A cohort study to detect aneuploidies and autosomal partial deletions and duplications using NIPT in 42,910 pregnant women (singleton) with different clinical features

#Summary

Background: Since the discovery of cell-free DNA (cfDNA) in maternal plasma, it has opened a new approach for non-invasive prenatal testing. With the progress of whole genome sequencing, NIPT has discovered partial deletion/duplication diseases of all autosomal regions. This study reviewed the effectiveness of NIPT as a screening test for aneuploidy and CNV (copy number variation) in 42,910 single pregnancies.

Methods: A total of 42,910 single pregnancies with different clinical characteristics were recruited. Cell-free fetal DNA was directly sequenced. Chromosomal aneuploidy and all autosomal partial deletion/duplication diseases were analyzed.

Results: A total of 534 pregnancies (1.24%) had abnormal results detected by NIPT, and 403 pregnancies had undergone prenatal diagnosis. The positive predictive value (PPV) for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidy (SCA), and other chromosome aneuploidies were 79.23% and 54.84%, respectively. They were 13.79%, 33.04% and 9.38%. The PPV of CNV was 28.99%. The PPV for CNV≦5 Mb was 20.83%, 5-10 Mb 50.00%, >10 Mb 27.27%. The PPV of NIPT also differs depending on pregnancy characteristics.

Conclusion: Our data are potentially important in demonstrating the utility of NIPT profiling not only for common whole-chromosome aneuploidies but also for CNVs. However, this modern method is still in its infancy for CNV. Clinical validation studies with accurate detection rates and false-positive rates in clinical practice are still needed.

Keywords:Non-invasive prenatal testing (NIPT), chromosomal aneuploidy, whole autosomal partial deletion/duplication disease (MMS), clinical features, positive predictive value (PPV)

Consideration of the above paper

According to a paper by Chen et al. published in 2019, trisomy 21 is 155/42910=1/276, trisomy 18 is 44/42910=1/975, and trisomy 13 is 33/42910=1/1300. It is occurring in proportion. This corresponds to the inspection items that we usually call the O set. In addition, the test content is the same as that carried out by the NIPT consortium in Japan. On the other hand, the frequency of occurrence of sex chromosomes is 147/42910=1/291, which is the second highest frequency after chromosome 21. This is what would be detected in the O+ set or A set in our inspection. Because developmental delays such as decreased intelligence are rare, they are not tested, but they are relatively common. In addition, all chromosomes included in the D+ set that we are conducting, all autosomal whole region partial deletion/duplication diseases have an incidence rate of (47+109)/42910=1/275, which is about the same as trisomy 21. . Until now, the D set, which detects partial deletion diseases of all autosomal regions only in specific regions 1.4, 5, 15, and 22, has not occurred very often, but It is reported that this happens frequently. However, for cases of aneuploidy of all chromosomes or chromosome 13, which occur less frequently, the positive predictive value is relatively low. If NIPT is positioned as a screening test, there may be some things that can’t be helped. Also, I have the impression that overall the number of positive cases is lower than at our facility. This is thought to be because in the population sampled in this paper, the proportion of pregnant women aged 35 years or older was 25.03%, which is lower than that at Hiro Clinic. At Hiro Clinic NIPT, the number seems to be even higher, but the above percentage of positive frequency is similar to what we feel.

Introduction

Since the discovery of cell-free fetal DNA (cffDNA) in triterminal plasma in 1997 [1], it has opened a new approach for non-invasive prenatal testing (NIPT). Since 2011, massively parallel sequencing (MPS) for embryonic polyploids has been available in more than 60 countries. NIPT, which uses cfDNA circulating in maternal blood, opens the door to early, accurate, and safe prenatal testing and has been used clinically for more than eight years [2]. 21, 18, 13 The weighted pooled detection and false positive rates of screening for monosomy reported as 90.3% (0.23%) and 93% (0.14%) [3].
A growing number of studies indicate that NIPT may reduce the incidence of unnecessary invasive procedures and iatrogenic fetal loss [4]. NIPT had many additional advantages over traditional biochemical and ultrasound screening, such as higher sensitivity and specificity and the ability to perform NIPT at earlier gestational ages. In China, NIPT is restarted for trisomy 21 (T21), T18, and T13 screening for patients at high risk for late pregnancy serological screening results [5]. Currently, an increasing number of pregnant women are choosing NIPT [6, 7].
This advanced prenatal screening method has other applications, including screening for all autosomal deletion/duplication diseases (MMS) caused by copy number variants (CNVs) smaller than 10 Mb. Although individually rare, MMS accounts for 1-2% of genital abnormalities in newborns and often imposes a heavy burden on families and society. Recently, further development and expansion of NIPT has focused on MMS such as Hu et al. [8] and Liang et al. [9] demon-strated NIPT showed good results in some MMS.
However, there are many problems and challenges in clinical practice, and extensive verification is required to make a decision.
Accurate Detection Rate and False-Positive Rate The purpose of this study was to review the effectiveness of NIPT as a screening test for aneuploidy and CNV in 42,910 single pregnancies.

