57Vol. 8, No. 1

Copyright © 2008 by the Society for Biology of Reproduction

The effect of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 on chorioamnion

secretion of prostaglandins (PG)F2α and E2 in pigs

Barbara Jana1,2 Anna Kozłowska2, Aneta Andronowska2, Maria Jedlińska-Krakowska3

2Division of Reproductive Endocrinology and Pathophysiology, Institute of Animal Reproduction and Food Research of the Polish

Academy of Sciences, Olsztyn, 3Division of Pathophysiology, Department of Pathology and Pharmacology, Faculty of Veterinary

Medicine, University of Warmia and Mazury, Olsztyn, Poland

Received: 2 January 2008; accepted: 29 February 2008

SUMMARY

The aim of the present study was to determine the effect of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) on prostaglandin (PG)F2α and PGE2 secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-α, IL-1β, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-α, IL-1β and/or IL-6 on PGF2α and PGE2 secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of

ORIGINAL RESEARCH

1Corresponding author: Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn; e-mail: baja@pan.olsztyn.pl

58 Cytokines and PGs in porcine chorioamnion

pregnancy. Cytokine stimulation of PGE2 was more pronounced than that of PGF2α. In addition, an increase in PGF2α and/or PGE2 secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation. Reproductive Biology 2008 8 1: 57-68.Key words: chorioamnion, cytokines, PGF2α, PGE2, COX-2, pigs

INTRODUCTION

Tumor necrosis factor-α (TNF-α), interleukin (IL)-1 and IL-6 are cytokines associated with such pathological processes as inflammation or septic shock. The cytokines are synthesized and released not only by immunocompetent cells in response to lipopolysaccharides (LPS; [15, 19]) but also by many tissue and cell types including those of the female reproductive system [10, 12]. They are produced by bone-marrow derived cells, particularly macrophages and endometrial cells [8, 12, 39] as well as fetal membranes [7, 37]. Local synthesis of TNF-α, IL-1 and IL-6 in human uterus fluctuates during pregnancy [16, 37].

Prostaglandins (PG)F2α and E2 which are also produced in the reproductive tract are of great significance for the establishment and maintenance of pregnancy. They participate in several key processes such as implantation, decidualization, immunosupression or dilatation of uterine arteries and initiation of parturition [3, 31, 35]. In vitro studies have shown that TNF-α, IL-1 and IL-6 stimulate the production of PGF2α and PGE2 in human amnion and decidual cells [13, 14, 21, 22, 25]. Functional TNF-α and IL-1 receptors were found in human fetal membranes [1, 11] and their role in PG release in women was reported.

Similar to cytokines, LPS which are released by Gram-negative bacteria and are often used as a model for intrauterine infection research, stimulate PGF2α and PGE2 secretion from human amnion and decidua [27, 29, 30]. Both LPS- and cytokine-induced changes in PGs synthesis in fetal membranes and the uterus threaten pregnancy in women and some

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animal species. Moreover, TNF-α, IL-1, IL-6 and/or LPS were reported to stimulate the production of PGs in fetal membranes through increasing the expression of cyclooxygenase-2 (COX-2; the key enzyme in PGs production) and phospholipase A2 [6, 26, 27, 32, 36].

In animals including sows, the first trimester of pregnancy is the most important for successful pregnancy. This period includes the development of fetal membranes, the formation and growth of placenta and an increase in amniotic and allantoic fluid volume [17]. Thus, cytokines by modulating the fetal membrane PG secretion may indirectly affect the processes regulated by PGs in pigs. Since mechanisms involved in the control of PG release are not fully recognized, the present study was performed to examine the effect of cytokines on: 1/ PGF2α and PGE2 secretion from amniochorion, and 2/ COX-2 protein expression in fetal membranes collected on days 25, 30 and 40 of gestation in pigs.

MATERIALS AND METHODS

Animals and experimental design

The experimental procedures were approved by the Local Ethics Com-mittee. Fifteen primiparous crossbred (Large White×Landrace) gilts (7-8 months, 90-100 kg) were used in the study. Housed on a farm, the ani-mals were checked for estrous behavior in the presence of a boar. The gilts were bred at the onset of estrus (day 0) and then 12 h and 24 h later. Three days before slaughtering the animals were transported to the animal research facility and kept in individual stalls under ambient light and temperature. They were fed a commercial grain mixture and tap water ad libitum.

