The number of studies on prenatal gene screening has soared with increasing awareness of its potential clinical test applications. NIPT have become an emerging research field, which requires the systematic analysis of its trends and knowledge structure. With that in mind, the present study revealed an increase in publications and 7 hot spots of research on NIPT from a quantitative and qualitative analysis of view. As expected, the number of publications was less before 2011 and started to steady increased during the period of 2011-2020. The maximal number of publications was found in 2020. Currently, the United States is still the top country for NIPT publications. In terms of all countries for NIPT publications, the major contributions were from a small number of developed countries in North America, Europe, and China. This may be due to the fact that gene sequencing technology has been developed earlier in these countries and regions.
In this study, we found that the 10 most active journals published NIPT related publications (accounting for 29.30% of all 2160 publications), and thus were considered as the core journals in the NIPT field. The Prenatal Diagnosis ranked the top among the10 active journals. Methodologically, the present study used the clustering analysis and visualization method to identify hot topics on inputting the g-cluto software. Altogether, 7 research hot spots of NIPT were found, as follows:
1) NIPT, a new screening method of prenatal diagnosis (Cluster 0).
With the rapid development of high-throughput sequencing technology and Professor Dennis Lo's team found that there is fetal dissociation in the peripheral blood of pregnant women. NIPT has been widely used in prenatal congenital malformation screening because of its high sensitivity and low false positive rate. Before NIPT came into clinical application, pregnant Down's syndrome screening played an important role in the pregnancy examination of trisomy 21, trisomy 18 and trisomy 13. Although the detection rate of 21- trisomy, 18- trisomy and 13- trisomy can be increased to 80%-94%, the false positive rate was very high (about 5%), which causes a lot of unnecessary anxiety for pregnant women. NIPT provides additional choices for prenatal testing which has alreadly replaced the serological screening but can not replaced the amniocentesis karyotype analysis. The reasons will be analyzed in detail in the next paragraph.
2) Karyotype analysis of amniocentesis remains the gold standard for prenatal diagnosis of fetal chromosomal abnormalities which can not be replaced by NIPT (Cluster 0)
Various of factors will affect the accuracy of the NIPT results, for example, (1) lower concentration of cffDNA. cffDNA was existed in maternal peripheral blood, the percentage of which is generally at the range of 3%-3o%, with the average of 13%. Most current NIPT protocols utilize 4% as the lower percentage cutoff value to ensure a reliable result [16]; (2) the chromosome abnormalities of mother. Liu J et al. have reported a confirmatory amniocentesis and karyotyping test taken from 563 patients who have received a positive trisomy result by NIPT. and revealed 489 true and 74 false positives. In 6 of these 74 patients (8.1%), CNV-Seq were performed to these 74 patients, 6 of which revealed non-pathogenic maternal duplications (1.76–10.90 Mb) on the chromosome associated with the fetal trisomy. Maternal CNVsmay cause false-positive NIPT trisomy results, and there was a strong correlation of higher Z-scores with increasing size of maternal CNVs [17] ;(3) The Confined placental mosaicism (CPM). Mosaicism in general is a biological phenomenon when two or more chromosomally distinct cell lines arising from a single zygote are both found in an individual. As we know, cffDNA was derived from embryonic trophoblast cells rather than fetal sources, so CPM was one important cause of false positive results in NIPT. No matter what the reason caused NIPT results are positive, all should be verified by confirmed experiments. As NIPT can be performed starting at 10 weeks gestation, asking a couple to wait until after 16 weeks for an amniocentesis to be performed can represent an unacceptably long and stressful waiting time for parents. An earlier option for further testing would be with chorionic villus sampling (CVS), which can be performed at a much earlier gestational age than amniocentesis (11–13 weeks) [18]. No matter which test is chosen, further confirmation experiments are necessary.
3) NITP is based on high-throughput sequencing and statistical calculation analysis (Cluster 1)
Different from ultrasound and serum biochemical screening, NITP is based on high-throughput sequencing and statistical calculation analysis [19]. NIPT has been used to screen for the common fetal aneuploidies, trisomy 13, 18, 21 and expented to detect rare autosomal aneuploidies (RATs) and CNVs in recent years. For example, a retrospective analysis was performed on toltal 24,702 pregnant women who took the expented NIPT. The sensitivity of expanded NIPT was 100% (95% confidence interval[CI], 97.38-100%), 96.67%(95%CI, 82.78-99.92%), and 100%(95%CI, 66.37-100.00%), and the specificity was 99.92%(95%CI, 99.87-99.96%), 99.96%(95%CI, 99.91-99.98%), and 99.88% (95%CI, 99.82-99.93%) for the detection of trisomies 21, 18, and 13, respectively. Expanded NIPT detected 45, X, 47, XXX, 47, XXY, XYY syndrome, RATs, and CNVs with positive predictive values of 25.49%, 75%, 94.12%, 76.19%, 6.45%, and 50%, respectively [20]. The study demonstrated that the expanded NIPT detects fetal trisomies 21, 18, and 13 with high sensitivity and specificity. However, the accuracy of detecting SCAs, RATs, and CNVs is still relatively poor and needs to be improved.
