Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling.

OBJECTIVE: To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection. METHODS: Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow. RESULTS: An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell. CONCLUSION: Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.

Cell-free DNA analysis for noninvasive examination of trisomy.

BACKGROUND: Cell-free DNA (cfDNA) testing for fetal trisomy is highly effective among high-risk women. However, there have been few direct, well-powered studies comparing cfDNA testing with standard screening during the first trimester in routine prenatal populations. METHODS: In this prospective, multicenter, blinded study conducted at 35 international centers, we assigned pregnant women presenting for aneuploidy screening at 10 to 14 weeks of gestation to undergo both standard screening (with measurement of nuchal translucency and biochemical analytes) and cfDNA testing. Participants received the results of standard screening; the results of cfDNA testing were blinded. Determination of the birth outcome was based on diagnostic genetic testing or newborn examination. The primary outcome was the area under the receiver-operating-characteristic curve (AUC) for trisomy 21 (Down's syndrome) with cfDNA testing versus standard screening. We also evaluated cfDNA testing and standard screening to assess the risk of trisomies 18 and 13. RESULTS: Of 18,955 women who were enrolled, results from 15,841 were available for analysis. The mean maternal age was 30.7 years, and the mean gestational age at testing was 12.5 weeks. The AUC for trisomy 21 was 0.999 for cfDNA testing and 0.958 for standard screening (P=0.001). Trisomy 21 was detected in 38 of 38 women (100%; 95% confidence interval [CI], 90.7 to 100) in the cfDNA-testing group, as compared with 30 of 38 women (78.9%; 95% CI, 62.7 to 90.4) in the standard-screening group (P=0.008). False positive rates were 0.06% (95% CI, 0.03 to 0.11) in the cfDNA group and 5.4% (95% CI, 5.1 to 5.8) in the standard-screening group (P<0.001). The positive predictive value for cfDNA testing was 80.9% (95% CI, 66.7 to 90.9), as compared with 3.4% (95% CI, 2.3 to 4.8) for standard screening (P<0.001). CONCLUSIONS: In this large, routine prenatal-screening population, cfDNA testing for trisomy 21 had higher sensitivity, a lower false positive rate, and higher positive predictive value than did standard screening with the measurement of nuchal translucency and biochemical analytes. (Funded by Ariosa Diagnostics and Perinatal Quality Foundation; NEXT ClinicalTrials.gov number, NCT01511458.).

Clinical performance of non-invasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies.

OBJECTIVE: To evaluate the clinical performance of non-invasive prenatal testing for trisomy 21, 18, and 13 using targeted cell-free DNA (cfDNA) analysis. METHODS: Targeted cfDNA analysis using DANSR™ and FORTE™ with microarray quantitation was used to evaluate the risk of trisomy 21, 18, and 13 in blinded samples from 799 singleton, twin, natural, and IVF pregnancies. Subjects either had fetal chromosome evaluation by karyotype, FISH, QF-PCR, or karyotype for newborns with suspected aneuploidy at birth. The results of targeted cfDNA analysis were compared to clinical genetic testing outcomes to assess clinical performance. RESULTS: Targeted cfDNA analysis with microarray quantification identified 107/108 trisomy 21 cases (99.1%), 29/30 trisomy 18 cases (96.7%), and 12/12 trisomy 13 cases (100%). The specificity was 100% for all three trisomies. Combining this data with all published clinical performance studies using DANSR/FORTE methodology for greater than 23 000 pregnancies, the sensitivity of targeted cfDNA analysis was calculated to be greater than 99% for trisomy 21, 97% for trisomy 18, and 94% for trisomy 13. Specificity for each trisomy was greater than 99.9%. CONCLUSION: Targeted cfDNA analysis demonstrates consistently high sensitivity and extremely low false positive rates for common autosomal trisomies in pregnancy across quantitation platforms.

Assessment of fetal sex chromosome aneuploidy using directed cell-free DNA analysis.

