Adiponectin multimers in maternal plasma

Objective. Adiponectin is an anti-diabetic, anti-atherogenic, anti-inflammatory, and angiogenic adipokine that circulates in oligomeric complexes including: low molecular weight (LMW) trimers, medium molecular weight (MMW) hexamers, and high molecular weight (HMW) isoforms. The aim of this study was to determine whether there are changes in adiponectin multimers in pregnancy and as a function of maternal weight.

Study design. In this cross-sectional study, plasma concentrations of total, HMW, MMW, and LMW adiponectin were determined in women included in three groups: (1) normal pregnant women of normal body mass index (BMI) (n = 466), (2) overweight pregnant women (BMI ≥25; n = 257), and (3) non-pregnant women of normal weight (n = 40). Blood samples were collected once from each woman between 11 and 42 weeks of gestation. Plasma adiponectin multimer concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis.

Results. (1) The median HMW adiponectin concentration and the median HMW/total adiponectin ratio were significantly higher, and the median LMW adiponectin concentration was significantly lower in pregnant women than in non-pregnant women. (2) Among pregnant women, the median plasma concentration of total, HMW, and MMW adiponectin was significantly higher in normal weight women than in overweight patients. (3) Maternal HMW was the most prevalent adiponectin multimer regardless of gestational age or BMI status. (4) There were no significant differences in the median concentration of total, MMW, and LMW adiponectin and their relative distribution with advancing gestation.

Conclusion. Human pregnancy is characterized by quantitative and qualitative changes in adiponectin multimers, especially the most active isoform, HMW adiponectin.     

Maternal plasma visfatin in preterm labor

Objective.?Visfatin, a novel adipokine with diabetogenic and immunoregulatory properties, has been implicated in the pathophysiology of insulin resistance, as well as in various acute and chronic inflammatory disorders. We have previously reported that amniotic fluid concentrations of visfatin are higher in patients with preterm labor (PTL) and intra-amniotic infection than in patients with PTL without infection. The aim of this study was to determine whether spontaneous PTL with intact membranes and intra-amniotic infection/inflammation (IAI) is associated with changes in maternal plasma circulating visfatin concentrations.

Study design.?This cross-sectional study included patients in the following groups: (1) normal pregnant women (n = 123); (2) patients with an episode of PTL and intact membranes without IAI who delivered at term (n = 57); (3) PTL without IAI who delivered preterm (n = 47); and (4) PTL with IAI who delivered preterm (n = 57). Plasma visfatin concentrations were determined by ELISA. Non-parametric statistics were used for analysis.

Results.?(1) PTL with IAI leading to preterm delivery was associated with a higher median maternal plasma concentration of visfatin than normal pregnancy; (2) among patients with PTL, those with IAI had the highest median maternal concentration of visfatin; (3) the changes in maternal plasma visfatin remained significant after adjusting for maternal age, body mass index, gestational age at sampling, and birth weight.

Conclusion.?(1) PTL with IAI is characterized by high maternal circulating visfatin concentrations; (2) these findings suggest that visfatin plays a role in the regulation of the metabolic adaptations to insults resulting in PTL in the context of IAI.     

Amniotic fluid heat shock protein 70 concentration in histologic chorioamnionitis,term and preterm parturition

Objective. Heat shock protein (HSP) 70, a conserved member of the stress protein family, is produced in almost all cell types in response to a wide range of stressful stimuli, and its production has a survival value. Evidence suggests that extracellular HSP70 is involved in the activation of the innate and adaptive immune response. Furthermore, increased mRNA expression of HSP70 has been observed in human fetal membranes following endotoxin stimulation. This study was conducted to determine the changes in amniotic fluid HSP70 concentrations during pregnancy, term and preterm parturition, intra-amniotic infection (IAI), and histologic chorioamnionitis.

Study design. A cross-sectional study was conducted in 376 pregnant women in the following groups: (1) women with a normal pregnancy who were classified into the following categories: (a) women in the mid-trimester (14–18 weeks) who underwent amniocentesis for genetic indications and delivered normal infants at term (n=72); (b) women at term not in labor (n = 23); and (c) those at term in labor (n = 48). (2) Women with spontaneous preterm labor and intact membranes who were subdivided into the following categories: (a) preterm labor who delivered at term without IAI (n = 42); (b) preterm labor who delivered preterm without IAI (n = 57); and (c) preterm labor and delivery with IAI (n = 30). (3) Women with preterm prelabor rupture of membranes (PROM) with (n = 50) and without (n = 54) IAI. Among patients with preterm labor with intact membranes and preterm PROM who delivered within 72 hours of amniocentesis, placenta, umbilical cord, and chorioamniotic membranes were collected and assessed for the presence or absence of acute inflammatory lesions in the extraplacental membranes (histologic chorioamnionitis) and/or umbilical cords (funisitis). HSP70 concentrations in amniotic fluid were determined using a sensitive and specific immunoassay. Non-parametric statistics were used for analysis. A p value of <0.05 was considered statistically significant.

