1 INTRODUCTION

Birth defects, also known as congenital abnormalities, are the leading cause of neonatal morbidity and death globally.¹ Such defects refer to abnormalities in the individual, morphology, structure, and function (including metabolism, mentality, and intelligence) that occur in the uterus before the birth of the fetus but are not caused due to childbirth injuries. Such defects include congenital malformations, genetic metabolic defects, congenital disabilities (blindness, deafness, and dumbness), immune diseases, mental retardation, etc. Overall, birth defects affect approximately 1 in 33 children.² According to reports, primary prevention is not possible as the cause of around 60% of birth defects is unknown, and about 20% of congenital disease are due to genetic defects.³ The most common genetic disease that causes birth defects in newborns is chromosomal abnormalities, accounting for about 1/160 live birth.⁴ Chromosome abnormalities, in general, include an abnormal number of chromosomes and abnormal chromosome structure. 21-trisomy syndrome (Down syndrome), 18-trisomy syndrome (Edward syndrome), 13-trisomy syndrome (Patau syndrome), and sex chromosome aneuploidy (SCA) are due to a common abnormal number of chromosomes.⁵ The deletion or duplication of a tiny fragment of chromosomes is a common chromosomal structural abnormality, which is the main reason of birth defects in newborns. Nearly 300 such diseases have been found until now, such as DiGeorge, Prader–Willi, Angelman, and Williams syndrome, Williams–Beuren syndrome, 17q21.31 microdeletion syndrome, Prader–Willi and Angelman syndrome, etc.^{6, 7} At present, there is no effective treatment for chromosomal abnormalities. However, more and more technologies have been applied for the early diagnosis of chromosomal abnormalities, in order to achieve early prenatal intervention.

The gold standard for diagnosing fetal chromosomal abnormalities is the karyotype till now, which analyzes cells extracted from the amniotic fluid. Fetal chromosomal aneuploidy, polyploidy, abnormal balance structure, chimera, and deletions and duplications that are bigger than 10–20 Mb can be diagnosed through it. However, cell culture is required, and the method has many shortcomings such as long detection time, low throughput, and the inability to detect copy number variations (CNVs) below 5 Mb.^{8, 9}

In recent years, there has been wide usage of genome copy number variation sequencing (CNV-seq) technology based on low-depth whole-genome sequencing due to its high throughput, simple operation, and only a small sample required for the detection of chromosomal aberrations, including aneuploidy, microdeletion, microduplication, etc. Based on short tandem repeat (STR), the quantitative fluorescent polymerase chain reaction (QF-PCR) technology has high throughput, high speed, and high accuracy and the ability to detect the contamination of maternal blood.

In this study, prenatal screening (serological Down's screening, noninvasive DNA testing, ultrasonography, etc.) and prenatal diagnosis (CNV-seq, QF-PCR, chromosome karyotype analysis, etc.) were carried out on 657 pregnant women with indications for chromosome examination. Then conducted a comparative analysis to explore the respective advantages of the above-mentioned techniques and the possible clinical significance of the abnormal results.

2 MATERIALS AND METHODS

3 RESULTS

3.1 The classification of etiology

We classified the etiology of patients who underwent amniocentesis. Figure 1 showed that 389 cases (59.21%, 389/657) had a high risk of serological Down's syndrome screening: The number age below 20 years old (<20) was 3 (0.77%, 3/389); the number age between 20–29 years old (20–29) was 224 (57.58%, 224/389); the number age between 30–39 years old (30–39) was 162 (41.65%, 162/389). Abnormal B-ultrasound accounted for 106 cases (16.13%, 106/657): 20–29 was 54 (50.94%, 54/106); 30–39 was 48 (45.28%, 48/106); 40–49 was 4 (3.77%, 4/106). We observed a high risk of noninvasive prenatal DNA testing (NIPT) in 86 cases (13.09%, 86/657): <20 was 2 (2.33%, 2/86); 20–29 was 32 (37.21%, 32/86); 30–39 was 43 (50.00%, 43/86); 40–49 was 9 (10.47%, 9/86). A history of adverse pregnancy was seen in 13 cases (1.98%, 13/657): 20–29 was 6 (46.15%, 6/13); 30–39 was 7 (53.85%, 7/13). A family genetic history accounted for 6 cases (0.91%, 6/657): 20–29 was 2 (33.33%, 2/6); 30–39 was 3 (50.00%, 3/6); 40–49 was 1 (16.67%, 1/6). Other factors (taking medication during pregnancy, in vitro fertilization-embryo transform, etc.) were in 57 cases (8.68%, 57/657): <20 was 2 (3.51%, 2/57); 20–29 was 23 (40.35%, 23/57); 30–39 was 26 (45.61%, 26/57); 40–49 was 6 (10.53%, 6/57). Overall, the pregnancy week range was mainly concentrated below 25 weeks, among these, the pregnancy week range was below 20 weeks was 265 (40.33%, 265/657), 20–25 weeks was 246 (30.44%, 246/657), over 25 weeks was 146 (22.22%,146/657).

