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(1991), 12, 353-363

Prostaglandin Production by Human Chorion Laeve Cells in Response to Inflammatory Mediators

SARAH LUNDIN-SCHILLER MURRAY D. MITCHELL”

&

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Medical Center, Salt Lake Cio: Utah 84132, USA rrTo whom correspondence should be addressed Paper accepted 23.4.1991

SUMMARY Cytokines produced during an intrauterine infection may cause increased prostaglandin production and thus preterm labor. Previously, we have shown that cytokines increaseprostaglandinproduction by human amnion and decidual cells. In thepresent study we examined the effect of interleukin-lb (IL-ID), tumor necrosisfactor (TNF), and endotoxin on prostaglandin production by chorion laeve cells. Upon reaching con&rue at day 3 and after IO days in culture, human chorion laeve cells in primary culture were incubated with each substance. On day 3 of culture the chorion cells demonstrated limited responsiveness to the test agents with a maximum response of less than two times basal production. However, a@ 10 days the cultures were highly responsive to the test agents with maximum responses ofgreater than ten times basal production. We conclude that early in culture chorion cells may express fewer functional recqtors for the test agents. Alternatively, a subpopulation of chorion cells responsive to cytokines and endotoxin maypredominate by day 10 of culture. Chorion laeve, therefore, could contribute to the increased intrauterine prostaglandin production associated with infection-driven preterm labor.

INTRODUCTION There is growing evidence that intra-amniotic infection [defined by Romero et al (1988b) as the presence of microorganisms in amniotic fluid obtained by transabdominal amniocentesis] frequently leads to preterm labor in women. In a recent study 16 per cent of women presenting with preterm labor and intact membranes have intra-amniotic infections; of these women over 60 per cent failed to respond to tocolysis and proceeded to spontaneously rupture their membranes (Romero et al, 1988b). Increased eicosanoid production by intrauterine tissues is considered a key event in the initiation of labor in women (Mitchell, 1984) and several studies have shown a positive correlation between increased amniotic fluid 0143-4004/91/040353

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levels of eicosanoids and preterm labor associated with intra-amniotic infection. Amniotic fluid concentrations of prostaglandins are significantly higher in women with preterm labor and intra-amniotic infections than in women in preterm labor without infection (Romero et al, 1988d; Romero, Hobbins and Mitchell, 1989a). Amniotic fluid concentrations of leukotriene Bq, 15-hydroxyeicosatetraenoic acid and S-hydroxyeicosatetraenoic acid are also increased in women in preterm labor with intra-amniotic infections (Romero et al, 1988e; Romero et al, 1987). In an effort to understand the link between infection, elevated prostaglandin production and preterm labor several investigators have sought to determine if bacteria or bacterial products influence prostaglandin production by intrauterine tissues. Conditioned media from bacterial cultures as well as purified bacterial phospholipase AZ have been shown to stimulate amnion prostaglandin production (Bennett et al, 1987; Lamont, Rose and Elder, 1985). Furthermore, endotoxin, a component of the cell wall of gram negative bacteria, has been shown to stimulate both amnion and decidual prostaglandin biosynthesis (Romero, Hobbins and Mitchell, 1988a; Mitchell, Edwin and Romero, 1990). Endotoxin is elevated in the amniotic fluid ofwomen in preterm labor with infection (Romero et al, 1988~). Bacterial products, therefore, may be causal of preterm labor associated with infection. Other investigators have hypothesized that cytokines produced by host macrophages in response to infection may stimulate intrauterine prostaglandin biosynthesis. The cytokines, interleukin-l/3 (IL-l/?) and tumor necrosis factor (TNF), have been shown to stimulate prostaglandin production by human amnion and decidual cells (Romero et al, 1989d; Mitchell, Edwin and Romero, 1990). The concentrations of IL-l/3 and TNF required to induce a significant stimulation of prostaglandin production are within the ranges found in amniotic fluid of women with intra-amniotic infection (Romero et al, 1989b; Romero et al, 1989~). Thus, it seems likely that the biochemical links between intra-amniotic infection and preterm labor are inflammatory mediators and perhaps bacterial products which stimulate prostaglandin biosynthesis by intrauterine tissues. The role of the chorion laeve in infection-driven preterm labor, however, has not been explored. The chorion laeve exists at the fetal-maternal interface and can both synthesize and metabolize prostaglandins; and, thus, it may be an important site for regulating the availability of prostaglandins to the myometrium. In the present study, therefore, we evaluated the effects of IL- l/3, TNF, and bacterial endotoxin on prostaglandin production by chorion laeve. This goal required the use of chorion laeve cells in culture. Since confluence in our cultures was attained by 3 days, experiments were conducted at this time. In addition, since others have reported that certain secretory activities of chorionic cultures do not appear until much later in culture (e.g. prorenin, 4 to 10 days in culture; Acker et al, 1982) we also evaluated the prostaglandin production by our cultures after 10 days.

