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Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing "biological microfractures" of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta. [ABSTRACT FROM AUTHOR]

Problem: This study localized CD45+ immune cells and compared changes in their numbers between term, not in labor (TNIL) and term, labor (TL) human fetal membranes. Method of study: Fetal membranes (amniochorion) from normal TNIL and TL subjects were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry for evidence of total (CD45+) immune cells as well as innate immune cells (neutrophils, macrophages and NK cells) using specific markers. Fetal origin of immune cells was determined using polymerase chain reaction (PCR) for SRY gene in Y chromosome. Results: CD45+ cells were localized in human fetal membranes for both TNIL and TL. A threefold increase in CD45+ cells was seen in TL fetal membranes of (7.73% ± 2.35) compared to TNIL (2.36% ± 0.78). This increase is primarily contributed by neutrophils. Macrophages and NK cells did not change in the membranes between TNIL and TL. Leukocytes of fetal origin are present in the fetal membranes. Conclusion: The fetal membranes without decidua contain a small proportion of immune cells. Some of these immune cells in the fetal membrane are fetal in origin. There is a moderate increase of immune cells in the fetal membranes at term labor; however, it is unclear whether this is a cause or consequence of labor. Further functional studies are needed to determine their contribution to membrane inflammation associated with parturition. [ABSTRACT FROM AUTHOR]

Chorionic trophoblast cells (CTCs) are one of the principal components of the fetal membrane and join with the decidua to form a feto-maternal interface. Recent success in isolating CTCs dealt with two separate questions: (1) The necessity of highly enriched and defined media with inhibitors of oxidative stress and cell transition and their impact on growth and trophoblast phenotype, (2) The functional differences between CTCs and other placental trophoblast lineages of cells (placental cytotrophoblast cells [PTC], and extravillous trophoblast [EVT]). CTCs were cultured either in defined media with various inhibitors or in media from which inhibitors were removed individually. Cellular morphology and growth (microscopy and crystal violet staining) and cellular and molecular biological features (immunofluorescence staining for GATA3, cytokeratin [CK] 7, and vimentin) were assessed. Syncytialization of cells (forskolin treatment) and invasive properties of CTCs (cell invasion assay) were tested and compared with PTCs and EVTs (HTR8/SVneo), respectively. Removal of various growth-supporting agents from the media delayed cell growth and inclined towards cellular transition (increase in vimentin compared to CK7 or GATA3) compared to CTCs grown in complete and enriched media. The CTCs failed to syncytialize, contrasting with the high levels of membrane fusion observed in PTCs. Although CTCs express human leukocyte antigen G (HLA-G) like EVTs, they do not invade. CTCs require several specific constituents for in vitro growth and phenotype maintenance. CTCs are trophoblast lineage cells that barricade immune cell-enriched decidua without invading them. These properties support their location and function, which are distinct from PTCs and EVTs., (© The Author(s) 2025. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: The placenta is essential for fetal growth and survival and maintaining a successful pregnancy. The sterility of the placenta has been challenged recently; however, the presence of a placental microbiome has been controversial. We tested the hypothesis that the bacterial extracellular vesicles (BEVs) from Gram-negative bacteria as an alternate source of microbial DNA, regardless of the existence of a microbial community in the placenta., Methods: Placentae from the term, not in labor Cesareans deliveries, were used for this study, and placental specimens were sampled randomly from the fetal side. We developed a protocol for the isolation of BEVs from human tissues and this is the first study to isolate the BEVs from human tissue and characterize them., Results: The median size of BEVs was 130-140 nm, and the mean concentration was 1.8-5.5 × 10 10 BEVs/g of the wet placenta. BEVs are spherical and contain LPS and ompA. Western blots further confirmed ompA but not human EVs markers ALIX confirming the purity of preparations. Taxonomic abundance profiles showed BEV sequence reads above the levels of the negative controls (all reagent controls). In contrast, the sequence reads in the same placenta were substantially low, indicating nothing beyond contamination (low biomass). Alpha-diversity showed the number of detected genera was significantly higher in the BEVs than placenta, suggesting BEVs as a likely source of microbial DNA. Beta-diversity further showed significant overlap in the microbiome between BEV and the placenta, confirming that BEVs in the placenta are likely a source of microbial DNA in the placenta. Uptake studies localized BEVs in maternal (decidual) and placental cells (cytotrophoblast), confirming their ability to enter these cells. Lastly, BEVs significantly increased inflammatory cytokine production in THP-1 macrophages in a high-dose group but not in the placental or decidual cells., Conclusion: We conclude that the BEVs are normal constituents during pregnancy and likely reach the placenta through hematogenous spread from maternal body sites that harbor microbiome. Their presence may result in a low-grade localized inflammation to prime an antigen response in the placenta; however, insufficient to cause a fetal inflammatory response and adverse pregnancy events. This study suggests that BEVs can confound placental microbiome studies, but their low biomass in the placenta is unlikely to have any immunologic impact., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Menon, Khanipov, Radnaa, Ganguly, Bento, Urrabaz-Garza, Kammala, Yaklic, Pyles, Golovko and Tantengco.)