Prenatal Aneuploidy Screening Research Articles

High-resolution profiling of human cytomegalovirus cell-free DNA in human plasma highlights its exceptionally fragmented nature

Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma. CMV reads were identified in 120 (5.4%) samples. Median cfDNA fragment size derived from CMV was significantly shorter than cfDNA derived from human chromosomes (103 vs 172 bp, p < 0.0001), corresponding to the 3rd percentile of human cfDNA. Sequencing of cfDNA from seven plasma samples from transplant patients positive for CMV confirmed the extraordinarily short nature of CMV cfDNA fragment size with a median length of 149 bp. We further show that these high-resolution measurements of CMV DNA fragment size accurately predict measured discrepancies in serum viral load measurements by different qPCR assays. These results highlight the exceptionally fragmented nature of CMV cfDNA and illustrate the promise of plasma cfDNA sequencing for quantitating viral loads through detection of fragments that would be unrecoverable by qPCR.

Clinical Practice Guidelines for Prenatal Aneuploidy Screening and Diagnostic Testing from Korean Society of Maternal-Fetal Medicine: Cell-Free DNA Screening

In 2019, the Korean Society of Maternal-Fetal Medicine developed the first Korean clinical practice guidelines for prenatal aneuploidy screening and diagnostic testing. These guidelines were developed by adapting established clinical practice guidelines in other countries that were searched systematically, and the guidelines aim to assist in decision making of healthcare providers providing prenatal care and to be used as a source for education and communication with pregnant women in Korea. This article delineates clinical practice guidelines specifically for maternal serum screening for fetal aneuploidy and cell-free DNA (cfDNA) screening. A total of 19 key questions (12 for maternal serum and 7 for cfDNA screening) were defined. The main recommendations are: 1) Pregnant women should be informed of common fetal aneuploidy that can be detected, risks for chromosomal abnormality according to the maternal age, detection rate and false positive rate for common fetal aneuploidy with each screening test, limitations, as well as the benefits and risks of invasive diagnostic testing, 2) It is ideal to give counseling about prenatal aneuploidy screening and diagnostic testing at the first prenatal visit, and counseling is recommended to be given early in pregnancy, 3) All pregnant women should be informed about maternal serum screening regardless of their age, 4) cfDNA screening can be used for the screening of trisomy 21, 18, 13 and sex-chromosome aneuploidy. It is not recommended for the screening of microdeletion, 5) The optimal timing of cfDNA screening is 10 weeks of gestation and beyond, and 6) cfDNA screening is not recommended for women with multiple gestations. The guideline was reviewed and approved by the Korean Academy of Medical Sciences.

1057: Noninvasive prenatal screening for fetal aneuploidy in twin pregnancies: A clinical laboratory experience

1057: Noninvasive prenatal screening for fetal aneuploidy in twin pregnancies: A clinical laboratory experience

Informed Decision Making Regarding Prenatal Aneuploidy Screening

ObjectiveTo explore informed decision making for prenatal aneuploidy screening (PAS) by investigating the relationships among women’s understanding of PAS, their health literacy, and their satisfaction with their decisions regarding PAS. DesignA descriptive, cross-sectional, correlational study. SettingSoutheastern United States. ParticipantsNinety-five adult women who were at least 18 weeks pregnant; most were White non-Hispanic (n = 75, 78.9%) and college educated (n = 56, 59.0%). MethodsPotential participants were invited to complete an online survey that was distributed via e-mail to recipients of an electronic pregnancy and parenting newsletter. The survey included questions about women’s understanding of PAS, their health literacy, and their satisfaction with their decisions regarding PAS based on three validated instruments. I used descriptive statistics, bivariate correlational analysis, and multiple linear regression to analyze the data. ResultsHigher level of education (β =.225, p = .038) and higher scores from the Health Literacy Questionnaire’s scale Ability to Actively Engage With Health Care Providers (β =.317, p = .004) were significantly associated with increased understanding of PAS. Higher scores on two scales from the Health Literacy Questionnaire, Feeling Understood andSupportedby Health Care Providers (β =.329 , p = .004) and Ability to Find Good Health Information (β = .402, p = .013), were significantly associated with greater satisfaction with decisions regarding prenatal aneuploidy screening. Forty-two percent (n = 40) of participants did not report satisfaction with PAS. ConclusionA woman’s health literacy and her ability to actively engage with health care providers are critical for informed decision making about PAS. Findings highlight the need for woman-centered strategies to promote open and intentional communication about PAS.

