Prenatal Cell-free DNA Screening in Pregnancies With Diverse Genetic Risk Profiles Utilizing Targeted and Whole-exome Sequencing

This is a prospective, multicenter clinical study evaluating the clinical validity and utility of a next-generation non-invasive prenatal screening platform, COATE-seq (Coordinative Allele-Aware Target Enrichment Sequencing). COATE-seq is a cfDNA-based approach that integrates hybrid capture panel sequencing with whole-exome sequencing (WES) from a single maternal plasma sample, enabling detection of a broad spectrum of fetal genetic conditions. These include common and rare aneuploidies, clinically significant microdeletions, monogenic pathogenic variants, uniparental disomy (UPD), and molar pregnancies.

The platform leverages coordinated allelic modeling and deep sequencing to enhance sensitivity for low-allelic-fraction variants, including those in autosomal recessive and dominant genes. The panel-based component targets 56 monogenic conditions selected using the SEPH framework (Severe, Early-onset, Prevalent, High detectability), while the WES component interrogates approximately 19,000 protein-coding genes to enable secondary and exploratory analyses of ultra-rare or novel variants. Variant interpretation adheres to ACMG/AMP guidelines, and only pathogenic or likely pathogenic variants associated with severe outcomes are reported.

The study is designed to benchmark the performance of this integrated assay against conventional diagnostic standards, including karyotyping, chromosomal microarray (CMA), targeted gene sequencing, whole-exome sequencing, and whole-genome sequencing. Reference testing is performed prenatally or postnatally, and in select cases, UPD is confirmed via methylation-specific PCR or short tandem repeat (STR) analysis.

Data collected across the study include:

Clinical history and prenatal imaging findings

cfDNA sequencing results from both targeted panels and exome-based assays

Confirmatory diagnostic results

Pregnancy outcomes and postnatal phenotypes, where available

Performance metrics including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) will be calculated in subjects with complete cfDNA and diagnostic data. Additional analyses will assess detection rates stratified by clinical indication (such as abnormal ultrasound findings), quantify the incremental yield of WES compared to panel-only testing, and evaluate the downstream clinical impact on decision-making and pregnancy management.

COATE-seq incorporates a rigorously validated bioinformatics pipeline for fetal fraction estimation, detection of allelic imbalance, chromosomal dosage, copy number variation (CNV) analysis, and pathogenic variant annotation. Quality assurance procedures are applied throughout sample processing, sequencing, and data interpretation, including safeguards during plasma handling, DNA extraction, target capture, library construction, and analytical review.

The study complies with Good Clinical Practice (GCP) and relevant regulatory and ethical guidelines. Institutional Review Board (IRB) approval is obtained at all participating sites, and informed consent is secured prior to enrollment. The results of this study will inform best practices for expanded cfDNA screening in prenatal care and contribute to the evidence base supporting the clinical integration of comprehensive molecular testing for fetal genetic conditions. Scientific findings will be disseminated through peer-reviewed publications and conference presentations.

Inclusion criteria

• Adult pregnant woman (≥18 years old)
• Gestational age between 9+0 and 25+6 weeks
• Singleton pregnancy
• Pregnancy with indications for prenatal diagnosis due to:

• Increased nuchal translucency (NT) ≥3.5 mm: capped at 25% of total subjects
• Increased NT ≥3.5 mm AND presence of any other "soft marker" or structural anomaly: capped at 25% of total subjects
• Presence of structural anomaly: at least 50% of total subjects
• Agree to participate in the clinical study for being followed-up and accept at least one molecular diagnosis (diagnostic procedures performed on prenatal invasive specimens, product of conception, umbilical cord blood, or other specimens) and possible family member testing

Exclusion criteria

• Age under 18 years
• Gestational age is less than 9+0 weeks or greater than 25+6 weeks
• One parent or other family member has a known pathogenic variant linked to the fetal ultrasound finding(s)
• Conditions affecting the accuracy of cfDNA assay (e.g., maternal malignancy during pregnancy, maternal allogeneic blood transfusion, organ transplantation, or cell therapy within the past year)