% (Table 2). Furthermore, the cytogenetics analysis of placental chorionic cells (middle of fetal-side placental tissue) demonstrated a mos 46,XX, + 21,der(21;21)(q10;q10)[86]/46,XX[14] karyotype, indicating that 86% of cells had trisomy 21 (Fig. 2), consistent with the CNV-seq analysis results of the placental tissue from the middle of the fetal side. However, the cytogenetic analysis of the amniotic fluid returned a karyotype of 46,XX, + 21,der(21;21)(q10;q10) without mosaicism, and both parents showed normal karyotypes (Fig. 3).

Table 2 CNV-seq analysis results
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Fig. 2
figure 2

Morphology of placental chorionic cells and G-banded karyotypes. A Morphology of placental chorionic cells cultured on day 21 (× 40). B–E G-banded karyotypes of placental chorionic cells

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Fig. 3
figure 3

G-banded karyotypes of the fetus and his parent. A and B Fetus: 46,XX, + 21,der(21;21)(q10;q10); C and D Mother: 46,XX; E and F Father: 46,XY

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Case 2

A 35-year-old pregnant mother of two healthy children underwent a 17GA NIPT test that yielded a normal result (Table 1). An ultrasound examination at 22GA revealed fetal nasal bone dysplasia (Fig. 1B). After counseling, the couple underwent fetal testing by amniocentesis at 25 weeks, demonstrating a T21 fetal karyotype of 47,XY, + 21. In addition, the CMA results showed a pathogenic 15q11.2 microdeletion. The patient terminated her pregnancy at 27GA, and placental tissue was immediately collected for placental mosaicism analysis (Table 2). The CNV-seq analysis of the placental biopsies confirmed that the placental tissue had T21 mosaicism, with a chimeric ratio ranging from 17 to 30%, and the umbilical cord tissue had a chimeric ratio of 96% (Table 2). Notably, the placental tissue from the middle of the fetal side and the middle of the maternal side also showed T2 mosaicism, with chimeric ratios of 8% and 10%, respectively (Table 2).

Discussion and conclusions

There is growing evidence that fetal DNA circulating in the maternal blood largely arises from placental trophoblastic cells, although fetal tissues also provide a small contribution [20]. Since cell-free DNA (cfDNA) was identified, NIPT has been widely promoted for prenatal screening for T21, T18, and T13 [21]. However, many factors may affect NIPT results, such as placental chimerism, maternal obesity, and maternal cancer [22]. In general, FN results are likely caused by two factors. First, if the proportion of cffDNA does not meet a certain value, the positive signal may be indistinguishable from the background signal. Second, due to placental chimerism, the plasma cffDNA can be derived from an area of the placenta with either no chimerism or a low proportion of chimerism. Due to advances in cfDNA enrichment techniques, NIPT can achieve lower detection limits than previous approaches. Confined placental mosaicism is the main reason that leads to FP or FN results with NIPT [10]. Placental mosaicism refers to a karyotype difference between placentally and fetally-derived tissues [23]. In this study, we provide information about two rare cases of FN NIPT results with partial T21 caused by placental mosaicism. This situation should be known to clinical professionals, and patients should be informed that discordant NIPT results may occur.

In the first case of placental mosaicism, multiple plasma experiments and CNV-seq analyses of distinct areas of placental tissue revealed that the NIPT negative results are likely attributed to the low placental mosaicism. However, amniotic fluid cytogenetic analysis revealed 46,XX, + 21,der(21;21)(q10;q10) without mosaicism, and both parents had normal karyotypes. Accordingly, this 21q;21q rearrangement was a de novo fetal chromosomal 21q rearrangement. According to some related research reports, most 21q;21q rearrangements are isochromosomes [24], and Down syndrome resulting from a de novo isochromosome 21q is more likely to lead to a FN NIPT result than standard karyotypes (47,XN, + 21) [25, 26]. Interestingly, the karyotype of placental chorionic cells (derived from the placental tissue from the mid-fetal side) was 46,XX, + 21,der(21;21)(q10;q10)[86]/46,XX[14]. To the best of our knowledge, this study investigates placental mosaicism from a cytogenetic perspective for the first time [25]. These results indicate that placental mosaicism caused by 21q;21q rearrangements is almost certainly a biological cause of FNs.

Regarding placental mosaicism in the second case, the CNV-seq analysis revealed a low T21 mosaicism percentage in all the different regions of placental tissue tested. Unexpectedly, placental biopsies taken from the middle of the maternal side and the middle of the fetal side also had variable T2 mosaicism levels of 10% and 8%, respectively. Altogether, the percentage of total chimerism was less than 30% in all the placental biopsies. The above results indicated that the NIPT negative results are also likely attributed to the low placental mosaicism.

In order to examine the correlation between the mosaic proportions of placental tissue and the Z-score for T21 of the NIPT, a search was conducted for published cases of false negative NIPT results due to T21. Regrettably, the majority of FN NIPT cases did not identify the placental biopsy tissues. Ultimately, a total of five FN NIPT cases were collected (Table 3). The current study's results indicate that the Z-score for T21 of the NIPT does not consistently reflect the T21 level present in placenta (Table 3). These findings have implications for both clinicians and patients, as they highlight the complexity of cfDNA screening biology.

Table 3 Published cases of false negative NIPT results due to T21
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The cases discussed here emphasize the importance of and the necessity for the complementary ultrasonographic control when NIPT results are negative. Therefore, clinicians and patients must understand that NIPT is a screening test. Individuals with negative NIPT results should be provided regular ultrasound monitoring to prevent misdiagnoses and should undergo further prenatal diagnostics, if necessary.