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.

Culture and Maintenance of Immune Cells to Model Innate Immune Status at the Feto-maternal Interface.

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.

Endocrine-disrupting compounds and their impact on human placental function: evidence from placenta organ-on-chip studies.

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.

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.

Validation of vaginal microbiome proxies for in vitro experiments that biomimic Lactobacillus-dominant vaginal cultures.

The vaginal microbiome includes diverse microbiota dominated by Lactobacillus [L.] spp. that protect against infections, modulate inflammation, and regulate vaginal homeostasis. Because it is challenging to incorporate vaginal microbiota into in vitro models, including organ-on-a-chip systems, we assessed microbial metabolites as reliable proxies in addition to traditional vaginal epithelial cultures (VECs). Human immortalized VECs cultured on transwells with an air-liquid interface generated stratified cell layers colonized by transplanted healthy microbiomes (L. jensenii- or L. crispatus-dominant) or a community representing bacterial vaginosis (BV). After 48-h, a qPCR array confirmed the expected donor community profiles. Pooled apical and basal supernatants were subjected to metabolomic analysis (untargeted mass spectrometry) followed by ingenuity pathways analysis (IPA). To determine the bacterial metabolites' ability to recreate the vaginal microenvironment in vitro, pooled bacteria-free metabolites were added to traditional VEC cultures. Cell morphology, viability, and cytokine production were assessed. IPA analysis of metabolites from colonized samples contained fatty acids, nucleic acids, and sugar acids that were associated with signaling networks that contribute to secondary metabolism, anti-fungal, and anti-inflammatory functions indicative of a healthy vaginal microbiome compared to sterile VEC transwell metabolites. Pooled metabolites did not affect cell morphology or induce cell death (∼5.5%) of VEC cultures (n = 3) after 72-h. However, metabolites created an anti-inflammatory milieu by increasing IL-10 production (p = .06, T-test) and significantly suppressing pro-inflammatory IL-6 (p = .0001), IL-8 (p = .009), and TNFα (p = .0007) compared to naïve VEC cultures. BV VEC conditioned-medium did not affect cell morphology nor viability; however, it induced a pro-inflammatory environment by elevating levels of IL-6 (p = .023), IL-8 (p = .031), and TNFα (p = .021) when compared to L.-dominate microbiome-conditioned medium. VEC transwells provide a suitable ex vivo system to support the production of bacterial metabolites consistent with the vaginal milieu allowing subsequent in vitro studies with enhanced accuracy and utility.

Functional interferon-epsilon gene polymorphisms and sexually transmitted infections of the endometrium.

PROBLEM: Interferon-epsilon (IFNε) is the only type I IFN constitutively expressed in the female reproductive tract and fluctuates across the menstrual cycle in humans. Mouse models show that IFNε protects against Chlamydia trachomatis, Herpes Simplex Virus, HIV, and Zika in mice, but human studies are limited. Bacterial sexually transmitted infections (STI) can ascend to the upper genital tract and cause pelvic inflammatory disease (PID) and subsequent infertility. However, the host immunological mechanisms that play a role in the ascension and infection of the endometrium in individuals with clinically suspected PID are not elucidated. METHOD OF STUDY: This pilot investigation determined if IFNε gene variants are associated with bacterial vaginosis (BV) and endometrial infection with C. trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium using biospecimens from 154 self-report Black individuals who participated in the PID Evaluation and Clinical Health (PEACH) study. RESULTS: The T allele for rs2039381 was associated with endometrial STI infection (OR 2.7, 95% CI: 1.0-7.1) and the C allele for rs1125488 was inversely associated with BV (OR: .2, 95% CI: .05-.8). CONCLUSIONS: Few studies have examined IFNε gene variants, our study raises the possibility that IFNε gene variants may be a potential host contributor to STI pathogenesis.

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 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.

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.

Chlamydia trachomatis antigen induces TLR4-TAB1-mediated inflammation, but not cell death, in maternal decidua cells.