OBJECTIVE: To examine the performance of chromosome-selective sequencing of cell-free (cf) DNA in maternal blood for assessment of fetal sex chromosome aneuploidies. METHODS: This was a case-control study of 177 stored maternal plasma samples, obtained before fetal karyotyping at 11-13 weeks of gestation, from 59 singleton pregnancies with fetal sex chromosome aneuploidies (45,X, n = 49; 47,XXX, n = 6; 47,XXY, n = 1; 47,XYY, n = 3) and 118 with euploid fetuses (46,XY, n = 59; 46,XX, n = 59). Digital analysis of selected regions (DANSR™) on chromosomes 21, 18, 13, X and Y was performed and the fetal-fraction optimized risk of trisomy evaluation (FORTE™) algorithm was used to estimate the risk for non-disomic genotypes. Performance was calculated at a risk cut-off of 1:100. RESULTS: Analysis of cfDNA provided risk scores for 172 (97.2%) samples; 4 samples (45,X, n = 2; 46,XY, n = 1; 46,XX, n = 1) had an insufficient fetal cfDNA fraction for reliable testing and 1 case (47,XXX) failed laboratory quality control metrics. The classification was correct in 43 (91.5%) of 47 cases of 45,X, all 5 of 47,XXX, 1 of 47,XXY and 3 of 47,XYY. There were no false-positive results for monosomy X. DISCUSSION: Analysis of cfDNA by chromosome-selective sequencing can correctly classify fetal sex chromosome aneuploidy with reasonably high sensitivity.

Clinical experience of noninvasive prenatal testing with cell-free DNA for fetal trisomies 21, 18, and 13, in a general screening population.

OBJECTIVE: Evaluate noninvasive prenatal testing (NIPT) with cell-free DNA as a screening method for trisomies 21, 18, and 13 in an obstetrical clinical practice setting. METHODS: Observational study of pregnant women who underwent prenatal screening for fetal trisomy from 30 July 2012 to 1 December 2012. NIPT was offered to all patients in addition to first trimester combined screening (FTS). RESULTS: The cohort included 289 women with mean age of 32.3 years (range: 17.8-42.0) who underwent testing at 13.0 gestational age weeks (range: 10.1-20.7). NIPT results were provided for 98.6% of patients at a mean reporting time of 9.3 calendar days. With NIPT, all patients had a risk less than 1:10 000 for trisomy 21, 18, or 13. With FTS, 4.5% of patients had screening results indicating an increased risk for trisomy 21. One patient who had an elevated trisomy 21 risk with FTS elected to have an amniocentesis, which revealed a euploid fetus. CONCLUSIONS: NIPT has the potential to be a highly effective screening method as a standard test for risk assessment of fetal trisomies 21, 18, and 13 in general pregnant populations.

Non-invasive prenatal testing with cell-free DNA: US physician attitudes toward implementation in clinical practice.

OBJECTIVE: The aim of this study was to assess awareness, potential adoption, and current utilization of non-invasive prenatal testing (NIPT) analysis for common fetal aneuploidies among obstetricians. METHODS: A 36-item web-based survey was designed to assess the current practice of fetal aneuploidy screening and knowledge and utilization of NIPT for fetal trisomy. Practicing obstetricians in the United States were invited via email to participate in the survey. RESULTS: Of the 101 obstetricians that completed the survey (27% academic-based, 73% private practice), 97% offer screening to high-risk patients and 91% offer screening to average-risk patients. With regard to current screening tests, the top three advantages were as follows: recommendation by professional societies, no risk to the pregnancy, and long history/experience with the test, whereas the top three limitations were as follows: patient anxiety, risks of follow-up invasive testing, and high false positives. NIPT had been used by 32% of respondents and 22% were familiar with NIPT and the associated clinical data. The majority of physicians predicted that they would offer NIPT to high-risk women (86.1%) and average-risk women (76.2%) within 12 months. CONCLUSION: Obstetricians plan to increase their utilization of NIPT and expect that the majority of both high-risk and average-risk patients will be offered NIPT as an option.

COSMOS: a platform for real-time morphology-based, label-free cell sorting using deep learning.

