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.

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.

Antenatal creatine supplementation reduces persistent fetal lung inflammation and oxidative stress in an ovine model of chorioamnionitis.

Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. The objective of the study was to evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero proinflammatory stimulus. Fetal lambs (n = 51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mg·kg-1·h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline 7 days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a two-way ANOVA. Fetal creatine supplementation increased lung creatine content by 149% (PCr < 0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS < 0.0001) and increased levels of CD45+ leukocytes (PLPS < 0.0001) and MPO+ (PLPS < 0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr = 0.045) and MPO+ cells (PCr = 0.012) in the lungs and reduced thiol oxidation in plasma (PCr < 0.01) and lung tissue (PCr = 0.02). In conclusion, fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.NEW & NOTEWORTHY We evaluated the effect of antenatal creatine supplementation to reduce pulmonary inflammation and oxidative stress in the fetal lamb lungs arising from lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal creatine supplementation increased lung creatine content and had no adverse effects on systemic fetal physiology and overall lung architecture. Importantly, fetuses that received creatine had significantly lower levels of inflammation and oxidative stress in the lungs, suggesting an anti-inflammatory and antioxidant benefit of creatine.

Clinical utility of gastric fluid cytokine levels in preterm infants for predicting histological chorioamnionitis.

BACKGROUND: The risk of various complications, such as neonatal death, early onset sepsis, and chronic lung disease, is increased in infants born to mothers with chorioamnionitis (CAM). However, predicting the diagnosis of histological CAM (hCAM) in the early postnatal period is challenging for clinicians due to pathological considerations. Therefore, an early diagnostic tool for hCAM is needed. Gastric fluid at birth is considered a suitable biomarker for predicting the intrauterine environment because most of its components are from amniotic fluid, and the sampling technique is less invasive. This study aimed to evaluate the clinical utility of cytokines in the gastric fluid of preterm infants at birth as predictors of hCAM. METHODS: We retrieved gastric fluid and serum from 21 preterm infants with a gestational age of ≤ 32 weeks within 1 h after birth and used cytometric bead array to measure the concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma. We compared the cytokine concentrations in the gastric fluid and serum of the preterm infants born to mothers with or without hCAM. RESULTS: The gastric fluid, serum IL-6, and serum IL-10 concentrations were significantly higher in the hCAM group than that in the non-hCAM group. The best cutoff values for predicting hCAM was > 2,855 pg/mL and > 315 pg/mL for IL-6 in the gastric fluid and serum, respectively. Receiver operating characteristic curves showed that gastric fluid IL-6 concentrations correlated more strongly with the presence of hCAM than serum IL-6 concentrations. CONCLUSION: IL-6 in the gastric fluid at birth may be a more promising biomarker for predicting the presence of hCAM than that in serum. IL-6 concentration analysis in the gastric fluid at birth might help to diagnose hCAM immediately after birth and improve the prognosis of preterm infants.

Anaphylaxis-induced premature uterine contractions: a case report and literature review.

BACKGROUND: Preterm labor is caused by multiple etiologies, including intra-amniotic infection and/or intra-amniotic inflammation, vascular disorders, cervical disease, decidual senescence, and breakdown of maternal-fetal tolerance. Accumulating evidence in vivo and in vitro has shown that an allergic reaction, including anaphylaxis, can induce preterm uterine contractions. This report describes a case of a pregnant woman who developed anaphylaxis and regular uterine contractions after the ingestion of a strawberry-coated biscuit. We also review the mechanism of allergic reaction (hypersensitivity)-induced preterm labor. Case presentation A 31-year-old woman (gravida 1, para 0) at 30+2 weeks of gestation was admitted to the labor and delivery unit with regular uterine contractions and anaphylactic symptoms after she ingested a strawberry-coated biscuit as a snack. The uterine contractions resolved after the treatment of anaphylaxis by administering antihistamines and epinephrine. The patient subsequently delivered at 39+3 weeks of gestation. The amniotic fluid profile showed no infection or inflammation. A postpartum skin-prick test confirmed a positive type 1 hypersensitivity reaction to the strawberry-coated biscuit. CONCLUSIONS: We report a case of anaphylaxis-induced uterine contractility in which uterine contractions subsided after the treatment of anaphylaxis. The absence of intra-amniotic infection and/or intra-amniotic inflammation and the cause of the anaphylaxis were confirmed. Our findings indicate that maternal allergic reactions may be one of the mechanisms of preterm labor.

Low- and high-level information analyses of transcriptome connecting endometrial-decidua-placental origin of preeclampsia subtypes: A preliminary study.

BACKGROUND: Existing proposed pathogenesis for preeclampsia (PE) was only applied for early onset subtype and did not consider pre-pregnancy and competing risks. We aimed to decipher PE subtypes by identifying related transcriptome that represents endometrial maturation and histologic chorioamnionitis. METHODS: We utilized eight arrays of mRNA expression for discovery (n=289), and other eight arrays for validation (n=352). Differentially expressed genes (DEGs) were overlapped between those of: (1) healthy samples from endometrium, decidua, and placenta, and placenta samples under histologic chorioamnionitis; and (2) placenta samples for each of the subtypes. They were all possible combinations based on four axes: (1) pregnancy-induced hypertension; (2) placental dysfunction-related diseases (e.g., fetal growth restriction [FGR]); (3) onset; and (4) severity. RESULTS: The DEGs of endometrium at late-secretory phase, but none of decidua, significantly overlapped with those of any subtypes with: (1) early onset (p-values ≤0.008); (2) severe hypertension and proteinuria (p-values ≤0.042); or (3) chronic hypertension and/or severe PE with FGR (p-values ≤0.042). Although sharing the same subtypes whose DEGs with which significantly overlap, the gene regulation was mostly counter-expressed in placenta under chorioamnionitis (n=13/18, 72.22%; odds ratio [OR] upper bounds ≤0.21) but co-expressed in late-secretory endometrium (n=3/9, 66.67%; OR lower bounds ≥1.17). Neither the placental DEGs at first-nor second-trimester under normotensive pregnancy significantly overlapped with those under late-onset, severe PE without FGR. CONCLUSIONS: We identified the transcriptome of endometrial maturation in placental dysfunction that distinguished early- and late-onset PE, and indicated chorioamnionitis as a PE competing risk. This study implied a feasibility to develop and validate the pathogenesis models that include pre-pregnancy and competing risks to decide if it is needed to collect prospective data for PE starting from pre-pregnancy including chorioamnionitis information.

CD47 and CD36 expressions in the placenta of mothers with chorioamnionitis.

INTRODUCTION: Chorioamnionitis is the inflammation of the placenta and is histologically defined as the presence of neutrophilic infiltration into the chorio-amnion membrane with and without involvement of the umbilical cord. Currently, the inflammatory mediators involved in the eliciting of inflammatory response is still largely under investigation. CD47 and CD36 are pro-inflammatory molecules that are still under investigation. The aim of this study was to determine the expressions of CD47 and CD36 in the placenta of mothers with chorioamnionitis. MATERIALS AND METHODS: This was a cross-sectional study, involving a total of 100 cases that comprised of acute subchorionitis (stage I, n=20), acute chorioamnionitis (stage II, n=20), acute necrotising chorioamnionitis (stage III, n=20) and non-chorioamnionitis placenta as control (n=40). All tissue blocks were retrieved from the archived pathology record over a period of 4 years. CD36 and CD47 immunohistochemistry were performed on all cases and their expression in various cell types on the placenta were analysed. RESULTS: CD36 was expressed only on the foetal vascular endothelial cells. Interestingly, CD47 showed positive staining on the neutrophils and its expression was significantly different between maternal inflammatory response stage II chorioamnionitis (n=13/20, p<0.001) with stage I and stage III chorioamnionitis. DISCUSSION: Our study showed CD47 was expressed in the neutrophils and it was associated with poorer perinatal outcomes and it may have a role in the pathogenesis of chorioamnionitis.

Inflammatory biomarkers in the cervicovaginal fluid to identify histologic chorioamnionitis and funisitis in women with preterm labor.

OBJECTIVE: We investigated the association between altered levels of inflammatory proteins in the cervicovaginal fluid (CVF) and acute histologic chorioamnionitis (HCA) and funisitis in women with preterm labor (PTL). METHODS: In this study, a total of 134 consecutive singleton pregnant women with PTL (at 23+0-34+0 weeks) who delivered preterm (at  < 37 weeks) and from whom CVF samples were collected at admission were retrospectively enrolled. The CVF levels of haptoglobin, interleukin-6/8, kallistatin, lipocalin-2, matrix metalloproteinase (MMP)-8, resistin, S100 calcium-binding protein A8, and serpin A1 were determined using enzyme-linked immunosorbent assay. The placentas were histologically analyzed after delivery. RESULTS: Multiple logistic regression analyses showed significant associations between elevated CVF interleukin-8 and resistin levels and acute HCA after adjusting for baseline covariates (e.g., gestational age at sampling). CVF haptoglobin, interleukin-6/8, kallistatin, MMP-8, and resistin levels were significantly higher in women with funisitis than in those without, whereas the baseline covariates were similar between the two groups (P > 0.1). The area under the receiver operating characteristic curves of the aforementioned biomarkers ranged from 0.61 to 0.77 regarding each outcome. Notably, HCA risk significantly increased with increasing CVF levels of interleukin-8 and resistin (P for trend  < 0.05). CONCLUSIONS: Haptoglobin, interleukin-6/8, kallistatin, MMP-8, and resistin were identified as potential inflammatory CVF biomarkers predictive of acute HCA and funisitis in women with PTL. Moreover, the risk severity of acute HCA may be associated with the degree of the inflammatory response in the CVF (particularly based on interleukin-8 levels).

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.

THE FEATURES OF LIMITED PROTEOLYSIS IN PLACENTAL FIBRINOID IN COMBINATION WITH INFLAMMATION AND IRON DEFICIENCY ANEMIA OF PREGNANT WOMEN.

OBJECTIVE: The aim: To establish the features of limited proteolysis in fibrinoid of the chorionic and basal plates of the placenta in acute and chronic chorioamnionitis, as well as basal deciduitis on the background of iron deficiency anemia in pregnant women. PATIENTS AND METHODS: Materials and methods: The histochemical procedure was performed using the ninhydrin-Schiff response to free amino groups of proteins by the method of A. Yasuma and T. Ichikava, and Bonheg bromophenol blue. RESULTS: Results: With iron deficiency anemia of pregnant women, the relative units of optical density in the chorionic plate were 0.312±0.0026, and with basal one - 0.310±0.0024 (with indicators of physiological pregnancy 0.285±0.0024 and 0.289±0.002.1). In the observations of acute chorioamnionitis, the quantitative indicators were 0.311±0.0024, chronic one - 0.311±0.0024, and with inflammation on the background of anemia of pregnant women - 0.315±0.0031 and 0.339±0.0036, respectively. With acute basal deciduitis - 0.316±0.0027, chronic one - 0.326±0.0034, and with inflammation of the basal plate of the placenta on the background of anemia of pregnant women - 0.320±0.0031 and 0.341±0.0038, respectively. CONCLUSION: Conclusions: With anemia of pregnant women, the processes of limited proteolysis are intensified in accordance with the indicators of optical density of histochemical staining in the fibrinoid of the chorionic and basal plates of the placenta compared with physiological pregnancy. In case of acute and chronic forms of chorioamnionitis and basal deciduitis, quantitative indicators of optic density of histochemical staining increase compared with physiological preg¬nancy. Comorbid anemia of pregnant women activates the processes of limited proteolysis only in the chronic form of chorioamnionitis and basal deciduitis.

Clinical chorioamnionitis at term is characterized by changes in the plasma concentration of CHCHD2/MNRR1, a mitochondrial protein.

OBJECTIVE: Mitochondrial dysfunction was observed in acute systemic inflammatory conditions such as sepsis and might be involved in sepsis-induced multi-organ failure. Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2), also known as Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1), a bi-organellar protein located in the mitochondria and the nucleus, is implicated in cell respiration, survival, and response to tissue hypoxia. Recently, the reduction of the cellular CHCHD2/MNRR1 protein, as part of mitochondrial dysfunction, has been shown to play a role in the amplification of inflammatory cytokines in a murine model of lipopolysaccharide-induced systemic inflammation. The aim of this study was to determine whether the plasma concentration of CHCHD2/MNRR1 changed during human normal pregnancy, spontaneous labor at term, and clinical chorioamnionitis at term. METHODS: We conducted a cross-sectional study that included the following groups: 1) non-pregnant women (n = 17); 2) normal pregnant women at various gestational ages from the first trimester until term (n = 110); 3) women at term with spontaneous labor (n = 50); and 4) women with clinical chorioamnionitis at term in labor (n = 25). Plasma concentrations of CHCHD2/MNRR1 were assessed by an enzyme-linked immunosorbent assay. RESULTS: 1) Pregnant women at term in labor with clinical chorioamnionitis had a significantly higher plasma CHCHD2/MNRR1 concentration than those in labor without chorioamnionitis (p = .003); 2) CHCHD2/MNRR1 is present in the plasma of healthy non-pregnant and normal pregnant women without significant differences in its plasma concentrations between the two groups; 3) there was no correlation between maternal plasma CHCHD2/MNRR1 concentration and gestational age at venipuncture; and 4) plasma CHCHD2/MNRR1 concentration was not significantly different in women at term in spontaneous labor compared to those not in labor. CONCLUSIONS: CHCHD2/MNRR1 is physiologically present in the plasma of healthy non-pregnant and normal pregnant women, and its concentration does not change with gestational age and parturition at term. However, plasma CHCHD2/MNRR1 is elevated in women at term with clinical chorioamnionitis. CHCHD2/MNRR1, a novel bi-organellar protein located in the mitochondria and the nucleus, is released into maternal plasma during systemic inflammation.

The fetal response to maternal inflammation is dependent upon maternal IL-6 in a murine model.

BACKGROUND: The induction of maternal inflammation in mice leads to fetal injury that is believed to be IL-6 dependent. The fetal inflammatory response, defined by elevated fetal or amniotic fluid IL-6, has been described as a potential mechanism for subsequent fetal injury. The role of maternal IL-6 production and signaling in the fetal IL-6 response is currently unclear. METHODS: Genetic and anti-IL-6 antibody strategies were used to systematically block the maternal IL-6 response during inflammation. Chorioamnionitis was induced using intraperitoneal injection of lipopolysaccharide (LPS) at mid gestation (E14.5) and late gestation (E18.5). This model was used in pregnant C57Bl/6 dams, IL6-/- dams, C57Bl/6 dams treated with anti-IL-6 (blocks both classical and trans-signaling) or anti-gp130 antibodies (blocks trans-signaling only) and IL6+/- dams. Six hours following LPS injection, maternal serum, placental tissue, amniotic fluid and fetal tissue or serum were collected. A bead-based multiplex assay was used to evaluate levels of IL-6, KC, IL-1ß, TNF, IL-10, IL-22, IFN-γ, IL-13 and IL-17A. RESULTS: Chorioamnionitis in C57Bl/6 dams was characterized by elevated maternal serum levels of IL-6, KC and IL-22 with litter loss during mid gestation. The fetal response to maternal inflammation in C57Bl/6 mice was primarily characterized by elevated levels of IL-6, KC and IL-22 in the placenta, amniotic fluid and fetus during both mid and late gestation. A global IL-6 knockout (IL6-/-) eradicated the maternal, placental, amniotic fluid and fetal IL-6 response to LPS during mid and late gestation and improved litter survival, while minimally influencing the KC or IL-22 responses. Blocking maternal classical IL-6 signaling in C57Bl/6 dams at the time of LPS exposure diminished the maternal, placental, amniotic fluid and fetal IL-6 response during mid and late gestation, while blocking maternal IL-6 trans-signaling only affected fetal IL-6 expression. To evaluate whether maternal IL-6 was crossing the placenta and reaching the fetus, IL-6+/- dams were utilized in the chorioamnionitis model. IL-6+/- dams mounted a systemic inflammatory response following injection with LPS, characterized by elevated IL-6, KC and IL-22. IL-6-/- pups born to IL6+/- dams had decreased amniotic fluid levels of IL-6 and undetectable levels of fetal IL-6 compared to IL-6+/+ littermate controls. CONCLUSION: The fetal response to systemic maternal inflammation is dependent upon maternal IL-6 signaling, but maternal IL-6 is not crossing the placenta and reaching the fetus at detectable levels.

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.

Proteomic analysis of plasma to identify novel biomarkers for intra-amniotic infection and/or inflammation in preterm premature rupture of membranes.

To identify potential plasma biomarkers associated with microbial invasion of the amniotic cavity (MIAC) and/or intraamniotic inflammation (IAI) in women with preterm premature rupture of membranes (PPROM). This retrospective cohort study included 182 singleton pregnant women with PPROM (23-33 weeks) who underwent amniocentesis. Plasma samples; all subjects were chosen from these participants and were analyzed using label-free liquid chromatography-tandem mass spectrometry for proteome profiling using a nested case-control study design (cases with MIAC/IAI vs. non-MIAC/IAI controls [n = 9 each]). Three identified target molecules for MIAC/IAI were further verified by ELISA in the study cohort (n = 182). Shotgun proteomic analysis revealed 17 differentially expressed proteins (P < 0.05) in the plasma of MIAC/IAI cases. In particular, the levels of FCGR3A and haptoglobin, but not LRP1, were found to be increased in the plasma of patients with MIAC, IAI, and both MIAC/IAI compared with those without these conditions. Moreover, these differences remained significant after adjusting for gestational age at sampling. The area under the curves of plasma FCGR3A and haptoglobin ranged within 0.59-0.65 with respect to each of the three outcome measures. Plasma FCGR3A and haptoglobin were identified as potential independent biomarkers for less-invasively detecting MIAC/IAI in women with PPROM.

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.

Wnt5a-Flt1 activation contributes to preterm altered cerebral angiogenesis after prenatal inflammation.

OBJECTIVE: Intraventricular hemorrhage (IVH) causes morbidity and mortality in preterm infants and prenatal exposure to inflammation contributes to brain injury. Moreover, prenatal exposure to severe inflammation increases the risk of IVH in preterm neonates. The current study investigated whether intrauterine exposure to inflammation affects cerebral angiogenesis and its underlying mechanisms. METHODS: Wnt5a, flt1, and vascular endothelial growth factor (VEGF)-A levels in cord blood serum (stored in a bio-bank) of the enrolled patients were measured via enzyme-linked immunosorbent assay. A preterm prenatal inflammation exposure model was established in rats by intraperitoneal injection intraperitoneally during pregnancy. Angiogenesis of cerebral tissue was analyzed using immunohistochemistry. Wnt5a, flt1, and VEGF-A expression levels were measured via immunohistochemistry, immunofluorescence, or western blotting. The correlation between Wnt5a and flt1 expression and the cerebral vessel area was also analyzed. RESULTS: The Wnt5a and flt1 levels in the cord blood serum were significantly higher in the amnionitis group than in the non-amnionitis group. The VEGF-A level in the cord blood serum was significantly lower in the amnionitis group. In the rat model, preterm rats in the prenatal inflammation group exhibited increased microglial cell infiltration and decreased vessel area and diameter in the cerebral tissue compared to the control group. Wnt5a was located in microglial cells, and Wnt5a and flt1 expression in brain tissue significantly increased after prenatal lipopolysaccharide (LPS) exposure. VEGF-A expression declined after prenatal LPS exposure. The cerebral vessel area was negatively correlated with Wnt5a and flt1 expression. CONCLUSION: Disordered cerebral angiogenesis is associated with increased Wnt5a-Flt1 activation in microglial cells after exposure to intrauterine inflammation.

20α-Hydroxysteroid Dehydrogenase Expression in the Human Myometrium at Term and Preterm Birth: Relationships to Fetal Sex and Maternal Body Mass Index.

The mechanism by which human labor is initiated in the presence of elevated circulating progesterone levels remains unknown. Recent evidence indicates that the progesterone-metabolizing enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), encoded by the gene AKR1C1, may contribute to functional progesterone withdrawal. We found that AKR1C1 expression significantly increased with labor onset in term myometrium, but not in preterm myometrium. Among preterm laboring deliveries, clinically diagnosed chorioamnionitis was associated with significantly elevated AKR1C1 expression. AKR1C1 expression positively correlated with BMI before labor and negatively correlated with BMI during labor. Analysis by fetal sex showed that AKR1C1 expression was significantly higher in women who delivered male babies compared to women who delivered female babies at term, but not preterm. Further, in pregnancies where the fetus was female, AKR1C1 expression positively correlated with the mother's age and BMI at the time of delivery. In conclusion, the increase in myometrial AKR1C1 expression with term labor is consistent with 20α-HSD playing a role in local progesterone metabolism to promote birth. Interestingly, this role appears to be specific to term pregnancies where the fetus is male. Upregulated AKR1C1 expression in the myometrium at preterm in-labor with clinical chorioamnionitis suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. The link between AKR1C1 expression and maternal BMI may provide insight into why maternal obesity is often associated with dysfunctional labor. Higher myometrial AKR1C1 expression in male pregnancies may indicate fetal sex-related differences in the mechanisms that precipitate labor onset at term.

Matrix metalloproteinases in preterm prelabor rupture of membranes in the setting of chorioamnionitis: A scoping review.

Fetal or gestational membranes extend from the placenta to enclose the fetus and amniotic fluid. While the membranes spontaneously rupture at term in normal pregnancies, they can rupture prematurely before the onset of labor, termed preterm prelabor rupture of membranes (PPROM). PPROM can be triggered by bacterial infection or sterile inflammation in the membranes, known as chorioamnionitis (CAM). The membranes derive their tensile strength from a collagen-rich extracellular matrix (ECM); as such, understanding the enzymes and processes that can degrade the membrane ECM are of paramount importance. Matrix metalloproteinases (MMPs) are a class of enzymes capable of degrading collagen and other components of the ECM, and can be induced by inflammation. We used a scoping review to address the question of how MMP activity is associated with PPROM, particularly their induction due to sterile or nonsterile CAM. We have found that the most studied MMPs in PPROM were MMPs 2, 8, and 9. Additionally, some MMPs are constitutively active, while others are induced by inflammation. Mechanistic studies of the pathways that induce MMP activation are sparse, and this area is ripe for future studies. Targeting MMP activation could be a future strategy to delay or prevent PPROM.

Preterm labor with and without chorioamnionitis is associated with activation of myometrial inflammatory networks: a comprehensive transcriptomic analysis.

BACKGROUND: The onset of preterm labor is associated with inflammation. Previous studies suggested that this is distinct from the inflammation observed during term labor. Our previous work on 44 genes differentially expressed in myometria in term labor demonstrated a different pattern of gene expression from that observed in preterm laboring and nonlaboring myometria. We found increased expression of inflammatory genes in preterm labor associated with chorioamnionitis, but in the absence of chorioamnionitis observed no difference in gene expression in preterm myometria regardless of laboring status, suggesting that preterm labor is associated with different myometrial genes or signals originating from outside the myometrium. Given that a small subset of genes were assessed, this study aimed to use RNA sequencing and bioinformatics to assess the myometrial transcriptome during preterm labor in the presence and absence of chorioamnionitis. OBJECTIVE: This study aimed to comprehensively determine protein-coding transcriptomic differences between preterm nonlaboring and preterm laboring myometria with and without chorioamnionitis. STUDY DESIGN: Myometria were collected at cesarean delivery from preterm patients not in labor (n=16) and preterm patients in labor with chorioamnionitis (n=8) or without chorioamnionitis (n=6). Extracted RNA from myometrial tissue was prepared and sequenced using Illumina NovaSeq. Gene expression was quantified by mapping the sequence reads to the human reference genome (hg38). Differential gene expression analysis, gene set enrichment analysis, and weighted gene coexpression network analysis were used to comprehensively interrogate transcriptomic differences and their associated biology. RESULTS: Differential gene expression analysis comparing preterm patients in labor with chorioamnionitis with preterm patients not in labor identified 931 differentially expressed genes, whereas comparing preterm patients in labor without chorioamnionitis with preterm patients not in labor identified no statistically significant gene expression changes. In contrast, gene set enrichment analysis and weighted gene coexpression network analysis demonstrated that preterm labor with and without chorioamnionitis was associated with enrichment of pathways involved in activation of the innate immune system and inflammation, and activation of G protein-coupled receptors. Key genes identified included chemotactic CYP4F3, CXCL8, DOCK2, and IRF1 in preterm labor with chorioamnionitis and CYP4F3, FCAR, CHUK, and IL13RA2 in preterm labor without chorioamnionitis. There was marked overlap in the pathways enriched in both preterm labor subtypes. CONCLUSION: Differential gene expression analysis demonstrated that myometria from preterm patients in labor without chorioamnionitis and preterm patients not in labor were transcriptionally similar, whereas the presence of chorioamnionitis was associated with marked gene changes. In contrast, comprehensive bioinformatic analysis indicated that preterm labor with or without chorioamnionitis was associated with innate immune activation. All causes of preterm labor were associated with activation of the innate immune system, but this was more marked in the presence of chorioamnionitis. These data suggest that anti-inflammatory therapy may be relevant in managing preterm labor of all etiologies.

Preterm labor with intact membranes: a simple noninvasive method to identify patients at risk for intra-amniotic infection and/or inflammation.

OBJECTIVE: To develop a noninvasive scoring system to identify patients at high risk for intra-amniotic infection and/or inflammation, which would reduce the need for amniocentesis. METHODS: This prospective cohort study comprised patients admitted with preterm labor and intact membranes (20-34 weeks of gestation) who underwent a transabdominal amniocentesis and for whom concentrations of quantitative cervical fetal fibronectin and of maternal serum C-reactive protein (CRP) were determined. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined as an amniotic fluid matrix metalloproteinase-8 concentration >23 ng/mL. Multivariate logistic regression analysis was performed to identify intra-amniotic infection/inflammtion with noninvasive parameters that had a significant relationship with univariate analysis. With four parameters identified by multivariate analysis, we generated a noninvasive risk scoring system. RESULTS: Of the study population consisting of 138 singleton pregnant women, (1) the overall rate of intra-amniotic infection/inflammation was 28.3% (39/138); (2) four parameters were used to develop a noninvasive risk scoring system [i.e. cervical fetal fibronectin concentration (score 0-2), maternal serum CRP concentration (score 0-2), cervical dilatation (score 0-2), and gestational age at presentation (score 0-1)]; the total score ranges from 0 to 7; 3) the area under the curve of the risk score was 0.96 (95% confidence interval (CI), 0.92-0.99), significantly higher than that of each predictor in the identification of intra-amniotic infection/inflammation (p < .001, for all); 4) the risk score with a cutoff of 4 had a sensitivity of 94.9% (37/39), a specificity of 90.9% (90/99), a positive predictive value of 80.4% (37/46), a negative predictive value of 97.8% (90/92), a positive likelihood ratio of 10.4 (95% CI, 5.6-19.5), and a negative likelihood ratio of 0.06 (95% CI, 0.15-0.22) in the identification of intra-amniotic infection/inflammation. CONCLUSIONS: (1) The combination of four parameters (concentrations of cervical fetal fibronectin and maternal serum CRP, cervical dilatation, and gestational age) was independently associated with intra-amniotic infection and/or inflammation; and (2) the risk scoring system comprised of the combination of 4 noninvasive parameters was sensitive and specific to identify the patients at risk for intra-amniotic infection and/or inflammation.