THE EFFECT OF INTRAUTERINE PROGESTERONE TREATMENT ON THE ENDOMETRIAL PROSTAGLANDIN F CONTENT IN THE RABBIT*

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1 FERTILITY AND STERILITY Copyright 1977 The American Fertility Society Vol. 2, No.5, May 1977 Printed in U.s.A. THE EFFECT OF INTRAUTERINE PROGESTERONE TREATMENT ON THE ENDOMETRIAL PROSTAGLANDIN F CONTENT IN THE RABBIT* ANTONIO SCOMMEGNA, M.D.t JOHN ILEKIS, B.S. GUNTHER SOTREL, M.D.t FREDERICK J. AULE'ITA, PH.D. Department of Obstetrics and Gynecology, Michael Reese Hospital and Medical Center, and The Pritzker School of Medicine of the University of Chicago, Chicago, Illinois The effect on rabbit endometrial prostaglandin F cased by progesterone delivered directly to the ters was investigated. For grops of animals were sed in the experiment: (1) no treatment (control); (2) an empty Silastic capsle (as an intraterine device [IUD]) was inserted in one horn and the other horn was sham-operated; (3) a Silastic capsle releasing 150 p.g ofprogesteronelday was placed in one horn and the other horn was sham-operated; () a Silastic capsle releasing progesterone was placed in one horn and the opposite horn received an empty Silastic capsle. In grop 1, which received no treatment, no difference was noted. In grop 2, the prostaglandin content of the horn containing an empty IUD was significantly higher than that of the sham-operated horn. In grop 3, the same significant difference was noted between the prostaglandin content of the IUD-containing, progesterone-treated horn and the sham-operated horn. In grop, no significant difference was observed between the horn containing an inert IUD and that containing a progesterone-releasing device. The addition of progesterone to an IUD does not significantly affect the elevated prosta- glandin content of the endometrim cased by an inert IUD. In 1971, Chadhril first proposed that local release of prostaglandins from the endometrim was responsible for the contraceptive action of the intraterine device (IUD). However, this contention has not been proven. There is sbstantial evidence in laboratory rodents,2. sheep,s and hmans 6 7 that the presence of an intraterine foreign body increases the local biosynthesis of prostaglandins E and F as measred in terine washings, terine venos blood, total terine tisse, or isolated endometrim. Indomethacin, a potent inhibitor of prostaglandin synthesis, abol- Accepted December 23,1976. *Spported by the Program for Applied Research on Fertility Reglation (PARFR [AID]). treprint reqests: Antonio Scommegna, M.D., Department of Obstetrics and Gynecology, Dr. Sidney Klein Maternity Hospital, 29th Street and Ellis Avene, Chicago, Ill tpresent address: Department of Obstetrics and Gynecology, Boston City Hospital, Boston, Mass ishes the expected increase in prostaglandin content of the endometrim after the insertion of an IUD bt does not reverse its contraceptive action in rats2 and mice.3 In rabbits, indomethacin treatment does reverse the contraceptive effect of the IUD. The prostaglandin content of the endometrim is a cyclic, hormone-dependent event. oll In animals, this cyclicity can still be observed in blood from the terine vein,s bt not in the peripheral blood. 12 However, cyclic changes of the more stabile 15-keto metabolite can be detected in plasma dring the menstral cycle. 13 In women, the prostaglandin content of the endometrim, expressed as nanograms per nit weight of wet tisse, is considerably higher in the progesteronedominated phase of the cycle, with a frther increase after the decrease in the progesterone level, namely dring menstration.9 11 The increase dring the secretory phase is not demon-

