BACKGROUND: Next-generation sequencing for copy number vari-ants is often used as a follow-up investigation of unusual fetal ultrasound results and is capable of detecting copy number variations with a reso-lution of w0.1 Mb. In a prenatal setting, observation and subsequent management of pregnancies with a fetal variant of uncertain significance remains problematic for counseling.OBJECTIVE: This study aimed to follow the decision-making processes in pregnancies with a fetal variant of uncertain significance and pro-spectively assess copy number variation interpretations and implications under the newer 2020 American College of Medical Genetics and Ge-nomics guidelines.STUDY DESIGN: In a single prenatal unit, prospective chromosome testing using copy number variation sequencing for 8030 fetuses with unexpected noninvasive findings identified 139 pregnancies with a copy number variation classified as a variant of uncertain significance according to the 2015 American College of Medical Genetics and Genomics guidelines current at the time. Parent-of-origin testing was subsequently performed to determine if the copy number variation was inherited or de novo. All couples were offered specialized genetic counseling to assist in pregnancy management decisions. For the continued pregnancies that reached term, newborns were clinically assessed for evidence of any disease at 0 to 10 months and/or at 2 to 4 years of age.RESULTS: Of the 139 variants of uncertain significance found, most (78%) were inherited with no evidence of disease in the carrier parent. On the basis of primary ultrasound findings combined with results from noninvasive prenatal screening tests, most inherited variant of uncertain significance pregnancies were continued, whereas most pregnancies involving de novo variants of uncertain significance were terminated. From clinical follow-up of the 113 live births, only 5 showed any evidence of a phenotype that was not apparently related to the original variant of un-certain significance. Prospective reanalysis of the 139 variants of uncer-tain significance using recent 2020 American College of Medical Genetics and Genomics guidelines changed the status of 24 variants of uncertain significance, with 15 reclassified as benign and 9 as pathogenic. However, the 5 children born with an inherited variant of uncertain significance reclassified as pathogenic showed no evidence of a disease phenotype on clinical follow-up.CONCLUSION: The severity of fetal ultrasound findings combined with results from parent-of-origin testing were the key drivers in pregnancy management decisions for patients. According to birth outcomes from continued pregnancies, most variants of uncertain significance proved to be apparently benign in nature and potentially of low risk of adverse disease outcome. There was a discordance rate of 17% for variant of uncertain significance scoring between the 2015 and 2020 American College of Medical Genetics and Genomics guidelines for defining a variant of uncertain significance, suggesting that difficulties remain for predicting true pathogenicity. Nonetheless, with increasing knowledge of population copy number variation polymorphisms, and a more complete assessment for alternative genetic causes, patients having prenatal assessments should feel less anxious when a fetal variant of uncertain significance is identified. Key words: copy number variation sequencing, parent-of-origin test, pregnancy decisions, pregnancy outcomes, prenatal diagnosis