Molecular Human Reproduction, Vol. 5, No. 9, 880-884,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular aspects of pregnancy |
Expression of cyclo-oxygenase types-1 and -2 in human myometrium throughout pregnancy
1 The London Myometrial Group Division of Paediatrics, Obstetrics and Gynaecology, Imperial College School of Medicine, Institute of Obstetrics and Gynaecology, Queen Charlottes and Chelsea Hospital, Goldhawk Road, London W6 OXG, and 2 Fetal Health Research Group, Department of Obstetrics and Gynaecology, Guy's, King's and St Thomas', School of Medicine, St Thomas' Hospital, London SE1 7EH, UK
Abstract
Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2) in human myometrium throughout pregnancy and to test the hypothesis that COX in the myometrium may play a role in labour onset. Expression of COX-1 and COX-2 at the mRNA level was analysed using reverse transcriptase–polymerase chain reaction (RT–PCR) and at the protein level using Western blotting. No significant changes of COX-1 RNA or protein expression were observed either with gestational age or labour. COX-2 mRNA and protein expression increased at term with significant up-regulation occurring prior to the onset of labour (P < 0.005). These data would suggest that up-regulation of COX-2, rather than COX-1, mediates increased prostaglandin synthesis in human myometrium at term. The increased COX-2 expression observed preceded labour onset, suggesting that COX-2 has a role in labour onset, rather than its presence merely a consequence of labour.
cyclo-oxygenase/labour/myometrium/pregnancy/prostaglandins
Introduction
Preterm labour occurs in up to 10% of all births but is associated with 85% of perinatal deaths in otherwise normal babies. The aetiology of preterm labour remains largely unknown, since the mechanisms involved in the onset of normal labour at term are not fully understood. Throughout pregnancy the uterus undergoes profound physiological changes, expanding to accommodate the growing fetus, whilst remaining in a quiescent state. The myometrium is resistant to activation, which is associated with incomplete cell–cell coupling, resulting in low basal smooth muscle tone, and decreased uterine activity. No single factor has been demonstrated as responsible for these pregnancy-associated changes. It is likely that there is a balance of `pro-pregnancy' factors, such as progesterone, maintaining uterine quiescence and `pro-labour' factors such as gap junctions, calcium-sensitive potassium channels (Khan et al., 1998
), and connexins, that result in the onset of uterine contractions, cervical dilation and delivery of the fetus (for review see Garfield et al., 1998).
Prostaglandins play a central role in human parturition. Labour is associated with increased prostaglandin synthesis within the uterus (Turnbull, 1977) prostaglandins are used for the induction of labour and prostaglandin synthesis inhibitors are the most effective tocolytic agents (Keirse, 1995). Prostaglandins act to mediate cervical ripening and to stimulate uterine contractions (Crankshaw and Dyal, 1994) and indirectly to increase fundally dominant myometrial contractility by up-regulation of oxytocin receptors and synchronization of contractions (Garfield et al., 1987; Liggins, 1989). Prostaglandins are formed from the precursor arachidonic acid which itself is a substrate for at least three enzyme groups. The cyclo-oxygenase (COX) or prostaglandin endoperoxide synthase pathway produces prostaglandin endoperoxides, which are then synthesized to prostaglandins by specific synthase enzymes. The fetal membranes are a major source of prostaglandins within the human uterus and much attention has been focused upon the regulation of prostaglandin synthesis within the fetal membranes and its association with the onset of labour. Fetal membrane prostaglandin synthesis increases in association with labour (Skinner and Challis, 1985). In amnion, COX-2 mRNA expression exceeds that of COX-1 by a factor of 100 (Slater et al., 1994) and the large increase in synthesis of prostaglandin E2 is associated with increased expression of COX-2 at term (Hirst et al., 1995; Slater et al., 1995). Increased expression of COX-2 and prostaglandin production in fetal membranes precedes labour onset (Slater et al., 1999). This would imply COX-2 in the fetal membranes has a role in labour onset as opposed to being merely a consequence of labour. The chorion is a major site of prostaglandin dehydrogenase (PGDH) (Cheung and Challis, 1989; Cheung et al., 1990, 1992). It has been suggested that there may be a down-regulation of PGDH activity in association with the onset of labour that would facilitate prostaglandin E2 access to the myometrium (Van Meir et al., 1997).
