FRTLTY AND STRLTY Copyright 1978 The American Fertility Society Vol. 3, No.6, December 1978 Printed in U.S.A. VAGNAL PROGSTRON FOR CONTRACPTON ARN VCTOR, M.D.* THODOR M. JACKANCZ, PH.D.t LOF D. B. JOHANSSON, M.D.:j: Department of Obstetrics and Gynecology, University Hospital, Uppsala, Sweden, and Biomedical Division, The Population Council, The Rockefeller University, New York, New York 121 Silastic vaginal rings impregnated with progesterone (P) or progesterone and estradiol () were used in nine women for thirty 21-day cycles to study the effect on ovarian function. The average daily rates of release of P and from the rings were 2.2 mgt day and 22 p.gl day, respectively. n anovulatory treatment cycles the mean plasma level of P was.9 ng/ml. levels fluctuated in the range seen in normally cycling women due to endogenous production. n a menopausal woman the levels increased to 5 to 1 pg/ml during treatment andfsh levels declined. Ovulation occurred in 18 (6%) of the treated cycles, and resulted in one pregnancy. Although the results rule out the use of these rings for contraceptive purposes, they indicate that at higher dosages the Silastic vaginal ring offers a mode of administration of natural steroids to be used in hormonal contraception. Fertil Steril3:631, 1978 Although the first suggestion that ovarian hormones could be used for contraceptive purposes stems from the early 192s 1 and it is well known that progesterone (P) can inhibit ovulation in various animals, to our knowledge only one study has been published utilizing progesterone to achieve anovulation for contraceptive purposes in women. 2 n that study Pincus2 used cyclic oral administration of 3 mg of P daily and was able to verify ovulation either by laparotomy or by indirect means in about 25% of treatment cycles. The reason for the frequent incidence of ovulation despite the high dose is probably due to poor bioavailability of P given orally.3 P is well absorbed both vaginally and rectally. 4 The difficulty of administering P by these routes is probably the reason why it has not been further tested for contraceptive use. With the advent of the Silastic contraceptive rings for the vaginal administration of steroids 5 a new opportunity emerged for studying the natural steroids as contraceptives. n several studies the contraceptive ring has been shown to be well accepted by most women. 6 7 The present investigation was undertaken to determine whether a relatively low dose ofp, alone or in combination with estradiol (), administered from Silastic vaginal rings would inhibit ovulation and thus provide a safe contraceptive for women. Received June 13, 1978; accepted July 27, 1978. *Reprint requests: Arne Victor, M.D., Department of Obstetrics and Gynecology, University Hospital, S-75 14 Uppsala, Sweden. tbiomedical Division, The Population Council, The Rockefeller University, New York, N.Y. :j:department of Obstetrics and Gynecology, University Hospital, Uppsala, Sweden. 631 MATRALS AND MTHODS Preparation of Rings. Micronized P and medical grade Silastic 382 (Dow Corning Corporation, Midland, Mich.) were mixed in a 1:4 ratio. A 2-gm aliquot was removed, mixed with stannous octoate catalyst, transferred to a 3-ml plastic syringe, and introduced into a brass mold with a ring cavity of
632 VCTOR T AL. 3.2 mm, 54 mm in diameter. After curing for 3 minutes, the ring was removed, placed in a second mold, and coated with Silastic to yield a ring of 9 mm x 6 mm dimensions containing about 29 mg ofp. Up to this point both types of rings were prepared in the same way. Those rings which would contain both and P were cut and covered with a.3-mm thick coating of a Silastic- (14%) mixture (approximately 2 mg of ). Finally, a.3-mm thick piece of silicone tubing was slipped on as an outer coating and the cut ends of the ring were rejoined with Dow Corning medical adhesive A. Those rings containing only P were covered with a.3-mm thick coating of Silastic and also covered with.3-mm thick Silastic tubing. n Vitro Diffusion. For in vitro studies, the ring was placed in 4 ml of a 1:75 Zephiran solution (benzalkonium chloride) and incubated at 37 C in a waterbath shaker. The solution was changed daily and its absorption measured at 24 nm to assay P. was determined by radioimmunoassay essentially by the method ofthorneycroft and Stone. 