Results. Immunoreactive HSP70 was detected in 88% (332/376) of amniotic fluid samples. The median amniotic fluid HSP70 concentration was significantly higher in women at term without labor than in those in the mid-trimester (term no labor: median 34.9 ng/mL, range 0–78.1 ng/mL vs. mid-trimester; median 6.6 ng/mL, range 0–20.8 ng/mL; p<0.001). Among patients with spontaneous preterm labor and preterm PROM, those with IAI had a significantly higher median amniotic fluid HSP70 concentration than those without IAI (preterm labor with IAI: median 82.9 ng/mL, range 0–500 ng/mL vs. preterm labor without IAI: median 41.7 ng/mL, range 0–244 ng/mL; p = 0.001; preterm PROM with IAI: median 86.5 ng/mL, range 0–428 ng/mL vs. preterm PROM without IAI: median 55.9 ng/mL, range 14.9–299.9 ng/mL; p = 0.007). There was no significant difference in the median amniotic fluid HSP70 concentration between patients with preterm labor who delivered preterm without IAI and those who delivered at term (p = 0.6). However, among patients with preterm labor without IAI, there was an inverse relationship between amniotic fluid concentration of HSP70 and the amniocentesis-to-spontaneous delivery interval (Spearman's Rho = ?0.26; p = 0.02). Patients with histologic chorioamnionitis/funisitis had a significantly higher median amniotic fluid HSP70 concentration than those without inflammation (inflammation: median 108.7 ng/mL, range 0–500 ng/mL vs. without inflammation: median 67.9 ng/mL, range 7.1–299.9 ng/mL; p = 0.02). Women at term in labor had a median amniotic fluid concentration of HSP70 significantly higher than those not in labor (term in labor: median 60.7 ng/mL, range 0–359.9 ng/mL vs. term not in labor: median 34.9 ng/mL, range 0–78.1 ng/mL; p = 0.02).

Conclusions. Intra-amniotic infection, histologic chorioamnionitis, and term parturition are associated with elevated amniotic fluid HSP70 concentrations. HSP70 plays a role in the host defense mechanism by activating the innate arm of the immune response in women with intrauterine infection. The mechanisms of preterm and term parturition in humans may involve extracellular HSP70.     

Amniotic fluid soluble human leukocyte antigen-G in term and preterm parturition,and intra-amniotic infection/inflammation

Objective.?Circulating soluble human leukocyte antigen-G (sHLA-G) has been associated with pregnancy complications, and determination of sHLA-G concentrations in amniotic fluid (AF) has been reported in normal pregnancies. Our aim was to determine if the AF concentrations of sHLA-G change with advancing gestation, spontaneous labor at term, and in patients with spontaneous preterm labor (PTL) with intact membranes, as well as in those with preterm prelabor rupture of membranes (PROM), in the presence or absence of intra-amniotic infection/inflammation (IAI).

Study design.?This cross-sectional study included the following groups: (1) mid-trimester (n?=?55); (2) normal pregnancy at term with (n?=?50) and without (n?=?50) labor; (3) spontaneous PTL with intact membranes divided into: (a) PTL who delivered at term (n?=?153); (b) PTL who delivered preterm without IAI (n?=?108); and (c) PTL with IAI (n?=?84); and (4) preterm PROM with (n?=?46) and without (n?=?44) IAI. sHLA-G concentrations were determined by ELISA. Non-parametric statistics were used for analysis.

Results.?(1) Among patients with PTL, the median AF sHLA-G concentration was higher in patients with IAI than in those without IAI or women that delivered at term (p?<?0.001 for both comparisons); (2) Similarly, patients with preterm PROM and IAI had higher median AF sHLA-G concentrations than those without IAI (p?=?0.004); (3) Among patients with PTL and delivery, those with histologic chorioamnionitis and/or funisitis had a higher median AF sHLA-G concentration than those without histologic inflammation (p?<?0.001); and (4) The median AF sHLA-G concentration did not change with advancing gestational age.

Conclusions.?AF sHLA-G concentrations are elevated in preterm parturition associated to IAI as well as in histologic chorioamnionitis. We propose that sHLA-G may participate in the regulation of the host immune response against intra-amniotic infection.     

Pentraxin 3 in maternal circulation: An association with preterm labor and preterm PROM,but not with intra-amniotic infection/inflammation

Objective.?Pentraxin 3 (PTX3) is an acute-phase protein that has an important role in the regulation of the innate immune response. The aim of this study was to determine if maternal plasma PTX3 concentration changes in the presence of intra-amniotic infection and/or inflammation (IAI) in women with preterm labor (PTL) and intact membranes, as well as those with preterm prelabor rupture of membranes (preterm PROM).

Study design.?This cross-sectional study included women in the following groups: (1) nonpregnant (n?=?40); (2) uncomplicated pregnancies in the first (n?=?22), second (n?=?22) or third trimester (n?=?71, including 50 women at term not in labor); (3) uncomplicated pregnancies at term with spontaneous labor (n?=?49); (4) PTL and intact membranes who delivered at term (n?=?49); (5) PTL without IAI who delivered preterm (n?=?26); (6) PTL with IAI (n?=?65); (7) preterm PROM without IAI (n?=?25); and (8) preterm PROM with IAI (n?=?77). Maternal plasma PTX3 concentrations were determined by ELISA.

Results.?(1) Maternal plasma PTX3 concentrations increased with advancing gestational age (r?=?0.62, p?<?0.001); (2) women at term with spontaneous labor had a higher median plasma PTX3 concentration than those at term not in labor (8.29?ng/ml vs. 5.98?ng/ml, p?=?0.013); (3) patients with an episode of PTL, regardless of the presence or absence of IAI and whether these patients delivered preterm or at term had a higher median plasma PTX3 concentration than normal pregnant women (p?<?0.001 for all comparisons); (4) similarly, patients with preterm PROM, with or without IAI had a higher median plasma PTX3 concentration than normal pregnant women (p?<?0.001 for both comparisons); and (5) among patients with PTL and those with preterm PROM, IAI was not associated with significant changes in the median maternal plasma PTX3 concentrations.

Conclusions.?The maternal plasma PTX3 concentration increases with advancing gestational age and is significantly elevated during labor at term and in the presence of spontaneous preterm labor or preterm PROM. These findings could not be explained by the presence of IAI, suggesting that the increased PTX3 concentration is part of the physiologic or pathologic activation of the pro-inflammatory response in the maternal circulation during the process of labor at term or preterm.     

