Prostaglandin secretion by human endometrium in vitro

Prostaglandin secretion by human endometrium in vitro B. K. TSANG T. C. 001 Ottawa, Ontm·io, Canada Late proliferative (days 9 to 14) and midsecretory (days 18 to 24) human endometria were maintained in organ culture in the presence of 17,8-estradiol and/or pi'OgEJSterone or mefenamic acid. 17,8-estradiol (0.1 to 1,000 ng/ml) increased prostSQiandin F (PGF) secretion by both proliferative and secretory endometria. Progesterone ( 1,000 ng/ ml) ·markedly inhibited PGF secretion by proliferative but not secretory endomettlu.m. 17{retttadiof and prog$$terone had no effect on prostaglandin E (PGE) secretion by both types of endometrium. Secretion of both PGF and PGE by proliferative and secretory endometria was inhibited by mefenamic acid (1 ~g/ml). This was more apparent with PGF secretion and was detectable within the first 2 hours of culture. These findings indicate that the human endometrium is a rich source of PGF and PGE and that the production of PGF is regulated by gonadal steroids. These results support the concept that one mechanism of action of mefenamic acid in the treatment of dysmenorrtlea is inhibi11on of prostaglandin production. (AM. J. 0BSTET. GYNECOL. 142:626, 1982.)

of the physiologic role of prostaglandins (PGs) in normal uterine function is incomplete. Recent studies have suggested that PGs produced within the uterus may be responsible for the constriction of endometrial spiral arterioles, resulting in the process of menstruation, and that imbalance in the synthesis and/or the metabolism of these lipids may be associated with menstrual disorders such as primary dysmenorrhea and menorrhagia. 1- 13 Various doubleblind clinical studies demonstrating the beneficial effects of PG synthetase inhibitors in the treatment of primary dysmenorrhea and menorrhagia also tend to support the hypothesis that overproduction of and/or PRESENT KNOWLEDGE

From the Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Ottawa, and the Metabolic Research Laboratory, Division of Metabolism and Endocrinology, Ottawa Civic Hospital. This work was supported in part by grants from Parke-Davis Canada Inc. and the University Medical Research Fund, Deparlmlmt of Obstetrics and Gynecology, University of Ottawa. Presented at the Thirty-seventh Annual Meeting of The Society of Obstetricians and Gynaecologists of Canada, Quebec City, Quebec, Canada, june 16-20, 1981. Reprint requests: Dr. B. K. Tsang, Reproductive Bwlogy Unit, Department of Obstetrics and Gynecology, University of Ottawa, Ottawa Civic Hospit,al, 105J Carling Ave., Ottawa, Ontario, Canada KJY 4E9. 62~

hypersensitivity to PGs may be causally related to these menstrual disorders .14-t 2 Studies with various animal species have shown that uterine PG synthesis and/or metabolism is under the control of ovarian steroids. It has been demonstrated in sheep, 23 • 24 rats, 25 • 26 guinea pigs,27 hamsters,27 and rhesus monkeys 28 that estrogen increases the uterine synthesis of PG. Administration of 17,8-estradiol antiserum29 or antiestrogen 30 is associated with a reduction in PG production. Progesterone, on the contrary, appears to be inhibitory in this regard, when given either alone or in combination with estrogen. 2,;· 31 In the human, the cyclic nature of changes in PG concentrations within the endometrium also suggests a similar role of ovarian steroids, although the nature and mechanism of regulation by these hormones are not well understood. 2 • 11 ' 32 In the present studies we have examined the regulation of endometrial PG synthesis by ovarian steroids in vitro by maintaining human proliferative and secretory endometria in organ culture in a chemically defmed medium containing 17,8-estradiol and/ or progesterone and assessing their PG biosynthetic capacity. With t:hese culture conditions we have also studied the influence of mefenamic acid, an anti-PG agent used clinically in the treatment of primary dysmenorrhea and menorrhagia, on the synthesis of endometrial PG in vitro. 0002-9378/82/060626+08$00.80/0© 1982 The C. V. Mosby Go.