8 n Vivo Release of Steroid. After the rings were used in clinical trials, each ring was sliced into discs no thicker than 1 mm and put into a Soxhlet extraction apparatus. The rings were extracted exhaustively with 1,2-dichloroethane and aliquots were removed for assay of the remaining steroid. P was determined by measurement of ultraviolet absorbance at 24 nm. was analyzed by the Kober assay as modified by Brown et al.9 The average daily release ofp and was calculated by dividing the total drug loss by the number of days the ring was used. 4 December 1978 Subjects. Nine healthy, fertile women with regular menstrual cycles volunteered for the study. Four women used a ring containing only P and four the combination ring. One woman used both types of rings. The rings were introduced into the vagina immediately after cessation of normal menstrual bleeding but never later than on the 7th day of menstruation. They were left in the vagina for 21 days and removed for 7 days before being reinserted. ach ring was used for two to four 21-day cycles. The participants in the study were informed that the ring was not a contraceptive and asked to use condoms for contraception during the study. To verify absorption of the steroids from the rings, one ring containing P and was used by a 65-year-old menopausal woman for 2 weeks. All women kept daily bleeding records. Venous blood samples were obtained at least two or three times weekly from all subjects, starting about 2 weeks before the expected day of the menstruation and continuing throughout treatment and until the first menstrual bleeding about 1 month after termination of treatment, unless ovulation occurred during the last treatment cycle. All blood samples were analyzed for their and P concentration, using previously described radioimmunoassays. 8 1 11 The samples from the menopausal woman were also analyzed for FSH by radioimmunosorbent technique. 12 RSULTS Figures 1 and 2 show the results of the in vitro diffusion study. nitially the rings released about 3 mg ofp/day which decreased to about 1.5 mg after 9 days of incubation. The release decreased p - t;:._. 25 5 75 FG. 1. n vitro release of P from four rings containing P or P +. Arrows indicate days when the ring had been removed from the incubation bath for 7 days to mimic the 7 -day period during which the woman removes the ring from the vagina. The higher values on these days are probably due to a diffusion ofp from the core of the ring to the Silastic covering.
Vol. 3, No.6 VAGNAL PROGSTRON"FOR CONTRACP'ON 633 2 - "' 'tj!: w 1 25 5 75 1 FG. 2. n vitro release of from two P + rings. Values obtained after a 7-day removal from the incubation bath were not different from those obtained on other days (c.f. Fig. 1). from about 18 /Lg/day intially to about 4 /Lg/day after 9 days. There was a close correspondence between the average in vivo release of P from the rings and the release calculated from the in vitro data for corresponding periods (2.16 ±.11 mg/day versus 2.37 ±.11 mg/day). For the average in vivo release was higher (22± 69/Lg/day) than the in vitro release calculated for corresponding time periods (152 ± 16 /Lg/day). - n the menopausal woman (Fig. 3) treated for 2 weeks with a combination ring the plasma P concentrations increased to a level between 1.5 and 2 ng/ml during treatment. The concentration after an initial peak of 25 pg/ml stabilized at a level between 5 and 1 pg/ml, which was clearly above pretreatment concentrations. During treatment her follicle-stimulating hormone (FSH) level declined but never reached premenopausal values. All of the other women except one had normal ovulatory control cycles preceding treatment as judged by plasma P concentrations of more than 5 VR stradiol 18 mgj Progesterone 289 mg L.L. G5yrs " 3 15' ' 2 2' ::1: :22... 1 tr 'tj 1ii l 'ti l W1()() 5 ' a... o 5 1 15 2 ZZZZ2 FG. 3. Plasma concentrations of P,, and FSH in a menopausal woman using a P + ring for 2 weeks. ng/ml in at least one blood sample. The woman who did not ovulate during her control cycle had been ovulatory in three previous untreated control cycles. All women who did not ovulate during the last treatment cycle ovulated during the following recovery cycles, including the subject who did not ovulate during her pretreatment control cycle. n anovulatory treatment cycles the mean plasma concentration of P varied between. 7 and 1.6 ng/ml (mean.9 ng/ml) with individual values ranging from.4 to 2. nglml. The values varied considerably during treatment. Peaks similar to the preovulatory peak were encountered in all subjects. These peaks were often followed by an increase in P indicating ovulation (c.f. Figs. 4 to 6). Ovulatory plasma P patterns were seen in 18 of the 3 treatment cycles (6%); Ovulation occurred BF. VR 28!lrrQ p 14.# 12 \ fjj J:1 111() : 8 j : t 1 1 2112112 3 FG. 4. Plasma concentrations of P and in a woman using a ring containing P for three cycles. Open bars indicate treatment periods. Hatched bars indicate spotting or bleeding (double height of bar).
