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Induction of labor and cervical maturation using Mifepristone (RU 486) in the late pregnant rat. Influence of a cyclooxygenase inhibitor (Diclofenac)
PROSTAGLANDINS INDUCTION OF LABOR AND C E R V I C A L M A T U R A T I O N U81NG MTFEPRISTONE (RU 486) IN THE LATE PREGNANT RAT. INFLUENCE OF A CYCLOOXYGENASE INHIBITOR (DICLOFENAC)
D. Cabrol, B. Carbonne, A. Bienkiewicz, E. Dallot, A.E. AIJ, and L. Cedard 166 and Clinique Universitaire Baudelocque 123, Boulevard de Port-Royal; 75014 Paris, France
INSERMU
ABSTRACT The mechanism of action of RU 486 (Mifepristone), an antiprogesterone compound, on labor induction and on cervical maturation, is still not weil documented. We have investigated the effect of RU 486, alone and in association with a cyclooxygenase inhibitor (Diclofenac) on the induction of preterm delivery and on concomitant changes in the distribution of cervical glycosaminoglycans (GAGs) in pregnant Wistar rats: a control group (n = 18), a RU 486 treated group (n = 36), and a RU 486 and Diclofenac treated group (n = 15). The results of this study confirm the ability of this antiprogestemne treaknent to induce preterm delivery in the rat. This effect was antagonized by cyclooxygenase inhibition, suggesting that the action of RU 486 on labor induction could be mediated by prostaglandins. The absence of an increase in plasma prostaglandin E2 (PGE2) levels in RU 486 treated animals could be explained by Iocal uterine changes in prostaglandin concentrations. Mifepristone also induced sorne of the biochemical features of cervical maturation (i.e. increased hydration and hyaluronic acid concentration). This effect was not inhibited in Diclofenac treated anirnals suggesting that factors other than prostaglandins play a role in this phenomenon. INTRODUCTION Cervical maturation is one of the essential features of normal parturition. Previous studies have shown that changes in the distribution of glycosaminoglycans in the cervix uteri accompanying cervical maturation are influenced by eicosanoids and especially prostaglandin E2 (PGE2) (1,2). Steroids play an important part in labor initiation and exert an influence on prostanoid metabolism (3). Progestemne in some species such as the rat is required for the maintenance of pregnancy and the decrease in its level, concomitant with estrogen increase is believed to initiate parturition. It has been shown that progesterone decreases prostaglandin synthesis through the inhibition of phospholipase A2 (PLA2) (3 - 5). RU 486 (Mifepristone, RousseI-Uclaf), an antiprogesterone compound acting at the receptor level, induces preterm delivery in the rat (6, 7). However, the mechanism of this induction is still not weil documented. It is possible that, by removing PLA2 inhibition, Mifepristone might increase the concentration of free arachidonic acid and thus prostaglandin synthesis. In JULY 1991 VOL. 42 NO. 1
71
PROSTAGLANDINS order to invesUgate this hypothesis, we have studied the effect of RU 486, alone or in association with a cyclooxygenase inhibitor (Diclofenac, CibaGeigy) (8) on the induction of labor and on changes in the distribution of cervical glycosaminoglycans, which has been described as one of the main features characterising cervical maturation (9 - 11), in pregnant Wistar rats. Since it has been suggested that the inhibition of PG synthesis by Diclofenac couid divert arachidonic acid towards other metabolic pathways, i.e. 5, 12 and 15 lipoxygenase, epoxygenase or acyl transferase (12, 13), we have determined plasma PGE2 and leukotriene B4 (LTB4) levels in order to verify this hypothesis. M A T E R I A L AND M E T H O D S Material Three groups of pregnant Wistar rats (Janvier, Le Genest, France), submitted to 14 hrs of light and~10 hrs of darkness daily, were used in this series of experiments. Under these conditions, spontaneous delivery occurs from day 22 of gestation at 12:00 to day 23 at 12:00 (6). • G r o u p I (n = 18) served as a control group. Rats were injected subcutaneously with 0.5 ml normal saline solution per day at 8:00 a.m. on days 19, 20 and 21 of gestation. At 8:00 a.m. on the 21st day of gestation, this group received orally 0.7 ml of a 1% carboxymethylcellulose (CMTC: vehicle) solution. • G r o u p II (n = 36). Rots were injected subcutaneously with 0.5 ml normal saline solution per day at 8:00 a.m. on days 19, 20 and 21 of gestation. At 8:00 a.m. on the 21st day of gestation this group was treated with 10 mg RU 486 orally in 0.7 ml of a 1% CMTC suspension. • G r o u p III (n = 15). Rats were injected subcutaneously with 1.25 mg Diclofenac in 0.5 ml of normal saline solution on days 19, 20 and 21 of gestation, and 10 mg RU 486 orally in 0.7 ml of a 1% CMTC suspension at 8:00 a.m. on the 21st day of gestation. On day 22 at 8:00 a.m., 24 hours after CMTC or RU 486 administration, the number of rats which had expeled at least one fetus in each group was noted. The rats were killed and the blood was collected for determination of plasma PGE2 and LTB4 levels. The cervices were removed and washed in normal saline solution to remove blood and mucus, then weighed and immediately frozen and stored at -80°C prior to the determinaUon of cervical glycosaminoglycan concentrations. Methods • MQasuremQnt of cQrvical olyqosaminoalvcan~: The glycosaminoglycans were isolated by the technique previously described by Picard et al. (14). Following delipidation and dehydration with acetone, the dry weight of the sample was determined and the proteins were digested with 1% Pronase (Boehringer, Germany) at 56°C for 48 h. After heat-inactivation of the Pronase (100°C for 1 min) the samples were centrifuged and dialysed. Glycosaminoglycans were purified by precipitation
