Macrophage inhibitory cytokine 1 in fetal membranes and amniotic fluid from pregnancies with and without preterm labour and premature rupture of membranes

The placenta and fetal membranes are the site of expression of macrophage inhibitory cytokine (MIC-1), a member of the transforming growth factor (TGF)-beta superfamily. We hypothesized that MIC-1 may act as an immune regulator in pregnancy complications associated with intrauterine inflammation. Decidual cells, chorionic trophoblasts and amnion epithelial cells were identified by immunohistochemistry as the predominant MIC-1-containing cell type in term membranes. Amnion and choriodecidual explants all produced MIC-1 in culture, the latter having the greatest production rate (206 +/- 74.5 pg/mg tissue/24 h, n=6; mean +/- SEM). Production was not responsive to stimulation by pro-inflammatory cytokines. MIC-1 was detectable in 217 transabdominal amniotic fluid (AF) samples taken from 15 to 41 weeks gestation, concentrations ranging from 0.9-51.1 ng/ml. AF MIC-1 concentrations in pregnancies with premature rupture of membranes (PROM) or preterm labour, either with or without microbial invasion of the amniotic cavity, were not significantly different from those delivered at term either with or without labour. Treatment with MIC-1 (0.25-25 ng/ml) did not alter production of interleukin-6 or -8 by amnion or choriodecidual cells in vitro. We conclude that AF MIC-1 is derived from the fetal membranes and decidua, but that MIC-1 is unlikely to be involved in the pathophysiology of preterm birth or PROM.     

Matrix Metalloproteinase-13 (MMP-13, Collagenase-3) is Highly Expressed in Human Tooth Pulp

Matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) participate into extracellular matrix degradation in physiological and pathological conditions. We hypothesized that MMP expression in pulp tissue changes in response to caries attack and investigated the gene expression profiles of MMPs and TIMPs in pulp tissue of sound and carious teeth with cDNA microarray. cDNA microarray demonstrated an extremely high MMP-13 (collagenase-3) mRNA expression in pooled pulp samples of sound and carious teeth, with less pronounced expression of MMP-16 (MT3-MMP) and TIMP-1. Real-time quantitative polymerase chain reaction of individual pulp samples revealed a wide range of the MMP-13 expression level between pulp samples with possible downregulation of MMP-13 expression during caries progression. Western blot and immunohistochemical staining confirmed the presence of MMP-13 with no observable differences between sound and carious teeth pulp tissues. The results reveal that MMP-13 is expressed and synthesized in pulp tissue, an interesting feature considering the very limited expression of MMP-13 in normal adult tissues. Further studies with a larger sample size are needed to clarify the changes in MMP-13 expression during caries progression.     

胎膜早破羊水与新生儿咽拭子细菌培养分析

目的评估胎膜早破与分娩间隔时间相关的新生儿和母亲感染结局。方法对住院胎膜早破孕妇33例按破膜距临产的时间分为4组：〈12h、12～24h、25～48h、〉48h,与未破膜组孕妇30例均进行羊水和新生儿咽拭子细菌培养。采用非参数检验进行统计学处理。结果胎膜早破超过48h羊水与新生儿咽拭子细菌培养阳性率为60%、40%,胎膜早破24～48h阳性率为20%、0,小于24h均为阴性。对照组羊水与新生儿咽拭子菌培养均为阴性。结论随破膜时间延长,母婴感染率增加。破膜后48h内分娩,能获得最好母婴结局。     

Effect of experimental genital mycoplasmosis on production of matrix metalloproteinases in membranes and amniotic fluid of Sprague-Dawley rats

