Parental willing to take invasive diagnosis after Expanded Noninvasive Prenatal Testing (expanded NIPT) in a cohort of 24768 cases

The main aims of the study were to investigate the performance of expanded noninvasive prenatal test (expanded NIPT) in screening for common trisomies, sex chromosomal aneuploidies (SCAs), rare autosomal aneuploidies (RATs) and copy number variations (CNVs) and parental willing to taking invasive prenatal diagnosis after expanded NIPT in China. Results Of the 24,736 cases, successful follow-up was conducted in 92.2% (411/446) cases. The sensitivity of expanded NIPT test was 98.61%,90.91% and 100% and specificity was 99.91%,99.95% and 99.87% for the detection of trisomies 21, 18 and 13 respectively. Expanded NIPT detected 45, XO, 47, XXX, 47, XXY, XYY syndrome, RATs and CNVs with positive predictive values of 57.39%, 19.61%, 75%, 79.41%, 77.78%, 10% and 56.25% respectively. The women carrying fetuses with T21/T18/T13 underwent invasive prenatal diagnosis and terminated their pregnancies at higher rates than those positive for SCAs, RATs and CNVs. demonstrates that and 13 with high specificity. The and is still relatively poor and needed to be a positive expanded NIPT result, the women at high for common trisomies are more willing to take invasive prenatal diagnosis and terminate their pregnancies. and CNVs and set up a precious genetic counseling protocol for those expanded NIPT-positive cases.


Conclusions
Our study demonstrates that the expanded NIPT detects fetal trisomies 21,18 and 13 with high sensitivity and specificity. The accuracy of detecting SCAs, RATs and CNVs is still relatively poor and needed to be improved. With a positive expanded NIPT result, the women at high risk for common trisomies are more willing to take invasive prenatal diagnosis and terminate their pregnancies.
* the first two authors contribute equally to this study.

Background
Noninvasive prenatal testing (NIPT) was firstly introduced to screen for fetal trisomy 21 in 2011 and went globally rapidly [1]. Up to date, millions of NIPT tests have been conducted throughout the world, and the clinical performance of expanded NIPT to detect fetal trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13) and SCAs are now well recognized [2][3][4][5][6]. In recent years, the test has been expanded to detect RATs and CNVs, however, there are few reports on the application of those expanded NIPT to the detection of RATs and CNVs [7][8][9][10][11]. Many factors affect the decisions to take 4 further prenatal diagnosis, and to continue or terminate pregnancy, including the type of fetal disease, the level of prenatal genetic counseling, the quality of life of the pregnant woman, cultural factors, multiple cross-disciplinary approaches, etc [12]. On receiving different positive expanded NIPT results, it remains unclear the attitude of pregnant women towards invasive prenatal diagnosis and continuation of pregnancy. Therefore, a systematic review of expanded NIPT test results, diagnostic tests and pregnancy outcomes of all screening results is needed in a large-scale population, which allows for comprehensively evaluating the clinical performance of the test and parental willing to taking invasive prenatal diagnosis.
In the present study, we analyzed the clinical data of a cohort of 24,768 participants who underwent expanded NIPT tests at the Women and Children's Hospital from January 2013 to April 2019 in Xiamen of Fujian province, China. We reviewed the expanded NIPT results, the diagnostic invasive test results and clinical follow-up information to evaluate the performance of expanded NIPT in screening for trisomies 21, 18, 13, SCAs, RATs and CNVs. We also retrospectively analyzed the clinical data of 533 pregnant women with positive expanded NIPT results and examined their clinical treatments, results of prenatal diagnosis and pregnancy outcomes. This study revealed expanded NIPT detected trisomies 21, 18, 13, 47, XXX, 47, XXY, XYY syndrome with a high true positive value but showed a relatively poor performance in detecting 45, XO, RATs and CNVs. The women at high risk for common trisomies are more willing to take invasive prenatal diagnosis and terminate their pregnancies.