2 52 SCOMMEGNA ET AL. May 1977 strated if prostaglandins are expressed per dry weight of endometrim. 10 Nevertheless, the idea that progesterone sppresses and estrogen stimlates prostaglandin biosynthesis of the endometrim is most appealing, since there is an additional increase in endometrial prostaglandin concentration after the premenstral decrease in progesterone. This hypothesis has been confirmed by Demers et al., who measred the prostaglandin F content in the terine flid of castrated rhess monkeys. In these animals estrogen treatment cased an increase in prostaglandin content above control levels, while the addition of progesterone to the estrogen indced a slight decrease. Even more convincing are the experiments by Cane and Villee,1 who sed tisse cltres of hman endometrim obtained at hysterectomy. The addition of estrogen to the cltre medim increased the prostaglandin synthesis in the system, whereas the addition of progesterone alone inhibited sch synthesis. Since the pioneering work of Pickles et al.,ll endometrial prostaglandins have been implicated in the etiology of dysmenorrhea. IS Significant nmbers of women wearing intraterine devices complain of menstral cramps. In view of the increased endometrial prostaglandin concentration in the presence of an IUD and the implication of these componds in dysmenorrhea, it is possible that endometrial prostaglandins are responsible for IUD-indced terine cramps. Progesterone-releasing IUDs, while more effective than inert IUDs in preventing pregnancy,16 seem to be associated with a lower incidence of menstral crampsy In fact, 6% of women complained of severe menstral cramps prior to the insertion of a progesterone IUD. After 6 months of se only 2% of these patients still complained of severe dysmenorrhea. In view of the postlated inhibitory effect of progesterone on endometrial prostaglandin synthesisi it is hypothesized that the progesterone released from the IUD at the endometrim cases a decrease in endometrial prostaglandin, thereby decreasing the incidence of terine cramps. The present experiment was designed to investigate the effect in the rabbit of the locally delivered progesterone on the expected increase in endometrial prostaglandin F cased by the presence of an intraterine foreign body. MATERIALS AND METHODS Sexally matre New Zealand White female rabbits, hosed for at least 2 weeks prior to srgery, were sed. A 30-mm length of Silastic medical grade tbing (Dow-Corning Corporation, Midland, Mich.) with an otside diameter of 3.1 mm and a wall thickness of 0. mm was sed to manfactre the progesterone IUD releasing system. The capsles were filled with 3. ±.1 mg of progesterone (mean ± standard error) and the ends were closed with a 2-mm layer of Silastic medical adhesive silicone type A (Dow Corning). It has been shown that sch capsles release in vivo approximately 150 JLglday of progesterone.1 Silastic capsles of eqal length and thickness bt withot progesterone were sed as inert IUDs. Under halothane anesthesia, a longitdinal mid ventral incision was made and the terine horns were exposed. A small incision was made on the antimesometrial area of the terine horns, and the IUDs were inserted free in the terine lmina. The IUDs were secred in place with a silk stre which was sed also to close the terine incision. For the sham procedre, IUDs (either active or inert) were inserted throgh the incision in the terine horn and immediately removed. A silk stre was sed to close each horn. The rabbits were randomly divided into for experimental grops: Three animals in grop 1 received no treatment (no srgery). Grop 2 inclded five animals. A blank IUD (inert) was inserted in one horn of each animal and a sham operation was performed in the opposite horn. In grop 3 a progesterone IUD (active) was placed in one horn of each animal and a sham operation was performed in the opposite horn. This grop had six animals. Grop contained five animals. A progesterone IUD (active) was placed in one horn of each animal and a blank IUD (inert) was placed in the opposite horn. Each rabbit served as its own control, the IUD insertion or sham procedre having been done on opposite terine horns (Le., progesterone IUD-left, blank IUD-right) as indicated for each of the three experimental grops. Three weeks after srgery the animals were killed with an overdose of Nembtal given intracardially; the terine horns were qickly excised and frozen immediately on a Dry Ice-acetone bath. The terine tisse was transferred to a Petri dish and stored at - 20 C ntil analyzed. Also, at necropsy, the ovaries were examined and no corpora ltea were observed. At the time of analysis, the tisse was thawed, the terine horns were separated, and the endometrim was niformly stripped from each horn,