MATERIALS Tissue culture media (Ham’s F12/Dulbecco’s Modified Eagles Medium 1: 1 [Ham’s F12/ DME] and Dulbecco’s Modified Eagles Medium) and fetal calf serum (FCS) were obtained from Irvine Scientific, Santa Ana, CA. Collagenase B was purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN. Percoll was obtained from Pharmacia, Pleasant Hill, CA. Tissue culture plates (24-16 mm well plates) were purchased from Costar, Cambridge, MA. IL-l/l and TNF-alpha were purchased from R&D Systems, Minneapolis, MN. Bacterial endotoxin from E. coli was purchased from Sigma, St Louis, MO. Antibodies

J,undin-Schiller, Mitchell: Regulation of Chorion Prostaglandin Production

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for prostaglandin E2 (PGEZ), prostaglandin Fz, (PGFZ,), and 6-keto-prostaglandin F1, (6keto-PGFt,) were purchased from Advanced Magnetics, Inc. (Cambridge, MA). Tritiated prostaglandins were obtained from Amersham Corporation (Arlington Heights, IL). ,411 other chemicals were of reagent grade.

METHODS Tissue Placentae were obtained at term at the time of elective Cesarean section before the onset of labor after uncomplicated pregnancies. These studies were approved by the Institutional Review Board. Histology Samples of tissue for sectioning were taken: (1) prior to removing amnion and decidua, (2) after stripping off the amnion only, and (3) after removing both the amnion and decidua. Tissue was fixed in 3.7 per cent formaldehyde in phosphate-buffered saline, pH 7.4. Tissue was then embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard methods by the University of Utah Medical Center Pathology Department’s Histology Laboratory. Photographs of tissue sections were taken with a Nikon camera through a 10x objective mounted on a Nikon Diaphot-TMD inverted microscope. For micrographs of cultures, unfixed cells were photographed with a Nikon camera through a 10x phase-contrast objective mounted on a Nikon Diaphot-TMD inverted microscope. Cell culture The amnion was stripped off the chorion laeve and decidual tissue adherent to the chorion laeve was removed by sharp dissection. The results of our dissection procedure are displayed in Figure 1. Chorion laeve cells were dispersed and isolated as described by Gibb, Riopel and Lavoie (1986). The chorion laeve was incubated at 37°C in collagenase B (50 mg/ml in DME) for approximately 2 h. The supernatant was filtered twice over gauze and the cells were pelleted by centrifugation at 800 xgfor 10 min. The cells were washed with fresh DME, layered over discontinuous Percoll gradients (60,40,20, and 5 per cent Percoll in DME), and then centrifuged at 900 xg for 20 min. The cells migrating to the 20-40 per cent interface were collected, washed, and plated at a density of 300,000 cells/well in 24-16 mm well plates in Ham’s F12/DME plus 10 per cent FCS. The cultures were maintained in Ham’s F12/ D&lE plus 10 per cent FCS at 37°C in a humidified atmosphere containing 5 per cent CO? in dir. Radioimmunoassays Prostaglandins were measured directly in the tissue culture media. Standard curves were prepared with the same volume of media that had not been on cells as was being measured from the experimental samples. PGE2 was measured as previously described (Magness et al, 1985), except that antibody was purchased from Advanced Magnetics Inc. 11 -Deoxy- 13,14dihydro- 15-keto- 11,16-cycle-prostaglandin E2 (PGEMII) was measured as previously described (Mitchell et al, 1982b); except after alkalinization and incubation, media were neutralized and assayed directly. PGFZ, (Gibb et al, 1988) and 6-keto-PGF,, (Strickland et al, 1982) were measured using specific antibodies from Advanced Magnetics (Cambridge, MA). 13,14-Dihydro-15-keto-prostaglandin F zn (PGFM) (Strickland, Brennecke and