BACKGROUND: During gestation, the decidua is an essential layer of the maternal-fetal interface, providing immune support and maintaining inflammatory homeostasis. Although Chlamydia (C.) trachomatis is associated with adverse pregnancy outcomes the pathogenic effects on maternal decidua contributing to adverse events are not understood. This study examined how C. trachomatis antigen affects cell signaling, cell death, and inflammation in the decidua. METHODS: Primary decidua cells (pDECs) from term, not-in-labor, fetal membrane-decidua were cultured using the following conditions: (1) control - standard cell culture conditions, (2) 100 ng/ml or (3) 200 ng/ml of C. trachomatis antigen to model decidual cell infection in vitro. Differential expression of Toll-like receptor (TLR) 4 (receptor for C. trachomatis antigen), signaling pathway markers phosphorylated TGF-Beta Activated Kinase 1 (PTAB1), TAB1, phosphorylated p38 mitogen-activated protein kinases (Pp38 MAPK), and p38 MAPK (western blot), decidual cell apoptosis and necrosis (flow cytometry), and inflammation (ELISA for cytokines) were determined in cells exposed to C. trachomatis antigen. T-test was used to assess statistical significance (p < 0.05). RESULTS: C. trachomatis antigen significantly induced expression of TLR4 (p = 0.03) and activation of TAB1 (p = 0.02) compared to controls. However, it did not induce p38 MAPK activation. In addition, pDECs maintained their stromal cell morphology when exposed to C. trachomatis antigen showing no signs of apoptosis and/or necrosis but did induce pro-inflammatory cytokine interleukin (IL)-6 (100 ng/ml: p = 0.02 and 200 ng/ml: p = 0.03), in pDECs compared to controls. CONCLUSION: Prenatal C. trachomatis infection can produce antigens that induce TLR4-TAB1 signaling and IL-6 inflammation independent of Pp38 MAPK and apoptosis and necrosis. This suggests that C. trachomatis can imbalance decidual inflammatory homeostasis, potentially contributing to adverse events during pregnancy.

Testing of drugs using human feto-maternal interface organ-on-chips provide insights into pharmacokinetics and efficacy.

Objectives: To improve preclinical drug testing during pregnancy, we developed multiple microfluidic organ-on-chip (OOC) devices that represent the structure, functions, and responses of the two feto-maternal interfaces (FMis) in humans (fetal membrane [FMi-OOC] and placenta [PLA-OOC]). This study utilized feto-maternal interface OOCs to test the kinetics and efficacy of drugs during pregnancy. Study design: The FMi-OOC contained amnion epithelial, mesenchymal, chorion trophoblast, and decidual cells. The PLA-OOC contained cytotrophoblasts (BeWo), syncytiotrophoblasts (BeWo + forskolin), and human umbilical vein endothelial cell lines. Therapeutic concentrations of either pravastatin or rosuvastatin (200 ng mL-1), a model drug for these experiments, were applied to either decidua (in FMi-OOC) and syncytiotrophoblasts (in PLA-OOC) chambers under normal and oxidative stress conditions (induced by cigarette smoke extract [CSE 1 : 25]) to evaluate maternal drug exposure during normal pregnancy or oxidative stress (OS) associated pathologies, respectively. We determined statin pharmacokinetics and metabolism (LC-MS/MS), drug-induced cytotoxicity (LDH assay), and efficacy to reduce OS-induced inflammation (multiplex cytokine assay). Results: Both OOCs mimicked two distinct human feto-maternal interfaces. The drugs tested permeated the maternal-fetal cell layers of the FMi-OOC and PLA-OOC within 4 hours and generated cell and time-specific statin metabolites from various cell types without causing any cytotoxicity. OS-induced pro-inflammatory cytokines were effectively reduced by statins by increasing anti-inflammatory cytokine response across the FMi-OOC and PLA-OOC. Conclusion: Two distinct feto-maternal interface OOCs were developed, tested, and validated for their utility to conduct preclinical trials during pregnancy. We demonstrated that the placenta and fetal membranes-decidual interface both are able to transport and metabolize drugs and that the safety and efficacy of a drug can be determined using the anatomical structures recreated on OOCs.

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.

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP).

