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Influence of indomethacin delivered from an intrauterine silastic device on basal levels and synthesizing capability of rat uterus for prostacyclin
Prostaglandins Leukotrienes and Medicine 14: 191-197, 1984
INFLUENCE OF INDOMETHACIN DELIVERED FROM AN INTRAUTERINE SILASTIC DEVICE ON BASAL LEVELS AND SYNTHESIZING CAPABILITY OF RAT UTERUS FOR PROSTACYCLIN. P.V. Peplow, P.R. Hurst. Department of Anatomy, University of Otago, Dunedin, New Zealand. (reprint requests to PVP) ABSTRACT The intrauterine release of indomethacin (approximately 12Wg over 23 days) from a unilateral silastic device did not cause any significant reduction in basal levels of PGI (determined as 6-keto PGFlc) in the tissue of the IUD horn. However, ti+ is tissue was found to have an increased potential for PG12 synthesis when compared to that of the uterine horn with a plain device. The implications of these findings regarding a possible effect of indomethacin on the myometrium are discussed. INTRODUCTION The delivery of low levels of nonsteroidal anti-inflammatory compounds such as indomethacin directly to the female reproductive tract from an intrauterine device (IUD) may provide a way of increasing the efficiency of the IUD, since indomethacin has been shown to interfere with implantation by preventing decidualization of the uterus (l-3) and also by possibly affecting the blastocyst (4-6). Moreover, the local release of indomethacin from the IUD or a vaginal suppository may overcome problems of uterine dysfunction (such as abnormal bleeding and pain) frequently found in women with intrauterine devices (7). Delivery of small quantities of the drug may not result in those side-effects (e.g. gastrointestinal disturbances) experienced by women taking larger doses of the drug orally. In a previous study designed to provide basic information relating to such a medicated delivery system in the rat, we described the effect of indomethacin released from a silastic intrauterine device on the basal levels and synthesizing capacity of the uterus for PGF and PGE (8). Both of these substances are produced mainly by the enaa ometrium (9,lO). Now we wish to report on the basal levels of prostacyclin, measured as its major stable metabolite 6-keto PGF which is considered to be produced principally by the myometrium1?;1,12). This should allow an evaluation of whether indomethacin from an intraluminal depot is reaching the myometrium in sufficient quantity to inhibit prostaglandin synthesis and thereby alter such levels. The production capability of the uterus has also been assessed by an in vitro technique using tissue homogenates with the amounts of prostaglandin present in the culture medium at the 191
end of a 2 hr incubation period being adjusted for the basal levels in the tissue. The work described will be relevant to further studies directed towards elucidating the roles of certain pGs in reproductive physiology through the local administration of specific inhibitors of PG synthesis to the uterus. METHODS Twenty-two adult female Wistar rats (approximately 250g body weight and matched for age) having regular estrous cycles were used. They were housed under a 14:lO hr regimen of light and darkness and given free access to food and water. Eleven of these animals were fitted with a unilateral silastic rod not incorporating any indomethacin (referred to as 'plain' rod), while the other eleven were fitted with silastic rods containing 3% w/w indomethacin. The preparation and sterilization of the rods (l.Ocm length, 0.09cm i-d.), covered by a vinyl sleeve (0.17cm o.d.), were performed as previously described (13). The rods were inserted on the first or second day after estrus by anaesthetizing the animals with sodium pentobarbitone and making a small incision in one horn, at about half-way along its length, to allow the device to be placed into the Lumen of the horn. The device was secured to the uterine wall and the incision closed by a short nylon suture. After the operation the animals were rested for 1 week and vaginal smears were taken daily. At around 21 days after fitting the devices, the animals were killed at 10.00 a.m. on the day of estrus, and the uterine horns removed. In this and subsequent manipulation, the horns were held only very gently with forceps to minimize any trauma-induced stimulation of PG synthesis that would raise Each horn was divided into two parts; for the horn with basal levels. the device, the rod was associated with either the caudal or cranial part. The parts were trimmed of fat and mesometrium, and cleaned on filter paper. Each part was slit longitudinally to allow the escape of luminal fluid and to permit the removal of the device. The luminal surface of the uterus was also blotted on filter paper. The separate parts of the uterus were immediately weighed and homogenized in 5ml Eagle's culture medium pH 7.4, or 5ml