PROBLEM: Preterm, premature rupture of membranes (PPROM) is a dire pregnancy outcome that is frequently associated with infection by the genital mycoplasmas, Mycoplasma hominis, Ureaplasma parvum, and U. urealyticum. One potential mechanism by which these microorganisms may cause PPROM is by increasing the concentration of matrix metalloproteinases (MMPs) in the membranes and amniotic fluid. We tested this hypothesis in a well-defined model system of genital infection with M. pulmonis, a natural reproductive pathogen of rats. METHOD OF STUDY: Timed-pregnant, specific pathogen-free, Sprague-Dawley rats were infected with 10(7) CFU M. pulmonis at gestation day (gd) 14. Controls received an equivalent volume (100 microL) of sterile medium. At gd 18, rats were euthanized, and membranes and amniotic fluids were harvested and stored at -70 degrees C until analysis. Proteinase activity of amniotic fluid and membranes was resolved on discontinuous 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography gels. Band intensity was determined using a digital gel documentation system and the manufacturer's software (Kodak). RESULTS: Gelatinolytic activity associated with a band similar in molecular weight to ProMMP-9 (92 kDa, the inactive precursor of MMP-9) was significantly increased in amniotic fluids and membranes harvested from M. pulmonis-treated pups at gd 18 when compared with tissues harvested from control pups. Both ProMMP-9 and ProMMP-2 (72 kDa, the inactive precursor of MMP-2) were increased in infected animals at gd 21. CONCLUSION: Our study suggests that the genital mycoplasmas can increase MMP-9 production in vivo.     

MMP-9和iNOS在胎膜早破胎膜组织中的表达及相关性研究

目的: 探讨基质金属蛋白酶-9(MMP-9)和诱导型一氧化氮合酶(iNOS)在胎膜早破胎膜组织中的表达及相关性。 方法: 选择2010年4月-2011年1月我院在临产前行剖宫产终止妊娠的孕妇60例,分为早产胎膜早破组(pPROM)、足月胎膜早破组(tPROM)和正常足月妊娠组(对照组),每组各20例,采用免疫组织化学方法检测胎膜中MMP-9和iNOS的 表达,分析其差异性及相关性;同时取静脉血及羊水,应用ELISA法进行MMP-9 定量检测;采用HE染色检测胎膜,分析MMP-9、iNOS与绒毛膜羊膜炎的关系。结果: MMP-9在tPROM组和pPROM组胎膜组织、母血及羊水中的表达水平均高于对照组(P<0.01),iNOS在pPROM组胎膜中的表达水平高于对照组(P<0.01)。绒毛膜羊膜炎孕妇胎膜中的MMP-9(P<0.05)和iNOS(P<0.01)表达水平均高于非绒毛膜羊膜炎,二者呈正相关(P<0.01)。结论: MMP-9和iNOS表达升高与pPROM及绒毛膜羊膜炎的发生相关;MMP-9和iNOS在PROM发病机制中存在关联作用。     

Maternal and neonatal interleukin-1 receptor antagonist genotype and pregnancy outcome in a population with a high rate of pre-term birth

Problem We evaluated associations between a length polymorphism in intron 2 of the gene coding for IL‐1ra (gene symbol IL1RN) and pregnancy outcome in a population with a high rate of preterm birth. Method of study Subjects were pregnant women in Maceio, Brazil and their newborns. DNA was tested for IL1RN genotypes and alleles by gene amplification using primer pairs that spanned the polymorphic region. Every subject completed a detailed questionnaire. Results The frequency of allele 2 (IL1RN*2) carriage was elevated in mothers with a spontaneous preterm birth (SPTB) in the current pregnancy (P = 0.02) and also with a prior preterm delivery (P = .01). Both SPTB with intact membranes (P = 0.01) and SPTB preceded by pre‐term pre‐mature rupture of membranes (P = .03) were associated with IL1RN*2 carriage. A previous fetal demise was more than twice as prevalent in mothers positive for two copies of IL1RN*2. Conclusion Maternal carriage of IL1RN*2 increases susceptibility to inflammation‐triggered spontaneous pre‐term birth.     

羊水中白细胞介素-8水平与绒毛膜羊膜炎的关系

目的探讨胎膜早破孕妇羊水中白细胞介素-8（IL-8）水平与绒毛膜羊膜炎的关系。方法用酶联免疫法（ELISA法）测定62例胎膜早破孕妇和46例正常足月妊娠未临产孕妇羊水中IL-8浓度;病理检查两组分娩后的胎膜组织,确定有无绒毛膜羊膜炎。结果胎膜早破组羊水中IL-8浓度高于对照组（P〈0.05）,破膜时间超过24h其羊水中IL-8水平明显高于破膜时间小于12h内的患者,绒毛膜羊膜炎组羊水中IL-8浓度高于对照组（P〈0.05）。结论胎膜早破及绒毛膜羊膜炎孕妇羊水中IL-8水平显著升高,可作为绒毛膜羊膜炎的早期诊断指标。     

ORIGINAL ARTICLE: Activation of the Alternative Pathway of Complement is a Feature of Pre‐Term Parturition but not of Spontaneous Labor at Term

