Neutrophils and monocyte toll like receptors 2 and 4 expression in preterm versus term delivery.

Pregnancy results in an increase in immune cells, especially monocytes, which enhances the innate immune system. The increase of inflammatory cytokines in pregnant women's amniotic fluid, can cause uterine contraction, is linked to preterm labor. These inflammatory responses are controlled by Toll-like receptors (TLRs), which are largely expressed on neutrophils and monocytes. This study aimed to determine the role of neutrophils and monocyte subsets, as well as their expression of TLR-2 and TLR-4 in women with preterm and full-term delivery. The study involved a total of 74 women, comprising of 29 preterm labor, 25 full-term labor, and 20 non-pregnant women. The distribution of three monocyte subsets, namely (CD14++CD16-), (CD14+CD16+), and (CD14-/dim CD16++) was measured. Also, the expression of TLR2 and TLR4 in monocytes and neutrophils was analyzed using flow cytometry. Non-classical monocytes and intermediate monocytes were significantly higher in the preterm group than the control and full-term groups (p=0.041, p=0.043, and p=0.004, p= 0.049, respectively). Women in the preterm group showed significantly TLR2 expression on nonclassical monocytes compared to the control and full-term groups (p=0.002, and p=0.010, respectively). Also, preterm group expression of TLR4 was significantly higher in classical monocytes and nonclassical monocytes in comparison to the control group (p=0.019, and p≤0.0001, respectively). Besides, TLR4 expression was significantly up regulated in the preterm group compared to full-term in non-classical monocyte subset (p < 0.0001). Moreover, the expression of TLR-4 in neutrophils from the preterm group was statistically higher than expression from the full-term labor and control groups (p < .0001 for both). Such findings highlight the important role of monocyte subsets and neutrophils in activating the innate immune system and initiating strong pro-inflammatory responses that induce preterm labor. Additionally, TLR4 and TLR2 expressions on non-classical monocytes may be used as a marker to assess the probability of preterm labor.

IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Methyl-CpG-binding protein 2 regulates CYP27A1-induced myometrial contraction during preterm labor.

Persistent and intense uterine contraction is a risk factor for preterm labor. We previously found that methyl-CpG-binding protein 2 (MeCP2), as a target of infection-related microRNA miR-212-3p, may play an inhibitory role in regulating myometrium contraction. However, the molecular mechanisms by which MeCP2 regulates myometrial contraction are still unknown. In this study, we found that MeCP2 protein expression was lower in myometrial specimens obtained from preterm labor cases, compared to those obtained from term labor cases. Herein, using RNA sequence analysis of global gene expression in human uterine smooth muscle cells (HUSMCs) following siMeCP2, we show that MeCP2 silencing caused dysregulation of the cholesterol metabolism pathway. Notably, MeCP2 silencing resulted in the upregulation of CYP27A1, the key enzyme involved in regulating cholesterol homeostasis, in HUSMCs. Methylation-specific PCR, chromatin immunoprecipitation, and dual luciferase reporter gene technology indicated that MeCP2 could bind to the methylated CYP27A1 promoter region and repress its transcription. Administration of siCYP27A1 in a lipopolysaccharide (LPS)-induced preterm labor mouse model delayed the onset of preterm labor. Human preterm myometrium and the LPS-induced preterm labor mouse model both showed lower expression of MeCP2 and increased expression of CYP27A1. These results demonstrated that aberrant upregulation of CYP27A1 induced by MeCP2 silencing is one of the mechanisms facilitating inappropriate myometrial contraction. CYP27A1 could be exploited as a novel therapeutic target for preterm birth.

Role of Platelet Activating Factor as a Mediator of Inflammatory Diseases and Preterm Delivery.