General characteristics of expanded NIPT test results
A total of 24,768 cases were included in this study, the majority of the women came from the Fujian province, China. Of these samples, 0.14% (32/24768) were rejected for further processing owing to inadequate volume, contamination, low fraction of fetal DNA. The mean age of the remaining cases was 32.53 years old, 30.9% (7, Fig. 1). The other 47 cases had no confirmed pregnancy outcomes    (Table 3). When stratified by CNV size, expanded NIPT detected eight cases whose CNVs size was greater than 10 Mb and eight samples whose CNVs size was less than 10 Mb. The PPV for individual CNVs > 10 Mb was 62.5%, but the performance decreased to 42.86% when the size of CNVs was smaller than 10 Mb (Table 3).    (> 10M) 21*** 9(42.86%) 12(57.14%) 5 5(100%) 0 Note: *<0.05, ***<0.005, fisher exact test. The differences in the rate of prenatal diagnosis and termination of pregnancy were compared between common aneuploidies group and any other group.

Discussion
In this study, 24,768 pregnant women were enrolled to investigate the clinical performance of the expanded NIPT test. The sensitivity and specificity of expanded NIPT test was over 99% for trisomies 21,18 and 13, which was consistent with previous studies [7][8][9][10][11]. Our results presented a comparable PPV for T21 and T18 but a relatively low PPV for T13 compared to other studies [7,13]. This could be because of the small number of T13-positive cases in our study, the difference in the number of T13 cases would increase the variation of PPV. Only 1.1% (267/24,736) of cases were classified as high-risk for common aneuploidies and needed to undergo diagnostic karyotyping analysis. The application of expanded NIPT tests avoided the implementation of more than 98% of invasive diagnostic procedures and reduced unnecessary fetal losses caused by invasive tests. The miscarriage rates were 1.4% after amniocentesis and 1.9% after chorionic villus sampling [14].Therefore, the test is a safe screening tool with high cost efficiency. In 35.85% (19/53) cases with unconfirmed high-risk expanded NIPT results, fetal anatomical anomalies were reported by regular ultrasound examination, these women underwent TOPs based on the abnormal ultrasound results. Ultrasound examination revealed 4 suspicious T21, 2 suspicious T18 and 1 suspicious T13 in the 19 cases. These results demonstrated that the number of true positive cases might be underestimated in this study.
In our study, expanded NIPT detected SCAs with a PPV of 57.4%, which is similar with Porreco's study [15] but much lower than the results reported by other studies [4,5]. The difference is to large extent attributable to the different analytical techniques used in the studies. Unlike the z-scores algorithm used in our study and Porreco's study [15], Mazloom constructed a classification algorithm for SCA detection and determined the accuracy of the classification algorithm on another independent cohort of maternal samples [4]. Hooks et al developed a chromosome-selective approach and devised a risk algorithm incorporating fetal fraction to evaluate the risk for SCAs [5]. In our study, expanded NIPT more accurately detected triple X, XXY and XYY syndrome than XO syndrome, which is in line with that reported by others [13,16,17]. The difference in the detection of different SCAs might be attributed to two main reasons. Firstly, there are 58 homologous genes on X and Y chromosome, 29 genes are located at the ends of sex chromosomes. The sequencing length of expanded NIPT is as short as 36 bases, therefore, an error in sequencing these homologous genes might easily occur on X and Y chromosomes [16]; Secondly, X chromosome mosaicism is another contributing maternal factor to the difference in pregnant women [18].
In recent years, the expanded NIPT test has been applied to the screening for other chromosomal abnormalities including fetal RATs and CNVs [19,20], however, the application of this expanded screening is still under debate in clinical settings. In our clinical practice, the PPV was as low as 10% for the detection of RATs and 50% for the detection of CNVs. By comparing the set of CNVs with the Online Mendelian Inheritance in Man database, 6 CNVs cases were pathogenic and 3 CNVs were unknown (Table S1). The results suggested the screening test was capable of successfully detecting fetal RATs and CNVs, but caused a large fraction of false-positive cases. Previous studies have claimed benefits [21]; however, we suggest that expanded NIPT hasn't demonstrated a sufficiently high true-positive rate in the detection of RATs and CNVs, particularly for RATs. Therefore, the application of expanded NIPT to screening for RATs and CNVs must be balanced with the miscarriage risk of using diagnostic testing due to the procedure itself.
In this study, in comparison to the women with positive results for positive SCA, RAT and CNV results, the rate of prenatal diagnosis for women with positive T21/T18/T13 results was significantly increased. These findings were in line with the Zhou's study [12]. The compliance of pregnant women was better because they know more about these three diseases. Due to the seriousness of the diseases, most pregnant women are willing to undergo prenatal diagnosis and almost all of the positive cases will terminate their pregnancy in China. Zhou's study reported women positive for Turner syndrome were more willing to continue their pregnancies, while we found women carrying fetuses with 47, XYY tended to continue their pregnancies, while women with other fetal SCAs were more likely to underdo TOP. Women with confirmed larger CNVs (> 10M) showed higher TOP rate than those with confirmed smaller CNVs(< 10M). These differences may have been related to the level of prenatal genetic counseling, the cognition level of pregnant women, economic situation, etc.
In conclusion, our study demonstrates that the expanded NIPT test detects fetal trisomies 21,18 and 13 with high sensitivity and specificity but shows a relatively low true positive value for SCAs, RATs