The gilts were euthanized by electrical shock on days 25, 30 and 40 of pregnancy (n=5 per day) and exsanguinated. The uteri were collected and transported on ice to the laboratory within 3 min. Fragments of the chorioamnion were carefully isolated from the uterus. These tissues were sliced (100 mg) and washed in sterile saline. Individual explants were placed into glass scintillation vials (20 ml) with 2.0 ml Medium-

60 Cytokines and PGs in porcine chorioamnion

199 containing 0.1% of bovine serum albumin (BSA), penicillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericin (2 µg/ml). After 30 min of preincubation (37°C, 5% CO2/air), the medium was changed and incubated for 16 h. The culture medium contained 0 (control), 1 or 10 ng/ml of human TNF-α, IL-1β and IL-6 alone or in a combination of all three cytokines at dose 1 or 10 ng/ml each (all reagents from Sigma, St. Louis, MO, USA). All treatments were performed in triplicates and each experiment was repeated three times.

The doses of cytokines and time of incubation were based on the results of Rajasingam et al. [27] and our preliminary study, respectively. Following incubation, the media were collected into tubes containing 10 µl stabilizing mixture (0.3 M EDTA, POCH Gliwice, Poland; 1% aspirin, Polfa, Starogard, Poland) and frozen at -20°C until PGs assay was completed. The tissue slices of fetal membranes were collected to determine COX-2 protein expression (Western blotting). The tissues were then homogenized on ice with a cold buffer (50 mmol/l Tris-HCl, pH 8.0; 150 mmol/l NaCl; 1% Triton X-100, 10 μg/ml aprotinin, 52 μmol/l leupeptin, 1 mmol/l pepstatin A, 1 mmol/l EDTA, 1 mol/l PMSF) and centrifuged (10 min, 2 500×g, 4°C). The supernatants were then centrifuged for one hour at 17 500×g and 4°C, and the resulting pellet was stored at -80°C for further analysis. The protein level was determined by Bradford’s method [5].

Enzymoimmunoassay of prostaglandins

The concentrations of PGF2α and PGE2 in the medium were measured by an enzyme immunoassay method [33]. The sensitivity of the assay for PGF2α and PGE2 were 0.016 and 0.14 ng/ml, respectively. Intra- and interassay coefficients of variation for PGF2α and PGE2 were 7.9 and 10.4% and 6.9 and 9.7%, respectively.

Western blotting for COX-2 protein

The fetal membrane portions (20 μg) were dissolved in sodium dodecyl sulphate (SDS), a gel-loading buffer (50 mmol/l Tris-HCl, pH 6.8; 4%

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SDS, 20% glycerol and 2% β-mercaptoethanol), heated (95°C, 4 min) and separated by 10% SDS-polyacrylamide gel electrophoresis. Separated pro-teins were electroblotted onto 0.45 μm nitrocellulose membrane in a trans-fer buffer (20 mmol/l Tris-HCl buffer, pH 8.2; 150 mmol/l glycine, 20% methanol, 0.05% SDS). The nonspecific binding sites were blocked by in-cubation with 5% fat-free dry milk in a TBS-T buffer at room temperature (RT) for 1.5 h. Nitrocellulose membrane was incubated overnight at 4°C with rabbit polyclonal anti-COX-2 antibodies (1:200; Cayman Chemical, USA). The COX-2 was detected by incubating the nitrocellulose mem-brane for 1.5 h at RT with secondary biotinylated goat anti-rabbit antibod-ies (1:3000; Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, CA, USA). COX-2 reaction was visualized by incubation with a 3,3’-di-aminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich Co., USA) for 10 min. Each analysis was repeated three times. The intensity of COX-2 protein expression was quantified by measuring optical density using KO-DAK 1D Image Analysis Software (USA).