During pregnancy, the placenta is established as a primary organ for drug transport at the maternal-fetal interface. The fetal membranes (FM) also form an interface with maternal tissues; however, their role in drug transport has not been previously investigated. Knowledge of drug transport across this feto-maternal interface along with the placenta can improve new drug development and testing for use during pregnancy. We also hypothesize that extracellular vesicles (exosomes 30-160 nm) released from the FM and placental cells may also contain drug transport proteins and might impact drug trafficking across the feto-maternal interfaces. The objectives were to (1) localize the breast cancer resistance protein (BCRP) in human FM; (2) determine the drug transport function of BCRP in chorion trophoblast cells (CTCs) of the FM; and (3) investigate the presence of BCRP in FM cell-derived exosomes, as a paracrine modifier of the tissue environment for transport functions. The gene and protein expressions of ABCG2/BCRP in FMs were determined by quantitative real-time PCR (qRT-PCR) and western blotting (WB) and were localized by immunohistochemistry (IHC). The surface expression of BCRP in FM cells was determined by flow cytometry. The functional role of BCRP was assessed by an EFFLUX dye multidrug resistance assay. The presence of BCRP in exosomes derived from CTCs and BeWo cells was examined using ExoView®. Data derived from CTCs are compared with placental trophoblast cells (BeWo). BCRP is expressed and localized in the fetal membrane, primarily in the chorion trophoblast cell layer and scarcely in the amnion epithelial layer (AEC), and primarily localized on both AEC and CTC cell surfaces. Efflux assay data showed that FM cells have similar drug resistance activity as BeWo cells, suggesting that FM also have drug transportation capabilities. BeWo- and CTC-derived exosomes expressed limited BCRP protein on the surface, so it was predominantly contained in the exosomal lumen. As far as we are aware, this is the first study to report BCRP expression in fetal membrane cells and as cargo in fetal membrane-derived exosomes. We report that fetal membrane cells are capable of drug transportation. Based on these results, investigational drug trials should include the FM and its exosomes as possible drug transportation routes in pregnancy.

Identifying Strategies for Robust Survivorship Program Implementation: A Qualitative Analysis of Cancer Programs.

PURPOSE: The Commission on Cancer seeks to promote robust survivorship programs among accredited cancer programs. In practice, cancer programs' survivorship programs range from cursory (eg, developing care plans without robust services) to robust (eg, facilitating follow-up care). To inform cancer programs' future efforts, in this study, we identified the implementation strategies that cancer programs used to achieve robust survivorship programs, distinguishing them from cursory programs. METHODS: We sampled 39 cancer programs across the United States with approaches to survivorship program implementation ranging from cursory to robust on the basis of LIVESTRONG survivorship care consensus elements. Within sampled cancer programs, we conducted in-depth semistructured interviews with a total of 42 health care professionals. We used template analysis to distinguish implementation strategies used in cancer programs with robust survivorship programs from strategies that yielded cursory survivorship programs. RESULTS: Cancer programs with robust survivorship programs established clear systems survivorship care and formal committees to improve the survivorship care processes. They sought buy-in from multiple stakeholders to leverage cancer program resources and defined clear roles with shared accountability among multidisciplinary groups. By contrast, cancer programs with cursory survivorship programs reported less consistency in survivorship care processes and lacked buy-in from key stakeholders. They had limited resources, faced persistent structural concerns, and had insufficient clarity in roles among team members. CONCLUSION: Accrediting bodies may consider incorporating the implementation strategies that robust survivorship programs have used as guidance for supporting cancer programs in operationalizing survivorship care and evaluating the use of these strategies during the accreditation and review process.

Molecular mechanisms of environmental toxin cadmium at the feto-maternal interface investigated using an organ-on-chip (FMi-OOC) model.

Human labor is associated with feto-maternal-derived signals that coordinate to initiate delivery. Exposure to environmental chemicals can prematurely trigger labor-initiating signals at the feto-maternal interface (FMi: decidua, amniochorion), leading to spontaneous preterm birth (PTB). Testing the association between environmental chemical exposure and PTB is difficult due to many limitations in vivo or in vitro. Physiological organ-on-chips (OOCs) are potential alternatives for studying mechanisms leading to PTB. The presented study tested the effect of maternal exposure to cadmium (Cd), an environmental toxin, using the FMi-OOC that incorporates maternal decidua cells and three different fetal cells (chorion, amnion mesenchymal, and amnion epithelial cells). Cd transport through the FMi and its impact on cell cycle, cell death, and inflammation were analyzed. Cd treatment resulted in significant cell death and a pro-inflammatory environment in the maternal decidua, but had minimal effect on the fetal chorion cells, and no effect in the fetal amnion cells compared to controls. The maternal response, but lack of fetal response, indicates that Cd-mediated adverse effects originate from maternal pathophysiology rather than fetal-derived triggers of preterm labor. This study demonstrates that the FMi-OOC can indeed predict the response of FMi upon exposure to chemicals, opening the possibility for using OOC models for environmental toxin screens.