Nearly 70% of preterm deliveries occur spontaneously, and the clinical pathways involved include preterm labor and preterm premature rupture of membranes. Prediction of preterm delivery is considered crucial due to the significant effects of preterm birth on health and the economy at both the personal and community levels. Although similar inflammatory processes occur in both term and preterm delivery, the premature activation of these processes or exaggerated inflammatory response triggered by infection or sterile factors leads to preterm delivery. Platelet activating factor (PAF) is a phosphoglycerylether lipid mediator of inflammation that is implicated in infections, cancers, and various chronic diseases and disorders including cardiovascular, renal, cerebrovascular, and central nervous system diseases. In gestational tissues, PAF mediates the inflammatory pathways that stimulate the effector mechanisms of labor, including myometrial contraction, cervical dilation, and fetal membrane rupture. Women with preterm labor and preterm premature rupture of membranes have increased levels of PAF in their amniotic fluid. In mice, the intrauterine or intraperitoneal administration of carbamyl PAF activates inflammation in gestational tissues, thereby eliciting preterm delivery. This review summarizes recent research on PAF as an important inflammatory mediator in preterm delivery and in other inflammatory disorders, highlighting its potential value for prediction, intervention, and prevention of these diseases.

Investigating ticagrelor in a preclinical pipeline as a novel therapeutic to prevent preterm birth.

In brief: Preterm birth is the leading cause of perinatal morbidity and mortality, and new therapies that delay preterm birth and improve neonatal outcomes are urgently needed. This study investigates whether ticagrelor inhibits uterine contractility and inflammation in preclinical in vitro, ex vivo (human) and in vivo (mouse) studies, to explore the potential of repurposing ticagrelor for the prevention of preterm birth. Abstract: Preterm birth remains a significant global health challenge, affecting approximately 10% of pregnancies and resulting in one million deaths globally every year. Tocolytic agents, used to manage preterm labour, have considerable limitations including lack of efficacy, and adverse side effects, emphasising the urgent need for innovative solutions. Here, we explore repurposing an antiplatelet cardioprotective drug, ticagrelor, as a potential treatment to prevent preterm birth. Ticagrelor has demonstrated pleiotropic actions beyond platelet inhibition, including relaxant effects on smooth muscle cells and anti-inflammatory effects in models of diabetes and sepsis. As preterm birth is underscored by inflammatory processes triggering uterine contractions, these actions position ticagrelor as an attractive candidate for prevention or delay of preterm birth. Utilising primary human myometrial tissue, human myometrial cells, and a mouse model of preterm birth, we investigated ticagrelor's potential as a safe and effective therapy for preterm birth. We showed that ticagrelor did not reduce the frequency or strength of spontaneous muscle contractions of ex vivo myometrial tissue nor did it reduce in vitro inflammation-induced contractility in myometrial cells. Additionally, ticagrelor did not exhibit the anticipated anti-inflammatory effects in myometrial cell culture experiments. In our mouse model of preterm birth, ticagrelor neither improved the preterm birth rate or fetal survival outcomes. Gene expression of pro-inflammatory cytokines and contraction-associated proteins in postpartum mouse uteri were unaltered by ticagrelor. In conclusion, ticagrelor is not a strong candidate to continue investigations in clinical trial for the treatment of preterm labour and prevention of preterm birth.

Endocannabinoid regulation in the cervix during pregnancy: insights into molecular mechanisms of premature labor.

In brief: The cervix plays a crucial role not only in the maintenance of pregnancy but also during delivery, when it undergoes extensive changes. This study highlights the involvement of the endocannabinoidome in cervical remodeling, emphasizing its relevance in the shift from a nonpregnant to pregnant state and its potential contribution to preterm delivery in inflammatory contexts. Abstract: During pregnancy, the main role of the cervix is to isolate the fetus from outside pathogens and maintain the relatively closed system of uterine gestation. Conversely, toward the end of pregnancy, the cervix must be remodeled to increase flexibility and allow the delivery. This process is called cervical remodeling and dysregulation of the process plays a role in premature delivery. The endocannabinoidome plays an important role in several reproductive events; however, its function on cervical tissue throughout pregnancy is poorly understood. The goal of this study was to evaluate the presence and participation of the endocannabinoidome in lipopolysaccharide (LPS)-induced cervical changes. Therefore, we evaluated key components of the endocannabinoidome in cervical tissue from nonpregnant mice and pregnant mice with and without LPS treatment. Using mass spectrometric analysis, we found an increase in anandamide and 2-arachidonoylglycerol in the cervix of pregnant mice when compared to nonpregnant mice. We have also found a reduction in FAAH protein expression in these tissues. Furthermore, when treated with LPS, we observed a reduction in the cervical immunostaining with anti-CB1 and anti-CB2 antibodies. Likewise, using cervix explants from pregnant mice, we found that LPS significantly increased cervical metalloprotease activity and cyclooxygenase 2, which were subsequently modulated by cannabinoid receptor antagonists. Collectively, our findings suggest that an LPS-induced imbalance of cervix endocannabinoidome likely contributes to premature cervical remodeling, which is part of the key components that contribute to premature delivery.