Statistical analysis

Experimental data are presented as mean±SEM of each experiments performed in triplicates. The in vitro effect of TNF-α, IL-1β and/or IL-6 on PGs and COX-2 content was analyzed by one-way analysis of variance (ANOVA) for repeated measures followed by Dunett’s post-hoc test (GraphPad PRISM). Differences with probability of p<0.05 were considered significant.

RESULTS

The influence of cytokines on in vitro PGF2α and PGE2 production

The effect of cytokines on PG secretion is depicted in fig. 1. Higher doses of IL-1β (p<0.05) and IL-6 (p<0.001) stimulated PGF2α production by the chorioamnion collected on day 25 of pregnancy. A similar effect (p<0.01)

62 Cytokines and PGs in porcine chorioamnion

Figure 1. The effect of TNF-α, IL-1β, IL-6 or combination of the three cytokines on PGF2α (left panel) and PGE2 (right panel) secretion (means±SEM) by the porcine chorioamnion collected on days 25, 30 and 40 of pregnancy (n=5 per group). Tissue slices (100 mg) were incubated for 16 h (Medium 199, 37ºC, 5% CO2/air). The means marked with asterisks are significantly different from controls (C): *p<0.05, **p<0.01, ***p<0.001

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Figure 2. The effect of TNF-α, IL-1β, IL-6 or combination of the three cytokines on COX-2 protein expression (means±SEM) in the porcine chorioamnion collected on days 25, 30 and 40 of pregnancy (n=5 per group). Tissue slices (100 mg) were treated with cytokines for 16 h (Medium 199, 37ºC, 5% CO2/air). Densitometric analysis of bands is presented in arbitrary units. The means marked with asterisks are significantly different from controls (C): *p<0.05, **p<0.01, ***p<0.001

64 Cytokines and PGs in porcine chorioamnion

was found following treatment with both doses of IL-1β on day 30. On day 40 of pregnancy, the medium concentration of PGF2α was increased by both doses of IL-1β (p<0.05) and a higher dose of TNF-α (p<0.01).

In vitro PGE2 release by fetal membranes collected on day 25 of preg- nancy was increased (p<0.05) by two doses of IL-1β and IL-6 as well as by the combination of the three cytokines at higher dose. On day 30, the higher doses of all examined treatments including the combination of all cytokines and the lower dose of IL-1β stimulated (p<0.05) secretion of PGE2. All cytokines alone or combined at both applied doses augmented (p<0.05) PGE2 secretion by porcine chorioamnion on day 40 of pregnancy.

The effect of cytokines on COX-2 protein expression All porcine fetal membranes collected on days 25, 30 and 40 of pregnancy expressed COX-2 protein at a satisfactorily detectable level (fig. 2). The chorioamnion expression of COX-2 protein on day 25 of pregnancy was increased (p<0.05) by both doses of IL-1β and a higher dose of IL-6. The higher doses of all examined treatments including combination of all cytokines and the lower dose of IL-1β stimulated (p<0.05) COX-2 protein expression on day 30. On day 40 of pregnancy, all cytokines alone or combined at both applied doses except a lower dose of TNF-α augmented (p<0.05) the expression of COX-2 protein in porcine fetal membranes.

DISCUSSION

In the present study we demonstrated that TNF-α, IL-1β and IL-6 stimulat-ed both secretion of PGF2α and PGE2 and expression of COX-2 protein in the chorioamnion collected during the first trimester of pregnancy of gilts. The release of PGs appeared to depend on cytokine type, treatment dose and day of pregnancy. Until now, the effect of cytokines on PG production was studied primarily in human fetal membranes during late gestation in the context of decidual-amniochorionic infections and their consequences (e.g. preterm labour). These studies encompass stimulation of PGF2α cho-

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rion synthesis by IL-1β, augmentation of amnion production by IL-α, IL-β, IL-6, and TNF-α, and increase in chorion production of PGF2α or PGE2 metabolite by IL-6 or TNF-α, respectively [13, 14].