Cells are the singular building blocks of life, and a comprehensive understanding of morphology, among other properties, is crucial to the assessment of underlying heterogeneity. We developed Computational Sorting and Mapping of Single Cells (COSMOS), a platform based on Artificial Intelligence (AI) and microfluidics to characterize and sort single cells based on real-time deep learning interpretation of high-resolution brightfield images. Supervised deep learning models were applied to characterize and sort cell lines and dissociated primary tissue based on high-dimensional embedding vectors of morphology without the need for biomarker labels and stains/dyes. We demonstrate COSMOS capabilities with multiple human cell lines and tissue samples. These early results suggest that our neural networks embedding space can capture and recapitulate deep visual characteristics and can be used to efficiently purify unlabeled viable cells with desired morphological traits. Our approach resolves a technical gap in the ability to perform real-time deep learning assessment and sorting of cells based on high-resolution brightfield images.

Complications of term pregnancies beyond 37 weeks of gestation.

OBJECTIVE: To estimate when rates of pregnancy complications increase beyond 37 weeks of gestation. METHODS: We designed a retrospective, cohort study of all women delivered beyond 37 weeks of gestational age from 1992 to 2002 at a single community hospital. Rates of perinatal complications by gestational age were analyzed with both bivariate and multivariable analyses. Statistical significance was designated by P <.05. RESULTS: Among the 45673 women who delivered at 37 completed weeks and beyond, the rates of meconium and macrosomia increased beyond 38 weeks of gestation (P <.001), the rates of operative vaginal delivery, chorioamnionitis, and endomyometritis all increased beyond 40 weeks of gestation (P <.001), and rates of intrauterine fetal death and cesarean delivery increased beyond 41 weeks of gestation (P <.001). CONCLUSION: Risks to both mother and infant increase as pregnancy progresses beyond 40 weeks of gestation.

Clinical utility and cost of non-invasive prenatal testing with cfDNA analysis in high-risk women based on a US population.

OBJECTIVE: Evaluate the clinical and economic consequences of fetal trisomy 21 (T21) screening with non-invasive prenatal testing (NIPT) in high-risk pregnant women. METHODS: Using a decision-analytic model, we estimated the number of T21 cases detected, the number of invasive procedures performed, corresponding euploid fetal losses and total costs for three screening strategies: first trimester combined screening (FTS), integrated screening (INT) or NIPT, whereby NIPT was performed in high-risk patients (women 35 years or older or women with a positive conventional screening test). Modeling was based on a 4 million pregnant women cohort in the US. RESULTS: NIPT, at a base case price of $795, was more clinically effective and less costly (dominant) over both FTS and INT. NIPT detected 4823 T21 cases based on 5330 invasive procedures. FTS detected 3364 T21 cases based on 108 364 procedures and INT detected 3760 cases based on 108 760 procedures. NIPT detected 28% and 43% more T21 cases compared to INT and FTS, respectively, while reducing invasive procedures by >95% and reducing euploid fetal losses by >99%. Total costs were $3786M with FTS, $3919M with INT and $3403M with NIPT. CONCLUSIONS: NIPT leads to improved T21 detection and reduction in euploid fetal loss at lower total healthcare expenditures.

The fetal fraction of cell-free DNA in maternal plasma is not affected by a priori risk of fetal trisomy.

OBJECTIVE: To determine the relationship between a priori risk for fetal trisomy and the fraction of fetal cell-free DNA (cfDNA) in maternal blood. METHODS: A comparative analysis on fetal cfDNA amounts was performed in subjects stratified into a priori risk groups based on maternal age, prenatal screening results, or nuchal translucency measurement. RESULTS: Across the highest and lowest deciles within each group, there were no significant differences in the fetal cfDNA fraction. CONCLUSIONS: These data support the concept that non-invasive prenatal test performance as determined by fetal cfDNA fraction is not predicted to be different based on patient risk classification.

Prenatal carrier testing for fragile X: counseling issues and challenges.

