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.

Palmitate and group B Streptococcus synergistically and differentially induce IL-1ß from human gestational membranes.

Introduction: Rupture of the gestational membranes often precedes major pregnancy complications, including preterm labor and preterm birth. One major cause of inflammation in the gestational membranes, chorioamnionitis (CAM) is often a result of bacterial infection. The commensal bacterium Streptococcus agalactiae, or Group B Streptococcus (GBS) is a leading infectious cause of CAM. Obesity is on the rise worldwide and roughly 1 in 4 pregnancy complications is related to obesity, and individuals with obesity are also more likely to be colonized by GBS. The gestational membranes are comprised of several distinct cell layers which are, from outermost to innermost: maternally-derived decidual stromal cells (DSCs), fetal cytotrophoblasts (CTBs), fetal mesenchymal cells, and fetal amnion epithelial cells (AECs). In addition, the gestational membranes have several immune cell populations; macrophages are the most common phagocyte. Here we characterize the effects of palmitate, the most common long-chain saturated fatty acid, on the inflammatory response of each layer of the gestational membranes when infected with GBS, using human cell lines and primary human tissue. Results: Palmitate itself slightly but significantly augments GBS proliferation. Palmitate and GBS co-stimulation synergized to induce many inflammatory proteins and cytokines, particularly IL-1ß and matrix metalloproteinase 9 from DSCs, CTBs, and macrophages, but not from AECs. Many of these findings are recapitulated when treating cells with palmitate and a TLR2 or TLR4 agonist, suggesting broad applicability of palmitate-pathogen synergy. Co-culture of macrophages with DSCs or CTBs, upon co-stimulation with GBS and palmitate, resulted in increased inflammatory responses, contrary to previous work in the absence of palmitate. In whole gestational membrane biopsies, the amnion layer appeared to dampen immune responses from the DSC and CTB layers (the choriodecidua) to GBS and palmitate co-stimulation. Addition of the monounsaturated fatty acid oleate, the most abundant monounsaturated fatty acid in circulation, dampened the proinflammatory effect of palmitate. Discussion: These studies reveal a complex interplay between the immunological response of the distinct layers of the gestational membrane to GBS infection and that such responses can be altered by exposure to long-chain saturated fatty acids. These data provide insight into how metabolic syndromes such as obesity might contribute to an increased risk for GBS disease during pregnancy.

Stretch Causes cffDNA and HMGB1-Mediated Inflammation and Cellular Stress in Human Fetal Membranes.

Danger-associated molecular patterns (DAMPs) are elevated within the amniotic cavity, and their increases correlate with advancing gestational age, chorioamnionitis, and labor. Although the specific triggers for their release in utero remain unclear, it is thought that they may contribute to the initiation of parturition by influencing cellular stress mechanisms that make the fetal membranes (FMs) more susceptible to rupture. DAMPs induce inflammation in many different tissue types. Indeed, they precipitate the subsequent release of several proinflammatory cytokines that are known to be key for the weakening of FMs. Previously, we have shown that in vitro stretch of human amnion epithelial cells (hAECs) induces a cellular stress response that increases high-mobility group box-1 (HMGB1) secretion. We have also shown that cell-free fetal DNA (cffDNA) induces a cytokine response in FM explants that is fetal sex-specific. Therefore, the aim of this work was to further investigate the link between stretch and the DAMPs HMGB1 and cffDNA in the FM. These data show that stretch increases the level of cffDNA released from hAECs. It also confirms the importance of the sex of the fetus by demonstrating that female cffDNA induced more cellular stress than male fetuses. Our data treating hAECs and human amnion mesenchymal cells with HMGB1 show that it has a differential effect on the ability of the cells of the amnion to upregulate the proinflammatory cytokines and propagate a proinflammatory signal through the FM that may weaken it. Finally, our data show that sulforaphane (SFN), a potent activator of Nrf2, is able to mitigate the proinflammatory effects of stretch by decreasing the levels of HMGB1 release and ROS generation after stretch and modulating the increase of key cytokines after cell stress. HMGB1 and cffDNA are two of the few DAMPs that are known to induce cytokine release and matrix metalloproteinase (MMP) activation in the FMs; thus, these data support the general thesis that they can function as potential central players in the normal mechanisms of FM weakening during the normal distension of this tissue at the end of a normal pregnancy.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1ß. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1ß production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1ß production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

Transcriptomic analysis delineates preterm prelabor rupture of membranes from preterm labor in preterm fetal membranes.

