Detection of human cytomegalovirus cell-free DNA in pregnant women with symptomatically infected fetuses: proof-of-concept study

Objective: To evaluate the presence and levels of cytomegalovirus (CMV) cell-free DNA (cfDNA) fragments in women pregnant with a fetus with symptomatic congenital CMV (cCMV). Methods: The study comprised nine women whose fetuses were diagnosed with cCMV between June 2019 and July 2024 at 20 + 4 to 34 + 1 weeks' gestation (n = 8) or neonatally (n = 1) after primary or non-primary maternal infection. In eight women, cfDNA sequencing data from a single timepoint were analyzed, either retrospectively, on data generated from 11–13 weeks' gestation (n = 5) or prospectively, on data generated from 20–26 weeks' gestation (n = 3), upon the diagnosis of cCMV. In one woman (Case 6), CMV-cfDNA analysis was performed at four timepoints: at 12 + 5 weeks (routine non-invasive prenatal testing); 23 + 3 weeks (cCMV diagnosis); and 30 min and 12 h after termination of pregnancy (TOP) at 23 + 6 weeks. Results: CMV-cfDNA was detectable in all cases. Mostly low levels of CMV-cfDNA were observed in samples obtained at 11–13 weeks' gestation and consistently high levels of CMV-cfDNA were present in samples obtained at cCMV diagnosis. In Case 6, the level of maternal CMV-cfDNA decreased substantially in the samples tested after TOP, compared with samples obtained before TOP. Conclusions: Low levels of CMV-cfDNA detected between 11 and 13 weeks may be a biomarker for severe fetal cCMV. CMV-cfDNA analysis in the first trimester could be of added value in CMV screening, particularly for non-primary maternal infections that cannot be identified using other methods. However, as CMV-cfDNA is detectable in many pregnant women in the first trimester, further studies are needed to determine the predictive value of CMV-cfDNA as a biomarker for the development of severe fetal cCMV. High levels of CMV-cfDNA at fetal cCMV diagnosis and low levels directly after TOP suggest that the level of CMV-cfDNA in maternal plasma may not necessarily reflect an active maternal infection, but could indicate a placental infection.