Background:The fetal immune system undergoes pivotal development during gestation, preparing for postnatal antigenic challenges. Bacterial extracellular vesicles (bEVs), bioactive particles shed by bacteria, are emerging as modulators of host immunity. However, their role in shaping fetal intestinal immune development remains largely unexplored. Objectives:This study aimed to investigate the effects of bEV exposure on lymphoid and myeloid populations in the fetal murine gut, focusing on their role in priming intestinal immunity, promoting differentiation, and modulating immune cell phenotypes in both normal and germ-free (GF) environments. Materials and Methods:We used a murine model to evaluate the immune-modulating effects of bEVs during fetal development. bEVs were isolated from bacterial cultures and introduced into the amniotic sac of embryonic day 15.5 (E15.5) fetuses through intra-amniotic injection. Fetal and neonatal mice were either raised under conventional conditions (normal environment, NE) or in germ-free (GF) environments to assess microbiota-dependent effects. Immune profiling of fetal (E17) and postnatal (4 weeks) gut tissues was performed using high-dimensional mass cytometry (CyTOF) in both conventionally housed and germ-free (GF) mice. Clustering and differential expression analyses identified lymphoid and myeloid subpopulations, including progenitors, antigen-presenting cells, and intestinal stem cells (ISCs). secondary immune challenge (LPS or TSST-1) was conducted in postnatal bEV-primed mice to assess immune memory responses. Results:bEV exposure significantly increased the prevalence of CD45- CD24+ CD44+ ISCs, promoting intestinal renewal and defense via differentiation into Paneth and tuft cells. These ISCs exhibited potential antigen-presenting capabilities through MHC expression. CD45+ lymphoid progenitors were upregulated, highlighting their role in early differentiation pathways. Myeloid progenitors, particularly monocyte-dendritic progenitor subsets, showed a bias toward antigen-presenting phenotypes.Germ-free models revealed heightened sensitivity to bEVs, with pronounced activation of progenitors and a reduction in exhaustion markers. Interestingly, macrophage and neutrophil populations displayed dose-dependent modulation, with low bEV concentrations promoting their expansion and higher doses leading to reduced incidence. Our findings suggest that bEVs act as immune priming agents in the fetal gut, promoting progenitor expansion and differentiation while preparing the intestine for postnatal challenges. Differences in responses between NE and GF models emphasize the importance of environmental influences, including microbiota, on bEV-mediated immune modulation. Conclusion:bEVs play a pivotal role in shaping fetal intestinal immunity by priming lymphoid and myeloid progenitors and enhancing ISC function. These results open potential avenues for leveraging bEVs in immunomodulation and vaccine strategies. Future studies should explore the functional responses of bEV-primed cells and their translational relevance in humans.

Differences in male (M) and female (F) neonates’ premature birth outcomes and placental trophoblast inflammation have been observed but are unknown to occur within the fetal membrane trophoblast layer (chorion trophoblasts [CTC]). This study examined whether sex-based differences in gene expression and inflammatory marker expression can be observed in CTCs under control or infectious inflammatory conditions modeling preterm birth. CTCs from six different patient-derived fetal membrane samples (3M/3F) were cultured and divided into experimental (Lipopolysaccharide [LPS]) and control groups for 6, 12, or 24 h. RNA from CTCs was subjected to RNA-seq, while cytokine multiplex or ELISA detected pro-/anti-inflammatory cytokines, progesterone, and soluble HLA-G in cell supernatants. CTC-M and CTC-F showed sex, time, and stimulant-dependent differential gene expression profiles. Cytokine analysis demonstrated a significantly lower IL-6 production in control CTC-M than in CTC-F. No sex-dependent responses were observed after LPS treatment regarding cytokines. CTC-M produced significantly lower progesterone than CTC-F. The theories of sexual dimorphism linked to placental inflammation may not extend to CTCs. This study supports that the chorion acts as a “great wall” protecting the fetus by being refractory to insults. Further examination into the weaknesses of the chorion barrier and sex-dependent responses of fetal membranes is needed.

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human term placenta-derived cytotrophoblast immortalized cell line (hPTC(CTB)) derived from the basal plate. Human-term placenta-derived cytotrophoblast immortalized cell line cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTC(CTB) cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. Human-term placenta-derived cytotrophoblast immortalized cell line and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTC(CTB) generated are cells of higher physiological relevance and (2) provides a look into the distinction between the spatially distinct placental and fetal barrier trophoblasts cells, hPTC(CTB) and hFM-CTC, respectively.