Outcomes for non-operatively managed fracture neck of femur patients: A single-institution study.

BACKGROUND: The majority of neck of femur (NOF) fractures are treated operatively in the United Kingdom. The literature reports the advantages of operative management for these patients. However, whilst a subset is treated non-operatively, there is currently no clear guidance for the selection and subsequent management of these patients. This study aims to explore the incidence, demographics, inpatient stay, use of imaging and outcomes of patients who have non-operatively managed NOF fractures. METHODS: A 6-year retrospective review (2013-2019) of all non-operatively managed NOF fractures at a tertiary teaching hospital and major trauma centre was conducted. Electronic patient records, radiographs and National Hip Fracture Database (NHFD) data were used to obtain information. We noted demographic details, fracture classification, rationale for non-operative management, mortality, clinical frailty score (CFS), use of imaging and analgesia requirements. Patients who were repatriated or transferred to other sites for specialist surgery were excluded. RESULTS: 3.2% (99/3132) of NOF fractures were managed non-operatively. The two commonest reasons for non-operative management were either 'comfortable mobilisation' (n = 44) or 'patient frailty/medically unwell' (n = 50). 74% (37/50) of the patients in whom operative risk was thought to outweigh benefit died within the 30 days of admission and 1-year mortality for this group was 92% (46/50). Of the "comfortable mobilisation" subgroup only 18% (8/44) of this patient subgroup subsequently required surgical intervention for failed non-operative management. The 30-day mortality for this cohort was 6.8% with a 1-year mortality rate of 25% (11/44). CONCLUSION: For a select group of patients whose fractures are stable enough to allow them to mobilise comfortably, non-operative management resulted in a 25% 1-year mortality rate and average length of stay of 10.1 days. This is comparable to statistics for overall NOF fracture management in the literature according to the NHFD January 2021 report. 82% of this group of patients were successfully managed without an operation indicating that there is a place for the consideration of non-operative management in a small select subgroup of hip fracture patients with minimally displaced, stable fractures. Further analysis is necessary to assess the functional outcomes of this subgroup, as well as the potential cost implications.

Progesterone alters human cervical epithelial and stromal cell transition and migration: Implications in cervical remodeling during pregnancy and parturition.

The cervix undergoes extensive remodeling throughout pregnancy and parturition. This process involves both ECM collagen degradation and cellular remodeling, which includes cell proliferation, transition and migration. Progesterone (P4) has been used clinically to delay cervical ripening and prevent preterm birth (PTB). However, the mechanisms by which progesterone affects cell transition and the migration of cervical epithelial and stromal cells are not yet fully known. In this study, we documented the role of a gestational level of P4 in the cellular transition (epithelial-mesenchymal transition [EMT] and mesenchymal-epithelial transition [MET]), cell migration, and inflammatory responses of endocervical epithelial cells (EEC) and cervical stromal cells (CSC). EEC and CSC were treated with LPS and P4 for 6 days. The epithelial:mesenchymal ratio (regular microscopy and cell shape index analysis), shift in intermediate filaments (immunofluorescence microscopy and western blot analyses for cytokeratin [CK]-18 and vimentin), adhesion molecules and transcription factors (western blot analyses for E-cadherin, N-cadherin and SNAIL), were used to determine growth characteristics and EMT and MET changes in EEC and CSC under the indicated conditions. To test cell remodeling, scratch assays followed by cellular analyses as mentioned above were performed. Inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor α [TNFα]) and matrix metallopeptidase 9 (MMP9) were measured by ELISA. LPS promoted EMT (decreased cell shape index, decreased CK-18 and E-cadherin, increased vimentin, N-cadherin, and SNAIL), and increased IL-6 and MMP9 production by EEC. A gestational level of P4 prevented LPS-induced EMT in EEC and exhibited anti-inflammatory effect in both EEC and CSC. LPS slowed down wound healing in CSC but P4 treatment prevented the negative impact of LPS in CSC wound healing. These results may explain the cellular mechanisms by which P4 helps to stabilize the cervical epithelial barrier and preserve the mechanical and tensile strength of the cervical stromal layer, which are important in normal cervical remodeling processes during pregnancy.