(4) Genetic counseling and interpretation (Cluster 2)
As NIPT is widely used in detecting chromosomal aneuploidies, informed decision-making has become the major ethical concerns. Before agreeing to screening tests, parents need to be fully informed about the risks, benefits and possible consequences of such a test. This includes subsequent choices for further tests they may face, and the implications of both false positive and false negative screening tests. The decisions that may be faced by expectant parents inevitably engender a high level of anxiety at all stages of the screening process, and the outcomes of screening can be associated with considerable physical and psychological morbidity [21]. A study investigated that the impact of genetic counselling has indicated that many women do not make informed decisions about prenatal genetic screening, and the introduction of genomic technologies has generally added to the ethical debate [22]. Appropriate pre- and post-testtest genetic counselling is recommended, and healthcare providers should include balanced information, as well as psychosocial support so that couples can get comprehensive understanding and make right decisions. Comprehensive pretest and posttest counseling is recommended and should address the importance of confirmatory testing and benefits of early diagnosis. Practice guidelines are needed to address provider responsibilities about postnatal testing.
(5) NIPT for monogenic diseases (Cluster 3)
NIPT has been widely adopted for the detection of fetal aneuploidies and CNVs in the past year. This paragraph will focus on the application of NIPT in the diagnosis of monogenic diseases. Initially, NIPT of monogenic disorders focused on detecting de novo or paternally inherited variants responsible for dominant monogenic disorders [23] Overall, the approaches of NIPT in recessive diseases are typically classified into two categories: relative mutation dosage (RMD) analysis [24] and relative haplotype dosage (RHDO) analysis [25]. The principle of RDM was quantitative comparisons between variant and wild-type alleles present in cfDNA. RDM was mainly detecting single nucleotide variants (SNVs) and small insertions/deletions (InDels) but cannot detect large InDels and CNVs; RHDO approach determines the relative proportions of variant and normal haplotypes in maternal plasma [26] and can theoretically detect most types of variants, including large InDels and CNVs [27]. However, RHDO analysis requires parental haplotype information and the detection cost was very expensive. The latest research reported a novel approach to detecting Thalassemia, which is based on population-based haplotyping-NIPT (PBH-NIPT) [28]. The PBN-NIPT provide a simple, fast, and inexpensive method. Compared with the traditional linked-read sequencing based NIPT method, the PBN-NIPT only cost 5-7 day to finish lab word and data analysis. Meanwhile, the detection cost is reduced from 1500 dollars/sample to 80 dollars/sample.
(6) Evaluation of NIPT in fetal sex determination(Cluster 3)
NIPT is a rapidly adopted prenatal testing method, which uses cell-free DNA to detect a range of genetic and chromosomal diseases, and determine fetal sex earlier, more easily and more reliably. Therefore, NIPT potentially expands the market for sex determination and sex selective abortion. For example, Ho et al. reported that fetal sex was correctly determined in 77 of 82 (93.9%) cell-free DNA samples of pregnant women of 6-39 gestational weeks, and confirmed that fetal sex was determined as early as 6 gestational weeks using cell-free DNA from maternal plasma [29]. Another study by Koumbaris and Kypri has developed an assay for the detection of fetal aneuploidies of sex chromosomes based on the targeted analysis of cell-free DNA, which can correctly identify fetal sex in all cases (95% CI, 99.4%-100%) [30]. Both studies indicate that cell-free DNA analysis exhibits high accuracy in fetal sex determination.
(7) Evaluation of NIPT in detecting sex chromosome abnormality (SCAs)(Cluster 4)
SCAs is estimated to be one in every 500 live births, between 75% and 90% of cases remain undiagnosed in their lifetime. Children with these disorders may present with dysfunction in speech, language, and motor development. A retrospective population‐based cohort study from 1986–2016 of pregnant women undergoing NIPT has provides a very meaningful set of data. With the emergence and application of NIPT detection technology, the percentage of prenatal diagnostic tests leading to a SCAs diagnosis increased significantly from 0.95% in 2010 to 2.93% in 2016[31]. This data showed that NIPT can effectively reduce the occurrence of fetal sex chromosome abnormality. However, NIPT also has limitations in detecting SCAs. For example, a study screening for SCAs in 9985 pregnancies by NIPT has showed the false positive rate of SCAs was 22.5%. Expecially in this case of 45, X0, the false positive rate is as high as 47% [32]. Therefore, screening for s SCAs by NIPT is still controversial.
Limitation and Expectation
Although NIPT based on cell-free DNA in maternal circulation has been accepted worldwide by the clinical community, several limitations such as maternal malignancy and fetoplacental mosaicism, maternal mosaicism, and a demised co-twin, preclude its full replacement of invasive prenatal diagnosis [33]. Many attentions have focused on both fetal nucleated red blood cells and trophoblasts [34]. A novel silicon-based nanostructured microfluidics platform has been reported to be feasible to capture circulating fetal nucleated red blood cells and extra villous cytotrophoblasts for cell-based noninvasive prenatal diagnosis [33]. We have enough confidence that NIPT will replace maternal serum screening (MSS) and invasive testing in the future. By that time, lacking of physician education on NIPT may become the major barriers for its implementation. Data from health care providers (HCPs) have indicated that counseling a patient after a failed cfDNA NIPT is challenging, reinforcing that this can possibility be covered by pre-test a counseling [35]. Along with the perfection of detecting techniques and the complete genetic counseling system, NIPT will provide more benifits for prenatal disgnosis.