The effects of interleukins 1α and 1β on prostaglandin production by cultured human fetal membranes

The effects of IL-1α and IL-1β on cultured human fetal membranes were studied. These cytokines are known to regulate prostaglandin synthesis by the separated components of the fetal membranes (amnion, chorion and decidua), but their effects on intact tissue are unknown. IL-1α increased PGE2 levels on the fetal side of the membrane, indicating increased production of prostaglandin from the amnion, but had little effect on levels of PGE2 on the maternal side of the membrane. Low levels of IL-1β (0.1 - 1.0 ng/ml) increased PGE2 levels on the fetal side of the meembrane, and also increased the production of PGE2 metabolites and PGF2α, suggesting that this cytokine stimulated the decidua as well as the amnion. High concentrations of both cytokines appeared able to stimulate prostaglandin production by the side of the membrane opposing that to which they were added, but it is not clear whether this was mediated by factors released by the stimulated membrane, or by direct transfer of small quantities of cytokines through the membrane. Taken together, these results indicate that IL-1β was a potent stimulator of the synthesis of prostaglandins by decidua and by amnion, whereas IL-1α only stimulated the amnion.     

Estradiol-17β and 2-hydroxyestradiol-17β-induced differential production of prostaglandins by cells dispersed from human intrauterine tissues at parturition

Prostaglandin (PGE, 6-keto PGF_1α) output by cells dispersed from human amnion and decidua in the presence of increasing levels (0–5000 ng/ml) of estradiol-17β (E₂) or 2-hydroxyestradiol-17β (2-OH E₂) was studied in relation to parturition. Tissues were obtained from women at term either before (CS) or after (SL) spontaneous labor and vaginal delivery. In the absence of estrogens, the output of both PGs from amnion increased significantly with labor. No significant increase in decidua PG output occurred with labor. Neither estrogen influenced CS amnion PG output. However, both E₂ and 2-OH E₂ stimulated SL amnion PGE output (2-OH E₂>E₂) while having no affect on 6-keto PGF_1α output. Only the highest dose of 2-OH E₂ stimulated PGE output in CS decidua, but both estrogens significantly inhibited 6-keto PGF_1α output in this tissue. In SL decidua only 2-OH E₂ significantly stimulated PGE, and neither estrogen affected 6-keto PGF_1α output. These results might suggest that estrogens modulate PG biosynthesis at the level of endoperoxide to primary PG conversion.     

The induction of cyclooxygenase-2 (COX-2) in intact human amnion tissue by interleukin-4

Infection is a major cause of preterm labor. Amniotic fluid from women in preterm labor associated with intrauterine infection contains increased concentrations of cytokines. The mechanism underlying this association may be a cytokine-mediated stimulation of amnion cell prostaglandin production. The biosynthesis of prostaglandins from arachidonic acid is regulated by the enzyme cyclooxygenase which exists in two forms; the constitutive form (COX-1) and the other mitogen inducible (COX-2). The purpose of this study was to evaluate the effect of the cytokine interleukin-4 (IL-4) on cyclooxygenase activity and PGE2 production in amnion. Amnion tissue was taken at caesarean section from term women not in labor and immediately incubated for 2 hours in media containing concentrations of IL-4 ranging from 1 to 100 ng/ml. An increase in both COX-2 enzyme and prostaglandin E₂ (PGE₂) production was observed for all concentrations of IL-4 greater than 25 ng/ml (P < 0.05, n = 8). No change in COX-1 was observed. Our data suggest that the cytokine IL-4 may be involved in the pathogenesis of premature labor by inducing COX-2 in amnion tissue resulting in increased production of PGE₂ and subsequent myometrial activity.     

Phospholipase A activity in human and ovine uterine tissues

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5 – 8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.     

Estradiol-17 beta and 2-hydroxyestradiol-17 beta-induced differential production of prostaglandins by cells dispersed from human intrauterine tissues at parturition

Prostaglandin (PGE, 6-keto PGF1 alpha) output by cells dispersed from human amnion and decidua in the presence of increasing levels (0-5000 ng/ml) of estradiol-17 beta (E2) or 2-hydroxyestradiol-17 beta (2-OH E2) was studied in relation to parturition. Tissues were obtained from women at term either before (CS) or after (SL) spontaneous labor and vaginal delivery. In the absence of estrogens, the output of both PGs from amnion increased significantly with labor. No significant increase in decidua PG output occurred with labor. Neither estrogen influenced CS amnion PG output. However, both E2 and 2-OH E2 stimulated SL amnion PGE output (2-OH E2 greater than E2) while having no affect on 6-keto PGF1 alpha output. Only the highest dose of 2-OH E2 stimulated PGE output in CS decidua, but both estrogens significantly inhibited 6-keto PGF1 alpha output in this tissue. In SL decidua only 2-OH E2 significantly stimulated PGE, and neither estrogen affected 6-keto PGF1 alpha output. These results might suggest that estrogens modulate PG biosynthesis at the level of endoperoxide to primary PG conversion.     