Myometrium expresses receptors for the E and F series prostaglandins (Matsumoto et al., 1997) linked to both inhibitory and excitatory mechanisms (Senior et al., 1993). Prostaglandin synthesis inhibitors decrease uterine activity and reduce contractility in in-vitro preparations of rat (Williams and Vane, 1975) and human (Johnson et al., 1975) myometrium. The principal prostaglandin product of human pregnant myometrium is prostacyclin (Bamford et al., 1980), which has no effect upon uterine contractility (Crankshaw and Dyal, 1994). Myometrium also synthesizes thromboxane and prostaglandins E2 and F2
, each of which is potentially oxytocic (Crankshaw and Dyal, 1994).
The aim of this study was to test the hypothesis that COX in the myometrium plays a role in the onset and/or maintenance of human labour. We studied the expression of COX-1 and COX-2 mRNA and protein, by reverse transcription–polymerase chain reaction (RT–PCR) and Western blotting, in human pregnant myometrium in the second and third trimesters of pregnancy and before and after labour at term.
Materials and methods
Tissue collection
Myometrial tissue ~0.5x2 cm was taken from the upper margin of the lower segment incision at Caesarean section. The local Research Ethics Committees of St Thomas' Hospital and Hammersmith Hospital Trust, UK, approved the study. All patients gave written informed consent. Patients age ranged from 18–40 years. The data were analysed in four groups: at term in non-labouring (38–41 weeks, n = 11), and labouring women (38–40 weeks, n = 5), and preterm in non-labouring women. The preterm patients were divided into two groups, early preterm (26–32 weeks, n = 8) and late preterm (32–36 weeks, n = 6). These are arbitrary groupings based upon the clinical distinction between preterm (>32 weeks) and very preterm (<32 weeks). The indications for Caesarean section in the non-labour groups were breech presentation, hypertensive disease, and previous Caesarean delivery. No patients were on medication. The non-labouring groups had no evidence of uterine contractions or cervical change. There was no evidence of uterine dysfunction. Myometrial tissue from labouring women was collected during emergency Caesarean section. In this group all patients had regular painful contractions with associated cervical dilation (>3 cm), and Caesarean section was indicated by fetal distress. There was no prior use of prostaglandins or oxytocin and the progress of labour had previously been normal.
RT–PCR
Myometrial tissue was immediately snap frozen in liquid nitrogen. Total RNA was isolated using a standard guanidine isothyocyamate technique (Chirgwin et al., 1979) and reverse transcribed into cDNA for use as the template for PCR. RNA samples (1 µg) were denatured at 70°C for 5 min. Reverse transcription was performed at 37°C for 60 min in a reaction volume of 20 µl containing random hexanucleotide primers, 0.2 µg (Pharmacia, UK), reverse transcriptase buffer, 0.1 mol/l dithiothreitol (DTT), 1 IU RNAse inhibitor, 1 mmol/l of each dNTP and 40 IU of mouse myeloma leukaemia virus reverse transcriptase (Promega, Southampton, UK). The reverse transcription reaction was stopped by heating at 90°C for 5 min. A 1/40 volume of the generated cDNA reaction was then used in subsequent amplification by PCR. PCR was performed in a 25 µl volume containing 1.5 mmol/l magnesium chloride, 0.2 mmol/l deoxynucleotide triphosphates (dNTPs), 125 ng of each sense and antisense primer, and 1 IU of Biotaq polymerase (Bioline UK). Following an initial denaturation step of 4 min at 94°C, the reaction cycles were denaturing at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s for the appropriate number of cycles. Primers used (Hla et al., 1986; Hla and Neilson, 1992) were; COX-2; 5'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3' (sense), 5'-AGA TCA TCT CTG CCT GAG TAT CTT-3' (antisense), COX-1; 5'-TGC CCA GCT CCT GGC CCG CCG CTT-3' (sense), 5'-CCA TGG CCC AAG GCC TTG-3' (antisense), glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3' (sense), 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (antisense). Aliquots of the PCR reaction were separated by agarose gel electrophoresis, transferred onto nylon membranes, and subsequently hybridized to radiolabelled COX-1, COX-2 or GAPDH probes to confirm PCR product identity.