Regulation of 20α-Hydroxysteroid Dehydrogenase Expression in Term Pregnant Human Myometrium Ex Vivo.

Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.

A multivariable prediction model for intra-amniotic infection in patients with preterm labor and intact membranes including a point of care system that measures amniotic fluid MMP-8.

OBJECTIVES: Among patients with preterm labor and intact membranes (PTL), those with intra-amniotic infection (IAI) present the highest risk of adverse perinatal outcomes. Current identification of IAI, based on microbiological cultures and/or polymerase chain reaction amplification of the 16S ribosomal RNA gene, delay diagnosis and, consequently, antenatal management. The aim to of the study was to assess the performance of a multivariable prediction model for diagnosing IAI in patients with PTL below 34.0 weeks using clinical, sonographic and biochemical biomarkers. METHODS: From 2019 to 2022, we prospectively included pregnant patients admitted below 34.0 weeks with diagnosis of PTL and had undergone amniocentesis to rule in/out IAI. The main outcome was IAI, defined by a positive culture and/or 16S ribosomal RNA gene in amniotic fluid. Based on the date of admission, the sample (n=98) was divided into a derivation (2019-2020, n=49) and validation cohort (2021-2022, n=49). Logistic regression models were developed for the outcomes evaluated. As predictive variables we explored ultrasound cervical length measurement at admission, maternal C-reactive protein, gestational age, and amniotic fluid glucose and matrix metalloproteinase-8 (MMP-8) levels. The model was developed in the derivation cohort and applied to the validation cohort and diagnostic performance was evaluated. Clinical management was blinded to the model results. RESULTS: During the study period, we included 98 patients admitted with a diagnosis of PTL. Of these, 10 % had IAI. The final model included MMP-8 and amniotic fluid glucose levels and showed an area under the receiver operating characteristic curve to predict the risk of IAI of 0.961 (95 % confidence interval: 0.860-0.995) with a sensitivity of 75 %, specificity of 93.3 %, positive likelihood ratio (LR) of 11.3 and negative LR of 0.27 in the validation cohort. CONCLUSIONS: In patients with PTL, a multivariable prediction model including amniotic fluid MMP-8 and glucose levels might help in the clinical management of patients undergoing amniocentesis to rule in/out IAI, providing results within a few minutes.

Evaluation of changes of apelin and apelin receptor (APJ) expression in cervix-uterus and placental axis in an LPS-induced preterm labor model.