356

Figure I. Cross-section

of fetal membranes and decidua prior to (a) and after dissection to isolate chorion laeve (b and c). (a) Section through amnion-chorion-decidua, filled arrow shows the amnion epithelium, open arrow shows decidual tissue. (b) Chorion-decidua after removal of the amnion, arrow shows connective tissue layer which lies between amnion epithelium and trophoblast layer of the chorion laeve, asterisk shows an atrophic villus common throughout the trophoblast layer. (c) Chorion laeve after sharp dissection of decidual tissue, arrow shows the trophoblast layer of the chorion laeve. Bar is equivalent to approximately 150,~.

Mitchell, 1982) and prostaglandin D2 (PGDZ) (Mitchell, were assayed as previously described.

Kraemer

and Strickland,

1982a)

Protein assay Cellular protein was assayed by the method of Lowry et al (195 1). Statistical analysis Data were analysed using Fisher’s Least Significant Difference Test. Each experiment was conducted in quadruplicate (n = 4) on cells from a minimum of seven different placentae (N). Figures were drawn using data from one representative experiment.

RESULTS Figure 1 illustrates the results of our dissection procedure to isolate the chorion laeve. As indicated by these sections prior to the Percoll gradients it is likely our tissue preparation includes cells from the trophoblast layer and the connective tissue layer of the chorion laeve.

I.vt~ditl-.S~.hiller~

F&w?. culture.

.MitchelI:

Regulation of Chorion Prostaglandin Production

Phase contrast micrographs of chorion laeve cell cultures. (a) Chorion laeve cultures ib) Chorion laeve cultures after 10 days in culture. Bar represents approximately 100,~.

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after 3 da)< in

Consistently, the majority of cells from all tissue preparations migrated to the 20-40 per cent interface of the gradients and these cells were used in all studies. At the S-20 per cent interface we observed primarily cellular debris. A small number of cells migrated to the 4060 per cent interface but these cells were not cultured. Any contaminating red blood cells migrated to the bottom of the gradient. In culture, the chorion cells attached to the dishes by 3 h and attained confluence by 3 days [Figure 2(a)]. At this stage in culture the cells appeared stellate, some with long cytoplasmic processes that can often be observed growing over other cells. The cells appeared to grow in a random arrangement. The cells continued to multiply throughout the 10 days of culture and became tightly packed in the dishes at which time the cells u-ere more elongated than previously observed but still irregularly shaped [Figure 2(b)]. In some areas of the culture, cells could be seen to grow on top of one another indicating a lack of contact inhibition. The time course of basal PGEZ production is presented in Figure 3. Production remained linear throughout the 24-h period. Subsequent experiments were terminated at 16 h. The major prostaglandins produced by the chorion cell cultures quantitatively were PGEZ and PGEMII with lesser amounts of PGFz,, PGFM, and 6-keto-Ft, (Figure 4). Very little, if any, PGD;! production occurs in these cultures (Figure 4). In some subsequent experiments PGE2 and PGEMII were both measured. PGEMII production was found to reflect PGEz production both basally and after stimulation with test agents; for clarity only PGEz production is presented. We observed a large variation in the basal prostaglandin production by tissues from different pregnancies (e.g. Table 1). In cultures prepared from tissue taken