Healthy women who carry a ''premutation'' in the FMR1 gene (or fragile X mental retardation protein) can pass on a further mutated copy of FMR1 to either male or female offspring, leading to fragile X syndrome (FXS). Premutation carriers do not have manifestations of FXS in cognitive deficits, behavioral abnormalities, or classic physical features, but are at increased risk for development of the ''fragile X-associated disorders'': premature ovarian insufficiency and fragile X-associated tremor and ataxia syndrome. When considering widespread prenatal carrier screening programs for fragile X, significant resources must be available for at-risk individuals, including counseling, accurate diagnostic options for fetal testing, and choice regarding continuation of a pregnancy. Further attention is needed to develop and utilize inexpensive screening tests with adequate sensitivity and specificity to reduce barriers to screening for the population. Recently newer methodologies for high-throughput and inexpensive screening assays, which correctly detect expanded alleles in premutation and full mutation patients with a high degree of sensitivity, show significant promise for reduction in cost with rapid turn around times. With the introduction of widespread screening, individuals will be made aware not only of their risk for offspring with FXS, but will also have knowledge of the potential risk to develop the adult-onset conditions- FXPOI and FXTAS. This introduces more complex counseling challenges. All individuals identified as carriers of intermediate or premutation alleles should be referred for genetic counseling to properly convey risks for allele expansion and to discuss possible future risks of fragile X-associated disease.

Nuchal translucency screening: how do women actually utilize the results?

OBJECTIVE: To examine how women use the nuchal translucency (NT) risk adjustment in decision-making for invasive prenatal diagnosis. STUDY DESIGN: Retrospective cohort study of 1083 consecutive NT screening exams. A screen-positive test was defined as a risk > or = 1/300. Primary outcome was what proportion of screen-positive or screen-negative women chose to undergo chorionic villus sampling or amniocentesis. RESULTS: Of the women tested, 79% (858/1083) were > or = 35-years-old and 88% (756/858) of these women had a decrease in age-related risk after NT. Of the screen-negative women, 31% (238/756) > or = 35 years of age chose to have invasive testing as compared to only 11.2% (25/223) of those < 35 years of age (p < 0.001). Of the screen-positive women, 71% (72/102) > or = 35 years of age and 100% (2/2) < 35 years of age chose to obtain invasive testing (p = 0.368). CONCLUSION: First-trimester NT screening for Down syndrome (DS) enables a significant number of women over age 35 to lower the risk for DS several fold and avoid the risks of invasive testing. However, despite significant reductions in age-specific mid-trimester DS risks, a relatively high proportion of women > or = 35 years of age still opted for invasive testing.

Attitudes toward prenatal screening and testing for Fragile X.

PURPOSE: Currently, the American Colleges of Medical Genetics and Obstetrics and Gynecology recommend screening in the prenatal setting only for individuals with specific family history indicators. Our aims were to study patient attitudes and psychologic impact of offering widespread screening for Fragile X in a prenatal setting. METHODS: Participants were recruited from pregnant women referred for "Prenatal Diagnosis Options" counseling by their primary provider in the first trimester of pregnancy. RESULTS: Pretest knowledge about Fragile X was limited; 33% had heard of Fragile X syndrome before enrollment. Postcounseling knowledge was similarly limited; only 30% accurately understood the 50% risk for girls. Participants were strongly in favor of being tested or screened, and did not experience undue anxiety related to Fragile X testing. Respondents hoped that knowledge of Fragile X in the general population would increase, and recommended that screening be offered during routine prenatal care. CONCLUSION: Fragile X screening in this setting was a favorable testing experience for the participants. Limited pretest knowledge and posttest retention of specific genetic information on Fragile X suggest that widespread screening will pose significant counseling and educational challenges, which should be addressed in such programs.

Screening for single gene genetic disease.

The screening and directed testing for genetic disease caused by single gene mutations is an expanding part of the overall scheme of prenatal care. In addition to reproductive choice, carrier screening and fetal diagnostic testing afford the important opportunity for preparation of the family and the delivery site for the birth of a fetus with a known genetic disorder. Increasingly the primary care provider in pregnancy bears the burden of engaging patients in discussions regarding available genetic tests appropriate to their family or personal history, their ethnic group, and with every patient for a limited but growing number of diseases. Ethnic-based risk identification and testing has expanded recently with, for example, the addition of familial dysautonomia for patients of Askhenazi ancestry. Widespread, or nearly universal, screening has emerged for cystic fibrosis and new initiatives are gaining momentum for prenatal maternal carrier screening for fragile X syndrome. The fruits of the human genome project will undoubtedly lead to the identification of more genes that underlie human disease. This will expand the menu of possible prenatal testing options and will raise the level of complexity in both counseling, testing logisitics and health care resource allocation.

