JCEM ONLINE Brief Report—Endocrine Research Evidence for a Spatial and Temporal Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion in Term and Preterm Parturition Deug-Chan Lee, Roberto Romero, Jung-Sun Kim, Wonsuk Yoo, JoonHo Lee, Pooja Mittal, Juan Pedro Kusanovic, Sonia S. Hassan, Bo Hyun Yoon, and Chong Jai Kim Perinatology Research Branch (D.-C.L., R.R., J.L.), National Institute of Child Health and Human Development/National Institutes of Health/Department of Health and Human Services, Bethesda, Maryland, 20892, and Detroit, Michigan, 48201; Departments of Pathology (J.-S.K., C.J.K.), Internal Medicine (W.Y.), and Obstetrics and Gynecology (P.M., J.P.K., S.S.H.), Wayne State University School of Medicine, Detroit, Michigan, 48201; and Department of Obstetrics and Gynecology (B.H.Y.), Seoul National University College of Medicine, Seoul 110-799, Korea Context: Prostaglandin-endoperoxide synthase 2 (PTGS2) is a key enzyme involved in parturition. PTGS2 mRNA was found to be differentially expressed between placental amnion (amnion over- lying the placental disc) and reflected amnion (amnion of the extraplacental chorioamniotic mem- branes) in term placentas. Objective: The aim was to evaluate the spatial and temporal regulation of PTGS2 expression in the amnion and the chorion-decidua. Design: PTGS2 expression was analyzed in the amnion and chorion-decidua obtained from 32 women: term not in labor (n ϭ 12), term in labor (n ϭ 12), and preterm labor (n ϭ 8), by immu- noblotting and densitometry. Prostaglandin E2 (PGE2) in the amnion and chorion-decidua was measured by a specific immunoassay. Results: Compared to preterm labor cases, PTGS2 expression increased at term before the onset of labor far more prominently in placental amnion (4.5-fold; P ϭ 0.002) than in reflected amnion (1.4-fold; P ϭ 0.007). There was a significant increase in PTGS2 expression in reflected amnion (2.9-fold; P Ͻ 0.01) but not in placental amnion with labor at term. PTGS2 expression was higher in reflected amnion than in chorion-decidua in labor at term (2.9-fold; P Ͻ 0.01). PTGS2 was barely detected in amnion and chorion-decidua with preterm labor. Expression of PGE2 showed a good correlation with PTGS2 expression (r ϭ 0.722; P Ͻ 0.001). Conclusion: PTGS2 expression in the amnion shows a distinct spatial and temporal regulation. Spontaneous labor at term and pathological preterm labor clearly differ in amniotic PTGS2 and PGE2 abundance. Our observations underscore the biological significance of the amnion and am- niotic fluid in human parturition. (J Clin Endocrinol Metab 95: E86–E91, 2010) rostaglandins play a critical role in the initiation of been subject to investigation (1–3). The expression of P human labor. The expression pattern of prostaglan- PTGS2, but not PTGS1, in the amnion and chorion- din-endoperoxide synthase 2 (PTGS2; cyclooxygenase 2), decidua is thought to be important for parturition at term a key enzyme in the synthesis of prostaglandins, by cho- (4). Preterm labor associated with intraamniotic infection rioamniotic membranes, decidua, and myometrium has is also characterized by dramatic elevation of amniotic ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: PGE2, Prostaglandin E2; PTGS2, prostaglandin-endoperoxide synthase 2; Printed in U.S.A. PTL, preterm labor; TIL, at term in labor; TNL, at term not in labor. Copyright © 2010 by The Endocrine Society doi: 10.1210/jc.2010-0203 Received January 26, 2010. Accepted May 11, 2010. First Published Online June 2, 2010 E86 jcem.endojournals.org J Clin Endocrinol Metab, September 2010, 95(9):E86–E91 J Clin Endocrinol Metab, September 2010, 95(9):E86–E91 jcem.endojournals.org E87 ␤ fluid concentration of prostaglandin E2 (PGE2) and PGF2␣ with a murine monoclonal anti- -actin antibody (Sigma-Aldrich (5). In conjunction with this, in vitro experimental studies Co., St. Louis, MO) as a loading control. Chemiluminescent have shown consistent and marked up-regulation of signals were detected using ChemiGlow West reagents (Alpha Innotech Corporation, San Leandro, CA). The PTGS2/␤-actin PTGS2 expression in amnion cells, trophoblasts, and de- ratio was analyzed by FluorChem SP densitometry using Alpha- cidual cells by proinflammatory cytokines such as IL-1␤ Ease FC Software (version 4.1.0; Alpha Innotech Corporation). and TNF␣ (6–8). We have recently shown that PTGS2 mRNA expression ELISA ␮ is significantly higher in the placental amnion (the amnion For the measurement of PGE2 content, 6.25 g of protein overlying the placental disc) than in the reflected amnion lysates (5 ␮l) from the amnion and chorion-decidua were added ␮ (the amnion of extraplacental chorioamniotic mem- to 95 l of assay buffer for immunoassay, respectively. Meclofe- namic acid (10 ␮g/ml; Sigma-Aldrich Co.) was added to all sam- branes) (9). This intriguing difference in the spatial PTGS2 ples. The assays were done using the Prostaglandin E2 Enzyme mRNA expression pattern led us to postulate that it might Immunoassay Kits (Assay Designs, Inc., Ann