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Lundin-.Schiller, Mitchell: Regulation of Chorion ProstaglandinProduction Table 1. Comparison motein

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Chorion 47 Chorion 48 Chorion 5 1 Chorion 59 Chorion 60 Chorion 61

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Figure 5, The effect of increasing concentrations of IL-1B on chorion cell PGE2 production in pg,+g protein/l6 h (mean + s.e.m., n = 4, N = 12 placentae). (a) Response ofcells after 3 days ofculture, control versus 1 ng/ml IL- l/j P < 0.05. (b) Response of cells after 10 days ofculture, control versus 0.1 ng/ml and higher concentrations of IL- l/j P< 0.001.

from the same pregnancy it was observed that basal production of prostaglandins decreased with time in culture (Table 1). The data are corrected for cellular protein but even without this correction basal production at 3 days in culture was significantly higher than at 10 days in culture. Early in culture the chorion cells were less responsive to all test agents than cells later in culture (Figures 5,6 and 7). Maximum stimulation of chorion PGEZ production induced bv the test agents was 2.5 times basal production whereas at day 10 of culture stimulation if greater than 10 times was frequently observed. After 10 days in culture the cells responded to all test agents with a concentration-dependent increase in PGE2 production (Figures 5,6 and 7). Significant stimulation occurred at 0.1 ng/ml IL-ID (P < O.OOl), 1 ng/ml TNF (P < O.Ol), and 1 pug/ml endotoxin (P < 0.001) at day 10 and at 1 ng/ml IL-lp (P < O.OS), 0.1 ng/ml TNF (P < O.OOl), and 10 pug/ml endotoxin (P < 0.05) for day 3 cultures.

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Figure 6. The effect of increasing concentrations

of TNF, on chorion ceil PGEp production in pg/pg protein/l6 h (mean f s.e.m., n = 4, N = 10 placentae). (a) Response of cells after 3 days of culture, control versus 0.1 ng/ml and higher concentrations of TNF, P i 0.001. (b)Response of cells after 10 days of culture, control versus 1 ng/ml and higher concentrations of TNF, P < 0.01.

600

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Figure 7. The effect of increasing concentrations of bacterial endotoxin on chorion cell PGEa production in pg/lcg protein/l6 h (mean f s.e.m., n = 4, N = 7 placentae). (a) Response of cells after 3 days of culture, control versus lOpg/ml endotoxin P < 0.05. (b) Response of cells after 10 days of culture, control versus 1 &ml endotoxin Pi 0.01.