ethanol (for measurement af PG basal levels) using a glass homogenizer with teflon plunger held in a high-speed electric drill (10 The homogenizer was washed with lml culture medium strokes over 1 min). or ethanol. The culture medium homogenates with washings were transferred This method is based on to stoppered glass tubes and incubated at 37OC. that described by Poyser and Scott (14) with PG production being terminated after 2 hr by the addition of 0.2N HCl (to bring samples to pH 4.51, and PGs extracted with ethyl acetate (3 x 3ml) at room temperature. The extracts were evaporated in vacua at a temperature not exceeding 450C and For the the residues taken up in 9ml O.OlM Tris-gelatin buffer pH 7.4. homogenizations in ethanol, the homogenates with washings were The supernatant was removed and the centrifuged at 4OC (l,OOOg, 15 min). The washing was centrifuged precipitate washed with 2ml ethanol. Both supernatants were (l,OOOg, 15 min) and the supernatant recovered. combined and evaporated to dryness in vacua at 40°C, and the residue taken into 2ml culture medium (pH 5.0). Extraction with ethyl acetate (3 x 3ml) was performed and the extracts evaporated to dryness in vacua. The residues were finally taken into lml Tris-gelatin buffer pH 7.4.
192
Measurementsof 6-keto PGF ylerez~)Ueat estrus for the uterine horns of experimental animals as th*g was the stage of the estrous cycle that had and PGE in animalswith been used previously in a study of uterine PGF In one of2@he two methods for the plain or indomethacin dwices (8). a double antibody procedure was used with radioinnnunoassayof 6-keto PGF gifts of rabbit anti-6-keto Pd"' serum from Professor G.C. Liggins, from Dr. K.H. Gibson University of Auckland and auth&tic 6-keto PGF (ICI Pharmaceuticals Division, Cheshire, U.K.),'gogether with [3Hl 6-keto tracer purchased from New England Nuclear, Boston, Massachusetts, ;za . The specificity of this antiserum has been previously investigated (relative cross-reactivities for PGF2u and PGE2 with this antiserum were 9 and 8% respectively) and the coefficients of variation obtained with this assay in our laboratory (e.g. interassay of 17%) were very similar to those reported by Liggins et al (15). The other procedure for 6-keto PGF radioimmunoassay involved a single antibody and kits were purchased @om Seragen Inc., Boston Massachusetts, USA; with this assay we obtained intra-and inter-assay coefficients of variation of 5 and 14% respectively. (The reported cross-reactivities in this assay against PGF 10 and 3% respectively.) We found good agreement between measurements made by the two methods. The method described above for measuring prostaglandin synthesis by uterine homogenates in vitro had been used previously in an investigation of PGF20 and PGE synthesis (8). While it is recognised that some PG synthesis must inevitably occur during the process of homogenization, measurements relating to PGFZa synthesis in vitro by uterine tissue from normal estrous rats (i.e. without an IUD) showed that most of the prostaglandin synthesis (at least 70%) occurred during the time that the homogenates were incubated. This was based on performing homogenizations in Eagle's medium pH 7.4 and immediately acidifying, followed by extraction and measurement oi PGF 2a' RESULTS No significant differences in basal levels of 6-keto PGF per 1OOmg wet uterine tissue) were found for the parts o animals with indomethacin devices compared to the equivalent parts of animals with plain devices (n =5, n2=6; Fig.1). However, significant increases occurred in 6-keto 4 GF production capability (in ng/lOOmg tissue/2 hr) for the parts of th&UD horn in animals with indomethacin devices compared to the corresponding parts of that horn in animals with plain devices (n1=6, n =5; ~~0.05, Fig.1). These differences were tested by a modified unpaired t-5 est, taking into account the unequal variances in two sets of results (shown by a variance ratio, F, test) and as a consequence the lowering in the number of degrees of freedom. No significant changes occurred in the 6-keto PGF production capability for the contralateral horns in the two groups oflsnimals. (In Fig.1 the production capability for PGF by the uterine horns of the two groups of animals has been shown for com$#rison with 6-keto PGFlu values.) As the difference in 6-keto PGF production capability (calculated per unit weight of tissue) shown for the'gorn with the indomethacin device compared to the horn with the plain device could be due partly to differences in intra- or extra-cellular fluid in the tissue causing changes in total protein per unit weight of the tissue, quotients of 6-keto PGFla production 193
capability/PGF2u production capability haye also been calculated for the different parts of the uterus. The standard error of quotient (B/A) has been calculated according to the equation: SE of B/A = B/A
where SE of B=b, and SE of A=a.