To determine the exponential phase of amplification, where product formation is related to starting template concentration, a PCR cycle profile was performed. An aliquot of cDNA, collected from each of the myometrial samples at 27–41 weeks of pregnancy, was pooled to create an `average' sample. PCR was performed using between 20 and 40 cycles with primers specific for COX-1, COX-2 and GAPDH. A 5 µl aliquot of each PCR was dot blotted onto nylon membrane (MSI), hybridized to specific radiolabelled probes, and washed under high stringency conditions. Dotted membrane filters were then placed in a scintillation counter and radioactivity determined as a function of counts per minute for each individual sample. The ratio of product concentration to template concentration was determined. The optimal cycle number was defined as the midpoint of cycle numbers giving a linear relationship between template and product concentrations. In these experiments, optimal cycle numbers were found to be 31 for each of COX-1 and COX-2 and 22 for GAPDH. These cycle numbers were therefore used in studies of COX expression. To control for RNA concentration, COX expression was calculated as a ratio to that of GAPDH.
Comparison of amplification efficiency
We have previously reported experiments showing that the efficiency of amplification of COX-1 and COX-2 is similar using these primers (Slater et al., 1994Slater et al., 1998). Briefly, COX-1 and COX-2 RT–PCR products were subcloned into the Bluescribe vector (Stratagene, La Jolla, CA, USA). The concentration of Bluescribe plasmid DNA containing either COX-1 or COX-2 PCR product inserts was determined spectrophotometrically, and compared visually by agarose gel electrophoresis. Stepwise (1 in 10) dilution series' were prepared for each plasmid to give equivalent concentrations of each target sequence and PCR was performed. Amplification efficiency was found to be similar for the two sequences.
Western analysis
Protein extracts were prepared from myometrial samples by homogenization in 10 vol T-Wash (50 mmol/l Tris buffer, 10 mmol/l EDTA, 1% Triton-100, with 10 mmol/l phenylmethylsulphonyl fluoride, 4 µg/ml pepstatin and 0.5 µg/ml leupeptin) for 30 s. The supernatant was separated from tissue debris by centrifugation at 1000 g, for 10 min, at 4°C. Protein concentrations were determined by Protein assay (Bio-Rad Laboratories, Richmond, CA, USA) and bovine serum albumin (BSA) reference standards. Electrophoresis was carried out on 20 µg aliquots of protein samples, in 2x loading buffer [4% sodium dodecyl sulphate (SDS), 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 mol/l Tris–HCl, pH 6.8]. Samples were boiled for 5 min, quenched on ice, and subsequently run on a 10% SDS–polyacrylamide gel.
Western blotting was carried out following electrophoretic transfer, in 25 mmol/l Tris, 192 mmol/l glycine and 20% v/v methanol, pH 8.3, on to Hybond ECL nitro-cellulose membrane (Amersham Life Science, Little Chalfont, UK). Membranes were blocked in 5% Marvel-0.1% Tween–phosphate-buffered saline (PBS), for 1 h at room temperature. Affinity-purified goat polyclonal antibodies, directed against the peptide immunogen, were used for COX-1 and COX-2 (COX-1 No. sc-1752, COX-2 No. sc-1745: Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:500, and incubated overnight at 4°C. Membranes were washed in 0.1% Tween–PBS and then incubated with anti-goat immunoglobulin G–horseradish peroxidase (IgG–HRP) secondary antibody at a dilution of 1:2000, for 1 h at room temperature. ECL Western blotting detection was carried out using standard protocols (Amersham Life Science). Protein band size was determined using Rainbow coloured protein molecular weight markers (Amersham Life Science). Antibody specificity was confirmed using positive controls. Human platelets were used as a positive control for COX-1 and interleukin-1ß stimulated human lymphocytes for COX-2. Western autoradiographs were quantified by digital densitometry using the Image Master VDS gel documentation system and Image Master VDS Software (Pharmacia Biotech). Protein bands were digitized, ensuring that the range of pixel densities did not extend to either the minimum or maximum values. Mean pixel density for each band was assessed using a sample gate of the same size. To allow comparisons between blots prepared on different occasions a single control sample was included on each blot. The final pixel density was adjusted to ensure that this control sample carried the same value for each blot.