BACKGROUND: Globally, preterm birth remains the leading cause of death in children younger than 5 years old. Spontaneous preterm birth is comprised of two events that may or may not occur simultaneously: preterm labor and preterm prelabor rupture of membranes (PPROM). To further explore the concept that spontaneous preterm birth can result from the initializing of two separate but overlapping pathological events, we compared fetal membrane tissue from preterm labor deliveries to fetal tissue from preterm labor with PPROM deliveries. We hypothesized that the fetal membrane tissue from preterm labor with PPROM cases will have an RNA-seq profile divergent from the fetal membrane tissue from preterm labor controls. METHODS: Chorioamnion, separated into amnion and chorion, was collected from eight gestationally age-matched cases and controls within 15 min of birth, and analyzed using RNA sequencing. Pathway enrichment analyses and functional annotations of differentially expressed genes were performed using KEGG and Gene Ontogeny Pathway enrichment analyses. RESULTS: A total of 1466 genes were differentially expressed in the amnion, and 484 genes were differentially expressed in the chorion (log2 fold change > 1, FDR < 0.05) in cases (preterm labor with PPROM), versus controls (preterm labor only). In the amnion, the most significantly enriched (FDR < 0.01) KEGG pathway among down-regulated genes was the extracellular matrix receptor interaction pathway. Seven of the most significantly enriched pathways were comprised of multiple genes from the COL family, including COL1A, COL3A1, COL4A4, and COL4A6. In the chorion, the most significantly enriched KEGG pathways in up-regulated genes were chemokine, NOD receptor, Toll-like receptor, and cytokine-cytokine receptor signaling pathways. Similarly, KEGG pathway enrichment analysis for up-regulated genes in the amnion included three inflammatory pathways: cytokine-cytokine interaction, TNF signaling and the CXCL family. Six genes were significantly up regulated in chorionic tissue discriminated between cases (preterm labor with PPROM) and controls (preterm labor only) including GBP5, CXCL9, ALPL, S100A8, CASP5 and MMP25. CONCLUSIONS: In our study, transcriptome analysis of preterm fetal membranes revealed distinct differentially expressed genes for PPROM, separate from preterm labor. This study is the first to report transcriptome data that reflects the individual pathophysiology of amnion and chorion tissue from PPROM deliveries.

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae.

The maternal decidua is a transient and dynamic tissue that functions as an immunoprivileged matrix related to nutritional and endocrine processes. The function of decidual cells is key to the success of embryo implantation and the maintenance of pregnancy with a positive maternal-fetal outcome. Therefore, establishing a method to optimize the isolation of primary decidual cells is essential. Our protocol described here provides a good yield of decidual cells in an optimized time.

Evaluation of Leukocyte Chemotaxis Induced by Human Fetal Membranes in an In Vitro Model.

Leukocyte infiltration into the maternal-fetal interface is a consequence of the robust inflammation in the gestational tissues during term labor and preterm labor with or without infection. During pregnancy, the fetal membranes act as a physical barrier that isolates the fetus into the amniotic cavity, keeping it in an optimal environment for its development. In addition, the fetal membranes possess immunological competencies such as the secretion of cytokines and chemokines in response to different stimuli. Clinical and experimental evidence indicates that these tissues are involved in the extensive chemotaxis of immune cells in normal or pathological conditions.Few studies have evaluated the chemotactic capacities of the fetal membranes considering that this tissue is composed of two adjacent tissues, the amnion and the chorion, which have different characteristics. Although these tissues function as a unit, their response is complex since there is an interaction between them, where each tissue contributes differently. The protocol described here allows us to evaluate the in vitro chemotactic capacities of fetal membranes in response to various applied stimuli, considering the contribution of each of their components (amnion and choriodecidua) using a Boyden chamber assay and phenotyping the chemo-attracted leukocytes by flow cytometry.

Culture of Human Fetal Membranes in a Two Independent Compartment Model: An Ex Vivo Approach.

