Efficiency of Non-Invasive Prenatal Testing in Detecting Fetal Copy Number Variation: A Retrospective Cohort Study.

Purpose: Screening of pathological copy number variations (CNVs) is important for early-diagnosis of hereditary disease. This study was designed to investigate the efficiency of non-invasive prenatal testing (NIPT) in detecting fetal CNVs. Methods: This retrospective analysis included fetuses with CNVs between January 2018 and December 2020. Karyotype analysis and CNV sequencing (CNV-seq) were performed. We then analyzed the positive predictive values of the subchromosomal microdeletions and microduplications. Results: Fifty-eight subjects with aberrant CNVs were screened after NIPT, among which 44 finally underwent amniocentesis. CNV-seq confirmed the presence of CNVs in 24 cases. This indicated that false positivity rate of NIPT was 45.5%. Among 24 cases with CNVs after CNV-seq, only 4 showed consistent findings with karyotype analysis, which showed that karyotyping analysis yielded a missed diagnosis rate of 83.3% for the genome CNV. Positive predictive value (PPV) was 50.0% for CNVs with a length of <5 Mb after NIPT screening. PPV for CNVs with a length of 5 Mb-10 Mb was 33.3%, while that for CNVs with a length of ≥10Mb was 60%. For CNVs duplication after NIPT, the PPV was 65.2%, while that for deletion was 36.4%. Conclusion: For CNVs detected after NIPT, it should be combined with ultrasonographic findings, karyotype analysis, CNV-seq or CMA to determine the pregnancy outcome. Expanding NIPT may increase the risk of unnecessary invasive surgery and unintended selective termination of pregnancy.

Is intermediate risk really intermediate? Comparison of karyotype and non-invasive prenatal testing results of pregnancies at intermediate risk of trisomy 21 on maternal serum screening.

The purpose of this study was to assess the additional contribution of karyotyping compared with genome-wide non-invasive prenatal testing (NIPT) for pregnancies at intermediate risk for trisomy 21 (T21), calculated using the maternal serum screening without major structural anomalies detected through sonography. Karyotype results of all pregnancies undergoing invasive prenatal diagnostic testing between January 2013 and March 2022 were obtained from a large hospital-based laboratory. Pregnancies with no major structural anomalies on ultrasound (including soft markers) and an intermediate risk for T21 on maternal serum screening were included in this study. The additional contribution of karyotyping for abnormal karyotype results was calculated after excluding results that could theoretically be identified with genome-wide NIPT. Among the 511 pregnancies analyzed, 13 (2.54%) were found to have abnormal karyotype results, 9 (1.76%) of which could theoretically have been detected with genome-wide NIPT. Within the cohort, 6/263 (2.28%) of women aged 35 years and older, and 3/248 (1.20%) of women younger than 35 years had results that could have been detected with genome-wide NIPT. After excluding results detectable using genome-wide NIPT, the additional contribution of karyotyping was found as 4/502 (0.79%) for the entire cohort, 2/257 (0.77%) for women aged 35 years and older, 2/245 (0.81%) for women younger than 35 years. Of the 511 examined pregnancies at intermediate risk for T21 by maternal serum screening, genome-wide NIPT would have failed to detect 4 of 13 abnormal karyotype results. The findings hold importance in guiding couples' informed decision-making processes regarding their choice of genetic screening and diagnostic testing in case of intermediate risk for T21.

Clinical strategy study on prenatal screening and diagnostic model for Down syndrome.