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PROSTAGLANDINS with 1% cetylpyridinium chloride (CPC) dissolved in 0.03 M NaCI. The glycosaminoglycan-CPC complexes were isolated by centrifugation, and then dissolved in a 0.1% CPC/1.25 M NaCI solution. They were then reprecipitated using ethanol and CPC alternately. After electrophoretic separation on a cellulose acetate strip, qualitative and quantitative analyses of glycosaminoglycans were performed, according to the method of Gardais et al. (15). Each glycosaminoglycan was characterized by its position of migration on the electrophoretic strip and also by its differential susceptibility to specific mucopolysaccharidases: hyaluronidase and chondroitinases AC and ABC (Sigma) or nitrous acid. • MQ~~urement of PGE2 levels: Plasma PGE2 levels were determined by radioimmunoassay. Antisera and [1251] PGE2 analogue tracers were obtained from NEN (Du Pont). For technical reasons, PGE2 levels were measured on only 5 rats of the control group, 11 rats in the RU 486 treated group and 7 rats in the RU 486 and Diclofenac treated group. • Me~surement of LTB4 levels: Plasma LTB4 concentration was measured on the same group of animals as described in the previous paragraph. Radioimmunoassay was performed after extraction of LTB4 in a solvant isopropanol/ether mixture (1:4). Antisera and PH] LTB4 were obtained from NEN (Du Pont). • St~ti~tical analvses: The RU 486 treated animals were distributed for analysis into two subgroups: rats which had expeled at the time of sacrifice and those which had not. The difference between data concerning the outcome of pregnancy in each group was assessed using X 2 test. Mean concentrations of glycosaminoglycans, mean dry weight/wet weight ratios and mean PGE2 and LTB4 levels in each group were compared using the Student's t test. The correlation between cervical water content and hyaluronic acid concentration was assessed by the coefficient of correlation r. RESULTS The efficiency of RU 486 alone and in association with Diclofenac on the indu¢tion of preterm delivery is shown in table 1. In the control group, no deliveries occurred before 8:00 a.m. on day 22 of gestation In the RU 486 treated group, 18 out of the 36 rats had expeled at least one fetus 24 hrs after RU 486 administration. However, one can assume that all animals in this group had uterine contractions, since it has been previously shown that electrical activity of the myometrium appears 20 hrs before the birth of the first fetus in the rat (16), and that, under the same conditions of RU 486 administration, all animals expel before 14:00 on day 22 (6). In the Di¢lofenac and RU 486 treated group, no expulsions were observed at the time of sacrifice.
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73
PROSTAGLANDINS T a b l e 1: outcome of pregnancy at 8:00 a.m. on day 22 of gestation
ùf ++f
NUMBER OF RATS WHICH EXPELED
N U M B E R OF RATS WHICH DID NOT EXPEL
0
18
(n = 36)
18
18
" RU 486 and D I C L O F E N A C TREATED G R O U P (n = 15)
0
15
C O N T R O L GROUP
(n = 18)
RU 486 TREATED GROUP
** : X2 test; p<0.001 Changes in cervical hydration and GAG distribution are shown in table 2. Increased cervical water content expressed as a decrease in the dry weight / wet weight ratio was observed in all treated groups. The fractional concentration of hyaluronic acid was also significantly increased when RU 486 was administered alone as weil as in the RU 486 and Diclofenac treated group (figure 1). However, hyaluronic acid concentration and cervical hydration were not statistically correlated (r = -0.856). GAG concentrations were expressed as mg/g of dry weight in order to eliminate errors of interpretation due to cervical hydration. T a b l e 2: glycosaminoglycan concentrations in the cervix (results are means _+S,D,) Control group (n = 18) Dry weight/wet weight ratio (%)
RU 486 and RU 486 treated group after expulslon without expulsion Diclofenac treated group(n= 16I (n - 18) (n = 18)
18.43 ± 1.46
15.43 + 1.48=,-,
16.46 ± 0.89 * * ,
15.55 ± 1.21 * * *
Total GAG (mg/g dry weight)
5.67+0.94
6.09 ±1.05
5.93+1.15
5.70+1.71
Hyaluronlc acid (mg/g dry weight)
1.35:1:0.40
1.73 ± 0.51 =
1.80 ± 0.44 1=
Heparan sulphate (mg/g dry weight)
1.50 ± 0.75
1.82 ± 0.91
1.30 + 0.81
(mg/g dry welght)
1.82 + 0.09
1.69 + 0.39
1.91 +0.25
1.90 ± 0.39
3hond roitin sulph. ! (mg/g dry welght)
1.00 + 0.22
0.86 + 0.20
0.92 ± 0.32
1.04 + 0.33
Dermatan sulphate
2.07 ± 1.08 , 0.69 + 0 . 3 6 ,
Student's t test: ,: p < 0.05; - * : p < 0.01; ,-,:. p < 0.001 w h e n c o m p a r e d to the control group.