Results

Patient background

From April 2015 to December 2018, a total of 42,931 maternal blood samples were collected from Ningbo Women’s and Children’s Hospital, China. Twenty-one cases failed, resulting in a failure rate of 0.05%. Therefore, the total sample included in this study was 42,910. The gestational age ranged from 12+0 to 26+6, the age ranged from 18 to 49 years, and there were 10,742 women with older maternal age (35 years or older). The clinical characteristics of the 42,910 cases are shown in Table 1. Of these 42,910 specimens, 348 pregnant women required resampling due to low fetal DNA concentration in plasma, and the resampling rate was 0.81% (348/42910), and all resamplings resulted in NIPT results. obtained (Table 1).

Prenatal test results for all pregnancies

Before NIPT, pregnant women undergo traditional tube screening tests including fetal ultrasound (including color ultrasound and 3-dimensional color ultrasound) and maternal serum biomarkers. Ultrasonography showed that 202 (0.47%) fetuses were structurally abnormal and there were 5749 (13.4%) fetuses with increased NT (NT ≥ 3 mm). Measurement of maternal serum biomarkers suggested 2318 (5.4%) high-risk pregnancies, 15,863 (36.97%) critical-risk pregnancies, and no clinical indication in 8024 (18.70%) pregnancies. (Table 1).

NIPT results for T21, T18, T13, and SCA

The flowchart is shown in Figure 1. A total of 42,931 specimens were recruited and 42,910 NIPT results were obtained.

Abnormal results in 534 cases (1.24%). Of these 534 cases, 155 of trisomy 21 (T21), 44 of T18, 33 of T13, 147 of sex chromosome aberrations (SCA), and other chromosomal aneuploidies (T21, T18, T13, sex chromosome (excluding aneuploidies) and 109 of CNVs.
Karyotypes were obtained to verify the abnormal results of NIPT prediction. The total abnormal results for T21, T18, T13, and SCA were 379. Of these 379 cases, 302 underwent prenatal diagnostic testing, resulting in 161 true positives (103 for T21, 17 for T18, 4 for T13, and 37 for SCA) and 141 false positives (FP ) was confirmed. Additionally, the positive predictive value (PPV) of each test was evaluated. For trisomy 21, the PPV was 79.23%, for trisomy 18 54.84%, for trisomy 13 13.79%, and for SCA 33.04% (Table 2 and Figure 1).

NIPT results for CNV and other chromosomal aneuploidies

In addition, since this technology is genome-wide sequencing, we analyzed CNVs and other chromo-some aneuploidies. The total cases of CNV abnormalities are as follows.
Of the 109 cases, 20 were true positives, 49 were false positives, and 50 were unconfirmed. The number and size of CNVs on each chromosome were evaluated. CNVs were classified into three groups according to length: CNV≦Mb, CNV within 5-10 Mb, and CNV >10 Mb. PPV of each group was also evaluated. The total PPV of CNV was 28.99%. The PPV for CNVs below 5 Mb is 20.83%, 50.00% for CNVs between 5 and 10 Mb, and 27.27% for CNVs above 10 Mb. The total number of cases of other chromosomal aneuploidies was 46, including 3 true positives, 29 false positives, and 14 unconfirmed cases. The PPV of other chromosomal aneuploidies was 9.38%. Among other chromosomal aneuploidies, Chr7 aneuploidy is the largest group. Although all Chr7 aneuploidies predicted by NIPT are trisomy 7, all confirmed patients (total number of 9) were confirmed to be false positives (Tables 3 and 4).