3 Vol. 2, No.5 EFFECT OF PROGESTERONE ON ENDOMETRIAL PROSTAGLANDIN F 53 blotted dry, and weighed. The endometrial tisse was placed in a glass homogenizer containing 1 ml of ice-chilled normal saline. The glass homogenizer was placed in an ice bath to prevent heating dring homogenization. The endometrim was homogenized for 1 minte; the homogenate was dilted with ice-cold saline so that the mixtre contained approximately 10 mg of endometrial tisse/ml. The mixtre was agitated vigorosly before aliqoting. A portion of the homogenized tisse mixtre (1 ml) was transferred to a 15-ml extraction tbe kept cool in an ice bath for prostaglandin analysis; 1 ml was transferred to a 12 x 75 mm glass cltre tbe and stored at C for protein analysis. The techniqe described by Aletta et al. 19 was sed for the prostaglandin F (PGF) radioimmnoassay. Tritim-labeled PGF:za with a specific activity of 9.2 Cilmmole was prchased from New England Nclear Corporation, Boston, Mass., and prified monthly on a silicic acid colmn. 19 The PGF antiserm was kindly donated by Dr. Robert Skarnes, Worcester Fondation for Experimental Biology, Shrewsbry, Mass. The antiserm was prepared in rabbits by sing PGF 2" bovine serm albmin conjgate as the antigen; the antiserm was sed at a 1:00 diltion. This antiserm exhibits no significant cross-reactivity with the prostaglandins of the A and E series nor with the 15-keto PGE metabolite. An 1% cross-reaction was noted with the PGF 2" metabolite and 77% with PGF 1". Becase of this significant cross-reaction with PGF 1" and since chromatographic separation does not separate PGF 1 " and PGF 2", the reslts are expressed as total PGF. To the 1-ml aliqot of tisse homogenate, 1000 cpm of3h-pgf 2" were added to determine recovery losses throgh the method. To adjst the ph to 3.5 to.0, 0.1 ml of 0.05 N HCI was added. The sample was extracted twice with nanograde ethyl acetate and centrifged at 1200 x g for 5 mintes to separate the aqeos and organic layers; the ethyl acetate extracts were transferred to a conical centrifge tbe and dried at 55 C nder nitrogen. Prostaglandin F was isolated by silicic acid colmn chromatography and assayed as described by Aletta et au9 The protein content of the homogenate was assayed with the techniqe described by Lowry et al. 20 All determinations and PGF and protein assays were carried ot in dplicate. Reslts are 100,, '-.,,,,, ",... ell A. CI Z ;;) o ID o PROSTAGLANDIN F2" (pe( FIG. 1. Evidence of parallelism in radioimmnoassay crves between standard prostaglandin Fza ( ) and aliqots of endometrial tisse extracts (*- - -*).

4 5 SCOMMEGNA ET AL. May 1977 PGF,,, added pg PGF 20' recovered fl pg 11.5 ± ± ± ± ± 6. ameans of 10 determinations ± standard deviation. expressed as picograms of PGF per microgram of protein. The significance of the difference between grops was evalated by the se of Stdents' t-test. RESULTS Validation of Prostaglandin F Assay from Endometrial Tisse. For validation of the assay, the endogenos tisse prostaglandin and standard prostaglandin F mst react with the antibody identically, and nder the conditions of the assay other sbstances in the tisse mst not affect the determination. As a necessary condition for identical reactivities of standard and tisse nknown the concentration in the nknown tisse extract mst decrease linearly with diltion so that the concentration in the ndilted tisse is independent of the diltion at which the tisse is assayed. This is eqivalent to having sperimposable crves for standards and tisse nknowns. Figre 1 represents a standard crve of PGF 2" and endometrial tisse extract assayed at varios diltions. The parallelism of the two crves indicates that the antigen in the endometrial tisse is immnologically identical with the standard. However, becase the antiserm sed does crossreact with PGF 1 and with PGF2" and its