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2.
Viruses ; 13(8)2021 08 11.
Article in English | MEDLINE | ID: covidwho-1355047

ABSTRACT

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3'-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.


Subject(s)
COVID-19/blood , COVID-19/virology , Cell-Free Nucleic Acids/blood , MicroRNAs/blood , RNA, Viral/blood , SARS-CoV-2/genetics , COVID-19/complications , COVID-19/genetics , Female , Genome, Viral , High-Throughput Nucleotide Sequencing , Humans , Male , MicroRNAs/genetics , RNA, Viral/genetics , Severity of Illness Index , Viral Nonstructural Proteins/genetics
3.
Sci Rep ; 11(1): 15701, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1341012

ABSTRACT

Coronavirus disease 19 (COVID-19) presents with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels and evolution of the damage associated molecular patterns (DAMPS) cell free DNA (cfDNA), extracellular histone H3 (H3) and neutrophil elastase (NE), and the immune modulators GAS6 and AXL in relation to clinical parameters, ICU scoring systems and mortality in patients (n = 100) with severe COVID-19. cfDNA, H3, NE, GAS6 and AXL were increased in COVID-19 patients compared to controls. These measures associated with occurrence of clinical events and intensive care unit acquired weakness (ICUAW). cfDNA and GAS6 decreased in time in patients surviving to 30 days post ICU admission. A decrease of 27.2 ng/mL cfDNA during ICU stay associated with patient survival, whereas levels of GAS6 decreasing more than 4.0 ng/mL associated with survival. The presence of H3 in plasma was a common feature of COVID-19 patients, detected in 38% of the patients at ICU admission. NETosis markers cfDNA, H3 and NE correlated well with parameters of tissue damage and neutrophil counts. Furthermore, cfDNA correlated with lowest p/f ratio and a lowering in cfDNA was observed in patients with ventilator-free days.


Subject(s)
Biomarkers/blood , COVID-19/pathology , Aged , COVID-19/mortality , COVID-19/virology , Cell-Free Nucleic Acids/blood , Critical Illness , Female , Histones/analysis , Histones/blood , Humans , Intensive Care Units , Intercellular Signaling Peptides and Proteins/blood , Kaplan-Meier Estimate , Leukocyte Elastase/blood , Male , Middle Aged , Prognosis , SARS-CoV-2/isolation & purification
4.
J Extracell Vesicles ; 10(8): e12092, 2021 06.
Article in English | MEDLINE | ID: covidwho-1261767

ABSTRACT

The clinical manifestations of COVID-19 vary broadly, ranging from asymptomatic infection to acute respiratory failure and death. But the predictive biomarkers for characterizing the variability are still lacking. Since emerging evidence indicates that extracellular vesicles (EVs) and extracellular RNAs (exRNAs) are functionally involved in a number of pathological processes, we hypothesize that these extracellular components may be key determinants and/or predictors of COVID-19 severity. To test our hypothesis, we collected serum samples from 31 patients with mild COVID-19 symptoms at the time of their admission for discovery cohort. After symptomatic treatment without corticosteroids, 9 of the 31 patients developed severe/critical COVID-19 symptoms. We analyzed EV protein and exRNA profiles to look for correlations between these profiles and COVID-19 severity. Strikingly, we identified three distinct groups of markers (antiviral response-related EV proteins, coagulation-related markers, and liver damage-related exRNAs) with the potential to serve as early predictive biomarkers for COVID-19 severity. As the best predictive marker, EV COPB2 protein, a subunit of the Golgi coatomer complex, exhibited significantly higher abundance in patients remained mild than developed severe/critical COVID-19 and healthy controls in discovery cohort (AUC 1.00 (95% CI: 1.00-1.00)). The validation set included 40 COVID-19 patients and 39 healthy controls, and showed exactly the same trend between the three groups with excellent predictive value (AUC 0.85 (95% CI: 0.73-0.97)). These findings highlight the potential of EV COPB2 expression for patient stratification and for making early clinical decisions about strategies for COVID-19 therapy.