During pregnancy, the fetal membranes composed of the amnion and chorodecidua constitute a selective barrier separating two distinct environments, maternal and fetal. These tissues have the function of delimiting the amniotic cavity. Their histological complexity gives them physical, mechanical, and immunological properties to protect the fetus. Although the study of the amnion, chorion, and decidua separately provides knowledge about the functions of the fetal membranes, the protocol we describe in this chapter has the advantage of maintaining the biological and functional complexity of these tissues. In addition, this experimental model allows the researcher to recreate various pathological scenarios because this model allows for differential stimulation of the amnion or choriodecidua.

Feto-Maternal Interface Organ-on-Chip: A New Technology to Study Ascending Infection.

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.

Infection of Fetal Membranes with Mycobacterium tuberculosis and Tissue Processing to Isolate RNA for Expression Analysis.

This chapter outlines the methodology employed to infect the chorionic and amniotic membranes with Mycobacterium tuberculosis during pregnancy. Particularly, congenital tuberculosis, a rare and serious condition associated with cases in neonates and reactivation of latent tuberculosis in pregnant mothers, is interesting to study. Understanding the mechanisms of infection and the response of fetal membranes is crucial for developing effective treatments in these cases, which will promote better neonatal and maternal health in situations of tuberculosis during pregnancy. Establishing a standardized infection model in the chorioamniotic membranes is imperative, followed by a treatment protocol for isolating both cellular and mycobacterial RNA. This will enable the expression analysis during the maternal-fetal interface interaction with M. tuberculosis. The proposed methodology might be invaluable for qRT-PCR, microarrays, and sequencing research.

Role of NF-κB p65/TNF-α in Cell Apoptosis in the Fetal Membranes of Pregnant Women with Preterm Premature Rupture of Membranes.

OBJECTIVE: This study aimed to investigate the roles of nuclear factor-kappa B p65 (NF-[Formula: see text]B p65) and tumor necrosis factor-α (TNF-α) in cell apoptosis occurring in the fetal membranes of pregnant women who experience preterm premature rupture of membranes (PPROM). METHODS: This was a case-control study involving 57 pregnant women who delivered in the obstetric department of Affiliated Loudi Hospital, Hengyang Medical School, University of South China, from June 2021 to June 2022. Samples of fetal membrane tissue were collected from pregnant women with PPROM (n=27) and pregnant women who had normal deliveries (control group; n=30). The membrane tissue morphology of both groups was observed, and the expression of NF-[Formula: see text]B p65, p-NF-[Formula: see text]B p65, TNF-α, and caspase-3 was detected. Apoptosis in fetal membranes was examined. RESULTS: Morphological evaluation of the fetal membrane tissues obtained from patients with PPROM revealed an abnormal structure with a thin collagen fiber layer and cells with a largely vacuolar cytoplasm. There was a positive correlation between the expression of p-NF-[Formula: see text]B p65/NF-[Formula: see text]B p65 and cell apoptosis (r1 =0.89, R2 =0.805, P=0.00). Furthermore, TNF-α was positively correlated with fetal membrane cell apoptosis (r2 =0.93, R2=0.881, P=0.00). CONCLUSION: NF-[Formula: see text]B p65 is involved in the occurrence of PPROM by promoting the expression of TNF-α, which upregulates caspase-3 to cause apoptosis of fetal membrane cells.

Induction of human-fetal-membrane remodeling in-vitro by the alpha hemolysin of Escherichia coli.

