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During pregnancy, two fetomaternal interfaces, the placenta-decidua basalis and the fetal membrane-decidua parietals, allow for fetal growth and maturation and fetal-maternal crosstalk, and protect the fetus from infectious and inflammatory signaling that could lead to adverse pregnancy outcomes. While the placenta has been studied extensively, the fetal membranes have been understudied, even though they play critical roles in pregnancy maintenance and the initiation of term or preterm parturition. Fetal membrane dysfunction has been associated with spontaneous preterm birth (PTB, < 37 weeks gestation) and preterm prelabor rupture of the membranes (PPROM), which is a disease of the fetal membranes. However, it is unknown how the individual layers of the fetal membrane decidual interface (the amnion epithelium [AEC], the amnion mesenchyme [AMC], the chorion [CTC], and the decidua [DEC]) contribute to these pregnancy outcomes. In this study, we used a single-cell transcriptomics approach to unravel the transcriptomics network at spatial levels to discern the contributions of each layer of the fetal membranes and the adjoining maternal decidua during the following conditions: scheduled caesarian section (term not in labor [TNIL]; n = 4), vaginal term in labor (TIL; n = 3), preterm labor with and without rupture of membranes (PPROM; n = 3; and PTB; n = 3). The data included 18,815 genes from 13 patients (including TIL, PTB, PPROM, and TNIL) expressed across the four layers. After quality control, there were 11,921 genes and 44 samples. The data were processed by two pipelines: one by hierarchical clustering the combined cases and the other to evaluate heterogeneity within the cases. Our visual analytical approach revealed spatially recognized differentially expressed genes that aligned with four gene clusters. Cluster 1 genes were present predominantly in DECs and Cluster 3 centered around CTC genes in all labor phenotypes. Cluster 2 genes were predominantly found in AECs in PPROM and PTB, while Cluster 4 contained AMC and CTC genes identified in term labor cases. We identified the top 10 differentially expressed genes and their connected pathways (kinase activation, NF-κB, inflammation, cytoskeletal remodeling, and hormone regulation) per cluster in each tissue layer. An in-depth understanding of the involvement of each system and cell layer may help provide targeted and tailored interventions to reduce the risk of PTB.

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Chorion trophoblasts (CTCs) and immune cell-enriched decidua (DECs) comprise the maternal-fetal membrane interface called the chorio-decidual interface (CDi) which constantly gets exposed to maternal stressors without leading to labor activation. This study explored how CTCs act as a barrier at CDi. The roles of human leukocyte antigen (HLA)-G and progesterone receptor membrane component 2 (PGRMC2) in mediating immune homeostasis were also investigated. The CDi was recreated in a two-chamber microfluidic device (CDi-on-chip) with an outer chamber of primary DECs and immune cell line-derived innate immune cells and an inner chamber of wild-type or PGRMC2 or HLA-G knockout immortalized CTCs. To mimic maternal insults, DECs were treated with lipopolysaccharide, poly(I:C), or oxidative stress inducer cigarette smoke extract. Expression levels of inflammation and immunity genes via targeted RNA sequencing, production of soluble mediators, and immune cell migration into CTCs were determined. In CDi-on-chip, decidua and immune cells became inflammatory in response to insults while CTCs were refractory, highlighting their barrier function. HLA-G and PGRMC2 are found to be vital to immune homeostasis at the CDi, with PGRMC2 serving as an upstream regulator of inflammation, HLA-G expression, and mesenchymal-epithelial transition, and HLA-G serving as a frontline immunomodulatory molecule, thus preventing fetal membrane compromise.

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Preterm birth remains an important global problem, and an important contributor to under-5 mortality. Reducing spontaneous preterm birth rates at the global level will require the early identification of patients at risk of preterm delivery in order to allow the initiation of appropriate prophylactic management strategies. Ideally these strategies target the underlying pathophysiologic causes of preterm labor. Prevention, however, becomes problematic as the causes of preterm birth are multifactorial and vary by gestational age, ethnicity, and social context. Unfortunately, current screening and diagnostic tests are non-specific, with only moderate clinical risk prediction, relying on the detection of downstream markers of the common end-stage pathway rather than identifying upstream pathway-specific pathophysiology that would help the provider initiate targeted interventions. As a result, the available management options (including cervical cerclage and vaginal progesterone) are used empirically with, at best, ambiguous results in clinical trials. Furthermore, the available screening tests have only modest clinical risk prediction, and fail to identify most patients who will have a preterm birth. Clearly defining preterm birth phenotypes and the biologic pathways leading to preterm birth is key to providing targeted, biomolecular pathway-specific interventions, ideally initiated in early pregnancy Pathway specific biomarker discovery, together with management strategies based on early, mid-, and-late trimester specific markers is integral to this process, which must be addressed in a systematic way through rigorously planned biomarker trials.

