Pan-viral metagenomic sequencing demonstrates that cryptic viral infection is rarely observed in villitis of unknown etiology.

Introduction: Aberrant maternal immune responses are implicated in villitis of unknown etiology (VUE), but the underlying cause of this loss of tolerance, including cryptic causative or precipitating infections, has been difficult to define. Herein, we performed pan-viral metagenomic sequencing of placentas with VUE to investigate the possibility of cryptic viral infection as a contributing factor in this inflammatory pathology.

Methods: Placentas evaluated at a single tertiary medical center between 2010 and 2024 were included in this study. Overall, the cohort included infectious villitis due to cytomegalovirus (CMV; n = 4), VUE (n = 25), and a reference group composed of pathologically unremarkable placentas (n = 17). Total nucleic acid was extracted from formalin-fixed paraffin embedded (FFPE) placental tissues and subjected to pan-viral metagenomic sequencing (PVMS) to identify viral-associated reads.

Results: PVMS detected reads mapping to CMV in 4 (of 4) of CMV cases. For VUE cases, 22 (of 25) had no identifiable viral reads, while 1 case demonstrated CMV reads and two had papillomavirus reads. The control samples demonstrated no identifiable reads in 13 (of 17) samples, while 3 cases had reads mapping to human papillomavirus 16 and one case had reads mapping to human Herpesvirus 6.

Discussion: Utilizing PVMS, we did not identify cryptic viral sequences in 88 % of morphologic VUE cases. In one clinical VUE case, CMV sequences were identified, suggesting a misclassification of infectious villitis. Both papillomavirus and herpesvirus sequences have previously been identified in the placenta, with unknown clinical significance. Overall, these findings exclude active viral infection as a potential etiology of VUE.