J-W
This should indicate more reliably whether an alteration in production The capability for PGI has occurred in the indomethacin-treated horn. quotients (mean f 8E) for the two parts of the horn with an indomethacin device were 3.73 f 1.44 (part associated with device) and 5.53 + 1.92 (remaining part), compared with quotients of 0.78 + 0.34 and 0.82 _+0.36 for the equivalent parts of the horn with a plain device. Testing of the quotients between the two groups of animals by a modified t-test, and again taking into account the lowering in the number of degrees of freedom due to unequal variances, showed that the quotients of corresponding parts of the IUD horns were different (0.05
Bf :~.‘..I~ RATS WITH INDOHETHAClN
120
c
RATS WITH PLAIN DEVICES
IUO horn Fig. 1.
DEVICES
contC;m~;ero I
IUD horn
contra~terol
6-keto PGFlc basal levels (upper histogram) and synthesizing capability of uteri of animals with plain or indomethacin devices for 6-keto PGF, and PGF_ [measured as PG release in vitro. lower histograi!iy. Devi% were present for 24 f 2, 22 + 1 days respectively. Differences between corresponding parts of uteri in the two groups were tested for significance by modified t-test (* denotes pcO.05). 194
this study basal leyels of PGX2 (measured as its win metablitet 6-k&o PGF ') were found to be unaltered in the indomethacin-treated IUD
In
This, although intrauterine delivery of indomethacin reduced the horn. and PGE (produced by the endometrium)(a), it did not basal levels of PGF Few &dies have been performed on the uterine myometrial affect PGI . activity ii cyclic rats and its alteration by different prostaglandins. However, all of the prostaglandins tested induced contractions in the nonpregnant rat uterus and a relative order of potency of F2c>E2>12 was established (16,17). More extensive investigations have been carried out on the uterine activity in pregnant rats resulting in an order of potency In the present and previous studies, the reduced rostaglandin synthesis reflected by the decreased basal and PGE, but not of PG12, found for the indomethacinlevels of PGF influenced IUp horn might therefore be associated with a lowering of This would be consistent with an increased contractions in this horn. retention of unsecured indomethacin-containingdevices that has been observed in cyclic rats (20). Two possible interpretations have been considered in regard to the apparent lack of effect of intraluminally-delivered indomethacin on basal PGI levels in the rat uterus. Firstly, inadequate quantities of indome2hacin In this case the levels of PGI synthesis may be reaching the myometrium. in vivo in the two experimental IUD horns are assumed to be the game and give rise to similar basal levels of PGI for these horns. A second possibility, however, is to consider tha3 some indomethacin reaches the The myometrium and that it suppresses PGI2 synthesis to some degree. indomethacin-lowered level of synthesis in this IUD horn is then assumed to be the same as that in the horn with a plain device, leading to similar basal levels. If the inhibitory effect of such indomethacin on PG12 synthesis in vivo were to be blocked or interfered with in some way, then a higher production of PGI for the horn with the indomethacin device would be expected compared to i! hat for the horn with a plain device. Lending support to this possibility are the findings from the second part of the present study where a higher production of PGI by homogenates in vitro for the horn with the indomethacin device was ghown over that for the horn with a plain device. In the incubation mixtures, dilution has resulted in extremely low concentrations of any indomethacin present (cl00 pg/ml), and any inhibitory effects on prostaglandin synthesis due to indomethacin have been eliminated. A final direct determination of whether indomethacin is reaching the myometrium will require the use of radiolabelled drug and an investigation of its localization in the uterus following delivery from an intrauterine device using autoradiography or a tissue isolation technique. Should this confirm that the myometrium is reached by indomethacin, then the biochemical changes in the IUD horn that are associated with the increased PGI synthesizing capacity found in response to sustained intraluminal 3. elivery of drug would merit further study. ACKNCWLEDGEMSNTS This study was supported by a grant from the Medical Research Council of New Zealand. The advice of G.F.S. Spears, Department of Preventive and Social Medicine, University of Otago in regard to the statistical analyses is gratefully acknowledged. 195
REFERENCES 1.