Differences in PPV depending on pregnancy characteristics Table 5 shows differences in PPV of NIPT depending on pregnancy characteristics. The total PPV of T21 is 79.23%, and the PPV of T21 fetuses in women with older maternal age is 89.29%, and in high-risk groups of serological screening, as follows

86.67%, the serious risk of the serological screening group is 71.74%, and the PPV of NIPT in increasing the NT group is the highest, 100%. Similarly, the PPV of the increased NT group was also the highest in predicting SCA fetuses. Fetal T18 fetal PPV is 100% for fetal structural abnormalities by B-ultrasound group, increased NT group, and high risk for serological screening, whereas for fetal T13 and CNV prediction; It is worth noting that the PPV of the serological screening group and high risk of fetal structural abnormalities by the B-ultrasound group was the highest, respectively.

Inspection

NIPT has been widely used for T21, T18, and T13 prenatal screening in the past few years. However, to date, large-scale clinical trials focusing on the efficiency of subchronic mosomal copy number variants (CNVs), which are typically less than 5 Mb in size, have still not been conducted [8, 9] . In addition, there are some concerns regarding clinical performance [10, 11]. Therefore, we hope that this study, which includes 42,910 cases, will provide data support for these questions.

In this study, NIPT was evaluated using positive predictive value (PPV). The PPV for T21 was 79.23%, and for T18 and T13 it was 54.84%, 13.79%, and 33.04%. Additionally, PPVs of other chromosomal aneuploidies and CNVs were also analyzed. The PPV of other chromosomal aneuploidies was 9.38%, and the CNV was 28.99%. In recent studies, the PPV range for T21 was 65–94%, T18 was 47–85%, and T13 was 12–62% [12–14]. falls within this range. Interestingly, the PPV of CNV was 28.99%, which was obviously higher than T13. Previous clinical validation studies have reported that the detection performance of specific MMS varies, with positive predictive value (PPV) being slightly low to moderate [9].
Recently, more relaxed guidelines have been proposed, allowing routine screening for MMS in young women, where full autosomal full-region deletions are more common than aneuploidies. [15] Based on the results of this retrospective study of more than 42,000 pregnancies, NIPT represents a suitable screening method for MMS of central nervous system origin. In this study, the PPV for CNV within 5-10 Mb was the highest (50.00%), and the PPV for CNV below 5 Mb was the lowest (20.83%). The PPV of CNVs>10 Mb (32%) and CNVs<10 Mb (19%) was also low, but reasonable, for potential screening of genome-wide fetal CNVs, according to Liang’s paper (reference) [9]. This indicates that sufficient sensitivity and specificity of the test is possible. PPV depends not only on the sensitivity and specificity of the test but also on the prevalence of the disease [16]. PPV for CNV <10 Mb is 31% in this study (data not shown in table, PPV = (5 + 6)/[(5 +)].

6)+19+6)] Much higher than Liang Guanglie’s paper. Moreover, a previous study reported that the total PPV of CNV was 9.2% [17], and the PPV of the present study was much higher.
The PPV for other chromosomal aneuploidies was low at 9.38%, similar to that reported in the paper by Liang (ref.). [9]). The reason is that these aneu-polyploids are not very abundant, and many of them have a high rate of focal placental mosaicism (CPM). NIPT is performed using cell-free fetal DNA, and the primary source of cell-fetal DNA in the maternal circulation is apoptosis of placental cells from cell-trophoblasts [18], which are not necessarily representative of the fetus. It is thought that. The situation in which chromosomal abnormalities are found only in the placenta, but not in the fetus, is known as CPM [19], with an observed incidence of approximately 1–2% [20]. NIPT is a screening test. For pre-counseling for NIPT, selected women should be fully informed about accuracy, reliability, false-positive, and false-negative rates. After counseling, confirmation of all positive findings by invasive prenatal diagnosis is strongly recommended with respect to current NIPT guidelines [21]. In addition, all women who were carrying a suspected fetus with a confirmed or potentially pathogenic fetal chromosomal abnormality were scheduled to undergo a genetic counseling session to discuss pregnancy management options. .
In addition, although we have further investigated the different PPV of NIPT according to pregnancy characteristics, the results in this section require support from more clinical data. Different pregnancy characteristics show different PPVs, with the PPV of NIPT being highest at T21 and much lower for other aneuploidies [22]. Older maternal age (usually over 35 years) is a risk factor for T21. Therefore, the PPV of mothers in old age is much higher than in the group without clinical indication. Also, the PPV of high risk in the serological screening group was higher than that in the critical risk group, which is consistent with Yu’s paper [23].

