Health economic evaluation of noninvasive prenatal testing and serum screening for down syndrome.

BACKGROUND: Down syndrome (DS), also known as trisomy 21 (T21), is the most common genetic disorder associated with intellectual disability. There are two methods commonly used for prenatal testing of DS: serum screening (SS) for biomarkers in maternal serum and noninvasive prenatal testing (NIPT) for aneuploidy by cell-free DNA (cfDNA) in maternal plasma. However, cost-effectiveness analyses of these two methods are mostly based on data derived from simulations with various models, with theoretical values calculated. In this study, we statistically analyzed clinical DS screening data and pregnancy outcomes during the follow-up of pregnant women in Zhuhai City, China. The economics of the two mainstream prenatal DS screening methods was evaluated from a public health perspective.
METHODS: A retrospective analysis was performed on the data of 17,363 pregnant women who received SS and NIPT during gestation in Zhuhai from 2018 to 2019, and a cost-effectiveness analysis was performed with four screening strategies. In strategy I, all pregnant women received SS, and those with T21 risk ≥1/270 had invasive prenatal diagnosis (IPD). In strategy II, all pregnant women received SS, those with T21 risk ≥ 1/270 had IPD, and those with 1/270 > T21 risk ≥ 1/1,000 had NIPT; then, women at high risk based on NIPT also had IPD. In strategy III, all pregnant women received SS, and those with T21 risk ≥1,000 had NIPT; then, women at high risk based on NIPT results had IPD. In strategy IV, all pregnant women received NIPT and those at high risk based on NIPT results had IPD. Finally, to assess the cost and effectiveness of DS screening, the total costs were calculated as the sum of screening and diagnosis as well as the direct and indirect economic burden during the average life cycle of DS patients.
RESULTS: A total of 22 of the 17,363 (1/789) pregnant women had DS, of which only one woman was over 35 years of age. SS detected 1,024 cases at high risk of T21 (≥1/270), 8 cases were true positive, with a positive predictive value of 0.78% and a detection rate of 36.4%. NIPT detected 27 cases at high risk of T21 (Z ≥ 3) and 22 cases of DS, with a positive predictive value of 81.5% and a detection rate of 100%. Strategy I had the largest total cost of 65.54 million CNY, strategy II and III had similar total costs of 40 million CNY, and strategy IV had the lowest total cost of 14.91 million CNY. By comparison, the screening strategy with NIPT alone had the highest health economic value for DS.
CONCLUSIONS: SS was greatly affected by nuchal translucency and the accuracy of gestational age measured by ultrasonography. Unstandardized ultrasonography was an important reason for the low DS detection rate with SS. The influence of interfering factors on NIPT was much lower than in SS. NIPT can be used as an alternative to SS and as a primary screening strategy of prenatal DS screening for secondary prevention and control of birth defects. NIPT greatly decreased the frequency of IPD and the miscarriages associated with IPD, saved the limited medical and health resources, and greatly increased DS detection rate. Therefore, NIPT has great social and economic benefits.

Introduction

Down syndrome (DS) is caused by chromosome 21 trisomy (T21) and represents the most common genetic disorder associated with intellectual disability. The 200–300 genes on chromosome 21 and multiple epigenetic factors have been associated with the clinical manifestations of DS [1]. The incidence of DS is approximately 1/800 worldwide, 1/500 in the United States [2], and 14.7/10,000 in China [3]. DS has imposed a heavy economic and emotional burden on society and families. Secondary prevention and control of DS during gestation has become an important and urgent public health issue in all countries and regions.

Commonly used prenatal DS screening methods include serum screening (SS) and noninvasive prenatal testing (NIPT). As early as the 1980s, Cuckle et al. [4] used alpha-fetoprotein in maternal serum for prenatal DS screening. In 1999, Wald et al. [5] proposed a new algorithm for risk assessment in sequential screening for DS, which integrated pregnancy-associated plasma protein A (PAPP-A) and fetal nuchal translucency (NT) measured by ultrasonography in the first trimester with alpha-fetoprotein, unconjugated estriol (uE3), free beta-human chorionic gonadotropin (F β-hCG), and inhibin A in the second trimester. At a false positive rate of 1%, this screening strategy yielded a DS detection rate of up to 85%, while single screening in the first trimester or triple screening in the second trimester had DS detection rates of 72% and 46%, respectively. With the rapid development of next generation sequencing (NGS), maternal plasma DNA sequencing has revolutionized prenatal DS screening [6]. At the Chinese University of Hong Kong, Prof. Y M Dennis Lo and colleagues first reported the presence of cell-free DNA (cfDNA) in maternal plasma in 1997. In theory, analysis of fetal genetic materials [7] allows for noninvasive and risk-free genetic detection, such as direct detection with cfDNA in maternal plasma for evaluating the risk of fetal DS, rather than indirectly with maternal serum biomarkers. This opens a new era of NIPT.

The cost of SS and NIPT are very different; NIPT can be >10-fold more expensive than SS in some regions. The clinical application of SS and NIPT is dependent on price, affordability, and government subsidies. Various models have been established for cost-effectiveness analysis (CEA) of NIPT in first-line screening and second-tier investigation [8-11]. In this study, we retrospectively analyzed the results of SS and NIPT and the pregnancy outcomes during follow-up of 17,363 pregnant women in Zhuhai City, China. We also performed a health economic evaluation of four screening strategies from a public health perspective. Our results provide a reference for the selection of screening strategies suitable for the situations in various regions.