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PROSTAGLANDINS A tendancy for dermatan sulphate concentration to decrease, was observed only in RU 486 treated animals which had expeled their fetuses. However the difference was not statistically significant. RU 486 and Diclofenac treated animals had Iow cervical concentrations of heparan sulphate (figure 2). Total glycosaminoglycans and chondroitin 4 and 6 sulphates remained unchanged in all treated groups. F i q u r e 1" Dry weight / wet weight ratio compared to hyaluronic acid concentrations (results are means + S.D.). Absence of correlation between these two parameters (r = - 0.856). ***I~[RU 486+ Diclofenac *** Im[ RU486 (Notexpeled)
~
tt
="* I m [ RU486 (Expeled) I I
[
CONTROL I
I
I
I
I
I
I
I
I
20 15 1 0.5 1 1.5 2 2.5 3 H y a l u r o n i c acid (mg/g) Dry w e i g h t / w e t w e i g h t (%) Student's t test: ~: p < 0.05; --: p < 0.01; -=*: p < 0.001 when compared to the control group. F i o u r e 2: heparan sulphate concentration (results are means + S.D.) mg/g dry weight
3
2
!j~ !j~ I~ ~°-'°up U
|
~
~
RU 486 treated group with expulslon RU 486 treated group without expulsion RU 486 and diclofenac treated group
0 * * * : p < 0.001 (Student'st test)
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75
PROSTAGLANDINS Cyclooxygenase inhibition using Diclofenac was responsible for a decrease in plasma PGE2 concentration. On the other hand, RU 486 administration did not increase PGE2 levels (table 3). There was no statistically significant difference in plasma LTB4 levels between treated groups and the control group (Table 3). T a b l e 3: plasma PGE2 and LTB4 levels (results are means _+S.D.) Control (n = 5)
RU 4 8 6 RU 4 8 6 without with expulsion expulsion
RU 486 + Diclofenac
(n-5)
(n=6)
(n =7) 0.47 + 0.33 (')
PG E2 (ng/ml)
1.25 _+ 0.35
1.12 + 0.42
0.95 + 0.50
LT B4 (ng/ml)
4.06 + 1.95
3.51 + 1.27
4.53 + 1.08
4.2 + 1.17
=: p < 0,05; Student's t test DISCUSSION The results of this study are consistent with previously pubUshed data reporting the efficiency of RU 486 in inducing preterm delivery in the rat (6, 7). Furthermore, we have observed that this effect is antagonized by Diclofenac (a cyclooxygenase inhibitor). This result suggests that the influence of RU 486 on labor induction could be mediated by prostaglandins. This hypothesis is compatible with clini¢al studies where an increased uterine sensitivity to prostaglandins after RU 486 treatment in the human and other primates has been demonstrated (17, 18, 19). In vitro studies have shown increased PGF2(x synthesis in short term cultures of human endometrial cells after incubation with RU 486 (20). However, in this series of experiments, no increase was observed in plasma PGE2 concentrations of RU 486 treated animals even after fetal expulsion. The absence of any modification in the plasma levels of PGE and PGF after RU 486 treatment has also been reported in the human (21). This absence of systemic changes in prostaglandin concentrations was not totally unexpected since prostanoids are generally considered to be Iocal factors (22). Further investigations are necessary to assess Iocal changes in uterine and cervical concentrations of prostaglandins under the same conditions. A n o t h e r possibility is that RU 486 administration in vivo could modify uterine sensitivity to prostaglandins through changes in prostaglandin receptor concentrations. This point requires further investigation. In addition to uterine contractility, the second important aspect of normal parturition is cervical maturation. In this study, cervical hydration and hyaluronic acid concentration were increased in all Mifepristone treated groups. These two phenomenons are observed in normal late pregnancy and are considered to be important features of cervical maturation. The fact that some features of cervical maturation were observed whereas animals did not expel (notably in RU 486 and Diclofenac treated group), is another
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JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS illustration of the dissociation of myometrial and cervical functions under experimental conditions. In the ewe, it has been shown that administration of Iow doses of PGE2 induces cervical softening without inducing myometrial contractions (23, 24). These results could suggest that cervical maturation requires Iower levels of prostaglandins than myometrial activity. Cervical maturation may also be mediated by other factors such as relaxin which facilitates cervical softening (25) as weil as inhibits the activity of the myometrium induced by prostaglandins in the rat (26). It has been previously suggested that increased cervical water content could be positively related to an increase in hyaluronic acid concentration in the cervix (9). The fact that, in this series of experiments, these two parameters were not statistically correlated, suggests that cervical hydration could also be related to other factors such as vasodilatation and increased vascular permeability, which could be, at least in part, controlled by phospholipid metabolites. Moreover, previous studies, in hysterectomized and ovariectomized rats where the vacularized uterine cervix is left intact, had shown that PGE2 is able to induce increased cervical hydration and hyaluronic acid concentrations (1). The same effects were paradoxically observed after the inhibition of prostaglandin synthesis using Diclofenac. It has been hypothesized that the inhibition of the cyclooxygenase pathway by the administration of Diclofenac may divert arachidonate substrate towards the production of other vasoactive metabolites (e.g. Platelet Activating Factor; 12, 15 Hydroperoxyeicosatetraenoic acids; 12, 15 Hydroxyeicosatetraenoic acids and leukotrienes) (12, 13). We partly addressed this point by assaying plasma LTB4 levels. No increase was observed in LTB4 concentrations in the plasma of animals treated by Diclofenac. However, LTB4 could act like other eicosanoids through a Iocal increse in concentration without an increase in the plasma concentration. On the other hand, other leukotrienes or other arachidonic acid metabolites could be involved in the observed connective tissue changes. The decrease in cervical dermatan sulphate concentrations, which has been described as a good indicator of cervical maturation (9, 11), was not observed in our experiments. We only noticed a slight and non significant decrease between control and RU 486 treated animals which had expeled their fetuses. However, the animals of the control group were killed only a few hours before the beginning of normal delivery and might therefore show some features of cervical maturation, since this phenomenon is believed to precede the onset of parturition. We would perhaps have observed more dramatic effects of RU 486 on the biochemical changes of the cervix compared to the control group, if RU 486 had been administered earlier in pregnancy or if the delay between RU 486 administration and sacrifice had been increased. This point is currently being investigated. Our results confirm previously published data showing the strong influence of prostaglandins upon heparan sulphate metabolism in the cervix. It has been shown that PGE2 administration in the rat increases cervical heparan sulphate concentration (1), and that the inhibition of prostaglandin synthesis decreases its concentration (13). In this series, Diclofenac again provoked a decrease in heparan sulphate concentration which was probably due to the inhibition of PGE2 induced increased synthesis.
