BACKGROUND: Nonimmune hydrops fetalis (NIHF) presents as life-threatening fluid collections in multiple fetal compartments and may be led by numerous etiologies. AIM: To establish the diagnostic yield of exome sequencing for single-gene disorders in unexplained NIHF and to evaluate the clinical utility of data reanalysis. METHODS: A series of 53 unexplained cases of NIHF were enrolled, including 39 cases met a strict definition of NIHF and 14 cases with increased nuchal translucency (NT) and/or cystic hygroma in combination with other fluid collections. Trio ES from fetal samples and parental blood was performed, and clinical reports were returned by geneticists and genetic counselors. Multidisciplinary team forums were conducted for accurate diagnoses and improved patient management. The clinical follow-up assessments were conducted, and the reanalysis was performed for cases with a non-positive result. RESULTS: Diagnostic variants were identified in 22.6% (12/53) of the cases, and variants of potential clinical significance were detected in an additional 13.2% (7/53) of the cases. Of them, three possible diagnoses (3/41, 7.3%) were obtained during reanalysis. Notably, half of the diagnosed cases were from the group exhibiting only skin edema and increased NT and/or cystic hygroma. The diagnostic rate in this group was 42.8% (6/14), while in the classically defined NIHF group, the rate was 15.4% (6/39). The pregnancy termination and live birth rates of the cases with positive genetic testing results were found to be statistically significantly different from those with negative results (91.7% vs. 53.6% and 8.3% vs. 36.6%, P <
0.05 for both). CONCLUSION: ES provides high incremental diagnostic yield for NIHF after standard-of-care testing, and reevaluating non-diagnostic exomes in light of updated knowledge can maximize diagnostic yield. Identifying the etiology of NIHF facilitates prenatal diagnosis, improves the management of NIHF cases and predicts recurrence risk in future pregnancies.