Subject(s)
COVID-19/blood , COVID-19/physiopathology , Cell-Free Nucleic Acids/blood , Coatomer Protein/blood , Extracellular Vesicles/chemistry , Biomarkers/blood , COVID-19/immunology , Humans , Retrospective Studies , SARS-CoV-2/physiology , Severity of Illness Index
5.
J Extracell Vesicles ; 10(8): e12092, 2021 06.
Article in English | MEDLINE | ID: covidwho-1258075

ABSTRACT

The clinical manifestations of COVID-19 vary broadly, ranging from asymptomatic infection to acute respiratory failure and death. But the predictive biomarkers for characterizing the variability are still lacking. Since emerging evidence indicates that extracellular vesicles (EVs) and extracellular RNAs (exRNAs) are functionally involved in a number of pathological processes, we hypothesize that these extracellular components may be key determinants and/or predictors of COVID-19 severity. To test our hypothesis, we collected serum samples from 31 patients with mild COVID-19 symptoms at the time of their admission for discovery cohort. After symptomatic treatment without corticosteroids, 9 of the 31 patients developed severe/critical COVID-19 symptoms. We analyzed EV protein and exRNA profiles to look for correlations between these profiles and COVID-19 severity. Strikingly, we identified three distinct groups of markers (antiviral response-related EV proteins, coagulation-related markers, and liver damage-related exRNAs) with the potential to serve as early predictive biomarkers for COVID-19 severity. As the best predictive marker, EV COPB2 protein, a subunit of the Golgi coatomer complex, exhibited significantly higher abundance in patients remained mild than developed severe/critical COVID-19 and healthy controls in discovery cohort (AUC 1.00 (95% CI: 1.00-1.00)). The validation set included 40 COVID-19 patients and 39 healthy controls, and showed exactly the same trend between the three groups with excellent predictive value (AUC 0.85 (95% CI: 0.73-0.97)). These findings highlight the potential of EV COPB2 expression for patient stratification and for making early clinical decisions about strategies for COVID-19 therapy.


Subject(s)
COVID-19/blood , COVID-19/physiopathology , Cell-Free Nucleic Acids/blood , Coatomer Protein/blood , Extracellular Vesicles/chemistry , Biomarkers/blood , COVID-19/immunology , Humans , Retrospective Studies , SARS-CoV-2/physiology , Severity of Illness Index
6.
JCI Insight ; 6(4)2021 02 22.
Article in English | MEDLINE | ID: covidwho-1150281

ABSTRACT

BackgroundMitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether circulating cell-free MT-DNA quantitation could be used to predict the risk of poor COVID-19 outcomes remains undetermined.MethodsWe measured circulating MT-DNA levels in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at hospital presentation. Our primary outcome was mortality. Intensive care unit (ICU) admission, intubation, vasopressor, and renal replacement therapy requirements were secondary outcomes. Multivariate regression analysis determined whether MT-DNA levels were independent of other reported COVID-19 risk factors. Receiver operating characteristic and area under the curve assessments were used to compare MT-DNA levels with established and emerging inflammatory markers of COVID-19.ResultsCirculating MT-DNA levels were highly elevated in patients who eventually died or required ICU admission, intubation, vasopressor use, or renal replacement therapy. Multivariate regression revealed that high circulating MT-DNA was an independent risk factor for these outcomes after adjusting for age, sex, and comorbidities. We also found that circulating MT-DNA levels had a similar or superior area under the curve when compared against clinically established measures of inflammation and emerging markers currently of interest as investigational targets for COVID-19 therapy.ConclusionThese results show that high circulating MT-DNA levels are a potential early indicator for poor COVID-19 outcomes.FundingWashington University Institute of Clinical Translational Sciences COVID-19 Research Program and Washington University Institute of Clinical Translational Sciences (ICTS) NIH grant UL1TR002345.


