Abstract
Purpose
This study aimed to establish suitable threshold values for biochemical indicators in low-risk pregnant women who underwent second trimester screening and design strategies for consecutive prenatal testing to increase trisomy 21 detection.
Methods
This study examined singleton pregnant women who underwent double, triple, or quadruple screening in the second trimester over six years. To obtain adequate detection efficiency for low-risk pregnancies, threshold values for serum biochemical indicators were established, and a cost-effectiveness assessment of the improved contingent screening strategy was conducted.
Results
Participants were included in serum double- (n = 88,550), triple- (n = 29,991), and quadruple-screening (n = 15,004) groups. Threshold values were defined as having a free beta subunit of human chorionic gonadotropin (free β-hCG) multiple of the median (MoM) ≥ 2.50, alpha-fetoprotein (AFP) MoM ≤ 0.50, or unconjugated estriol (uE3) MoM ≤ 0.70 for low risk. Low-risk pregnancies, comprising 1.35% (988/73,183), 4.45% (1,171/26,286), and 11.91% (1,559/13,085) of the double-, triple-, and quadruple-screening groups, respectively, underwent further non-invasive prenatal screening. In the double-, triple-, and quadruple-screening groups, we detected 11.76% (2/17), 40.00% (2/5), and 66.67% (2/3) of trisomy 21 cases with false negative results, respectively, with the overall detection rates of 85.00% (85/100), 90.63% (29/32), and 95.24% (20/21), respectively, and decreased ratio of overall costs of 5.26%, 16.63%, and 24.36%, respectively.
Conclusion
Utilizing threshold values of AFP, free β-hCG, and uE3 to trigger further non-invasive prenatal screening may increase trisomy 21 detection in pregnancies deemed low risk in the second trimester while reducing the overall costs of screening strategies.
Keywords: Trisomy 21, Contingent screening strategy, Second trimester screening, Biochemical indicator
What does this study add to the clinical work
| Utilizing threshold values of AFP, free β-hCG, and uE3 to trigger further NIPS may increase trisomy 21 detection in pregnancies deemed low risk in the second trimester while reducing the overall costs of screening strategies. Compared to the universal NIPS strategy, our method may facilitate contingent screening for trisomy 21, particularly in low-resource settings, where one would like to minimize NIPS uptake. |
Introduction
In recent decades, screening mothers’ serum biochemically for trisomy 21 has become a standard procedure for obstetric care in many nations. Double, triple, and quadruple combinations of the free beta subunit of human chorionic gonadotropin (free β-hCG), alpha-fetoprotein (AFP), inhibin A, and unconjugated estriol (uE3) have been used in the second trimester to screen for trisomy 21, with detection rates (DRs) of 60–80% and a 5% false-positive rate (FPR) [1–3]. Subsequently, many countries have moved screening from the second trimester to the first trimester using free β-hCG and pregnancy-associated plasma protein A (PAPP-A) together with nuchal translucency (NT), known as first-trimester combined screening [2]. Compared with serum biochemical screening in the second trimester, the advantages of first-trimester combined screening include earlier diagnosis and safer termination of the pregnancy [4]. However, the test performance of first-trimester combined screening varies between countries, regions, and programs, as NT measurements depend on individual quality and skill control [5]. Serum biochemical screening during the second trimester remains a suitable choice for institutions that cannot effectively perform NT measurements. Thus, in China, first-trimester combined screening has not been widely used as frontline screening, whereas double and triple screening in the second trimester remain widely used [6].
In the past decade, several centers have applied non-invasive prenatal screening (NIPS) based on a risk classification of maternal serum biochemical [7]. According to specialist recommendations, NIPS should be used instead of traditional screening techniques for fetal trisomy 21 in all pregnant women with singleton gestation [8]. Some studies have supported this recommendation in terms of accuracy and reliability [9–12]. NIPS may be safer and more effective than traditional serum biochemical screening; however, because NIPS is more expensive in China, it is unlikely to replace traditional serum biochemical screening. Contingent screening may be a suitable tactic to balance the cost and effectiveness of new testing technologies [13]. However, contingent screening strategies, such as serum biochemical indicators, cut-off criteria, frontline screening methods, and the selection of secondary screening tests, vary greatly among countries [14–19].
The maternal serum biochemical screening strategy is widely used in China. According to the technical standards and norms for prenatal screening and diagnosis in China, high-risk (≥ 1:270) women are provided with invasive prenatal diagnosis, intermediate-risk (1:1000–1:271) women are provided with NIPS, and low-risk (< 1:1000) women are offered continued standard prenatal care. Using this traditional contingent screening strategy, we found that the majority of undetected low-risk trisomy 21 fetuses occurred in pregnant women with abnormal biochemical indicators [20]. Our previous research has shown that, for pregnant women at low risk in the first trimester, with multiple of the median (MoM) threshold values of ≥ 2.75 for free β-hCG or ≤ 0.5 for PAPP-A, the overall DR for trisomy 21 was improved by 10.64% following serum biochemical screening and 12.77% following first-trimester combined screening [20].
Our research aimed to create a sequential prenatal diagnosis strategy in the second trimester and determine suitable threshold values for serum biochemical indicators in pregnant women at low risk in accordance with the current screening strategy, with the goal of increasing the rate of trisomy 21 detection.
