Are fetal microchimerism and circulating fetal extracellular vesicles important links between spontaneous preterm delivery and maternal cardiovascular disease risk?

Trafficking and persistence of fetal microchimeric cells (fMCs) and circulating extracellular vesicles (EVs) have been observed in animals and humans, but their consequences in the maternal body and their mechanistic contributions to maternal physiology and pathophysiology are not yet fully defined. Fetal cells and EVs may help remodel maternal organs after pregnancy-associated changes, but the cell types and EV cargos reaching the mother in preterm pregnancies after exposure to various risk factors can be distinct from term pregnancies. As preterm delivery-associated maternal complications are rising, revisiting this topic and formulating scientific questions for future research to reduce the risk of maternal morbidities are timely. Epidemiological studies report maternal cardiovascular risk as one of the major complications after preterm delivery. This paper suggests a potential link between fMCs and circulating EVs and adverse maternal cardiovascular outcomes post-pregnancies, the underlying mechanisms, consequences, and methods for and how this link might be assessed.

Fetal inflammatory response at the fetomaternal interface: A requirement for labor at term and preterm.

Human parturition at term and preterm is an inflammatory process synchronously executed by both fetomaternal tissues to transition them from a quiescent state t an active state of labor to ensure delivery. The initiators of the inflammatory signaling mechanism can be both maternal and fetal. The placental (fetal)-maternal immune and endocrine mediated homeostatic imbalances and inflammation are well reported. However, the fetal inflammatory response (FIR) theories initiated by the fetal membranes (amniochorion) at the choriodecidual interface are not well established. Although immune cell migration, activation, and production of proparturition cytokines to the fetal membranes are reported, cellular level events that can generate a unique set of inflammation are not well discussed. This review discusses derangements to fetal membrane cells (physiologically and pathologically at term and preterm, respectively) in response to both endogenous and exogenous factors to generate inflammatory signals. In addition, the mechanisms of inflammatory signal propagation (fetal signaling of parturition) and how these signals cause immune imbalances at the choriodecidual interface are discussed. In addition to maternal inflammation, this review projects FIR as an additional mediator of inflammatory overload required to promote parturition.

Histologic Evidence of Epithelial-Mesenchymal Transition and Autophagy in Human Fetal Membranes.

Preterm, prelabor rupture of the human fetal membranes (pPROM) is involved in 40% of spontaneous preterm births worldwide. Cellular-level disturbances and inflammation are effectors of membrane degradation, weakening, and rupture. Maternal risk factors induce oxidative stress (OS), senescence, and senescence-associated inflammation of the fetal membranes as reported mechanisms related to pPROM. Inflammation can also arise in fetal membrane cells (amnion/chorion) due to OS-induced autophagy and epithelial-mesenchymal transition (EMT). Autophagy, EMT, and their correlation in pPROM, along with OS-induced autophagy-related changes in amnion and chorion cells in vitro, were investigated. Immunocytochemistry staining of cytokeratin-18 (epithelial marker)/vimentin (mesenchymal marker) and proautophagy-inducing factor LC3B were performed in fetal membranes from pPROM, term not in labor, and term labor. Ultrastructural changes associated with autophagy were verified by transmission electron microscopy of the fetal membranes and in cells exposed to cigarette smoke extract (an OS inducer). EMT and LC3B staining was compared in the chorion from pPROM versus term not in labor. Transmission electron microscopy confirmed autophagosome formation in pPROM amnion and chorion. In cell culture, autophagosomes were formed in the amnion with OS treatment, while autophagosomes were accumulated in both cell types with autophagy inhibition. This study documents the association between pPROMs and amniochorion autophagy and EMT, and supports a role for OS in inducing dysfunctional cells that increase inflammation, predisposing membranes to rupture.

A Microphysiological Device to Model the Choriodecidual Interface Immune Status during Pregnancy.