Materials and methods

Subjects

17,363 case of pregnant women who participated voluntarily in the public health service program of prenatal testing for prevention and control of birth defects in Zhuhai from 2018 to 2019 received both SS and NIPT during the same gestation. For those at high risk (≥1/270) and intermediate risk (1/1,000 to 1/270) for T21 in SS, amniocentesis was recommended as an invasive prenatal diagnosis (IPD) to analyze fetal karyotype. Those at high risk based on NIPT were recommended for amniocentesis or cordocentesis for fetal karyotype analysis. Those who received IPD were called at one week and one month after operation and one month after delivery to follow up the pregnancy outcomes. Alternatively, the Maternal and Child Health Information System of Zhuhai Health Bureau was queried for the outcomes. Those who did not receive IPD were called one month after the expected date of delivery or the information system was queried to follow up pregnancy outcomes. All examinations were approved by the ethics committee and signed by pregnant women with informed consent. All pregnant women do not include minors.

Noninvasive prenatal testing

The cfDNA in maternal plasma was detected using the BIGSEQ 500 (MGI, China) high-throughput sequencing system, and fetal chromosome aneuploidy was analyzed via HALOS software. The NIFTY® reagent was purchased from BGI (China). Quality control of the data used in bioinformatics analysis were as follows: ≥3.5% fetal DNA per sample, 38–42% GC, ≥5.2 M original data, ≥3.5 M valid data, and a cutoff Z score of 3.

Serum screening

The test was performed on an Auto DELFIA 1235 automatic time-resolved fluoroimmunoassay (TRFIA) system (PerkinElmer, USA) with TRFIA reagent. In first-trimester screening, risk was assessed using PAPP-A and F β-hCGin maternal peripheral venous serum, as well as fetal NT measured by ultrasonography. In second-trimester screening, risk assessment was based on alpha-fetoprotein, F β-hCG, and uE3 in maternal serum. Risk assessment was done using the Lifecycle 4.0 software, with a cutoff value of 1/270 for high risk and a cutoff value of 1/1,000 for intermediate risk for T21.

Chromosome karyotype analysis

Approximately 10 mL of amniotic fluid was drawn from pregnant women at G18–G24 W, centrifuged to remove the supernatant, and the precipitated amniotic fluid cells were cultured in sterile cell culture medium (GIBCO, USA). The 0.5–1 mL heparin-anticoagulated cord blood from pregnant women after G24 W was incubated in blood lymphocyte culture medium. At the metaphase of mitosis, chromosomes were subjected to conventional Giemsa-banded karyotyping at a resolution of 320–400 bands. Karyotype images were acquired using an Imager Z2 automatic chromosome karyotype analyzer (Zeiss, Germany), and the karyotype description was based on the International System for Human Cytogenomic Nomenclature (ISCN 2016).

Health economic evaluation

All test and follow-up results were subjected to retrospective analysis using the four following screening strategies. CEA was conducted based on the price and fee schedules of Zhuhai and the per capita gross domestic product (GDP) of Guangdong Province in 2019. The economic costs of DS mainly include DS screening, IPD diagnosis, IPD-related miscarriages, and the economic burden of the patient’s family due to the disease. The economic burden of DS can be direct and indirect. Direct economic burden refers to the direct medical costs and direct non-medical costs derived from the treatment of DS, and the costs of development services and special education of DS patients. Indirect economic burden is the loss of labor productivity in DS patients and in their family members, which is estimated by the reduction of effective working time and productivity. Zeng et al. [12] at Central South University estimated that the economic burden of DS was about 1.1 million CNY per case based on the per capita GDP of 23,798 CNY in Hunan Province in 2010. According to the China Statistical Yearbook 2020 [13], the per capita GDP of Guangdong Province in 2019 was 94,172 CNY. Therefore, the economic cost for each missed case of DS in Zhuhai was approximately 4.35 million CNY. In addition, according to the medical service price schedule from the Zhuhai Price Bureau, the cost of SS was 120 CNY/test, the cost of NIPT was 855 CNY/test, the cost of IPD (including abdominal paracentesis, ultrasound-guided abdominal paracentesis, and chromosome karyotype analysis) was 2,500 CNY/test, and the average cost of IPD-related miscarriages was 2,000 CNY/person. In the following formulas, A is the total number of pregnant women screened, B is the number of pregnant women at high risk of T21 in SS (≥1/270), C is the number of pregnant women at intermediate risk of T21 in SS (1/1,000 to 1/270), D is number of pregnant women at high risk of T21 in NIPT, E is the number of missed cases of DS, and F is the number of IPD-related miscarriages. The values of D and E are dependent on the screening strategy. All costs calculated in this study were expressed in CNY.

Strategy I: All pregnant women received SS and those at high risk of T21 had IPD.

Cost = A × 120 + B × 2,500 + E × 4,350,000 + F × 2,000 (CNY)

Strategy II: All pregnant women received SS, those with T21 risk ≥1/270 had IPD, and those with 1/270 > T21 risk ≥1/1,000 received NIPT; women at high risk based on NIPT results had IPD.

Cost = A × 120 + C × 855 + (B + D) × 2,500 + E × 4,350,000 + F×2,000 (CNY)

Strategy III: All pregnant women received SS, and those with T21 risk ≥1/1,000 had NIPT; women at high risk based on NIPT results had IPD.

Cost = A × 120 + (B + C) × 855 + D × 2,500 + E × 4,350,000 + F × 2,000 (CNY)

Strategy IV: All pregnant women received NIPT and those at high risk for T21 had IPD.

Cost = A × 855 + D × 2,500 + E × 4,350,000 + F × 2,000 (CNY)

The flowchart of screening with the above four strategies is shown in Fig 1.

Fig 1

Flowchart of four screening strategies.

NIPT, noninvasive prenatal testing; IPD, invasive prenatal diagnosis.