In the current study, TNF-α, IL-1β and IL-6 stimulated chorioamnion in vitro release of both PGF2α and PGE2 , however stimulation of PGE2

was more pronounced. The latter finding may result from a more potent effect exerted on the expression of PGE2 synthase (PGES) than of PGF2α synthase. The timing of PGE2 response to cytokines observed on days 25, 30 and 40 of pregnancy is parallel with the period of initial expansion of chorioallantoic membranes and establishment of intimate contact be-tween placenta and endometrial surface [17]. In addition, the increase in chorioamnion PGE2 secretion observed on day 30 and 40 of pregnancy may, through the increase in the blood vessel permeability, initiate and/or elevate an accumulation of amniotic fluid [17].

An excess of intrauterine PGF2α secretion may be harmful for develop-ing fetuses. It was reported that in vivo intra-amniotic administration of LPS or Escherichia coli during the second half of gestation in pigs induces a high level of IL-8 or TNF-α in amniotic fluid [34, 38]. LPS was also found to increase TNF-α and IL-8 expression in the amniotic epithelium [38]. LPS and cytokines, in turn, are known to stimulate PGF2α and PGE2 secretion from human amnion and decidua [29, 30]. The large amount of intrauterine PGF2α, characteristic for intra-amniotic infections, results in vasoconstriction in the fetal blood supply, and may lead to intrauterine embryo/fetal death [9].

The amount of PGF2α and PGE2 released from porcine chorioamnion in response to cytokines increased gradually with gestational age i.e. from day 25 to day 40. A similar phenomenon was observed for basal chorioam-nion release of PGE2 during later stages of pregnancy in pigs [28]. Such an increase may be caused by growing availability of arachidonic acid [18], a substrate for PGs synthesis, in developing fetal membranes.

We did not observe any additive effect of cytokines on PG production. Similar results were reported previously for the rat uterus [2]. This may be due to a post-receptor convergence on the same secondary signal path-ways. Such a mechanism was described for both TNF-α- and IL-1-induced

66 Cytokines and PGs in porcine chorioamnion

stimulation of the human immunodeficiency virus enhancer which occurs through the activation of NF-κB [23]. It is possible that a similar signalling pathway is involved in the activation of COX-2 or other PG-synthesizing enzymes.

In the present study, an increase in PGF2α and/or PGE2 release was usually associated with the augmentation of COX-2 protein expression. The increases in the PGs secretion and COX-2 expression were demonstrated previously in intact human fetal membranes [6, 36] and amnion-derived AV3 cell line [26]. In a few cases, however, we have not observed the simultaneous increase in PGE2 release and the COX-2 protein expression (figs. 1 and 2) in porcine fetal membranes. This lack of parallelism may be caused by the cytokine effect on the expression of PGES, an COX-2 downstream enzyme. PGES was immunolocalized in human amnion epithelium [20] and PGES mRNA was found to be increased by TNF-α and IL-1β in the bovine endometrium and human myometrium, respectively [4, 24]. The exact mechanism by which TNF-α, IL-1β or IL-6 affect metabolism of PGs in porcine fetal membranes requires further examination.

In conclusion, the presented data demonstrate for the first time that TNF-α, IL-1β and IL-6 stimulated secretion of PGF2α and PGE2 by chori-oamnion collected on days 25, 35 and 40 of pregnancy of gilts. Our results support the notion concerning the possible role of cytokines in modulat-ing production of PGs by fetal membranes during the first trimester of gestation. However, further studies should be performed to elucidate the mechanism of PG production in response to cytokines.

REFERENCES

1. Alnaif B, Benzie RJ, Gibb W 1994 Studies on the action of interleukin-1 on term human fetal membranes and decidua. Canadian Journal of Physiology and Pharmacology 72 133-139.

2. Arslan A, Zingg HH 1996 Regulation of COX-2 gene expression in rat uterus in vivo and in vitro. Prostaglandins 52 463-481.

3. Ashworth MD, Ross JW, Hu J, White FJ, Stein DR, Desilva U, Johnson GA, Spencer TE, Geisert RD 2006 Expression of porcine endometrial prostaglandin synthase during the estrous cycle and early pregnancy, and following endocrine disruption of pregnancy. Biology of Reproduction 74 1007-1015.

67Jana et al.