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77
PROSTAGLANDINS T h e p h a r m a c o l o g i c a l a p p r o a c h in this study is p e r h a p s s o m e w h a t distant from physiological conditions. Nevertheless, t h e s e results s u g g e s t that the efficiency of an a n t i p r o g e s t e r o n e treab'nent (RU 486) for l a b o u r induction in the late p r e g n a n t rat is at least in part d u e to the influence of prostaglandins on the m y o m e t r i u m and cervix. T h e y also suggest that cervical maturation in late p r e g n a n c y is controlled not only by prostaglandins but also p r o b a b l y by other phospholipid metabolites (1, 13) and/or other factors. REFERENCES 1 - Cabrol D., Dubois P., Sedbon E., Dallot E., LegagneuxJ., Amlchot G., Cedard L, Sureau C.: Prostaglandin E2 - induced changas in the distribution of glycosamlnoglycans In the isolated rat uterine cervix. Eur J Obstet Gynecol Reprod BioL 2._.66:359, 1987. 2 - Uldbjerg N., Ekrnan G., Malrnstrom A., Sporrong B., Ulmsten U., Wingerup L: Biochemical and morphological changas of human cervix after Iocal application of Prostaglandin E2 in pregnancy. Lancet. 267, 1981. 3 - Abel M.H., Balrd D.T.: The effect of beta estradlol and progesterone on prostaglandin production by human endometrium malntalned in organ culture. Endocrinology. 106: 1599, 1980. 4 - Glmeno M.F., Franchi A.M., Gonzalez E.T., Gimeno A.L: Uptake and release of 3H prostaglandins E2 and F2 alpha in uterine strips isolated from ovariectomlzed rats. Influence of in vitro progasterone. Prostaglandins. 33: 51,1987. 5 - Peplow P.V., Hurst P.R.: Prostaglandin productlon by isolated cells of rat uterus incubated with or without progesterone. Prostaglandins Leukotdenas and EssentiaJ Fatty Aclds. 35: 57, 1989. 6 - Bosc M~I., Germain G., Nicolle A., Mouren M., Philibert D., Baulieu E.E.: Control of birth in rats by RU 486, an antlprogasterone compound. J Reprod Fert. 79: 1, 1987. 7 - Garfield R.E., Gase J.M., Baulieu E.E.: Effect of the antlprogesterone RU 486 on preterm birth in the rat. Am J Obstet Gynecol. 157: 1281, 1987. 8 - Brogden R.N., Heel R.C., Pakas G.E., Speight T.M., Avery G.S.: Diclofenac sodium: a review of its pharmacological propertias and therapeutlc use in rheumatic disasses and paln of varying origins. Drugs. 20: 24, 1980. 9 - Cabrol D., Huszar G., Romero R., Naftolin F.: Gestational changes in the rat uterlne cervlx: protein, collagen and glycosaminoglycan content. In: The cervix in pregnancy and labour. (Elwood D.A., Anderson A.B.M. eds.) Churchill Uvlngstone, Edinburgh, 1981. p.34. 10 - Cabrol D., Dallot E., Cedard L, Sureau C.: Pregnancy related changas In the dlstrlbution of glycosamlnoglycans in the cervlx and corpus of the human uterus. Eur J Obstet Gynecol Reprod Biol. 20: 289, 1985. 11 - Golichowski A.M.: Biochemlcal basis of cervlcal maturation. In: The physiology and blochemistry of the uterus in pregnancy and labor. (Huszar G., ed.) CRC press, Boca Raton,1986. p.261. 12 - Walker J.l_: The regulatory functlon of prostaglandin In the release of histamlns and SRS-A from passively sensltlzed human lung tissue. Adv Biosci. _9:.235, 1973.
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PROSTAGLANDINS 13 - Cabrol D., Dallot E., Bienkiewicz A., El AIJ A., Sedbon E., Cedard / : Cyclooxygenase and lipoxygenase inhibitors - induced changes in the distribution of glycosaminoglycans In the pregnant rat uterine cervlx. Prostaglandins. 39: 515. 1990. 14 - Picard J., Gardais A., Vedei M.: Glycoprotéines sulfates de rœuf de poule et de roviducte. Biochim. Biophys Acta. 320: 427, 1972. 15 - Gardais A., Picard J., Tarasse C.: Micro fractionnement et micro dosage des glycosaminoglycanes par électrophorèse sur bande d'acétate de cellulose gélatinisee. J Chromatog. 4_.22:396, 1969. 16 - Germain G., Legrand C., Maltier J. P.: Implication of adrenoreceptors In the control of electrical activity of the preparturient uterus in the rat. In: Uterine Contractility. (Bottari S, Thomas J. P., Vokaer A., Vokaer R. eds) Masson, Paris / New York, 1984, p. 207. 17 - Cameron I.T., Michie A.F., Baird D.T.: Therapeutic abortion in early pregnancy with antiprogestin RU 486 alone or in combination with prostaglandin analogue (Gemeprost). Contraception. 34: 489, 1986. 18 - Haiuska G.J., Stanczyk F.Z., Cook M.J. Novy M.J.: Temporal changes in uterine activity and prostaglandin response to RU 486 in rhesus macaques in late gestation. Am J Obstet Gynecol. 157: 1487, 1987. 19 - Swahn M.L., Ugocsai G., Bygdeman M., KovacsL., Belsey E.M., Van Look P.F.A.: Effect of oral prostaglandin E2 on uterine contractility and outcome of treatment in women receiving RU 486 (Mlfepristone) for termination of early pregnancy. Human Reprod. 4_: 21,1989. 20 - Kelly R.W., Healy D.L., Cameron M.J., Cameron I.T., Baird D.T.: RU 486 stimulation of PGF2 alpha productlon in Isolated endometrlal cells in short term culture. In: The antiprogestin steroid RU 486 and human fertility control. (Baulieu E.E. and Segal S.J., eds.) Plenum Press, New York,1985. p. 259. 21 - Hill N.C.W., Selinger M., Ferguson J., Lopez Bemal A., Mac Kenzie I.Z.: The physiologlcal and clinical effects of progesterone inhibition with RU 38,486 in the second trimester. 7th international Conference on prostaglandins and Related Compounds. Fiorence, 1990. Abstract Book, p. 160. 22 - Noort W.A., ed., Prostanoid excretion in term and preterm gastation. Offsetdrukkerij Haveka BV. Alblasserdam, 1989. p. 9. 23 - Stys S. J., Dresser B. I_, Otte T. E., Clark K. E.: Effect of prostaglandin E2 on cervical compliance in pregnant ewes. Am J Obstet Gynecol. 140: 415, 1981. 24 - Fitzpatrick R. J., Ugglns G. C.: Effects of prostaglandins on the cervix of pregnant women and sheep. In: Dilatation of the uterlne cervl~ (Naftolln F., Stubblefield P. G., eds), Raven Prsss, New York, 1980. p. 287. 25 - Hollingsworth M., Isherwood C. N. M., Foster R. W.: The effects of oestradiol benzoate, progesterone relaxin and ovariectomy on cervical extensibility in the late pregnant rat. J Reprod Fertil. 56: 471, 1979. 26 - Porter D. G., Downing S. J. D., Bradshaw J, M. C.: Inhibition of oxytocin or PGF2c¢driven myometdal activity by relaxin in the rat is oestrogen - dependent. J Endocrinol. 89: 399, 1981. Editor:
H.R.