Volume

1·42 :'\umber 6, Part l

Prostaglandin secretion by endometrium

627

Material and methods Endometrial and myometrial samples used in the prescnt studies were collected from the uteri of eight patients undergoing either hysterectomy for benign diseases or diagnostic curettage during either the late proliferative (davs ~~to J . l) or midsecretory (days IH to 2 I) pkhc of the menstrual cycle, as judged by the date of the last menstmal period. The status of the specimens was confirmed by histologic examination. The womcn (aged ::?:l to :l9 years) had regular menstrual ncles and an a\·erage cycle length of 29 days (range, 25 t< 1 :ll . \\ith about three or four pieces (150 to 250 f.tg ol protein) per dish, and cultured in 1.5 ml of the above medium with \·arious concentrations of 17{3est radio) ( 0 to I ,000 ng/ml) and/or progesterone (0 to I ,000 ng/ml) or mefenamic acid ( l f.tg/ml). Each experiment \\·as performed on a specimen obtained from one patient. The culture-; were maintained for either two 2"l-hour or three 2-hour periods at 36° C in 957c air: :J'.{ carbon dioxide. In experiments where PG secretion \\·as assessed over the three 2-hour periods, endometrial ti!'.sues were preincubated for at least 15 hours in the same medium prior to the start of each experiment to achieve constancy in the rate of PG secretiotJ. At the end of each culture period, medium was hanested and stored at -40° C f()r less than 24 hours pending extraction and subsequent determination of PGs bv radioimrnunoassays. \fedia collened from the cultures were extracted for PGs bv the method of Jaffe and associates." 3 To extract PGs from endometrial tissue at the end of the culture period, 1.0 ml of ab•;olute ethanol was added to each culture dish immediately following the final medium collection and allowed to stand overnight. Tissue resid ttes were then separated by centrifugation for subsequent protein analvsis. Duplicate aliquots of the ethyl acetate and ethanolic extracts were assayed f
Control

E2 (1 )JG

P4

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Fig. l. Prostaglandin secretion by human proliferative endo-

metrium (days 9 to 14) in vitro in the presence of 17{3estradiol (E 2 , I J.<.g/ml) and/or progesterone (P4 , I J.<.g/ml) during 48 hours of culture. Data represent results from three experiments. Each histogram is the mean ± SE of II or 12 cultures, maintained during the first(open mlumn) and second (shaded rolumn) 24-hour periods.

Table I. PGF secretion by human endometrial and myometrial tissues in vitro PGF secretion (pgiJLg protein; mean ± SE; First 24-hour culture

Endometrium

Second 24-hour culture

18.43

:!:

3.20

11.28 ± 5.10

0.48

:!:

0.09

0.22 ± 0.04

(n = 4)

Myometrium (n = 5)

antiserum used for PG F assay did not differentiate hetween the different PGs in the F se1·ies. Standard curves constructed for the PG assays covered the range 2.5 to 640 pg with 5 pg being the minimum amount that could he detected reliably. The values of PGF and PGE were expressed in picograms per microgram of cell protein. The intra-assay coefficients of variation for PGF and PGE were 2.47c and 3.6%, respective!\, and

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Fig. 3. Effect of various concentrations of E 2 and P4 on endometrial PGF secretion during 48 hours of culture. Data represent results from five experiments with endometrium from the midsecretory phase (days 18 to 24) of the menstrual cycle. Each point is the mean± SE of 16 to 18 cultures, maintained in the presence of 17,8-estradiol (o,e) or prog~sterone (o,•) during the first(--) and second(----) 24"hour periods.

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the recovery of the two classes of PG after extraction ranged between 75% and 88%. Protein was determined by the method of Lowry and associates. 35 Hormones added to the culture medium were 17{3estradiol and progesterone (Sigma Chemical Co., St. Louis, Missouri). Ham's FlO medium was obtained from Flow Laboratories (Mississauga, Ontario). L-Glutamine (No. 503L) and penicillin-streptomycin (No. 514L) were obtained from GIBCO Laboratories (Burlington, Ontario). Mefenamic acid was a gift from Dr. Andrew Darke, Parke-Davis Canada Inc. (Scarborough, Ontario). Data were analyzed by means of analysis of variance

and Duncan's New Multiple Range Test (36). Log-drithmic transformation of data was carried out prior to analysis where heterogeneity of variance was indicated.