P-glycoprotein (P-gp) is primarily reported in chorion trophoblast cells (CTC) for efflux drug transport. Its presence is reported in the neighboring amnion mesenchymal cells (AMC) and decidual cells (DEC), suggesting limited efflux capabilities of these cells. To test the hypothesis that CTC-derived extracellular vesicles (EVs) containing P-gp as cargo could induce P-gp expression in AMC and DEC, we examined CTC-EVs' impact on transitioning cells to perform efflux functions. Gene expression levels of P-gp were analyzed in fetal membrane cells through quantitative real-time polymerase chain reaction (qRT-PCR), and protein levels were assessed using western blot analysis. CTC-derived EVs were isolated and characterized for size, shape, and quantity using nanoparticle tracking analysis, and the presence of P-gp was confirmed. Flow cytometry was utilized to evaluate CTC-EVs' effect on inducing P-gp expression in cells lacking it. The functional activity of P-gp in AMC and DEC was assessed through a multidrug resistance assay after exposure to CTC-derived P-gp-containing EVs. To validate our findings, decidual cells with P-gp knockdown (KD) using the CRISPR/Cas9 approach were treated with CTC-derived EVs containing P-gp and tested for restoration. The gene and protein expression levels of P-gp in the amniochorion layers and their derived EVs were determined (Fig. 1A and 1B). Although the expression levels of EVs within the cells were low, both amniotic epithelial cells (AEC) and CTC-derived EVs carried P-gp. Treatment with CTC-derived EVs containing P-gp led to increased P-gp expression in AMC and DEC, resulting in enhanced drug efflux and a decrease in drug resistance (Fig. 1C). Furthermore, P-gp expression was reduced in trophoblast cells with P-gp KD but was restored upon treatment with chorion-derived EVs. This study demonstrates that EV-mediated delivery of P-gp can induce drug efflux functions in cells in a paracrine fashion. The modulation of P-gp by EVs highlights the interplay between EVs and the cells of the feto-maternal interface drug transport mechanisms.

Human fetal membranes (amniochorion) that line the intrauterine cavity consist of two distinct cell layers; single-layer amnion epithelial cells (AEC) and multilayer chorion trophoblast cells (CTC). These layers are connected through a collagen-rich extracellular matrix. Cellular remodeling helps support membrane growth and integrity during gestation and helps to maintain pregnancy. Preterm prelabor rupture of the human amniochorionic (fetal) membrane (pPROM) is antecedent to 40% of all spontaneous preterm birth. Oxidative stress (OS) induced activation of the p38 MAPK due to various maternal risk exposures and the amniochorion cells’ senescence are reported pathological features of pPROM. Our transcriptomics analysis implicated dysregulated autophagy and epithelial-mesenchymal transition (EMT) in fetal membranes from pPROM. The molecular interplay between OS-induced p38 MAPK activation, autophagy, and EMT was investigated in AECs and CTCs to better understand the involvement of autophagy and EMT. We report the differential impact of OS on the autophagic machinery in AECs and CTCs, resulting in distinct cell fates. In AECs, OS-induced p38 MAPK activation causes autophagosome accumulation and reduced autophagic flux mediated by decreased ULK1 activity and kinase activity, leading to senescence. In CTCs, induction of autophagy has a limited effect; however, inhibition of autophagy led to SQSTM1-mediated EMT of trophoblast cells. Autophagy, EMT, and senescence were associated with proinflammatory changes. Thus, AECs and CTCs respond differently to OS via differential autophagy response, partly mediated via p38 MAPK. Besides senescence, OS-induced autophagy dysregulation in amniochorion cells may play a mechanistic role in pPROM pathophysiology.

Multiple cell lines have been utilized over time in studying placental biology. Still, most of them rely on choriocarcinoma cells or immortalized trophoblast cells that may not be entirely comparable with actual human placental trophoblast cells. Term placentas can be a source of primary villous trophoblasts. However, challenges remain in isolating them and maintaining them in extended culture. This manuscript describes our three-phase protocol utilizing enzymatic/mechanical digestion, modified Percoll gradient density separation, and immunopurification using magnetic beads. The resulting trophoblast culture remains viable for an extended period and highly pure after initial passaging.

