Between 18 April 2017 and 30 September 2019, 36,456 blood samples were sent for NIPT. The patient and pregnancy characteristics are shown in Table 1. The cohort included 46% elevated-risk and 54% low-risk patients. The mean gestational age at the time of blood draw was 12 weeks and 2 days (range 91/7-326/7). In 76.2%of the patients, blood was drawn in the first trimester of pregnancy. Maternal age ranged from 18 to 48 years, with a mean of 35.4 years. The average fetal DNA fraction was 9.54% ± 3.72%. Among all pregnancies, 5% (1807/36456) were conceived using assisted reproductive technologies (ART) and 0.04% (16/36456) were conceived through egg donation. The latter were excluded from the average maternal age calculations.
Table 1
Demographic characteristics of women requesting cell-free DNA (cfDNA) screening for common trisomies and sex chromosome aneuploidies.
Characteristics
|
n (%) unless otherwise stated
|
Low risk NIPT results
|
35955 (98.6)
|
Singleton pregnancies (%)
|
35650 (97.8)
|
Twin pregnancies (%)
|
800 (2.2)
|
Triplet pregnancies,(%)
|
6 (0.02)
|
ART Pregnancies(%)
|
1807 (5)
|
Twin pregnancies (%)
|
403 (22.3)
|
Egg donation (%)
|
16 (0.9)
|
Maternal age (yrs) mean (range)*
|
35.4 (18-48)
|
<35(%)
|
19693 (54)
|
≥35(%)
|
16763 (46)
|
Gestational age (wks+days) mean (range)
|
12+2 (9+1-32+6)
|
Samples drawn in first trimester (< 13+6 wks)
|
27780 (76.2%)
|
Fetal fraction mean± SD
|
9.55±3.72
|
Fetal fraction first trimester mean± SD
|
9.53±3.69
|
ART =Artificial Reproductive Technology
*We excluded pregnancies with egg donation.
|
Among the cohort of 36456, 2.2% (800) were twin pregnancies and 0.02% (6) were triplet pregnancies. Of the 800 twin pregnancies, 718 (89.7%) were dichorionic and 82 (10.3%) were monochorionic. The mean maternal age among twin pregnancies was 38.2 (range 34.3–45.0) years, and therefore slightly higher than that in the whole cohort. The mean gestational age at sampling in twin pregnancies was similar to that in the whole cohort: 12 weeks and 3 days (range 101/7-196/7). Approximately 50% (403/800) of twin cases were conceived using assisted reproductive techniques.
Overall, 35955 (98.6%) patients received a low-risk NIPT result, and 501(1.4%) received a high-risk NIPT result (including SCA) in singleton pregnancies. The high-risk results were 254 T21, 69 T18, 33 T13 and 145 SCA. Thus, SCA comprised 28.9% of the abnormal results. Elevated risk results for classic trisomies were found in 1.5% (12/806) of multiple pregnancies and 1% (344/35650) of singleton pregnancies.
Test Results and Pregnancy Outcome Data (Tables 2 and 3)
Table 2
NIPT Performance for Detecting Trisomies 21, 18, and 13 and Sex Chromosome Aneuploidies: cases with high risk NIPT results, including singleton and twin pregnancies.
|
Loss to follow-up
|
No Confirmatory Testing
|
Confirmatory Diagnostic Testing
|
Nipt results
|
n
|
|
IUFD (n)
|
TOP (n)
|
TP (n)
|
FP (n)
|
PPV% (95% CI)
|
T21
|
254
|
1
|
4
|
0
|
247
|
2
|
99.2 (99.1, 99.3)
|
T18
|
69
|
|
0
|
1
|
62
|
6
|
91.2 (91.0, 91.6)
|
T13
|
33
|
|
1
|
0
|
27
|
5
|
84.4 (84.5, 85.2)
|
SCA
|
145
|
|
8
|
2
|
117
|
18
|
86.7 (86.2, 87.9)
|
Total
|
501
|
1
|
13
|
3
|
453
|
31
|
93.6 (91.44, 95.76)
|
CI, confidence interval; IUFD, intrauterine fetal demise; n, number; NIPT, non-invasive prenatal test; PPV, positive predictive value; SCA, sex chromosome aneuploidy; TOP, termination of pregnancy; T, trisomy; TP, true-positive; FP, false positive |
Table 3
NIPT Performance for Detecting Trisomies 21, 18, 13 and Sex Chromosome Aneuploidies: all cases with cytogenetic or clinical follow-up.