Evidence of the involvement of caspase-1 under physiologic and pathologic cellular stress during human pregnancy: A link between the inflammasome and parturition

Objective. Caspase-1 is a component of the NALP3 inflammasome, a cytosolic multiprotein complex that mediates the processing of pro-inflammatory caspases and cytokines. The inflammasome represents the first line of defense against cellular stress and is a crucial component of innate immunity. Caspase-1 is the enzyme responsible for the cleavage and activation of interleukin (IL)-1β, which is a potent pro-inflammatory cytokine, and plays a central role in the mechanisms leading to labor (preterm and term) particularly in the context of intrauterine infection/inflammation. In addition, caspase-1 cleaves IL-18 and IL-33. The objectives of this study were to determine whether there is a relationship between amniotic fluid concentrations of caspase-1 and gestational age, parturition (term and preterm), and intra-amniotic infection/inflammation (IAI).

Study design. A cross-sectional study was conducted including 143 pregnant women in the following groups: (1) mid-trimester of pregnancy (n = 18); (2) term not in labor (n = 25); (3) term in labor (n = 28); (4) preterm labor (PTL) who delivered at term (n = 23); (5) PTL without IAI who delivered preterm (n = 32); (6) PTL with IAI who delivered preterm neonates (n = 17). Caspase-1 concentrations in amniotic fluid were determined by a specific and sensitive immunoassay. Non-parametric statistics were used for analysis.

Results. (1) Caspase-1 was detected in amniotic fluid of women at term, but in none of the mid-trimester samples. (2) Patients in labor at term had a significantly higher median amniotic fluid concentration of caspase-1 than women at term not in labor (term in labor: 10.5 pg/mL, range 0.0–666.0 vs. term not in labor: 5.99 pg/mL, range 0.0–237.4; p < 0.05). (3) Among patients with spontaneous PTL, those with IAI (median 41.4 pg/mL, range 0.0–515.0) had a significantly higher median amniotic fluid caspase-1 concentration than those without IAI who delivered preterm (median 0.0 pg/mL, range 0.0–78.4) and than those who delivered at term (median 0.0 pg/mL, range 0.0–199.5); p < 0.001 for both comparisons.

Conclusions. (1) The presence and concentration of caspase-1 in the amniotic fluid varies as a function of gestational age. (2) Women with spontaneous labor at term had a higher median caspase-1 amniotic fluid concentration than women at term without labor. This suggests that the inflammasome may be activated in spontaneous parturition at term. Since most women with labor do not have intra-amniotic infection, we propose that cellular stress during labor accounts for activation of the inflammasome. (3) Preterm labor associated with infection/inflammation was also associated with a high concentration of caspase-1, suggesting that infection may induce caspase-1 production and activation of the inflammasome. (4) The sequential activation of the inflammasome and caspase-1, leading to interleukin-1β processing and secretion, is a candidate pathway leading to the activation of the common pathway of parturition.     

The anti-inflammatory limb of the immune response in preterm labor,intra-amniotic infection/inflammation,and spontaneous parturition at term: A role for interleukin-10

Objective. The anti-inflammatory limb of the immune response is crucial for dampening inflammation. Spontaneous parturition at term and preterm labor (PTL) are mediated by inflammation in the cervix, membranes, and myometrium. This study focuses on the changes in the amniotic fluid concentrations of the anti-inflammatory cytokine interleukin (IL)- 10. The objectives of this study were to determine whether there is a relationship between amniotic fluid concentrations of IL-10 and gestational age, parturition (at term and preterm), and intra-amniotic infection/inflammation (IAI).

Study design. A cross-sectional study was conducted including 301 pregnant women in the following groups: (1) mid-trimester of pregnancy who delivered at term (n = 112); (2) mid-trimester who delivered preterm neonates (n = 30); (3) term not in labor without IAI (n = 40); (4) term in labor without IAI (n = 24); (5) term in labor with IAI (n = 20); (6) PTL without IAI who delivered at term (n = 31); (7) PTL without IAI who delivered preterm (n = 30); (8) PTL with IAI who delivered preterm (n = 14). IL-10 concentrations in amniotic fluid were determined by a specific and sensitive immunoassay. Non-parametric statistics were used for analysis.

Results. (1) IL-10 was detectable in amniotic fluid and its median concentration did not change with gestational age from mid-trimester to term. (2) Patients in labor at term had a significantly higher median amniotic fluid IL-10 concentration than that of patients at term not in labor (p = 0.04). (3) Women at term in labor with IAI had a significantly higher median amniotic fluid IL-10 concentration than that of patients at term in labor without IAI (p = 0.02). (4) Women with PTL and IAI who delivered preterm had a significantly higher median amniotic fluid concentration of IL-10 than those without IAI who delivered preterm and than those who delivered at term (p = 0.009 and p < 0.001, respectively). (5) Among patients with preterm labor without IAI, those who delivered preterm had a significantly higher median amniotic fluid IL-10 concentration than those who delivered at term (p = 0.03).

Conclusions. The anti-inflammatory cytokine IL-10 is detectable in the amniotic fluid of normal pregnant women. Spontaneous parturition at term and in preterm gestation is associated with increased amniotic fluid concentrations of IL-10. IAI (preterm and at term) is also associated with increased amniotic fluid concentrations of IL-10. We propose that IL-10 has a role in the regulation of the immune response in vivo by initiating actions that dampen inflammation.     

Adiponectin in amniotic fluid in normal pregnancy,spontaneous labor at term,and preterm labor: A novel association with intra-amniotic infection/inflammation

Objective.?Adiponectin, an anti-inflammatory and anti-diabetogenic adipokine, has an important regulatory effect on both the innate and adaptive limbs of the immune response. The objective of this study was to determine whether adiponectin is present in amniotic fluid (AF) and if its concentration changes with gestational age, in the presence of labor, and in the presence of intra-amniotic infection (IAI) in patients with spontaneous preterm labor (PTL) and intact membranes.