Statistical analysis
Data were analysed in groups, estimating the mean and SE respectively, before comparisons were made using analysis of variance (ANOVA). Differences between groups were assessed by post-hoc analysis using Fisher's exact test with the StatView 4.5 statistics software (Abacus Concepts Inc. Berkeley, CA, USA). P < 0.05 was considered to be statistically significant.
Results
COX-1 and COX-2 were expressed at both mRNA and protein levels in all myometrial samples examined. No significant differences of GAPDH expression were observed in any of the samples examined. Western analysis showed COX-2 protein at a size of 72 kDa and COX-1 protein at 74 kDa. COX-1 and COX-2 expression data were analysed in four groups: early preterm (26–32 weeks, n = 8); late preterm (33–37 weeks, n = 6); term not in labour (38–41 weeks, n = 11); and term in labour (38–40 weeks, n = 5).
Cycle profiles (n = 3) were performed on pooled myometrial samples and the number of PCR cycles used to measure both COX-1 and COX-2 in this study was 31. Figure 1 depicts a representative cycle profile. It was observed that whilst the relative abundance of COX-1 to COX-2 mRNA in myometrium was of the same order of magnitude, in general it appeared that COX-1 was expressed at levels slightly higher than those of COX-2.
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A decrease of COX-1 mRNA expression was observed in association with labour compared with all other groups; however, this decrease was not statistically significant (Figure 2a
). No further changes in COX-1 expression at either the mRNA or protein level were seen between any of the gestational age groups, or in association with labour (Figure 2a,b).
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Expression of COX-2 mRNA and protein was significantly (P < 0.05) higher in the term in-labour group compared with both early and late preterm groups. Expression of COX-2 was also higher in the term non-labour group compared with either of the preterm groups, however, only increased protein values reached statistical significance (P < 0.05). No differences were observed for COX-2 mRNA expression between either the early or late preterm groups. COX-2 protein expression was significantly higher in late preterm compared with early preterm (P < 0.05). No significant differences were observed in association with labour (Figure 3a,b
). Linear regression analysis of COX expression by gestational age showed no significant association between expression of either COX-1 or COX-2 and gestational age (data not shown).
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Discussion
During pregnancy the uterus expands to accommodate the growing fetus and placenta whilst remaining relatively quiescent, and the cervix remains firm and closed. At term these changes need to be reversed to allow the uterus to contract and the cervix to soften and dilate (for review see Garfield et al., 1998). It has been suggested that labour is the result of the activation of a `cassette of contraction associated proteins' which might include gap junction proteins, oxytocin and prostaglandin receptors (Lye, 1994). We have previously shown that, at the mRNA level, it is principally COX-2 which is expressed in both amnion and chorion decidua and that its expression increases with the onset of labour, whilst the expression of COX-1 in both tissues is unchanged (Slater et al., 1995Slater et al., 1998). We postulated that the central prostaglandin synthetic enzyme cyclo-oxygenase (COX) might also be a member of the `cassette of contraction associated proteins' and this is supported by these findings of significant up-regulation of COX-2 expression at the mRNA level in myometrium in late pregnancy and in association with labour.