Exploring efficient and easily implementable prenatal screening strategies aims at birth defect prevention and control. However, there have been limited economic evaluations of non-invasive prenatal screening (NIPS) strategies in China. Furthermore, these studies were predominantly confined to local or geographically proximate provinces and lacked universality and representativeness. This study assesses the health economics of current prenatal screening strategies and NIPS as first-line screening programs, analyzing their efficacy to determine an optimal strategy. From the perspective of health economics, cost-effectiveness, cost-benefit, and single-factor sensitivity were conducted for five different screening strategies using a decision tree model. Among pregnant women aged < 35 years who underwent only one screening for foetal Down syndrome (DS), the detection rate, false positive rate and positive predictive value of NIPS for foetuses with DS were superior to those of the other four serological screening methods. Although applying NIPS as first-line screening method yields the highest efficacy and benefits, it currently lacks cost-effectiveness when compared to serological screening and sequential NIPS screening strategies.

Non-invasive prenatal screening: Testing motivations and decision making in the low-risk population.

Non-invasive prenatal screening provides a risk assessment for aneuploidies by utilizing cell-free DNA (cfDNA). It is recommended that cell-free DNA screening (cfDNA screening) be offered to all pregnant people regardless of a priori risk for aneuploidy. In the absence of an increased risk, alternative motives for electing cfDNA screening and different levels of informed decision making may arise. Therefore, our study aimed to characterize low-risk patients' motivations for cfDNA screening election, determine how often informed decisions are being made, and compare motivations between informed and uninformed decision makers. A survey that included a modified, validated measure of informed choice (MMIC) and questions to assess patients' motivations for cfDNA screening was offered at four MFM clinics following genetic counseling. It was found that 44% of participants (n = 100) made an uninformed decision about testing. Participants with private insurers were 4.25 times more likely to make an informed decision (95% CI = 1.10-16.37). Informed decision makers scored avoiding invasive procedures higher (p = 0.007) and ranked doing what family/friends desire lower (p = 0.005) than uninformed decision makers. While most participants scored receiving information about genetic conditions highest, 12% of participants reported fetal sex disclosure as a priority. However, this was not found to be associated with uninformed decision making. This study ultimately established that following genetic counseling, a low-risk population shared motivations with high-risk populations which highlights the importance of complete pre-test counseling for all. Future research should investigate the effect of modifying variables, such as socioeconomic status, on the performance of informed choice measures and critically evaluate the parameters that determine informed choice.

Combined Z-scores to assess the impact of rare autosomal trisomies that results in non-invasive prenatal screening on pregnancy outcomes.

OBJECTIVE: This study aimed to combine Z-scores to evaluate the effects of rare autosomal trisomies (RATs) in non-invasive prenatal screening (NIPS) on pregnancy outcomes at a single center. METHODS: We retrospectively collected the clinical data of women with high-risk RATs results using NIPS at a single center between January 2017 and December 2021. NIPS-positive results were separated into three groups based on the Z-value of RATs (Group1: 6 ≤ Z < 10; Group2: 10 ≤ Z < 15; Group 3: Z ≥ 15). Pregnancy outcomes of women with RATs were compared with the low-risk NIPS group. RESULTS: Overall, 83 RATs were identified in 23,321 NIPS results at our center. Prenatal diagnosis was conducted for 55 patients, and no case was confirmed, with a positive predictive value (PPV) of zero. Fifteen of these patients had adverse pregnancy outcomes, including delivered preterm and/or birth weight (9/15, 60.0 %), structural abnormalities (4/15, 26.7 %), miscarriage (1/15, 6.7 %), and intrauterine death (1/15, 6.7 %). There were 8 (8/22, 36.4 %) adverse pregnancy outcomes in Group 3, which was significantly higher than that in the low-risk NIPS group (p < 0.01). No significant difference was observed between the control group and Group 1 and Group 2 (p > 0.01). CONCLUSIONS: Clinicians should pay more attention to the RATs results when the Z-score is ≥ 15. The data are available for clinicians to guide the prenatal diagnosis of RATs and pregnancy management.

Relationships among maternal monosomy X mosaicism, maternal trisomy, and discordant sex chromosome aneuploidies.