Infection and inflammation are two major risk factors for preterm birth. There is a lack of physiologically-relevant human models of the two feto-maternal interfaces (FMi; placenta and fetal membranes with decidua) to test anti-inflammatory drug efficacy and functions in an in vitro setting. We used a fetal membrane-placenta organ-on-chip (FMi-PLA-OOC) to mimic infectious inflammation of the FMiand conduct pre-clinical drug testing (cytotoxicity, mechanism of action, and efficacy) using a novel anti-inflammatory compound, exosomal-derived interleukin-10 (eIL-10), engineered to facilitate drug passage across FMi barriers. FMi-PLA-OOC contained cells from human umbilical cord endothelium, cyto- and syncytiotrophoblasts, decidua, chorion, and amnion (mesenchyme and epithelium) in seven cell chambers connected through collagen-filled microchannels.The decidual cells were treated with lipopolysaccharide (LPS; 100ng/mL) followed by a co-treatment of LPS and eIL-10 (500ng/mL) (Fig 1A).LPS and eIL-10 propagation and P-NF-kB expression were determined by immunostaining, functional IL-10 signaling (Quant blue assay), and inflammation (multiplex cytokine assay). Maternal-to-fetal propagationof LPS occurred within 6 days without inducing cytotoxicity of cells in the FMi-PLA-OOC (Fig 1B-C). LPS induced P-NF-kB expression (Fig 1D) and induced pro-inflammatory cytokines (IL-6, IL-8, TNF-a) across the fetal membrane and placenta layers (Fig 1E; [black *-p< 0.05 normalized to control]). eIL-10 did not induce cytotoxicity (Fig 1B) within the maternal or fetal cells and propagated to distal chambers within 3 days (Fig 1C). LPS+eIL-10 co-treatment induced IL-10 signaling pathways (Fig 1F), reduced P-NF-kB expression (Fig 1D), and suppressed LPS-induced pro-inflammatory cytokine production in the fetal membranes and placenta (red *-p< 0.05 normalized to LPS) (Fig 1E). Using thismodel, we successfully tested the ability of eIL-10 to suppress LPS-induced inflammation at the FMi. FMi-PLA-OOC is an alternative to animal models for preclinical testing of drugs during pregnancy.

Progesterone and its receptors (progesterone receptor membrane components-1/2 [PGRMC1/2] and nuclear progesterone receptors A/B [PRA/B]) play a crucial role in pregnancy maintenance by promoting decidualization, immunomodulation, uterine quiescence, and cervical integrity. The paracrine signaling functions they may provide when propagated via uterine cell-derived exosomes are unclear. Lysates of myometrial and decidual cells grown in a 2D culture were prepared, and extracellular vesicles (exosomes) were isolated from media using differential ultracentrifugation and size exclusion chromatography. Immunocytochemistry and western blots localized receptors within cells and exosomes. ZetaView determined exosome size, concentrations, and exosomal-enriched CD markers. Immunocytochemistry and western blot analysis of myometrial and decidual cell lysates confirmed the expression of both membrane (PGRMC1/PGRMC2) and genomic receptors (PRA/PRB). Myometrial and decidual cell-derived exosomes were around 110-130nm (Fig.1A; Fig.1C), concentrated within 109 -1010 per/ml (Fig.1A; Fig.1C), and expressed CD63 or CD9 (Fig.1B; Fig.1D). Myometrial exosomes predominantly expressed genomic receptors PRA/B (Fig.1B), while decidual exosomes expressed PGRMC1 (Fig.1D). These differences highlight the potential distinction in progesterone paracrine signaling and could be later used to target either cell type depending on the pathology. We confirmed the expression of progesterone receptors and report their differential expressions in exosomes from myometrial and decidua cells. Given that a subset of pregnant women does not respond to progesterone treatment due to functional progesterone withdrawal, PR-containing exosomes could be utilized to deliver these receptors to gestational tissues during pregnancy. Potentially making them responsive to progesterone. Functional studies are still needed to understand the role of exosome-derived progesterone receptors.