Materials and methods
Study design
This study retrospectively analyzed women who underwent double, triple, or quadruple serum screening in the second trimester of their singleton pregnancies. We gathered information on pregnancy outcomes and the double-, triple-, and quadruple-screening findings and examined how the three different screening tests performed. To assess the risk of having fetuses with trisomy 21, gravidas were split into three categories: high risk (≥ 1:270), intermediate risk (1:1000–1:271), and low risk (< 1:1000). The intermediate-risk group was further subjected to NIPS, whereas the high-risk group underwent invasive testing. To obtain adequate detection efficiency, the threshold values of serum biochemical indicators were modified for the low-risk group. In low-risk gravidas with abnormal biochemical indicators, consecutive NIPS was indicated, whereas routine prenatal care was continued for other pregnancies. For the new strategy, it was assumed that NIPS had a DR of 99% and a FPR of 0.5% [21] and that both invasive prenatal diagnosis and NIPS would be fully utilized.
Study population
Between January 2015 and December 2020, singleton pregnancies at the Department of Medical Genetics/Prenatal Diagnostic Center of West China Second University Hospital were screened for common fetal chromosomal disorders through double, triple, or quadruple screening. The participants provided written informed consent, and the study was approved by Sichuan University’s Institutional Ethics Committee. The inclusion criteria were as follows: (1) pregnant women aged > 16 years, and (2) gestational weeks, as determined by biparietal diameter or crown-rump length, between 15 and 20 weeks plus 6 days. The exclusion criteria were as follows: (1) co-twin mortality or multiple pregnancies, (2) gestations with stillbirths or termination of pregnancy but no chromosomal results, (3) pregnancies with known chromosomal abnormalities prior to screening, and (4) absence of follow-up.
Clinical follow-up assessments
All pregnancy results were documented. In cases of live delivery, clinical follow-up evaluations were performed by phone or through a review of medical records 12 weeks after the expected date of pregnancy. For non-live births, fetal chromosomes were validated using amniotic fluid or fetal tissue samples by copy number variation sequencing (CNV-seq), chromosomal microarray analysis (CMA), or karyotyping.
Screening method
Serum free β-hCG, AFP, and uE3 were detected through time-resolved immunofluorescence analysis according to the manufacturer’s instructions (Perkin Elmer, USA). Inhibin A was detected through chemiluminescent immunoassay according to the manufacturer’s instructions (Beckman, USA). Based on maternal age and serum biochemical indicators, risk was calculated using a previously reported method [22]. Measurements of biochemical indicators were converted into MoM for gestational age, adjusted for maternal weight, insulin-dependent diabetes mellitus, smoking status, and race [23]. The serum free β-hCG and AFP were detected for double screening, and the risk of double screening was calculated using Lifecycle software (Perkin Elmer). The serum free β-hCG, AFP, and inhibin A were detected for triple screening, and the risk of triple screening was calculated using Prenatal Screening Software (TCSoft, China). The serum free β-hCG, AFP, inhibin A, and uE3 were detected for quadruple screening, and the risk of quadruple screening was calculated using Prenatal Screening Software (TCSoft).
Cost analysis
Based on the price standards of the Sichuan Price Bureau in 2015–2020, the cost of double screening, triple screening, quadruple screening, NIPS, prenatal diagnosis fees (including amniocentesis and testing fees), and life-course costs of missed detection of trisomy 21 were set at $22.49, $33.73, $44.98, $359.82, $624.89 (average cost of CNV-seq and CMA with karyotyping), and $747,481.26 [24], respectively. Costs in Chinese yuan were converted into USD at the average of the 2015–2020 exchange rate (6.67 Chinese yuan = 1.00 USD).
Statistical analysis
SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA) was used for the data analysis. Categorical variables and descriptive data are presented as n (%), whereas continuous variables are presented as median (interquartile range). The DR and odds of being affected given a positive result (OAPR)are the performance metrics for the prenatal screening algorithm. To compare the two groups, the Wilcoxon rank-sum test was used. Statistical significance was set at P < 0.05.
Results
Study population characteristics
In total, 88,550, 29,991, and 15,004 singleton pregnant women who underwent double, triple, or quadruple screening, respectively, were included in our study. The double-, triple-, and quadruple-screening groups showed no significant differences in smoking ratio, insulin-dependent diabetes, method of conception, gestational age, age distribution, or maternal weight (Table 1). The MoMs of AFP and uE3 were significantly lower (P < 0.001), whereas those of free β-hCG and inhibin A in fetuses diagnosed with trisomy 21 were significantly greater (P < 0.001) than those in the unaffected group (Table 2).
Table 1.