OBJECTIVE: To explore the impact of maternal factors on the false-positive fetal sex chromosome aneuploidies (SCAs) results obtained through noninvasive prenatal screening (NIPS). METHODS: We retrospectively analyzed pregnant women with high-risk SCAs as revealed using NIPS between January 2017 and December 2022. Clinical data such as results of invasive prenatal diagnoses, copy number variation sequencing (CNV-seq) and pregnancy outcomes were analysed. RESULTS: Overall, 177 (0.6 %) women with SCA-positive results were collected from 27,941 patients who had undergone NIPS. Among them, 110 (62.2 %) pregnant women chose prenatal diagnosis and 39 (35.5 %) cases were confirmed. For the women with monosomy X false-positive results from the NIPS, 53.1 % (17/32) were found to be maternal mosaicism monosomy X. In cases with 47, XXX false-positive results, 60 % (6/10) of them were maternal 47,XXX (5 cases) or maternal mosaicism 47,XXX (1 case). One (1/6, 16.7 %) case of maternal mosaicism monosomy X was detected in the false positive results of 47, XXY/47, XYY revealed. The incidence rate of maternal sex chromosome abnormalities was positively correlated with the Z-score of ChrX. When the Z-score of ChrX ≥ 15, more than 50 % of pregnant women were found to be maternal sex chromosome abnormalities, and when Z-score ≥ 30, the incidence rate was as high as 100 %. CONCLUSIONS: Maternal monosomy X mosaicism and trisomy X respectively played an important role in the discordance of 45, X and 47, XXX revealed by NIPS. CNV-seq was recommended for the pregnant women at risk of maternal sex chromosome abnormalities, which could help clinicians to provide more accurate and efficient advice during genetic counseling and to guide appropriate prenatal diagnosis strategy for the next pregnancy.

Residual risk of clinically significant copy number variations in fetuses with nasal bone absence or hypoplasia after excluding non-invasive prenatal screening-detectable findings.

BACKGROUND: It remains controversial whether prenatal screening or diagnostic testing should be offered to fetuses with nasal bone (NB) absence or hypoplasia, and there are no studies comparing the yield of chromosomal microarray analysis (CMA) to non-invasive prenatal screening (NIPS). The aim of this study was to evaluate the residual risk of clinically significant copy number variations (CNVs) in fetuses with NB absence or hypoplasia after excluding theoretically NIPS-detectable abnormalities, and to assess their clinical outcomes. METHODS: This prospective study encompassed 400 fetuses with NB absence or hypoplasia undergoing CMA testing between 2015 and 2022. Clinically significant CMA findings were categorized into three subgroups, including three-NIPS-detectable (trisomies 21, 18 and 13), five-NIPS-detectable (trisomies 21, 18 and 13 and sex chromosome aneuploidies) and genome-wide NIPS-detectable (variants over 7 Mb). We calculated the theoretical residual risk and compared it with the results of a control cohort of low-risk pregnancies. We further evaluated their clinical outcomes. RESULTS: The overall diagnostic yield in our cohort was 7.8% (31/400). The detection rate of clinically significant CMA findings in fetuses with non-isolated NB absence or hypoplasia was significantly higher than that in fetuses with isolated NB absence or hypoplasia (20.0% vs. 6.6%, P =.005). The theoretical residual risks in all NIPS models were significantly higher when compared with the control cohort. The normal infant rate in fetuses with normal CMA results was 97.9% (323/330), and a significant higher incidence was observed in fetuses with isolated NB absence or hypoplasia compared with non-isolated NB absence or hypoplasia (98.4% vs. 91.7%, P =.028). CONCLUSIONS: The residual risk of clinically significant CNVs in fetuses with NB absence or hypoplasia following the exclusion of theoretically NIPS-detectable findings was higher than that in low-risk pregnancies. This risk should be considered in genetic counseling to make a more comprehensive and precise choice regarding prenatal genetic testing.

A new contingent screening strategy increased detection rate of trisomy 21 in the first trimester.