4 DISCUSSION

Each year, there have been roughly 135 million newborns worldwide, of which 3% suffer from major structural birth defects. This brings a serious economic and spiritual burden to society and the family.¹³ Therefore, diagnosing fetal chromosomal aberrations quickly and accurately has become more and more important to eliminate the mother's anxiety and reduce the birth rate of abnormal fetuses.

Till now, karyotype analysis is the gold standard for prenatal diagnosis of fetal chromosomal aberrations. However, there are many shortcomings, such as long detection time, cell culture requirement, low resolution (<5 Mb), and misdiagnosis due to maternal blood contamination.¹⁴ CNV-seq technology has been widely used in detecting chromosomal aberrations such as aneuploidy, microdeletion, and microduplication due to its many advantages, such as high throughput and easy operation. However, it cannot identify maternal cell contamination. QF-PCR can diagnose common chromosomal aneuploidy within 24 h, through qualitative and quantitative analysis of the polymorphism of STR genetic markers, adopt multiplex PCR amplification and capillary electrophoresis separation technology. One of the advantages of QF-PCR is that it can identify maternal contamination. However, it cannot detect chromosome structure abnormalities, chromosome polyploidy, and mosaics with a mosaic ratio that is <20%.¹⁵

Among the 657 amniotic fluid specimens in this study, while diagnosing aneuploidy in 5 chromosomes (13, 18, 21, X, and Y), we found 33 cases of trisomy 21, 5 cases of trisomy 18, 1 case of trisomy 13, 4 cases of XXX, 7 cases of XXY, and 6 cases of XYY. The coincidence rate between CNV-seq and QF-PCR was 100%. For all chromosomal structure and number abnormalities, the rate of QF-PCR and CNV-seq was the same at 79.60%. The rate of abnormality indicated by CNV-seq but not QF-PCR was 19.48%, whereas that indicated by QF-PCR but not CNV-seq was 1.22%.

Among the 9 cases abnormalities that could not be detected by CNV-seq but QF-PCR, 5 cases showed D13S634 site microduplication, accounting for 55.56%. In the remaining, D21S1411, D13S628, D18S386, and D21S1445 site microduplication were seen in 1 case each, accounting for 11.1%, respectively. Although the clinical significance was still not clear, it still showed guiding significance for clinical auxiliary diagnosis and future prenatal chromosomal aberration detection.

Among the 141 CNVs that could not be detected by QF-PCR but CNV-seq, 17.73% were polymorphic, the pathogenicity of 20.57% was unknown, 22.7% were possibly benign, 14.18% were explicitly benign, 10.64% were possibly pathogenic, and 14.18% were pathogenic. Regarding the size of chromosomal CNVs variant fragments, 4.26% were larger than 5 Mb, and 95.74% were <5 Mb, which was less than the resolution of the karyotype. If only the karyotype analysis was performed, it might lead to a missed diagnosis.

There were 389 cases (59.21%, 389/657) in this study who underwent amniocentesis due to a high risk of serological Down's syndrome screening, which accounted for the largest proportion. Serological Down's syndrome screening determines the fetus's risk factor that may suffer from 21-trisomy syndrome (Down Syndrome), 18-trisomy syndrome (Edward Syndrome) and neural tube defects (NTD). In combination with the age, weight, and gestational age of the pregnant woman, it analyzes the concentration of fetal alpha-fetal protein, chorionic gonadotropin, and free estriol in the maternal serum.¹⁶ CNV-seq and QF-PCR only detected 4 cases of trisomy 21 among the 328 cases, which were at high risk of trisomy 21 by serological Down's syndrome screening, with a coincidence rate of 1.22% (4/328). Among the population with a high risk of trisomy 18, no positive case was detected, and the accuracy was relatively low.