Pregnancy characteristics of women with three screening tests
| Characteristic | Double screening | Triple screening | Quadruple screening |
|---|---|---|---|
| Number | 88,550 | 29,991 | 15,004 |
| Age distribution | |||
| Maternal age at expected date of delivery < 35 years (%) | 87,158 (98.43%) | 29,352 (97.87%) | 14,766 (98.41%) |
| Maternal age at expected date of delivery ≥ 35 years (%) | 1392 (1.57%) | 639 (2.13%) | 238 (1.59%) |
| Maternal age (years) at expected date of delivery (IQR) | 26.69 (24.07–29.25) | 26.80 (24.18–29.48) | 29.42 (27.20–31.85) |
| Method of conception (%) | |||
| Spontaneous | 88,139 (99.54%) | 29,819 (99.43%) | 14,505 (96.67%) |
| Assisted | 411 (0.46%) | 172 (0.57%) | 499 (3.33%) |
| Median maternal weight (IQR) | 54 (49–60) | 54 (49–60) | 54.5 (50–60) |
| Median gestational age (days) at blood sample (IQR) | 120 (115–127) | 122 (116–129) | 115 (112–120) |
| Insulin-dependent diabetes (‰) | 32 (0.36‰) | 12 (0.40‰) | 8 (0.53‰) |
| Smoker (%) | 270 (0.30%) | 170 (0.57%) | 187 (1.25%) |
| Incidence of Down syndrome (%) | 100 (0.11%,1/886) | 32 (0.11%,1/937) | 21 (0.14%,1/714) |
*Data are given as median (interquartile range) or n (%). IQR interquartile range
Table 2.
Characteristics of biomarkers of singleton pregnancies affected by trisomy 21 and unaffected pregnancies
| Characteristic | Double screening | Triple screening | Quadruple screening | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Unaffected | Trisomy 21 | P | Unaffected | Trisomy 21 | P | Unaffected | Trisomy 21 | P | |
| Number | 88,450 | 100 | 29,959 | 32 | 14,983 | 21 | |||
| Free β-hCG MoM | 0.98 (0.66–1.51) | 2.75 (1.57–4.09) | < 0.001 | 1.01 (0.68–1.56) | 2.55 (1.46–4.21) | < 0.001 | 0.97 (0.68–1.60) | 2.68 (1.41–4.71) | < 0.001 |
| AFP MoM | 0.99 (0.81–1.23) | 0.73 (0.63–0.96) | < 0.001 | 1.00 (0.81–1.24) | 0.76 (0.64–0.94) | < 0.001 | 0.99 (0.80–1.23) | 0.71 (0.55–0.93) | < 0.001 |
| Inhibin A MoM | 0.96 (0.74–1.25) | 1.95 (1.48–2.69) | < 0.001 | 0.95 (0.73–1.25) | 2.24 (1.40–2.92) | < 0.001 | |||
| uE3 MoM | 1.03 (0.86–1.22) | 0.74 (0.66 ~ 0.92) | < 0.001 | ||||||
Data are given as median (interquartile range). β-hCG beta-human chorionic gonadotropin, AFP alpha-fetoprotein, uE3 unconjugated estriol, MoM multiples of the median
Performance of the traditional contingent screening strategy
Patients in the double-screening group were categorized as high risk in 4,997 cases (5.64%), intermediate risk in 10,370 cases (11.71%), and low risk in 73,183 cases (82.65%). Within the triple-screening cohort, patients were categorized as high risk in 1,302 cases (4.34%), intermediate risk in 2,403 cases (8.01%), and low risk in 26,286 cases (87.65%). Patients in the quadruple-screening group were categorized as high risk in 760 cases (5.07%), intermediate risk in 1,159 cases (7.72%), and low risk in 13,085 cases (87.21%) (Table 3).
Table 3.
Performance of three screening tests with traditional contingent screening strategy
| Risk stratification | Double screening | Triple screening | Quadruple screening | ||||||
|---|---|---|---|---|---|---|---|---|---|
| N (%) | Trisomy 21 (%) | OAPR | N (%) | Trisomy 21 (%) | OAPR | N (%) | Trisomy 21 (%) | OAPR | |
| High risk (risk ≥ 1/270) | 4,997 (5.64%) | 60 (60.00%) | 1:83 | 1,302 (4.34%) | 22 (68.75%) | 1:59 | 760 (5.07%) | 16 (76.19%) | 1:48 |
| Intermediate risk (1/1000 ≤ risk < 1/270) | 10,370 (11.71%) | 23 (23.00%) | 1:451 | 2,403 (8.01%) | 5 (15.63%) | 1:481 | 1,159 (7.72%) | 2 (9.52%) | 1:580 |
| Low risk (risk < 1/1000) | 73,183 (82.65%) | 17 (17.00%) | 1:4305 | 26,286 (87.65%) | 5 (15.63%) | 1:5257 | 13,085 (87.21%) | 3 (14.29%) | 1:4362 |
OAPR Odds of being affected given a positive result
The percentage of high- and intermediate-risk cases in the double-screening group was 17.35% (15,367/88,550), and the percentage of trisomy 21 diagnoses was 83% (83/100). The OAPR was 1:83 and 1:451 for high- and intermediate-risk cases, respectively. In the triple-screening group, 12.35% (3,705/29,991) were classified as high- and intermediate-risk, and 84.37% (27/32) were diagnosed as trisomy 21 cases. The OAPR was 1:59 and 1:481 for high- and intermediate-risk cases, respectively. The percentage of high- and intermediate-risk cases in the quadruple-screening group was 12.79% (1,919/15,004), whereas the percentage of trisomy 21 diagnosis was 85.71% (18/21). The OAPR was 1:48 and 1:580 for high- and intermediate-risk cases, respectively (Table 3).
In the double-screening group, low-risk cases accounted for 82.65% (73,183/88,850), and low-risk cases of trisomy 21 accounted for 17.00% (17/100). Low-risk cases comprised 87.65% (26,286/29,991) of the triple-screening group, whereas low-risk trisomy 21 cases comprised 15.63% (5/32). In the quadruple-screening group, low-risk cases accounted for 87.21% (13,085/15,004), and low-risk cases of trisomy 21 accounted for 14.29% (3/21) (Table 3).