metabolite(s), the reslts obtained shold be considered as total PGF expressed in PGF2" eqivalents. Recovery of known amonts of PGF2" added to the endometrial samples was also investigated. The reslts shown in Table 1 indicate that prostaglandin F 2" added to the endometrial homogenate TABLE 2. Replicate Determinations of Endometrial PGF2" Content PGF,,, pgl pg protein 1.7 ± ± ± ± ± ± ± 0.06 No. of determinations TABLE 3. Replicate Determinations of PGF2 " Content of Endometrial Tisse Assayed Immediately after Collection or Frozen (-2(1' C) and Assayed Months Later Endometrial tisse Fresh Frozen 3.7 ± ± 0.2 pgl pg protein 2.5 ± ± 0.1 a Means of three determinations ± standard deviation. in amonts varying between 100 and 2500 pg was recovered with a satisfactory degree of precision. Replicate determinations were sed to evalate the precision of the assay. The reslts (Table 2) show that replicate determinations were within 10% of the mean. Virtally identical reslts were obtained when the tisse was assayed immediately after collection and when the tisse was frozen (- 20" C) and assayed months later (Table 3). Reslts in Rabbits. The reslts (means ± standard error) are shown in Figre 2. In grop 1 (no treatment) no difference was seen between the prostaglandin content of the left horn and that of the right horn, the reslts being 1.6 ± 0.7 and 1.7 ± 0.7 pg of prostaglandin, respectively W = 3). In grop 2 (sham operation verss inert IUD) a significant difference <P < 0.05) was seen between the two horns. The sham-operated horns contained 1.5 ± 0.3 pg of prostaglandin and the horns with the inert IUD contained 3.6 ± 1.2 pg of prostaglandin W = 5). In grop 3 (sham operation verss progesterone IUD) the same significant difference was noted <P < 0.05). The sham-operated horns con c no. t. horns e rfl Q. E mean!sem 'E 6 6 Ii: Q. " 5 5. E :;. 0 'ii Q Q C ,., E.....: " II: II: FIG.2. Rabbit endometrial prostaglandin F in control grop, inert IUD grop, and progesterone IUD (J'rog IUD) grop.

5 Vol. 2, No. 5 EFFECT OF PROGESTERONE ON ENDOMETRIAL PROSTAGLANDIN F 55 c 'i E 0. 5 rfl no.t. horns mean ± sem e 5 'e ---. CJ Il. " E l! '" 0 's = = E C 0.. Ci '" 0.: Ii: '" FIG. 3. Rabbit endometrial prostaglandin F in terine horns; reslts in horns of varios grops bt with similar treatment were pooled (J>rog IUD, progesterone IUD). tained 2.5 ± 0.7 pg of prostaglandin and the progesterone-treated horns contained 5.7 ± 1.2 pg of prostaglandin <N = 6). In grop, in which inert IUDs were compared with progesterone IUDs, no significant difference was observed. The prostaglandin contents were 7.1 ± 2.2 and 6. ± 1.5 pg, respectively, in the inert IUD-bearing horns and progesterone IUD-bearing horns <N = 5). The reslts obtained in the control, sham-operated, inert IUD-bearing, and progesterone IUDbearing horns were pooled and the vales obtained are shown in Figre 3. Sham srgery did not increase the content of endometrial prostaglandin over the control vales. Inert IUDs cased an increase in endometrial prostaglandin as compared with the control horn. The addition of progesterone to the IUD did not significantly affect the content of endometrial prostaglandin, which remained elevated as compared with that of control horns. DISCUSSION These reslts show that progesterone delivered locally to the ters at a rate of approximately 150 /-Lg/day has no effect on the prostaglandin content of the endometrim. The increase in endometrial prostaglandin content cased by the intraterine foreign body, as demonstrated in this experiment as well as by others, was not abolished by the high doses of progesterone, which were expected to inhibit prostaglandin synthesis. The introdction of an IUD into the endometrial cavity cases infiltration by netrophils and 7 6 macrophages. It has been shown by Bray et al. 6 that sch macro phages are capable of prodcing prostaglandins and might be the principal sorce of the increased prostaglandin content of the endometrim after IUD insertion. The contraceptive effectiveness of an inert IUD in the rabbit is probably related to prostaglandin prodction, since indomethacin administration does reverse the contraceptive effectiveness of the IUD. The addition of progesterone to a Silastic capsle markedly enhances its contraceptive properties in the rabbit. 