Cannabinoids stimulate prostaglandin production by human gestational tissues through a tissue- and CB1-receptor-specific mechanism

Endocannabinoids have been implicated in the mechanisms of implantation, maintenance of pregnancy, and parturition in women. Intrauterine prostaglandin production and actions are also critical in each of these mechanisms. Hence, we have evaluated the effects of cannabinoids on prostaglandin biosynthesis by human gestational membranes. Explants of term amnion and choriodecidua were established and treated with the endogenous endocannabinoids 2-arachidonoyl glycerol and anandamide, as well as the synthetic cannabinoid CP55,940, to determine their ability to modulate PGE(2) production. The explants were also treated with CP55,940 in the presence of either SR141716A (a potent and selective antagonist of the cannabinoid receptor CB1) or NS398 [a cyclooxygenase (COX)-2 inhibitor] to determine whether any observed stimulation of PGE(2) production was mediated through the CB1-receptor and/or COX-2 activity. All three cannabinoids caused a significant increase in PGE(2) production in the amnion but not in the choriodecidua. However, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. The enhanced PGE(2) production caused by CP55,940 was abrogated by cotreatment with either SR141716A or NS398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by CB1 agonism and COX-2. Data from Western blotting show that cannabinoid treatment results in the upregulation of COX-2 expression. This study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labor and membrane rupture.     

Study of oxytocin receptor: II. oxytocin and prostaglandin F2 alpha receptors in human myometria and amnion-decidua complex during pregnancy and labor

Oxytocin receptors (OXT-R) and prostaglandin F2 alpha receptors (PGF2 alpha-R) in human myometrium, amnion and decidua during pregnancy and at parturition were examined in an effort to clarify their role in the initiation and maintenance of uterine contractions. The number of binding sites for OXT in myometria showed an increase as gestation advance (Ist trimester v.s. at term; 205 +/- 90 v.s. 671 +/- 98 fmol/mg protein, N = 5, p less than 0.01), and a rapid decrease following the onset of labor (254 +/- 60 fmol/mg protein, N = 5, p less than 0.02). On the other hand the number of PGF2 alpha-R, remained unchanged throughout pregnancy and in labor. This myometrial PGF2 alpha binding capacity was approximately 1/20 to 1/30 that of the OXT binding, while binding affinity was almost equal. The OXT-R both in amnion and decidua, which was 1/6 to 1/7 that in myometrium, showed no significant changes throughout pregnancy or after the onset of labor. Binding affinity for each tissue was almost the same and appeared to increase towards term but no statistical significance was detected. Present data confirmed the presence of OXT as well as PGF2 alpha receptors in the three functionally distinct entities of pregnant human uterus; myometrium, amnion, and decidua. Among the components, the OXT binding increased only in the myometrium during pregnancy, suggesting this tissue specifically responds to OXT. In contrast, there was a constant binding in myometria for PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal Fibronectin Signaling Induces Matrix Metalloproteases and Cyclooxygenase-2 (COX-2) in Amnion Cells and Preterm Birth in Mice

Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E₂ synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes.     

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.     

Elevated Soluble Triggering Receptor Expressed on Myeloid Cells (sTREM)-1 Levels in Maternal Serum during Term and Preterm Labor

Background

Infection and inflammation are important mechanisms leading to preterm birth. Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) belongs to a family of cell surface receptors that seems to play an important role in fine-tuning the immune response. It has been demonstrated that sTREM-1 is involved in bacterial infection as well as in non-infectious inflammatory conditions. Few studies have investigated serum sTREM-1 expression during preterm labor. Therefore, the purpose of this study was to assess sTREM-1 concentrations in maternal serum during term and preterm labor.