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.

Effects of a gestational level of estradiol on cellular transition, migration, and inflammation in cervical epithelial and stromal cells.

PROBLEM: Estrogen (E2) is one of the main steroid hormones associated with pregnancy and parturition. High levels of E2 increase uterine contractions, promote fetal membrane weakening, and induce degradation of the cervical extracellular matrix (ECM). Current evidence supports the role of E2 in epithelial-to-mesenchymal transition (EMT) and inflammation in different cell types; however, its effects on the cellular components of the cervix are still unknown. METHOD OF STUDY: In this study, we assessed the effects of gestational levels of E2 in: (a) the cellular transition of endocervical epithelial cells (EEC) and cervical stromal cells (CSC) in vitro using immunocytochemical staining and Western blot analyses for EMT markers (cytokeratin-18, E-cadherin, N-cadherin, SNAIL, and vimentin); (b) cell migration using in vitro scratch assays; (c) inflammatory cytokine (interleukin 1ß and TNF-α) and MMP9 production under untreated and lipopolysaccharide (LPS)-treated conditions using immunoassays. RESULTS: E2 treatment and co-treatment with LPS as a proxy for infection maintained the metastate of EEC (expression of both cytokeratin and vimentin) and the mesenchymal state of CSC. E2 delayed wound healing, which mimics the tissue remodeling process, in EEC and CSC. E2 led to persistently elevated levels of vimentin throughout the EEC wound healing process. E2 did not affect inflammatory cytokine production by EEC and CSC but increased MMP9 production by EEC. CONCLUSION: Collectively, these results show that third trimester levels of E2 may permit localized inflammation, increase MMP-9 production, and cause an EMT-mediated impairment of the remodeling process in the cervix in vitro. These data suggest a potential contribution of E2 in cervical ripening.

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

Current intervention strategies have not been successful in reducing the risks of adverse pregnancy complications nor maternal and fetal morbidities associated with pregnancy complications. Improving pregnancy and neonatal outcomes requires a better understanding of drug transport mechanisms at the feto-maternal interfaces, specifically the placenta and fetal membrane (FM). The role of several solute carrier uptake transporter proteins (TPs), such as the organic anion transporting polypeptide 2B1 (OATP2B1) in transporting drug across the placenta, is well-established. However, the mechanistic role of FMs in this drug transport has not yet been elucidated. We hypothesize that human FMs express OATP2B1 and functions as an alternate gatekeeper for drug transport at the feto-maternal interface. We determined the expression of OATP2B1 in term, not-in-labor, FM tissues and human FM cells [amnion epithelial cell (AEC), chorion trophoblast cell (CTC), and mesenchymal cells] using western blot analyses and their localization using immunohistochemistry. Changes in OATP2B1 expression was determined for up to 48 h after stimulation with cigarette smoke extract (CSE), an inducer of oxidative stress. The functional role of OATP2B1 was determined by flow cytometry using a zombie violet dye substrate assay. After OATP2B1 gene silencing, its functional relevance in drug transport through the feto-maternal interface was tested using a recently developed feto-maternal interface organ-on-a-chip (OOC) system that contained both FM and maternal decidual cells. Propagation of a drug (Rosuvastatin, that can be transported by OATP2B1) within the feto-maternal interface OOC system was determined by mass spectrometry. FMs express OATP2B1 in the CTC and AEC layers. In FM explants, OATP2B1 expression was not impacted by oxidative stress. Uptake of the zombie violet dye within AECs and CTCs showed OATP2B1 is functionally active. Silencing OATP2B1 in CTCs reduced Rosuvastatin propagation from the decidua to the fetal AEC layer within the feto-maternal interface-OOC model. Our data suggest that TPs in FMs may function as a drug transport system at the feto-maternal interface, a function that was previously thought to be performed exclusively by the placenta. This new knowledge will help improve drug delivery testing during pregnancy and contribute to designing drug delivery strategies to treat adverse pregnancy outcomes.