On the other hand, advanced maternal age may not be a risk indicator for T18 and T13. Similarly, unlike aneuploidy, the most common CNVs are not related to maternal age, so older mothers do not have higher PPV values.
CNVs are increasingly recognized as important contributors to human disease, present in approximately 1.7% of structurally normal pregnancies [24]. Chromosomal microarray analysis (CMA) is a powerful tool for detecting small invisible chromosomal deletions or duplications, and as a first-step diagnostic tool for some pa-thient with well-defined syndromes. Recommended [26, 27]. However, CMA has many limitations. Sampling for CMA requires invasive testing, which is associated with risks such as miscarriage, miscarriage, and intrauterine infection [28][29], or can identify variants of unknown significance. Some women may refuse it. NIPT’s detection of subchronic mosomal copy number variants (CNVs) has shown good performance in some MMS [30], and in recent years, NIPT expanded for MMS has resulted in a significant number of reports. exists [8,9,31]. However, NIPT is a screening test, and a clinically valid test is still needed regarding its accurate detection rate and false-positive rate for a large number of clinical samples.
In this study, follow-up results are negative. According to the People’s Republic of China Health Commission guidelines, follow-up started at 12 weeks after delivery. Follow-up should include the subject’s pregnancy outcome and the health of the newborn. The main follow-up information for newborns is whether the newborn is T21, T18, or T13. Our follow-up began at 3 months of age and strictly followed national guidelines for this study. At follow-up, parents who complain of a newborn with a congenital anomaly should undergo further genetic diagnosis. Furthermore, regarding CNV, although we considered positive results, we hope that this study can provide validity of NIPT as a screening test for aneuploidy and CNV.

Conclusion

In conclusion, this study included a large prospective group of pregnant women with different clinical characteristics. This data is potentially important in demonstrating the utility of using NIPT profiling not only for general chromosomal aneuploidies but also for CNVs. However, this modern method is still in its infancy for CNV. Clinical validation studies with accurate eradication rates and false-positive rates are still needed in clinical practice.

Materials and methods

Patient

Pregnant women were collected consecutively. Pregnant women from April 2015 to December 2018 came to Ningbo Women’s and Children’s Hospital for prenatal testing. A total of 42,910 pregnant women were recruited. A signed consent form was obtained from each participant before blood collection. Inclusion criteria were (1) gestational age between 12+0 and 26+6, (2) single pregnancy, and (3) body mass index (BMI) <100. Exclusion criteria were: (1) pregnant women with chromosomal abnormalities or abnormalities, (2) multiple pregnancy, (3) pregnant women who underwent stem cell therapy and transplant surgery, (4) received allogeneic blood products within 1 year, and (5) ) received immunotherapy within 4 weeks.

Serological screening and ultrasound examination

Allocation decision

Maternal peripheral blood (5 ml) was collected into ethyl-enediaminetetraacetic acid (EDTA) tubes at 12+0 to 26+6 weeks of gestational age. Immediately after blood collection, it was stored at 4°C. Plasma was isolated within 8 hours using a two-step centrifugation protocol as previously described (ref.). [6]). Cell-free DNA extraction, library construction, sequencing, and bioinformatics analysis were performed according to previous studies (ref.). [6]). For high-throughput sequencing of non-fetal DNA fragments, use the JingXin BioelectronSeq 4000 system (CFDA Registration Permit NO.). 20153400309) Semiconductor sequencer. Sequencing reads were filtered and aligned to the human reference genome (hg19). A combination of GC correction method and Z-score testing method was used to identify autosomal aneuploidy in the fetus. Here, each chromosome with an absolute value of Z score greater than 3 was marked with chromosomal aneuploidy or whole autosomal whole region partial deletion/duplication disease.

