Low High Unclear
The high-risk classification was done on the basis of first- and second-trimester screening and/or other individual risk factors, including abnormal serum screening findings, advanced maternal age, family history of chromosomal anomaly, previous aneuploid pregnancy, sonographic markers or ultrasound anomalies. Advanced maternal age was the main or only indication for NIPT in seven studies [46, 52, 57, 61-63, 70]. NIPT was predominantly indicated on the basis of FCT results in five studies [48, 49, 60, 66]. The threshold for high-risk classification in these studies ranged from 1 in 200 to 1 in 300. Several studies included women with anxiety, not suitable for invasive tests, or who just wished to have NIPT for other reasons [50, 52, 60-63, 67, 68, 70]. The proportion of women with no risk indication was 15% or less in all studies.
Four of the 25 studies used the Harmony® prenatal test (Ariosa Diagnostics Inc., San Jose, CA, USA) [52, 63, 67, 69]. The rest of studies used the WGS method. Four specified using the rama test or sending samples to Natera (San Carlos, CA, USA) [49, 56, 60, 66], one offered Pano-rama and Prendia (Genesupport, Switzerland) [123], two reported usuing MaterniT 21 or sending samples to Sequenom Laboratories [55, 70], two used the Verifi™ test (Illumina Inc., San Diego, CA, USA) [50, 68], one sent samples to LifeCodexx [62], three sent samplers to Berry Genomics [57, 58, 65] and two sent samples to BGI Laboratories in China [47, 64]. Eight other studies per-formed in-house testing using WGS methods; five used Illumina sequencing platforms [48, 50, 61]
and two used the Ion PGM and the Torrent™ Personal Genome Machine™ (PGM) System (Life Technologies, CA, USA) [46, 59]. Three other NIPT-based studies reported that NIPT was per-formed by Berry Genomics, China [57, 58, 65], and one study sent samples to LabGenomics Clin-ical Laboratory (Korea) [54]. Three studies failed to report the testing method [53, 64, 67]. Twelve studies reported that NIPT was blinded to reference standard results [46, 50, 54, 57, 61, 62, 64, 65, 68-70].
The reference standard (full karyotyping results obtained with CVS or amniocentesis) was per-formed in all pregnancies in 18 of the 27 studies [46-49, 54, 57, 59, 61, 62, 65-70, 123]. In the rest, invasive testing was used only for verification of positive cases; negative cases were verified by follow-up.
Risk of bias and QoE
All but two of the studies [68, 70] on the high-risk pregnant population were judged “at risk of bias”
and/or as having “concerns regarding applicability” (one or more domains were judged as “high” or
“unclear”). Twenty of the 25 studies (80%) had a high or uncertain risk of bias in the flow and timing domain. With respect to the reference standard, 11 of the 25 studies (36%) had an unclear or high risk of bias. The proportion of studies with uncertain/high risk of bias in the index domain rose to approximately 16% (4 of 25). Twenty-one studies had an unclear or high risk of bias with respect to the patient selection domain (Figure 4).
With regard to applicability, almost all studies had a low concern for the index test and reference standard domains. However, 19 of the 25 studies (76%) had an unclear/high concern regarding applicability for the patient selection domain because of of the uncertainties regarding patient en-rolment (Figure 5).
Evidence assessment of individual studies can be reviewed in Appendix 1 (Table A8).
Figure 4: Risk of bias assessed by the QUADAS-2 tool for noninvasive prenatal testing in women with high-risk singleton pregnancies
Figure 5: Concern regarding applicability of the use of the QUADAS-2 tool for noninvasive prenatal testing in women with high-risk singleton pregnancies
According to the GRADE approach, the QoE of NIPT accuracy for T21 was moderate for sensitivity and low for specificity as all studies were judged “at risk of bias” or as having “concerns regarding of applicability”. However, for T18 and T13, the QoE was low or even very low because of the presence of risk of bias, publication bias and/or imprecision of sensitivity estimations. Tables of GRADE assessment can be found in Appendix 1 (Tables A9–A12).