Results

Basic information

The data were collected from 19,465 pregnant women and 2,102 were lost to follow-up due to incomplete records of postpartum visits in the Maternal and Child Health Information System, change of telephone number, or unsuccessful telephone follow up, with a loss rate of 10.8%. Finally, a total of 17,363 pregnant women were enrolled, with a mean gestation period of G9–G30 W and a mean age of 28.9 ± 3.7 years, of which 796 (4.6%) were ≥35 years old. A total of 22 DS cases were detected, of which only one was 37 years old, and the remaining were <35, with an incidence of 1/789. One pregnant woman with IPD had a miscarriage within two weeks after the procedure.

A total of 8,765 pregnant women participated in first-trimester screening, with a mean gestational age of 12.1 ± 0.7 W and a mean age of 29.1 ± 3.4 years. A total of 8,598 pregnant women participated in second-trimester screening, with a mean gestational age of 16.4 ± 1.2 W and a mean age of 28.7 ± 4.0 years. Of the 22 DS cases, 8 were detected in pregnant women with a T21 risk ≥1/270, giving a positive detection rate of 36.4% (8/22). Of the 14 missed DS cases, 10 were in the first trimester and 4 in the second trimester, and 6 had a T21 risk between 1/1,000 and 1/270. When the risk cutoff was expanded to 1/1,000, the additional 6 cases led to a positive detection rate of only 63.6% (14/22). Of 16 ture DS cases in first-trimester screening, for the 10 cases missed calculation without NT and with the serum indicators PAPP-A and F β-hCG identified 4 cases, and the detection rate was 62.5% (10/16) (Table 1) (Fisher exact test; P = 0.289).

Table 1

List of Serum screening results.

parameter	Number of pregnant women in first-trimester screening (%)	Number of pregnant women in second-trimester screening (%)	Total number of pregnant women screened (%)
T21 risk ≥ 1/270	361 (4.12)	663 (7.71)	1,024 (5.90)
T21 risk < 1/270	7,496 (85.52)	6,379 (74.19)	13,875 (79.91)
1/1,000 ≤ T21 risk < 1/270	908 (10.36)	1,556 (18.10)	2,464 (14.19)
Detected casesa	6 (0.06)	2 (0.02)	8 (0.05)
Undetected casesa	10 (0.11)	4 (0.05)	14 (0.08)
FPR	4.05%	7.69%	5.85%
PPV	1.66%	0.30%	0.78%
NPV	99.88%	99.95%	99.91%
Total	8,765 (100.0)	8,598 (100.0)	17,363 (100.0)

FPR, false positive rate; PPV, positive predictive value; NPV, negative predictive value.

a the number of detected cases is the number of DS cases with risk ≥1/270, and the number of missed cases is the number of DS cases with risk <1/270.

NIPT screening showed 27 cases at high risk for T21, 22 confirmed cases, and 5 false positive cases. There were no reports of missed cases in the follow-up (Table 2). The results of SS screening and the fetal karyotype of IPD corresponding to the 27 high-risk cases in NIPT screening are shown in Table 3.

Table 2

List of Noninvasive prenatal testing results.

parameter	Number of pregnant women screened for T21 (%)		Total
	First trimester (≤13+6 W)	Second and third trimesters (≥14 W)	Number of pregnant women screened for T21 (%)
Low risk	4,483 (99.87)	12,853 (99.84)	17,336 (99.84)
High riska	6 (0.13)	21 (0.16)	27 (0.16)
Detected cases	6 (0.13)	16 (0.12)	22 (0.13)
Undetected cases	0 (0)	0 (0)	0 (0)
FPR	0	0.11%	0.03%
PPV	100%	76.2%	81.5%
NPV	100%	100%	100%
Total	4,489 (100.0)	12,874 (100.0)	17,363 (100.0)

FPR, false positive rate; PPV, positive predictive value; NPV, negative predictive value.

aZ ≥ 3 indicates high risk in NIPT.

Table 3

Serum screening and fetal karyotype of cases at high risk in noninvasive prenatal testing.

Case number	Age (years)	Gestational weeks in NIPT	cfDNA (%)	Z valuea	Gestational weeks in SS	T21 risk in SSb	Fetal karyotype
Case 1	30	18	9.64	17.78	16	1/97	47, XNc, +21
Case 2	26	17	9.14	11.17	16	1/58	46, XY, der(14;21)(q10;q10), +21
Case 3	32	19	11.75	21.52	16	1/703	47, XX, +21
Case 4	34	18	9.48	4.01	16	1/778	46, XX
Case 5	26	20	9.14	10.81	17	1/812	47, XY, +21
Case 6	30	20	8.27	17.27	16	1/761	47, XX, +21
Case 7	28	22	8.84	4.85	19	1/3,708	46, XY
Case 8	23	17	11.78	14.69	16	1/1,002	47, XX, +21
Case 9	32	16	13.36	10.01	13	1/205	47, XY, +21
Case 10	37	16	10.49	29.99	12	1/37	47, XNc, +21
Case 11	31	13	11.98	12.96	12	1/6	47, XX, +21
Case 12	31	13	12.37	17.93	12	1/5	47, XNc, +21
Case 13	33	12	5.51	4.48	12	1/104	47, XX, +21
Case 14	32	13	10.10	10.84	12	1/5	47, XX, +21
Case 15	31	14	12.08	15.50	12	1/377	47, XX, +21
Case 16	30	14	11.71	13.75	12	1/586	47, XY, +21
Case 17	30	12	6.71	8.55	12	1/595	47, XX, +21
Case 18	24	16	21.12	12.19	12	1/1,677	46, XX
Case 19	22	26	10.26	12.18	12	1/11,033	46, XY
Case 20	30	20	8.10	10.77	12	1/2,001	47, XY, +21
Case 21	32	15	8.08	13.72	12	1/1,002	47, XX, +21
Case 22	31	17	7.83	5.59	13	1/5,749	47, XY, +21
Case 23	31	19	11.71	3.98	12	1/1,873	47, XY, +21[9]/46, XY[41]
Case 24	28	13	9.50	17.42	12	1/1,096	47, XX, +21
Case 25	30	20	10.37	14.88	12	1/2,581	47, XX, +21
Case 26	27	15	13.34	10.75	12	1/1,569	47, XY, +21
Case 27	31	21	11.06	3.61	11	1/1,292	46, XX