INTRODUCTION: Almost 80% of urinary tract infections during pregnancy are caused by uropathogenic strains of Escherichia coli. Alpha-hemolysin, toxin secreted by them, has a fundamental role in this pathology development. Considering that urinary tract infections are related with premature rupture of fetal membranes, we proposed to evaluate the effects that alpha-hemolysin induces on human-fetal-membranes. METHODS: Thirteen fetal membranes obtained from elective cesarean sections (>37 weeks) were mounted in a transwell-device generating two independent chambers. To mimic an ascendant-urinary-tract infection, membranes were incubated with different concentrations of pure alpha-hemolysin from the choriodecidual side during 24h. Extensive histological analyses were performed and transepithelial electrical-resistance were determined. Cell viability, metalloproteinase activity and cyclooxygenase-2- gene expression was estimated by lactate-dehydrogenase-release assay, zymography and RT-qPCR, respectively. Finally, four fetal membranes were treated with hemolysin preincubated with polyclonal anti-hemolysin antibodies. Cell viability and metalloproteinase activity were monitored. RESULTS: After 24 h of treatment, fetal membranes evidenced a structural damage and a decrease in membrane resistance that progressed as the concentration of alpha hemolysin increased. While the amniotic-epithelial-layer remained practically unaffected, the chorion cells manifested an increase in vacuolization and necrosis. In addition, the extracellular matrix exhibited collagen-fiber disorganization, a marked decrease in fiber content, and became thicker in presence of the toxin. Cyclooxigenase-2 expression and metalloproteinase activity were also higher in the treated groups than in untreated ones. Finally, a preincubation of hemolysin with specific antibodies prevented the cytotoxicity on the chorion cells and the increase in metalloproteinase activity. DISCUSSION: Hemolysin induces structural and molecular changes associated with the remodeling of human-fetal-membranes in-vitro.

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.

Membrane inflammasome activation by choriodecidual Ureaplasma parvum infection without intra-amniotic infection in a Non-Human Primate model†.

Intrauterine infection is a significant cause of neonatal morbidity and mortality. Ureaplasma parvum is a microorganism commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms of early stage ascending reproductive tract infection remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes we utilized a non-human primate (NHP) model of choriodecidual U. parvum infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at ~105-112 days gestation and choriodecidual inoculation with U. parvum (105 CFU/mL, n =4) or sterile media (controls; n = 4) starting at 115-119 days, repeated at 5-day intervals until C-section at 136-140 days (term=167 days). The average inoculation to delivery interval was 21 days, and Ureaplasma infection of the amniotic fluid (AF) was undetectable in all animals. Choriodecidual Ureaplasma infection resulted in increased fetal membrane expression of MMP-9 and PTGS2, but did not result in preterm labor or increased concentrations of AF pro-inflammatory cytokines. However, membrane expression of inflammasome sensors, NLRP3, NLRC4, AIM2, and NOD2, and adaptor ASC (PYCARD) gene expression were significantly increased. Gene expression of IL-1ß, IL-18, IL-18R1  , CASPASE-1, and pro-CASPASE-1 protein increased with Ureaplasma infection. Downstream inflammatory genes MYD88 and NFκB (Nuclear factor kappa-light-chain-enhancer of activated B cells) were also significantly upregulated. These results demonstrate that choriodecidual Ureaplasma infection, can cause activation of inflammasome complexes and pathways associated with pPROM and preterm labor prior to microbes being detectable in the AF.

The dependence between glycodelin and selected metalloproteinases concentrations in bovine placenta during early gestation and parturition with and without retained foetal membranes.

Pregnancy course depends on the appropriate connection between the mother and the developing foetus. Pregnancy is completed when the placenta is timely expelled. Placental retention is one of the possible pregnancy complications. Extracellular matrix, including adhesive proteins and enzymes that can break down collagens, seems to be responsible for it. The aim of the present study was to examine the impact of one of the adhesive proteins - glycodelin (Gd) - on selected metalloproteinases degrading collagens (MMP2, MMP3, MMP7). Placental tissues from healthy pregnant cows collected during early-mid pregnancy (2nd month n = 7, 3rd month n = 8, 4th month n = 6) and in cows that properly released placenta (NR; n = 6) and cows with retained foetal membranes (R; n = 6) were experimental material. The concentrations of glycodelin and protein content of selected metalloproteinases were measured by ELISA in the maternal and foetal placental homogenates as well as in the culture of epithelial cells derived from the maternal part of the placenta. The presence of these protein molecules was confirmed by Western Blotting. In the bovine placenta, the concentrations of examined proteins exhibit significant changes during placental formation. Gd, MMP3 and MMP7 concentrations decrease with pregnancy progress (between the 2nd and 4th month), while MMP2 concentrations were on the same level in this period. During parturition, concentrations of Gd and MMP3 were significantly higher in the R group compared to the NR group. In parallel, MMP2 concentrations did not show significant differences between the groups (NR vs R), and MMP7 concentrations decreased significantly in the maternal part of the placenta in cows with retained foetal membranes (R). Obtained results show correlations between the gestational age and proteins' (Gd, MMP3, MMP7) concentration, both in the maternal and foetal part of the placenta.