Arbor, MI). The be an important feature of human parturition. This study interassay coefficient of variation was 8.1%, and the intraassay was conducted 1) to evaluate the spatiotemporal regula- coefficient of variation was 6.7%. tion patterns of amniotic PTGS2 expression in human pregnancy in the context of advancing gestational age and Immunohistochemistry the presence of labor; and 2) to compare PTGS2 and PGE To determine PTGS2 expression patterns, immunohisto- 2 chemistry was performed in TNL (n ϭ 3), TIL (n ϭ 3), and PTL expression between the amnion and the chorion-decidua. (n ϭ 3) cases. Formalin-fixed, paraffin-embedded, 5-␮m-thick sections of the chorioamniotic membranes were placed on silanized slides and stained using a Ventana Discovery auto- Materials and Methods matic staining system (Ventana Medical Systems, Tucson, AZ). Immunostaining was performed using 1:50 diluted, Tissue samples mouse monoclonal anti-Cox-2 antibody (clone no. 29; Santa Cruz Biotechnology, Inc.). Placental amnion, reflected amnion, and chorion-decidua tis- sues were obtained as previously described (9) and flash-frozen using liquid nitrogen. The tissue samples were obtained from the Statistical analysis following groups of patients: 1) women at term not in labor Kruskal-Wallis tests were used to test for differences among (TNL; n ϭ 12); 2) women at term in labor (TIL; n ϭ 12); and the groups. When differences existed, Mann-Whitney U-tests 3) women with spontaneous preterm labor (PTL) without clin- were used to investigate which pairs were significantly different. ical and histological chorioamnionitis (n ϭ 8). Three cases in the The median protein expressions among placental amnion, re- PTL group were delivered by cesarean section due to a nonre- flected amnion, and chorion-decidua in each group were com- assuring fetal heart rate pattern during labor, and five PTL cases pared using Friedman tests and Wilcoxon signed ranks tests. The were delivered vaginally. The samples were stored at Ϫ80 C until correlation between the expression of PTGS2 and PGE2 was use. All patients provided written informed consent, and the tested using the Spearman rho test. SPSS version 15.0 (SPSS Inc., Institutional Review Board of the Eunice Kennedy Shriver Na- Chicago, IL) was employed. All P values are two-sided, and P tional Institute of Child Health and Human Development, Na- values of less than 0.05 were considered to be significant. tional Institutes of Health, Department of Health and Human Services approved the collection and use of materials for research purposes. Results Protein extraction The median gestational ages of TNL, TIL, and PTL cases Each tissue was liquid nitrogen-pulverized using a mortar and were 39 wk (range, 38–40 wk), 39 wk (range, 37–40 wk), pestle, and protein was isolated using T-PER Tissue Protein Ex- and 32 wk (range, 28–34 wk), respectively. A distinct traction Reagents with a protease inhibitor cocktail (Thermo feature of PTGS2 expression observed in immunoblotting Scientific, Rockford, IL). Protein concentrations were deter- mined using the BCA Protein Assay Kit (Thermo Scientific). was its relative abundance in the amnion compared with the chorion-decidua (Fig. 1, A and B). Immunoblotting Densitometric analysis of PTGS2 expression for TNL Ten micrograms of proteins were electrophoresed in 10% cases showed higher expression of PTGS2 in the placental SDS-PAGE gels under nonreducing conditions and transferred amnion than in the reflected amnion [median PTGS2/␤- onto a polyvinylidene difluoride membrane. Membranes were actin ratio, 1.44 (range, 0.23–3.42) vs. 0.48 (range, 0.31– blocked with 5% nonfat dry milk in Tris-buffered saline con- 1.51); P Ͻ 0.01] (Fig. 1, A and C). PTGS2 expression in taining 0.1% Tween 20, incubated with 1:250 diluted mouse placental and reflected amnion at TNL was 4.5-fold (P ϭ monoclonal anti-Cox-2 antibody (clone no. 29; Santa Cruz Bio- ϭ technology, Inc., Santa Cruz, CA) for1hatroom temperature, 0.002) and 1.4-fold (P 0.007) higher, respectively, than and then probed with peroxidase-conjugated antimouse IgG (1: in those with PTL. Interestingly, increases in PTGS2 ex- 5000; Santa Cruz Biotechnology, Inc.). Membranes were probed pression were more prominent in reflected amnion than in E88 Lee et al. PTGS2 Expression in the Amnion J Clin Endocrinol Metab, September 2010, 95(9):E86–E91 placental amnion in TIL cases com- pared with those of TNL cases. There- fore, there was no difference in the PTGS2/␤-actin ratio between the pla- cental amnion and the reflected amnion in tissues derived from women who had undergone labor [median, 1.73 (range, 0.44–5.94) vs. median, 1.38 (range, 0.36–6.03)] (Fig. 1, A and C). PTGS2 expression in the placental amnion (median PTGS2/␤-actin ratio, 0.32; range, 0.26–0.50) and the re- flected amnion (median PTGS2/␤-actin ratio, 0.34; range, 0.27–0.58) of PTL cases was significantly lower compared with those of the placental amnion and the reflected amnion from women at term in spontaneous labor (P Ͻ 0.001 for each).