Karyotype analysis and amniocentesis

Women with positive NIPT results were recommended to undergo amniotic fluid karyotyping for further verification. Amniocentesis was performed as routinely described. Karyotype analysis was performed according to the International System for Human Cytogenetic Nomenclature Guidelines [34].

Follow-up of negative cases

A follow-up survey was conducted for NIPT negative cases. According to the guidelines of the Mission of the National Health Commission of the People’s Republic of China, follow-up was started at 12 weeks after delivery. Follow-up should include the subject’s pregnancy outcome and the health of the newborn. The main follow-up information for newborns is whether the newborn is T21, T18, or T13. Our follow-up began at 3 months of age and strictly followed national guidelines for this study. At follow-up, parents who complain of a newborn with a congenital anomaly should undergo further genetic diagnosis. Patients who were lost to follow-up were excluded from the analysis.

Statistical analysis

SPSS 20.0 software was used for statistical analysis. Measured data were expressed as mean ± standard deviation (x).
±SD), adoption rate (%) of count data, positive predictive value = number of true positives/all positive cases.

Abbreviation

cfDNA: Cell-free DNA; CMA: Chromosome microarray analysis; CNV: Copy number variant; MMS: Whole autosomal partial deletion/duplication disease; NIPT: Non-invasive prenatal testing; NT: Nuchal half Transparent;PPV:Positive Prevalence

Thank you

Not available.

Author contributions

All authors substantially participated in this research and manuscript preparation. YC, QY, and XM collected all clinical data and performed all molecular genetic analyses. WL participated in data analysis and wrote the manuscript. MH was involved in the molecular genetic analysis. WL designed the study, drafted and revised the manuscript. All authors approved the final article.

Fund transfer

Mizue Health Science and Technology Plan (number) 2018KY720

Availability of data and materials

The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Ethical acknowledgment and consent to participate

This study was approved by the Ethics Committee of Ningbo Women’s and Children’s Hospital.

Consent to publication

The authors declare that they have no competing interests and the patient in this case report consented for publication.

Contrary interests

The authors declare that they have no competing interests.

Author details

Ningbo Women’s Hospital, No.339, China, Ningbo 315010, Haizhou County Ningbo, Kaitong Street, 1 Ningbo Women’s and Children’s Hospital, Liuting Street, China, Ningbo 315010. 2CapitalBio・Technology Company, China, Beijing 101111 3Beijing CapitalBio Medical Laboratory, Beijing 101111, China.