L undin-,Ychiller, Mitchell: Regulation

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DISCUSSION The chorion laeve cells in culture attached to the dishes rapidly (by 3 h) which is consistent with the findings of Gibb, Riopel and Lavoie (1986) and Acker et al (1982). Morphologically, our cultures are similar to those described by Acker et al (1982) and to the cells grown in 10 per cent FCS in Gibb, Riopel and Lavoie (1986) (Figure 2). We tested the steroidogenic capacity of our cells early in culture (unpublished observations) and found that they possessed 3-/?-hydroxysteroid dehydrogenase (3/3HSD) activity as well as alkyl steroid sulfatase and estrogen sulfatase activity with the 3pHSD activity being greater than the sulfatase activities. These results are also similar to the findings of Gibb, Riopel and Lavoie (1986) for chorionic cells in culture. Though we did not test the steroidogenic capacity of our cells later in culture, Gibb, Riopel and Lavoie (1986) observed that over time chorion cultures grown in the presence of 10 per cent FCS expressed increased 3PHSD activity and decreased sulfatase activities. This phenomenon was attributed to either an effect of culture conditions on the sulfatase enzymes or a loss of a subpopulation of cells which possessed predominantly sulfatase activity. In this study we have demonstrated that chorion laeve cells in vitro produce PGEZ, PGFZ,,, and to a lesser extent prostacyclin (6-keto-PGFI,) and metabolize PGE2 and PGF2, into their respective inactive metabolites. These findings vary somewhat from those of Gibb et al was found to be produced by (1988). In that study little PGFz,, PGFM, or 6-keto-PGF1, chorion cell cultures. Consistent with their findings, however, our cultures also produce predominantly PGE2. The finding of significant quantities of PGEMII and PGFM by our cultures is consistent with the observations that intact chorion laeve tissue has very high 15 hydroxyprostaglandin dehydrogenase activity (Keirse and Turnbull, 1976; Okazaki et al, 1981). Basal production ofprostaglandins diminishes with time in culture while responsiveness to inflammatory mediators and bacterial endotoxin increases. The decline in basal production with time in culture is similar to that seen in cultured amnion cells (Bala, Thakur and Bleasdale, 1990; unpublished observations) and also to the chorion cultures by Gibb et al (1988). This may result from reduced activity or expression of enzymes such as phospholipases and cyclooxygenase. Indeed, studies have shown that basal phospholipase C activity in cultured amnion cells decreases with time in culture (Bala, Thakur and Bleasdale, 1990). The increased responsiveness of cells after 10 days in culture was a somewhat unexpected result. We speculate that this may be due either to a loss of functional receptors following enzymatic dispersion of the tissue which recover after several days in culture or that a subpopulation of chorion cells which are responsive to cytokines increase in number relative to other cells after several days in culture. Furthermore, it is possible that when basal prostaglandin production is relatively high, as in the day 3 cultures, it is simply not possible for the cells to generate further free arachidonic acid or enzymes necessary to produce additional prostaglandins. It should be noted that the enhanced responsiveness of cells after 10 days in culture is based on calculations of fold or percentage increases in prostaglandin production in response to test substances. Given the high basal rate of prostaglandin production after 3 days in culture the small percentage increases in production in response to test substances at this time may be greater in absolute amounts than after 10 days in culture. Evidence is accumulating which suggests that the chorion laeve may be an important source of hormonal signals which regulate amnion and decidual prostaglandin production and perhaps its own prostaglandin production. Vl.e have shown that chorion-conditioned medium is stimulatory to amnion cell PGEz

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biosynthesis (Lundin-Schiller, Gibb and Mitchell, 1990). We have also observed that IL- I/? stimulated or unstimulated chorion cells produce large amounts of interleukin-6 (IL-6) (unpublished observations). Others have shown that chorionic cells produce prorenin and renin (Symonds, Stanley and Skinner, 1968; Acker et al, 1982; Higashimori et al, 1989). We have also found that both IL-6 (100 ng/ml) and renin (0.1 U/ml) will stimulate amnion cell PGEZ biosynthesis. Mitchell and Challis (1988) proposed that changes in substrate supply and activities of steroidogenic enzymes in the chorion laeve at term result in increased local concentrations of estrogens and decreased concentrations of progesterone. This change in the estrogen to progesterone ratio may then favor prostaglandin biosynthesis within the fetal membranes and decidua. In this study, we have shown that the chorion laeve itself may be a source of elevated prostaglandin levels found in amniotic fluid during infection-driven preterm labor. Thus, elucidation of the mechanism of parturition may require more complete understanding of the regulation of protein hormone, steroid hormone and prostaglandin biosynthesis by the chorion laeve. In summary, this study demonstrates that both qualitatively and quantitatively the responses of chorion cell cultures change over time. Qualitative changes in the responsiveness of these cells makes interpretation of the data difficult. However, overall these data suggest that the chorion laeve does have the capacity to respond to inflammatory mediators with an increase in prostaglandin production and may contribute to the increased prostaglandin levels seen with intra-amniotic infection.

ACKNOWLEDGEMENTS This work was supported editorial assistance.

by NIH grant #HD20779.