Study design.?This cross-sectional study included 468 patients in the following groups: (1) women in the mid-trimester of pregnancy (14–18 weeks) who underwent amniocentesis for genetic indications and delivered a normal neonate at term (n?=?52); (2) normal pregnant women at term with (n?=?49) and without (n?=?41) spontaneous labor; (3) patients with an episode of PTL and intact membranes who were classified into: (a) PTL who delivered at term (n?=?149); (b) PTL who delivered preterm (<37 weeks gestation) without IAI (n?=?108); and (c) PTL with IAI (n?=?69). Adiponectin concentration in AF was determined by ELISA.

Results.?(1) The median AF adiponectin concentration at term was significantly higher than in the mid-trimester (35.6?ng/ml, interquartile range [IQR] 26.4–52.7 vs. 29.9?ng/ml, IQR 19.9–35.2; p?=?0.01); (2) among women with PTL and intact membranes, the median AF adiponectin concentration was significantly higher in patients with IAI than in those without IAI who delivered either at term (54.3?ng/ml, 39.0–91.8 vs. 50.1?ng/ml, 33.2–72.8; p?=?0.02) or preterm (47.6?ng/ml, 32.6–74.6; p?=?0.01); and (3) among women at term, there was no significant difference in the median AF adiponectin concentration between those with and without labor (33.7?ng/ml, IQR 21.7–53.9 vs. 35.6?ng/ml, IQR 26.4–52.7; respectively p?=?0.5).

Conclusions.?(1) Adiponectin is a physiologic constituent of AF; and (2) adiponectin concentrations in AF are increased significantly with advancing gestation and in the presence of IAI. Collectively, these findings suggest that adiponectin plays a dynamic role in normal gestation and in the presence of IAI.     

Changes in amniotic fluid concentration of thrombin–antithrombin III complexes in patients with preterm labor: Evidence of an increased thrombin generation

Objective.?Preterm labor is associated with excessive maternal thrombin generation, as evidenced by increased circulating thrombin–antithrombin (TAT) III complexes concentration. In addition to its hemostatic functions, thrombin has uterotonic properties that may participate in the mechanism leading to preterm birth in cases of intrauterine bleeding. Thrombin also has a proinflammatory role, and inflammation is associated with increased thrombin generation. The aim of this study was to determine whether intra-amniotic infection/inflammation (IAI) is associated with increased amniotic fluid (AF) thrombin generation in women with preterm and term deliveries.

Study design.?This cross-sectional study included the following groups: (1) mid-trimester (n = 74); (2) term not in labor (n = 39); (3) term in labor (n = 25); (4) term in labor with IAI (n = 22); (5) spontaneous preterm labor (PTL) who delivered at term (n = 62); (6) PTL without IAI who delivered preterm (n = 59); (7) PTL with IAI (n = 71). The AF TAT III complexes concentration was measured by enzyme linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis.

Results.?(1) TAT III complexes were identified in all AF samples; (2) patients with PTL who delivered preterm, with and without IAI, had a higher median AF TAT III complexes concentration than those with an episode of PTL who delivered at term (p < 0.001, p = 0.03, respectively); (3) among patients with PTL without IAI, elevated AF TAT III complexes concentration were independently associated with a shorter amniocentesis-to-delivery interval (hazard ratio, 1.5; 95% CI, 1.07–2.1); (4) among patients at term, those with IAI had a higher median AF TAT III complexes concentration than those without IAI, whether in labor or not in labor (p = 0.02); (5) there was no significant difference between the median AF TAT III complexes concentration of patients at term with and without labor; (6) patients who had a mid-trimester amniocentesis had a lower median AF TAT III complexes concentration than that of patients at term not in labor (p < 0.001).

Conclusions.?We present herein a distinct difference in the pattern of intra-amniotic thrombin generation between term and preterm parturition. PTL leading to preterm delivery is associated with an increased intra-amniotic thrombin generation regardless of the presence of IAI. In contrast, term delivery is associated with an increased intra-amniotic thrombin generation only in patients with IAI.     

Amniotic fluid fetal hemoglobin in normal pregnancies and pregnancies complicated with preterm labor or prelabor rupture of membranes

Objective. Hemoglobin and its catabolic products have been associated with amniotic fluid (AF) discoloration and intra-amniotic infection/inflammation (IAI). However, the origin of AF hemoglobin (maternal or fetal) has not been determined. The aims of this study were to determine if fetal hemoglobin can be detected in AF obtained from normal pregnancies, and whether there is an association between AF fetal hemoglobin concentrations and gestational age, spontaneous labor (term and preterm), preterm prelabor rupture of membranes (PPROM) and IAI.

Study design. This cross-sectional study included pregnant women in the following groups: (1) mid-trimester (n = 60); (2) term not in labor (n = 21); (3) term in labor (n = 47); (4) spontaneous preterm labor with intact membranes (PTL) without IAI who delivered at term (n = 89); (5) PTL without IAI who delivered preterm (n = 74); (6) PTL with IAI (n = 78); (7) PPROM with (n = 48) and (8) without IAI (n = 48). AF fetal hemoglobin concentrations were determined by ELISA. Non-parametric statistics were used for analyses.

Results. (1) Fetal hemoglobin was detected in 80.4% of all AF samples; (2) women at term not in labor had a higher median AF fetal hemoglobin concentration than those at mid-trimester (p = 0.008); (3) labor at term was not associated with a significant difference in the median AF fetal hemoglobin concentration; (4) the median AF fetal hemoglobin concentration was not significantly different among the three PTL groups or between the PPROM groups; (5) women with PTL and IAI had a lower AF fetal hemoglobin percentage of the total hemoglobin than those without IAI who delivered preterm (p = 0.03) or at term (p < 0.001); (6) The median AF fetal hemoglobin concentration was higher in pregnancies complicated with PTL or PPROM than in women at term (p < 0.001 for all comparison).