Results When cultured in medium containing no exogenous steroid hormone, endometrium secreted 40 to 50 times more PGF than myometrium (Table I). This greater ability of the endometrium to secrete PGF could not have been due to tissue damage alone since endometrial and myometrial cultures were both prepared and carried out in a similar manner. Fig. 1 demonstrates the influence of 17{3-estradiol and progesterone, alone or in combination, on PGF and PGE secretion in vitro by endometria collected from three patients during the proliferative phase. 17{3-Estradiol, at a concentration of l J,tig/ml, had minimal effect (p > 0.05) on PGF secretion by the endometrium during the first 24 hours of culture but significantly (p < 0.005) doubled its secretory activity during

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Prostaglandin secretion by endometrium

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Table II. Endometrial PGF levels following 48-hour culture in the absence and the presence of li{3200

est radio!, progesterone, I i {3-estradiol and progesterone. or mefenamic acid Endometrial PGF ln!fls !pglf,Ig protein: mran ± SE:

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0.07 1.45 14.20 ± 1.90 1.60 :t 0.29 1.97 ± 0.38

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the -;econd culture period. On the contran. progesterone (I ~-tg/m]) markedly inhibited the basal (p < 0.001) and 17{3-estradiol-·stimulated (p < 0.001) PG 1- secretion, when present alone and with I 7{3-estradiol in the culture. respecti1·eh. !'he amount ol PGE ~ecreted ll\ the endometrium 11as only about one fifteenth that ol' PGF and was not sigTlificantlv altered bv 17{3-estradiol and/or progesterone. Hence, the secretion ratio •>f the two PC series (P(;F/PGEJ f(Jilowed a pattern ~imilar to that of PGF secretion and was significantlv increased (p < 1).0]) b) 17{3-estradiol and inhibited (p < 0.001) ln progester~ one (Fig. I). In contrast to proliferative endometrium, sc
Fig. 4. Steroidal interaction in the regulation of PGF secretion bl' human secretor) endometrium (dal''> IH to 2.J) during .JH hour~ of culture. Data represent results from five experiments. Each point is the mean cumulative PGF secretion of 16 to I~ cultures. The numbers in parenthese;, indicate the concentration of the steroid added to the medium in nanogTams per milliliter.

ng/ml (Figs.:) and .J). 17,B~Estradiol ( 1.000 ng/ml) and progoterone ( 1,000 ng/ml) in combination al~o had little effect on PGF secretion, but the presence of the two steroids at lower concentrations ( 17 {3~estradiol. 0.;) ng/ ml: progesterone, I 0 ng/ml) markedlv increased the amount of the PG secreted (Fig.+). While PGE secretion bv the -;ecretorv enclomet rium \\'as nnlv 'lightly decrea~.ed by 17{3-estradiol and pro~ gesterone, alone or in combination, the PG ratio was >ignificantly (p < 0.05) ele\atecl CW9( to I we;) h\ the~e ~teroids (Fig. 2). Data in Table I I and Fig. 5 depict the dt
630

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secretory phase (Table II and Fig. 5), while 17{3estradiol appeared to elicit a biphasic response similar to that of PGF secretion, tissue PGF levels were markedly lowered following the 48-hour culture in the presence of progesterone. The secretion of PG by both proliferative and secretory endometria was sensitive to the action of mefenamic acid, an inhibitor of the enzyme cyclooxygenase (Fig. 6). Although secretion of both PGF and PGE was decreased by mefenamic acid during the 48-hour culture, the degree of inhibition appeared to be more apparent with the secretion of PGF than with PGE, as also indicated by a marked drop in i:he PG ratio. This inhibition of PG F secretion was accompanied by a significant (p < 0.01) lowering of the endometrial PGF levels after the 48-hour culture period (Table II) and could be detected within the first 2 hours of incubation with the inhibitor (Table III).