NIPT, noninvasive prenatal testing; SS,serum screening.

aZ ≥ 3 indicates high risk in NIPT.

brisk ≥1/270 indicates high risk inSS; and 1/1,000 ≤ risk < 1/270 indicates intermediate risk in SS.

c N indicates that invasive prenatal diagnosis was conducted in other institutions and fetal gender was not reported.

It can be seen from Tables 1 and 2 that although the NPV of NIPT is similar to that of SS, it is far superior to SS in terms of detection rate, IPD rate, PPV and FPR. as shown in Fig 2.

Fig 2

Comparison of various indicators in the statistical results of NIPT and SS.

Health economic evaluation of DS screening

A hypothetical retrospective analysis was performed on the data of four screening strategies from a public health perspective. In strategy IV, all pregnant women received NIPT and those at high risk had IPD, which was the optimal screening strategy with the lowest total cost. In strategies II and III, SS was performed for primary screening and NIPT for secondary screening, and those at high risk received IPD. Both strategies showed missed DS cases, and the total costs were approximately 2.7-fold that of strategy IV. In strategy I, DS detection with SS and IPD led to the highest number of missed cases, the highest total cost (50.63 million CNY, or 4.4-fold, more than strategy IV), and an additional cost of 2,916 CNY per case (Table 4).

Table 4

Costs of DS screening with four screening strategies.

Screening strategy	Total number of pregnant women screened (A)	SS ≥ 1/270 (B)	SS 1/1,000–1/270 (C)	High risk in NIPT (D)	Missed DS cases in SS (E)	Number of IPD-related miscarriages (F)	Total costs (CNY)
I	17,363	1,024	−	−	14	0	65,543,560
II	17,363	1,024	2,464	8a	8	1	41,572,280
III	17,363	1,024	2,464	15b	8	1	39,905,300
IV	17,363	−	−	27	0	0	14,912,865

SS, serum screening; DS, Down syndrome; NIPT, noninvasive prenatal testing; IPD, invasive prenatal diagnosis.

a denotes the number of cases at high risk in NIPT in C.

b denotes the number of cases at high risk in NIPT in B and C.

Discussion

SS is divided into first-trimester screening at G9–13+6 W and second-trimester screening at 14–20+6 W. The gestational age in the model was estimated by biparietal diameter of the fetal skull measured by ultrasonography, which was included in risk calculation in the first trimester. In another study, the use of gestational age estimated by ultrasonography significantly reduced the variation of indices within one week of pregnancy. These data suggest that routine use of ultrasonography for gestational age estimation increased DS detection rate from 58% to 67% with a false positive rate of 5% [14]. However, there are multiple subjective factors such as detection technique, measurement method, clinical experience, and expertise of ultrasound technicians. The lack of standardized measurement in primary hospitals easily leads to large variation in the estimation of NT and gestational age, which in turn affects the accuracy of DS risk calculation. The use of NT in risk calculation for DS screening in the first trimester and the use of gestational age estimated by ultrasonography in the second trimesters led to a detection rate of SS much lower than theoretical value. In this study, double screening without NT detected 62.5% of DS cases in the first trimester, while screening with NT only detected 36.4% of DS cases. Although there was no significantly difference between the two detection rates (P = 0.289), it might be caused by insufficient samples. In this study, the <40% detection rate of triple screening in the second trimester was much lower than rates reported in various regions, which may be attributable to the great variation in gestational age measured by ultrasonography.

Accumulating screening data have shown that DS screening with NIPT has unparalleled advantages over SS regarding PPV, FPV, and IPD. The data in this study also confirms this point. For example, we observed much higher PPV of NIPT (81.5%) than SS (0.78%). Moreover, SS is applicable only to pregnant women at G9–G20+6 W, while NIPT is applicable at more than 21 weeks of gestation until last trimester. However, the NIPT technical specifications issued by the Health and Family Planning Commission of China in 2016 [15] recommended NIPT as a secondary strategy to SS. In the specification, NIPT is applicable to pregnant women whose SS shows a fetal aneuploidy risk between the high-risk cutoff value and 1/1,000 or who miss the optimal time for SS (≤G20+6 W) but are required to perform risk assessment for trisomy 21, trisomy 18, and trisomy 13, or who have contraindications for IPD.

Because of religious beliefs, legal regulations, medical payment and other factors, different regions adopt different strategies when choosing state-sponsored DS screening. In the public health service program, cost is the most important factor preventing NIPT from replacing SS as a first-line prenatal DS screening strategy. Additionally, the cost-effectiveness ratio of the screening strategy is the most important consideration for local health administrations. Some countries effectively regulate the supply of NIPT on grounds of cost-effectiveness and reliability, there is disagreement regarding the implementation of NIPT in different nations [16], and health facilities worldwide have carried out various CEAs of SS and NIPT for DS screening. However, almost all these analyses were based on calculation with statistical models assuming SS as a first-line screening test and NIPT as a secondary strategy.