Cell-free DNA fetal fraction in twin gestations in single-nucleotide polymorphism-based noninvasive prenatal screening.

ObjectivesThe performance of noninvasive prenatal screening (NIPS) for fetal aneuploidy in twin pregnancies is dependent on the amount of placentally derived cell‐free DNA, the “fetal fraction (FF),” present in maternal plasma. We report FF values in monozygotic (MZ) and dizygotic (DZ) pregnancies.MethodsWe reviewed FF in pregnancies at 10 to 20 completed weeks gestational age based on single‐nucleotide polymorphism (SNP)‐based NIPS where zygosity was routinely established in twin pregnancies. The cohort included 121 446 (96.3%) singleton, 1454 (1.2%) MZ, and 3161 (2.5%) DZ pregnancies. For DZ twins, individual FFs were measured.ResultsCombined FF for DZ and MZ fetuses were 35% and 26% greater than singletons, respectively. The individual FF contributions from each fetus in DZ twins were, on average, 32% less than singletons. FF in DZ twin pairs were moderately correlated (Pearson correlation coefficient.66). When a threshold of 2.8% FF was applied to define uninterpretable results, 1.7% (2102/121 446) of singletons, 0.8% (11/1454) of MZ pairs, and 5.6% (178/3161) of DZ pairs were uninterpretable.ConclusionFor optimal aneuploidy NIPS in twin pregnancies, zygosity should be established and in DZ twins FF for both fetuses should be determined to identify those cases where results can be reliably interpreted.

A 2-year review of publicly funded cell-free DNA screening in Ontario: utilization and adherence to funding criteria.

Ontario offers a publicly funded modified contingent model of prenatal screening for aneuploidy in which cell-free DNA (cfDNA) screening is covered for pregnancies at higher risk of fetal aneuploidy. The objective of this study was to review utilization of provincially funded cfDNA screening and adherence to the criteria laid out in Ontario prenatal screening guidelines. This was a descriptive cohort study using data collected by Ontario's prescribed maternal and child registry. The study population included all pregnant individuals who received cfDNA screening from January 2016 to December 2017. The most common criteria for provincially funded cfDNA screening were advanced maternal age ≥40 years (37.7%), positive multiple marker screen (34.1%), modifying risk factors such as ultrasound soft markers (7.1%), and previous aneuploidy (5.5%). The audit demonstrated that 2.9% of funded cfDNA screens tests did not meet funding criteria, and that 11.4% of self-paid cfDNA screens could have been publicly funded. Reviewing and auditing the application of criteria for funded cfDNA screening using prescribed registry data allows an opportunity to identify areas where targeted education may improve adherence to standardized screening protocols, and provides a basis for reassessment of the funding model.

The association of HBB-related significant hemoglobinopathies and low fetal fraction on noninvasive prenatal screening for fetal aneuploidy

Objectives HBB-related significant hemoglobinopathies have been anecdotally associated with low fetal fraction on noninvasive prenatal screening (NIPS). We sought to compare the difference in fetal fraction using NIPS in women with HBB-related significant hemoglobinopathies (HSH) and women with normal hemoglobin. Study design This is a retrospective case-control study. Cases were women with a diagnosis of HSH using NIPS from a commercial laboratory. The comparison group was women with hemoglobin AA from a tertiary care center database. We tested for differences in median fetal fraction using quantile regression analysis, adjusting for maternal body weight and gestational age. Results This study includes 35 women with clinically significant HSH and a comparison group of 636 women with hemoglobin AA. Adjusting for gestational age and body weight, the median fetal fraction was 4.1 point lower in the HSH than in the comparison group (β − 4.1; 95% −5.7 to −2.5, p < .05). The rate of no-calls due to low fetal fraction was significantly higher in the clinically significant HSH group than in the comparison group [HSH: n = 9/35, 25.7% versus comparison: n = 32/636, 5.0% (p < .001)]. Conclusion Women with HSH were more likely to have a lower fetal fraction and ultimately a five-fold higher no-call rate. What’s already known about this topic? Low fetal fraction is one of the most common causes of no-call result in noninvasive prenatal screening High maternal weight, early gestational age and fetal aneuploidies are associated with low fetal fraction What does this study add? HBB-related significant hemoglobinopathies are associated with low fetal fraction Reduction in fetal fraction due to HBB-related significant hemoglobinopathies may also result in higher no-call rate

Detection of fetal trisomy and single gene disease by massively parallel sequencing of extracellular vesicle DNA in maternal plasma: a proof-of-concept validation

BackgroundDuring human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.MethodsMaternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.ResultsEVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.ConclusionsWe proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.