Subject(s)
COVID-19/diagnosis , Cell-Free Nucleic Acids/blood , DNA, Mitochondrial/blood , Severity of Illness Index , Aged , Aged, 80 and over , Biomarkers/blood , COVID-19/mortality , COVID-19/therapy , COVID-19/virology , Female , Follow-Up Studies , Hospital Mortality , Humans , Intensive Care Units/statistics & numerical data , Male , Middle Aged , Prospective Studies , ROC Curve , Renal Replacement Therapy/statistics & numerical data , Respiration, Artificial/statistics & numerical data , Risk Factors , SARS-CoV-2/isolation & purification , Vasoconstrictor Agents/therapeutic use
8.
JCI Insight ; 6(7)2021 04 08.
Article in English | MEDLINE | ID: covidwho-1112383

ABSTRACT

INTRODUCTIONThe clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODSWe conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTSWe found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSIONcfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19-induced tissue injury.FUNDINGIntramural Targeted Anti-COVID-19 grant, NIH.


Subject(s)
COVID-19 , Cell-Free Nucleic Acids , Multiple Organ Failure , Organ Specificity/genetics , SARS-CoV-2 , Biomarkers/analysis , Biomarkers/blood , COVID-19/blood , COVID-19/complications , COVID-19/diagnosis , COVID-19/mortality , Cell-Free Nucleic Acids/analysis , Cell-Free Nucleic Acids/blood , Cohort Studies , DNA Methylation , Female , Humans , Male , Middle Aged , Multiple Organ Failure/blood , Multiple Organ Failure/diagnosis , Multiple Organ Failure/etiology , Outcome Assessment, Health Care , Prognosis , Prospective Studies , Reproducibility of Results , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity , Severity of Illness Index , United States/epidemiology
10.
J Clin Invest ; 131(7)2021 04 01.
Article in English | MEDLINE | ID: covidwho-1076050

ABSTRACT

BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.


Subject(s)
COVID-19/blood , COVID-19/virology , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/genetics , RNA, Viral/blood , RNA, Viral/genetics , SARS-CoV-2 , Adolescent , Adult , Aged , Animals , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/statistics & numerical data , CRISPR-Cas Systems , Child , Child, Preschool , Disease Models, Animal , Female , Humans , Infant , Longitudinal Studies , Macaca mulatta , Male , Middle Aged , Pandemics , SARS-CoV-2/genetics , Sensitivity and Specificity , Time Factors
12.
Transplant Proc ; 53(4): 1202-1206, 2021 May.
Article in English | MEDLINE | ID: covidwho-1014862

ABSTRACT

Kidney transplant recipients who develop coronavirus disease 2019 (COVID-19) are at increased risk of life-threatening illness, which often requires reducing immunosuppression despite the potential risk of causing an allograft rejection. Herein, we describe the clinical presentation and course of a kidney transplant recipient who acquired COVID-19 and was hospitalized with severe symptoms and hypoxemia. Upon admission, the patient was found to have elevated de novo donor-specific antibodies (DSA) yielding a positive cytotoxicity crossmatch and concurrent elevated plasma donor-derived cell-free DNA (dd-cfDNA) level, indicating a possible ongoing rejection despite improvement in his serum creatinine. Because of persistent positive COVID-19 tests and stable serum creatinine, a kidney allograft biopsy was initially deferred and his dd-cfDNA and DSA were monitored closely postdischarge. Three months later, because of persistent elevated dd-cfDNA and positive DSA, a kidney allograft biopsy was performed, which showed chronic active antibody-mediated rejection. Accordingly, the patient was treated with intravenous immunoglobulin and his maintenance immunosuppressive regimen was increased.