BACKGROUND: Although the traditional contingent screening strategy is effective, there are still undetected low-risk trisomy 21. This study aims to define appropriate cut-off values of serum biochemical markers at low-risk and develop a strategy for sequential prenatal testing associated with first-trimester screening to increase the detection rate of trisomy 21. METHODS: This was a 9-year retrospective analysis of singleton pregnant women who underwent serum biochemical screening or combined first-trimester screening (CFTS) in the first trimester. For the low-risk group, the cut-off values of the serum biochemical markers were adjusted to determine the appropriate detection efficiency. Gravidas with abnormal serum biochemical markers at low-risk were advised to undergo further non-invasive prenatal screening (NIPS), whereas others continued with routine prenatal care. RESULTS: When cut-off values of free beta subunit of human chorionic gonadotropin (free ß-hCG) multiples of the median (MoM) or pregnancy-associated plasma protein A (PAPP-A) MoM were defined with ≥ 2.75 or ≤ 0.5, 7.72% (2,194/28,405) in the serum biochemical screening group and 12.36% (4,005/32,403) in CFTS group could be detected as abnormal results for further NIPS. Finally, 55.56% (5/9) and 85.71% (6/7) of trisomy 21 cases with false-negative results were detected, and the overall detection rate for trisomy 21 was improved by 10.64% (5/47) and 12.77% (6/47), respectively. CONCLUSIONS: The new contingent screening strategy can increase the detection rate of trisomy 21 compared with the traditional contingent screening strategy.

Maternal, neonatal, pregnancy outcome characteristics of pregnant women with high plasma cell-free DNA concentration in non-invasive prenatal screening: a retrospective analysis.

Objective: Cell-free DNA (cfDNA) is a useful biomarker in various clinical contexts. Herein, we aimed to identify maternal characteristics and pregnancy outcomes associated with a failed NIPS test due to high cfDNA concentrations. Methods: A retrospective study of cases with high plasma cfDNA concentration in pregnant women in which NIPS test was performed (from 174,318 cases). We reported the detection of 126 cases (118 with complete clinical information) in which the high amount of cfDNA did not allow the performance of NIPS and study the possible causes of this result. Results: 622 (0.35%) of 174,318 pregnant women had failed the NIPS test, including 126 (20.3%) cases with high plasma cfDNA concentrations. The failed NIPS due to high plasma cfDNA concentrations was associated with maternal diseases and treatment with low-molecular-weight heparin (LMWH). Further follow-up of the 118 pregnant women in the case group revealed that the pregnancy outcomes included 31 premature deliveries, 21 abortions. The cfDNA concentrations of pregnant women with preterm deliveries were 1.15 (0.89, 1.84), which differed significantly from those who had full-term deliveries. Conclusions: Among pregnant women with high cfDNA concentrations, systemic autoimmune diseases, pregnancy complications and LMWH were associated with increased incidence of failed NIPS test. High maternal cfDNA concentrations may not be associated with chromosomal abnormalities in the fetus. However, they should be alerted to the possibility of preterm births and stillbirths. Further clinical studies on pregnant women with high cfDNA concentrations are required.

Pregnant people's views and knowledge on prenatal screening for fetal trisomy in the absence of a national screening program.

Multiple non-invasive prenatal tests (NIPT) are available to screen for risk of fetal trisomy, however, there is no national prenatal screening program in Republic of Ireland. This study aimed to analyze pregnant people's opinions on availability, cost, and knowledge of NIPT for fetal aneuploidy. An anonymous questionnaire on prenatal screening tests and termination of pregnancy was distributed to patients attending antenatal clinics at a tertiary hospital. Descriptive analyses and chi-squared tests were completed. Among respondents, 62% (200/321) understood the scope of prenatal screening tests, with 77% (251/326) and 76% (245/323) correctly interpreting low- and high-risk test results, respectively. Only 26% (83/319) of participants had heard of NIPT. Chi-square tests showed a higher proportion of these people were ≥40 years old (p-value, <0.001), had post-graduate education (p-value, <0.001), or attended private clinics (p-value <0.001). Over 91% (303/331) of participants said every pregnant person should be offered prenatal screening tests for aneuploidy and 88% (263/299) believed these should be free. While pregnant Irish individuals have reasonable understanding of screening test interpretation, most were unaware of screening options. Additionally, participants' views on availability and associated cost of tests show the need for a national prenatal screening program, including education on fetal aneuploidy. These findings have relevance for countries without screening policies and are pertinent for broader maternity services.