Western analysis showed increases in COX-2 protein concentrations between preterm and term tissues, paralleling that seen at the mRNA level. The differences in expression at the protein level were not as great as those seen at the mRNA level and appeared to occur at earlier gestational ages. This may reflect the `suicide' nature of the COX enzyme. When COX is not catalysing the synthesis of prostaglandins, for example in the circulating platelet, it is relatively stable. During synthesis of prostaglandins, however, COX undergoes destruction after a limited number of reactions (Marshall et al., 1979) leading to an effectively short half-life. Any stimulus to prostaglandin production must therefore increase synthesis of new COX protein, to replace that destroyed during catalysis. It is possible that the increase in COX-2 protein which we have seen to occur at 33–37 weeks, without an associated increase in mRNA, represents the accumulation of protein in anticipation of an increase in prostaglandin synthesis associated with labour at term. Synthesis of oxytocic prostaglandins, during labour, would consume COX protein whose replacement would require either increased COX gene transcription or increased mRNA stability. This hypothesis would be consistent with our finding a greater increase in mRNA than protein concentrations at term prior to onset of labour.
In myometrium, prostacyclin is the principle prostaglandin synthesized (Bamford et al., 1980). In this study we found that COX-1 is expressed in myometrium with a relative abundance similar to that of COX-2 whereas in the fetal membranes COX-2 expression greatly exceeds that of COX-1 (Slater et al., 1994). Expression of the `constitutive' COX-1 enzyme in myometrium probably reflects constitutive prostacyclin synthesis. Prostacyclin is a vasodilator and may contribute to the necessary increases in myometrial blood flow during pregnancy. Myometrial COX-2 expression increases with a pattern similar to that of the chorion–decidua suggesting common mechanisms of control (Slater et al., 1998). In-situ hybridization and immunocytochemistry were used to show that human pregnant myometrium expresses both COX-1 and COX-2 (Zuo et al., 1994). These authors found that COX-1 expression decreased with advancing gestational age but did not change with labour and that COX-2 expression increased with gestational age but decreased with labour. Their findings are not entirely consistent with our results. However, their conclusions were based upon qualitative scoring of histological sections which did not permit statistical analysis. Myometrial samples in this study were taken from the lower segment of the uterus. The lower uterine segment develops more rapidly late in pregnancy and undergoes significant changes at term. It would be of interest to know if the same changes also occurred in the fundal segment of the uterus. Access to these fundal samples is very limited. However, these data still suggest a gestational stage-related trend for increased prostaglandin synthetic capacity in human myometrium. Our current data, together with those from previous studies showing increased COX-2 expression in fetal membranes with the onset of labour (Hirst et al., 1995; Slater et al., 1995, 1999), support the overall hypothesis that it is COX-2 and not COX-1 whose expression is increased in both the myometrium and the fetal membranes with the onset of labour and which catalyses the formation of oxytocic prostaglandins.
Acknowledgments
Funding from Wellbeing and the Tommy's Campaign supported this work.