Castracane VD, Saksena SK, Shaikh AA. Effect of IUDs, prostaglandins and indomethacin on decidual cell reaction in the rat. Prostaglandins 6: 397, 1974.
2.
Tobert JA. A study of the possible role of prostaglandins in decidualization using a nonsurgical method for the instillation of fluids into the rat uterine lumen. J Reprod Fert 47: 391, 1976.
3.
Sanan&s N, Baulieu E-E, Le Goascogne C. in decidualization of the rat uterus.
4.
Holmes PV, Gordashko BJ. Evidence of prostaglandin involvement in blastocyst implantation. J Embryo1 exp Morph 55: 109, 1980.
5.
Biggers JD, Leonov BV, Baskar JF, Fried J. Inhibition of hatching of mouse blastocysts in vitro by prostaglandin antagonists. Biol Reprod 19: 519, 1978.
6.
Hurst PR, MacFarlane DW. Further effects of nonsteroidal anti-inflammatory compounds on blastocyst hatching in vitro and implantation rates in the mouse. Biol Reprod 25: 777, 1981.
7.
Ylikorkala 0, Kauppila A, Siljander M. Anti-prostaglandin therapy in prevention of side effects of intrauterine contraceptive devices. Lancet 1: 393, 1978.
8.
Peplow PV, Hurst PR. Influence of local release of indomethacin from a silastic device on uterine prostaglandins F and E. Prostaglandins Leukotrienes and Medicine 9: 437, 1882.
9.
Differential production of Liggins GC, Seamark RF. campos GA, PGF and 6-keto-PGF by the rat endometrium and myometrium in response to oxyto& catecholamines and calcium ionophore. Prostaglandins 20: i97, 1980.
A role for prostaglandins J Endocr 89: 25, 1981.
10.
Release of prostaglandins by the I.U.C.D. Chaudhuri G. Prostaglandins 3: 773, 1973.
11.
Differential prostaglandin production by Downing I, Williams KI. Br J Pharmac 61: microsomal fractions of rat pregnant uterus. 158P, 1977.
12.
Factors affecting prostacyclin El Tahir KEH, Williams KI. Br J Pharmac 71: formation by the rat pregnant myometrium. 641, 1980.
13.
&I intrauterine silastic system for the Peplow PV, Hurst PR. sustained release of indomethacin. Prostaglandins and Medicine 7: 563, 1981.
14.
Prostaglandin and thromboxane production by Poyser NL, Scott FM. the rat uterus and ovary in vitro during the oestrous cycle. J Reprod Fert 60: 33, 1980.
15.
Production rates of Liggins GC, Campos GA, Roberts CM, Skinner SJ. prostaglandin F, 6-keto-PGF and thromboxane B by perifused human endometrium. Prostag lp1 andins 19: 461, 1960.
196
16.