Although preterm birth is among the preventable causes of maternal and infant death, its mechanism has not yet been clarified. When evaluated in terms of the results, the psycho-social burden of mother-infant losses and the costs of rehabilitation, care, and treatment for postpartum sequelae are high. When evaluated in terms of its causes, infection/inflammation has an important place. Therefore, it is essential to understand the role of pro- and anti-inflammatory proteins in the process. In our study, apelin and apelin receptor (APJ) expression in the cervix-uterus and placental axis were evaluated at tissue and protein levels in pregnant and non-pregnant control, sham, PBS, and LPS groups in the infection model in which LPS induction was performed by midline laparotomy, in CD-1 mice. The evaluation of this axis regarding apelin and apelin receptor in the preterm birth model is new in the literature. Apelin is expressed more intensely in uterine epithelial cells than in the cervix. In the placenta, expression is more intense in the junctional zone compared to other zones. Apelin protein levels decrease significantly in the cervix and placenta whereas it increases in the uterus. While no change was observed in the expression of the apelin receptor at the tissue and protein level in the cervix and uterus, it increased in both aspects in the placenta in the invasive procedure groups. We propose that the decrease in apelin protein due to LPS in the preterm delivery model may be related to the effort to compensate for the balance deteriorated in the pro-inflammatory direction with post-transitional modification at the tissue level. The tendency of apelin to increase with pregnancy has led to the conclusion that it is necessary for a healthy pregnancy. Although the apelin receptor does not change with inflammation, it is necessary to investigate the mechanisms associated with its stress and trauma-induced increase, since it increases in the invasive procedure group.

Prostaglandin F2α requires activation of calcium-dependent signalling to trigger inflammation in human myometrium.

Introduction: Preterm birth is one of the major causes of neonatal morbidity and mortality across the world. Both term and preterm labour are preceded by inflammatory activation in uterine tissues. This includes increased leukocyte infiltration, and subsequent increase in chemokine and cytokine levels, activation of pro-inflammatory transcription factors as NF-κB and increased prostaglandin synthesis. Prostaglandin F2α (PGF2α) is one of the myometrial activators and stimulators. Methods: Here we investigated the role of PGF2α in pro-inflammatory signalling pathways in human myometrial cells isolated from term non-labouring uterine tissue. Primary myometrial cells were treated with G protein inhibitors, calcium chelators and/or PGF2α. Nuclear extracts were analysed by TranSignal cAMP/Calcium Protein/DNA Array. Whole cell protein lysates were analysed by Western blotting. mRNA levels of target genes were analysed by RT-PCR. Results: The results show that PGF2α increases inflammation in myometrial cells through increased activation of NF-κB and MAP kinases and increased expression of COX-2. PGF2α was found to activate several calcium/cAMP-dependent transcription factors, such as CREB and C/EBP-ß. mRNA levels of NF-κB-regulated cytokines and chemokines were also elevated with PGF2α stimulation. We have shown that the increase in PGF2α-mediated COX-2 expression in myometrial cells requires coupling of the FP receptor to both Gαq and Gαi proteins. Additionally, PGF2α-induced calcium response was also mediated through Gαq and Gαi coupling. Discussion: In summary, our findings suggest that PGF2α-induced inflammation in myometrial cells involves activation of several transcription factors - NF-κB, MAP kinases, CREB and C/EBP-ß. Our results indicate that the FP receptor signals via Gαq and Gαi coupling in myometrium. This work provides insight into PGF2α pro-inflammatory signalling in term myometrium prior to the onset of labour and suggests that PGF2α signalling pathways could be a potential target for management of preterm labour.

Thymic stromal lymphopoietin participates in the host response to intra-amniotic inflammation leading to preterm labor and birth.

The aim of this study was to establish the role of thymic stromal lymphopoietin (TSLP) in the intra-amniotic host response of women with spontaneous preterm labor (sPTL) and birth. Amniotic fluid and chorioamniotic membranes (CAM) were collected from women with sPTL who delivered at term (n = 30) or preterm without intra-amniotic inflammation (n = 34), with sterile intra-amniotic inflammation (SIAI, n = 27), or with intra-amniotic infection (IAI, n = 17). Amnion epithelial cells (AEC), Ureaplasma parvum, and Sneathia spp. were also utilized. The expression of TSLP, TSLPR, and IL-7Rα was evaluated in amniotic fluid or CAM by RT-qPCR and/or immunoassays. AEC co-cultured with Ureaplasma parvum or Sneathia spp. were evaluated for TSLP expression by immunofluorescence and/or RT-qPCR. Our data show that TSLP was elevated in amniotic fluid of women with SIAI or IAI and expressed by the CAM. TSLPR and IL-7Rα had detectable gene and protein expression in the CAM; yet, CRLF2 was specifically elevated with IAI. While TSLP localized to all layers of the CAM and increased with SIAI or IAI, TSLPR and IL-7Rα were minimal and became most apparent with IAI. Co-culture experiments indicated that Ureaplasma parvum and Sneathia spp. differentially upregulated TSLP expression in AEC. Together, these findings indicate that TSLP is a central component of the intra-amniotic host response during sPTL.