Extracellular vesicles (EVs) are essential for communication at the fetal-maternal interface. EVs represent the physiologic state of the cell at the time of its release. This study evaluated the changes in the serum EV characteristics and their protein cargo in women with preeclampsia (PE) treated with aspirin (ASA). Previously collected maternal samples from at-risk people receiving 81 mg vs. 162 mg aspirin, based on the development of PE or not. Samples were collected between 28-32 weeks (12-20 weeks after aspirin initiation). The optimal method of ultra-centrifugation (UC) followed by size exclusion chromatography was chosen for EV isolation and characterization. EVs from women not on ASA (control), on 81 mg, or 162 mg ASA were isolated and characterized (size and quantity [ZetaView], shape [transmission electron microscopy], markers [Exoview]). Mass spectrometry-based proteomics experiments are performed using a stochastic sampling approach (data-dependent acquisition) for EV proteome cargo analysis. Ultracentrifugation and size exclusion chromatography yielded EVs’ highest number and quality. EV size and concentrations among groups were similar (Fig. 1A-B). TEM images validated intact and expected EVs’ size and shape (Fig. 1C). Average gestational age at collection was 35 weeks for the control group (n=7), 29 weeks for 81 mg group (n=32), and 28 weeks for 162 mg group (n=13). ExoView analysis confirmed known EV markers of CD9, CD81, and CD63 (Fig. 1D) that were not different between groups. The proteomic analysis did not show any differences in total maternal serum EV proteins regardless of the dose of treatment or untreated groups. Total serum EVs were not different between different doses of ASA-treated and untreated PE groups, as our current approach did not distinguish between fetal vs. maternal EVs. PE is a disease of the placenta and vasculature and therefore, we postulate that placental-specific (fetal) EV characterization is required to identify prognostic markers of ASA treatment.

Cell-free RNAs and extracellular vesicles (EVs) are valuable biomarkers in liquid biopsies, but they are prone to preanalytical variabilities such as nonstandardized centrifugation or ex vivo blood degradation. Herein, we report a high-throughput and label-free inertial microfluidic device (ExoArc) for isolation of platelet-free plasma from blood for RNA and EV analysis. Unlike conventional inertial microfluidic devices widely used for cell sorting, a submicrometer size cutoff (500 nm) was achieved which completely removed all leukocytes, RBCs, platelets, and cellular debris based on differential lateral migration induced by Dean vortices. The single-step operation also reduced platelet-associated miRNAs (∼2-fold) compared to centrifugation. We clinically validated ExoArc for plasma miRNA profiling (39 samples) and identified a 7-miRNA panel that detects non-small cell lung cancer with ∼90% sensitivity. ExoArc was also coupled with size exclusion chromatography (SEC) to isolate EVs within 50 min with ∼10-fold higher yield than ultracentrifugation. As a proof-of-concept for EV-based transcriptomics analysis, we performed miRNA analysis in healthy and type 2 diabetes mellitus (T2DM) subjects (n = 3 per group) by coupling ExoArc and ExoArc+SEC with quantitative polymerase chain reaction (RT-qPCR) assay. Among 293 miRNAs detected, plasmas and EVs showed distinct differentially expressed miRNAs in T2DM subjects. We further demonstrated automated in-line EV sorting from low volume culture media for continuous EV monitoring. Overall, the developed ExoArc offers a convenient centrifugation-free workflow to automate plasma and EV isolation for point-of-care diagnostics and quality control in EV manufacturing.