Cost-effectiveness analysis of prenatal population-based fragile X carrier screening.

OBJECTIVE: To investigate the cost-effectiveness of a widespread prenatal population-based fragile X carrier screening program. STUDY DESIGN: A decision tree was designed comparing screening versus not screening for the fragile X mental retardation protein 1 premutation in all pregnant women. Baseline values included a prevalence of fragile X mental retardation protein 1 premutations of 3.3 per 1000, a premutation expansion rate of 11.3%, and a 99% sensitivity of the screening test. The cost of the screening test was varied from 75 US dollars to 300 US dollars. A sensitivity analysis of the probabilities, utilities, and costs was performed. RESULTS: The screening strategy would lead to the identification of 80% of the fetuses affected by fragile X annually. Assuming the cost of 95 US dollars per test and only one child, the program would be cost effective at 14,858 US dollars per quality-adjusted life-year. The screening strategy remained cost effective up to 140 US dollars per test and 1 child per woman or for 2 children per woman up to a cost of 281 US dollars per test. CONCLUSION: Population-based screening for the fragile X premutation may be both clinically desirable and cost effective. Prospective pilot studies of this screening modality are needed in the prenatal setting.

Meconium-stained amniotic fluid is associated with puerperal infections.

OBJECTIVE: The purpose of this study was to determine whether meconium-stained amniotic fluid is associated with puerperal infection and whether the quality of the meconium is further associated with this risk. STUDY DESIGN: We designed a retrospective cohort study of all deliveries beyond 37 weeks gestational age from 1992 to 2002 at a single community hospital. Data were collected on rates of chorioamnionitis, endomyometritis, quality of amniotic fluid, and length of labor and analyzed with bivariate and multivariate analyses. RESULTS: We found that, among the 43,200 women who were delivered at term, 18.9% of the women had meconium staining (8.8% thin, 5.5% moderate, 4.6% thick). Compared with deliveries with clear amniotic fluid, those with meconium-stained amniotic fluid had higher rates of chorioamnionitis (2.3% vs 4.1%, P<.001) and endomyometritis (1.0% vs 1.7%, P<.001). Further, the severity of meconium staining was associated with increased rates of infection. CONCLUSION: We found that the presence and severity of meconium-stained amniotic fluid is associated with puerperal infection even when being controlled for confounders.

Rapid prenatal diagnosis in translocation carriers by interphase FISH with chromosome-specific subtelomere probes.

Interphase fluorescence in situ hybridization (FISH) analysis can provide rapid preliminary analysis of chromosome aneuploidy from direct amniocyte and chorionic villus sample (CVS) preparations. Typically, interphase FISH is used in screening for numerical abnormalities of chromosomes X, Y, 13, 18, and 21. More recently, FISH probe sets became available for the subtelomeric region of each chromosome, allowing screening for terminal chromosome rearrangements. The purpose of the current study was to evaluate the use of dual-color interphase FISH analysis with chromosome-specific subtelomere probes for rapid prenatal diagnosis in 14 pregnancies from 12 different translocation carriers. Interphase FISH analysis was performed on direct CVS or amniocyte preparations from 12 reciprocal translocation and two Robertsonian translocation pregnancies with the appropriate chromosome-specific subtelomere probes for each chromosome involved in the translocation. Analysis of the interphase FISH probe signals predicted balanced or normal segregants in each case, thus rapidly excluding a chromosomally unbalanced segregant. Subsequent metaphase analysis showed normal karyotypes in seven fetuses and balanced translocations in the remaining seven. This series illustrates the utility of interphase FISH analysis with chromosome-specific subtelomere probes for rapid prenatal diagnosis in cases of parental reciprocal translocations and Robertsonian translocations.