Comparison of various prostaglandins (PGs) Whalley ET, White SK. on the in vitro longitudinal uterine smooth muscle of the rat Br J Pharmac 68: 15OP, 1979. and guinea-pig.
17.
Whalley ET, White SK. Effect of PGF2c, PGE2 and ICI 81008 on the in vivo and in vitro uterus of non-pregnant rats and guineaBr J Pharmac 69: 309P, 1980. pigs.
18.
Prostaglandins, thromboxanes and the Phillips CA, Poyser NL. pregnant rat uterus at term. Br J Pharmac 73: 75, 1981.
19.
The contribution of prostaglandin Vane JR, Williams KI. production to contractions of the isolated uterus of the rat. Br J Pharmac 48: 629, 1973.
20.
von Dadelszen P, Hurst PR, Peplow PV. Effect of indomethacin incorporation on the retention of silastic intrauterine devices Contraception 26: 159, 1982. in the rat.
197
INFLUENCE OF INDOMETHACIN DELIVERED FROM AN INTRAUTERINE SILASTIC DEVICE ON BASAL LEVELS AND SYNTHESIZING CAPABILITY OF RAT UTERUS FOR PROSTACYCLIN. P.V. Peplow, P.R. Hurst. Department of Anatomy, University of Otago, Dunedin, New Zealand. (reprint requests to PVP) ABSTRACT The intrauterine release of indomethacin (approximately 12Wg over 23 days) from a unilateral silastic device did not cause any significant reduction in basal levels of PGI (determined as 6-keto PGFlc) in the tissue of the IUD horn. However, ti+ is tissue was found to have an increased potential for PG12 synthesis when compared to that of the uterine horn with a plain device. The implications of these findings regarding a possible effect of indomethacin on the myometrium are discussed. INTRODUCTION The delivery of low levels of nonsteroidal anti-inflammatory compounds such as indomethacin directly to the female reproductive tract from an intrauterine device (IUD) may provide a way of increasing the efficiency of the IUD, since indomethacin has been shown to interfere with implantation by preventing decidualization of the uterus (l-3) and also by possibly affecting the blastocyst (4-6). Moreover, the local release of indomethacin from the IUD or a vaginal suppository may overcome problems of uterine dysfunction (such as abnormal bleeding and pain) frequently found in women with intrauterine devices (7). Delivery of small quantities of the drug may not result in those side-effects (e.g. gastrointestinal disturbances) experienced by women taking larger doses of the drug orally. In a previous study designed to provide basic information relating to such a medicated delivery system in the rat, we described the effect of indomethacin released from a silastic intrauterine device on the basal levels and synthesizing capacity of the uterus for PGF and PGE (8). Both of these substances are produced mainly by the enaa ometrium (9,lO). Now we wish to report on the basal levels of prostacyclin, measured as its major stable metabolite 6-keto PGF which is considered to be produced principally by the myometrium1?;1,12). This should allow an evaluation of whether indomethacin from an intraluminal depot is reaching the myometrium in sufficient quantity to inhibit prostaglandin synthesis and thereby alter such levels. The production capability of the uterus has also been assessed by an in vitro technique using tissue homogenates with the amounts of prostaglandin present in the culture medium at the 191
end of a 2 hr incubation period being adjusted for the basal levels in the tissue. The work described will be relevant to further studies directed towards elucidating the roles of certain pGs in reproductive physiology through the local administration of specific inhibitors of PG synthesis to the uterus. METHODS Twenty-two adult female Wistar rats (approximately 250g body weight and matched for age) having regular estrous cycles were used. They were housed under a 14:lO hr regimen of light and darkness and given free access to food and water. Eleven of these animals were fitted with a unilateral silastic rod not incorporating any indomethacin (referred to as 'plain' rod), while the other eleven were fitted with silastic rods containing 3% w/w indomethacin. The preparation and sterilization of the rods (l.Ocm length, 0.09cm i-d.), covered by a vinyl sleeve (0.17cm o.d.), were performed as previously described (13). The rods were inserted on the first