In 2014, the UK National Health Service (NHS) [8] used a pre-existing model to assess and compare the costs and outcomes of NIPT for DS. They found that NIPT as a contingent test would be cost neutral or cost saving compared with current DS screening if the cost of NIPT was <400 GBP and the screening risk cutoff was 1:150. NIPT as first-line testing would achieve more favorable outcomes but at a greater cost. Therefore, further research is needed to determine whether NIPT can be promoted as a first-line screening strategy in public health services. In the Netherlands, Beulen et al. [11] developed a decision-analytic model for CEA of prenatal DS screening in clinical practice. They found that the introduction of NIPT increased DS detection rate and decreased IPD, thereby decreasing IPD-related miscarriages and the cost of DS per case. Because of the high cost of NIPT as a new technology, it is not feasible to use NIPT as a primary screening test, but it should be used as an optional test for pregnancies at high risk for T21. In the United States in 2015, Evans et al. [17] performed a decision tree analysis and suggested that NIPT as a primary screening was not cost-effective, and the cost was lowest when it was used as a contingent strategy, especially with a risk cutoff of 1/1,000. Although the cost of a hybrid strategy was lower than that of NIPT as a primary strategy, the cost was higher than that of NIPT as a contingent strategy. The cost of NIPT in the United States was 1,017 USD/test.

In 2019, Zhang et al. [10] used a microsimulation decision-analytic model to conduct a sample survey of 45,605 pregnant women in British Columbia, Canada. They concluded that NIPT screening was more effective and more expensive, and NIPT at 200 USD or less was more cost-effective as a first-line screening strategy. Xu et al. [18] of Fudan University conducted a survey of physicians and experts from 25 medical facilities in Shanghai, Hunan Province, Zhejiang Province, and Shandong Province, China, as well as a literature search of relevant data. A decision-analytic model was established in a simulated cohort of 10,000 pregnant women using TreeAge Pro software, and a CEA of NIPT was performed from a societal perspective. The study found that NIPT was the most effective when used as a universal screening strategy because it detected more DS cases, and NIPT was the safest and most cost-effective as a contingent screening strategy. The cost evaluation of the study did not include direct non-medical costs and non-direct costs, which in fact bring the greatest economic burden of DS on society and families.

Our institute provides public health services such as prenatal screening of DS for more than 20,000 pregnant women each year in Zhuhai. Pregnant women have the option of either or both SS and NIPT tests, and the costs are covered by a special fund of Zhuhai Municipal Government and the maternity insurance in the social medical security fund. If the fetus is diagnosed with DS, before the fetus is born, the pregnant woman has the right to decide whether to terminate the pregnancy or give birth to the fetus. The data used for health economic evaluation in this study were based on a statistical analysis of the clinical test results and the postnatal follow-up outcomes of pregnant women who received both tests during gestation. Only 1 of the 22 DS cases in 17,363 pregnancies was identified in a woman with advanced age (>35 years), which was because most participants with advanced age received IPD instead of SS and NIPT. Based on the data in Zhuhai, it was estimated that SS as a single test or SS as a first-line test and NIPT as a secondary test had much higher total costs than NIPT as a first-line screening strategy. With the rapid development of NGS and the decrease in sequencing costs, NIPT will become increasingly inexpensive, and the cost difference will be exacerbated. Moreover, the impact of subjective factors on NIPT was much lower than on SS. Therefore, secondary prevention and control of birth defects using NIPT instead of SS as a first-line test for prenatal DS screening can greatly reduce the frequency of IPD and IPD-related miscarriages, save the limited healthcare resources, greatly improve DS detection rate, and thus bring significant social and economic benefits. If the pregnancy woman with a DS fetus finally decides to have the baby, the parents still benefit from screening in being able to prepare for the birth of a child with disabilities, but it would affect the economic costs and benefits of the outcome.

26 May 2021

PONE-D-21-11149

Health economic evaluation of noninvasive prenatal testing and serum screening for Down syndrome

PLOS ONE

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Reviewer #1: Dear authors,

Thanks for your submission. I just have some minor comments;

1- Please mention the full name of hormone uE3 before using the abbreviation at first mention in the document (line 106).

2- Titles of tables 1 and 2 need to be more informative.

3- In the discussion you mention that the DS risks calculated with or without the NT were significantly different in the first trimester (line 238), but i couldn't find such comparison in results section.

4- In the discussion you mention in line 253 that cfDNA is undetectable in maternal blood until 2 h after delivery. What is the significance of this information in relation to you discussion argument?

Finally the manuscript is generally acceptable and worth publishing after these minor rivisions

Thanks,

Reviewer #2: General Comments

The paper is interesting. The message is straight forward, and the results presented support the conclusion. The authors successfully demonstrate how NIPT can be used as a non-invasive primary step for the diagnosis Down Syndrome. They also provide evidence showing that it may be more cost effective and accurate than serum screening. However, the results are not discussed in depth and I see several other problems with the manuscript.

Firstly, there seems to be a lack of detail in the methods. While many of the details are ratified within the results, the Methods and Materials section at times is somewhat ambiguous, especially regarding the number of study participants and the included tests.

Secondly, the first page of the discussion, while interesting in context of the history of down syndrome screening, would probably fit better within the introduction. This first portion of the discussion makes the section very long and while it provides some context, it does not provide information that supports the presented results.

Thirdly, the paper would be well served by including graphical representation of the results, rather than including all results within tables. The one figure that was included was illegible.