n = 36000
|
TP
|
FP
|
TN
|
FN
|
Sensitivity
TP/(TP + FN)
% (95% Cl)
|
Specificity
TN/(TN + FP)
% (95% Cl)
|
PPV
TP/(TP + FP)
% (95% Cl)
|
NPV
TN/(TN + FN)
% (95% Cl)
|
T21
|
247
|
2
|
35410
|
0
|
100
247/247
|
99.99
35410/35412
|
99.20
247/249
|
100
35410/35410
|
|
|
|
|
|
(98.47, 100.0)
|
(99.98, 100.0)
|
(98.10, 99.30)
|
(99.99, 100.0)
|
T18
|
62
|
6
|
35591
|
0
|
100
62/62
|
99.98
35591/35597
|
91.20
62/68
|
100
35591/35591
|
|
|
|
|
|
(94.17, 100.0)
|
(99.96, 100.0)
|
(84.54, 97.86)
|
(99.99, 100.0)
|
T13
|
27
|
5
|
35627
|
0
|
100
27/27
|
99.99
35627/35632
|
84.40
27/32
|
100
35627/35627
|
|
|
|
|
|
(87.54, 100.0)
|
(99.97, 100.0)
|
(83.15, 96.90)
|
(99.99, 100.0)
|
SCA
|
117
|
18
|
35524
|
0
|
100
117/117
|
99.95
35524/35542
|
86.7
117/135
|
100
35524/35524
|
|
|
|
|
|
(96.82, 100.0)
|
(99.92, 99.97)
|
(81.0, 92.38)
|
(99.99, 100.0)
|
All
|
453
|
31
|
35175
|
0
|
100
453/453
|
99.91
35175/35206
|
93.6
453/484
|
100
35175/35175
|
|
|
|
|
|
(99.19, 100.0)
|
(99.88, 99.94)
|
(91.44, 95.76)
|
(99.99, 100.0)
|
CI, confidence interval; T21, trisomy 21; T18, trisomy 18; T13 trisomy 13; SCA, sex chromosomal aneuploidy; TP, true positive; FP, false postive; FN,false negative; NPV, Negative Predictive Value |
Follow-up data were available in 98.7% of cases; in 35516/35955 (98.8%) of women with low-risk results, 472/489 (96.5%) of women with singleton pregnancies and high-risk results, and 12/12 (100%) of women with multiple pregnancies and high-risk results. In the cohort with low-risk NIPT results and follow-up data, we received the results of invasive testing for 120 women (0.3%) and clinical outcome data based on newborn physical exam via physician or parent reporting for 35396 women (99.7%); among the 120 cases with diagnostic testing results, none reported T21, T18, or T13. In the 35396 cases with normal NIPT results and clinical outcome data, 35055 had live births with a normal newborn physical examination, 153 experienced a pregnancy loss, 96 women opted for termination of pregnancy (TOP) because of foetal abnormalities at ultrasound, 71 chose TOP for personal reasons, and 21 had neonatal demise or other rare diseases (Fig. 1). No false negative results were reported to the laboratory.
Confirmatory testing results were available in 484/501 (96.6%) of the cases with high-risk NIPT results (Fig. 1): 410 amniocentesis, 69 chorionic villus sampling, and 5 cases with products of conception after termination/miscarriage. The 17 cases without confirmatory test results were all singleton pregnancies: in 16 cases, intrauterine fetal death (IUFD) occurred or the pregnancy was terminated because of ultrasound anomalies without confirmatory testing, and in one case, no follow-up was available. In total, 448/484 high-risk NIPT results were concordant with karyotype or SNP array in chorionic villi or amniotic fluid cells, and 5/484 were concordant in products of conception after termination/miscarriage. There were 31/484 (0.08%) false positives: 2 T21, 6 T18, 4 T13 and 18 SCA in singleton pregnancies and 1 T13 in a twin pregnancy. Ten were ascertained in chorionic villi and 21 in amniotic fluid cells.