Performance of the improved contingent screening strategy
The threshold values of abnormal biochemical indicators were defined as AFP MoM ≤ 0.50 or free β-hCG MoM ≥ 2.50 to achieve an OAPR similar to that of intermediate-risk individuals and a greater trisomy 21 DR in the double-screening group (Table 4). In addition to the biochemical indicators in the double-screening group, the serum biochemical indicator inhibin A was added to the triple-screening group. With inhibin A removed from the cut-off value of the abnormal biochemical indicators, the low-risk group in the triple-screening group had the greatest OAPR when the threshold values of the abnormal biochemical indicators were divided (Table 4). The quadruple-screening group included uE3 in addition to the biochemical indicators in the triple-screening group. With a cut-off value of uE3 MoM ≤ 0.70, free β-hCG MoM ≥ 2.50, or AFP MoM ≤ 0.50, without adding inhibin A as the threshold value of abnormal biochemical indicators, the OAPR was the highest in the low-risk group (Table 4).
Table 4.
Performance of different biochemical indicator MoM in three screening tests
| Screening test | Serum indicator | Abnormal serum indicator* | Trisomy 21** | OAPR | |||||
|---|---|---|---|---|---|---|---|---|---|
| Free β-hCG MoM | AFP MoM | Inhibin A MoM | uE3 MoM | n | % | n | % | ||
| Double screening | 2.00 | 0.40 | 2431 | 3.32 | 2 | 11.76 | 1:1216 | ||
| 2.00 | 0.50 | 2851 | 3.90 | 3 | 17.65 | 1:950 | |||
| 2.00 | 0.60 | 4878 | 6.67 | 3 | 17.65 | 1:1626 | |||
| 2.00 | 0.70 | 9868 | 13.48 | 6 | 35.29 | 1:1645 | |||
| 2.00 | 0.80 | 17,853 | 24.40 | 9 | 52.94 | 1:1984 | |||
| 2.25 | 0.40 | 1164 | 1.59 | 1 | 5.88 | 1:1164 | |||
| 2.25 | 0.50 | 1584 | 2.16 | 2 | 11.76 | 1:792 | |||
| 2.25 | 0.60 | 3612 | 4.94 | 2 | 11.76 | 1:1806 | |||
| 2.25 | 0.70 | 8607 | 11.76 | 5 | 29.41 | 1:1721 | |||
| 2.25 | 0.80 | 16,596 | 22.68 | 8 | 47.06 | 1:2075 | |||
| 2.50 | 0.40 | 568 | 0.78 | 1 | 5.88 | 1:568 | |||
| 2.50 | 0.50 | 988 | 1.35 | 2 | 11.76 | 1:494△ | |||
| 2.50 | 0.60 | 3016 | 4.12 | 2 | 11.76 | 1:1508 | |||
| 2.50 | 0.70 | 8011 | 10.95 | 5 | 29.41 | 1:1602 | |||
| 2.50 | 0.80 | 16,000 | 21.86 | 8 | 47.06 | 1:2000 | |||
| 2.75 | 0.40 | 290 | 0.40 | 0 | 0 | – | |||
| 2.75 | 0.50 | 710 | 0.97 | 1 | 5.88 | 1:710 | |||
| 2.75 | 0.60 | 2738 | 3.74 | 1 | 5.88 | 1:2738 | |||
| 2.75 | 0.70 | 7735 | 10.57 | 4 | 23.53 | 1:1934 | |||
| 2.75 | 0.80 | 15,732 | 21.50 | 8 | 47.06 | 1:1967 | |||
| 3.00 | 0.40 | 163 | 0.22 | 0 | 0 | – | |||
| 3.00 | 0.50 | 583 | 0.80 | 1 | 5.88 | 1:583 | |||
| 3.00 | 0.60 | 2611 | 3.57 | 1 | 5.88 | 1:2611 | |||
| 3.00 | 0.70 | 7609 | 10.40 | 4 | 23.53 | 1:1902 | |||
| 3.00 | 0.80 | 15,608 | 21.33 | 8 | 47.06 | 1:1951 | |||
| Triple screening | 2.50 | 0.50 | – | 1171 | 4.45 | 2 | 40.00 | 1:586△ | |
| 2.50 | 0.50 | 1.50 | 3234 | 12.30 | 4 | 80.00 | 1:809 | ||
| 2.50 | 0.50 | 1.75 | 2049 | 7.80 | 2 | 40.00 | 1:1025 | ||
| 2.50 | 0.50 | 2.00 | 1554 | 5.91 | 2 | 40.00 | 1:777 | ||
| 2.50 | 0.50 | 2.25 | 1364 | 5.19 | 2 | 40.00 | 1:682 | ||
| 2.50 | 0.50 | 2.50 | 1267 | 4.82 | 2 | 40.00 | 1:634 | ||
| 2.50 | 0.50 | 2.75 | 1227 | 4.67 | 2 | 40.00 | 1:614 | ||
| 2.50 | 0.50 | 3.00 | 1208 | 4.60 | 2 | 40.00 | 1:604 | ||
| Quadruple screening | 2.50 | 0.50 | – | – | 856 | 6.54 | 1 | 33.33 | 1:856 |
| 2.50 | 0.50 | – | 0.40 | 870 | 6.65 | 1 | 33.33 | 1:870 | |
| 2.50 | 0.50 | – | 0.50 | 898 | 6.86 | 1 | 33.33 | 1:898 | |
| 2.50 | 0.50 | – | 0.60 | 1044 | 7.98 | 1 | 33.33 | 1:1044 | |
| 2.50 | 0.50 | – | 0.70 | 1559 | 11.91 | 2 | 66.67 | 1:780△ | |
| 2.50 | 0.50 | – | 0.80 | 2624 | 20.05 | 3 | 100.00 | 1:875 | |
*The number of abnormal serum indicator in low risk. **The number of trisomy 21 of abnormal serum indicator in low risk. β-hCG beta-human chorionic gonadotropin, AFP alpha-fetoprotein, uE3 unconjugated estriol, MoM multiples of the median, OAPR Odds of being affected given a positive result