or second day after estrus by anaesthetizing the animals with sodium pentobarbitone and making a small incision in one horn, at about half-way along its length, to allow the device to be placed into the Lumen of the horn. The device was secured to the uterine wall and the incision closed by a short nylon suture. After the operation the animals were rested for 1 week and vaginal smears were taken daily. At around 21 days after fitting the devices, the animals were killed at 10.00 a.m. on the day of estrus, and the uterine horns removed. In this and subsequent manipulation, the horns were held only very gently with forceps to minimize any trauma-induced stimulation of PG synthesis that would raise Each horn was divided into two parts; for the horn with basal levels. the device, the rod was associated with either the caudal or cranial part. The parts were trimmed of fat and mesometrium, and cleaned on filter paper. Each part was slit longitudinally to allow the escape of luminal fluid and to permit the removal of the device. The luminal surface of the uterus was also blotted on filter paper. The separate parts of the uterus were immediately weighed and homogenized in 5ml Eagle's culture medium pH 7.4, or 5ml ethanol (for measurement af PG basal levels) using a glass homogenizer with teflon plunger held in a high-speed electric drill (10 The homogenizer was washed with lml culture medium strokes over 1 min). or ethanol. The culture medium homogenates with washings were transferred This method is based on to stoppered glass tubes and incubated at 37OC. that described by Poyser and Scott (14) with PG production being terminated after 2 hr by the addition of 0.2N HCl (to bring samples to pH 4.51, and PGs extracted with ethyl acetate (3 x 3ml) at room temperature. The extracts were evaporated in vacua at a temperature not exceeding 450C and For the the residues taken up in 9ml O.OlM Tris-gelatin buffer pH 7.4. homogenizations in ethanol, the homogenates with washings were The supernatant was removed and the centrifuged at 4OC (l,OOOg, 15 min). The washing was centrifuged precipitate washed with 2ml ethanol. Both supernatants were (l,OOOg, 15 min) and the supernatant recovered. combined and evaporated to dryness in vacua at 40°C, and the residue taken into 2ml culture medium (pH 5.0). Extraction with ethyl acetate (3 x 3ml) was performed and the extracts evaporated to dryness in vacua. The residues were finally taken into lml Tris-gelatin buffer pH 7.4.
192
Measurementsof 6-keto PGF ylerez~)Ueat estrus for the uterine horns of experimental animals as th*g was the stage of the estrous cycle that had and PGE in animalswith been used previously in a study of uterine PGF In one of2@he two methods for the plain or indomethacin dwices (8). a double antibody procedure was used with radioinnnunoassayof 6-keto PGF gifts of rabbit anti-6-keto Pd"' serum from Professor G.C. Liggins, from Dr. K.H. Gibson University of Auckland and auth&tic 6-keto PGF (ICI Pharmaceuticals Division, Cheshire, U.K.),'gogether with [3Hl 6-keto tracer purchased from New England Nuclear, Boston, Massachusetts, ;za . The specificity of this antiserum has been previously investigated (relative cross-reactivities for PGF2u and PGE2 with this antiserum were 9 and 8% respectively) and the coefficients of variation obtained with this assay in our laboratory (e.g. interassay of 17%) were very similar to those reported by Liggins et al (15). The other procedure for 6-keto PGF radioimmunoassay involved a single antibody and kits were purchased @om Seragen Inc., Boston Massachusetts, USA; with this assay we obtained intra-and inter-assay coefficients of variation of 5 and 14% respectively. (The reported cross-reactivities in this assay against PGF 10 and 3% respectively.) We found good agreement between measurements made by the two methods. The method described above for measuring prostaglandin synthesis by uterine homogenates in vitro had been used previously in an investigation of PGF20 and PGE synthesis (8). While it is recognised that some PG synthesis must inevitably occur during the process of homogenization, measurements relating to PGFZa synthesis in vitro by uterine tissue from normal estrous rats (i.e. without