Non-invasive cell-free DNA prenatal screening for trisomy 21 as part of primary screening strategy in twin pregnancies.

OBJECTIVES: The performance of non-invasive prenatal screening using cell-free DNA testing in maternal blood in twin pregnancies is still under-evaluated, while serum marker-based strategies yield poor results. This study aims at assessing the performance of non-invasive prenatal screening for trisomy 21 in twin pregnancies as a first-tier test. The secondary objectives were to assess the failure rate and associated factors. METHODS: This retrospective cohort study included twin pregnancies for which non-invasive prenatal screening using cell-free DNA was performed as the primary screening strategy between May 2017 and October 2019. We used the NIPT VeriSeq® test for in vitro diagnosis and set a fetal fraction cut-off of 4% for monochorionic pregnancies and 8% for dichorionic ones. Clinical data and pregnancy outcome was collected from either physicians or midwives through a questionnaire or were retrieved directly on site. We calculated the performance of non-invasive cell free DNA screening for trisomy 21 and analyzed failure rate and factors. RESULTS: We included 2577 multiple pregnancies among which 1885 (84.8%) were retained after excluding vanishing twins and pregnancies without follow-up. Overall, there were six confirmed trisomy 21 cases (0.32%). For trisomy 21, sensitivity was 100% (95% CI, 61-100%) and the false-positive rate 0.2% (95% CI, 0.07-0.6%). The primary failure rate was 4.6% with 4% due to insufficient fetal fraction. After a new blood draw (59.8% of failed cases), failure rate was only 1.5%. Body mass index and chorionicity were significantly correlated with the risk of failure. CONCLUSION: This study adds further evidence on the high performance of NIPS in twins, as part of the primary screening strategy for trisomy 21, at an extremely low false-positive rate. This article is protected by copyright. All rights reserved.

Mild TSC phenotype and non-penetrance associated with a frameshift variant in TSC2 prompts caution in evaluating pathogenicity of frameshift variants.

INTRODUCTION: Technological advances in genetic testing, particularly the adoption of noninvasive prenatal screening (NIPS) for single gene disorders such as tuberous sclerosis complex (TSC, OMIM# 613254), mean that putative/possible pathogenetic DNA variants can be identified prior to the appearance of a disease phenotype. Without a phenotype, accurate prediction of variant pathogenicity is crucial. Here, we report a TSC2 frameshift variant, NM_000548.5(TSC2):c.4255_4256delCA, predicted to result in nonsense-mediated mRNA decay (NMD) and cessation of TSC2 protein production and thus pathogenic according to ACMG criteria, identified by NIPS and subsequently detected in family members with few or no symptoms of TSC. Due to the lack of TSC-associated features in the family, we hypothesized that the deletion created a non-canonical 5' donor site resulting in cryptic splicing and a transcript encoding active TSC2 protein. Verifying the predicted effect of the variant was key to designating pathogenicity in this case and should be considered for other frameshift variants in other genetic disorders. METHODS: Phenotypic information on the family members was collected via review of the medical records and patient reports. RNA studies were performed using proband mRNA isolated from blood lymphocytes for RT-PCR and Sanger sequencing. Functional studies were performed by transient expression of the TSC2 variant proteins in cultured cells, followed by immunoblotting. RESULTS: No family members harboring the variant met any major clinical diagnostic criteria for TSC, though a few minor features non-specific to TSC were present. RNA studies supported the hypothesis that the variant caused cryptic splicing, resulting in an mRNA transcript with an in-frame deletion of 93 base pairs r.[4255_4256del, 4251_4343del], p.[(Gln1419Valfs*104), (Gln1419_Ser1449del)]. Expression studies demonstrated that the canonical function of the resulting truncated TSC2 p.Gln1419_Ser1449del protein product was maintained and similar to wildtype. CONCLUSION: Although most frameshift variants are likely to result in NMD, the NM_000548.5(TSC2):c.4255_4256delCA variant creates a cryptic 5' splice donor site, resulting in an in-frame deletion that retains TSC2 function, explaining why carriers of the variant do not have typical features of TSC. The information is important for this family and others with the same variant. Equally important is the lesson that predictions can be inaccurate, and that caution should be used when designating frameshift variants as pathogenic, especially when phenotypic information to corroborate testing results is unavailable. Our work demonstrates that functional RNA- and protein-based confirmation of the effects of DNA variants improves molecular genetic diagnostics.