△The highest OAPR
For trisomy 21, gravidas were grouped into four groups according to the improved contingent screening strategy: high risk (≥ 1:270), intermediate risk (1:1000–1:271), abnormal biochemical indicators (free β-hCG MoM ≥ 2.50, AFP MoM ≤ 0.50, or uE3 MoM ≤ 0.70) in low-risk cases, and others (Table 5). Gravidas in the intermediate-risk and abnormal biochemical indicator groups, constituting 1.35% (988/73,183), 4.45% (1,171/26,286), and 11.91% (1,559/13,085) of the women in the double-, triple-, and quadruple-screening groups, respectively, received NIPS, those in the high-risk group received an invasive test, and others received regular prenatal care. In the double-, triple-, and quadruple-screening groups, 11.76% (2/17), 40.00% (2/5), and 66.67% (2/3) trisomy 21 cases with false-negative results, respectively, were detected, with an overall DR for trisomy 21 of 85.00% (85/100), 90.63% (29/32), and 95.24% (20/21), respectively (Table 5). The number of false negatives for trisomy 21 decreased to 15, 3, and 1 in the double-, triple-, and quadruple-screening groups, respectively. The percentage of patients with abnormal biochemical indicators in the double-screening group was 1.12% (988/88,550), whereas the percentage of trisomy 21 cases with false-negative findings was 2.00% (2/100). The percentage of patients with abnormal biochemical indicators in the triple-screening group was 3.90% (1,171/29,991), whereas the percentage of trisomy 21 cases with false-negative findings was 6.25% (2/32). The percentage of patients with abnormal biochemical indicators in the quadruple-screening group was 10.39% (1,559/15,004), whereas the percentage of trisomy 21 cases with false-negative findings was 9.52% (2/21).
Table 5.
Performance of three screening tests with improved contingent screening strategy
| Risk stratification | Double screening | Triple screening | Quadruple screening | ||||||
|---|---|---|---|---|---|---|---|---|---|
| n (%) | Trisomy 21 (%) | OAPR | n (%) | Trisomy 21 (%) | OAPR | n (%) | Trisomy 21 (%) | OAPR | |
| High risk (risk ≥ 1/270) | 4,997 (5.64%) | 60 (60.00%) | 1:83 | 1302 (4.34%) | 22 (68.75%) | 1:59 | 760 (5.07%) | 16 (76.19%) | 1:48 |
| Intermediate risk (1/1000 ≤ risk < 1/270) | 10,370 (11.71%) | 23 (23.00%) | 1:451 | 2403 (8.01%) | 5 (15.63%) | 1:481 | 1159 (7.72%) | 2 (9.52%) | 1:580 |
| Abnormal biochemical indicators (free β-hCG MoM ≥ 2.50 or AFP MoM ≤ 0.50 or uE3 MoM ≤ 0.70(risk < 1/1000)) | 988 (1.12%) | 2 (2.00%) | 1:494 | 1171 (3.90%) | 2 (6.25%) | 1:586 | 1559 (10.39%) | 2 (9.52%) | 1:780 |
| Others (free β-hCG MoM < 2.50 and AFP MoM > 0.50 and uE3 MoM > 0.70 (risk < 1/1000)) | 72,195 (81.53%) | 15 (15.00%) | 1:4813 | 25,115 (83.74%) | 3 (9.38%) | 1:8372 | 11,526 (76.82%) | 1 (4.76%) | 1:11,526 |
β-hCG beta-human chorionic gonadotropin, AFP alpha-fetoprotein, MoM multiples of the median, OAPR Odds of being affected given a positive result
Cost of the contingent screening strategy
The costs of the traditional and improved contingent screening strategies are shown in Table 6. For the traditional contingent screening strategy, the overall cost of the double-, triple-, and quadruple-screening groups was 21,585,074 USD, 6,434,756 USD, and 3,813,021 USD, respectively. For the improved contingent screening strategy, the overall cost of the double-, triple-, and quadruple-screening groups was 20,448,5738 USD, 5,364,892 USD, and 2,884,017 USD, respectively. The overall costs of the improved contingent screening strategy were all lower than those of the traditional strategies, with overall costs of the double-, triple-, and quadruple-screening groups decreasing by 5.26%, 16.63%, and 24.36%, respectively.
Table 6.