Acceptance: June 30, 2019 Acceptance: November 15, 2019

References

• Lo, Y.M., et al. Presence of fetal DNA in maternal plasma and serum. 1997. 350(9076): p. 485-487.
• Minear, M.A., et al., Global perspectives on clinical adoption of NIPT. 2015. 35(10): p. 959-967.
• Gil, M.M., et al., Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis. 2015. 45(3): p. 249-266.
• Zhang, H., et al., Non-invasive premental testing for trisomies 21, 18, 13: Clinical experience of 146,958 pregnancies. 2015. 45(5): p. 530-538.
• McCullough RM et al. Non-invasive prenatal chromosomal aneuploidy testing—clinical experience: 100,000 clinical specimens. PLoS One. 2014;9(10):e109173.
• Hu, H, et al., Clinical experience with non-invasive prenatal chromosomal aneuploidy testing in 190,277 patient specimens. 2016. 16(8): p. -.
• Mccullough, R.M.et al., Non-invasive premental chromosomal aneuploidy testing – Clinical experience: 100,000 clinical specimens 2014. 9(10): p. e109173.
• Hu, H., et al., Noninvasive premental testing for chromosomal aneuploidies and subchromosomal microdeleations/microduplication in a cohort of 8141 Single pregnancy. 2019. 13(1): p. 14.
• Liang D, et al. Clinical utility of non-invasive prenatal screening for expanded chromosomal disease syndromes. Genet Med. 2019.
• Rose NC, Benn P, Milunsky A. Current controversies in prenatal diagnosis 1: Should NIPT routinely include all autosomal partial deletions and duplications? Pregnancy diagnosis. 2016;36(1):10–4.
• Evans, M.I., et al., Noninvasive prenatal screening or advanced diagnostic testing: caveat emptor. 2016. 215(3): p. 298-305.
• Neofytou MC et al. Target capture enrichment assay for non-invasive prenatal testing of large and small subchromosomal deletions and duplications. PLoS One. 2017;12(2):e0171319.
• Yaron Y et al. Current status of testing for microdeletion syndromes and rare autosomal trisomies using cell-free DNA technology Obstetrics and Gynecology 2015;126(5):1095–9.
• Mary E et al. Cell-free DNA analysis for non-invasive testing of trisomy. N Engl J Med. 2015;372:1589–97.
• Verma, I.C., R. Dua-Puri, and S.J.F. M. Bijarnia-Mahay, ACMG 2016, Update on non-invasive prenatal testing for fetal aneuploidy: Implications for India. 2017. 4(1): p. 1-6.
• Lutgendorf, M.A., et al., Noninvasive prefative testing: limitations and open questions. 2014. 16(4): p. 281-285.
• Schwartz, S. et al. Clinical experience of non-invasive prenatal testing using cell-free DNA and follow-up with clinical testing for positive autosomal partial deletions in 349 cases. 2018.
• Tjoa, M.L., et al., Trophoblastic oxidative stress and release of cell-free fetoplacental DNA. 2006. 169(2): p. 400-404.
• J.A.J.O.M.G.P.C.S.i.M.G. Wapner, Trapped placental mosaicism and its impact on confirmation of NIPT results. 2016. 172(2): p. 118-122.
• Grati, F.R., et al., Fetoplacental mosaicism: potential implications for false-positive and false-negative non-invasive prenatal screening results. 2014. 16(8): p. 620.
• Gregg, A.R. et al., Noninvasive premental screening fetal aneuploidy, 2016 update: American College of Medical Genetics and Genomics position statement. 2016. 18(10): p. 1056-1065.
• Norton ME et al. Cell-free DNA analysis for non-invasive testing of trisomies. 2015;372(17):1589–97.
• Yu, B, et al., Comprehensive evaluation of the clinical value of prenatal screening for fetal free DNA in maternal blood. 2017. 96(27): p. e7114.
• Girirajan, S., C.D. Campbell, and E.E.J.A.R.o.G. Eichler, Human copy number variation and complex genetic diseases. 2011. 45(1): p. 203-226.
• Wapner RJ et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med. 2012;367(23):2175–84.
• Miller, D.T., et al., Consensus statement: Chromosomal microarrays are a first-line clinical diagnostic test for individuals with developmental disorders or congenital abnormalities. 2010. 86(5): p. 749-764.
• Melanie, M. and H.G.i.M. Louanne, Array-based techniques and recommendations for their use in medical genetics practice for the detection of chromosomal abnormalities. 2010. 12(11): p. 742-745.
• Niederstrasser, S.L., et al. Fetal loss after invasive prenatal testing: a comparison of transabdominal chorionic villus sampling, transcervical chorionic sampling, and amniocentesis. 2016. 37(S 01).
• Tabor A, Alfirevic Z. Update procedure-related risks for prenatal diagnostic techniques. Fetal Diagnosis 2010;27(1):1–7.
• Srinivasan A et al. Non-invasive detection of fetal subchromosomal abnormalities through deep sequencing of maternal plasma. Am J Hum Genet. 2013;92(2):167–76.
• Martin K et al. Clinical experience using non-invasive prenatal testing based on single nucleotide polymorphisms for five clinically important autosomal full-region partial deletions. Clin Genet 2018;93(2):293–300.
• Nicolaides, K.H., %J American Journal of Obstetrics and Gynecology, Nuchal translucency, and other ultrasound markers of chromosomal abnormalities in early pregnancy. 2004. 191(1): p. 45-67.
• Ching-Hua, H., et al., Extended first-trimester screening using multiple sonographic markers and maternal serum biochemistry: a five-year prospective study. 2014. 35(4): p. 296-301.
• Gonzalez Garcia, J.R. and J.P.J.B. Mezaespinoza, International system for cytogenetic nomenclature (ISCN). 2006. 108(12): p. 3952.

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