Evaluation of the clinical effects of non-invasive prenatal screening for diseases associated with aneuploidy and copy number variation.

BACKGROUND: To explore and compare the clinical effects of high-resolution non-invasive prenatal screening (NIPS-Plus) for common/uncommon chromosomal aneuploidy and microdeletion/microduplication syndromes (MMS). METHODS: The current prospective study included a total of 25,380 pregnant women who performed NIPS-Plus, and amniocentesis was performed on women with MMS with the screening results to diagnose patients with suspected MMS. RESULTS: There were 415 samples with positive results for NIPS-Plus, included 275 with aneuploidy and 140 with MMS. After diagnosis by amniocentesis, 188 cases were confirmed as true positive, included46 cases of T21, 9 cases of T18, 1 case of T13, 34 cases of SCA, 41 cases of other chromosomal euploidy and 57 cases of MMS. In addition, no false negative cases were found, MMS was classified with 5 Mb with the cutoff value, and the PPV of different fragment size was counted, respectively. CONCLUSION: We found that the corresponding PPV was 44.66% with the fragment of copy number variation (CNV) being less than or equal to 5 Mb, and when it was greater than 5 Mb, the PPV was 29.73%, which suggested that NIPS-Plus was more suitable for screening the PPV of small fragment abnormalities. NIPS-Plus has a good application effect in routine aneuploidy screening and had the best detection effect for T21; moreover, it performed well in screening of MMS and had better detection effect on MMS with CNV fragment length less than 5 Mb. Based on the current results, we suggested that NIPS-Plus should be used as a comprehensive elementary prenatal screening method for all pregnant women, but for MMS caused by abnormal large fragment CNV, the detection method and efficiency still need to be improved.

A call for public funding of invasive and non-invasive prenatal testing.

For decades, prenatal screening and genetic testing strategies were limited, requiring less complex decisions. Recently, however, several new advanced technologies were introduced, including chromosomal microarray analysis (CMA) and non-invasive prenatal screening (NIPS), bringing about the need to choose the most appropriate testing for each pregnancy. A worrisome issue is that opposed to the wide implementation and debates over public funding of NIPS, currently invasive testing is still recommended only in selected pregnancies with increased risk for chromosomal aberrations (according to screening tests or sonographic anomalies). This current decision-making regarding public funding for invasive and screening testing might compromise informed consent and patient's autonomy. In this manuscript, we compare several characteristics of CMA vs. NIPS, namely: the accuracy and the diagnostic scope, the risks for miscarriage and for clinically uncertain findings, the timing for testing, and pretest counselling. We argue that it must be recognized that one size might not fit all, and suggest that both options should be presented to all couples through early genetic counseling, with public funding for the specific selected test.

What knowledge is required for an informed choice related to non-invasive prenatal screening?