We would like to thank Mary Ann Isenhart

for her expert

REFERENCES Acker, G. M., Galen, F. X., Devaux, C., Foote, S., Papernitz, E., Pesty, A., Menard, J. & Corval, P. (1982) Human chorionic cells in primary culture: a model for renin biosynthesis. Journal of Clinical Endocrinology and Metabolism, 55, 902-909. Bala, G. A., Thakur, N. R. & Bleasdale, J. E. (1990) Characterization of the major phosphoinositide-specific phospholipase C of human amnion. Biology of Rtproduction, 43,704711. Bennett, P. R., Rose, M. P., Myatt, L. & Elder, M. G. (1987) Preterm labor: stimulation of arachidonic acid metabolism in human amnion cells by bacterial products. American journal of Obstetri’rsand G.vecology, 156,649655. Bourne, G. (1962) The Human Amnion and Chorion. Chicago: Year Book Medical Publishers, Inc. Gibb, W., Riopel, L. & Lavoie, J.-C. (1986) Primarv cultures of cells from human chorion laeve: steroid metabolism and properties ofcells grown in defined media supplemented with 0.1 per cent or 10 per cent fetal calf serum. Journal of Clinical Endoerinolo~ and Metabolism, 62, 1124-l 129. Gibb, W., Riopel, L., Collu, R., Ducharme, J. R., Mitchell, M. D. & Lavoie, J. C. (1988) Cyclooxygenase products formed by primary cultures of cells from human chorion laeve: influence of steroids. CanadianJournal of Physiology and Pharmacology, 66, 788-793. Higashimori, K., Mizuno, K., Nakajo, S., Boehm, F. H., Marcotte, P. A., Egan, D. A., Holleman, W. H., Heusser, C., Poisner, A. M. & Inagami, T. (1989) Pure human inactive renin. 3ournal ofBiological Chemistry, 254,14662-14667. Keirse, M. J. N. C. & Tumbull, A. C. (1976) The fetal membranes as a possible source of amniotic fluid prostaglandins. BritishJournal of Obstetrics and Gynaecologx 83, 146-151. Lamont, R. F., Rose, M. & Elder, M. G. (1985) Effect of bacterial products on prostaglandin E production by amnion cells. The Lancer, ii, 1331-1333. Lundin-Schiller, S., Gibb, W. & Mitchell, M. D. (1990) Amnion cell prostaglandin EZ stimulatory activity in chorion-laeve conditioned medium. Biochimica et Biophysics Acta, 1053, 15 1-155.