Citation Vaisbuch E, Romero R, Erez O, Mazaki‐Tovi S, Kusanovic JP, Soto E, Dong Z, Chaiworapongsa T, Kim SK, Ogge G, Pacora P, Yeo L, Hassan SS. Activation of the alternative pathway of complement is a feature of pre‐term parturition but not of spontaneous labor at term. Am J Reprod Immunol 2010; 63: 318–330 Problem Plasma concentrations of fragment Bb (FBb) are a marker for activation of the alternative pathway of the complement system. High concentrations of FBb in maternal blood, as early as the first trimester, are associated with subsequent spontaneous pre‐term delivery <34 weeks of gestation. The aim of this study was to determine whether spontaneous pre‐term labor (PTL) with intact membranes, intra‐amniotic infection/inflammation (IAI) or labor at term are associated with alterations in circulating maternal FBb concentrations. Method of study This cross‐sectional study included women in the following groups: (i) non‐pregnant (n = 40); (ii) normal pregnancy (gestational age range 20–36, 6/7 weeks, n = 63); (iii) women at term not in labor (n = 70); (iv) women at term in spontaneous labor (n = 59); (v) patients with an episode of PTL who delivered at term (n = 62); (vi) PTL without IAI who delivered pre‐term (n = 30); and (vii) PTL with IAI who delivered pre‐term (n = 67). Maternal plasma FBb concentrations were determined by ELISA. Results (i) Among patients with PTL, those who had a pre‐term delivery either with IAI (1.21 μg/mL, IQR 0.77–2.16) or without IAI (1.13 μg/mL, IQR 0.92–2.08) had a higher median maternal plasma FBb concentration than those who delivered at term (0.86 μg/mL, IQR 0.64–1.57; P = 0.007 and P = 0.026, respectively); (ii) there was no difference in the median plasma FBb concentration between patients with and without IAI who delivered pre‐term (P = 0.9); (iii) in contrast, spontaneous labor at term was not associated with a significant change in the maternal plasma FBb concentration (P = 0.8); (iv) maternal plasma concentration of FBb did not differ significantly between normal pregnant women and the non‐pregnant controls (P = 0.8) and were not correlated with advancing gestational age (r = ?0.28, P = 0.8). Conclusion (i) Pre‐term parturition is associated with activation of the alternative complement pathway in maternal circulation; (ii) such activation is not detectable in spontaneous labor at term; (iii) IAI does not explain the activation of the alternative pathway of complement in PTL. Collectively, these observations suggest that pre‐term and term labors have fundamental differences in the regulation of innate immunity.     

Preterm labor and preterm premature rupture of membranes have a different pattern in the involved compartments of acute histologoic chorioamnionitis and/or funisitis: Patho‐physiologic implication related to different clinical manifestations

It is unknown whether histo‐topographic findings about the involved compartments (i.e., choriodecidua, amnion, chorionic‐plate) of acute‐histologic chorioamnionitis (acute‐HCA) and/or funisitis according to the presence or absence of intra‐amniotic inflammation (IAI) and/or fetal inflammatory response syndrome (FIRS) are different between preterm labor and intact membranes (PTL) and preterm premature rupture of membranes (preterm‐PROM). The involved compartments of acute‐HCA and/or funisitis were examined in 161 singleton preterm‐births (<34 weeks) due to PTL (n = 88) and preterm‐PROM (n = 73). The study‐population was divided into IAI(?)/FIRS(?), IAI(+)/FIRS(?), and IAI(+)/FIRS(+) groups according to the presence or absence of IAI (amniotic‐fluid MMP‐8 ≥ 23 ng/ml) and/or FIRS (umbilical‐cord plasma CRP ≥ 200 ng/ml). Histological inflammation was not detected in any‐compartment except choriodecidua in IAI(?)/FIRS(?) group with PTL while inflammation appeared in all‐compartment0s (choriodeciduitis‐46.2 %; amnionitis‐23.1 %; funisitis‐30.8 %; chorionic‐plate inflammation‐7.7 %) in IAI(?)/FIRS(?) group with preterm‐PROM. IAI(+)/FIRS(?) group had a significantly higher frequency of inflammation in each‐compartment than IAI(?)/FIRS(?) group in PTL (each‐for P < 0.01), but not preterm‐PROM (each‐for P > 0.1). However, IAI(+)/FIRS(+) group had a significantly higher rate of inflammation in each compartment than IAI(+)/FIRS(?) group in both PTL and preterm‐PROM (each‐for P < 0.05). We first demonstrated that PTL and preterm‐PROM had a different pattern in the involved compartments of acute‐HCA and/or funisitis in the IAI(?)/FIRS(?‐) group and in the change of involved compartments from IAI(?)/FIRS(?) to IAI(+)/FIRS(?)     