The antioxidant N-acetyl cysteine inhibits cytokine and prostaglandin release in human fetal membranes stimulated ex vivo with lipoteichoic acid or live group B streptococcus.

BACKGROUNDS: Infection during pregnancy is a significant public health concern due to the increased risk of adverse birth outcomes. Group B Streptococcus or Streptococcus agalactiae (GBS) stands out as a major bacterial cause of neonatal morbidity and mortality. We aimed to explore the involvement of reactive oxygen species (ROS) and oxidative stress pathways in pro-inflammatory responses within human fetal membrane tissue, the target tissue of acute bacterial chorioamnionitis. METHODS: We reanalyzed transcriptomic data from fetal membrane explants inoculated with GBS to assess the impact of GBS on oxidative stress and ROS genes/pathways. We conducted pathway enrichment analysis of transcriptomic data using the Database for Annotation, Visualization and Integrated Discovery (DAVID), a web-based functional annotation/pathway enrichment tool. Subsequently, we conducted ex vivo experiments to test the hypothesis that antioxidant treatment could inhibit pathogen-stimulated inflammatory responses in fetal membranes. RESULTS: Using DAVID analysis, we found significant enrichment of pathways related to oxidative stress or ROS in GBS-inoculated human fetal membranes, for example, "Response to Oxidative Stress" (FDR = 0.02) and "Positive Regulation of Reactive Oxygen Species Metabolic Process" (FDR = 2.6*10-4 ). There were 31 significantly changed genes associated with these pathways, most of which were upregulated after GBS inoculation. In ex vivo experiments with choriodecidual membrane explants, our study showed that co-treatment with N-acetylcysteine (NAC) effectively suppressed the release of pro-inflammatory cytokines (IL-6, IL-8, TNF-α) and prostaglandin PGE2, compared to GBS-treated explants (p < .05 compared to GBS-treated samples without NAC co-treatment). Furthermore, NAC treatment inhibited the release of cytokines and PGE2 stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS) in whole membrane explants (p < .05 compared to LTA or LPS-treated samples without NAC co-treatment). CONCLUSIONS: Our study sheds light on the potential roles of ROS in governing the innate immune response to GBS infection, offering insights for developing strategies to mitigate GBS-related adverse outcomes.

Preterm membranes are mechanically more resistant than term membranes.

OBJECTIVE: To compare the biomechanical properties of fetal preterm membranes (20 + 0 weeks to 30 + 0 weeks) to those of the term (37 + 0 to 41 + 0 weeks). METHOD: Amnion and chorion were manually separated and samples were cut to the required geometry. Rectangular samples with (mode 1) and without (uniaxial) a notch, were tested for tearing energy, critical elongation, and tangent stiffness. Suture retention and inter-suture distance testing investigated the effect of suture placement. RESULTS: From the 15 preterm and 10 term placentas studied, no notable differences were observed in uniaxial testing. Mode 1 fracture testing showed a difference in tearing energy between the preterm and term chorion (0.025 ± 0.005 vs. 0.017 ± 0.005 J/m-1 ; p = 0.027) but not in the amnion (0.030 ± 0.017 vs. 0.029 ± 0.009 J/m-1 ; p = 0.895). Both preterm amnion and chorion showed a higher critical elongation compared with term (1.229 ± 0.057 vs. 1.166 ± 0.046; p = 0.019 and 1.307 ± 0.049 vs. 1.218 ± 0.058; p = 0.012). Preterm amnion had a higher suture retention strength than its term counterpart (0.189 ± 0.065 vs. 0.121 ± 0.031 N; p = 0.023). In inter-suture distance tests, no significant interaction was observed beyond 3 mm, but the preterm chorion showed less interaction at 1-2 mm distances. CONCLUSION: Preterm membranes have equivalent or superior tensile properties to term membranes. The chorion appears to contribute to the mechanical integrity of fetal membranes, particularly in preterm stages.

Anatomy of the fetal membranes: insights from spinning disk confocal microscopy.