New Insights on the Biomechanics of the Fetal Membrane.

During pregnancy, the Fetal Membrane (FM) is subjected to mechanical stretching that may result in preterm labor. The structural integrity of the FM is maintained by its collagenous layer. Disconnection and reconnection of molecular bonds between collagen fibrils is the fundamental process that governs the irreversible mechanical and supramolecular changes in the FM. At a critical threshold strain, bundling and alignment of collagen fibrils alter the super-molecular structure of the collagenous layer. Recent studies indicate that these changes are associated with inflammation and/or expression of specific proteins that are known to be related to uterine contractions and labor. The potential healing of stretching-induced damages in the FM by mediators involved in mechano-transduction is discussed.

ß3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for ß3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the ß2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that ß2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by ß2 receptors is unable to provide meaningful tocolysis. The failure of ß2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The ß3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of ß2 agonists as tocolytics and suggests a non-canonical signaling role for ß3AR in myometrium. The addition of the ß3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to ß3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

Single cell profiling at the maternal-fetal interface reveals a deficiency of PD-L1+ non-immune cells in human spontaneous preterm labor.

The mechanisms that underlie the timing of labor in humans are largely unknown. In most pregnancies, labor is initiated at term (≥ 37 weeks gestation), but in a signifiicant number of women spontaneous labor occurs preterm and is associated with increased perinatal mortality and morbidity. The objective of this study was to characterize the cells at the maternal-fetal interface (MFI) in term and preterm pregnancies in both the laboring and non-laboring state in Black women, who have among the highest preterm birth rates in the U.S. Using mass cytometry to obtain high-dimensional single-cell resolution, we identified 31 cell populations at the MFI, including 25 immune cell types and six non-immune cell types. Among the immune cells, maternal PD1+ CD8 T cell subsets were less abundant in term laboring compared to term non-laboring women. Among the non-immune cells, PD-L1+ maternal (stromal) and fetal (extravillous trophoblast) cells were less abundant in preterm laboring compared to term laboring women. Consistent with these observations, the expression of CD274, the gene encoding PD-L1, was significantly depressed and less responsive to fetal signaling molecules in cultured mesenchymal stromal cells from the decidua of preterm compared to term women. Overall, these results suggest that the PD1/PD-L1 pathway at the MFI may perturb the delicate balance between immune tolerance and rejection and contribute to the onset of spontaneous preterm labor.

Further Evidence that an Episode of Premature Labor Is a Pathologic State: Involvement of the Insulin-Like Growth Factor System.

INTRODUCTION: Approximately 47% of women with an episode of preterm labor deliver at term; however, their infants are at greater risk of being small for gestational age and for neurodevelopmental disorders. In these cases, a pathologic insult may disrupt the homeostatic responses sustaining pregnancy. We tested the hypothesis of an involvement of components of the insulin-like growth factor (IGF) system. METHODS: This is a cross-sectional study in which maternal plasma concentrations of pregnancy-associated plasma protease (PAPP)-A, PAPP-A2, insulin-like growth factor-binding protein 1 (IGFBP-1), and IGFBP-4 were determined in the following groups of women: (1) no episodes of preterm labor, term delivery (controls, n = 100); (2) episode of preterm labor, term delivery (n = 50); (3) episode of preterm labor, preterm delivery (n = 100); (4) pregnant women at term not in labor (n = 61); and (5) pregnant women at term in labor (n = 61). Pairwise differences in maternal plasma concentrations of PAPP-A, PAPP-A2, IGFBP-1, and IGFBP-4 among study groups were assessed by fitting linear models on log-transformed data and included adjustment for relevant covariates. Significance of the group coefficient in the linear models was assessed via t-scores, with p < 0.05 deemed a significant result. RESULTS: Compared to controls, (1) women with an episode of premature labor, regardless of a preterm or a term delivery, had higher mean plasma concentrations of PAPP-A2 and IGFBP-1 (each p < 0.05); (2) women with an episode of premature labor who delivered at term also had a higher mean concentration of PAPP-A (p < 0.05); and (3) acute histologic chorioamnionitis and spontaneous labor at term were not associated with significant changes in these analytes. CONCLUSION: An episode of preterm labor involves the IGF system, supporting the view that the premature activation of parturition is a pathologic state, even in those women who delivered at term.