孕晚期羊水量的减少与妊娠结局的临床分析

目的探讨羊水量减少孕妇发生妊娠不良结局的危险。方法2007年3月至2007年9月在新疆医科大学第一附属医院住院分娩的羊水减少的孕妇60例,羊水指数（AFI）5.1cm-8cm为羊水量减少,研究组和对照组均进行产前检查,包括：胎心监护,S/D比值测定,B超检查。妊娠结局包括分娩孕周,新生儿出生体重,胎盘钙化,分娩方式,羊水污染,新生儿窒息,围生儿死亡。结果羊水减少组新生儿体重明显低于羊水正常组（P〈0.05）,2组羊水污染有统计学意义（P〈0.05）,羊水减少组剖宫产率高于羊水正常组,2组终止妊娠时间,新生儿窒息的差异无统计学意义（P〉0.05）,2组均无围生儿死亡。羊水减少（5.1-6.5）A1组与羊水减少（6.6-8.0）A2组相比较,终止妊娠时间、分娩方式,羊水污染,新生儿窒息、新生儿出生体重及围生儿死亡的发生,差异无统计学意义（P〉0.05）,胎盘钙化A1组和A2组有统计学意义（P〈0.05）。结论羊水量的减少是妊娠不良结局的一个危险信号,为减少妊娠不良结局的发生,应重视对羊水量减少孕妇的监测。     

The role of CD4+CD25bright regulatory T cells in the maintenance of pregnancy, premature rupture of membranes, and labor

PURPOSE: The aim of this study was to evaluate the changes of the regulatory T cell subset in peripheral blood caused by gestational age and premature rupture of membranes (PROM) with or without labor to verify the role of regulatory T cells in pregnancy. PATIENTS AND METHODS: We investigated regulatory T cell distribution in the peripheral blood of pregnancies during the first trimester (group I, n=2), the second trimester (group II, n=12), and the third trimester without PROM and labor (group III, n=15). In addition, we evaluated pregnancies in the third trimester complicated by PROM (group IV, n=4) and labor with no complication by PROM (Group V, n=5). Comparisons were made with non-pregnant controls (group VI, n=4) using flow cytometry. RESULTS: During uncomplicated pregnancy, the CD4(+)CD25(bright) regulatory T cell population decreased with advancing gestational age (group I=3.35+/-0.47, group II=2.91+/-1.44, group III=2.81+/-1.36, group VI=2.52+/-0.71, p=NS). When we compared group IV with group III and V to evaluate the changes of the regulatory T cells with PROM, the CD4(+)CD25(bright) regulatory T cell population was significantly decreased in group IV compared to group III (p=0.001) and group V (p=0.026). CONCLUSION: The present results revealed that the regulatory T cell population increased in early pregnancy but decreased in pregnancies complicated by PROM, indicating that regulatory T cells might be related to the maintenance of pregnancy.     

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid

Cell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.     

Granulocyte-macrophage colony-stimulating factor levels in amniotic fluid before the onset of labor and during labor do not differ in normal pregnancies

PROBLEM: Granulocyte-macrophage colony-stimulating factor (GM-CSF) at the implantation site may regulate invasion and differentiation of placental trophoblast. We evaluated whether GM-CSF levels in amniotic fluid during labor contributing to subsequent delivery differed from those before the onset of labor in normal pregnancies. METHOD OF STUDY: This study enrolled 36 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these pregnancies, 18 were women during labor that led to subsequent term delivery (labors). The other 18 were women without labor underwent cesarean section (controls). These two groups (18 labors and 18 controls) were compared. The average gestational age at entry was 38-39 weeks of gestation. The women's ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the GM-CSF levels were compared between two groups. The GM-CSF level was determined by the enzyme-linked immunosorbent assay method. RESULTS: There was no significant increase in GM-CSF levels in amniotic fluid during labor compared with that before the onset of labor. CONCLUSIONS: The GM-CSF in amniotic fluid may not promote the onset of labor at term and/or term labor contributing to subsequent delivery may not induce the production and secretion of GM-CSF into amniotic cavity.     