Behrman
J U L Y 1991 VOL. 42 NO. 1
Received:
11-9-90
Accepted:
4-2-91
79
D. Cabrol, B. Carbonne, A. Bienkiewicz, E. Dallot, A.E. AIJ, and L. Cedard 166 and Clinique Universitaire Baudelocque 123, Boulevard de Port-Royal; 75014 Paris, France
INSERMU
ABSTRACT The mechanism of action of RU 486 (Mifepristone), an antiprogesterone compound, on labor induction and on cervical maturation, is still not weil documented. We have investigated the effect of RU 486, alone and in association with a cyclooxygenase inhibitor (Diclofenac) on the induction of preterm delivery and on concomitant changes in the distribution of cervical glycosaminoglycans (GAGs) in pregnant Wistar rats: a control group (n = 18), a RU 486 treated group (n = 36), and a RU 486 and Diclofenac treated group (n = 15). The results of this study confirm the ability of this antiprogestemne treaknent to induce preterm delivery in the rat. This effect was antagonized by cyclooxygenase inhibition, suggesting that the action of RU 486 on labor induction could be mediated by prostaglandins. The absence of an increase in plasma prostaglandin E2 (PGE2) levels in RU 486 treated animals could be explained by Iocal uterine changes in prostaglandin concentrations. Mifepristone also induced sorne of the biochemical features of cervical maturation (i.e. increased hydration and hyaluronic acid concentration). This effect was not inhibited in Diclofenac treated anirnals suggesting that factors other than prostaglandins play a role in this phenomenon. INTRODUCTION Cervical maturation is one of the essential features of normal parturition. Previous studies have shown that changes in the distribution of glycosaminoglycans in the cervix uteri accompanying cervical maturation are influenced by eicosanoids and especially prostaglandin E2 (PGE2) (1,2). Steroids play an important part in labor initiation and exert an influence on prostanoid metabolism (3). Progestemne in some species such as the rat is required for the maintenance of pregnancy and the decrease in its level, concomitant with estrogen increase is believed to initiate parturition. It has been shown that progesterone decreases prostaglandin synthesis through the inhibition of phospholipase A2 (PLA2) (3 - 5). RU 486 (Mifepristone, RousseI-Uclaf), an antiprogesterone compound acting at the receptor level, induces preterm delivery in the rat (6, 7). However, the mechanism of this induction is still not weil documented. It is possible that, by removing PLA2 inhibition, Mifepristone might increase the concentration of free arachidonic acid and thus prostaglandin synthesis. In JULY 1991 VOL. 42 NO. 1
71
PROSTAGLANDINS order to invesUgate this hypothesis, we have studied the effect of RU 486, alone or in association with a cyclooxygenase inhibitor (Diclofenac, CibaGeigy) (8) on the induction of labor and on changes in the distribution of cervical glycosaminoglycans, which has been described as one of the main features characterising cervical maturation (9 - 11), in pregnant Wistar rats. Since it has been suggested that the inhibition of PG synthesis by Diclofenac couid divert arachidonic acid towards other metabolic pathways, i.e. 5, 12 and 15 lipoxygenase, epoxygenase or acyl transferase (12, 13), we have determined plasma PGE2 and leukotriene B4 (LTB4) levels in order to verify this hypothesis. M A T E R I A L AND M E T H O D S Material Three groups of pregnant Wistar rats (Janvier, Le Genest, France), submitted to 14 hrs of light and~10 hrs of darkness daily, were used in this series of experiments. Under these conditions, spontaneous delivery occurs from day 22 of gestation at 12:00 to day 23 at 12:00 (6). • G r o u p I (n = 18) served as a control group. Rats were injected subcutaneously with 0.5 ml normal saline solution per day at 8:00 a.m. on days 19, 20 and 21 of gestation. At 8:00 a.m. on the 21st day of gestation, this group received orally 0.7 ml of a 1% carboxymethylcellulose (CMTC: vehicle) solution. • G r o u p II (n = 36). Rots were injected subcutaneously with 0.5 ml normal saline solution per day at 8:00 a.m. on days 19, 20 and 21 of gestation. At 8:00 a.m. on the 21st day of gestation this group was treated with 10 mg RU 486 orally in 0.7 ml of a 1% CMTC suspension. • G r o u p III (n = 15). Rats were injected subcutaneously with 1.25 mg Diclofenac in 0.5 ml of normal saline solution on days 19, 20 and 21 of gestation, and 10 mg RU 486 orally in 0.7 ml of a 1% CMTC suspension at 8:00 a.m. on the 21st day of gestation. On day 22 at 8:00 a.m., 24 hours after CMTC or RU 486 administration, the number of rats which had expeled at least one fetus in each group was noted. The rats were killed and the blood was collected for determination of plasma PGE2 and LTB4 levels. The cervices were removed and washed in normal saline solution to remove blood and mucus, then weighed and immediately frozen and stored at -80°C prior to the determinaUon of cervical glycosaminoglycan concentrations. Methods • MQasuremQnt of cQrvical olyqosaminoalvcan~: The glycosaminoglycans were isolated by the technique previously described by Picard et al. (14). Following delipidation and dehydration with acetone, the dry weight of the sample was determined and the proteins were digested with 1% Pronase (Boehringer, Germany) at 56°C for 48 h. After heat-inactivation of the Pronase (100°C for 1 min) the samples were centrifuged and dialysed. Glycosaminoglycans were purified by precipitation
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JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS with 1% cetylpyridinium chloride (CPC) dissolved in 0.03 M NaCI. The glycosaminoglycan-CPC complexes were isolated by centrifugation, and then dissolved in a 0.1% CPC/1.25 M NaCI solution. They were then reprecipitated using ethanol and CPC alternately. After electrophoretic separation on a cellulose acetate strip, qualitative and quantitative analyses of glycosaminoglycans were performed, according to the method of Gardais et al. (15). Each glycosaminoglycan was characterized by its position of migration on the electrophoretic strip and also by its differential susceptibility to specific mucopolysaccharidases: hyaluronidase and chondroitinases AC and ABC (Sigma) or nitrous acid. • MQ~~urement of PGE2 levels: Plasma PGE2 levels were determined by radioimmunoassay. Antisera and [1251] PGE2 analogue tracers were obtained from NEN (Du Pont). For technical reasons, PGE2 levels were measured on only 5 rats of the control group, 11 rats in the RU 486 treated group and 7 rats in the RU 486 and Diclofenac treated group. • Me~surement of LTB4 levels: Plasma LTB4 concentration was measured on the same group of animals as described in the previous paragraph. Radioimmunoassay was performed after extraction of LTB4 in a solvant isopropanol/ether mixture (1:4). Antisera and PH] LTB4 were obtained from NEN (Du Pont). • St~ti~tical analvses: The RU 486 treated animals were distributed for analysis into two subgroups: rats which had expeled at the time of sacrifice and those which had not. The difference between data concerning the outcome of pregnancy in each group was assessed using X 2 test. Mean concentrations of glycosaminoglycans, mean dry weight/wet weight ratios and mean PGE2 and LTB4 levels in each group were compared using the Student's t test. The correlation between cervical water content and hyaluronic acid concentration was assessed by the coefficient of correlation r. RESULTS The efficiency of RU 486 alone and in association with Diclofenac on the indu¢tion of preterm delivery is shown in table 1. In the control group, no deliveries occurred before 8:00 a.m. on day 22 of gestation In the RU 486 treated group, 18 out of the 36 rats had expeled at least one fetus 24 hrs after RU 486 administration. However, one can assume that all animals in this group had uterine contractions, since it has been previously shown that electrical activity of the myometrium appears 20 hrs before the birth of the first fetus in the rat (16), and that, under the same conditions of RU 486 administration, all animals expel before 14:00 on day 22 (6). In the Di¢lofenac and RU 486 treated group, no expulsions were observed at the time of sacrifice.