Subject(s)
COVID-19/diagnosis , Graft Rejection/prevention & control , Kidney Transplantation , Antibodies/blood , Antibodies/immunology , COVID-19/complications , COVID-19/virology , Cell-Free Nucleic Acids/blood , Creatinine/blood , Graft Rejection/diagnosis , HLA-DR7 Antigen/immunology , Humans , Immunoglobulins, Intravenous/therapeutic use , Immunosuppressive Agents/blood , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Oxygen Inhalation Therapy , SARS-CoV-2/isolation & purification , Tacrolimus/blood , Tacrolimus/therapeutic use
13.
Cells ; 9(12)2020 12 12.
Article in English | MEDLINE | ID: covidwho-971834

ABSTRACT

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to an adaptive immune response in the host and the formation of anti-SARS-CoV-2 specific antibodies. While IgG responses against SARS-CoV-2 have been characterized quite well, less is known about IgA. IgA2 activates immune cells and induces inflammation and neutrophil extracellular trap (NET) formation which may contribute to organ injury and fatal outcome in SARS-CoV-2-infected patients. SARS-CoV-2 spike protein specific antibody levels were measured in plasma samples of 15 noninfected controls and 82 SARS-CoV-2-infected patients with no or mild symptoms, moderate symptoms (hospitalization) or severe disease (intensive care unit, ICU). Antibody levels were compared to levels of C-reactive protein (CRP) and circulating extracellular DNA (ecDNA) as markers for general inflammation and NET formation, respectively. While levels of SARS-CoV-2-specific IgG were similar in all patient groups, IgA2 antibodies were restricted to severe disease and showed the strongest discrimination between nonfatal and fatal outcome in patients with severe SARS-CoV-2 infection. While anti-SARS-CoV-2 IgG and IgA2 levels correlated with CRP levels in severely diseased patients, only anti-SARS-CoV-2 IgA2 correlated with ecDNA. These data suggest that the formation of anti-SARS-CoV-2 IgA2 during SARS-CoV-2 infection is a marker for more severe disease related to NET formation and poor outcome.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Extracellular Traps/immunology , Immunoglobulin A/blood , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Aged, 80 and over , Biomarkers/blood , C-Reactive Protein/immunology , COVID-19/epidemiology , Case-Control Studies , Cell-Free Nucleic Acids/blood , Female , Humans , Male , Middle Aged , SARS-CoV-2 , Severity of Illness Index , Young Adult
14.
Discov Med ; 29(157): 129-137, 2020.
Article in English | MEDLINE | ID: covidwho-812954

ABSTRACT

Sepsis is a life-threatening clinical condition demanding accurate and rapid diagnosis of the culprit pathogen, thereby to improve prognosis. Pathogen determination through blood culture is the gold standard for diagnosis but has limitations due to low sensitivity. Recently, circulating DNAs derived from pathogenic organisms were found in the plasma of patients with sepsis and were further proved to be more sensitive biomarkers for the diagnosis of the pathogen origin in sepsis. However, the fundamental molecular characteristics of circulating DNA in patients with sepsis remain unclear. Here, we used specific PCR and Sanger sequencing to verify the microbiology culture results via the corresponding plasma circulating DNA. We analyzed the composition and molecular characteristics of circulating DNA in septic patients using next-generation sequencing technology. We showed the presence of pathogen-derived circulating DNA in the plasma of patients with sepsis. The sizes of circulating DNA fragments derived from pathogenic bacteria showed a skewed unimodal distribution, while those derived from host cells showed a normal unimodal distribution. Lengths of fragments at peak concentration for both origins ranged from 150 bp to 200 bp, and reads mapping to pathogenic bacteria genome distributed uniformly on the reference. Our findings have improved our understanding of microbial circulating DNA in patients with sepsis as a potential methodology for the accurate diagnosis of sepsis, especially in light of an urgent need for such a diagnosis associated with the COVID-19 infection.


Subject(s)
Bacterial Infections/microbiology , Cell-Free Nucleic Acids/blood , DNA, Bacterial/blood , Sepsis/microbiology , Adult , Aged , Bacterial Infections/complications , Bacterial Infections/diagnosis , Betacoronavirus , COVID-19 , COVID-19 Testing , Cell-Free Nucleic Acids/analysis , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Culture Techniques , DNA, Bacterial/analysis , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Neoplasms/complications , Pandemics , Pneumonia, Viral , Polymerase Chain Reaction , SARS-CoV-2 , Sepsis/complications , Sepsis/diagnosis , Sequence Analysis, DNA
15.
Mol Diagn Ther ; 24(3): 251-262, 2020 06.
Article in English | MEDLINE | ID: covidwho-634822

