The Hidden Fetal Genome after Negative Exome Sequencing: Incremental Diagnostic and Clinical Utility of Short-read and Long-read Genome Sequencing in Fetuses with Structural Anomalies – A Systematic Review

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BSTRACT

Despite major advances in prenatal exome sequencing (ES), many fetuses with structural anomalies still do not receive a molecular diagnosis. Growing evidence suggests that these unresolved cases may contain pathogenic variants within the “hidden fetal genome,” including complex structural rearrangements, repeat expansions, deep intronic and regulatory variants, balanced chromosomal abnormalities, mobile element insertions, and other genomic changes that conventional ES often misses. This systematic review examined the additional diagnostic and clinical value of short read and long read whole genome sequencing (WGS) after nondiagnostic prenatal ES in fetuses with structural anomalies. A systematic literature search was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. Eligible publications included original research, systematic reviews, and implementation studies assessing prenatal genome sequencing after standard cytogenetic and exome-based testing. Twenty-nine studies met the predefined eligibility criteria and were included in the qualitative synthesis. Overall, the evidence shows that short read WGS expands genomic analysis beyond coding regions and improves detection of pathogenic copy number variants, noncoding variants, and selected structural alterations. Long read sequencing provides further advantages by identifying complex genomic architecture, repeat expansions, balanced rearrangements, phased alleles, and difficult-to-map genomic regions that often remain undetected with short read methods. In addition to increasing diagnostic yield, genome sequencing supports prenatal counseling, pregnancy management, delivery planning, neonatal care, and recurrence risk assessment, demonstrating its growing importance in maternal fetal medicine. However, challenges involving variant interpretation, limited prenatal phenotyping, cost, infrastructure, and equitable access continue to limit broader clinical adoption. Across the included studies, incremental diagnostic yield varied according to fetal phenotype and sequencing strategy but consistently favored genome sequencing over ES for selected unresolved cases. Combining complementary genome sequencing approaches within multidisciplinary prenatal diagnostic pathways may reduce the hidden fetal genome and strengthen precision prenatal genomics, although prospective multicenter studies remain necessary to establish the most effective strategies for clinical implementation.