4. Astle S, Newton R, Thornton S, Vatish M, Slater DM 2007 Expression and regulation of prostaglandin E synthase isoforms in human myometrium with labour. Molecular Human Reproduction 13 69-75.

5. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 248-254.

6. Brown NL, Alvi SA, Elder MG, Bennett PR, Sullivan MHF 1998 Interleukin-1β and bacterial endotoxin change the metabolism of prostaglandins E2 and F2α in intact term fetal membranes. Placenta 19 625-630.

7. Bry K, Hallman M, Lappalainen U 1994 Cytokines released by granulocytes and mononuclear cells stimulate amnion cell prostaglandin E2 production. Prostaglandins 48 389-399.

8. Chabot V, Lambert RD, Laforest JP, St-Jaques S, Matte JJ, Guay F, Pallin MF, Lessard M 2004 Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts. Animal Reproduction Science 81 137-149.

9. Cort N, Kindahl H, Einarsson S 1986 The effect of a gram-negative bacterial endotoxin and cloprostenol on the plasma levels of 15-keto-13, 14-dihydro-PGF2α, progesterone, oestradiol-17β, oestrone sulphate and luteinizing hormone in non-pregnant and 60-day-pregnant gilts. Animal Reproduction Sciences 10 147-162.

10. Dudley DJ 1997 Pre-term labor: an intra-uterine inflammatory response syndrome? Journal of Reproductive Immunology 30 93-109.

11. Fortunato SJ, Menon R, Swan KF 1994 Expression of TNF-alpha and TNFR p55 in cultured amniochorion. American Journal of Reproductive Immunology 32 188-193.

12. Hunt JS 1993 Expression and regulation of the tumor necrosis factor-α in the female reproductive tract. Reproduction Fertility and Development 5 141-153.

13. Kent AS, Sullivan MH, Sun MY, Zosmer A, Elder MG 1993 Effects of interleukin-6 and tumor necrosis factor-alpha on prostaglandin production by cultured human fetal membranes. Prostaglandins 46 351-359.

14. Kent AS, Sun MY, Sullivan M, Elder MG 1993 The effects of interleukins 1 alpha and 1 beta on prostaglandin production by cultured human fetal membranes. Prostaglandins 46 51-59.

15. Kijubu DA, Fletcher BS, Varnum BC, Lim RW, Uerschman UR 1991 TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin/cyclooxygenase homologue. Journal of Biological Chemistry 266 12866-12872.

16. Kim YJ, Ahn JJ, Woo BH 1998 The effect of cytokine mediators on prostaglandin inhibition by human decidual cells. American Journal of Obstetrics and Gynecology 179 146-149.

17. Knight JW, Bazer FW, Thacher WW, Franke DE, Wallace DE 1977 Conceptus development in intact and unilaterally hysterectomized-ovariectomized gilts: interrelations among hormonal status, placental development, fetal fluids and fetal growth. Journal of Animal Science 44 620-637.

18. Ledwozyw A, Kadziołka A 1989 Swine fetal membrane and myometrium lipids in ontogenesis (in Polish). Polskie Archiwum Weterynaryjne 29 93-105.

19. Lee SH, Soyoola E, Chanmungan P, Hart S, Sun W, Zhong H, Liou S, Simmons D, Hwang D 1992 Selective expression of mitogen-inducible cyclooxygenase in macrophagas, stimulated with lipopolysaccharide. Journal of Biological Chemistry 267 25934-25938.

20. Meadows JW, Eis AL, Brockmam DE, Myatt L 2003 Expression and localization of prostaglandin E synthase isoforms in human fetal membranes in term and preterm labor. Journal of Clinical Endocrinology and Metabolism 88 433-439.

21. Mitchell MD, Dudley DJ, Edwin SS, Sciller SL 1991 Interleukin-1 stimulates prostaglandin produc-tion by human amnion and decidual cells. European Journal of Pharmacology 192 189-191.

68 Cytokines and PGs in porcine chorioamnion

22. Mitchell MD, Edwin SS, Lundin-Schiller S, Silver RM, Smotkin D, Trautman MS 1993 Mech-anism of interleukin-1 beta stimulation of human prostaglandin biosynthesis: mediation via novel inducible cyclooxygenase. Placenta 14 615-625.

