In recent years, NIPT has been proven superior in screening for SP fetal chromosomal aneuploidy disorders by a large number of studies at home and abroad and it was recognized as an almost perfect screening method[12–14]. Galeva Slavyana[23]et al reported that the screening effect of twins and singleton was similar, especially the effectiveness of T21 was better. This has given hope to the medical community and the public that NIPT can also be applied in ART. However, few studies have reported data on NIPT in ART compared to NIPT in SP. More importantly, existing studies have focused on T21, T18 and T13, with even fewer studies on SCA. Marco La Verde[18]et al. reported that NIPT was performed on 36,456 singleton and twin pregnancies, and the results showed that NIPT had high accuracy and was suitable for both singleton and twin pregnancies, of which the ART group of 1807 cases (403 cases twin) results were shown the sensitivity and specificity of T21 and T18 were 100%, T13 and SCA were 99.94% and 99.83%, respectively. Yang Cuiyu[24]et al. reported that NIPT was performed on 474 cases ART pregnant twins, and the results showed that the positive predictive values of T21 and T18 were 80.00% (4/5) and 100.00% (1/1).
In this study, 97 out of 94757 samples were excluded from the subsequent result analysis due to multiple factors. According to our study of 91280 samples in the SP group and 3477 samples in the IVF group, the PPV, sensitivity and specificity of T21 in single fetuses in the SP group were 89.12%, 99.53% and 100.00%, and those in the IVF group were 66.67%, 100.00% and 100.00%, and the PPV of screening T21 was within 65–94%[25]. In this study, the PPV value of T21 in IVF singleton was smaller than that in SP group, but there was no significant difference between the two groups (66.67%vs. 89.12%, p = 0.09) (Table 3). The specificity of twin T21 in SP group and the PPV, sensitivity and specificity of twin T21 in IVF group were 100.00%, consistent with previous studies[12, 14, 17]. However, the PPV of T21 twins in IVF group was 100.00%, which was higher than that of single twins in the two groups, which may be related to the bias of the results caused by the small sample size of twins. Although there was a lack of data on T21 for twins in the SP group and the two groups were not comparable, the PPV of T21 of twins in the IVF group was 100.00%, which was higher than that of singleton in the two groups, possibly because of the small sample size for twins. For T18 and T13, the data of the two groups were not comparable due to the lack of data from the IVF group, but their sensitivity and specificity were both ≥ 99.97%, consistent with previous studies[12, 17]. It has been reported that the sensitivity and specificity of NIPT in detecting T21, T18, and T13 are 98.0%-99.6% and 98.8%-99.9%[14]. In the SP group, the PPV, sensitivity and specificity of singleton T18 were 51.85%, 100.00% and 99.99%, and those of T13 were 21.74%, 100.00% and 99.98%. However, the PPV of T13 was lower, which was similar to the results of previous studies[25]. This may account for the low number of T13 positive cases. And according to our results, overall, there was no significant difference in the combined PPV of T21/T18/T13 between the SP group and the IVF group in singleton pregnancies (77.85%vs.57.14%, p = 0.20) (Table 3), consistent with previous studies[15, 25, 26]. In conclusion, NIPT has a high detection rate, sensitivity and specificity in screening chromosomal aneuploidies in singleton pregnancies, and is an ideal screening method for SP pregnant women and IVF pregnant women. However, large-scale clinical data are needed to demonstrate the effectiveness of NIPT in twin pregnancies in future studies.
In this study, the PPV of singleton SCA was 40.00% in the SP group and 50.00% in the IVF group, and there was no significant difference between the two groups (p = 0.57) (Table 3), consistent with previous studies[26, 27]. Among the twins, the lack of data on SCA in the IVF group led to no comparability between the two groups, but the PPV of SCA in the SP group was 100.00%, which was probably because of the lack of data in the twins. In this study, we did not calculate the sensitivity and specificity of SCA. The reasons for this are as follows: firstly, the infants with SCA were not found to have obvious phenotypic abnormalities in the clinical physical examination at birth, and most fetuses with SCA were not diagnosed in the neonatal period[28]. Secondly, the follow-up time of this study was limited, and the follow-up time was up to one month after birth, so the analysis of false negative
Table 5
Details of prenatal diagnosis results and pregnancy outcomes of 27 high-risk NIPT cases in IVF group.