In total, 800 twin pregnancies and 6 triplet pregnancies were tested (Table 4). Follow-up was available in all cases; confirmatory testing was performed by amniocentesis in all cases. Twelve dichorionic twin pregnancies were reported as having a high risk of being affected: 8 T21, 2 T18 and 1 T13. The T21 and T18 high-risk results were confirmed by follow-up testing, and the T13 was a false positive NIPT result. Of the six triplet pregnancies, one was reported as high risk for T21 by NIPT, which was confirmed in the amniotic fluid. No false negative cases were reported among the multiple pregnancies.
Table 4
NIPT Performance for Detecting Trisomies 21, 18, 13 and Sex Chromosome Aneuploidies in 800 Twin and 6 Triplet Pregnancies.
Twins n = 800
|
TP
|
FP
|
TN
|
FN
|
Sensitivity
TP/(TP + FN)
%(95% Cl)
|
Specificity
TN/(TN + FP)
%(95% Cl)
|
PPV
TP/(TP + FP)
% (95% Cl)
|
NPV
TN/(TN + FN)
% (95% Cl)
|
Trisomy 21 (8)
|
8
|
0
|
792
|
0
|
100
8/8
|
100
792/792
|
100
8/8
|
100
792/792
|
|
|
|
|
|
(59.7,100.0)
|
(99.39, 100.0)
|
(59.7, 100.0)
|
(99.39, 100.0)
|
Trisomy 18 (2)
|
2
|
0
|
798
|
0
|
100
2/2
|
100
798/798
|
100
2/2
|
100
798/798
|
|
|
|
|
|
(19.79, 100)
|
(99.40, 100)
|
(19.78, 100.0)
|
(99.40, 100.0)
|
Trisomy 13 (1)
|
0
|
1
|
799
|
0
|
|
99.77
799/800
|
|
100
799/799
|
|
|
|
|
|
|
(99.13, 100.0)
|
|
(99.99, 100.0)
|
Triplets n = 6
|
|
|
|
|
|
|
|
|
Trisomy 21 (1)
|
1
|
0
|
0
|
0
|
|
|
|
|
CI, confidence interval; TP, true positive; FP, false positive; FN, false negative. |
In 1807 assisted reproductive technology (ART) pregnancies, including 403 twin pregnancies and 16 egg donation pregnancies (Table 5), the test's performance showed 100% sensitivity and specificity for T21 and T18 and a slightly lower specificity for T13 (99.94%) and SCA (99.83%).
Table 5
NIPT Performance for Detecting Trisomies 21, 18, 13 and Sex Chromosome Aneuploidies in ART Pregnancies.
ART pregnancies
n = 1807
|
TP
|
FP
|
TN
|
FN
|
Sensitivity
TP/(TP + FN)
%(95% Cl)
|
Specificity
TN/(TN + FP)
%(95% Cl)
|
PPV
TP/(TP + FP)
%(95% Cl)
|
NPV
TN/(TN + FN)
%(95% Cl)
|
T21
|
9
|
0
|
1798
|
0
|
100
9/9
|
100
1798/1798
|
100
9/9
|
100
1798/1798
|
|
|
|
|
|
(62.88,100.0)
|
(99.73, 100.0)
|
(62.88, 100.0)
|
(99.79, 100.0)
|
T18
|
2
|
0
|
1805
|
0
|
100
2/2
|
100
1805/1805
|
100
2/2
|
100
1805/1805
|
|
|
|
|
|
(19.79,100.0)
|
(99.73, 100.0)
|
(19.78, 100.0)
|
(99.73, 100.0)
|
T13
|
0
|
1
|
1806
|
0
|
|
99.94
1806/1807
|
-
|
100
1806/1806
|
|
|
|
|
|
|
(99.51, 99.98)
|
|
(99.99, 100.0)
|
SCA
|
7
|
3
|
1797
|
0
|
100
7/7
|
99.83
1797/1800
|
70
7/10
|
100
1797/1797
|
|
|
|
|
|
(64.57,100.0)
|
(99.23, 99.91)
|
(41.58, 98.42)
|
(99.99, 100.0)
|
CI, confidenceinterval; ART, Assisted Reproductive Technology; T21, trisomy 21; T18, trisomy 18; T13 trisomy 13; TP, true positive; FP, false positive; FN,false negative; PPV, Positive Predictive Value |
Of the 254 cases reported as high-risk for T21, 5 singleton pregnancies did not have confirmatory testing. Four of these cases ended in intrauterine foetal death (IUFD), and in one case, we did not have additional data. In 247 (97.2%) cases with confirmatory testing, T21 was confirmed in all cases, and 2 were false positives. This led to a specificity of 99.99% (95% CI 99.98, 100.0) and a PPV of 99.2 (95% CI 98.1, 99.3) (247/249). If we were to consider the 5 cases without confirmatory testing as false positives, which is unlikely for the four cases ending in IUFD since T21 is known to be associated with a high risk of IUFD, the PPV for T21 for the entire cohort would drop to 97.2 % (247/254).The PPV for T21 for the multiple pregnancy cohort was 100% (CI 59.7, 100.0) (9/9), and there were no positive cases without follow-up data. There were no discordant negative cases, resulting in a sensitivity of 100% (95% CI 98.47,100.0) and NPV of 100% (95% CI 99.99, 100.0) (Tables 2,3 and S1).