During human pregnancy the chorion (fetal) lines decidua (maternal) creating the feto-maternal interface. Despite their proximity, resident decidual immune cells remain quiescent during gestation and do not invade the chorion. Infection and infiltration of activated immune cells toward the chorion are often associated with preterm birth. However, the mechanisms that maintain choriodecidual immune homeostasis or compromise immune barrier functions remain unclear. To understand these processes, a two-chamber microphysiological system (MPS) was created to model the human choriodecidual immune interface under normal and infectious conditions in vitro. This MPS has outer (fetal chorion trophoblast cells) and inner chambers (maternal decidual + CD45+ cells [70:30 ratio]) connected by microchannels. Decidual cells were treated with LPS to mimic maternal infection, followed by immunostaining for HLA-DR and HLA-G, immune panel screening by imaging cytometry by time of flight, and immune regulatory factors IL-8 and IL-10, soluble HLA-G, and progesterone (ELISA). LPS induced a proinflammatory phenotype in the decidua characterized by a decrease in HLA-DR and an increase in IL-8 compared with controls. LPS treatment increased the influx of immune cells into the chorion, indicative of chorionitis. Cytometry by time of flight characterized immune cells in both chambers as active NK cells and neutrophils, with a decrease in the abundance of nonproinflammatory cytokine-producing NK cells and T cells. Conversely, chorion cells increased progesterone and soluble HLA-G production while maintaining HLA-G expression. These results highlight the utility of MPS to model choriodecidual immune cell infiltration and determine the complex maternal-fetal crosstalk to regulate immune balance during infection.

Establishment and comparison of human term placenta-derived trophoblast cells†.

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 (hPTCCTB) 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 hPTCCTB 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 hPTCCTB 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, hPTCCTB and hFM-CTC, respectively.

Development of oxidative stress-associated disease models using feto-maternal interface organ-on-a-chip.

Oxidative stress (OS) and inflammation arising from cellular derangements at the fetal membrane-decidual interface (feto-maternal interface [FMi]) is a major antecedent to preterm birth (PTB). However, it is impractical to study OS-associated FMi disease state during human pregnancy, and thus it is difficult to develop strategies to reduce the incidences of PTB. A microfluidic organ-on-chip model (FMi-OOC) that mimics the in vivo structure and functions of FMi in vitro was developed to address this challenge. The FMi-OOC contained fetal (amnion epithelial, mesenchymal, and chorion) and maternal (decidua) cells cultured in four compartments interconnected by arrays of microchannels to allow independent but interconnected co-cultivation. Using this model, we tested the effects of OS and inflammation on both fetal (fetal → maternal) and maternal (maternal → fetal) sides of the FMi and determined their differential impact on PTB-associated pathways. OS was induced using cigarette smoke extract (CSE) exposure. The impacts of OS were assessed by measuring cell viability, disruption of immune homeostasis, epithelial-to-mesenchymal transition (EMT), development of senescence, and inflammation. CSE propagated (LC/MS-MS analysis for nicotine) over a 72-hour period from the maternal to fetal side, or vice versa. However, they caused two distinct pathological effects on the maternal and fetal cells. Specifically, fetal OS induced cellular pathologies and inflammation, whereas maternal OS caused immune intolerance. The pronounced impact produced by the fetus supports the hypothesis that fetal inflammatory response is a mechanistic trigger for parturition. The FMi disease-associated changes identified in the FMi-OOC suggest the unique capability of this in vitro model in testing in utero conditions.

A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

Modeling ascending Ureaplasma parvum infection through the female reproductive tract using vagina-cervix-decidua-organ-on-a-chip and feto-maternal interface-organ-on-a-chip.

Genital mycoplasmas can break the cervical barrier and cause intraamniotic infection and preterm birth. This study developed a six-chamber vagina-cervix-decidua-organ-on-a-chip (VCD-OOC) that recapitulates the female reproductive tract during pregnancy with culture chambers populated by vaginal epithelial cells, cervical epithelial and stromal cells, and decidual cells. Cells cultured in VCD-OOC were characterized by morphology and immunostaining for cell-specific markers. We transferred the media from the decidual cell chamber of the VCD-OOC to decidual cell chamber in feto-maternal interface organ-on-a-chip (FMi-OOC), which contains the fetal membrane layers. An ascending Ureaplasma parvum infection was created in VCD-OOC. U. parvum was monitored for 48 h post-infection with their cytotoxicity (LDH assay) and inflammatory effects (multiplex cytokine assay) in the cells tested. An ascending U. parvum infection model of PTB was developed using CD-1 mice. The cell morphology and expression of cell-specific markers in the VCD-OOC mimicked those seen in lower genital tract tissues. U. parvum reached the cervical epithelial cells and decidua within 48 h and did not cause cell death in VCD-OOC or FMi-OOC cells. U. parvum infection promoted minimal inflammation, while the combination of U. parvum and LPS promoted massive inflammation in the VCD-OOC and FMi-OOC cells. In the animal model, U. parvum vaginal inoculation of low-dose U. parvum did not result in PTB, and even a high dose had only some effects on PTB (20%). However, intra-amniotic injection of U. parvum resulted in 67% PTB. We report the colonization of U. parvum in various cell types; however, inconsistent, and low-grade inflammation across multiple cell types suggests poor immunogenicity induced by U. parvum.