In r.he present study, an organ culture system has been used to swdy the production of PG by human endometrium. We have demonstrated that the rate of PG synthesis is dependent upon the stage of the menstrual cycle during which the specimens are obtained and is higher with late proliferative endometrium than with the midsecretory counterpart. Although our findings are at variance with earlier studies measuring PG concentrations in excised endometrial tissue.~· 4 · 1 L :e they are in good agreement with the results of Liggins and associates 37 who demonstrated that the PG production rate of perfused human endomeu·ia from late proliferative and premenstrual phases were higher than those from secretory and early proliferative phases. The reasons for the differences in these results are unclear, but differences in experimental design in these studies may be responsible, particularly with regard to the degree of tissue injun and trauma. It is known that concentrations of PG in excised tissues do not accurately retiect PG concentrations in \ iYO and that high concentrations of PG found in excised tissue~ may be due to its rapid formation in response to surgical trauma and tissue injury.aH It is likely that tissue culture systems used in the present studies as well as by others' 17 · a~. 411 might have reduced tissue injury and, thus, the subsequent release of PG. This possibility is supported bv our observations that the rate of PG secretion by the endometrium cultured in the absence of exogenous hormone was relatively constant during the two 24-hour periods (Figs. l, 2, and 4). Moreover, the present study also demonstrates that the greater ability of endometrium (as compared to mrometrium) to secrete PGF could not be solely due to .. tissue damage .. since the myometrial cultures, prepared by procedures identical to those for the endometrium, were capable of secreting only one fiftieth the amount of PG synthesized by endometrium. Although PGE is known to be the predominant product synthesized from the action of prostaglandin synthetase, PGF2a has frequently been identified in significant amounts among the PGs within the reproductive tract. In addition to the substrate concentration of fatty acid 41 and the availability and concentration of reducing agents, the relative amount of the two series of PG present in a biological system depends largely on the balance in the activities between the reductase and isomerase for endoperoxide breakdown. 41 - 44 The results of this study indicate that human proliferative and secretory endometria produce more PGF than PGE in vitro. Moreover, while the secretion of PGE was not significantly affected by steroidal treatment in \'itro, PGF secretion was markedly altered bv the presence of

Volume 142 '>umber n. P"rt I

Prostaglandrn secretion by endometrium

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Fig. 6. Cumulative prostaglandin secretion bv human endometrium from late proliferative (davs 9 to It: o,e) and midsecretory (days 18 to 24: o.•) phases of the memtrual c1de during 48 hours of culture in the presence(·---) and absence(--) of mefenamic acid (I /Lg/ml). Data represent results from three (proliferative endometrium) or five (secretory endometrium) experiments. Each point is the mean ::': SE of II or 12 (proliferatin· endometrium) or 16 to 18 (secretory endometrium) cultures.

I 7f3-estradiol and/ or progesterone in the culture media. Whether the reduction of endoperoxides to PG F is indeed one of the enzymatic steps in the biosynthesis of PG that is responsive to steroidal regulation remains to be determined. Our results are, however. consistent with earlier findings that the secretion rate of PGF. but not of PGE. is under the influence of gonadal steroids."'' Since PGF and PGE are known to exert opposite effects on smooth muscle tone, 42 hormonal intluence on the production of these PGs and therebv the phvsiologic end result may best be examined bv determining the production ratio of these PGs (PGF/ PCE). This ratio mav be an important index to con-

sider 11·hen the pathophvsiolog~· of certain menstrual disorders is studied. In the present studv we have demonstrated that 17{3-estradiol stimulates PGF production bv both proliferative and secretorv endometrium. \Vhile an increase in PGF production bv secretor\' endometrium was evident \\·ithin the first 24 hours of culture. significant increase in the production of PGF !)\ proliferatiYe endometrium could not be detected until the second clay of culture. This observation is consistent with the suggestion that prior exposure to progesterone during the luteal phase of the cycle increases the responsi\·eness of endometrium to 17{3-estradiol.'·-·· ""· 4" The effect of '"progesterone priming" rna\ al-;o be

632

Tsang and Ooi \ m.