Of the 69 cases reported as high-risk for T18, 68 had confirmatory results: 62 (91.3%) were true positive and 6 cases were false positives. One singleton pregnancy was terminated because of ultrasound anomalies without follow-up testing. This led to a specificity of 99.98% (95% CI 99.96, 100) and a PPV of 91.2% (95% CI 84.54,97.86) (62/68). If we considered the case without confirmatory testing a false positive, which is unlikely in view of the ultrasound anomalies, the PPV would drop to 89.8% (62/69).The number of T18 cases in the multiple pregnancy cohort was too low (2) to calculate a PPV. No false negative T18 cases were reported. The T18 sensitivity was 100% (95% CI 94.17, 100.0) and the NPV was 100% (95% CI 99.99, 100.0) (Tables 2,3 and S1).
Of the 33 cases reported as high-risk for T13, 27 were confirmed by diagnostic testing, one ended infetal demise without a confirmatory test, and 5 were false positives, resulting in a specificity of 99.99% (95% CI 99.97,100.0) and a PPV of 84.4% (95% CI 83.15, 96.90) (27/32). If we were to consider the case without confirmatory testing a false positive, which is unlikely since T13 is known to be associated with a high risk of IUFD, the PPV would drop to 81.8% (27/33). The number of T13 cases in the multiple pregnancy cohort was too low (1) to calculate a PPV. No false negative T13 cases were reported. Thus, the T13 sensitivity was 100% (95%CI 87.54, 100.0), with an NPV of 100% (95% CI 99.99, 100) (Tables 2,3 and S1). In four out of the twelve false positive results for classic trisomies, a vanishing twin was identified (Table S1).
NIPT indicated 145 singleton pregnancies with SCA. Eight 45,X pregnancies ended in fetal demise without confirmatory testing, and 2 pregnancies were terminated without confirmation (Table 2). A cytogenetic analysis of chorionic villi or amniotic fluid was performed in 135 cases and confirmed 117 positive cases; 18 cases were false-positive. This resulted in an overall specificity of 99.95% (95% CI 99.92, 99.97) and a PPV of 87.7% (95% CI 81.0, 92.38).The specificity was lowest for 45,X and highest for 47,XYY. No false negatives were reported, but this was not expected, since, with the exception of some cases of Turner syndrome, newborns with an SCA do not have a phenotype that prompts karyotyping. Therefore, we did not tabulate the sensitivity or NPV (Table 6). The 145 SCAs included 69 cases of monosomy X, which was 13.8% of the total number of abnormal results. Fifty-two monosomy X cases were confirmed at follow-up karyotyping; 9 were false positives, and 8 cases ended with IUFD without cytogenetic confirmation. Two of the 52 confirmed cases were mosaic in the foetus: 1 case of 45,X[68]/46,X,i(X)(q10)[32] and 1 case of 45X/46XY. One of the 52 confirmed cases was a de novo partial deletion: 46,X,del(X)(q?).NIPT indicated 24 cases of 47,XXX, which was 4.79% of the abnormal results, with 20 cases confirmed at follow-up karyotyping and 4 false positives. NIPT indicated 42 cases with 47,XXY, 8.38% of the abnormal results, with 35 of those being confirmed at follow-up karyotyping and 5 false positives. Two had TOP before the gestational age of 90 days, the legal gestational age for termination of pregnancy for social reasons in Italy [23], and remained without cytogenetic confirmation. In the cases where NIPT indicated 47,XXY,karyotyping in amniotic fluid identified one case with 48,XXYY and one case with 49,XXXXY. NIPT also indicated 10 cases with 47,XYY, 1.99% of the abnormal results, and all were confirmed by follow-up karyotyping (Table 6). Finally, we identified 11 cases of maternal sex chromosome aneuploidy. Maternal karyotyping was recommended because of high LLR and/or NCV_X scores: 6 of these were mosaic for 45,X0, 4 were identified as 47,XXX, and one was a mosaic 47,XXX/45,X0 (Table S2). None of these women had conceived pregnancy naturally. Overall, 29% of the abnormal results were anomalies of the sex chromosomes; 51%were trisomy 21 and 20% were trisomy 18 or 13.