Fetal membrane inflammation is one of the drivers of pPROM. Epithelial-mesenchymal transition (EMT) of amnion epithelial and chorion trophoblast cells (CTC) at cellular-level generate inflammation that can weaken the membranes. In amnion, progesterone (P4) through receptor membrane components (PGRMC 1 and 2) restricts EMT to avoid inflammation. CTCs at the fetomaternal interface are vulnerable to maternal risk factors that can cause EMT. This study tested the influence of PGRMC 1 and 2 on EMT of CTCs in response to stressors associated with pPROM. PGRMC1 or PGRMC2 knock out (KO) CTCs were prepared by CRISPR/Cas9 (Fig 1). The cells were treated with stressors Bafilomycin (BafA1) and TGF-β (EMT inducers), immunogens lipopolysaccharide (LPS), chlamydial antigen, and Ureaplasma-conditioned media (UP) to simulate various infections known to be associated with pPROM. Primary endpoints included morphologic evidence of EMT, cell-shape index, N-Cadherin to E-Cadherin ratio, TNF-α, IL-6, and progesterone production (P4). Progesterone from chorion self regulates, providing antiinflammatory barrier function. In PGRMC1 KO, there was no difference in EMT compared to wild type (WT) under normal conditions (Fig 1). In PGRMC2 KO, only LPS, but no other agent, induced EMT compared with WT (p=0.045) and an increase in P4 (p=0.033). Inflammatory cytokines (TNF-α, IL-6) significantly increased in PGRMC1 and PGRMC2 KO in response to BafA1, TGF-β, LPS, and UP (p< 0.05) (Table 1). P4 and PGRMC do not impact CTC EMT. CTCs are refractory to EMT in response to risk factors of pPROM (LPS, Chlamydia and U parvum antigens) and inducers of EMT (TGF-β). Increases in proinflammatory cytokines in PGRMC KO compared to WT suggest P4-PGRMC axis regulating CTC inflammation, which is independent of EMT. CTCs are resistant to EMT to maintain its barrier functions. Chorionic inflammation, independent of EMT, is regulated by P4/PGRMC mediated autocrine signaling. Risk factors of pPROM do not induce chorion cell derangements and membrane dysfunctions are likely resulting from EMT of the amnion cells.

Pregnant women and their fetuses are often excluded from clinical trials due to missing drug-related pre-clinical trial information at the human feto-maternal interface (FMi). The two interfaces-placenta/decidua and fetal membranes/decidua are gatekeepers of drug transport; however, testing their functions is impractical during pregnancy. Limitations of current in-vivo/in-vitro models have hampered drug development and testing during pregnancy. Hence, major complications like preterm births and maternal and neonatal mortalities remain high. Advancements in organ-on-chip (OOC) platforms to test drug kinetics and efficacy and novel extracellular vesicle-based fetal drug delivery are expected to accelerate preclinical trials related to pregnancy complications. Here we report the development and testing of a humanized multi-organ fetal membrane/placenta (fetal)-decidua (maternal) interface OOC (FMi-PLA-OOC) that contains seven cell types interconnected through microchannels to maintain intercellular interactions as seen in-utero. Cytotoxicity, propagation, mechanism of action, and efficacy of engineered extracellular vesicles containing anti-inflammatory interleukin (IL)-10 (eIL-10) were evaluated to reduce FMi inflammation associated with preterm birth. A healthy and disease model (lipopolysaccharide-infectious inflammation) of the FMi-PLA-OOC was created and co-treated with eIL-10. eIL-10 propagated from the maternal to fetal side within 72-h, localized in all cell types, showed no cytotoxicity, activated IL-10 signaling pathways, and reduced lipopolysaccharide-induced inflammation (minimized NF-kB activation and proinflammatory cytokine production). These data recapitulated eIL-10s’ ability to reduce inflammation and delay infection-associated preterm birth in mouse models, suggesting FMi-PLA-OOC as an alternative approach to using animal models. Additionally, we report the utility of eIL-10 that can traverse through FMis to reduce inflammation-associated pregnancy complications.

Infection and inflammation are risk factors for spontaneous preterm birth. However, the spread of infection and inflammatory mediators at the two feto-maternal interfaces (FMi; placenta and fetal membranes with decidua [Fig 1A]) are difficult to study due to the limitations of animal models and the lack of comprehensive in-vitro models. We developed a humanized multi-organ fetal membrane-placenta organ-on-chip (FMi-PLA-OOC) (Fig 1B-C), established physiologic and pathologic models of the FMi, and determined the responses of FMis to infection and inflammation. FMi-PLA-OOC contained cells from human umbilical cord endothelium, cyto- and syncytiotrophoblasts, decidua, chorion, and amnion (mesenchyme and epithelium) in seven cell chambers connected by arrays of collagen-filled microchannels.To validate FMi-PLA-OOC, we determined cell viability (cytotoxicity - lactate dehydrogenase [LDH] assay) and confirmed that in utero cellular characteristics (immunocytochemistry [ICC]) were maintained during culture on-chip. We also determined lipopolysaccharide (LPS; 100ng/mL) propagation (immunostaining) and inflammation (multiplex cytokine assay). Cell markers and viability in the FMi-PLA-OOC were similar to in-utero FMi, validating the physiological relevance of the device (Fig 1D-E). LPS propagated from maternal to fetal cells within 6 days(Fig 1F), inducing an increase in pro-inflammatory cytokine concentrations (IL-6, IL-8, TNF-a) and a decrease in anti-inflammatory cytokine production (IL-10) across the fetal membrane and placenta layers(Fig 1G; [Heat map is normalized to control. *- p< 0.05]). Healthy and disease models of FMis on a chip demonstrated differential responses of the placental and fetal membrane to infection and inflammation. This platform provides broad utility in obstetrics research.