Methods

This case control study included 176 singleton pregnancies in the following groups: patients in (1) preterm labor, delivered before 34 weeks (PTB) (n = 52); (2) GA matched controls, not in labor, matched for gestational age (GA) with the PTB group (n = 52); (3) at term in labor (n = 40) and (4) at term not in labor (n = 32). sTREM-1 concentrations were determined by enzyme-linked immunoassay.

Results

sTREM-1 was detected in all serum samples. Median sTREM-1 concentrations were significantly higher in women with PTB vs. GA matched controls (367 pg/ml, interquartile range (IQR) 304–483 vs. 273 pg/ml, IQR 208–334; P<0.001) and in women at term in labor vs. at term not in labor (300 pg/ml, IQR 239–353 vs. 228 pg/ml, IQR 174–285; P<0.001). Women with PTB had significantly higher levels of sTREM-1 compared to women at term in labor (P = 0.004). Multiple regression analysis, with groups recoded as three key covariates (labor, preterm and rupture of the membranes), showed significantly higher sTREM-1 concentrations for labor (+30%, P<0.001) and preterm (+15%, P = 0.005) after adjusting for educational level, history of PTB and sample age.

Conclusions

sTREM-1 concentrations in maternal serum were elevated during spontaneous term and preterm labor and sTREM-1 levels were significantly higher in preterm labor.     

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.     

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.     

Tissue plasminogen activator and its receptor in the human amnion, chorion, and decidua at preterm and term

The plasminogen activator system consists of two proteins: tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act upon their specific receptors to generate plasmin from plasminogen located on the cell surface. Plasmin then acts directly and indirectly to degrade the components of the extracellular matrix (ECM). This process is likely to be important in the normal turnover of the ECM of fetal membranes and in its premature weakening in preterm premature rupture of the fetal membranes. Quantitative Northern analysis and in situ hybridization have shown that the decidua expresses mRNA for tPA. However, the immunolocalized tPA protein was most strongly associated with the amnion and chorion, as was its receptor annexin II, suggesting that the amnion and chorion are the targets for decidual tPA. At term, decidual tPA expression was unaffected by labor, and the tPA receptor was elevated both before and after labor. At preterm, the converse was found: decidual tPA expression was significantly (p < 0. 05) up-regulated by labor, but the tPA receptor was not. The results suggest that the generation of plasmin at term would be controlled by an increased concentration of the tPA receptor in the amnion and chorion, whereas at preterm a pathological increase in plasmin would be generated by an overexpression of tPA, initiated by labor.     

Local stimulation of prostaglandin production by corticotropin-releasing hormone in human fetal membranes and placenta

Corticotropin-releasing hormone is produced by the human placenta and fetal membranes, but its physiological significance is not established. We examined the possibility that CRH might affect prostaglandin output by these intra-uterine tissues. Primary cultures of amnion, chorion, decidua and placenta were established from tissue obtained from women at term elective cesarean section were maintained in the presence of increasing concentrations of synthetic hCRH. PG output at 48h was measured by radioimmunoassay. hCRH stimulated PGE2 output by amnion, chorion and placenta, but not by decidual tissue. PGF2 alpha output was stimulated in amnion, decidua and placenta but not chorion, whereas output of 13, 14-dihydro-15-keto PGF2 alpha was stimulated in all four tissues. We conclude that hCRH stimulates prostaglandin output by human placenta, decidua and the fetal membranes, raising the possibility of paracrine or autocrine interactions between CRH and prostaglandins in vivo.     

Does formyl-methionyl-leucyl-phenylalanine exert a physiological role in labor in women?

The classical chemotactic receptor for N-formyl peptides has traditionally been associated with polymorphonuclear and mononuclear phagocytes; however, several recent reports indicate that this receptor is also expressed in non-myeloid cells. In this study we have investigated the presence of binding sites for formyl-methionyl-leucyl-phenylalanine (fMLP) in human amniotic membranes of laboring and nonlaboring women; we have also evaluated the effect of the peptide on prostaglandin E (PGE) release from the same tissue. Our results demonstrate the presence of specific, saturable binding sites for 3H-fMLP; Scatchard plot analysis suggests the presence of both high- and low-affinity binding sites in laboring amnion, while only the low-affinity receptors were evident in nonlaboring tissue. N-t-butoxycarbonyl-methionyl-leucyl-phenylalanine (Boc-MLP), a formyl peptide receptor antagonist, inhibited 3H-fMLP binding in both preparations. In addition, fMLP was able to significantly increase PGE synthesis in perifused amnion fragments from laboring and nonlaboring women. This effect was counteracted by Boc-MLP treatment. The presence of specific binding sites for fMLP in amniotic tissue and their differing expression in laboring versus nonlaboring membranes, together with the action of the peptide on PGE synthesis, all suggest a physiological role for fMLP in labor.     