634 VCTOR T AL. December 1978 '---=,2"': 5, _, ' ' 1 211l 2112 &l 3JO o 2 Ol. u 1 _g <fj w? 6 FG. 5. Plasma levels of P and during treatment and the following recovery cycle in a woman using a ring containing P + for three cycles. For further explanation see legend to Figure 4. in 9 of 17 (53%) cycles treated with the ring containing only P and' in 9 of 13 cycles (77%) treated with the combination ring. The subject with an anovulatory pretreatment cycle ovulated during her first treatment cycle. One subject (Fig. 4) became pregnant during her last treatment cycle in spite of mechanical contraception. The pregnancy ended in a normal term baby. The height of the ovulatory P increases during treatment did not differ from the pre- or post-treatment cycles. Thirty-six bleeding events were encountered during treatment as counted from the 1st day of treatment to 7 days after. Fifteen of these could be considered to be regular postovulatory bleedings; eight followed a marked peak in plasma and three occurred on removal of the ring. n ten instances bleeding could not be related to any of these events. Only one subject complained of side effects (Fig. 5). She experienced pronounced breast tenderness that was related in time to the marked peaks which were not associated with ovulation. KW 14 l VR 282 rrv P 3 g 12 l <fj. 1 ; " o e Ci. 8 ' 2- ' 2'?' 6 p 8'.. e :Q 4 1ii 1 -;;; a.. w 9 6 121 "2112112 FG. 6. Plasma levels of P and during four treatment cycles in a woman using a ring containing only P. For further explanation see legend to Figure 4. DSCUSSON The production rate of P has been calculated to be.75 to 2.5 mg/day during the follicular phase and 15 to 5 mg/day during the luteal phase.13 14 With the radioimmunoassay methods presently used for P, follicular phase levels are mostly below.5 ng/mi.b During anovulatory treatment cycles with the Pring, the mean plasma level ofp was.9 ng/ml (range,.7 to 1.6 ng/ml). Knowing that the mean release ofp from the ring was 2.2 mg/day one might suspect that the calculated value for P production by the ovary during the follicular phase is too high. The results of the present study when compared with the calculated production rates would indicate a very good, almost complete, absorption of P released from the ring. On the other hand, Nillius and Johansson4 in their study of P absorption from various sites found that the vaginal dose of P would have to be about 4-fold that of P given intramuscularly to give comparable plasma levels of P. These data indicate that the production rate of P during the follicular phase is even lower than has been calculated, 13 14 but can also be due to differences in the mode of administering the drug into the vagina (vaginal suppository4 or vaginal ring [present study]). Although ovulation occurred in 6% of the cycles studied it seems justifiable to conclude that even at this low dosage and with the relatively low plasma levels of P encountered, treatment inhibited ovulation in a significant number of cycles. The addition of to the ring did not significantly affect the incidence of ovulation. With similar rings containing d-norgestrel as the gestagen the addition of decreases the incidence of breakthrough bleeding and spotting. 6 n this small series of cycles this was not the case. The inability of these rings to inhibit ovulation and spontaneous peaks is the most likely reason for the apparent lack of effect on bleeding control by the addition of to the ring. The pregnancy encountered in this study indicates that P does not offer the "local" protective effect of synthetic gestagens used in low-dose gestagen contraceptive pills; however, this conception may also have been due to passage of spermatozoa during the 7 days after ring removal. With low-dose gestagen pills the frequency of ovulation during treatment is about the same as in the present study.15 At plasma levels higher than those encountered in this study P has been found to decrease the luteinizing hormone (LH) response to LH-releasing hormone in humans16 and to inhibit the periovulatory LH and FSH peaks. 17 The peaks not followed by an increase in P occurring