PURPOSE: The fetal membranes are essential for the maintenance of pregnancy, and their integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies. METHODS: In this proof of principle study, we collected fetal membranes from women undergoing vaginal delivery or cesarean section. Small membrane sections were then fixed, stained for nucleic acids, actin, and collagen using fluorescent probes, and subsequently imaged in three dimensions using a spinning disk confocal microscope. RESULTS: Four fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro) fractures could be identified in several samples. CONCLUSION: The presented method proves to be well suited to determine whether and how the occurrence of membrane (micro) fractures and cellular jamming correlate with the timing of membrane rupture and the mode of delivery. In future measurements, this method could be combined with mechanical probing techniques to compare optical and mechanical sample information.

Could assisted reproductive techniques affect equine fetal membranes and neonatal outcome?

Embryo transfer (ET) and intracytoplasmic sperm injection (ICSI) are widely used in equine species, but their effects on fetal adnexa and neonates have not been investigated yet. The aim of this study was to retrospectively evaluate whether pregnancies obtained by ET or ICSI could be associated with the presence of macroscopic alterations of fetal membranes (FM) and umbilical cord (UC) and if the use of these techniques could influence neonatal outcome. Sixty-six light breed mares hospitalized at the Veterinary Teaching Hospital, University of Bologna, for attending delivery were included in the study. Mares were divided into Artificial Insemination (AI; 32/66 mares, 48 %), Embryo Transfer (ET; 12/66 mares, 18.2 %) and Intracytoplasmic Sperm Injection (ICSI; 22/66 mares, 33 %) groups. All the medical reports of mares and their foals were reviewed and data about mare, pregnancy, foaling, fetal membranes, umbilical cord and foal were recorded. The occurrence of dystocia resulted statistically different between AI group and ICSI group (p = 0.0066), and between AI group and ET group (p = 0.044). Macroscopic examination of FM revealed alterations in 30/66 mares (46 %): 8/32 in AI (25 %), 7/12 in ET (58 %) and 15/22 in ICSI (68 %) with significant lower incidence in AI compared to ET (p = 0.04) and ICSI (p = 0.002) groups. Alterations reported were chorionic villi hypoplasia, chorioallantois edema, allantois cysts, necrotic areas and greenish-grey concretions. Total length of UC resulted significantly shorter in ICSI group (49 ± 9 cm; p < 0.03) compared to AI (60 ± 17 cm) and ET (59 ± 15 cm). However, there were no differences in the incidence of foals' diseases at birth and in foals' survival among groups (p > 0.05). The results demonstrate that transfer of in vivo or in vitro produced embryos may lead to alterations of placental development, as observed in other species, without being associated with a higher incidence of neonatal morbidity and mortality. Further studies about trophoblast development, FM histological evaluation, and placental gene expression should be carried out to clarify the mechanisms underlying the placental alterations.

CD8+ T cells in fetal membranes display a unique phenotype, and their activation is involved in the pathophysiology of spontaneous preterm birth.

Preterm labor/birth is the leading cause of perinatal mortality and morbidity worldwide. Previous studies demonstrated that T cells were crucial for maintaining maternal-fetal immune tolerance during the first trimester of pregnancy; however, their phenotypes and functions in labor and delivery remain largely unknown. We recruited three cohorts of women at delivery for T-cell immunophenotyping in the placentas, fetal membranes, umbilical cord blood, and maternal peripheral blood. Our data showed a differential enrichment of T cells during the third trimester of human pregnancy, with CD4+ T cells being more observable within the umbilical cord blood, whereas CD8+ T cells became relatively more abundant in fetal membranes. CD4+ and CD8+ T cells derived from fetal membranes were dominated by effector memory T cells and exhibited extensive expression of activation markers but decreased expression of homing receptor. In comparison with term births, fetal membrane CD8+ T cells, especially the central memory subset, were significantly increased in frequency and showed more profound activation in spontaneous preterm birth patients. Finally, using an allogeneic mouse model, we found that T-cell-activation-induced preterm birth could be alleviated by the depletion of CD8+ T but not CD4+ T cells in vivo. Collectively, we showed that CD8+ T cells in fetal membranes displayed a unique phenotype, and their activation was involved in the pathophysiology of spontaneous preterm birth, which provides novel insights into the immune mechanisms of preterm birth and potential targets for the prevention of this syndrome. © 2023 The Pathological Society of Great Britain and Ireland.