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73
PROSTAGLANDINS T a b l e 1: outcome of pregnancy at 8:00 a.m. on day 22 of gestation
ùf ++f
NUMBER OF RATS WHICH EXPELED
N U M B E R OF RATS WHICH DID NOT EXPEL
0
18
(n = 36)
18
18
" RU 486 and D I C L O F E N A C TREATED G R O U P (n = 15)
0
15
C O N T R O L GROUP
(n = 18)
RU 486 TREATED GROUP
** : X2 test; p<0.001 Changes in cervical hydration and GAG distribution are shown in table 2. Increased cervical water content expressed as a decrease in the dry weight / wet weight ratio was observed in all treated groups. The fractional concentration of hyaluronic acid was also significantly increased when RU 486 was administered alone as weil as in the RU 486 and Diclofenac treated group (figure 1). However, hyaluronic acid concentration and cervical hydration were not statistically correlated (r = -0.856). GAG concentrations were expressed as mg/g of dry weight in order to eliminate errors of interpretation due to cervical hydration. T a b l e 2: glycosaminoglycan concentrations in the cervix (results are means _+S,D,) Control group (n = 18) Dry weight/wet weight ratio (%)
RU 486 and RU 486 treated group after expulslon without expulsion Diclofenac treated group(n= 16I (n - 18) (n = 18)
18.43 ± 1.46
15.43 + 1.48=,-,
16.46 ± 0.89 * * ,
15.55 ± 1.21 * * *
Total GAG (mg/g dry weight)
5.67+0.94
6.09 ±1.05
5.93+1.15
5.70+1.71
Hyaluronlc acid (mg/g dry weight)
1.35:1:0.40
1.73 ± 0.51 =
1.80 ± 0.44 1=
Heparan sulphate (mg/g dry weight)
1.50 ± 0.75
1.82 ± 0.91
1.30 + 0.81
(mg/g dry welght)
1.82 + 0.09
1.69 + 0.39
1.91 +0.25
1.90 ± 0.39
3hond roitin sulph. ! (mg/g dry welght)
1.00 + 0.22
0.86 + 0.20
0.92 ± 0.32
1.04 + 0.33
Dermatan sulphate
2.07 ± 1.08 , 0.69 + 0 . 3 6 ,
Student's t test: ,: p < 0.05; - * : p < 0.01; ,-,:. p < 0.001 w h e n c o m p a r e d to the control group.
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JULY 1991 VOL. 42 NO. 1
PROSTAGLANDINS A tendancy for dermatan sulphate concentration to decrease, was observed only in RU 486 treated animals which had expeled their fetuses. However the difference was not statistically significant. RU 486 and Diclofenac treated animals had Iow cervical concentrations of heparan sulphate (figure 2). Total glycosaminoglycans and chondroitin 4 and 6 sulphates remained unchanged in all treated groups. F i q u r e 1" Dry weight / wet weight ratio compared to hyaluronic acid concentrations (results are means + S.D.). Absence of correlation between these two parameters (r = - 0.856). ***I~[RU 486+ Diclofenac *** Im[ RU486 (Notexpeled)
~
tt
="* I m [ RU486 (Expeled) I I
[
CONTROL I
I
I
I
I
I
I
I
I
20 15 1 0.5 1 1.5 2 2.5 3 H y a l u r o n i c acid (mg/g) Dry w e i g h t / w e t w e i g h t (%) Student's t test: ~: p < 0.05; --: p < 0.01; -=*: p < 0.001 when compared to the control group. F i o u r e 2: heparan sulphate concentration (results are means + S.D.) mg/g dry weight
3
2
!j~ !j~ I~ ~°-'°up U
|
~
~
RU 486 treated group with expulslon RU 486 treated group without expulsion RU 486 and diclofenac treated group
0 * * * : p < 0.001 (Student'st test)
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75
PROSTAGLANDINS Cyclooxygenase inhibition using Diclofenac was responsible for a decrease in plasma PGE2 concentration. On the other hand, RU 486 administration did not increase PGE2 levels (table 3). There was no statistically significant difference in plasma LTB4 levels between treated groups and the control group (Table 3). T a b l e 3: plasma PGE2 and LTB4 levels (results are means _+S.D.) Control (n = 5)
RU 4 8 6 RU 4 8 6 without with expulsion expulsion
RU 486 + Diclofenac
(n-5)
(n=6)
(n =7) 0.47 + 0.33 (')
PG E2 (ng/ml)
1.25 _+ 0.35
1.12 + 0.42
0.95 + 0.50
LT B4 (ng/ml)
4.06 + 1.95
3.51 + 1.27
4.53 + 1.08
4.2 + 1.17
=: p < 0,05; Student's t test DISCUSSION The results of this study are consistent with previously pubUshed data reporting the efficiency of RU 486 in inducing preterm delivery in the rat (6, 7). Furthermore, we have observed that this effect is antagonized by Diclofenac (a cyclooxygenase inhibitor). This result suggests that the influence of RU 486 on labor induction could be mediated by prostaglandins. This hypothesis is compatible with clini¢al studies where an increased uterine sensitivity to prostaglandins after RU 486 treatment in the human and other primates has been demonstrated (17, 18, 19). In vitro studies have shown increased PGF2(x synthesis in short term cultures of human endometrial cells after incubation with RU 486 (20). However, in this series of experiments, no increase was observed in plasma PGE2 concentrations of RU 486 treated animals even after fetal expulsion. The absence of any modification in the