Ureaplasma parvum infection induces inflammatory changes in vaginal epithelial cells independent of sialidase.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

mRNA and Protein Expression in Human Fetal Membrane Cells: Potential Biomarkers for Preterm Prelabor Rupture of the Fetal Membranes?

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.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (

Stress signaler p38 mitogen-activated kinase activation: a cause for concern?

Oxidative stress (OS) induced activation of p38 mitogen-activated kinase (MAPK) and cell fate from p38 signaling was tested using the human fetal membrane's amnion epithelial cells (AEC). We created p38 KO AEC using the CRISPR/Cas9 approach and tested cell fate in response to OS on an AEC-free fetal membrane extracellular matrix (ECM). Screening using image CyTOF indicated OS causing epithelial-mesenchymal transition (EMT). Further testing revealed p38 deficiency prevented AEC senescence, EMT, cell migration, and inflammation. To functionally validate in vitro findings, fetal membrane-specific conditional KO (cKO) mice were developed by injecting Cre-recombinase encoded exosomes intra-amniotically into p38αloxP/loxP mice. Amnion membranes from p38 cKO mice had reduced senescence, EMT, and increased anti-inflammatory IL-10 compared with WT animals. Our study suggested that overwhelming activation of p38 in response to OS inducing risk exposures can have an adverse impact on cells, cause cell invasion, inflammation, and ECM degradation detrimental to tissue homeostasis.

Organ-on-chip of the cervical epithelial layer: A platform to study normal and pathological cellular remodeling of the cervix.

Damage to the cervical epithelial layer due to infection and inflammation is associated with preterm birth. However, the individual and/or collective roles of cervical epithelial layers in maintaining cervical integrity remain unclear during infection/inflammation. To determine the intercellular interactions, we developed an organ-on-chip of the cervical epithelial layer (CE-OOC) composed of two co-culture chambers connected by microchannels, recapitulating the ectocervical and endocervical epithelial layers. Further, we tested the interactions between cells from each distinct region and their contributions in maintaining cervical integrity in response to LPS and TNFα stimulations. The co-culture of ectocervical and endocervical cells facilitated cellular migration of both epithelial cells inside the microchannels. Compared to untreated controls, both LPS and TNFα increased apoptosis, necrosis, and senescence as well as increased pro-inflammatory cytokine productions by cervical epithelial cells. In summary, the CE-OOC established an in vitro model that can recapitulate the ectocervical and the endocervical epithelial regions of the cervix. The established CE-OOC may become a powerful tool in obstetrics and gynecology research such as in studying cervical remodeling during pregnancy and parturition and the dynamics of cervical epithelial cells in benign and malignant pathology in the cervix.

Actions of Bisphenol A on Different Feto-Maternal Compartments Contributing to Preterm Birth.

Preterm birth remains to be one of the most prevalent obstetric complications worldwide. Since there are multiple etiological factors associated with this disease process, an integrative literature search in PubMed and Scopus databases on possible mechanism of action and effect of bisphenols on exposure on human or animal placental samples in preterm birth was conducted. From 2332 articles on initial literature search, 63 studies were included for full data extraction. Altogether, several pathways were shown to be possibly affected by bisphenols, leading to dysregulations in structural and endocrine foundation in the placenta, potential induction of senescence and failure of decidualization in the decidua, and possible propagation of inflammation in the fetal membranes. Combined, these actions may eventually counteract bisphenol-induced relaxation of the myometrium and promote contractility alongside fetal membrane weakening. In totality, these individual impairments in gestation-critical processes may lead to failure of maintenance of pregnancy, and thus effecting preterm birth.

Progesterone receptor membrane components: key regulators of fetal membrane integrity.