Cost of different contingent screening strategy
| Screening strategy | Screening test | Double screening | Triple screening | Quadruple screening | |||
|---|---|---|---|---|---|---|---|
| Cost | n | USD | n | USD | n | USD | |
| Traditional contingent screening strategy | Prenatal diagnosis* | 5049 | 3,155,070 | 1314 | 821,105 | 766 | 478,666 |
| NIPS | 10,370 | 3,731,333 | 2403 | 864,647 | 1159 | 417,031 | |
| Missed detection for T21 | 17 | 12,707,181 | 5 | 3,737,406 | 3 | 2,242,444 | |
| Serum screening | 88,550 | 1,991,490 | 29,991 | 1,011,596 | 15,004 | 674,880 | |
| Overall cost | 21,585,074 | 6,434,756 | 3,813,021 | ||||
| Improved contingent screening strategy | Prenatal diagnosis* | 5054 | 3,158,194 | 1320 | 824,855 | 774 | 483,665 |
| NIPS** | 11,358 | 4,086,836 | 3574 | 1,285,997 | 2,718 | 977,991 | |
| Missed detection for T21 | 15 | 11,212,219 | 3 | 2,242,444 | 1 | 747,481 | |
| Serum screening | 88,550 | 1,991,490 | 29,991 | 1,011,596 | 15,004 | 674,880 | |
| Overall cost | 20,448,738 | 5,364,892 | 2,884,017 | ||||
| Decrease ratio of overall cost | 5.26% | 16.63% | 24.36% | ||||
*The number of prenatal diagnosis included high risk of serum screening and NIPS (FPR of 0.5%). **The number of NIPS included intermediate risk and abnormal biochemical indicators of serum screening
Discussion
The MoMs of AFP, free β-hCG, inhibin A, and uE3 in pregnancies with and without trisomy 21 fetuses were consistent with those from a prior investigation [25]. In addition, double, triple, and quadruple screening identified 60.00% (60/100), 68.75% (22/32), and 76.19% (16/21) of trisomy 21 cases as high risk, respectively, in accordance with earlier research [2, 3, 8].
In China, owing to its low cost and mature experimental procedures, serum biochemical screening is still used for the first-tier screening of pregnancies. High-risk individuals are offered intrusive prenatal diagnosis; however, for intermediate-risk patients, NIPS is advantageous given its excellent detection performance [26–28]. Therefore, the DR of trisomy 21 was significantly increased by traditional contingent screening during the second trimester, which was based on serum biochemical screening. For gravidas with intermediate risk, NIPS contingent screening is an acceptable way to avoid intrusive procedures [29]. However, using contingent screening with double, triple, and quadruple screening, 17.00% (17/100), 15.63% (5/32), and 14.29% (3/21) of trisomy 21 instances, respectively, were still undetected.
We utilized the characteristics of serum biochemical indicators: free β-hCG MoM values increased by approximately two-fold, AFP MoM values decreased by 0.5-fold, inhibin A MoM values increased by two-fold, and uE3 MoM values decreased by approximately 30% in pregnant women with trisomy 21 fetuses when stratifying the MoM values of individual biochemical indicators in the low-risk group [25, 30]. Our study showed that the best DR for trisomy 21 occurred when abnormal biochemical indicators were classified as free β-hCG MoM ≥ 2.50, AFP MoM ≤ 0.50, or uE3 MoM ≤ 0.70 in the double-, triple-, and quadruple-screening groups, respectively. In the double-, triple-, and quadruple-screening groups, the low-risk group with abnormal biochemical indicators had an OAPR of 1:494, 1:586, and 1:780, respectively. As these OAPRs were within the intermediate-risk range (1:1000–1:271), we recommend further NIPS for low-risk patients with abnormal biochemical indicators. In low-risk patients, 1.38% (988/73,183) of cases in the double-screening group, 4.45% (1,171/26,286) of cases in the triple-screening group, and 11.91% (1,559/13,085) of cases in the quadruple-screening group were identified for further NIPS. Thus, in the double-, triple-, and quadruple-screening groups, 11.76% (2/17), 40% (2/5), and 66.67% (2/3) of trisomy 21 instances with false-negative results, respectively, were found. Otherwise, the double-, triple-, and quadruple-screening groups had an overall DR for trisomy 21 of 85.00% (85/100), 90.63% (29/32), and 95.24% (20/21), respectively.
According to our research data and other studies on traditional contingent screening, the efficiency of quadruple screening is better than that of triple screening, and triple screening is better than double screening [2, 3]. The screening efficiency of double screening was low, with a high DR of 60%. The traditional contingent screening DR was 83%, whereas the new contingent screening DR was 85%. Our study shows that in the improved contingent screening strategy of triple screening as the front-line screening method, only dividing the optimal threshold values for free β-hCG MoM and AFP MoM values can increase the overall DR by approximately 5%, resulting in a total DR of over 90%. The new contingent triple-screening strategy, as the front-line screening method with the added stratification of inhibin A MoM values, reduced the OAPR of abnormal biochemical indicators without improving the overall DR. When the improved contingent screening strategy of quadruple screening was used as the front-line screening method with stratification of uE3 MoM values (MoM ≤ 0.7) without stratification of inhibin A MoM values, the OAPR (1:780) was optimal, increasing the overall DR to 95.24% with only one missed case. Using this approach, the final DR was limited by that of the primary screening test. Therefore, it is recommended that quadruple or triple screening be prioritized as the first-line screening method in the second trimester.