ABSTRACT

This opinion article discusses the increasing attention paid to the role of activating damage-associated molecular patterns (DAMPs) in initiation of inflammatory diseases and suppressing/inhibiting DAMPs (SAMPs) in resolution of inflammatory diseases and, consequently, to the future roles of these novel biomarkers as therapeutic targets and therapeutics. Since controlled production of DAMPs and SAMPs is needed to achieve full homeostatic restoration and repair from tissue injury, only their pathological, not their homeostatic, concentrations should be therapeutically tackled. Therefore, distinct caveats are proposed regarding choosing DAMPs and SAMPs for therapeutic purposes. For example, we discuss the need to a priori identify and define a context-dependent "homeostatic DAMP:SAMP ratio" in each case and a "homeostatic window" of DAMP and SAMP concentrations to guarantee a safe treatment modality to patients. Finally, a few clinical examples of how DAMPs and SAMPs might be used as therapeutic targets or therapeutics in the future are discussed, including inhibition of DAMPs in hyperinflammatory processes (e.g., systemic inflammatory response syndrome, as currently observed in Covid-19), administration of SAMPs in chronic inflammatory diseases, inhibition of SAMPs in hyperresolving processes (e.g., compensatory anti-inflammatory response syndrome), and administration/induction of DAMPs in vaccination procedures and anti-cancer therapy.


Subject(s)
Inflammation/drug therapy , Inflammation/metabolism , Molecular Targeted Therapy/methods , Biomarkers/blood , COVID-19/drug therapy , Cell-Free Nucleic Acids/blood , Chronic Disease , Coronavirus Infections/drug therapy , HMGB1 Protein/blood , Homeostasis , Humans , Immunity, Innate/drug effects , Immunity, Innate/physiology , Pathogen-Associated Molecular Pattern Molecules/metabolism , S100 Proteins/blood , Vaccination
18.
Prenat Diagn ; 40(10): 1265-1271, 2020 09.
Article in English | MEDLINE | ID: covidwho-343193

ABSTRACT

OBJECTIVE: Advances in prenatal genetics place additional challenges as patients must receive information about a growing array of screening and testing options. This raises concerns about how to achieve a shared decision-making process that prepares patients to make an informed decision about their choices about prenatal genetic screening and testing options, calling for a reconsideration of how healthcare providers approach the first prenatal visit. METHODS: We conducted interviews with 40 pregnant women to identify components of decision-making regarding prenatal genetic screens and tests at this visit. Analysis was approached using grounded theory. RESULTS: Participants brought distinct notions of risk to the visit, including skewed perceptions of baseline risk for a fetal genetic condition and the implications of screening and testing. Participants were very concerned about financial considerations associated with these options, ranking out-of-pocket costs on par with medical considerations. Participants noted diverging priorities at the first visit from those of their healthcare provider, leading to barriers to shared decision-making regarding screening and testing during this visit. CONCLUSION: Research is needed to determine how to restructure the initiation of prenatal care in a way that best positions patients to make informed decisions about prenatal genetic screens and tests.


Subject(s)
Decision Making , Genetic Testing , Prenatal Care , Adult , Attitude to Health , Cell-Free Nucleic Acids/analysis , Cell-Free Nucleic Acids/blood , Female , Genetic Testing/economics , Genetic Testing/methods , Genetic Testing/standards , Humans , Mass Screening/economics , Mass Screening/organization & administration , Mass Screening/psychology , Mass Screening/standards , Maternal Serum Screening Tests/economics , Maternal Serum Screening Tests/psychology , Maternal Serum Screening Tests/standards , Office Visits/economics , Patient Participation/psychology , Patient Participation/statistics & numerical data , Perception , Pregnancy , Prenatal Care/economics , Prenatal Care/organization & administration , Prenatal Care/psychology , Prenatal Care/standards , Prenatal Diagnosis/economics , Prenatal Diagnosis/methods , Prenatal Diagnosis/psychology , Prenatal Diagnosis/standards , Risk Assessment , United States
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