23. Osborn L, Kunkel S, Nabel GI 1989 TNF-α and IL-1 stimulate the human immunodeficiency virus enhancer by activation of the NF-κB. Proceedings of the National Academy of Sciences USA 86 2336-2340.

24. Parent J, Fortier MA 2005 Expression and contribution of three different isoforms of prostaglandin E synthase in the bovine endometrium. Biology of Reproduction 73 36-44.

25. Pollard JK, Mitchell MD 1993 Tumor necrosis factor stimulates amnion prostaglandin biosynthesis primarily via an action on fatty acid cyclooxygenase. Prostaglandins 46 499-510.

26. Potter S, Hansen WR, Keelan JA, Mitchell MD 1999 Characterization of the amnion-derived AV3 cell line for use as a model for investigation of prostaglandin-cytokine interactions in human amnion. Prostaglandins & Other Lipid Mediators 61 373-379.

27. Rajasingam D, Bennet PR, Alvi SA, Elder MG, Sullivan HF 1998 Stimulation of prostaglandin pro-duction from intact human fetal membranes by bacteria and bacterial products. Placenta 19 301-306.

28. Rice GE, Christensen P, Dantzer V, Skadhauge E 1989 Gestational profile of prostaglandin E2 synthesis by porcine placenta and fetal membranes. Eicosanoids 2 235-240.

29. Romero R, Hobbins JC, Mitchell MD 1988 Endotoxin stimulates PGE2 production by human amnion. Obstetrics and Gynecology 71 227-228.

30. Romero R, Mazor M, Wu YK, Avila C, Oyarzun E, Mitchell MD 1989 Bacterial endotoxin and tumor necrosis factor stimulate prostaglandin production by human decidua. Prostaglandins Leukotriens and Essential Fatty Acids 37 183-186.

31. Scherle PA, Ma W, Lim H, Dey SK, Trzaskos JM 2000 Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of the early pregnancy. Journal of Biological Chemistry 24 37086-37092.

32. Shoji T, Yoshida S, Mitsunari M, Miyake N, Tsukihara S, Iwabe T, Harada T, Terakawa N 2007 Involvement of p38 MAP kinase in lipopolysaccharide-induced production of pro- and anti-inflammatory cytokines and prostaglandin E(2) in human choriodecidua. Journal of Reproductive Immunology 75 82-90.

33. Skarzynski DJ, Okuda K 2000 Different actions of noradrenaline and nitric oxide on the output of prostaglandins and progesterone in cultured bovine luteal cells. Prostaglandins & Other Lipid Mediators 60 35-47.

34. Splichalovà A, Spihal I, Trebichavsky I, Hojnà H 2004 Expression of inflammatory markers in pig amnion after intraamniotic infection with nonpathogenic or enteropathogenic Escherichia coli. Folia Microbiologica 49 751-756.

35. Stanfield KM, Bell RR, Lisowski AR, English ML, Saldeen SS, Khan KN 2003 Expression of cyclooxygenase-2 in ebryonic and fetal tissues during organogenesis and late pregnancy. Birth Defects Research Part A-Clinical and Molecular Teratology 67 54-58.

36. Sullivan MH, Alvi SA, Brown NL, Elder MG, Bennett PR 2002 The effects of a cytokine suppressive anti-inflammatory drug on the output of prostaglandin E(2) and interleukin-1 beta from human fetal membranes. Molecular Human Reproduction 8 281-285.

37. Tabibzadeh S 1991 Human endometrium: an active site of cytokine production and action. Endocrine Reviews 12 272-290.

38. Trebichavsky I, Spichal I, Zahradnickova M, Spichalova A, Mori Y 2002 Lipopolysacharide induces inflammatory cytokines in the pig amnion. Veterinary Immunology and Immunopathology 87 11-18.

39. Yu Z, Gordon JR, Kendall J, Tacker PA 1998 Elevation in tumor necrosis factor-alpha (TNF-alpha) messenger RNA levels in the uterus of pregnant gilts after oestrogen treatment. Animal Reproduction Science 50 57-67.