When using the cut-off division with abnormal MoM values, other factors potentially affecting biochemical indicators should also be considered. A systematic review and meta-analysis showed that the maternal free β-hCG level was slightly increased in pregnancies conceived by intracytoplasmic sperm injection [31]. The abnormality of biochemical indicators can be accidentally observed in cases with other chromosomal abnormalities, such as sex chromosome aneuploidies, microdeletions and microduplications, which are also related to adverse pregnancy outcomes [30]. Low serum uE3 level (< 0.4 MoM) is associated with an increased risk of aberrant CNVs [32].
This improved contingent trisomy 21 screening strategy, which uses serum biochemical screening in the second trimester as the first-line screening modality, may be applicable in regions with low healthcare and economic levels without increasing the technical complexity or burden. Compared to the universal NIPS strategy, our method may facilitate contingent screening for trisomy 21 particularly in low-resource settings where one would like to minimize NIPS uptake. Given the improved DR of trisomy 21, the health and economic costs can be lowered compared to traditional screening strategies. Among the three first-line serum screening methods, quadruple screening showed the largest reduction in cost (24.36%), followed by triple screening (16.63%) and double screening (5.26%).
In conclusion, using the appropriate biochemical indicator threshold values during screening in the second trimester can successfully identify trisomy 21 in low-risk cases. An additional NIPS test is indicated for low-risk individuals with abnormal biochemical indicators as part of the improved contingent screening method for trisomy 21, which can boost the trisomy 21 DR and reduce the overall cost of screening strategies compared to that of the traditional contingent screening strategy.
Acknowledgements
We express our gratitude to all physicians who participated in patient recruiting. We appreciate the efforts made by each team member to collect and ensure the integrity of the data.
Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Wei Luo, Ting Hu and Shanling Liu. The first draft of the manuscript was written by Wei Luo and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the National Key Research and Development Program of China (2022YFC2703302), Sichuan Province Science and Technology Support Program, China (2021YFS0078 and 2022YFS0078), and the Fundamental Research Funds for the Central Universities (SCU2022F4080).
Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethics approval
Approval was granted by Sichuan University’s Institutional Ethics Committee (No. 2024(073)).
Consent to participate
Informed consent was obtained from all individual participants included in the study.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Ting Hu, Email: huting4123@163.com.
Shanling Liu, Email: sunny630@126.com.
References
- 1.Hsu JJ, Hsieh TT, Soong YK, Spencer K (1997) Comparison of Down’s syndrome screening strategies in Asians combining serum free beta-hCG and alpha-fetoprotein with maternal age. Prenat Diagn 17:707–716 [PubMed] [Google Scholar]
- 2.Screening for Fetal Chromosomal Abnormalities (2020) ACOG practice bulletin summary, number 226. Obstet Gynecol 136:859–867 [DOI] [PubMed] [Google Scholar]
- 3.Palomaki GE, Knight GJ, McCarthy JE, Haddow JE, Donhowe JM (1997) Maternal serum screening for Down syndrome in the United States: a 1995 survey. Am J Obstet Gynecol 176:1046–1051 [DOI] [PubMed] [Google Scholar]
- 4.Prenatal CH, Markers SUM (2014) J Clin Med 3:504–520 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.van der Meij KRM, Henneman L, Sistermans EA (2023) Non-invasive prenatal testing for everybody or contingent screening? Prenat Diagn 43:443–447 [DOI] [PubMed] [Google Scholar]
- 6.Tu S, Rosenthal M, Wang D, Huang J, Chen Y (2016) Performance of prenatal screening using maternal serum and ultrasound markers for Down syndrome in Chinese women: a systematic review and meta-analysis. BJOG 123(Suppl 3):12–22 [DOI] [PubMed] [Google Scholar]
- 7.Bianchi DW, Parker RL, Wentworth J, Madankumar R, Saffer C, Das AF et al (2014) DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med 370:799–808 [DOI] [PubMed] [Google Scholar]
- 8.Dungan JS, Klugman S, Darilek S, Malinowski J, Akkari YMN, Monaghan KG et al (2023) Noninvasive prenatal screening (NIPS) for fetal chromosome abnormalities in a general-risk population: an evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG). Genet Med 25:100336 [DOI] [PubMed] [Google Scholar]
- 9.Xu L, Huang H, Lin N, Wang Y, He D, Zhang M et al (2020) Non-invasive cell-free fetal DNA testing for aneuploidy: multicenter study of 31 515 singleton pregnancies in southeastern China. Ultrasound Obstet Gynecol 55:242–247 [DOI] [PubMed] [Google Scholar]