Adherence of cell-free DNA noninvasive prenatal screens to ACMG recommendations

PurposeNoninvasive prenatal screening (NIPS) for fetal aneuploidy via cell-free DNA has been commercially available in the United States since 2011. In 2016, the American College of Medical Genetics and Genomics (ACMG) issued a position statement with specific recommendations for testing laboratories. We sought to evaluate adherence to these recommendations. MethodsWe focused on commercial laboratories performing NIPS testing in the United States as of 1 January 2018. Sample laboratory reports and other materials were scored for compliance with ACMG recommendations. Variables scored for common and sex chromosome aneuploidy detection included detection rate, specificity, positive and negative predictive value, and fetal fraction. Labs that performed analysis of copy-number variants and results for aneuploidies other than those commonly reported were identified. Available patient education materials were similarly evaluated. ResultsNine of 10 companies reported fetal fraction in their reports, and 8 of 10 did not offer screening for autosomal aneuploidies beyond trisomy 13, 18, and 21. There was inconsistency in the application and reporting of other measures recommended by ACMG. ConclusionsLaboratories varied in the degree to which they met ACMG position statement recommendations. No company adhered to all laboratory guidance.

EP04.01: The increase of chorionic villous sampling and the decline of amniocentesis in the era of prenatal aneuploidy screening in a referral centre

EP04.01: The increase of chorionic villous sampling and the decline of amniocentesis in the era of prenatal aneuploidy screening in a referral centre

Quality Assurance of Non-Invasive Prenatal Screening (NIPS) for Fetal Aneuploidy Using Positive Predictive Values as Outcome Measures.

Non-invasive prenatal screening (NIPS) based on the analysis of cell-free DNA in maternal plasma has been shown to have high sensitivity and specificity. We gathered follow-up information for pregnancies in women with test-positive NIPS results from 2014–2017 with quarterly assessments of positive predictive values (PPVs). A non-inferiority analysis with a minimum requirement of 70%/80% of expected performance for trisomy 21 and 18 was used to ensure testing met expectations. PPVs were evaluated in the context of changes in the population receiving testing. For all quarters, PPVs for trisomies 21 and 18 exceeded the requirement of > 70% of the reference PPV. Overall observed PPVs for trisomy 21, 18, 13 and monosomy X were similar for women aged <35 (90.9%, 95% Confidence Interval (CI) 88.6–92.7%) compared to women with advanced maternal age (94.5%, 95% CI 93.1–95.6%). Despite significant declines in test-positive rates from 1.18% to 0.62% for trisomy 21, and from 0.75% to 0.48% for trisomies 18, 13 and monosomy X combined, PPVs remained stable through the four-year interval. We conclude that quarterly evaluation of PPV provides an overview of past testing and helps demonstrate long-term consistency in test performance, even in the setting of increasing use by women with lower a priori risks.

A two-year experience of non-invasive prenatal testing (NIPT) at an urban tertiary medical center in South Korea

ObjectiveTo report our experience of implementing non-invasive prenatal testing (NIPT) in a tertiary urban academic medical center in South Korea. Materials and methodsAn observational retrospective study of singleton and twin pregnancies that underwent prenatal screening for fetal aneuploidy from July 2016 to April 2018 was conducted. Demographics of the study population electing NIPT versus those opting the integrated test were compared. We also assessed clinical significant factors influencing cfDNA fetal fraction in NIPT. ResultsAmong the 817 women who underwent serum screening tests during the study period, 490 women (60.0%) chose the integrated test while 327 women (40.0%) chose NIPT. Compared to the integrated test group, women in the NIPT group were older (mean age 34.7 ± 3.7 vs. 32.6 ± 3.4; p-value < 0.01), multiparous (47.1% vs. 39.8%; p-value = 0.046), and had higher rate of previous abortion history (28.4% vs. 21.6%; p-value = 0.033). A significant decrease in the number of invasive diagnostic tests was observed since the adoption of NIPT. The screen negative and positive rates of the integrated test group for fetal aneuploidy were 95.3% and 4.7%, respectively while those of the NIPT group were 95.9% and 1.2%, respectively. The rate of inadequate cfDNA fetal fraction was 3.0%. Low fetal fraction was associated with higher maternal age, body weight and BMI. ConclusionsThe implementation of NIPT has significantly affected the practice pattern of prenatal aneuploidy screening by replacing the integrated test and decreasing invasive diagnostic tests.