Assessment of the tocolytic nifedipine in preclinical primary models of preterm birth.

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy.

A key role for NLRP3 signaling in preterm labor and birth driven by the alarmin S100B.

Preterm birth remains the leading cause of neonatal morbidity and mortality worldwide. A substantial number of spontaneous preterm births occur in the context of sterile intra-amniotic inflammation, a condition that has been mechanistically proven to be triggered by alarmins. However, sterile intra-amniotic inflammation still lacks treatment. The NLRP3 inflammasome has been implicated in sterile intra-amniotic inflammation; yet, its underlying mechanisms, as well as the maternal and fetal contributions to this signaling pathway, are unclear. Herein, by utilizing a translational and clinically relevant model of alarmin-induced preterm labor and birth in Nlrp3-/- mice, we investigated the role of NLRP3 signaling by using imaging and molecular biology approaches. Nlrp3 deficiency abrogated preterm birth and the resulting neonatal mortality induced by the alarmin S100B by impeding the premature activation of the common pathway of labor as well as by dampening intra-amniotic and fetal inflammation. Moreover, Nlrp3 deficiency altered leukocyte infiltration and functionality in the uterus and decidua. Last, embryo transfer revealed that maternal and fetal Nlrp3 signaling contribute to alarmin-induced preterm birth and neonatal mortality, further strengthening the concept that both individuals participate in the complex process of preterm parturition. These findings provide novel insights into sterile intra-amniotic inflammation, a common etiology of preterm labor and birth, suggesting that the adverse perinatal outcomes resulting from prematurity can be prevented by targeting NLRP3 signaling.

Evidence for the participation of CHCHD2/MNRR1, a mitochondrial protein, in spontaneous labor at term and in preterm labor with intra-amniotic infection.

OBJECTIVE: Intra-amniotic inflammation (IAI), associated with either microbe (infection) or danger signals (sterile), plays a major role in the pathophysiology of preterm labor and delivery. Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2) [also known as Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1)], a mitochondrial protein involved in oxidative phosphorylation and cell survival, is capable of sensing tissue hypoxia and inflammatory signaling. The ability to maintain an appropriate energy balance at the cellular level while adapting to environmental stress is essential for the survival of an organism. Mitochondrial dysfunction has been observed in acute systemic inflammatory conditions, such as sepsis, and is proposed to be involved in sepsis-induced multi-organ failure. The purpose of this study was to determine the amniotic fluid concentrations of CHCHD2/MNRR1 in pregnant women, women at term in labor, and those in preterm labor (PTL) with and without IAI. METHODS: This cross-sectional study comprised patients allocated to the following groups: (1) mid-trimester (n = 16); (2) term in labor (n = 37); (3) term not in labor (n = 22); (4) PTL without IAI who delivered at term (n = 25); (5) PTL without IAI who delivered preterm (n = 47); and (6) PTL with IAI who delivered preterm (n = 53). Diagnosis of IAI (amniotic fluid interleukin-6 concentration ≥2.6 ng/mL) included cases associated with microbial invasion of the amniotic cavity and those of sterile nature (absence of detectable bacteria, using culture and molecular microbiology techniques). Amniotic fluid and maternal plasma CHCHD2/MNRR1 concentrations were determined with a validated and sensitive immunoassay. RESULTS: (1) CHCHD2/MNRR1 was detectable in all amniotic fluid samples and women at term without labor had a higher amniotic fluid CHCHD2/MNRR1 concentration than those in the mid-trimester (p = 0.003); (2) the amniotic fluid concentration of CHCHD2/MNRR1 in women at term in labor was higher than that in women at term without labor (p = 0.01); (3) women with PTL and IAI had a higher amniotic fluid CHCHD2/MNRR1 concentration than those without IAI, either with preterm (p < 0.001) or term delivery (p = 0.01); (4) women with microbial-associated IAI had a higher amniotic fluid CHCHD2/MNRR1 concentration than those with sterile IAI (p < 0.001); (5) among women with PTL and IAI, the amniotic fluid concentration of CHCHD2/MNRR1 correlated with that of interleukin-6 (Spearman's Rho = 0.7; p < 0.001); and (6) no correlation was observed between amniotic fluid and maternal plasma CHCHD2/MNRR1 concentrations among women with PTL. CONCLUSION: CHCHD2/MNRR1 is a physiological constituent of human amniotic fluid in normal pregnancy, and the amniotic concentration of this mitochondrial protein increases during pregnancy, labor at term, and preterm labor with intra-amniotic infection. Hence, CHCHD2/MNRR1 may be released into the amniotic cavity by dysfunctional mitochondria during microbial-associated IAI.