Sequencing And Bioinformatics Analysis
Details of the expanded NIPT methods have been published previously [22]. In brief, plasma was separated by sequential centrifugations of the blood sample at 1600 g at 4 °C for 10 min. Cell-free DNA was extracted from plasma and subjected to library construction. The quantity and quality of the library were examined by real-time polymerase chain reaction and size distribution analysis. Only qualified library was sequenced, and the data generated were analyzed using bioinformatics algorithms to detect trisomy 21 (T21), T18, T13, sex chromosomal aneuploidies (SCAs), rare autosomal aneuploidies (RATs) and large copy number variations (CNVs) as previously described [23].

Validation Of High-risk Screening Results
Each participant paid CNY1400 (~ USD178) to take the expanded NIPT test in the Women and Children's Hospital. For those participants who received high-risk results of T21, T18, T13, SCAs, RATs, they were recommended to take confirmatory invasive tests using amniocentesis, followed by karyotyping analysis. For those CNVs-positive cases, chromosomal microarray analysis (CMA) was performed for validation. Prenatal diagnosis was carried out according to our routine experimental method and completed in our prenatal diagnosis center. The costs of diagnostic tests were covered by an insurance. Moreover, CNY400,000 (~ USD50,990) would be paid for those women who had confirmed false negative results of T21, T13 and T18 as compensation. However, the insurance didn't cover the false negative results of SCAs, RATs and CNVs cases in the Women and Children's Hospital.

Clinical Follow-up Analysis
Routine prenatal care was recommended for participants who had low-risk screening results in 14 expanded NIPT tests. Ultrasound examination was performed regularly to detect fetal abnormalities. If abnormal fetal anomalies were observed, participants were suggested to take genetic counselling.
Some participants chose termination of pregnancy (TOP). For the other participants, a postnatal follow-up was conducted by telephone interview at 3 months after birth. The following information was collected, including the ultrasound examination report, final pregnancy outcomes, baby's gender and newborn physical examination. Pregnancies who had high-risk expanded NIPT results of T21, T18, T13, SCAs, RATs and CNVs were recommended for diagnostic tests using amniocentesis. The "true positive" and "false positive" referred to the high-risk expanded NIPT results that were concordant or discordant with diagnostic genetic testing respectively. The "true negative" cases were low-risk in the expanded NIPT test and validated by normal neonatal physical examination except for SCAs or diagnostic test analysis. The "false negative" referred to those cases who were reported to be aneuploidy-negative but present an aneuploidy karyotype which was validated by invasive genetic testing. Notably, the "true positive" and "false positive" CNVs were validated by the gold-standard CMA analysis. Participants who had high-risk screening results but didn't have confirmatory diagnostic results were excluded in the evaluation of the performance of expanded NIPT in screening for T21, T18, T13, SCAs, RATs and CNVs. Loss to follow-up referred to cases who were unreachable or rejected to be interviewed.

Statistical analysis
The data were analyzed using R3.2.0 (http://www.R-project.org).Differences in categorical variables were compared between two groups using fisher exact test, and P values ≤ 0.05 was considered as statistically significant.

Declarations
Ethics approval and consent to participate All the participants provided written inform consent. This study followed strictly confidentiality regulations on privacy protection and approved by the Institutional Review Board of the Women and Children's Hospital (KY-2019-037).

Consent for publication
All the participants provided written inform consent.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary files.

Competing interests
The authors declare there is no competing interests.

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