Notes
3 To whom correspondence should be addressed 
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J. A.Z. Loudon, C. L. Elliott, F. Hills, and P. R. Bennett Progesterone Represses Interleukin-8 and Cyclo-Oxygenase-2 in Human Lower Segment Fibroblast Cells and Amnion Epithelial Cells Biol Reprod, July 1, 2003; 69(1): 331 - 337. [Abstract] [Full Text] [PDF]
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R. M. Tribe, P. Moriarty, A. Dalrymple, A. A. Hassoni, and L. Poston Interleukin-1{beta} Induces Calcium Transients and Enhances Basal and Store Operated Calcium Entry in Human Myometrial Smooth Muscle Biol Reprod, May 1, 2003; 68(5): 1842 - 1849. [Abstract] [Full Text] [PDF]
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Y. Lee, V. Allport, A. Sykes, T. Lindstrom, D. Slater, and P. Bennett The effects of labour and of interleukin 1 beta upon the expression of nuclear factor kappa B related proteins in human amnion Mol. Hum. Reprod., April 1, 2003; 9(4): 213 - 218. [Abstract] [Full Text] [PDF]
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T. Schmitz, M.J. Leroy, E. Dallot, M. Breuiller-Fouche, F. Ferre, and D. Cabrol Interleukin-1{beta} induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts Mol. Hum. Reprod., January 1, 2003; 9(1): 1 - 8. [Abstract] [Full Text] [PDF]
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I. Osman, A. Young, M. A. Ledingham, A. J. Thomson, F. Jordan, I. A. Greer, and J. E. Norman Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term Mol. Hum. Reprod., January 1, 2003; 9(1): 41 - 45. [Abstract] [Full Text] [PDF]
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K. Cesen-Cummings, K. D. Houston, J. A. Copland, V. J. Moorman, C. L. Walker, and B. J. Davis Uterine Leiomyomas Express Myometrial Contractile-Associated Proteins Involved in Pregnancy-Related Hormone Signaling Reproductive Sciences, January 1, 2003; 10(1): 11 - 20. [Abstract] [PDF]
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D. Korita, N. Sagawa, H. Itoh, S. Yura, M. Yoshida, K. Kakui, M. Takemura, C. Yokoyama, T. Tanabe, and S. Fujii Cyclic Mechanical Stretch Augments Prostacyclin Production in Cultured Human Uterine Myometrial Cells from Pregnant Women: Possible Involvement of Up-Regulation of Prostacyclin Synthase Expression J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5209 - 5219. [Abstract] [Full Text] [PDF]
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S. Zervou, E. Karteris, E.W. Hillhouse, and R.W. Old Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells Mol. Hum. Reprod., July 1, 2002; 8(7): 597 - 605. [Abstract] [Full Text] [PDF]
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S. Mesiano, E.-C. Chan, J. T. Fitter, K. Kwek, G. Yeo, and R. Smith Progesterone Withdrawal and Estrogen Activation in Human Parturition Are Coordinated by Progesterone Receptor A Expression in the Myometrium J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2924 - 2930. [Abstract] [Full Text] [PDF]
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D. M. Slater, S. Zervou, and S. Thornton Prostaglandins and Prostanoid Receptors in Human Pregnancy and Parturition Reproductive Sciences, May 1, 2002; 9(3): 118 - 124. [Abstract] [PDF]
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D. Giannoulias, F. A. Patel, A. C. Holloway, S. J. Lye, H. H. Tai, and J. R. G. Challis Differential Changes in 15-Hydroxyprostaglandin Dehydrogenase and Prostaglandin H Synthase (Types I and II) in Human Pregnant Myometrium J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1345 - 1352. [Abstract] [Full Text] [PDF]
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 B. Hinz and K. Brune Cyclooxygenase-2---10 Years Later J. Pharmacol. Exp. Ther., February 1, 2002; 300(2): 367 - 375. [Abstract] [Full Text] [PDF]
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G. C.S. Smith, W. X. Wu, and P. W. Nathanielsz Lipoxygenase gene expression in baboon intrauterine tissues in late pregnancy and parturition Mol. Hum. Reprod., June 1, 2001; 7(6): 587 - 594. [Abstract] [Full Text] [PDF]
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T. Engstrøm, P. Bratholm, N. J. Christensen, and H. Vilhardt Effect of Oxytocin Receptor Blockade on Rat Myometrial Responsiveness to Prostaglandin F2{alpha} Biol Reprod, November 1, 2000; 63(5): 1443 - 1449. [Abstract] [Full Text]
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T.-L. Erkinheimo, K. Saukkonen, K. Narko, J. Jalkanen, O. Ylikorkala, and A. Ristimäki Expression of Cyclooxygenase-2 and Prostanoid Receptors by Human Myometrium J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3468 - 3475. [Abstract] [Full Text]
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 S. R Bartlett, R. Sawdy, and G. E Mann Induction of cyclooxygenase-2 expression in human myometrial smooth muscle cells by interleukin-1{beta}: involvement of p38 mitogen-activated protein kinase J. Physiol., October 15, 1999; 520(2): 399 - 406. [Abstract] [Full Text] [PDF]
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