589 - A congenital CMV infection and placental histopathology cohort study using spatial transcriptomics and a novel human placental villous model system to understand mechanisms of vertical transmission.

Publication Number: 589.4004

Nitin Arora, University of Alabama School of Medicine, Birmingham, AL, United States

Background: Congenital cytomegalovirus (CMV) is the most common congenital infection affecting 0.5-2% of neonates delivered each year in the United States. This pathogen is the leading non-genetic cause of sensorineural hearing loss and morbidity in children. Despite decades of research there is limited understanding of how CMV is transmitted across the human placenta from mother to baby.

Objective: The objective of our study is to examine the morphological changes in a 7 year cohort of human placentas collected from congenital CMV infected infants, evaluate the cell types infected, elucidate the gene pathways of relevance and further investigate the mechanism of CMV transmission in the placental villous core using a novel primary human placental mesenchymal stroma model system.

Design/Methods: From May 2018 to March 2024, there were 25 placental specimens obtained for evaluation, of which 19 were concerning for CMV infection. The pathology of these placental samples was reviewed to identify histopathological characteristics. IHC, IF, and ISH techniques were then utilized to identify CMV infection and replication products within placental specimens. Samples were then selected for transcriptomic evaluation (to be presented at PAS) to identify biologic pathways of relevance. Furthermore, an explant method for isolation of (pMSC) mesenchymal stem cells from chorionic villi of human term un-infected placentas was developed and infected with clinically relevant CMV strains.

Results: 19 infants that tested positive for CMV after delivery. 74% (14/19) of these infants had clinical findings and laboratory/imaging data evaluated using IHC, IF, and ISH of the placentas. The number of affected cells in each stained sample was assessed, 8 samples selected and a spatial transcriptomic evaluation performed. Following characterization of pMSC a statistically significant (n=3) dose (MOI) dependent increase in infection of pMSC cells was observed following infection with AD-169 and TR strains of CMV.

Conclusion(s): There is a scarcity of scientific data examining the complexity of transmission of CMV in the human placental villous core. The cells comprising this region of the placenta are most susceptible to CMV infection (Uenaka et al 2019). These findings were confirmed in our cohort. Following which a novel model system of pMSC cells was established. Furthermore, the biological pathways of relevance identified by our spatial transcriptomic assay (present at PAS) is being mechanistically examined by this model system.

Imaging and scoring of CMV infection
Immunohistochemistry staining of placental samples looking at  nuclear DAPI staining, CMV early replication protein IE1, and CMV late replication protein PP65, and a negative control sample. Immunofluorescence Z-stack with DAPI nuclear staining, CMV early replication protein IE1, and trophoblast CK-19 staining. In situ hybridization with DAPI nuclear and CMV DNA probe staining with positive and negative controls

Infection burden scoring system with number of infected cells and placental samples stained.

Placental mesenchymal stem cell (pMSC) characterization and isolation
Characterization of pMSC stem cell using morphology markers and immunoflorescence staining (Nanog, Vimentin and STRO-1).
(Completed but not shown due to abstract size limit considerations) Negative controls for staining and for endothelial cells (CD31), Hofbauer cells (CD163)
(Completed but not shown due to abstract size limit considerations) Day 7 and Day 14 of explant based methodology of cell isolation DIC images

Placental mesenchymal stem cell (pMSC) infectivity assay with TR strain of CMV.
Dose dependent increase in infection of pMSC cells with TR strain of CMV
(Completed but not shown due to abstract size limit considerations) Dose dependent increase in infection of pMSC cells with AD-169 strain of CMV (Completed but not shown due to abstract size limit considerations) Human foreskin fibroblast controls (HFF) for staining and infection.