noted from the present study when incubation of secretory endometrium with phvsiologic concentrations of both I i ,8-estradiol and progesterone resulted in the highest rate of PGF production (Fig. 4). However, when supra physiologic concentrations ( l ~-tg/ml) of both I i,B-estradiol and progesterone were present in the medium. PGF production by secreton endometrium was not stimulated and proliferative endometrial PGF production was markedly inhibited. While the nature of the priming action of progesterone remains obscure, it is possible that this effect of progesterone may be a dose-dependent phenomenon. It has been suggested that in addition to its inhibitorY action on PG biosvnthesis, mefenamic acid may act by blocking receptor sites for PG at the level of the target tissue. 1" This latter possibilitv has been emphasized hecause it arcounts for the rapid onset of relief of dvsmenorrhea. Our present observation of PG synthesis inhibition in \"itro b\ mefenamic acid within 2 hours provides further support for this mode of action and may account for its rapid onset of action independent of PG receptor blockade. Since mefenamic acid is known to act by inhibiting c~'Cio-oxygenase, an enzyme common to the biosynthetic pathways of both PGF and PG£, 42 the degree of the inhibition by mefenamic acid of PGF and PGE production should, in theory. be comparable. The present studY has. however, demonstrated that while endometrial secretion of both PGF and PGE was inhibited bv

J.

\-larch I 5, 191l2 Obstet. Gyneml.

mefenamic acid, the inhibitory effect. appeared to IJe more apparent on PGF secretion. :\'eYertheless. it has also been suggested that PGE maY be formed noiwnzymatically from PGH 2 .'l 9 The possibility that the relative insensitivity of endometrial PGE. compared to PGF, secretion to the action of mefenamic acid Jtl
REFERENCES l. Chan, W. Y., and Hill,]. C.: Determination of menstrual

2. 3.

4. 5.

6.

7.

8.

prostaglandin levels in non-dysmenorrheic and dysmenorrheic subjects, Prostaglandins 15:365, 1978. Downie,]., Poyser, N. L., and Wunderlich, M.: Levels of prostaglandins in human endometrium during the normal menstrual cycle,]. Physiol. 236:465, 1974. Halbert, D. R., Demero, L.. Fontana, J., and Jones, D. E. D.: Prostaglandin levels in endometrial jet wash specimens in patients with dysmenorrhea before and after indomethacin therapy. Prostaglandins 10:1047, 1975. Levitt, T. M., Tobon, H., and Josimovich, J. B.: Prostaglandin content of human endometrium, Fertil. Steril. 26:296, 1975. Lundstrom, V., Green, K., and Wiqvist, N.: Prostaglandins, indomethacin and dysmenorrhea, Prostaglandins 11:893, 1976. Lundstrom, V., Green, K., and Svanborg, K.: Endogenous prostaglandins in dysmenorrhea and the effect of prostaglandin synthetase inhibitors (PGSI) on uterine contractivity, Acta Obstet. Gynecol. Scand. 87:51, 1979. Maathuis,J. B., and Kelly, R. W.: Concentrations of prostaglandins F2a and E 2 in the endometrium throughout the human menstrual cycle, after the administration of clomiphene or an oestrogen-progestogen pill and in early pregnancy, J. Endocrinol. 77:361, 1978. Pickles, V. R., Hall, W. J., Best, F. A., and Smith, G. N.: Prostaglandins in endometrium and menstrual fluid from

9.

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normal and dysmenorrheic subjects, J. Obstet. Gynaecol. Br. Commonw. 72:185, 1965. Pulkkinen, M. 0., Henzl, M. R., and Caspo. A. 1.: The effect of naproxen-sodium on the prostaglandin concentrations of the menstrual blood and uterine '1et-washings: in dysmenorrheic women," Prostagl:~ndins 15:543, 1978. Shangold, M. M., Aksel, S., Schomberg, D. W .. and Hammond, C. B.: Plasma prostaglandin F2a levels in dysmenorrheic women, Fertil. Steril. 27:1171. 1976. Singh, E. C., Baccarini, 1. M., and Zuspan, F. P.: Levels of prostaglandins F2a and E 2 in human endometrium during the menstrual cycle, AM. J. OilsTET. GYNECOL. 1.!1: 1003, 1975. Wilks,]. W., Wentz, A. C., and Jones, G.: Prostaglandin F2a concentrations in blood of women during normal menstrual cycle and dysmenorrhea, J. Clin. Endocrinol. Metab. 37:469, 1973. Willman, E. A., Collins, W. P., and Clayton, S. G.: Studies in the involvement of prostaglandins in uterine symptomatology and pathology. Br. J. Obstet. Gynaecol. 83: 337, 1976. Anderson, A. B. M., Guillebaud, ]., Haynes, P. ]., and Turnbull, A. C.: Reduction of menstrual blood~loss by prostaglandin-synthetase inhibitors, Lancet 1:774, 1976. Budoff, P. W.: Use of mefenamic acid in the treatment of primary dysrnenorrhea,JAMA -4>1:2713, 1979. Chan, W. Y., Dawood, M. Y., and Fuchs, F.: Relief of

\'olume 142 'umher t), Part

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21. 22.