Conclusions. (1) The concentration of immunoreactive AF fetal hemoglobin increases with gestational age; (2) the median AF fetal hemoglobin concentration is higher in pregnancies complicated with PTL or PPROM than in term pregnancies; (3) among women with PTL or PPROM, the AF fetal hemoglobin concentrations were not associated with IAI; (4) however, women with PTL and IAI had a lower percentage of AF fetal hemoglobin of the total hemoglobin than those without IAI, suggesting different mechanisms of disease.     

Evidence in support of a role for anti-angiogenic factors in preterm prelabor rupture of membranes

Objective.?Vaginal bleeding, placental abruption, and defective placentation are frequently observed in patients with preterm prelabor rupture of membranes (PROM). Recently, a role of vascular endothelial growth factor (VEGF) and its receptor, VEGF receptor (VEGFR)- 1 has been implicated in the mechanisms of membrane rupture. The purpose of this study was to determine whether the soluble form of VEGFR-1 and -2 concentrations in amniotic fluid (AF) change with preterm PROM, intra-amniotic infection/inflammation (IAI), or parturition.

Study design.?This cross-sectional study included 544 patients in the following groups: (1) midtrimester (MT) (n?=?48); (2) preterm labor (PTL) leading to term delivery (n?=?143); (3) PTL resulting in preterm delivery with (n?=?72) and without IAI (n?=?100); (4) preterm PROM with (n?=?46) and without IAI (n?=?42); (5) term in labor (n?=?48); and (6) term not in labor (n?=?45). The concentrations of sVEGFR-1 and sVEGFR-2 were determined by ELISA. Non-parametric statistics and logistic regression analysis were applied.

Results.?(1) Preterm PROM (with and without IAI) had a lower median AF concentration of sVEGFR-1 than patients with PTL who delivered at term (p?<?0.001 for each comparison); (2) A decrease in AFsVEGFR-1 concentrations per each quartile was associated with PROM after adjusting for confounders (OR 1.8; 95%CI 1.4–2.3); (3) IAI, regardless of the membrane status, was not associated with a change in the median AF concentrations of sVEGFR-1 and sVEGFR-2 (p?>?0.05 for each comparison); and (4) Spontaneous term and PTL did not change the median sVEGFR-1 and sVEGFR-2 concentrations (p?>?0.05 for each comparison).

Conclusion.?(1) This is the first evidence that preterm PROM is associated with a lower AF concentration of sVEGFR-1 than patients with PTL intact membranes. These findings cannot be attributed to gestational age, labor, or IAI; and (2) AF concentrations of sVEGFR-2 did not change with preterm PROM, IAI, or labor at term and preterm.     

A role for CXCL13 (BCA-1) in pregnancy and intra-amniotic infection/inflammation

Objectives. CXCL13 is a potent chemokine, produced by mature and recently recruited macrophages to sites of inflammation, which has antimicrobial and anti-angiogenic properties. The purpose of this study was to: (1) determine whether CXCL13 is present in maternal serum, umbilical cord blood, and amniotic fluid (AF); (2) to determine if AF concentration changes with intra-amniotic infection/inflammation (IAI); and (3) to localize the production of CXCL13 in chorioamniotic membranes and umbilical cord.

Study design. A cross-sectional study on maternal serum was performed including patients in the following groups: (1) non-pregnant women (n = 20), (2) normal pregnant women (n = 49), (3) patients at term not in labor (n = 30), and (4) patients in spontaneous labor at term (n = 29). Umbilical cord blood was collected from term neonates with (n = 30) and without labor (n = 28). Amniotic fluid was obtained from patients in the following groups: (1) midtrimester (n = 65); (2) term not in labor (n = 22); (3) term in labor (n = 47); (4) preterm labor (PTL) with intact membranes leading to term delivery (n = 70); and (5) PTL leading to preterm delivery with IAI (n = 79) and without IAI (n = 60). CXCL13 concentrations were determined by enzyme-linked immunosorbent assay. Chorioamniotic membranes and umbilical cords were examined with immunohistochemistry. Non-parametric statistics were used for analysis.

Results. (1) CXCL13 was present in 100% of serum and cord blood samples, and 99% of AF samples (339/343). (2) Serum CXCL13 concentration was significantly higher in pregnant women when compared to non-pregnant women (median 313.3 pg/mL (interquartile range (IQR) 197.2–646.9) vs. 40.5 pg/mL (IQR 29.5–93.5), respectively; p < 0.001). (3) Serum CXCL13 concentration decreased with advancing gestational age (Spearman's Rho = ?0.424; p < 0.001). (4) There were no significant differences in the median serum CXCL13 concentration between women at term with and without labor (371.6 pg/mL (IQR 194.3–614.3) vs. 235.1 pg/mL (IQR 182.8–354.7), respectively; p = 0.6). (5) The concentration of CXCL13 in AF did not change with gestational age (p = 0.1). (6) Patients with PTL and delivery with IAI had a significantly higher median concentration of CXCL13 than those without IAI (median 513.2 pg/mL (IQR 199.7–2505.5) vs. 137.3 pg/mL (IQR 96.7–209.6), respectively; p < 0.001) and those who delivered at term (133.7 pg/mL (IQR 97.8–174.8); p < 0.001). (7) Spontaneous labor did not result in a change in the median AF concentration of CXCL13 (labor: 86.9 pg/mL (IQR 55.6–152.0) vs. no labor: 77.8 pg/mL (IQR 68.0–98.0); p = 0.8). (8) CXCL13 was immunolocalized to macrophages in fetal membranes and umbilical vein.

Conclusions. (1) We report for the first time the presence of CXCL13 in AF. (2) AF CXCL13 concentrations are dramatically increased in IAI. (3) Unlike other chemokines, AF and serum CXCL13 concentrations did not change with spontaneous parturition.     