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Spontaneous preterm birth (PTB) affects around 11% of births, posing significant risks to neonatal health due to the inflammation at the fetal-maternal interface (FMi). This inflammation disrupts immune tolerance during pregnancy, often leading to PTB. While organ-on-a-chip (OOC) devices effectively mimic the physiology, pathophysiology, and responses of FMi, their relatively low throughput limits their utility in high-throughput testing applications. To overcome this, we developed a three-dimensional (3D)-printed model that fits in a well of a 96-well plate and can be mass-produced while also accurately replicating FMi, enabling efficient screening of drugs targeting FMi inflammation. Our model features two cell culture chambers (maternal and fetal cells) interlinked via an array of microfluidic channels. It was thoroughly validated, ensuring cell viability, metabolic activity, and cell-specific markers. The maternal chamber was exposed to lipopolysaccharides (LPS) to induce an inflammatory state, and proinflammatory cytokines in the culture supernatant were quantified. Furthermore, the efficacy of anti-inflammatory inhibitors in mitigating LPS-induced inflammation was investigated. Results demonstrated that our model supports robust cell growth, maintains viability, and accurately mimics PTB-associated inflammation. This high-throughput 3D-printed model offers a versatile platform for drug screening, promising advancements in drug discovery and PTB prevention.

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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.

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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-hours, 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.

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BACKGROUND: Maternal-fetal immunology is intricate, and the effects of mRNA-S maternal vaccination on immune regulation at the maternal-fetal interface require further investigation. Our study endeavors to elucidate these immunological changes, enhancing our comprehension of maternal and fetal health outcomes. By analyzing immune profiles and cytokine responses, we aim to provide valuable insights into the impact of mRNA-S vaccination on the delicate balance of immune regulation during pregnancy, addressing critical questions in the field of reproductive pharmacology. OBJECTIVES: This investigation sought to examine the prospective influence of mRNA-S-based vaccines and extracellular vesicles (EVs) containing the Spike (S) protein at the maternal-fetal interface. Our primary emphasis was on evaluating their effects on maternal decidua cells and fetal chorion trophoblast cells (hFM-CTCs). METHODS: We validated the generation of EVs containing the S protein from small human airway epithelial cell lines (HSAECs) following mRNA-S vaccine exposure. We assessed the expression of angiotensin-converting enzyme 2 (ACE2) gene and protein in fetal membranes and the placenta, with specific attention to decidual cells and fetal membrane chorion cells. To assess cellular functionality, these cells were exposed to both recombinant S protein and EVs loaded with S proteins (eSPs). RESULTS: Our findings revealed that cells and EVs subjected to mRNA-S-based vaccination exhibited altered protein expression levels of S proteins. At the feto-maternal interface, both placental and fetal membrane tissues demonstrated similar ACE-2 expression levels. Among individual cellular layers, syncytiotrophoblast cells in the placenta and chorion cells in the fetal membrane exhibited elevated ACE-2 expression. Notably, EVs derived from HSAECs activated the MAPK pathway in decidual cells. Additionally, decidual cells displayed a substantial increase in gene expression of chemokines like CXCL-10 and CXCL-11, as well as proinflammatory cytokines such as IL-6 in response to eSPs. However, the levels of Ccl-2 and IL-1ß remained unchanged in decidual cells under the same conditions. Conversely, hFM-CTCs demonstrated significant alterations in the proinflammatory cytokines and chemokines with respect to eSPs. CONCLUSION: In conclusion, our study indicates that mRNA-S-based maternal vaccination during pregnancy may influence the maternal-fetal interface's COVID-19 interaction and immune regulation. Further investigation is warranted to assess safety and implications.

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INTRODUCTION: Pregnant women are therapeutic orphans as they are excluded from clinical drug development and therapeutic trials. We identify limitations in conducting clinical trials and propose two 'New Approach Methods'(NAMs) to overcome them. AREAS COVERED: NAMs have proven invaluable tools in basic and clinical research to understand human health and disease better, elucidate mechanisms, and study the efficacy and toxicity of therapeutics that have not been possible through animal-based methodologies. The lack of humanized experimental models of FMi and drugs that can safely and effectively cross FMi to reduce the risk of adverse pregnancy has hindered progress in the field of reproductive pharmacology. This report discusses two technological advancements in perinatal research and medicine to accelerate clinical trials during pregnancy. (1) We have developed a humanized microphysiologic system, an Organ-on-a-chip (OOC) platform, to study FMi and their utility in pharmacological studies, and (2) use of extracellular vesicles (EVs) as drug delivery vehicles that are immunologically inert and can cross the fetomaternal barriers. EXPERT OPINION: We provide an overview of NAMs that can accelerate preclinical trials and develop drugs to cross the feto-maternal barriers to reduce the risk of adverse pregnancy outcomes like preterm birth.

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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. An amnion membrane (AM) organ-on-chip (OOC) was utilized to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 hours flow culture to mimic fluid motion. Static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control for 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 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 flow culture environment is not necessary to mimic any in utero physiologic cellular conditions of fetal membrane-derived cells.