Noninvasive prenatal screening for twin pregnancy: an analysis of 2057 cases

To analyze the results of noninvasive prenatal screening (NIPS) for fetal chromosome aneuploidy in twin pregnancy. A total of 2057 women with twin-pregnancy between 12-26+6 weeks were recruited from Women's Hospital, Zhejiang University School of Medicine, Hangzhou Municipal Women's Hospital and Jiaxing Maternal and Child Health Hospital during February 2015 to August 2018. The cell-free DNA was extracted from the peripheral blood sample for DNA library, and non-invasive prenatal testing (NIPT) was performed by high-throughput sequencing technique. The fetal karyotype analysis or neonatal karyotype analysis was performed in pregnant women with fetal chromosome aneuploidy, and all subjects were followed up. The efficiency of NIPS testing for twin aneuploidy was calculated. NIPS revealed chromosome abnormalities in 11 out of 2057 twin pregnant women, 9 cases were confirmed chromosome abnormalities, 2 cases were normal and no false negative cases. In this screening, the detection rate, sensitivity, specificity, positive predictive value, false positive rate of NIPS were 100.00%, 100.00%, 99.90%, 81.82%, 0.10%. Those were 100.00%, 100.00%, 99.95%, 87.50% and 0.05% for trisomy 21, 100.00%, 100.00%, 100.00%, 100.00%, 0.00% for trisomy18, and the specificity and false positive rate for trisomy13 were 99.95% and 0.05%, respectively. NIPS can detect fetal chromosomal aneuploidy rapidly and accurately in twin pregnancies,and it is of value in clinical application.

Clinical application of noninvasive prenatal screening for sex chromosome aneuploidies in 50,301 pregnancies: initial experience in a Chinese hospital.

To evaluate the clinical performance of noninvasive prenatal screening (NIPS) for fetal sex chromosome aneuploidies (SCAs), pregnant women were recruited in this retrospective observational study. The NIPS test was undertaken using high-throughput gene sequencing. In total,50,301 pregnant women were analysed for demographic characteristics and medical history. Of them, 308 women (0.61%) had high risk for fetal SCAs, including 138 for 45,X, 111 for 47,XXY, 42 for 47,XXX, and 17 for 47,XYY. After the pre-test counselling, 182 participants chose to undergo invasive prenatal diagnosis, confirming 59 positive cases. The combined positive predictive value of NIPS was 32.42% (59/182), 18.39% (16/87), 44.4% (12/27), 39.29% (22/56), and 75% (9/12) for detecting SCAs, 45,X, 47,XXX, 47,XXY, and 47,XYY, respectively. NIPS can be a useful method to detect the fetal SCAs using high-throughput gene sequencing, though accuracy can still be improved, especially for 45,X. Although the value of NIPS compare favorably with those seen in traditional screening approaches for SCAs, it is important to highlight the limitations of NIPS while educating clinicians and patients.

Noninvasive prenatal testing: the aspects of its introduction into clinical practice

The last couple of years have witnessed the rapid development of prenatal molecular-based screening for fetal aneuploidies that utilizes the analysis of cell-free DNA circulating in the bloodstream of a pregnant woman. The present review looks at the potential and limitations of such testing and the possible causes of false-positive and false-negative results. The review also describes the underlying principles of data acquisition and analysis the testing involves. In addition, we talk about the opinions held by the expert community and some aspects of legislation on the use of noninvasive prenatal testing (NIPT) in clinical practice in the countries where NIPT is much more widespread than in Russia.

Ten years of prenatal aneuploidy screening program with combined first-trimester test in a multicentric unit

Ten years of prenatal aneuploidy screening program with combined first-trimester test in a multicentric unit

Knowledge of Prenatal Screening, Down Syndrome, Amniocentesis, and Related Factors among Iranian Pregnant Women: A Cross-Sectional Study