plasma levels of PGE and PGF after RU 486 treatment has also been reported in the human (21). This absence of systemic changes in prostaglandin concentrations was not totally unexpected since prostanoids are generally considered to be Iocal factors (22). Further investigations are necessary to assess Iocal changes in uterine and cervical concentrations of prostaglandins under the same conditions. A n o t h e r possibility is that RU 486 administration in vivo could modify uterine sensitivity to prostaglandins through changes in prostaglandin receptor concentrations. This point requires further investigation. In addition to uterine contractility, the second important aspect of normal parturition is cervical maturation. In this study, cervical hydration and hyaluronic acid concentration were increased in all Mifepristone treated groups. These two phenomenons are observed in normal late pregnancy and are considered to be important features of cervical maturation. The fact that some features of cervical maturation were observed whereas animals did not expel (notably in RU 486 and Diclofenac treated group), is another
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PROSTAGLANDINS illustration of the dissociation of myometrial and cervical functions under experimental conditions. In the ewe, it has been shown that administration of Iow doses of PGE2 induces cervical softening without inducing myometrial contractions (23, 24). These results could suggest that cervical maturation requires Iower levels of prostaglandins than myometrial activity. Cervical maturation may also be mediated by other factors such as relaxin which facilitates cervical softening (25) as weil as inhibits the activity of the myometrium induced by prostaglandins in the rat (26). It has been previously suggested that increased cervical water content could be positively related to an increase in hyaluronic acid concentration in the cervix (9). The fact that, in this series of experiments, these two parameters were not statistically correlated, suggests that cervical hydration could also be related to other factors such as vasodilatation and increased vascular permeability, which could be, at least in part, controlled by phospholipid metabolites. Moreover, previous studies, in hysterectomized and ovariectomized rats where the vacularized uterine cervix is left intact, had shown that PGE2 is able to induce increased cervical hydration and hyaluronic acid concentrations (1). The same effects were paradoxically observed after the inhibition of prostaglandin synthesis using Diclofenac. It has been hypothesized that the inhibition of the cyclooxygenase pathway by the administration of Diclofenac may divert arachidonate substrate towards the production of other vasoactive metabolites (e.g. Platelet Activating Factor; 12, 15 Hydroperoxyeicosatetraenoic acids; 12, 15 Hydroxyeicosatetraenoic acids and leukotrienes) (12, 13). We partly addressed this point by assaying plasma LTB4 levels. No increase was observed in LTB4 concentrations in the plasma of animals treated by Diclofenac. However, LTB4 could act like other eicosanoids through a Iocal increse in concentration without an increase in the plasma concentration. On the other hand, other leukotrienes or other arachidonic acid metabolites could be involved in the observed connective tissue changes. The decrease in cervical dermatan sulphate concentrations, which has been described as a good indicator of cervical maturation (9, 11), was not observed in our experiments. We only noticed a slight and non significant decrease between control and RU 486 treated animals which had expeled their fetuses. However, the animals of the control group were killed only a few hours before the beginning of normal delivery and might therefore show some features of cervical maturation, since this phenomenon is believed to precede the onset of parturition. We would perhaps have observed more dramatic effects of RU 486 on the biochemical changes of the cervix compared to the control group, if RU 486 had been administered earlier in pregnancy or if the delay between RU 486 administration and sacrifice had been increased. This point is currently being investigated. Our results confirm previously published data showing the strong influence of prostaglandins upon heparan sulphate metabolism in the cervix. It has been shown that PGE2 administration in the rat increases cervical heparan sulphate concentration (1), and that the inhibition of prostaglandin synthesis decreases its concentration (13). In this series, Diclofenac again provoked a decrease in heparan sulphate concentration which was probably due to the inhibition of PGE2 induced increased synthesis.