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1,owry. 0. H., Rosebrough, N. J., Farr, A. L. & Randall R. J. (1951) Protein measurement with folin phenol reagent.Journal ofBiological Chemistry, 193, 265-275. Magness, R. R., Osei-Boaten, K., Mitchell, M. D. & Rosenfeld, C. R. (1985) Invitro prostacyclin production h! ovine uterine and systemic arteries; effects of angiotensin II. j’oburnal of Clinical Investigation, 76, 2206-2212. Mitchell, B. F. & Challis, J. R. G. (1988) Estrogen and progesterone metabolism in human fetal membranes. In The l’hpiology and Biochemistry of Human Fetal Membranes (Ed.) Mitchell, RF.. pp. 5;28. Ithaca: Perinatoiog Press. Mitchell, M. D. (1984) The mechanism(s) of human parturition.3obumal ofDevelopmental Physiologx 6, 107-I 18. Mitchell, M. D., Edwin, S. & Romero, R. J. (1990) Prostaglandin biosynthesis by human decidual cells: effects ot inflammatory mediators. Prostaglandins, Leukottienes and Essential Fatty Acids, 41, 35-38. Ilitchell, M. D., Kraemer, D. L. & Strickland, D. M. (1982a) The h uman placenta: a major source ot prostaglandin Dz. Prostaglandins, Leukotrienes andMedicine, 8, 383-387. Mitchell, M. D., Ebenback, K., Kraemer, D. L., Cox, K., Cutrer, S. & Strickland, D. M. (1982b) A sensitive radioimmunoassay for 1 l-deoxy-13,14-dihydro-15-keto-l1,16-cyclo-prostaglandin Ez: Application as an index of pnrstaglandin E2 biosynthesis during human pregnancy and parturition. Prostaglandins. Leukotrrencr crud IIedi&e, 9, 549-557. Okazaki, T., Casey, M. L., Okita, J. R., MacDonald, P. C. &Johnston, J. M. (1981) Initiation ofparturition: XII: Rios! nthesis and metabolism of prostaglandins in human fetal membranes and uterine decidua. America~Jwnu~l qf‘Ob.~tetricsand G>~necologp,139, 373-38 1. Romero, R., Hobbins, J. C. & Mitchell, M. D. (1988a) Endotoxin stimulates prostaglandin Ea production h! human amnion. Obstetrics and Gynecology, 71, 227-228. Romero, R., Quintero, R., Emanian, M., Wan, M., Grzyboski, C., Hobbins, J. C. & Mitchell, M. D. (1987) \rachidonic acid lipox?genase metabolites in amniotic fluid ofwomen with intra-amniotic infection and pretcrm labor. .inreri~~an_~ownalqf Obstetrics and G,vnecology, 157, 1454-1460. Romero, R., Mazor, M., Wu, Y. K., Sirtori, M., Oyanun, E., Mitchell, M. D. 6i Hobbins, J. C. (IYkXh) Infection in the pathogenesis of preterm labor. Seminars in Perinatologp, 12, 262-279. Romero, R., Roslansky, P., Oyarzun, E., Wan, M., Emanian, M., Novitsky, T. J., Gould, M. J. & Hobbins, J. C. (1988~) Labor and infection: II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor. .4rneritu~~.?o;o+nal of Obstetrics and GJjnecologv, 158, 1044-1049. Romero, R., Wu, Y. K., Mazor, M., Hobbins, J. C. &Mitchell, M. D. (1988d) Amniotic fluid prostaglandin 12 in preterm labor. Prostag/andins. Leukotrienes and Essential Fat!lj Acids, 34, 141-145. Romero, R., Wu, Y. K., Mazor, M., Hobbins, J. C. & Mitchell, M. D. (1988e) ;Imniotic fluid ;hkdrouyeicosatetraenoic acid in preterm labor. Prostaglandins, 36, 179-189. Romero, R., Wu, Y. K., Sirtori, M., Oyarzun, E., Mazor, M., Hobbins, J. C. & Mitchell, M. D. (1989a) -\mniotic tluid concentrations of prostaglandin Fa,,, 13,1-t-dihydro-15-keto-prostaglandin FzIl and 1 I-decls\13,1-l-dihl-dro-15-keto-I 1,16-cycle-prostaglandin Ea in preterm labor. Prostaglandins, 37, 149-161. Romero, R., Brody, D. T., Oyarzun, E., Mazor, M., Wu, Y. K., Hobbins, J. C. & Durum, S. K. (IYXYh) and Infection and labor. III. Interleukin-1: a signal for the onset of parturition. .dmerirtrn Journal qf Obstetricu Gpzology, 160. 1117-1123. Romero, R., Manogue, K. R., Mitchell, M. D., Wu, Y. K., Oyarzun, E., Hobbins, J. C. & Cerami, A. (19NYc) Infection and labor. It-. Cachectin-tumor necrosis factor in the amniotic fluid of women with intraamniotic infection and preterm labor. AmericanJournal ofObstetrics and G.gneroloa, 161, 336-341. Romero. R.. Durum. S.. Dinarello. C. A.. Ovarzun. E.. Hobbins. 1. C. &Mitchell. M. D. 11989d) Interleukin- I stimulates’prostaglandin biosynthesis by’human amnion. ProstagliGdins, 37, 13-22.’ ~ Strickland, D. M., Brennecke, S. P. & Mitchell, M. D. (1982) Measurements of 13,1-l-dihydro-15-l\ctoprosraglandin Fj,, and 6-keto-prostaglandin F trr in plasma by radioimmunoassa\prior to extraction md chromatography. Prostaglandim, Leukotrienes and.Vledicine, 9,491-493. Symonds, E. M., Stanley, M. A. & Skinner, S. L. (1968) Production ofrenin by in \:itro cultures ofhuman chorion dnd uterine muscle. .\‘arr,rr. 217, 1152-1153.