an IUD) showed that most of the prostaglandin synthesis (at least 70%) occurred during the time that the homogenates were incubated. This was based on performing homogenizations in Eagle's medium pH 7.4 and immediately acidifying, followed by extraction and measurement oi PGF 2a' RESULTS No significant differences in basal levels of 6-keto PGF per 1OOmg wet uterine tissue) were found for the parts o animals with indomethacin devices compared to the equivalent parts of animals with plain devices (n =5, n2=6; Fig.1). However, significant increases occurred in 6-keto 4 GF production capability (in ng/lOOmg tissue/2 hr) for the parts of th&UD horn in animals with indomethacin devices compared to the corresponding parts of that horn in animals with plain devices (n1=6, n =5; ~~0.05, Fig.1). These differences were tested by a modified unpaired t-5 est, taking into account the unequal variances in two sets of results (shown by a variance ratio, F, test) and as a consequence the lowering in the number of degrees of freedom. No significant changes occurred in the 6-keto PGF production capability for the contralateral horns in the two groups oflsnimals. (In Fig.1 the production capability for PGF by the uterine horns of the two groups of animals has been shown for com$#rison with 6-keto PGFlu values.) As the difference in 6-keto PGF production capability (calculated per unit weight of tissue) shown for the'gorn with the indomethacin device compared to the horn with the plain device could be due partly to differences in intra- or extra-cellular fluid in the tissue causing changes in total protein per unit weight of the tissue, quotients of 6-keto PGFla production 193
capability/PGF2u production capability haye also been calculated for the different parts of the uterus. The standard error of quotient (B/A) has been calculated according to the equation: SE of B/A = B/A
where SE of B=b, and SE of A=a.
J-W
This should indicate more reliably whether an alteration in production The capability for PGI has occurred in the indomethacin-treated horn. quotients (mean f 8E) for the two parts of the horn with an indomethacin device were 3.73 f 1.44 (part associated with device) and 5.53 + 1.92 (remaining part), compared with quotients of 0.78 + 0.34 and 0.82 _+0.36 for the equivalent parts of the horn with a plain device. Testing of the quotients between the two groups of animals by a modified t-test, and again taking into account the lowering in the number of degrees of freedom due to unequal variances, showed that the quotients of corresponding parts of the IUD horns were different (0.05
Bf :~.‘..I~ RATS WITH INDOHETHAClN
120
c
RATS WITH PLAIN DEVICES
IUO horn Fig. 1.
DEVICES
contC;m~;ero I
IUD horn
contra~terol
6-keto PGFlc basal levels (upper histogram) and synthesizing capability of uteri of animals with plain or indomethacin devices for 6-keto PGF, and PGF_ [measured as PG release in vitro. lower histograi!iy. Devi% were present for 24 f 2, 22 + 1 days respectively. Differences between corresponding parts of uteri in the two groups were tested for significance by modified t-test (* denotes pcO.05). 194
this study basal leyels of PGX2 (measured as its win metablitet 6-k&o PGF ') were found to be unaltered in the indomethacin-treated IUD
In
This, although intrauterine delivery of indomethacin reduced the horn. and PGE (produced by the endometrium)(a), it did not basal levels of PGF Few &dies have been performed on the uterine myometrial affect PGI . activity ii cyclic rats and its alteration by different prostaglandins. However, all of the prostaglandins tested induced contractions in the nonpregnant rat uterus and a relative order of potency of F2c>E2>12 was established (16,17). More extensive investigations have been carried out on the uterine activity in pregnant rats resulting in an order of potency In the present and previous studies, the reduced rostaglandin synthesis reflected by the decreased basal and PGE, but not of PG12, found for the indomethacinlevels of PGF influenced IUp horn might therefore be associated with a lowering of This would be consistent with an increased contractions in this horn. retention of unsecured indomethacin-containingdevices that has been