Fragment Bb in amniotic fluid: evidence for complement activation by the alternative pathway in women with intra-amniotic infection/inflammation

Objective.?Fragment Bb is an activator of the alternative pathway of the complement system. Recently, increased first trimester maternal plasma concentrations of this fragment were reported in patients destined to have a spontaneous preterm delivery before 34 weeks of gestation. The aim of this study was to determine whether the amniotic fluid (AF) concentrations of fragment Bb change with gestational age, spontaneous labor (term and preterm) and in the presence of intra-amniotic infection/inflammation (IAI).

Study design.?This cross-sectional study included patients in the following groups: (1) mid-trimester (n = 64); (2) term in spontaneous labor (n = 70); (3) term not in labor (n = 43); (4) spontaneous preterm labor (PTL) who delivered at term (n = 76); (5) PTL without IAI who delivered preterm (n = 73); (6) PTL with IAI (n = 76); (7) preterm prelabor rupture of membranes (PROM) without IAI (n = 71); and (8) preterm PROM with IAI (n = 71). Fragment Bb concentration in AF was determined by an enzyme-linked immunoassay. Non-parametric statistics were used for analyses.

Results.?(1) Fragment Bb was detected in all AF samples (n = 544); (2) The median AF concentration of fragment Bb in patients at term not in labor was significantly higher than that of those in the mid-trimester [2.42 μg/ml, interquartile range (IQR) 1.78–3.22 vs. 1.64 μg/ml, IQR 1.06–3.49; p < 0.001]; (3) Among patients with PTL, those with IAI had a higher median AF fragment Bb concentration than that of woman without IAI, who delivered preterm (4.82 μg/ml, IQR 3.32–6.08 vs. 3.67 μg/ml, IQR 2.35–4.57; p < 0.001) and than that of women with an episode of PTL, who delivered at term (3.21 μg/ml, IQR 2.39–4.16; p < 0.001); (4) Similarly, among patients with preterm PROM, the median AF fragment Bb concentration was higher in individuals with IAI than in those without IAI (4.24 μg/ml, IQR 2.58–5.79 vs. 2.79 μg/ml, IQR 2.09–3.89; p < 0.001). (5) Among patients at term, the median AF fragment Bb concentration did not differ between women with spontaneous labor and those without labor (term in labor: 2.47 μg/ml, IQR 1.86–3.22; p = 0.97).

Conclusions.?(1) Fragment Bb, an activator of the alternative complement pathway, is a physiologic constituent of the AF, and its concentration increases with advancing gestational age; (2) AF concentrations of fragment Bb are higher in pregnancies complicated with IAI; and (3) labor at term is not associated with changes in the AF concentrations of fragment Bb. These findings suggest a role for fragment Bb in the host immune response against IAI.     

Amniotic fluid concentration of surfactant proteins in intra-amniotic infection

Objective. Pulmonary surfactant is a complex molecule of lipids and proteins synthesized and secreted by type II alveolar cells into the alveolar epithelial lining. Both lipid and protein components are essential for lung function in postnatal life. Infection is a well-established cause of preterm delivery, and several inflammatory cytokines play a role in the mechanisms of preterm parturition. An increased concentration of inflammatory cytokines in amniotic fluid or fetal plasma has been linked to the onset of preterm parturition and fetal/neonatal injury, including cerebral palsy and chronic lung disease. Experimental evidence indicates that inflammatory mediators also regulate surfactant protein synthesis, and histologic chorioamnionitis is associated with a decreased incidence of hyaline membrane disease in neonates. This study was conducted to determine if amniotic fluid concentrations of surfactant protein (SP)-A, SP-B, and SP-D change in patients with and without intra-amniotic infection (IAI).

Materials and methods. A case–control study was conducted to determine amniotic fluid concentrations of SP-A, SP-B, SP-D, and total protein in patients who had an amniocentesis performed between 18 and 34 weeks of gestation for the detection of IAI in patients with spontaneous preterm labor with intact membranes (n = 42) and cervical insufficiency prior to the application of cerclage (n = 6). Amniotic fluid samples were selected from a bank of biological specimens and included patients with (n = 16) and without (n = 32) IAI matched for gestational age at amniocentesis. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Each group was further subdivided according to a history of corticosteroid administration within 7 days prior to amniocentesis into the following subgroups: (1) patients without IAI who had received antenatal corticosteroids (n = 21), (2) patients with IAI who had received antenatal corticosteroids (n = 9), (3) patients without IAI who had not received antenatal corticosteroids (n = 11), and (4) patients with IAI who had not received antenatal corticosteroids (n = 7). Amniotic fluid was obtained by transabdominal amniocentesis. SP-A, SP-B, and SP-D concentrations in amniotic fluid were determined by enzyme-linked immunosorbent assay (ELISA). Non-parametric statistics were used for analysis.

Results. Women with IAI had a higher median amniotic fluid concentration of SP-B and of SP-B/total protein, but not other SPs, than those without IAI (both p = 0.03). Among patients who had received antenatal corticosteroids, the median amniotic fluid concentration of SP-B and of SP-B/total protein was significantly higher in patients with IAI than in those without IAI (SP-B, IAI: median 148 ng/mL, range 37.3–809 ng/mL vs. without IAI: median 7.2 ng/mL, range 0–1035 ng/mL; p = 0.005 and SP-B/total protein, IAI: median 14.1 ng/mg, range 4.3–237.5 ng/mg vs. without IAI: median 1.45 ng/mg, range 0–79.5 ng/mg; p = 0.003). Among women who had not received antenatal corticosteroids, the median amniotic fluid concentrations of SP-B and of SP-B/total protein were not significantly different between patients with and without IAI (SP-B, IAI: median 4 ng/mL, range 0–31.4 ng/mL vs. without IAI: median 3.4 ng/mL, range 0–37 ng/mL; p = 0.8 and SP-B/total protein, IAI: median 0.55 ng/mg, range 0–6.96 ng/mg vs. without IAI: median 0.59 ng/mg, range 0–3.28 ng/mg; p = 0.9). The median amniotic fluid concentrations of SP-A, SP-A/total protein, SP-D, and SP-D/total protein were not significantly different between patients with and without IAI whether they received antenatal corticosteroids or not (all p > 0.05).