- 10.Liu Y, Liu H, He Y, Xu W, Ma Q, He Y et al (2020) Clinical performance of non-invasive prenatal served as a first-tier screening test for trisomy 21, 18, 13 and sex chromosome aneuploidy in a pilot city in China. Hum Genom 14:21 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Yang L, Tan WC (2020) Prenatal screening in the era of non-invasive prenatal testing: a Nationwide cross-sectional survey of obstetrician knowledge, attitudes and clinical practice. BMC Pregnancy Childbirth 20:579 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Spingler T, Sonek J, Hoopmann M, Prodan N, Jonaityte G, Elger T et al (2024) Importance of a detailed anomaly scan after a cfDNA test indicating fetal trisomy 21, 18 or 13. Arch Gynecol Obstet 310:749–755 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Xu Y, Wei Y, Ming J, Li N, Xu N, Pong RW et al (2019) Cost-effectiveness analysis of non-invasive prenatal testing for Down syndrome in China. Int J Technol Assess Health Care 35:237–242 [DOI] [PubMed] [Google Scholar]
- 14.Huang T, Gibbons C, Rashid S, Priston MK, Bedford HM, Mak-Tam E et al (2020) Prenatal screening for trisomy 21: a comparative performance and cost analysis of different screening strategies. BMC Pregnancy Childbirth 20:713 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Prodan NC, Wiechers C, Geipel A, Walter A, Siegmann HJ, Kozlowski P et al (2022) Universal cell free DNA or contingent screening for trisomy 21: does It make a difference? A comparative study with real data. Fetal Diagn Ther 49:85–94 [DOI] [PubMed] [Google Scholar]
- 16.Wang S, Liu K, Yang H, Ma J (2022) a cost-effectiveness analysis of screening strategies involving non-invasive prenatal testing for trisomy 21. Front Public Health 10:870543 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Sanchez-Duran MA, Bernabeu Garcia A, Calero I, Ramis Fossas J, Illescas T, Aviles MT et al (2019) Clinical application of a contingent screening strategy for trisomies with cell-free DNA: a pilot study. BMC Pregnancy Childbirth 19:274 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Wanapirak C, Buddhawongsa P, Himakalasa W, Sarnwong A, Tongsong T (2019) Fetal Down syndrome screening models for developing countries; Part II: Cost-benefit analysis. BMC Health Serv Res 19:898 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Ye C, Duan H, Liu M, Liu J, Xiang J, Yin Y et al (2024) The value of combined detailed first-trimester ultrasound–biochemical analysis for screening fetal aneuploidy in the era of non-invasive prenatal testing. Arch Gynecol Obstet 310:843–853 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Luo W, He B, Han D, Yuan L, Tang J, Pang L et al (2023) A new contingent screening strategy increased detection rate of trisomy 21 in the first trimester. BMC Pregnancy Childbirth. 10.1186/s12884-023-06115-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Committee on practice bulletins-obstetrics CoG, the Society for Maternal-Fetal M (2016) Practice Bulletin No. 163: Screening for Fetal Aneuploidy. Obstet Gynecol 127:e123–e137 [DOI] [PubMed]
- 22.Malone FC, Ball RH (2005) First- and Second-Trimester Evaluation of Risk (FASTER) Research Consortium. First-trimester or second-trimester screening, or both, for Down’s syndrome. N Engl J Med 353:2001–2011 [DOI] [PubMed] [Google Scholar]
- 23.Malone FD, Canick JA, Ball RH, Nyberg DA, Comstock CH, Bukowski R et al (2005) First-trimester or second-trimester screening, or both, for Down’s syndrome. N Engl J Med 353:2001–2011 [DOI] [PubMed] [Google Scholar]
- 24.Wang NZS, Wang S, Kuang Y, Zu Y, Xie R, Yang M, Peng HLJ, He J (2022) Economic burden of Down syndrome patients and psychological and social discrimination to female caregivers in Changsha, China. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 39:11–15 [DOI] [PubMed] [Google Scholar]
- 25.Alldred SK, Takwoingi Y, Guo B, Pennant M, Deeks JJ, Neilson JP et al (2017) First and second trimester serum tests with and without first trimester ultrasound tests for Down’s syndrome screening. Cochrane Database Syst Rev 3:CD012599 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G (2012) Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol 207(374):e1–e6 [DOI] [PubMed] [Google Scholar]
- 27.Gil MM, Accurti V, Santacruz B, Plana MN, Nicolaides KH (2017) Analysis of cell-free DNA in maternal blood in screening for aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol 50:302–314 [DOI] [PubMed] [Google Scholar]
- 28.Norton ME, Jacobsson B, Swamy GK, Laurent LC, Ranzini AC, Brar H et al (2015) Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med 372:1589–1597 [DOI] [PubMed] [Google Scholar]
- 29.Bowden B, de Souza S, Puchades A, Williams K, Morgan S, Anderson S et al (2022) Implementation of non-invasive prenatal testing within a national UK antenatal screening programme: Impact on women’s choices. Prenat Diagn 42:549–556 [DOI] [PubMed] [Google Scholar]
- 30.Chitayat D, Langlois S, Wilson RD (2017) No. 261-prenatal screening for fetal aneuploidy in singleton pregnancies. J Obstet Gynaecol Can 39:e380–e394 [DOI] [PubMed] [Google Scholar]
- 31.Cavoretto P, Giorgione V, Cipriani S, Viganò P, Candiani M, Inversetti A et al (2017) Nuchal translucency measurement, freeβ-hCG and PAPP-A concentrations in IVF/ICSI pregnancies: systematic review and meta-analysis. Prenat Diagn 37:540–555 [DOI] [PubMed] [Google Scholar]
- 32.Zhang M, Gao Y, Liang M, Wang Y, Guo L, Wu D et al (2024) Correlation between maternal serum biomarkers and the risk of fetal chromosome copy number variants: a single-center retrospective study. Arch Gynecol Obstet 310:933–942 [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.