Table 6
NIPT Performance for detecting individual sex chromosome aneuploidies.
n = 145
|
TP
|
FP
|
TN
|
FN
|
Specificity
TN/(TN + FP)
%(95% Cl)
|
PPV
TP/(TP + FP)
%(95% Cl)
|
47,XXX
|
20
|
4
|
111
|
0
|
96.52
111/115
|
83.3
20/24
|
|
|
|
|
|
(93.12,98.92)
|
(68.40, 98.20)
|
47,XXYº
|
35
|
5
|
95
|
0
|
95.00
95/100
|
87.5
35/40
|
|
|
|
|
|
(89.62, 98.01)
|
(78.30, 97.90)
|
45, X°°
|
52
|
9
|
74
|
0
|
89.16
74/83
|
85.2
52/61
|
|
|
|
|
|
(81.09, 94.33)
|
(79.16,94.84)
|
47,XYY
|
10
|
0
|
125
|
0
|
100
125/125
|
100
10/10
|
|
|
|
|
|
(96.28, 100)
|
(96.28, 100.0)
|
CI, confidence interval; TP, true positive; FP, false positive; FN, false negative. |
° Karyotyping in amniotic fluid revealed one was a case of 48, XXYY and one was a case of 49, XXXXY |
°° Karyotyping in amniotic fluid revealed one partial deletion 46,X,del(X)(q?)dn and two mosaics: 45X/46XY and mos45,X[68]/46,X,i(X)(q10)[32] |
Test failure
In 1497 (4.1%) of the 36456 samples, the results could not be reported at the first attempt (Figure 2). In 10 (0.03%) cases, there were “administrative errors”, including problems with collection or transportation and haemolytic samples. In 1163 (3.2%) cases, the fetal fraction was below 4% and failed according to Italian regulations even though they had passed iFACT [24, 25]. An additional 280 cases failed iFACT; 32 samples classified as data outside of the expected range (DOER) and 12 samples failed because of fragment size out of the expected range. The test was repeated on a new sample in 1163 women with FF<4%, and in 6 of these women, the test had to be repeated twice before a result could be issued. In all women with initial failure because of FF < 4%, a result could be reported in a new sample where the FF eventually surpassed 4%. The 280 samples with iFACT failure and the 32 with DOER were repeated on the second tube drawn at the first sampling and stored; of them, 208 were reportable. A second blood sample was taken from 80 patients with a second iFACT failure; 12 patients with failure due to fragment size outside of the expected range and 10 patients with “administrative” reasons for primary failure. In the 24 samples with DOER that could not be reported after a second tube testing, the data of all autosomes were analysed; rare autosomal trisomies, large deletions and/or duplications (>7 MB), or complex maternal genomic profiles explaining QC failure were seen in all 24 samples (Figure 3). For 1443 out of 1453 repeated tests, a conclusive result for a classic trisomy or SCA could be issued. None of the failures affected twin or triplet pregnancies. In total, including repeated testing, a conclusive result was reported in 99.97% of singleton pregnancies and in all multiple pregnancies.