Apoptosis and related proteins during parturition in prostaglandin F receptor-deficient mice

This study investigated whether apoptosis and related proteins are involved in parturition by comparative observation of FP-deficient mice without labor and wild type mice with vaginal delivery. We examined the expression of apoptosis, Fas, FasL, active caspase-3 and bcl-2 proteins in the amnion, placenta and decidua. DNA laddering in the amnion, placenta and decidua tissue did not significantly differ between FP-deficient and wild type mice on day 18 of pregnancy. Similar TUNEL staining results were found in all tissues of FP-deficient mice compared with those of wild type mice. A higher intensity of apoptotic cells was found in the decidua basalis. The index of TUNEL-positive cells were not significantly different in the amnion, placenta and decidua of FP-deficient mice compared with that of wild type mice on day 18 of pregnancy. Specific bands for Fas were clearly observed in the amnion, placenta and decidua tissue. FasL specific bands were observed in the placenta and decidua, but a few in amnion tissue. A great number of active caspase-3 specific bands were detected in decidua, while a few such bands were detected in the placenta and few bands in the amniotic tissue. Bands for bcl-2 were detected in the amnion, placenta and decidua tissue. The weakest band was in decidual tissue. Fas, FasL, active caspase-3, and bcl-2 specific bands did not show any significant differences between the two groups. These findings demonstrate that apoptosis, Fas, FasL, caspase-3, and Bcl-2 occur in mouse term placenta that is not involved in parturition.     

Phospholipase A activity in human and ovine uterine tissues.

Phospholipase A was assayed in crude lysosomal fractions of human decidua, amnion, chorion and myometrium. Activity was present in all the tissues and was highest in decidua and amnion which contained enzymes with pH optima of 6.5-8.0 and 7.2 respectively. Comparison of the activities in tissues obtained before labor with others obtained during labor showed no differences. In sheep, the fetal membranes contained a greater activity of phospholipase A than placenta and myometrium. Stimulation of the fetal adrenals with corticotrophin caused a marked increase in activity in both amnion and chorioallantois. It is concluded that the human amnion and the ovine amnion and chorioallantois could participate in the biosynthesis of prostaglandins by releasing stored arachidonic acid.     

Possible role for prostacyclin in human parturition

Human amnion, chorion, decidua and placenta produced 6-oxo-PGF_1α when superfused . Furthermore amnion, an avascular tissue, produced more 6-oxo-PGF_1α after labour than all other tissues investigated and its production of 6-oxo-PGF_1α was significantly greater after labour than before the onset of labour. These findings suggest that prostacyclin production by foetal membranes may have a role in the mechanisms controlling human parturition. Moreover, this is the first evidence for the production of prostacyclin by an avascular tissue.     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human fetal membranes and decidua vera

We have previously reported that platelet-activating factor (PAF) is present in human amniotic fluid obtained from women in labor. We have also demonstrated that PAF, lyso-PAF, and alkyl acyl-sn-glycero-3-phosphocholine (AA-GPC) are present in human amnion tissue. In the reported study, we have investigated the enzymes involved in PAF metabolism in amnion tissue and their regulation. A phospholipase A2 activity has been demonstrated in amnion tissue which cleaves alkyl acyl (long-chain) sn-glycero-3-phosphocholine. The enzyme activity is not altered by Ca2+ and is distinctly different from the phospholipase A2 that we have previously characterized in this tissue. Amnion tissue contains acetyltransferase activity which requires Ca2+ and is associated with the microsomal fraction. Acetylhydrolase is also present in the cytosolic fraction of amnion tissue. Acetylhydrolase activity has also been demonstrated in amniotic fluid. The affinities of acetyltransferase (for lyso-PAF) and acetylhydrolase (for PAF) were unaffected by Ca2+. In the presence of Ca2+, however, the specific activity of acetyltransferase was increased four- to fivefold while that of acetylhydrolase was unaffected. Acetyltransferase and acetylhydrolase activities in fetal membranes and decidua were similar and were unchanged with gestational age. The possible role of PAF in the initiation of human parturition is discussed.