However, among the 389 cases, which had a high risk of serological Down's screening, there were 78 cases (20.05%, 78/389) detected abnormal CNVs but without chromosomal aneuploidy abnormalities by CNV-seq. Therefore, when these pregnant women were aware of the high risk of serological Down's screening, if they only chosen NIPT to further verify whether there were 21-trisomy syndrome or 18-trisomy syndrome but gave up amniocentesis for prenatal diagnosis based on amniotic fluid, the result of NIPT should be that the fetus were normal, the existing CNVs would be missed.

However, judging from the abnormality of CNVs, out of 389 fetuses with a high risk of serological Down's screening (high risk of trisomy 21 and trisomy 18), only 4 cases (1.03%, 4/389) were diagnosed as trisomy 21. But there were 78 cases (20.05%, 78/389) existing 87 kinds of CNVs, which were characterized by microdeletion and microduplication (there might be two or more CNVs in the same specimen, so the number of CNVs was greater than the total number of samples), of which 13 cases were pathogenic, 39 cases were pathogenic possibly, benign possibly or the significance was unclear temporarily, 35 cases were known to be benign and polymorphic. And among these 13 cases CNVs, which were pathogenic, the fragment size of 2 cases were larger than 5 Mb, and 11 cases were <5 Mb, suggesting that if these 11 cases were diagnosed by karyotype analysis based on amniotic fluid, might cause missed diagnosis because of the detection limit (<5 Mb). This ratio was much higher than our understanding of the positive rate of serological Down's screening. Therefore, this result suggests that the high risk of serological Down's screening may not only due to the aneuploidy of chromosomes 21 and 18, but also the microdeletion or microduplication of all chromosomes (including chromosomes 21 and 18). In addition, we need to remind everyone that it might cause missed diagnosis if only NIPT was used for further detection for people who were at the high risk. Through amniocentesis, using CNV-seq and other high-resolution, high-coverage prenatal diagnosis technology for prenatal diagnosis, can detect the abnormalities of these CNVs that may cause disease to the greatest extent.

86 (13.09%, 86/657) cases underwent amniocentesis because a high risk of NIPT. It sequences free DNA fragments (including free fetal DNA) in maternal peripheral plasma using next-generation DNA sequencing technology and then analyzes the result by biologic information.¹⁷ Among the 86 cases in this study, the coincidence rate of chromosome 21 was 69.23% (27/39), the coincidence rate of chromosome 18 was 33.33% (3/9), the coincidence rate of chromosome 13 was 20.00% (1/5), and the coincidence rate of sex chromosomes was 39.39% (13/33). The accuracy rate was improved compared to serological Down's syndrome screening, but it is still a screening experiment that could not be made for a final diagnosis. In addition, the accuracy of NIPT decreased correspondingly in the following situations: if the pregnancy week was too early or too late, if the expected age was ≥35 years, if the pregnant woman was severely obese (body mass index >40), etc.¹⁸

In conclusion, QF-PCR can detect the contamination of maternal cells, it possesses high sensitivity and specificity in diagnosing aneuploidy in 5 chromosomes of 13, 18, 21, X, and Y within a short time, but it cannot detect abnormal chromosomal structure and low-proportion mosaicism. For these reasons, CNV-seq can be better supplemented, it can detect chromosomal aberrations such as microdeletion and microduplication, which were <5 Mb. However, QF-PCR is still required for distinguishing whether existing maternal cell contamination. In addition, it should be noted that CNVs such as microdeletion or microduplication of all chromosomes (including chromosomes 21 and 18) may cause a high risk of serological Down's screening except the existence aneuploidy of chromosomes 21, 18 and NTD, which may cause missed diagnosis if only NIPT was used for further detection. CNV-seq combined with QF-PCR can complement each other in order to diagnose fetal chromosomal abnormalities more efficiently and accurately, this combination may be considered as the first-line method of prenatal chromosome diagnosis.

AUTHOR CONTRIBUTIONS

All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

COMPETING INTERESTS

Authors state no conflict of interest.

INFORMED CONSENT

Not applicable.