observed in cyclic rats (20). Two possible interpretations have been considered in regard to the apparent lack of effect of intraluminally-delivered indomethacin on basal PGI levels in the rat uterus. Firstly, inadequate quantities of indome2hacin In this case the levels of PGI synthesis may be reaching the myometrium. in vivo in the two experimental IUD horns are assumed to be the game and give rise to similar basal levels of PGI for these horns. A second possibility, however, is to consider tha3 some indomethacin reaches the The myometrium and that it suppresses PGI2 synthesis to some degree. indomethacin-lowered level of synthesis in this IUD horn is then assumed to be the same as that in the horn with a plain device, leading to similar basal levels. If the inhibitory effect of such indomethacin on PG12 synthesis in vivo were to be blocked or interfered with in some way, then a higher production of PGI for the horn with the indomethacin device would be expected compared to i! hat for the horn with a plain device. Lending support to this possibility are the findings from the second part of the present study where a higher production of PGI by homogenates in vitro for the horn with the indomethacin device was ghown over that for the horn with a plain device. In the incubation mixtures, dilution has resulted in extremely low concentrations of any indomethacin present (cl00 pg/ml), and any inhibitory effects on prostaglandin synthesis due to indomethacin have been eliminated. A final direct determination of whether indomethacin is reaching the myometrium will require the use of radiolabelled drug and an investigation of its localization in the uterus following delivery from an intrauterine device using autoradiography or a tissue isolation technique. Should this confirm that the myometrium is reached by indomethacin, then the biochemical changes in the IUD horn that are associated with the increased PGI synthesizing capacity found in response to sustained intraluminal 3. elivery of drug would merit further study. ACKNCWLEDGEMSNTS This study was supported by a grant from the Medical Research Council of New Zealand. The advice of G.F.S. Spears, Department of Preventive and Social Medicine, University of Otago in regard to the statistical analyses is gratefully acknowledged. 195
REFERENCES 1.
Castracane VD, Saksena SK, Shaikh AA. Effect of IUDs, prostaglandins and indomethacin on decidual cell reaction in the rat. Prostaglandins 6: 397, 1974.
2.
Tobert JA. A study of the possible role of prostaglandins in decidualization using a nonsurgical method for the instillation of fluids into the rat uterine lumen. J Reprod Fert 47: 391, 1976.
3.
Sanan&s N, Baulieu E-E, Le Goascogne C. in decidualization of the rat uterus.
4.
Holmes PV, Gordashko BJ. Evidence of prostaglandin involvement in blastocyst implantation. J Embryo1 exp Morph 55: 109, 1980.
5.
Biggers JD, Leonov BV, Baskar JF, Fried J. Inhibition of hatching of mouse blastocysts in vitro by prostaglandin antagonists. Biol Reprod 19: 519, 1978.
6.
Hurst PR, MacFarlane DW. Further effects of nonsteroidal anti-inflammatory compounds on blastocyst hatching in vitro and implantation rates in the mouse. Biol Reprod 25: 777, 1981.
7.
Ylikorkala 0, Kauppila A, Siljander M. Anti-prostaglandin therapy in prevention of side effects of intrauterine contraceptive devices. Lancet 1: 393, 1978.
8.
Peplow PV, Hurst PR. Influence of local release of indomethacin from a silastic device on uterine prostaglandins F and E. Prostaglandins Leukotrienes and Medicine 9: 437, 1882.
9.
Differential production of Liggins GC, Seamark RF. campos GA, PGF and 6-keto-PGF by the rat endometrium and myometrium in response to oxyto& catecholamines and calcium ionophore. Prostaglandins 20: i97, 1980.
A role for prostaglandins J Endocr 89: 25, 1981.
10.
Release of prostaglandins by the I.U.C.D. Chaudhuri G. Prostaglandins 3: 773, 1973.
11.
Differential prostaglandin production by Downing I, Williams KI. Br J Pharmac 61: microsomal fractions of rat pregnant uterus. 158P, 1977.
12.
Factors affecting prostacyclin El Tahir KEH, Williams KI. Br J Pharmac 71: formation by the rat pregnant myometrium. 641, 1980.
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