Patients | Maternal age (years) | Gestational age (weeks) | Pregnancy type | NIPT result | Prenatal diagnosis result | Clinical outcome |
Case1 | 37 | 16 + 5 | Singleton | T13 | 46, XN | Delivery |
Case2 | 40 | 16 + 6 | Singleton | T18 | Abnormal ultrasound | Induced labor |
Case3 | 32 | 19 + 6 | Singleton | T18 | 46, XN | Delivery |
Case4 | 34 | 19 + 3 | Singleton | T21 | 47, XN + 21 | Induced labor |
Case5 | 33 | 17 + 2 | Singleton | T21 | 47, XN + 21 | Induced labor |
Case6 | 38 | 17 + 2 | Singleton | T21 | 47, XN + 21 | Induced labor |
Case7 | 28 | 18 + 5 | Twin | T21 | 47, XN + 21 | Induced labor |
Case8 | 30 | 12 + 3 | Singleton | T21 | 47, XN + 21 | Induced labor |
Case9 | 32 | 15 + 5 | Singleton | T21 | 46, XN | Delivery |
Case10 | 29 | 17 + 3 | Singleton | T21 | 46, XN | Delivery |
Case11 | 43 | 15 + 5 | Singleton | T21 | Refuse prenatal diagnosis | premature delivery |
Case12 | 32 | 15 + 6 | Singleton | 45, X | 45, X | Induced labor |
Case13 | 36 | 17 + 1 | Singleton | 45, X | 46, XN | Delivery |
Case14 | 32 | 16 + 5 | Singleton | 45, X | 46, XN | Delivery |
Case15 | 30 | 16 + 5 | Singleton | 45, X | 46, XN | Delivery |
Case16 | 31 | 16 + 5 | Singleton | 45, X | Refuse prenatal diagnosis | Delivery |
Case17 | 31 | 20 + 0 | Singleton | 45, X | Refuse prenatal diagnosis | Delivery |
Case18 | 31 | 18 + 0 | Singleton | 47, XXX | 46, XN | Delivery |
Case19 | 30 | 17 + 4 | Singleton | 47, XXX | 46, [][].14ps+ | Delivery |
Case20 | 24 | 17 + 3 | Singleton | 47, XXX | Refuse prenatal diagnosis | Loss to follow-up |
Case21 | 32 | 13 + 1 | Singleton | 47, XXX | Refuse prenatal diagnosis | Loss to follow-up |
Case22 | 27 | 14 + 3 | Twin | 47, XXX | Refuse prenatal diagnosis | Loss to follow-up |
Case23 | 30 | 18 + 1 | Singleton | 47, XXY | 47, XXY | Induced labor |
Case24 | 39 | 13 + 0 | Singleton | 47, XXY | 47, XXY | Delivery |
Case25 | 28 | 14 + 3 | Singleton | 47, XXY | 47, XXY | Delivery |
Case 26 | 30 | 18 + 1 | Singleton | 47, XXY | 46, XN | Delivery |
Case 27 | 29 | 17 + 1 | Twin | 47, XYY | Refuse prenatal diagnosis | Delivery |
results of SCA may not be accurate.
In addition, according to our study, the lower PPV of SCA compared with autosomal aneuploidies may be due to the lower prenatal diagnosis rate of women with high risk of SCA suggested by NIPT than those with high-risk of common trisomy (58.10%, 251/432; 81.45%, 325/399, p < 0.05). Through follow-up, because some pregnant women with high-risk of SCA chose to terminate their pregnancies, karyotype analysis could not be performed, resulting in the exclusion of these high-risk cases when calculating the PPV of SCA, which may result in a low PPV. In conclusion, NIPT is suitable for SP pregnant women and IVF pregnant women when screening for SCA in singleton pregnancies, but more large-scale clinical data are needed to evaluate the effectiveness of NIPT screening for SCA in twin pregnancies. However, for the sake of the health of IVF children, we recommend that pregnant women with a high-risk of SCA from NIPT undergo further prenatal diagnosis to determine the fetal karyotype to reduce the occurrence of sex chromosome birth defects. In addition, it is necessary to extend the follow-up time of children at high-risk of SCA to help early detection of children with sex chromosome abnormalities and take treatment measures as early as possible.