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77
PROSTAGLANDINS T h e p h a r m a c o l o g i c a l a p p r o a c h in this study is p e r h a p s s o m e w h a t distant from physiological conditions. Nevertheless, t h e s e results s u g g e s t that the efficiency of an a n t i p r o g e s t e r o n e treab'nent (RU 486) for l a b o u r induction in the late p r e g n a n t rat is at least in part d u e to the influence of prostaglandins on the m y o m e t r i u m and cervix. T h e y also suggest that cervical maturation in late p r e g n a n c y is controlled not only by prostaglandins but also p r o b a b l y by other phospholipid metabolites (1, 13) and/or other factors. REFERENCES 1 - Cabrol D., Dubois P., Sedbon E., Dallot E., LegagneuxJ., Amlchot G., Cedard L, Sureau C.: Prostaglandin E2 - induced changas in the distribution of glycosamlnoglycans In the isolated rat uterine cervix. Eur J Obstet Gynecol Reprod BioL 2._.66:359, 1987. 2 - Uldbjerg N., Ekrnan G., Malrnstrom A., Sporrong B., Ulmsten U., Wingerup L: Biochemical and morphological changas of human cervix after Iocal application of Prostaglandin E2 in pregnancy. Lancet. 267, 1981. 3 - Abel M.H., Balrd D.T.: The effect of beta estradlol and progesterone on prostaglandin production by human endometrium malntalned in organ culture. Endocrinology. 106: 1599, 1980. 4 - Glmeno M.F., Franchi A.M., Gonzalez E.T., Gimeno A.L: Uptake and release of 3H prostaglandins E2 and F2 alpha in uterine strips isolated from ovariectomlzed rats. Influence of in vitro progasterone. Prostaglandins. 33: 51,1987. 5 - Peplow P.V., Hurst P.R.: Prostaglandin productlon by isolated cells of rat uterus incubated with or without progesterone. Prostaglandins Leukotdenas and EssentiaJ Fatty Aclds. 35: 57, 1989. 6 - Bosc M~I., Germain G., Nicolle A., Mouren M., Philibert D., Baulieu E.E.: Control of birth in rats by RU 486, an antlprogasterone compound. J Reprod Fert. 79: 1, 1987. 7 - Garfield R.E., Gase J.M., Baulieu E.E.: Effect of the antlprogesterone RU 486 on preterm birth in the rat. Am J Obstet Gynecol. 157: 1281, 1987. 8 - Brogden R.N., Heel R.C., Pakas G.E., Speight T.M., Avery G.S.: Diclofenac sodium: a review of its pharmacological propertias and therapeutlc use in rheumatic disasses and paln of varying origins. Drugs. 20: 24, 1980. 9 - Cabrol D., Huszar G., Romero R., Naftolin F.: Gestational changes in the rat uterlne cervlx: protein, collagen and glycosaminoglycan content. In: The cervix in pregnancy and labour. (Elwood D.A., Anderson A.B.M. eds.) Churchill Uvlngstone, Edinburgh, 1981. p.34. 10 - Cabrol D., Dallot E., Cedard L, Sureau C.: Pregnancy related changas In the dlstrlbution of glycosamlnoglycans in the cervlx and corpus of the human uterus. Eur J Obstet Gynecol Reprod Biol. 20: 289, 1985. 11 - Golichowski A.M.: Biochemlcal basis of cervlcal maturation. In: The physiology and blochemistry of the uterus in pregnancy and labor. (Huszar G., ed.) CRC press, Boca Raton,1986. p.261. 12 - Walker J.l_: The regulatory functlon of prostaglandin In the release of histamlns and SRS-A from passively sensltlzed human lung tissue. Adv Biosci. _9:.235, 1973.
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PROSTAGLANDINS 13 - Cabrol D., Dallot E., Bienkiewicz A., El AIJ A., Sedbon E., Cedard / : Cyclooxygenase and lipoxygenase inhibitors - induced changes in the distribution of glycosaminoglycans In the pregnant rat uterine cervlx. Prostaglandins. 39: 515. 1990. 14 - Picard J., Gardais A., Vedei M.: Glycoprotéines sulfates de rœuf de poule et de roviducte. Biochim. Biophys Acta. 320: 427, 1972. 15 - Gardais A., Picard J., Tarasse C.: Micro fractionnement et micro dosage des glycosaminoglycanes par électrophorèse sur bande d'acétate de cellulose gélatinisee. J Chromatog. 4_.22:396, 1969. 16 - Germain G., Legrand C., Maltier J. P.: Implication of adrenoreceptors In the control of electrical activity of the preparturient uterus in the rat. In: Uterine Contractility. (Bottari S, Thomas J. P., Vokaer A., Vokaer R. eds) Masson, Paris / New York, 1984, p. 207. 17 - Cameron I.T., Michie A.F., Baird D.T.: Therapeutic abortion in early pregnancy with antiprogestin RU 486 alone or in combination with prostaglandin analogue (Gemeprost). Contraception. 34: 489, 1986. 18 - Haiuska G.J., Stanczyk F.Z., Cook M.J. Novy M.J.: Temporal changes in uterine activity and prostaglandin response to RU 486 in rhesus macaques in late gestation. Am J Obstet Gynecol. 157: 1487, 1987. 19 - Swahn M.L., Ugocsai G., Bygdeman M., KovacsL., Belsey E.M., Van Look P.F.A.: Effect of oral prostaglandin E2 on uterine contractility and outcome of treatment in women receiving RU 486 (Mlfepristone) for termination of early pregnancy. Human Reprod. 4_: 21,1989. 20 - Kelly R.W., Healy D.L., Cameron M.J., Cameron I.T., Baird D.T.: RU 486 stimulation of PGF2 alpha productlon in Isolated endometrlal cells in short term culture. In: The antiprogestin steroid RU 486 and human fertility control. (Baulieu E.E. and Segal S.J., eds.) Plenum Press, New York,1985. p. 259. 21 - Hill N.C.W., Selinger M., Ferguson J., Lopez Bemal A., Mac Kenzie I.Z.: The physiologlcal and clinical effects of progesterone inhibition with RU 38,486 in the second trimester. 7th international Conference on prostaglandins and Related Compounds. Fiorence, 1990. Abstract Book, p. 160. 22 - Noort W.A., ed., Prostanoid excretion in term and preterm gastation. Offsetdrukkerij Haveka BV. Alblasserdam, 1989. p. 9. 23 - Stys S. J., Dresser B. I_, Otte T. E., Clark K. E.: Effect of prostaglandin E2 on cervical compliance in pregnant ewes. Am J Obstet Gynecol. 140: 415, 1981. 24 - Fitzpatrick R. J., Ugglns G. C.: Effects of prostaglandins on the cervix of pregnant women and sheep. In: Dilatation of the uterlne cervl~ (Naftolln F., Stubblefield P. G., eds), Raven Prsss, New York, 1980. p. 287. 25 - Hollingsworth M., Isherwood C. N. M., Foster R. W.: The effects of oestradiol benzoate, progesterone relaxin and ovariectomy on cervical extensibility in the late pregnant rat. J Reprod Fertil. 56: 471, 1979. 26 - Porter D. G., Downing S. J. D., Bradshaw J, M. C.: Inhibition of oxytocin or PGF2c¢driven myometdal activity by relaxin in the rat is oestrogen - dependent. J Endocrinol. 89: 399, 1981. Editor:
H.R.
Behrman
J U L Y 1991 VOL. 42 NO. 1
Received:
11-9-90
Accepted:
4-2-91
79