Circulating Extracellular Vesicles microRNAs Are Altered in Women Undergoing Preterm Birth.

Preterm labor (PTL) and preterm premature rupture of membranes (PPROM) lead to high perinatal morbidity/mortality rates worldwide. Small extracellular vesicles (sEV) act in cell communication and contain microRNAs that may contribute to the pathogenesis of these complications. We aimed to compare the expression, in sEV from peripheral blood, of miRNAs between term and preterm pregnancies. This cross-sectional study included women who underwent PTL, PPROM, and term pregnancies, examined at the Botucatu Medical School Hospital, SP, Brazil. sEV were isolated from plasma. Western blot used to detect exosomal protein CD63 and nanoparticle tracking analysis were performed. The expression of 800 miRNAs was assessed by the nCounter Humanv3 miRNA Assay (NanoString). The miRNA expression and relative risk were determined. Samples from 31 women-15 preterm and 16 term-were included. miR-612 expression was increased in the preterm groups. miR-612 has been shown to increase apoptosis in tumor cells and to regulate the nuclear factor κB inflammatory pathway, processes involved in PTL/PPROM pathogenesis. miR-1253, miR-1283, miR378e, and miR-579-3p, all associated with cellular senescence, were downregulated in PPROM compared with term pregnancies. We conclude that miRNAs from circulating sEV are differentially expressed between term and preterm pregnancies and modulate genes in pathways that are relevant to PTL/PPROM pathogenesis.

The amniotic fluid proteome changes with term labor and informs biomarker discovery in maternal plasma.

The intra-uterine components of labor, namely, myometrial contractility, cervical ripening, and decidua/membrane activation, have been extensively characterized and involve a local pro-inflammatory milieu of cellular and soluble immune mediators. Targeted profiling has demonstrated that such processes extend to the intra-amniotic space, yet unbiased analyses of the proteome of human amniotic fluid during labor are lacking. Herein, we utilized an aptamer-based platform to characterize 1,310 amniotic fluid proteins and found that the proteome undergoes substantial changes with term labor (251 proteins with differential abundance, q < 0.1, and fold change > 1.25). Proteins with increased abundance in labor are enriched for immune and inflammatory processes, consistent with prior reports of labor-associated changes in the intra-uterine space. By integrating the amniotic fluid proteome with previously generated placental-derived single-cell RNA-seq data, we demonstrated the labor-driven upregulation of signatures corresponding to stromal-3 and decidual cells. We also determined that changes in amniotic fluid protein abundance are reflected in the maternal plasma proteome. Collectively, these findings provide novel insights into the amniotic fluid proteome in term labor and support its potential use as a source of biomarkers to distinguish between true and false labor by using maternal blood samples.