~:\.

21.

25.

dvsmenorrhea with the prostaglandin synthetase inhibitor ibuprofen: effect on prostaglandin levels in menstrual fluid, AM. J. 0BSTET. GYNECOL. 135:102, 1979. Da\ies, A. J., Anderson, A. B. M., and Turnbull, A. C.: Reduction hv naproxen of excessive menstrual bleeding in women usmg intrauterine devices, Obstet. Gynecol. 57:7 l, 1981. Guillehaud. J.. -\nderson, A. B. ~f.. and Turnbull, A. C.: Reduction In mefenamic acid of increased menstrual blood loss associated with intrauterine contraception. Br. .J. Ohstet. (;ynaecol. 85:53, 1978. Jakuhowie7, D. L., and Wood, C.: The use of the prostaglandin synthetase inhibitor mefenamic acid in the treatment of menonhagia. Aust. !'\. Z. J. Obstet. Gvnaecol. 18:135, 1978. \!orrison, .J. C., and Jennings, J. C.: Primary dvsmenorrhea treated "·ith indomethacin, South. ~fed. J. 72:425, 1979. Pulkkinen, ~I. 0., and Kaihola, H. L.: Mefenamic acid in chsmenorrhea, Acta Obstet. Gynecol. Scand. 56:75, 1977. Pulkkinen, M. 0., and Caspo, A. 1.: The effect of ibuprof,·n 'm the intrauterine pressure and menstrual pain of dvsmenorrheic patients, Prostaglandins 15:1055, 1978. Barcikowski, B., Carlson, J. C., Wilson, L., and McCracken, .J. A.: The effect of endogenous and exogenous estradiol-! 7{3 on the release of prostaglandin F2 , from the mine uterus. Endocrinology 95:1340, 1974. Roberts, .J. S., Barcikowski, B., Wilson, L., Skarnes, R. C., and ~kCracken. J. :\.: Hormonal and related factors aflccting the release of prostaglandin F2a from the uterus, J. Steroid Biochem. 6:1091. 1975. Ca~tracane. Y. D .. and jordan. V. C.: The effect ofestrc)r g<'n and progesterone on uterine prostaglandin biosynthc'i' in the m·ariectomized rat. Bioi. Reprod. 13:587. 1<)i!'l.

2ti. Ham, E. A .. Cirillo. V.J.. Zanetti, M. E., and Kuehl. F. A.: btrogen-dirccted "'nthesis of specific prostaglandins in uterus. Proc. :\atl. Acad. Sci. CSA 72:1420, 1975. 27. Po\'ser. :\. L.: Honnonal control of prostag]andin syntlwsis in reproductive processes. Pol. J. Pharmacol. !'harm. 30:171, 197tl. 2~. Demers. L. M ., Yoshinaga, K.. and Greep, R. 0.: Prostaglandin Fin n10nkev uterine fluid during the menstrual ndc and fiJIIowing steroid treatment. Prostaglandins 5:"d:\. 197-1. 2q, Caldwell. B. V., Tillson, S. A., Brock, W. A., and Speroff. L: The effects of exogenous progesterone and estradiol on prostaglandin F levels in ovariectomised ewes, Prostaglandins 1:217, 1972. :·Hi. Fenwick, L, Jones, R. L.. :'>Jador, B., Povser, :\'. L .. and \\'ibon. :'>1. H.: Production of prostaglandins by the pseudopregnant rat uterus in l'itro and the effect of tamoxifen "·ith tltc identification of 6 keto prostaglandin F 1a as a m<~jor product, Br..J. Pharrnacol. 59:191, 19tl0. :\I. Bait d, D. T.: Local utero-marian relationships. in Cricht<>tt, D. B., and Lamming. G. F.. , editors: Control of 0HtLttion. London. 1977, Butterworths, p. 21 i.

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