Conclusions. IAI was associated with an increased amniotic fluid concentration of SP-B in patients who received antenatal corticosteroids within 7 days prior to amniocentesis.     

Evidence of participation of soluble CD14 in the host response to microbial invasion of the amniotic cavity and intra-amniotic inflammation in term and preterm gestations

Objective: Endotoxin has been implicated in the mechanism responsible for the setting of infection in preterm labor. To exert its biological effects, endotoxin binds to a circulating protein known as lipopolysaccharide binding protein (LBP) and presents endotoxin monomers to CD14, which may be a membrane-bound receptor or a soluble molecule. The endotoxin- LBP-CD14 complex interacts with Toll-like receptor 4 and other regulatory proteins leading to cellular activation and an inflammatory response. The purpose of this study was to determine whether microbial invasion of the amniotic cavity (MIAC)/intra-amniotic inflammation (both preterm and term) and parturition at term are associated with changes in the amniotic fluid and umbilical plasma soluble concentrations of CD14 (sCD14). Study design: Amniotic fluid was retrieved by amniocentesis from 88 patients in the following groups: group 1, preterm labor with intact membranes with MIAC/intra-amniotic inflammation (n = 18) and without these conditions (n = 26); group 2, term gestations not in labor without MIAC/intra-amniotic inflammation (n = 11), in labor without MIAC/intra-amniotic inflammation (n = 12) and in labor with MIAC/intra-amniotic inflammation (n = 13); and group 3, patients who underwent genetic amniocentesis at mid-trimester (n = 8). A sample of cord blood was obtained after delivery in all patients except those in group 3. sCD14 was assayed with a sensitive and specific immunoassay. Non-parametric statistics were used for analysis. A p value of < 0.05 was considered significant. Results: sCD14 was detectable in 97% (85/88) of the amniotic fluid samples. Amniotic fluid sCD14 concentrations were lower in patients at term than in the mid-trimester of pregnancy (mid-trimester: median 482 ng/ml, range 258-838 ng/ml vs. term no labor: median 7 ng/ml, range 2-274 ng/ml, p = 0.01). Among patients with preterm labor with intact membranes, the median amniotic fluid sCD14 level of patients with MIAC/intra-amniotic inflammation was higher than in patients without these conditions (median 1568 ng/ml, range 98-5887 ng/ml vs. median 645 ng/ml, range 0-3961 ng/ml, respectively; p = 0.01). Among women at term in labor, those with MIAC/intra-amniotic inflammation had a higher median amniotic fluid sCD14 concentration than those without these conditions (median 85 ng/ml, range 2-1113 ng/ml vs. median 17 ng/ml, range 0-186 ng/ml; p = 0.01). MIAC/ intra-amniotic inflammation in women with preterm labor with intact membranes was associated with a higher median umbilical venous plasma sCD14 concentration (median 744 ng/ml, range 0-3620 ng/ml vs. median 0 ng/ml, range 0-2060 ng/ml; p = 0.04). sCD14 was undetectable in plasma from umbilical cords of all neonates born to women at term. An increase in amniotic fluid concentration of sCD14 was observed in cases of intrauterine infection, not only by Gram-negative bacteria, but also Gram-positive bacteria and Ureaplasma spp. Conclusion: sCD14 is a physiological constituent of amniotic fluid, and its concentrations at term are lower than in the mid-trimester. Intrauterine infection/inflammation is associated with a higher median amniotic fluid sCD14 concentration in both preterm and term parturition. Neonates born from mothers with preterm labor with intact membranes and MIAC/intra-amniotic inflammation had a higher median concentration of sCD14 in umbilical cord plasma than those without these conditions. sCD14 concentrations are increased in the amniotic fluid and umbilical cord blood even in the absence of a microbiologically proven Gram-negative infection. CD14 appears to participate in the host response to intrauterine infection even in cases involving genital mycoplasmas.     

Alteration of heat shock protein 70 expression levels in term and preterm delivery

Abstract

Objective: Heat shock protein 70 (Hsp70), a highly conserved cellular stress protein, is produced in every organism from bacteria to man. The purpose of this study was to determine the difference in Hsp70 concentrations between term and preterm deliveries.

Methods: In total, 30 healthy term delivery and 99 preterm delivery (PD) women were recruited, including 46 women with preterm labor and intact membranes (PTL) with or without an intra-amniotic infection (IAI) and 53 women with a preterm premature rupture of membranes (PPROMs) with or without IAI. The Hsp70 levels in the maternal and the umbilical cord sera were tested by an enzyme-linked immunosorbent assay (ELISA); the expression levels of Hsp70 in the placentas were determined by immunoblotting and real-time PCR.

Results: In contrast to the expression levels in normal term controls, Hsp70 expression levels were upregulated in PD; similar changes in Hsp70 expression levels were detected in PD maternal and umbilical sera. Patients with IAI were associated with a higher Hsp70 concentration than those without IAI.

Conclusion: These results suggest a probable contributing role of altered Hsp70 expression levels in the pathophysiology of PD.     