Thirdly, while it is stated at line 72 “We also performed a health economic evaluation of four screening strategies from a public health perspective.” very little discussion is given to the possible economic impact or outcomes of the different screening strategies. In table 4 it is demonstrated that strategy 4 (where NIPT is the only screening method) is approximately 25,000,000 CNY cheaper than the next cheapest strategy and over 50,000,000 CNY cheaper than strategy 1, a factor of over 4 times cheaper. At line 260 it is stated “In the public health service program, cost is the most important factor preventing NIPT from replacing SS as a first-line prenatal DS screening strategy. Additionally, the cost-effectiveness ratio of the screening strategy is the most important consideration for local health administrations.” If this is the case, and strategy 4 is considerably cheaper with promising outcomes why is this not being considered or implemented? There is no critical discussion present on this. Furthermore, there seems to be no discussion over the advantages or disadvantages of using NIPT over SS as the primary screening method, apart from NIPT being less susceptible to subjective factors (which are not outlined).

Finally, at line 312 “Therefore, secondary prevention and control of birth defects using NIPT instead of SS as a first-line test for prenatal DS screening can greatly reduce the frequency of IPD and IPD-related miscarriages”. While I agree that this conclusion is correct based on the presented results, can this conclusion be made in regards to IPD-related miscarriage when a single miscarriage was recorded?

Specific Points

INTRODUCTION

1. Line 53: “200-300 genes” is there no better precise estimate?

2. Line 65: Is there any trimester separation for NIPT or is it as applicable through pregnancy?

METHODS AND MATERIALS

3. Line 81: Did all women undergo both SS and NIPT or was it a mixture? Not clear.

4. No where in the methods do the authors specify the number of women that were tested. I assume it was 17363 as mentioned in the introduction.

5. Line 83: Are there breakpoints which categorise high risk in the NIPT test like described for SS?

6. Line 84: Is there a reason why only amniocentesis was recommended for SS while amniocentesis or cordocentesis was recommended for NIPT?

7. Again, were both SS and NIPT performed on all the women or SS on one proportion and NIPT on another? Based on the results I assume SS and NIPT were both performed on all patients. Needs to be outlined better within the Methods.

8. Line 85: How many patients underwent IPD?

9. Line 157: Figure 1 is essentially unreadable. Needs to be remade.

RESULTS

NIPT

10. Line 190: Table 2: “FPV” not consistent with “FPR, False Positive Rate”

11. Line 192: “*Z ≥ 3 indicates high risk in NIPT” Z is not outlined in NIPT methods section as risk assessment is in the SS methods section (Line 106 - 108).

DISCUSSION

12. Line 285: Is this true? There is no reference for this statement. The NIH priced NIPT in the USA as $700-1000 per test in 2014. One would expect this to be cheaper 7 years later.

**********

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27 Jun 2021

Journal Requirements:

1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response：Yes, the format of the manuscript has been edited to meet PLOS ONE's style requirements.

2.Thank you for including your ethics statement:  "All examinations were approved by the ethics committee of Zhuhai Center for Maternal and Child Healthcare and informed consent was obtained from pregnant women.The authors of the manuscript declare no conflicts of interest.".

Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Response：All pregnant women signed the informed consent form before the examination. The relevant content has been added in line 116.“All examinations were approved by the ethics committee and signed by pregnant women with informed consent. All pregnant women do not include minors.”

3.Please amend the manuscript submission data (via Edit Submission) to include authors Guoqing Wang, Huayu Luo.

Response：Data about Guoqing Wang and Huayu Luo has been amended via Edit Submission.

Reviewer #1:

1-Response to comment: Please mention the full name of hormone uE3 before using the abbreviation at first mention in the document (line 106).

Response：the full name of hormone uE3 is “unconjugated estriol”. It has been added in line 133.

2-Response to comment: Titles of tables 1 and 2 need to be more informative.

Response：The titles of tables 1 and 2 have been amended as “List of Serum screening results”(Line 209) and “List of Noninvasive prenatal testing results”(Line 218).

3- Response to comment: In the discussion you mention that the DS risks calculated with or without the NT were significantly different in the first trimester (line 238), but i couldn't find such comparison in results section.

Response：It has been mentioned in line 206.“For the 10 cases missed in first-trimester screening, calculation without NT and with the serum indicators PAPP-A and F β-hCG identified 4 cases, and the detection rate was increased to 62.5% (10/16) (Table 1).”

4- Response to comment: In the discussion you mention in line 253 that cfDNA is undetectable in maternal blood until 2 h after delivery. What is the significance of this information in relation to you discussion argument?

Response：We mean that the applicable gestational week of NIPT is longer than that of SS. It has been modified to“Moreover, SS is applicable only to pregnant women at G9–G20+6 W, while NIPT is applicable at more than 21 weeks of gestation until last trimester”in line 272.

Reviewer #2:

General Comments:

1- Response to comment: There seems to be a lack of detail in the methods. While many of the details are ratified within the results, the Methods and Materials section at times is somewhat ambiguous, especially regarding the number of study participants and the included tests.

Response：The number of study participants and the included tests have been added in lines 105-107:“17,363 case of pregnant women who participated voluntarily in the public health service program of prenatal testing for prevention and control of birth defects in Zhuhai from 2018 to 2019 received both SS and NIPT during the same gestation. ”In the "Noninvasive prenatal testing" and "Serum screening" section of Materials and Methods, there are detailed introductions about these two tests respectively.

2-Response to comment: The first page of the discussion, while interesting in context of the history of down syndrome screening, would probably fit better within the introduction. This first portion of the discussion makes the section very long and while it provides some context, it does not provide information that supports the presented results.

Response：Thank you for your reminder. In the revised manuscript, the general introduction about SS and NIPT on the first page of the discussion was moved to the introduction(line 78), and some sentences were deleted.