Macrophage colony-stimulating factor levels in amniotic fluid before and after the onset of labor do not differ in normal pregnancies

PROBLEM: Macrophage colony-stimulating factor (M-CSF) promotes placental growth and maintenance. M-CSF also regulates trophoblast invasion into the placental bed. We evaluated whether M-CSF levels in amniotic fluid during labor contributing to subsequent delivery differed from those before the onset of labor in normal pregnancies. METHOD OF STUDY: This study enrolled 48 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these pregnancies, 24 were women during labor: 22 led to subsequent term delivery (labors); two had premature delivery. The other 24 were women without labor underwent cesarean section (controls). These two groups (22 labors and 24 controls) were compared. The average gestational age at entry was 38 weeks of gestation. The women's ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the M-CSF levels were compared between two groups. The M-CSF level was determined by the sandwich enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The levels of M-CSF in amniotic fluid did not differ significantly between the women during labor and those without labor. CONCLUSIONS: M-CSF in amniotic fluid may not contribute to the onset of labor in term pregnancy and/or labor resulting in subsequent delivery may not induce the production and secretion of M-CSF into amniotic cavity.     

Interleukin-6 as a predictor of subclinical chorioamnionitis in preterm premature rupture of membranes

Citation Gulati S, Bhatnagar S, Raghunandan C, Bhattacharjee J. Interleukin‐6 as a predictor of subclinical chorioamnionitis in preterm premature rupture of membranes. Am J Reprod Immunol 2012; 67: 235–240 Problem One of the major challenges faced by the clinicians in preterm premature rupture of the membranes (PPROM) is to correctly identify when a significant chorioamnionitis is evolving and decide timely delivery of the fetus. Measuring interleukin‐6 levels in maternal serum can be useful for the identification of asymptomatic intrauterine infections in subjects with PPROM. Method of study A total of 75 pregnant women, of which 45 pregnant women presenting with PPROM between 24 and 34 weeks gestation and 30 healthy pregnant women without PPROM, were included in the study. Serum IL‐6 levels were determined by solid‐phase sandwich enzyme‐linked immunosorbent assay (Diaclone Research, Besancon, France). Results The mean serum IL‐6 value at admission in the control group was 2.48 ± 2.7 pg/mL and in the study group was 11.86 ± 14.5 pg/mL (P = 0.001). Mean serum IL‐6 concentrations at admission in subjects without histological chorioamnionitis were 3.98 ± 3.9 pg/mL and in those who had histological chorioamnionitis were 20.09 ± 16.8 pg/ml (P < 0.001). Conclusion Maternal serum IL‐6 levels were significantly elevated in subjects with PPROM with infectious morbidity as compared to those without infectious morbidity in the present study. There was a significant rise in maternal serum IL‐6 levels with increased duration of rupture of membranes and with evidence of histological chorioamnionitis and funisitis in the placenta.     

MMP-13 (collagenase 3) immunolocalisation during initial orthodontic tooth movement in rats

Matrix metalloproteinases (MMPs) are enzymes that play a central role in periodontal ligament (PDL) space remodelling during orthodontic tooth movement. It has previously been shown that messenger RNA levels of MMP-13 increase significantly following the application of orthodontic forces. The aim of the present study was to examine immunolocalisation of MMP-13 and to evaluate if this collagenase is time-dependently and differentially detected within the PDL following the application of orthodontic forces to create areas of compression and tension. This was achieved by placing elastic bands between the maxillary first and second molars of 16 male Sprague-Dawley rats (each weighing 120-200g) for 12 and 24h. The molar-bearing segments were dissected and processed for histological and immunohistochemical examination. Binding of a monoclonal antibody was used to evaluate MMP-13 localization using an indirect streptavidin/biotin immunperoxidase technique. MMP-13 was found to be inducible at the protein level by the application of forces. The PDL and osteoblast-lineage cells showed a time-dependent increase in immunolabelling of MMP-13. Immunolabelling of MMP-13 was detected initially on the compression side, and then on both the compression and the tension sides. Since this increase in MMP-13 immunolabelling occurred very early following the application of an orthodontic force in both PDL and alveolar bone, this would indicate that MMP-13 might play an important role during tooth movement.