Chromosome microarray analysis combined with karyotype analysis is a powerful tool for the detection in pregnant women with high-risk indicators

Amniotic fluids or fetal umbilical cord bloods from 3336 cases with prenatal diagnostic indicators were successfully analyzed by CMA and karyotyping. 2911 cases only had a single prenatal diagnosis indicator: 1044 cases were AMA; 234 cases were NT ≥ 2.5 mm; 260 cases were abnormal result on MSS; 281 cases were high-risk of NIPT; 509 cases were anomaly on US; 318 cases were APH; 265 cases were other indicator (Table 1). Among 2911 cases with single indicator, 129 cases (129/2911, 4.43%) were aneuploidy; 30 cases (30/2911, 1.03%) were balanced chromosome structural rearrangements (or balanced chromosome abnormalities, BCAs); 71 cases (71/2916, 2.44%) were pathogenic and likely pathogenic CNVs (Table 1).

The remaining 425 cases had two high-risk indicators. Especially the group of AMA combined another high-risk indicator occupied a large portion, with a total of 340 cases. In 425 cases with multiple indicators, 29 cases (29/425, 6.82%) were aneuploidy; 8 cases (8/425, 1.88%) were BCAs; 9 cases (9/425, 2.12%) were pathogenic and likely pathogenic CNVs (Table 1).

A total of 1044 cases with single AMA performed CMA and karyotype analysis. 1044 samples were divided into seven subgroups according to the maternal age (35, 36, 37, 38, 39, 40, ≥ 41). Aneuploidy rate increased as pregnancy age increased, while pathogenic or likely pathogenic CNVs were not related to pregnancy age (Table 2; Fig. 1A). The results indicated that 20 samples were pathogenic or likely pathogenic chromosome abnormalities, including 8 (8/1044, 0.77%) cases of aneuploidy and 12 (12/1044, 1.15%) case of pathogenic or likely pathogenic CNVs (Table 2).

In 340 cases of AMA accompanied by another indicator, 51 cases were AMA combined NT (≥ 2.5 mm), 2 cases were AMA combined MSS abnormal result, 35 cases were AMA combined NIPT high-risk, 70 cases were AMA combined US anomaly, 127 cases were AMA combined APH and 55 cases were AMA combined others (Table 1). The results revealed that 26 samples were pathogenic or likely pathogenic chromosome abnormalities, including 21 (21/340, 6.18%) case of aneuploidy and 5 (5/340, 1.47%) case of pathogenic or likely pathogenic CNVs (Table 1; Fig. 1C). The detection rate of aneuploidy in group AMA combined NT ≥ 2.5 mm was significantly higher than that in group AMA (p < 0.001); the detection rate of aneuploidy and pathogenic/likely pathogenic CNVs in the group of AMA combined NIPT high-risk were higher than that in the group of AMA (p < 0.001, p = 0.027) (Fig. 1C).

A total of 234 pregnancies were single NT (≥ 2.5 mm). The cases were divided into five types according to the specific value of NT (2.5–2.9, 3.0-3.4, 3.5–4.4, 4.5–5.4, ≥ 5.5 mm). The relationship between NT value and pathogenic or likely pathogenic chromosome abnormality rate was analyzed (Table 3; Fig. 1B). The result revealed that aneuploidy rate increased as NT value increased, while pathogenic or likely pathogenic CNVs were not related to NT value (Table 3; Fig. 1B).The data declared that 24 cases were pathogenic or likely pathogenic chromosome abnormalities, including 21 (21/234, 8.97%) case of aneuploidy and 3 (3/234, 1.28%) case of pathogenic or likely pathogenic CNVs (Table 3).

In 72 cases of NT ≥ 2.5 mm accompanied by another indicator, 51 cases were NT ≥ 2.5 mm combined AMA, 2 cases were NT ≥ 2.5 mm combined high-risk of NIPT, 10 cases were NT ≥ 2.5 mm combined US anomaly, 6 cases were NT ≥ 2.5 mm combined APH and 3 cases were NT ≥ 2.5 mm combined others (Table 1). The results suggested that 12 (12/72, 16.67%) cases were pathogenic or likely pathogenic chromosome abnormalities, including 9 (9/72, 12.50%) cases were aneuploidy and 3 cases (3/72, 4.17%) were pathogenic and likely pathogenic CNVs (Table 1; Fig. 1D ).

281 cases with single high-risk of NIPT were identified in this research. The high-risk of NIPT were divided into five types, chromosome 13 (Chr 13), chromosome 18 (Chr 18), chromosome 21 (Chr 21), sex chromosomes (Chr sex) and other chromosomes (Chr other) except Chr 13, Chr 18, Chr 21 and Chr sex. CMA and karyotype analysis were performed to verify the abnormal NIPT results. 25 cases were NIPT abnormal on Chr 13, including 2 confirmed cases of trisomy 13; 29 cases were NIPT abnormal on Chr 18, including 9 confirmed cases of trisomy 18 aneuploidy and 1 confirmed case of mosaic; 44 cases were NIPT abnormal on Chr 21, including 27 confirmed cases of trisomy 21 and 3 confirmed case of mosaic, and 2 cases of pathogenic or likely pathogenic CNVs; 100 cases were NIPT abnormal on Chr sex, including 40 confirmed cases of aneuploidy 6 confirmed case of mosaic, and 4 cases of pathogenic CNVs; 83 cases were NIPT abnormal on Chr other, including 2 confirmed cases of mosaic aneuploidy and 7 cases of pathogenic or likely pathogenic CNVs (Table 4). The false positive rate and the false negative rate of abnormal NIPT were 63.35% and 0%.

44 cases were abnormal NIPT result accompanied by another indicator. Among them, the group of NIPT high-risk combined NT ≥ 2.5 mm and the group of NIPT high-risk combined US anomaly had the highest accuracy, reaching 100% (2/2) and 80% (4/5) respectively (Table 1).

Among the 3336 cases studied in this study, 3222 underwent both CMA and karyotype analysis simultaneously, while 114 cases only underwent CMA due to 110 cases of gestational age > 26 weeks and 4 cases of amniotic fluid cell culture failed. Among the 3222 samples, 148 samples of common aneuploidy and 10 samples of mosaic aneuploidy were detected by CMA; while 144 samples of common aneuploidy, 4 samples of translocation aneuploidy and 13 samples of mosaic aneuploidy were detected by karyotype analysis. It should be noted that the 3 mosaic aneuploidy cases of the mosaic rate ≤ 20% have not been detected by CMA. In addition, 38 samples of BCAs were verified by karyotype analysis (Table 5). It’s purely coincidental that all pathogenic and likely pathogenic CNVs in this study appeared in the samples only undergoing CMA.