Accord to our study, NIPT showed high sensitivity and specificity in SP and IVF. Although NIPT is recognized as an ideal screening method for chromosomal aneuploidy, it cannot avoid the occurrence of false negatives and false positives. Among 94757 samples in our study, only one false negative case of T21 was found in the SP group through follow-up results. NIPT data of this case showed low-risk of T21, and the amniotic fluid karyotype analysis verified that it was mosaicism of 47, XY, + 21[67]/46, XY [16]. So, it can be seen that the reason for the false positive of this sample may be caused by the fetal chromosome karyotype of mosaicism, which is consistent with previous studies[15]. Lin Ying [29]et al. reported that mosaicism is an important factor affecting NIPT, and the mosaicism of the placenta may reduce the accuracy of the examination, and mosaicism is the main cause of false negative NIPS. In addition, NIPT detection is also affected by many factors. Since cffDNA was mainly derived from the trophoblast cells of the placental villi, and the continuous turnover of cytotrophoblasts induces apoptosis to release cffDNA into the maternal blood and does not fully represent the fetus, Therefore, differences genetic information between placental and fetal tissue may influence NIPT[7–9]. Zou Yang[30]et al. and Chen Mi[31]et al. reported that the development of ART led to a significant increase in the number of lost twin pregnancies, that residual cffDNA in deceased twins could persist for 16 weeks, and that residual cffDNA may affect NIPT outcomes. Jatsenko Tatjana[32]et al. reported that maternal malignancy can also affect NIPT. In conclusion, it is inevitable that NIPT is affected by many factors, although NIPT is a safe and efficient screening method, which can effectively avoid the occurrence of birth defects. However, NIPT is only a prenatal screening method and cannot replace invasive prenatal diagnosis. Therefore, for the health of children, it is recommended that pregnant women with low-risk of NIPT also receive professional genetic counseling and regular prenatal care.
The reasons for the failure of the 97 samples included low cffDNA, low FF, sample hemolysis, and pregnant women's refusal to collect blood again. According to our data analysis showed that the IVF group had a significantly higher detection failure rate than the SP group (0.63%vs. 0.08%, p < 0.05), in line with the results reported by Galeva Slavyana[23] et al., their study showed that IVF was the most important factor leading to the failure of the first cffDNA sampling test compared with SP in singleton and twin pregnancies, and IVF resulted in a 3.8-fold increased risk of test failure compared with SP. Scott Fergus Perry[9]et al. reported that FF was a key parameter affecting the performance of NIPT, and when FF < 4%, it has a greater impact on the results. However, FF was influenced by many factors. Galeva Slavyana [23]et al. reported that 23495 singleton pregnancies and 928 twin pregnancies were screened for fetal trisomy by cffDNA testing, and the results showed that maternal age, weight, gestational age, twins, mode of conception, and placental protein were independent predictors of cffDNA testing failure. And the risk of trial failure was higher in twins than in singleton pregnancies, mainly because the proportion of twins conceived through in vitro fertilization was higher. Qiao Longwei[[21]et al. reported that NIPT was performed on 2817 singleton fetuses (1409 males and 1408 females) and 86 twins, and the results showed that maternal age, BMI, cffDNA concentration, and the number of twins were negatively correlated with FF. In contrast, gestational age was positively associated with fetal fraction. In our study, the mean FF at the overall gestational age was lower in the IVF group than in the SP group (10.51%vs. 11.23%, p < 0.05) (Table 4), which was consistent with the study of Talbot Anna L[33]. According to our data, the proportion of twins (16.34%vs.1.34%, p < 0.05) (Table 1) in the IVF group and the overall average age (31.23 years vs.30.16 years, p < 0.05) (Table 1) of pregnant women were significantly higher than those in the SP group, and the proportion of twins in the IVF group was about 12 times higher than that in the SP group. There was no significant difference in mean maternal BMI between the IVF and SP groups (23.52 kg/m2vs.24.14 kg/m2, p = 0.48) (Table 1). The mean FF at total gestational weeks was lower in the IVF group than in the SP group. In summary, the significantly higher detection failure rate in the IVF group than in the SP group may be explained by the lower overall FF, the older maternal age, the mode of conception and the significantly higher proportion of twins in the IVF group.