Pro-pregnancy hormone progesterone (P4) helps to maintain a quiescent status of uterine tissues during gestation. However, P4's functional role in maintaining fetal membrane (amniochorion) integrity remains unclear. P4 functions through its membrane receptors (progesterone receptor membrane components (PGRMCs)) as fetal membrane cells lack nuclear receptors. This study screened the differential expression of PGRMCs in the fetal membranes and tested P4-PGRMC interactions under normal and oxidative stress (OS) conditions expected that can disrupt P4-PGRMC interactions impacting fetal membrane stability resulting in parturition. Human fetal membranes were collected from term and preterm deliveries (N = 5). Immunohistochemistry and western blot localized and determined differential expression of P4 receptors. Primary amnion epithelial, mesenchymal (AMCs), and chorion cell were treated with P4 alone or co-treated (P4 + OS induced by cigarette smoke extract (CSE)). Proximity ligation assay (PLA) documented P4-receptor binding, whereas P4 enzyme-linked immunosorbent assay documented culture supernatant levels. Immunohistology confirmed lack of nuclear progesterone receptors; however, confirmed expressions of PGRMC 1 and 2. Term labor (P = 0.01) and preterm rupture (P = 0.01) are associated with significant downregulation of PGRMC2. OS-induced differential downregulation of PGRMCs in both amnion and chorion cells (all P < 0.05) and downregulates P4 release (AMCs; P = 0.01). The PLA showed preferential receptor-ligand binding in amnion and chorion cells. Co-treatment of P4 + CSE did not reverse CSE-induced effects. In conclusion, P4-PGRMCs interaction maintains fetal membranes' functional integrity throughout pregnancy. Increased OS reduces endogenous P4 production and cell type-dependent downregulation of PGRMCs. These changes can lead to fetal membrane-specific "functional progesterone withdrawal," contributing to the dysfunctional fetal membrane status seen at term and preterm conditions.

Oxidative stress promotes cellular damages in the cervix: implications for normal and pathologic cervical function in human pregnancy†.

A physiologic increase in reactive oxygen species throughout pregnancy is required to remodel the cervix. Oxidative stress can cause cellular damage that contributes to dysfunctional tissue. This study determined the oxidative stress-induced cell fate of human cervical epithelial and cervical stromal cells. We treated the ectocervical and endocervical epithelial cells and cervical stromal cells with cigarette smoke extract, an oxidative stress inducer, for 48 h. Cell viability (crystal violet assay); cell cycle, apoptosis, and necrosis (flow cytometry); senescence (senescence-associated ß-galactosidase staining); autophagy (staining for autophagosome protein, microtubule-associated protein 1 light chain 3B); stress signaler p38 mitogen-activated protein kinases pathway activation (western blot analyses); and inflammation by measuring interleukin-6 (enzyme-linked immunosorbent assay) were conducted after 48 h of cigarette smoke extract treatment. Oxidative stress induced reactive oxygen species production in cervical cells, which was inhibited by N-acetylcysteine. Oxidative stress promoted cell cycle arrest and induced necrosis in cervical cells. High senescence and low autophagy were observed in cervical stromal cells under oxidative stress. Conversely, senescence was low and autophagy was high in endocervical epithelial cells. Oxidative stress induced p38 mitogen-activated protein kinases (p38MAPK) activation in all cervical cells but only increased interleukin-6 production by the ectocervical epithelial cells. Inhibition of interleukin-6 production by a p38 mitogen-activated protein kinases inhibitor confirmed the activation of an oxidative stress-induced pathway. In conclusion, oxidative stress can promote cell death and sterile inflammation that is mediated by p38 mitogen-activated protein kinases activation in the cellular components of the cervix. These cellular damages may contribute to the normal and premature cervical ripening, which can promote preterm birth.

Cross talk: trafficking and functional impact of maternal exosomes at the feto-maternal interface under normal and pathologic states†.

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (ecto), endocervical epithelial cells (endo), and cervical stromal cells (stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual cells, chorion trophoblast cells (CTC), chorion mesenchymal cells (CMC), amnion mesenchymal cells (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. Enzyme-linked immunosorbent assay for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

Microvesicles and exosomes released by amnion epithelial cells under oxidative stress cause inflammatory changes in uterine cells†.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 107 control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P < 0.05). AEC produced cup-shaped exosomes of 90-150 nm and circular MVs of 160-400 nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P < 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human ß-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response.

BACKGROUND: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. METHODS: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. RESULTS: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. CONCLUSION: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract.