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This study aimed to determine the association between umbilical cord leucine-rich alpha-2 glycoprotein (LRG) and fetal infection and investigate the underlying mechanism of LRG elevation in fetuses. We retrospectively reviewed the medical records of patients who delivered at Osaka University Hospital between 2012 and 2017 and selected those with histologically confirmed chorioamnionitis (CAM), which is a common pregnancy complication that may cause neonatal infection. The participants were divided into two groups: CAM with fetal infection (CAM-f[+] group, n = 14) and CAM without fetal infection (CAM-f[-] group, n = 31). Fetal infection was defined by the histological evidence of funisitis. We also selected 50 cases without clinical signs of CAM to serve as the control. LRG concentrations in sera obtained from the umbilical cord were unaffected by gestational age at delivery, neonatal birth weight, nor the presence of noninfectious obstetric complications (all, p > 0.05). Meanwhile, the LRG levels (median, Interquartile range [IQR]) were significantly higher in the CAM-f(+) group (10.37 [5.21-13.7] μg/ml) than in the CAM-f(-) (3.61 [2.71-4.65] μg/ml) or control group (3.39 [2.81-3.93] μg/ml; p < 0.01). The area under the receiver operating characteristic (ROC) curve of LRG for recognizing fetal infection was 0.92 (optimal cutoff, 5.08 μg/ml; sensitivity, 86%; specificity, 88%). In a mouse CAM model established by lipopolysaccharide administration, the fetal LRG protein in sera and LRG mRNA in the liver were significantly higher than those in phosphate-buffered saline (PBS)-administered control mice (p < 0.01). In vitro experiments using a fetal liver-derived cell line (WRL68) showed that the expression of LRG mRNA was significantly increased after interleukin (IL)-6, IL-1β, and tumor necrosis factor- alpha (TNF-α) stimulation (p < 0.01); the induction was considerably stronger following IL-6 and TNF-α stimulation (p < 0.01). In conclusion, LRG is an effective biomarker of fetal infection, and fetal hepatocytes stimulated with inflammatory cytokines may be the primary source of LRG production in utero., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: TN received a grant from SEKISUI MEDICAL CO., LTD. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: To evaluate maternal serum C-reactive protein (CRP) concentrations in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) in relation to the presence of microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI)., Methods: Two hundred and eighty-seven women with singleton pregnancies complicated by PPROM between 2014 and 2016 were included in this study. Maternal blood and amniotic fluid samples were collected at the time of admission. Maternal serum CRP concentration was measured using a high-sensitivity immunoturbidimetric assay. Interleukin-6 (IL-6) concentration was measured using a point-of-care test. MIAC was diagnosed based on a positive polymerase chain reaction result for Ureaplasma species, Mycoplasma hominis, and/or Chlamydia trachomatis and for the 16S rRNA gene. IAI was characterized by an amniotic fluid IL-6 concentration of ≥ 745 pg/mL., Result: Women with MIAC and IAI had higher maternal serum CRP concentrations than did women without (with MIAC: median 6.9 mg/L vs. without MIAC: median 4.9 mg/L; p = 0.02; with IAI: median 8.6 mg/L vs. without IAI: median 4.7 mg/L; p < 0.0001). When women were split into four subgroups based on the presence of MIAC and/or IAI, women with the presence of both MIAC and IAI had higher maternal serum CRP than did women with IAI alone, with MIAC alone, and women without MIAC and IAI (both MIAC and IAI: median: 13.1 mg/L; IAI alone: 6.0 mg/L; MIAC alone: 3.9 mg/L; and without MIAC and IAI: median 4.8 mg/L; p < 0.0001). The maternal serum CRP cutoff value of 17.5 mg/L was the best level to identify the presence of both MIAC and IAI, with sensitivity of 47%, specificity of 96%, positive predictive value of 42%, negative predictive value of 96%, and the positive likelihood ratio of 10.9., Conclusion: The presence of both MIAC and IAI was associated with the highest maternal serum CRP concentrations. Maternal serum CRP concentration in women with PPROM at the time of admission can rule out the presence of the combined condition of both MIAC and IAI, therefore, it may serve as a non-invasive screening tool to distinguish between women with PPROM who are at high or at low risk for the presence of both MIAC and IAI.

Background: Histological chorioamnionitis (HCA) is related to perinatal morbidity. However, there is no definite diagnostic method for detecting chorioamnionitis before delivery., Methods: We evaluated whether the delta neutrophil index (DNI) was an effective early marker of HCA in patients with preterm premature rupture of membranes (PPROM). We retrospectively evaluated 149 women diagnosed with PPROM (gestational age, 20+0 to 36+6 weeks) at Severance Hospital from January 2013 to December 2014. The women were categorized into the following two groups: (a) PPROM without HCA and (b) PPROM with HCA. The maternal white blood cell (WBC) count, neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP) level, and DNI were measured at admission. The DNI has been reported to reflect the fraction of circulating immature granulocytes associated with infection., Results: Of the 149 patients, 87 were included in the PPROM without HCA group and 62 were included in the PPROM with HCA group. The interval between admission and delivery was significantly shorter in the PPROM with HCA group than in the PPROM without HCA group. There was no significant difference in the maternal WBC count. The serum CRP level, NLR, and DNI were significantly lower in the PPROM without HCA group than in the PPROM with HCA group, while the lymphocyte count was significantly lower in the PPROM with HCA group than in the PPROM without HCA group. A predictive equation was generated by combining the DNI, lymphocyte count, and CRP level, and the sensitivity and specificity for predicting a placental inflammatory response were 69.1% and 70.5%, respectively., Conclusions: The DNI could be a predictive marker for HCA in patients with PPROM. Our predictive equation involving the DNI, lymphocyte count, and CRP level may be helpful for predicting the placental inflammatory response in patients with PPROM.

Objective: This study evaluated maternal C-reactive protein (CRP) as a predictor of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) in women with preterm prelabor rupture of the membranes (PPROM) before and after 32 weeks of gestation., Methods: This study was a prospective observational cohort study of 386 women. Maternal serum CRP concentrations were evaluated, and amniotic fluid samples were obtained via transabdominal amniocentesis at the time of admission. Placentas underwent histopathological examination after delivery. MIAC was defined based on a positive PCR for Ureaplasma species, Mycoplasma hominis and Chlamydia trachomatis and/or positive 16S rRNA gene amplification. HCA was defined based on the Salafia classification., Results: Maternal CRP was significantly higher in women with MIAC and HCA (median 9.0 mg/l) than in women with HCA alone (median 6.9 mg/l), MIAC alone (median 7.4 mg/l) and without MIAC or HCA (median 4.5 mg/l) (p<0.0001). CRP was a weak predictor of the occurrence of MIAC and HCA before and after 32 weeks of gestation. Only the 95th percentile of CRP and PPROM before 32 weeks exhibited a false-positive rate of 1%, a positive predictive value of 90% and a positive likelihood ratio of 13.2 to predict MIAC and HCA. However, the low sensitivity of 15% limits the clinical utility of this detection., Conclusion: CRP is a poor predictor of the occurrence of MIAC and HCA, even at early gestational ages.

Chorioamnionitis (inflammation of the fetal membranes) is strongly associated with preterm birth and in utero exposure to inflammation significantly impairs contractile function in the preterm lamb diaphragm. The fetal inflammatory response to intra-amniotic (IA) lipopolysaccharide (LPS) is orchestrated via interleukin 1 (IL-1). We aimed to determine if LPS induced contractile dysfunction in the preterm diaphragm is mediated via the IL-1 pathway. Pregnant ewes received IA injections of recombinant human IL-1 receptor antagonist (rhIL-1ra) (Anakinra; 100 mg) or saline (Sal) 3 h prior to second IA injections of LPS (4 mg) or Sal at 119d gestational age (GA). Preterm lambs were killed after delivery at 121d GA (term = 150 d). Muscle fibres dissected from the right hemi-diaphragm were mounted in an in vitro muscle test system for assessment of contractile function. The left hemi-diaphragm was snap frozen for molecular and biochemical analyses. Maximum specific force in lambs exposed to IA LPS (Sal/LPS group) was 25% lower than in control lambs (Sal/Sal group; p=0.025). LPS-induced diaphragm weakness was associated with higher plasma IL-6 protein, diaphragm IL-1β mRNA and oxidised glutathione levels. Pre-treatment with rhIL-1ra (rhIL-1ra/LPS) ameliorated the LPS-induced diaphragm weakness and blocked systemic and local inflammatory responses, but did not prevent the rise in oxidised glutathione. These findings indicate that LPS induced diaphragm dysfunction is mediated via IL-1 and occurs independently of oxidative stress. Therefore, the IL-1 pathway represents a potential therapeutic target in the management of impaired diaphragm function in preterm infants.

Background: The relationship between histological chorioamnionitis and haematological and biochemical markers in mothers and infants at delivery, and in infants postnatally, is incompletely characterised. These markers are widely used in the diagnosis of maternal and neonatal infection. Our objective was to investigate the effects of histological chorioamnionitis (HCA) on haematological and biochemical inflammatory markers in mothers and infants at delivery, and in infants post-delivery., Methods: Two hundred and forty seven mothers, delivering 325 infants, were recruited at the only tertiary perinatal centre in Western Australia. Placentae were assessed for evidence of HCA using a semi-quantitative scoring system. Maternal high sensitivity C-reactive protein (hsCRP), procalcitonin, and umbilical cord hsCRP, procalcitonin, white cell count and absolute neutrophil count were measured at delivery. In infants where sepsis was clinically suspected, postnatal CRP, white cell count and absolute neutrophil count were measured up to 48 hours of age. The effect of HCA on maternal, cord and neonatal markers was evaluated by multivariable regression analysis., Results: The median gestational age was 34 weeks and HCA was present in 26 of 247 (10.5%) placentae. Mothers whose pregnancies were complicated by HCA had higher hsCRP (median 26 (range 2-107) versus 5.6 (0-108) mg/L; P<0.001). Histological chorioamnionitis was associated with higher umbilical cord hsCRP (75(th) percentile 2.91 mg/L (range 0-63.9) versus 75(th) percentile 0 mg/L (0-45.6); P<0.001) and procalcitonin (median 0.293 (range 0.05-27.37) versus median 0.064 (range 0.01-5.24) ug/L; P<0.001), with a sustained increase in neonatal absolute neutrophil count (median 4.5 (0.1-26.4)×10(9)/L versus 3.0 (0.1-17.8)×10(9)/L), and CRP up to 48 hours post-partum (median 10 versus 6.5 mg/L) (P<0.05 for each)., Conclusion: Histological chorioamnionitis is associated with modest systemic inflammation in maternal and cord blood. These systemic changes may increase postnatally, potentially undermining their utility in the diagnosis of early-onset neonatal infection.

Background: Chronic chorioamnionitis is found in more than one-third of spontaneous preterm births. Chronic chorioamnionitis and villitis of unknown etiology represent maternal anti-fetal cellular rejection. Antibody-mediated rejection is another type of transplantation rejection. We investigated whether there was evidence for antibody-mediated rejection against the fetus in spontaneous preterm birth., Methods and Findings: This cross-sectional study included women with (1) normal pregnancy and term delivery (n = 140) and (2) spontaneous preterm delivery (n = 140). We analyzed maternal and fetal sera for panel-reactive anti-HLA class I and class II antibodies, and determined C4d deposition on umbilical vein endothelium by immunohistochemistry. Maternal anti-HLA class I seropositivity in spontaneous preterm births was higher than in normal term births (48.6% vs. 32.1%, p = 0.005). Chronic chorioamnionitis was associated with a higher maternal anti-HLA class I seropositivity (p<0.01), significant in preterm and term birth. Villitis of unknown etiology was associated with increased maternal and fetal anti-HLA class I and II seropositivity (p<0.05, for each). Fetal anti-HLA seropositivity was closely related to maternal anti-HLA seropositivity in both groups (p<0.01, for each). C4d deposition on umbilical vein endothelium was more frequent in preterm labor than term labor (77.1% vs. 11.4%, p<0.001). Logistic regression analysis revealed that chronic chorioamnionitis (OR = 6.10, 95% CI 1.29-28.83), maternal anti-HLA class I seropositivity (OR = 5.90, 95% CI 1.60-21.83), and C4d deposition on umbilical vein endothelium (OR = 36.19, 95% CI 11.42-114.66) were associated with preterm labor and delivery., Conclusions: A major subset of spontaneous preterm births has a signature of maternal anti-fetal cellular and antibody-mediated rejections with links to fetal graft-versus-host disease and alloimmune reactions.