ABSTRACTBackground:The primary goal of prenatal aneuploidy screening is the early detection of pregnancies at high risk of Down syndrome (DS). The present study aimed to assess the knowledge about the prenatal screening test (PST), DS, and amniotic fluid test (AFT) among Iranian pregnant women. Methods:The present cross-sectional study was conducted from April 2013 to July 2014 in Mostafa Khomeini Educational Hospital, Tehran, Iran. The participants were recruited among pregnant women referred to the Obstetrics and Gynecology Clinic of the hospital for their routine medical evaluations. A total of 471 consecutive women fulfilling the inclusion criteria were enrolled in the study. The data collection instruments included a 9-item socio-demographic information form and a 23-item knowledge questionnaire. The latter included 9 items on DS, 10 items on PST, and 4 items on AFT. The data were analyzed using the SPSS software (version 20.0) with the Kruskal-Wallis and Mann-Whitney tests. P<0.05 was considered statistically significant. Results:The mean score of total knowledge, PST, DS, and AFT was 9.93±4.86, 5.26±2.19, 3.25±2.42, and 1.41±1.39, respectively. There was a significant correlation between the mean score of total knowledge and the age of the mother (P=0.019), gestational age (P=0.001), educational level of the mother and spouse (P=0.001), history of birth abnormality (P=0.03), history of birth abnormality in the family (P=0.02), and sources of information (P=0.001). Conclusion: The results showed that the mean score of knowledge in pregnant women was less than half of the total scores, and total score was related to various parameters. Intervention factors that could contribute to improved knowledge and informed decision-making among pregnant women should be considered.

Canadian Pregnant Women's Preferences Regarding NIPT for Down Syndrome: The Information They Want, How They Want to Get It, and With Whom They Want to Discuss It.

This study sought to assess Canadian pregnant women's and their partners' preferences for information about non-invasive prenatal testing (NIPT). Pregnant women and their partners across Canada were surveyed as part of the Personalized Genomics for prenatal Aneuploidy Screening Using maternal blood (PEGASUS) study. A total of 882 pregnant women and 395 partners participated. Women preferred being informed by a physician (77.2%). They preferred getting information ahead of time, except for information about resources for families with Down syndrome, which they preferred getting with test results. More than half thought that written consent is important (63.7%) and could decide whether to do NIPT on the day they received the information (54.9%). Women preferred to be informed of results by telephone (43.7%) or in person (28%), but they preferred in person if they were considered at high risk for Down syndrome on the basis of the results (76%). The partner was the person whose input was considered most important (62.6%). Partners' preferences were similar, except that partners tended to want information later (at the time of the test or with the results) and felt that their opinion was not considered as highly by health professionals. Canadian women want information about NIPT early, in person, by a knowledgeable physician. Partners also want to be informed and involved in the decision-making process.

Fetal cardiac examination can affect patients' preference on invasive tests: A new data on maternal anxiety indicated karyotyping.

Background:Prenatal screening for aneuploidies has seen great changes over the last 2 decades. But there is still no non-invasive diagnostic test. Therefore, prenatal invasive procedures are still being routinely performed due to maternal anxiety. The association of cardiac anomalies and abnormal findings with aneuploidies has been known for a long time. This prospective study was done to evaluate abnormal fetal cardiac examination (FCE) findings on patients undergoing diagnostic invasive procedures due to maternal anxiety and to assess the predictive value of abnormal cardiac findings on abnormal karyotype.Materials and methods:Patients who underwent prenatal diagnostic invasive tests due to maternal anxiety indication between March 2013 and September 2016 were included in this study. FCE was performed in the study group immediately prior to invasive tests. Findings of fetal cardiac examination are classified as normal, major–minor cardiac anomalies and soft markers. Fetal karyotypes were compared among groups depending on cardiac findings.Results:One hundred eighty-two invasive procedures were performed because of maternal anxiety during this period. There were 29 abnormal findings detected on FCE. A total of 7 abnormal karyotypes were detected. FCE was abnormal in 5 of the abnormal karyotypes (71.4%). The presence of a major cardiac anomaly was most predictive for abnormal karyotype (LR+: 96,67, LR-: 0,34). No association was detected between the presence of minor cardiac anomalies and abnormal karyotype. Normal FCE appeared to be a good predictive factor for normal karyotype (LR-: 0.20).Conclusions:This is the first study evaluating the power of early fetal cardiac examination findings on fetal aneuploidies. This study suggested that the application of fetal cardiac examination findings to genetic counseling for screening aneuploidies may be efficient on patients’ preference about invasive tests. Due to the small number of abnormal findings and karyotypes detected (not the large study group), further studies on large study groups are needed to confirm these results.

Korean physicians’ attitudes toward the prenatal screening for fetal aneuploidy and implementation of non-invasive prenatal testing with cell-free fetal DNA

Korean physicians’ attitudes toward the prenatal screening for fetal aneuploidy and implementation of non-invasive prenatal testing with cell-free fetal DNA