Background:Fetal inflammatory response mediated by the influx of immune cells and activation of pro-inflammatory transcription factor NF-κB in feto-maternal uterine tissues is the major determinant of infection-associated preterm birth (PTB, live births < 37 weeks of gestation).Objective:To reduce the incidence of PTB by minimizing inflammation, extracellular vesicles (EVs) were electroporetically engineered to contain anti-inflammatory cytokine interleukin (IL)-10 (eIL-10), and their efficacy was tested in an ascending model of infection (vaginal administration of E. coli) induced PTB in mouse models.Study design:EVs (size: 30-170 nm) derived from HEK293T cells were electroporated with recombinant IL-10 at 500 volts and 125 Ω, and 6 pulses to generate eIL-10. eIL-10 structural characters (electron microscopy, nanoparticle tracking analysis, ExoView [size and cargo content] and functional properties (co-treatment of macrophage cells with LPS and eIL-10) were assessed. To test efficacy, CD1 mice were vaginally inoculated with E. coli (1010CFU) and subsequently treated with either PBS, eIL-10 (500ng) or Gentamicin (10mg/kg) or a combination of eIL-10+gentamicin. Fetal inflammatory response in maternal and fetal tissues after the infection or treatment were conducted by suspension Cytometer Time of Flight (CyTOF) using a transgenic mouse model that express red fluorescent TdTomato (mT+) in fetal cells.Results:Engineered EVs were structurally and functionally stable and showed reduced proinflammatory cytokine production from LPS challenged macrophage cells in vitro. Maternal administration of eIL-10 (10 µg/kg body weight) crossed feto-maternal barriers to delay E. coli-induced PTB to deliver live pups at term. Delay in PTB was associated with reduced feto-maternal uterine inflammation (immune cell infiltration and histologic chorioamnionitis, NF-κB activation, and proinflammatory cytokine production).Conclusions:eIL-10 administration was safe, stable, specific, delayed PTB by over 72 hrs and delivered live pups. The delivery of drugs using EVs overcomes the limitations of in-utero fetal interventions. Protecting IL-10 in EVs eliminates the need for the amniotic administration of recombinant IL-10 for its efficacy.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 10 4 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60 days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28 days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

Ureaplasma, a genus of the order Mycoplasmatales and commonly grouped with Mycoplasma as genital mycoplasma is one of the most common microbes isolated from women with infection/inflammation-associated preterm labor (PTL). Mycoplasma spp. produce sialidase that cleaves sialic acid from glycans of vaginal mucous membranes and facilitates adherence and invasion of the epithelium by pathobionts, and dysregulated immune response. However, whether Ureaplasma species can induce the production of sialidase is yet to be demonstrated. We examined U. parvum-infected vaginal epithelial cells (VECs) for the production of sialidase and pro-inflammatory cytokines.Immortalized VECs were cultured in appropriate media and treated with U. parvum in a concentration of 1 × 105 DNA copies/ml. After 24 h of treatment, cells and media were harvested. To confirm infection and cell uptake, immunocytochemistry for multi-banded antigen (MBA) was performed. Pro-inflammatory cytokine production and protein analysis for sialidase confirmed pro-labor pathways.Infection of VECs was confirmed by the presence of intracellular MBA. Western blot analysis showed no significant increase in sialidase expression from U. parvum-treated VECs compared to uninfected cells. However, U. parvum infection induced 2-3-fold increased production of GM-CSF (p = 0.03), IL-6 (p = 0.01), and IL-8 (p = 0.01) in VECs compared to controls.U. parvum infection of VECs induced inflammatory imbalance associated with vaginal dysbiosis but did not alter sialidase expression at the cellular level. These data suggest that U. parvum's pathogenic effect could be propagated by locally produced pro-inflammatory cytokines and, unlike other genital mycoplasmas, may be independent of sialidase.