3- Response to comment: The paper would be well served by including graphical representation of the results, rather than including all results within tables. The one Fig that was included was illegible.

Response：This is a great suggestion. We have sorted out part of the data and shown it in Fig 2. The resolution of Fig 1 has been adjusted to 400dpi.

4-Response to comment: While it is stated at line 72 “We also performed a health economic evaluation of four screening strategies from a public health perspective.” very little discussion is given to the possible economic impact or outcomes of the different screening strategies. In table 4 it is demonstrated that strategy 4 (where NIPT is the only screening method) is approximately 25,000,000 CNY cheaper than the next cheapest strategy and over 50,000,000 CNY cheaper than strategy 1, a factor of over 4 times cheaper. At line 260 it is stated “In the public health service program, cost is the most important factor preventing NIPT from replacing SS as a first-line prenatal DS screening strategy. Additionally, the cost-effectiveness ratio of the screening strategy is the most important consideration for local health administrations.” If this is the case, and strategy 4 is considerably cheaper with promising outcomes why is this not being considered or implemented? There is no critical discussion present on this.

Furthermore, there seems to be no discussion over the advantages or disadvantages of using NIPT over SS as the primary screening method, apart from NIPT being less susceptible to subjective factors (which are not outlined).

Response：The question you mentioned is the meaning of our article. Because almost all health economics evaluations were based on calculation with statistical models assuming SS as a first-line screening test and NIPT as a secondary strategy（Line 285）, there has been a lack of objective and actual data from clinical practice on large populations for reference to health administration departments. Because NIPT is currently available in the private sector in most country, the price of NIPT is expensive. This is also the main reason why the model data believes that SS is more advantageous than NIPT in cost-benefit analysis. However, the price of NIPT in Zhuhai City is much lower than that in other places due to the intervention of the government. So the total cost of NIPT as a first-line screening has been greatly reduced. But not every private sector is willing to reduce the price of NIPT to such a cheap price.

We have Added “The data in this study also confirms this point” in line 270. And in line 272 of the discussion, We also wrote that “Moreover, SS is applicable only to pregnant women at G9–G20+6 W, while NIPT is applicable at more than 21 weeks of gestation until last trimester”, which is also one of the advantages of NIPT. We also added Fig 2 in “Results” to show that NIPT has more advantages than SS.(Line 232)

5-Response to comment: Finally, at line 312 “Therefore, secondary prevention and control of birth defects using NIPT instead of SS as a first-line test for prenatal DS screening can greatly reduce the frequency of IPD and IPD-related miscarriages”. While I agree that this conclusion is correct based on the presented results, can this conclusion be made in regards to IPD-related miscarriage when a single miscarriage was recorded?

Response：The evidence of this conclusion is not because there was only one miscarriage, but the number of people with high risk of SS requiring IPD is much greater than that of NIPT, so the probability of miscarriage is greatly increased. This is a conclusion drawn from probability.

Specific Points:

Introduction

1. Response to comment: Line 53: “200-300 genes” is there no better precise estimate?

Response：There is no accurate number of genes associated with Down syndrome. Because the technology is constantly developing, new related genes are constantly being discovered, and the reported data in various places is sometimes not updated timely.

2. Response to comment: Line 65: Is there any trimester separation for NIPT or is it as applicable through pregnancy?

Response：NIPT is applicable at more than 12 weeks of gestation until last trimester. However, considering that high-risk pregnant women need to set aside enough time for interventional prenatal diagnosis, it is usually only recommended to have a maximum of 30 weeks of pregnancy.

Materials and methods:

3. Response to comment: Line 81: Did all women undergo both SS and NIPT or was it a mixture? Not clear.

Response：Yes. All pregnant women received both NIPT and SS during the same pregnancy. (Line 107)

4. Response to comment: No where in the methods do the authors specify the number of women that were tested. I assume it was 17363 as mentioned in the introduction.

Response：Yes,you are right. It is mentioned in line 192 of the results.“Finally, a total of 17,363 pregnant women were enrolled, ” We have also added the number of women that were tested in “Subjects” of “Materials and methods”.(Line 105)

5.Response to comment: Line 83: Are there breakpoints which categorise high risk in the NIPT test like described for SS?

Response：Yes. It is mentioned in line 124 as“and a cutoff Z score of 3”.

6. Response to comment: Line 84: Is there a reason why only amniocentesis was recommended for SS while amniocentesis or cordocentesis was recommended for NIPT?

Response：The suitable gestational week for SS is 9-20+6 weeks, and the suitable gestational week for NIPT can be from 12 weeks to the third trimester. Generally, the most suitable gestational week for drawing amniotic fluid for karyotype analysis is 16-25 weeks. After 25 weeks, amniotic fluid cell culture is difficult to succeed, so cord blood is drawn for cell culture and karyotype analysis. Because the risk of miscarriage caused by amniocentesis is smaller than that of cord blood puncture, it is generally recommended to draw amniotic fluid for prenatal diagnosis for pregnant women at high risk of SS.

However, some pregnant women may have been taking NIPT for more than 25 weeks, and it is no longer suitable to draw amniotic fluid for karyotyping, so it is recommended to draw cord blood. Although in the third trimester of DS diagnosis, in addition to the method of karyotyping, DNA of amniotic fluid cells can also be extracted to analyze chromosome data, but because of the high cost of testing, karyotyping of cord blood cells is still the first choice.

7. Response to comment: Again, were both SS and NIPT performed on all the women or SS on one proportion and NIPT on another? Based on the results I assume SS and NIPT were both performed on all patients. Needs to be outlined better within the Methods.