Non-invasive prenatal screening (NIPS) using cell-free DNA is a screening test for fetal aneuploidy offered by a variety of prenatal healthcare providers. Guidelines for genetic screening consistently recommend that providers facilitate informed choices, which have been associated with better psychological and clinical outcomes than uninformed choices. The multidimensional measure of informed choice (MMIC) is a widely used and theory-based measure that combines knowledge, values, and behavior to classify decisions as either informed or uniformed. We implemented a previously validated version of the MMIC for women offered NIPS to describe the choices made by women receiving prenatal care at the Vanderbilt University Medical Center. The survey included the Ottawa Decisional Conflict scale, an outcome measure used for validation of choice categorization. We found that most women (87%) made an informed choice about NIPS. Of the women categorized as uninformed, 67% had insufficient knowledge, and 33% had an attitude discordant with their decision. The vast majority of respondents (92.5%) underwent NIPS and had a positive attitude toward screening (94.3%). Ethnicity (p = 0.04) and education (p = 0.01) were found to be significantly associated with informed choice. Decisional conflict was extremely low among all participants, with only 5.6% of all participants demonstrating any form of decisional conflict, and all being categorized as having made an informed choice. This study suggests that pre-test counseling by a genetic counselor results in high rates of informed choice and low-decisional conflict amongst women offered NIPS by genetic counselors, though more research is required to determine if rates of informed choice remain high when NIPS is offered by other prenatal providers.

Reappraisal of evolving methods in non-invasive prenatal screening: Discovery, biology and clinical utility.

Non-invasive prenatal screening (NIPS) offers an opportunity to screen or determine features associated with the fetus. Earlier, prenatal testing was done with cytogenetic procedures like karyotyping or fluorescence in-situ hybridization, which necessitated invasive methods such as fetal blood sampling, chorionic villus sampling or amniocentesis. Over the last two decades, there has been a paradigm shift away from invasive prenatal diagnostic methods to non-invasive ones. NIPS tests heavily rely on cell-free fetal DNA (cffDNA). This DNA is released into the maternal circulation by placenta. Like cffDNA, fetal cells such as nucleated red blood cells, placental trophoblasts, leukocytes, and exosomes or fetal RNA circulating in maternal plasma, have enormous potential in non-invasive prenatal testing, but their use is still limited due to a number of limitations. Non-invasive approaches currently use circulating fetal DNA to assess the fetal genetic milieu. Methods with an acceptable detection rate and specificity such as sequencing, methylation, or PCR, have recently gained popularity in NIPS. Now that NIPS has established clinical significance in prenatal screening and diagnosis, it is critical to gain insights into and comprehend the genesis of NIPS de novo. The current review reappraises the development and emergence of non-invasive prenatal screen/test approaches, as well as their clinical application, with a focus, on the scope, benefits, and limitations.

Synthesis of positive plasmas with known chromosomal abnormalities for validation of non-invasive prenatal screening.

Non-invasive prenatal screening (NIPS) is a DNA sequencing-based screening test for fetal aneuploidies and possibly other pathogenic genomic abnormalities, such as large deletions and duplications. Validation and quality assurance (QA) of this clinical test using plasmas with and without targeted chromosomal abnormalities from pregnant women as negative and positive controls are required. However, the positive plasma controls may not be available for many laboratories that are planning to establish NIPS. Limited synthetic positive plasmas are commercially available, but the types of abnormalities and the number/quantity of synthetic plasmas for each abnormality are insufficient to meet the minimal requirements for the initial validation. We report here a method of making synthetic positive plasmas by adding cell-free DNA (cfDNA) isolated from culture media of prenatal cells with chromosomal abnormalities to the plasmas from non-pregnant women. Thirty-eight positive plasmas with various chromosomal abnormalities, including autosomal and sex chromosomal aneuploidies, large deletions and duplications, were synthesized. The synthetic plasmas were characterized side-by-side with real positive plasmas from pregnant women and commercially available synthetic positive plasmas using the Illumina VeriSeq NIPT v2 system. All chromosomal abnormalities in the synthetic plasmas were correctly identified with the same testing sensitivity and specificity as in the real and commercial synthetic plasmas. The findings demonstrate that the synthetic positive plasmas are excellent alternatives of real positive plasmas for validation and QA of NIPS. The method described here is simple and straightforward, and can be readily used in clinical genetics laboratories with accessibility to prenatal cultures.