5.2.3 NIPT as an add-on to FCT for the high-and intermediate-risk singleton pregnancy population
The only DTA study [71] which focused on high-and intermediate-risk population (risk of 1 in 2500) reported on 4012 women with singleton pregnancies who had undergone contingent NIPT (com-bined serum screening during 11–13 weeks’ gestation followed by NIPT). The risk of 1 in 100 or greater was selected to define the high-risk group. This group was offered the options of CVS, NIPT or no testing. The risk of 1 in 2500 was chosen to select patients who would be offered the options
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testing was performed in 2.7% of the study population. The median maternal age of high- and in-termediate-risk women was 36.1 and 34.8 years, respectively.
Risk of bias and QoE
This study had a high risk of bias for the flow and timing domain. Regarding applicability, low con-cern was identified in the index test and reference standard domains. Evidence assessment of individual studies can be found in Appendix 1 (Table A8).
According to the GRADE approach, the only DTA study performed in the high- or intermediate-risk pregnant population showed a moderate QoE of NIPT sensitivity and specificity for T21. The QoE of NIPT for T18 and T13 sensitivity was low because of high imprecision of pooled sensitivity, whilst NIPT specificity was moderate for these trisomies. Tables of GRADE assessment can be found in Appendix 1 (Tables A9–A12).
5.2.4 NIPT as an add-on to FCT for the high risk population twin pregnancy population The six DTA studies (cross-sectional design) which reported on twin pregnancies had samples size which ranged from 12 to 565 patients. Sarno et al. [39] included singleton and twin pregnan-cies, but only twin pregnancies are taken into account in this section. One of the studies enrolled pregnant women from the general population, with or without prior screening results, who were prospectively tested by NIPT after ART treatment [73] and the other four assessed women for whom NIPT was indicated because of high risk. The indication for screening in this last group was based on advanced age, abnormal ultrasound findings, previous affected pregnancy or positive serum screening (n=1) [72]; abnormal serum screening, sonographic markers or anxiety (n=1) [75]; prior first- and second-trimester combined screening or ultrasound examination (n=1) [74]; and positive traditional screening results and decision not to have invasive testing (n=1) [76]. In two of the studies [74, 75] 53%–58% of the patients had also undergone ART treatment. In the four studies which reported on chorionicity, more than 80% of twins were dichorionic diamniotic.
The median maternal age of enrolled women ranged from 29.8 to 36.8 years. Although all of the studies included first- and second-trimester pregnancies, the median gestational age was around 12–13 weeks in all but one of the studies [75], indicating that most of the patients were analysed during the first trimester.
One of the studies used the Harmony® prenatal test (Ariosa Diagnostics Inc., San Jose, CA, USA) [74]. The rest were based on WGS; three used BGI technology [73, 75, 76] and one used the Verifi™ test (Illumina Inc., San Diego, CA, USA) [72].
Huang et al. [75] confirmed all results with karyotyping. Tan et al. [73] used karyotyping to confirm positive cases and obtained clinical outcomes of negative cases from telephone interview 1 month after delivery. The rest confirmed cases by invasive diagnostic procedures and newborn testing/
physical examinations. Fosler et al. [72] also considered ultrasound evaluations for verification pur-poses.
[75]. As can be observed in Figure 6, five of the six studies (83%) had a low risk of bias for the index test. Four studies (66.7%) showed a high or unclear risk of bias due to the reference standard domain as neonatal examination was used as the reference standard in negative NIPT cases and ultrasound findings were used as the reference standard for all NIPT cases. Most of the studies included had high risk of bias due to flow and timing, mainly due to cases excluded from the analy-sis.
All studies had a low concern regarding applicability for the reference standard and index test. With respect to the patient selection domain, three studies had high concern for different reasons; that is, 100% of pregnant women included had a pregnancy by ART, opted to have invasive testing included or NIPT indications do not match the review question (Figure 7).
Evidence assessment of individual studies can be found in Appendix 1 (Table A8).
Figure 6: Risk of bias assessed by the QUADAS-2 tool for noninvasive prenatal testing in women with high-risk twin pregnancies
Figure 7: Concern regarding applicability of the use of the QUADAS-2 tool for twin pregnancy population studies
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