Resistin in amniotic fluid and its association with intra-amniotic infection and inflammation

Objective. Intra-amniotic infection/inflammation (IAI) is one of the most important mechanisms of disease in preterm birth. Resistin is an adipocytokine that has been linked to insulin resistance, diabetes, obesity and inflammation. The objective of this study was to determine if resistin is present in amniotic fluid (AF) and if its concentration changes with gestational age, in the presence of labour, and in IAI in patients with spontaneous preterm labour (PTL) and intact membranes, preterm prelabour rupture of membranes (PPROM) and clinical chorioamnionitis.

Study design. This cross-sectional study included 648 patients in the following groups: (1) women in the mid-trimester of pregnancy (14–18 weeks) who underwent amniocentesis for genetic indications and delivered a normal neonate at term (n = 61); (2) normal pregnant women at term with (n = 49) and without (n = 50) spontaneous labour; (3) patients with an episode of PTL and intact membranes who were classified into: (a) PTL who delivered at term (n = 153); (b) PTL who delivered preterm (<37 weeks gestation) without IAI (n = 108); and (c) PTL with IAI (n = 84); (4) women with PPROM with (n = 47) and without (n = 44) IAI; and (5) patients with clinical chorioamnionitis at term with (n = 22) and without (n = 30) microbial invasion of the amniotic cavity. Resistin concentration in AF was determined by enzyme-linked immunoassay. Non-parametric statistics were used for analyses.

Results. (1) Resistin was detected in all AF samples; (2) the median AF resistin concentration at term was significantly higher than in the mid-trimester (23.6 ng/mL vs. 10 ng/mL; p < 0.001); (3) among patients with PTL, the median AF resistin concentration was significantly higher in patients with IAI than in those without IAI (144.9 ng/mL vs. 18.7 ng/mL; p < 0.001) and those with PTL and intact membranes who delivered at term (144.9 ng/mL vs. 16.3 ng/mL; p < 0.001); (4) patients with PPROM with IAI had a significantly higher median AF resistin concentration than those without IAI (132.6 ng/mL vs. 13 ng/mL; p < 0.001); (5) no significant differences were observed in the median AF resistin concentration between patients with spontaneous labour at term and those at term not in labour (28.7 ng/mL vs. 23.6 ng/mL; p = 0.07); and (6) AF resistin concentration ≥37 ng/mL (derived from a receiver-operating characteristic curve) had a sensitivity of 85.4% and a specificity of 94.3% for the diagnosis of intra-amniotic inflammation.

Conclusions. Resistin is a physiologic constituent of the AF, and its concentrations in AF: (1) are significantly elevated in the presence of IAI; (2) increase with advancing gestation; and (3) do not change in the presence of spontaneous labour at term. We propose that resistin may play a role in the innate immune response against intra-amniotic infection.     

Exodus-1 (CCL20): evidence for the participation of this chemokine in spontaneous labor at term, preterm labor, and intrauterine infection

AIM: CCL20, also known as MIP-3 alpha, is a chemokine that participates in chemotaxis of immature dendritic cells, effector/memory T-cells, and B-lymphocytes. The objectives of this study were to determine whether CCL20 can be detected in amniotic fluid (AF) and if AF concentration of this chemokine changes with advancing gestational age, parturition (term and preterm), and intra-amniotic infection/inflammation (IAI). METHODS: A cross-sectional study was conducted including the following groups: (1) mid-trimester of pregnancy (n=65); (2) term not in labor (TNL; n=22); (3) term in labor (TIL; n=47); (4) spontaneous preterm labor (PTL) who delivered at term (n=57); (5) spontaneous PTL without IAI who delivered preterm (n=71); and (6) spontaneous PTL with IAI (n=38). AF CCL20 concentrations were determined using ELISA. RESULTS: (1) The median AF CCL20 concentration in TNL was higher than that of mid-trimester patients; (2) Women in spontaneous labor at term had a higher median AF concentration of CCL20 than patients at term not in labor; (3) Patients with spontaneous PTL and IAI had a significantly higher median AF concentration of CCL20 than those without IAI who delivered preterm and those who delivered at term. Moreover, women with spontaneous PTL without IAI who delivered preterm had a significantly higher median AF concentration than those with PTL who subsequently delivered at term. CONCLUSIONS: (1) CCL20 is a physiologic constituent of AF and its concentration increases as term approaches; (2) spontaneous labor (term and preterm) in the absence of IAI is associated with increased bioavailability of AF CCL20 suggesting that an increase in CCL20 is part of the common pathway of human parturition; (3) patients with IAI had dramatic elevations in the AF CCL20 concentrations suggesting that this chemokine participates in the host response to infection or other stimuli associated with intra-amniotic infection.     

Amniotic fluid cathepsin-G in pregnancies complicated by the preterm prelabor rupture of membranes

Objective: The aim of this study was to evaluate the amniotic fluid cathepsin-G concentrations in women with preterm prelabor rupture of membranes (PPROM) based on the presence of the microbial invasion of the amniotic cavity (MIAC) and/or intra-amniotic inflammation (IAI).

Methods: A total of 154 women with singleton pregnancies complicated by PPROM were included in this study. Amniotic fluid samples were obtained by transabdominal amniocentesis. Amniotic fluid cathepsin-G concentrations were assessed by ELISA. MIAC was determined using a non-cultivation approach. IAI was defined as an amniotic fluid bedside interleukin-6 concentration?≥?745?pg/mL.

Results: Women with MIAC had higher amniotic fluid cathepsin-G concentrations than women without MIAC (with MIAC: median 82.7?ng/mL, versus without MIAC: median 64.7?ng/mL; p?=?0.0003). Women with IAI had higher amniotic fluid cathepsin-G concentrations than women without this complication (with IAI: median 103.0?ng/mL, versus without IAI: median 66.2?ng/mL; p?p?Conclusions: The presence of either microbial-associated or sterile IAI was associated with increased amniotic fluid cathepsin-G concentrations in pregnancies complicated by PPROM. Amniotic fluid cathepsin-G appears to be a potential marker of IAI.