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Immune cells at the feto-maternal interface play an important role in pregnancy; starting at implantation, maintenance of pregnancy, and parturition. The role of decidual immune cells in induction of labor still needs to be understood. Published reports on this topic show heterogeneity in methods of cell isolation, assay, analysis and cellular characterization making it difficult to collate available information in order to understand the contribution of immune cells at term leading to parturition. In the present study, available literature was reviewed to study the differences in immune cells between the decidua basalis and decidua parietalis, as well as between immune cells in term and preterm labor. Additionally, immune cells at the decidua parietalis were isolated from term not in labor (TNL) or term in labor (TL) samples and characterized via flow cytometry using a comprehensive, high-dimensional antibody panel. This allowed a full view of immune cell differences without combining multiple studies, which must include variation in isolation and analysis methods, for more conclusive data. The ratio of cells found in decidua parietalis in this study generally matched those reported in the literature, although we report a lower percentage of natural killer (NK) cells at term. We report that CD4 expression on CD8- NK cells decreased in term labor compared to not in labor samples, suggesting that natural killer cells may be migrating to other sites during labor. Also, we report a decrease in CD38 expression on CD8+ CD57+ T cells in labor, indicative of cytotoxic T cell senescence. Our study provides a comprehensive status of immune cells at the decidua-chorion interface at term.

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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.

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Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device's fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.

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The inflammatory process leading to human labor is mostly facilitated by immune cells, which can be studied by isolating and characterizing primary immune cells from the feto-maternal interface. However, difficulty and inconsistency in sampling approaches of immune cells and short lifespan in vitro prevent their usage in mechanistic studies to understand the maternal-fetal immunobiology. To address these limitations, existing cell line models can be differentiated into immune-like cells for use in reproductive biology experiments. In this chapter, we discussed cell culture methods of maintaining and differentiating HL-60, THP-1, and NK-92 cells to obtain neutrophil-like, macrophage-like, and decidual natural killer-like cells, respectively, which can then be used together with intrauterine cells to elucidate and investigate immune mechanisms that contribute to parturition.

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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.

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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.

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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.

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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.

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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.

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The effects of endocrine-disrupting compounds (EDCs) on the placenta, a critical gestational organ for xenobiotic protection, are well reported; however, models to determine the role of EDCs in placental disruption are limited. An advanced 2nd-trimester human placenta organ-on-chip model (2TPLA-OOC) was developed and validated, with six representative cells of the maternal and the fetal interface interconnected with microchannels. Various EDCs (150 ng mL-1 each of bisphenol A, bisphenol S, and polybrominated diphenyl ethers-47 and -99) were gradually propagated across the chip for 72 hours, and their various effects were determined. Cigarette smoke extract (CSE), an environmental risk factor, was used as a positive control. EDCs produced overall oxidative stress in the placental/decidual cells, induced cell-specific endocrine effects, caused limited (<10%) apoptosis/necrosis in trophoblasts and mesenchymal cells, induced localized inflammation but an overall anti-inflammatory shift, did not change immune cell migration from stroma to decidua, and did not affect placental nutrient transport. Overall, (1) the humanized 2TPLA-OOC recreated the placental organ and generated data distinct from the trophoblast and other cells studied in isolation, and (2) at doses associated with adverse pregnancies, EDCs produced limited and localized insults, and the whole organ compensated for the exposure.

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Ureaplasma parvum is a mycoplasma commonly associated with female reproductive pathologies, such as preterm birth and infertility. It can survive intracellularly and utilize exosomes to propagate infection and its virulence factors. This study explored the differential protein composition of exosomes derived from normal and U. parvum-infected cells. We also investigated the impact of U. parvum on exosome biogenesis in ectocervical epithelial cells. Ectocervical epithelial (ECTO) cells were infected with U. parvum, and immunocytochemical staining was performed using U. parvum-specific marker multiple banded antigen (mba) and exosome marker CD9. NanoLC-MS/MS analysis was conducted to identify differentially expressed proteins in exosomes. Ingenuity Pathway Analysis (IPA) was performed to identify affected canonical pathways and biological functions associated with the protein cargo of exosomes. Western blot analysis of ECTO cells validated the proteomic findings in ECTO cells. U. parvum exhibited colonization of ECTO cells and colocalization with CD9-positive intraluminal vesicles. Proteomic analysis revealed decreased protein abundance and distinct protein profiles in exosomes derived from U. parvum-infected ECTO cells. Differentially expressed proteins were associated with clathrin-mediated endocytosis and various signaling pathways indicative of infection, inflammation, and cell death processes. Additionally, U. parvum infection altered proteins involved in exosome biogenesis. In ECTO cells, U. parvum infection significantly decreased clathrin, ALIX, CD9, and CD63 and significantly increased TSG101, Rab5, Rab35, and UGCG. These findings contribute to our understanding of the infection mechanism and shed light on the importance of exosome-mediated communication in the pathophysiology of diseases affecting the cervix, such as cervicitis and preterm birth.

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