21 Since this effect is obtained withot change in endometrial prostaglandin content as compared with an inert IUD, it appears that intraterine progesterone cases its contraceptive effect by a mechanism other than prostaglandin release. Hypermatrational changes in the endometrim as well as direct deleterios effects of progesterone on the blastocysts have been postlated 22 to explain the contraceptive effectiveness. The lack of decrease in endometrial prostaglandin content in the rabbit with chronic administration of intraterine progesterone does not preclde sch a decrease taking place in women in the presence of intraterine progesterone. The significant decrease in the occrrence of dysmenorrhea in women wearing an intraterine progesterone delivery system sggests that sch a decrease does indeed take place. Stdies are now in progress to investigate this matter. REFERENCES 1. Chadhri G: Intraterine device: possible role of prost a glandins. Lancet 1:0, Chadhri G: Release of prostaglandins by the I. U.C.D. Prostaglandins 3:773, La IF, Saksena SK, Chang MC: Prostaglandin F in the terine horns of mice with intraterine devices. J Reprod Fertil 37:29, 197. Saksena SK, Harper MJK: Prostaglandin-mediated action of intraterine devices: F-prostaglandins in the terine horns of pregnant rabbits with nilateral intraterine devices. Fertil Steril 25:121, Spilman CH, Dby RT: Prostaglandin-mediated lteolytic effect of an intraterine device in sheep. Prostaglandins 2:159, Bray MA, Gordon D, Morley J: Macrophages on intraterine contraceptive devices prodce prostaglandins. Natre 257:227, Green K, Hagenfeldt K: Prostaglandins in the hman endometrim: gas chromatographic-mass spectrometric qantitation before and after IUD insertion. Am J Obstet Gynecol 122:611, Demers LM, Y oshinaga K, Greep RO: Prostaglandin F in monkey terine flid dring the menstral cycle and following steroid treatment. Prostaglandins 5:513, 197

6 56 SCOMMEGNA ET AL. May Downie J, Poyser NL, Wnderlich M: Levels of prostaglandins in hman endometrim dring the normal menstral cycle. J Physiol (Lond) 236:65, Levitt MJ, Tobon H, Josimovich JB: Prostaglandin content of hman endometrim. Fertil Steril 26:296, Pickles VR, Hall WJ, Best FA, Smith GN: Prostaglandins in endometrim and menstral flid from normal and dysmenorrhoeic sbjects. J Obstet Gynaecol Br Commonw 72:15, Wilks JW, Wentz AC, Jones GS: Prostaglandin F 2,. concentrations in the blood of women dring normal menstral cycles and dysmenorrhea. J Clin Endocrinol Metab 37:69, Grieves SA, Kendall JZ, Liggins GC: Measrement of prostaglandin F 2,. (PGF 2,.) and its 15-keto metabolites in the hman menstral cycle (abstr). New Zealand J Med 7:367, Cane EM, Villee CA: The synthesis of prostaglandin F by hman endometrim in organ cltre. Prostaglandins 9:21, Halbert DR, Demers LM, Darnell Jones DE: Dysmenorrhea and prostaglandins. Obstet Gynecol Srvey 31:77, Brenner PF, Cooper DL, Mishell DR Jr: Clinical stdy of a progesterone-releasing intraterine contraceptive device. Am J Obstet Gynecol 121:70, Pharris B, Erickson R, Tillsen S, Scommegna A: The mechanism for redction of dysmenorrhea by progesterone: a hypothesis. Scientific exhibit presented at the Annal Meeting of the American College of Obstetrics and Gynecology, Dallas, Tex, May 10-13, Lifchez AS, Scommegna A: Diffsion of progestogens throgh Silastic rbber implants. Fertil Steril 21:26, Aletta FJ, Asman RM, Caldwell BV: Development and standardization ofradioimmnoassays for prostaglandins E, F and A. Clin Chern 20:150, Lowry OH, Rosebrogh NH, Farr AL, Randall RJ: Protein measrement with the Folin phenol reagent. J BioI Chern 193:265, Seshadri B, Gibor Y, Scommegna A: Antifertility effects of intraterine progesterone in the rabbit. Am J Obstet Gynecol 109:536, Scommegna A, Pandya GN, Christ M, Lee AW, Cohen MR: Intraterine administration of progesterone by a slow releasing device. Fertil Steril 21:201, 1970