Response：Yes,you are right. All pregnant women received both NIPT and SS during the same pregnancy.(Line 107)

8. Response to comment: Line 85: How many patients underwent IPD?

Response：Regrettably, because some pregnant women did IPD not in our hospital, we can only see the final pregnancy outcome through the follow-up of the information system,and it,s not certain whether the pregnant woman had IPD. So there is no exact data on the total IPD.

9. Response to comment: Line 157: Fig 1 is essentially unreadable. Needs to be remade.

Response：Well, We have remade Fig 1. The resolution of Fig 1 has been adjusted to 400dpi.

Results

10. Response to comment: Line 190: Table 2: “FPV” not consistent with “FPR, False Positive Rate”

Response：We are very sorry for our negligence of it. It should be "FPR". It has been corrected now.(Line 218: Table 2)

11. Response to comment: Line 192: “*Z ≥ 3 indicates high risk in NIPT” Z is not outlined in NIPT methods section as risk assessment is in the SS methods section (Line 106 - 108).

Response：It has been described as“≥3.5% fetal DNA per sample, 38–42% GC, ≥5.2 M original data, ≥3.5 M valid data, and a cutoff Z score of 3”. (line 124)

Discussion

12. Response to comment: Line 285: Is this true? There is no reference for this statement. The NIH priced NIPT in the USA as $700-1000 per test in 2014. One would expect this to be cheaper 7 years later.

Response：Yes, the price of NIPT in the United States in 2021 may be cheaper. However, this sentence in this manuscript is derived from the results section of the abstract of Reference 17 “Utilizing a primary cffDNA screening strategy,the cost per patient was 1017 US$.” We have already stated the year of publication in the manuscript is 2015.(Line 300) We regret that we did not find a newer price of NIPT in US.

18.Response to comment: PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Response：I choose no.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 Nov 2021

15 Dec 2021

1- the first mentioning of uE3 is now moved to line 81 (this part was not included in your original submission), hence you should the full name of this abbreviation accordingly.

Response：the full name of hormone uE3 is “unconjugated estriol”. It has been added in line 83.

2- Your response to my 3rd comment is inadequate : (It has been mentioned in line 206.“For the 10 cases missed in first-trimester screening, calculation without NT and with the serum indicators PAPP-A and F β-hCG identified 4 cases, and the detection rate was increased to 62.5% (10/16) (Table 1).”). This doesn't necessarily imply significance. There has to be a p-value of at least <0.05 based on a suitable statistical test to conclude significant difference.

Response：We added statistical test result in line 205, and analyzed the reason in line 262 of discussion.

3-Have the authors made all data underlying the findings in their manuscript fully available?

Response：We have added “Availability of data and materials” in line 340.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Feb 2022

Health economic evaluation of noninvasive prenatal testing and serum screening for Down syndrome

PLOS ONE

Dear Dr. Xiao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers are now happy with the technical aspects of the paper. I am not the original editor responsible for this paper, so I apologize, but I am going to request two small additional considerations. First, this is a topic where there are ethical issues involved, concerning the rights of those living with disabilities. I request the authors to refer briefly to this literature in the Introduction (a couple of sentences plus references would suffice).

Second, it would be helpful in the Discussion section to note that the results for China may not transfer readily to other countries. In some other countries, late detection of Down's syndrome may be too late for termination of the pregnancy to be legally permitted. Even where it is legally permitted, not all parents may exercise the choice to terminate the pregnancy. I don't know if this is an option in China (would Zhuhai Municipality permit the parents to continue on with the pregnancy?). In these circumstances the parents still benefit from screening in being able to prepare for the birth of a differently-abled child, but it would affect the economic costs and benefits of the outcome. Again, a couple of sentences qualifying the findings would suffice.

Please submit your revised manuscript by Apr 07 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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25 Mar 2022

Response:

1- This is a topic where there are ethical issues involved, concerning the rights of those living with disabilities. I request the authors to refer briefly to this literature in the Introduction (a couple of sentences plus references would suffice).

Response：Because of religious beliefs, legal regulations, medical payment and other factors, different regions adopt different strategies when choosing state-sponsored DS screening. In China, termination of pregnancy is legally permitted when the fetus is diagnosed to be disabled before birth. It has been description in line 278.

2- It would be helpful in the Discussion section to note that the results for China may not transfer readily to other countries. In some other countries, late detection of Down's syndrome may be too late for termination of the pregnancy to be legally permitted. Even where it is legally permitted, not all parents may exercise the choice to terminate the pregnancy. I don't know if this is an option in China (would Zhuhai Municipality permit the parents to continue on with the pregnancy?). In these circumstances the parents still benefit from screening in being able to prepare for the birth of a differently-abled child, but it would affect the economic costs and benefits of the outcome. Again, a couple of sentences qualifying the findings would suffice.

Response：If the fetus is diagnosed with DS, before the fetus is born, the pregnant woman has the right to decide whether to terminate the pregnancy or give birth to the fetus.We have added your comments to lines 283, 324 and 339.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 Mar 2022

Health economic evaluation of noninvasive prenatal testing and serum screening for Down syndrome

PONE-D-21-11149R3

Dear Dr. Xiao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLOS ONE

Additional Editor Comments (optional):

please accept 2 small edits to the text (for English-language speakers): in line 337, delete the word "born" (it is ok to say "have the baby") and instead of "disability child" in line 338 please say "child with disabilities": in the literature on disability, the convention now is to put the person first, followed by the term describing their abilities.

Reviewers' comments:

5 Apr 2022

PONE-D-21-11149R3

Health economic evaluation of noninvasive prenatal testing and serum screening for Down syndrome

Dear Dr. Xiao:

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