Clinical utility of expanded non-invasive prenatal screening compared with chromosomal microarray analysis in over 8000 pregnancies without major structural anomaly.

OBJECTIVES: To evaluate the theoretical added value of two types of non-invasive prenatal screening (NIPS) expansions in pregnancies without major structural anomalies over the commonly used NIPS for chromosomes 13, 18, 21, X and Y (5-NIPS) and to compare them with the added value of chromosomal microarray analysis (CMA). METHODS: This was a retrospective cohort study based on CMA results of all pregnancies with normal ultrasound (including pregnancies with soft markers and with abnormal maternal serum screening) that had undergone amniocentesis between January 2013 to February 2022 and were registered in the database of the Rabin Medical Center genetic laboratory. We calculated the theoretical yield of 5-NIPS and compared the added value of expanded 5-NIPS for common microdeletions (1p36.3-1p36.2, 4p16.3-4p16.2, 5p15.3-5p15.1, 15q11.2-15q13.1 and 22q11.2) and genome-wide NIPS (including variants > 5 Mb) with the added value of CMA in the overall cohort and in subgroups according to indication for invasive testing. RESULTS: Among the 8605 examined pregnancies, 122 (1.4%) clinically significant CMA results were demonstrated. Of these, 44 (36.1%) were theoretically detectable on 5-NIPS, with the rates of 1.56% in 642 pregnancies with abnormal maternal serum screening, 0.63% in 318 pregnancies with soft markers, 0.62% in 4378 women with advanced maternal age (≥ 35 years) and 0.15% in 3267 women younger than 35 years. In addition to aneuploidies detectable on 5-NIPS, three (0.03%) cases detectable on 5-NIPS expanded for common microdeletions and nine (0.10%) cases detectable on genome-wide NIPS (excluding common microdeletions) were identified in the overall cohort. The added value of expanded NIPS tools over 5-NIPS was significantly lower compared with that of CMA, for the overall cohort and subgroups. CONCLUSIONS: 5-NIPS and even genome-wide NIPS would miss 63.9% and 54.1% of clinically significant CMA findings, respectively. The added value of 5-NIPS expanded to detect common microdeletions over 5-NIPS is about 0.035%, and the overall added value of genome-wide NIPS aimed at large CNVs is about 0.14%, both much lower compared with the added value of CMA (0.91%). These findings should assist healthcare practitioners in guiding couples towards informed decision-making regarding the choice between prenatal invasive testing and NIPS. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

A new and improved method of library preparation for non-invasive prenatal testing: plasma to library express technology.

OBJECTIVES: This study aims to develop a novel library preparation method, plasma to library express technology (PLET), to construct next-generation sequencing (NGS) libraries directly from plasma without cell-free DNA (cfDNA) isolation. METHODS: Peripheral blood samples (600) were obtained from a retrospective cohort of 300 pregnant women prior to invasive diagnostic testing. The samples were subsequently distributed between library preparation methodologies, with 300 samples prepared by PLET and 300 by conventional methods for non-invasive prenatal testing (NIPT) to screen for common trisomies using low-pass whole genome next generation sequencing. RESULTS: NIPT conducted on PLET libraries demonstrated comparable metrics to libraries prepared using conventional methods, including 100% sensitivity and specificity. CONCLUSIONS: Our study demonstrates the potential utility of PLET in the clinical setting and highlights its significant advantages, including dramatically reduced process complexity and markedly decreased turnaround time.