Vol. 3, No.6 VAGNAL PROGSTRON FOR CONTRACPTON 635 frequently in this study can be interpreted as coming from ripening follicles. Similar peaks are often encountered in women on continuous low-dose gestagen treatment. 1 s By increasing the dose of gestagen the frequency of peaks diminished, 19 probably by preventing follicular development. n the rhesus monkey it has been shown that low doses (.5 mg/day) of P do not inhibit follicular development, whereas higher doses (2. or 5. mg/day) do. 2 t is thus probable that with a dose within the luteal phase ovarian production rate of P, which can be achieved by changing the design of the ring, a contraceptive effect ofp could be found. The use of a natural gestagen for contraception can be anticipated to reduce the side effects of contraceptive treatment as compared with the synthetic gestagens. Therefore it seems worth while to extend the studies of the P-releasing contraceptive ring. Acknowledgment. This work was undertaken as part of the contraceptive development program sponsored and coordinated by the nternational Committee for Contraception Research of the Population Council, New York, N.Y. RFRNCS 1. Simmer HH: On the history of hormonal contraception.. Ludwig Huberlandt (1885-1932) and his concept of "hormonal sterilization." Contraception 1:3, 197 2. Pincus G: Some effects of progesterone and related compounds upon reproduction and early development in mammals. Acta ndocrinol [Suppl) (Kbh) 28:18, 1956 3. Kincl FA, Ciaccio LA, Benagiano G: ncreasing oral bioavailability of progesterone by formulation. J Steroid Biochem 9:83, 1978 4. Nillius SJ, Johansson DB: Plasma levels of progesterone after vaginal, rectal and intramuscular administration of progesterone. Am J Obstet Gynecol 11:97, 1971 5. Mishell DR Jr, Talas M, Parlow AF, Mayer DL: Contraception by means of a Silastic vaginal ring impregnated with medroxyprogesterone acetate. Am J Obstet Gynecol 17:1, 197 6. Mishell DR, Jr, Moore D, Roy S, Brenner PF, Page MA: inical performance and endocrine profiles with contraceptive vaginal rings containing a combination of estradiol and d-norgestrel. Am J Obstet Gynecol 13:55, 1978 7. Victor A, Johansson DB: Contraceptive rings: selfadministered treatment governed by bleeding. Contraception 16:137, 1977 8. Thorneycroft H, Stone SC: Radioimmunoassay of serum progesterone in women receiving oral contraceptive steroids. Contraception 5:129, 1972 9. Brown JB, Machaughtan C, Smith MA, Smyth B: Further observations on the Kober colour and trich fluorescence reactions in the measurement of oestriol, oestrone and oestradiol. J ndocrinol4:175, 1968 1. dqvist L, Johansson DB: Radioimmunoassay of estrone and estradiol in human and bovine peripheral plasma. Acta ndocrinol (Kbh) 71:716, 1972 11. Lindberg BS, Lindberg P, Martinsson K, Johansson DB: Radioimmunological methods for the determination of estrone, estradiol-1713 and estriol in pregnancy plasma. Acta Obstet Gynecol Scand [Suppl) 32:5, 1974 12. Wide L, Nillius SJ, Gemzell C, Roos P: Radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. Acta ndocrinol [Suppl) (Kbh) 174:8, 1973 13. Lin TJ, Billiar RB, Little B: Metabolic clearance rate of progesterone in the menstrual cycle. J in ndocrinol Metab 35:879, 1972 14. Baird DT: Ovarian steroid secretion and metabolism in women. n The ndocrine Function of the Human Ovary, dited by VHT James, M Serio, G Giusti. London, Academic Press, 1976, p 125 15. Larsson-Cohn U, JohanBBon DB, Gemzell C: ffects of continuous administration of.3 mg of norethindrone on the plasma levels of progesterone and the urinary excretion of pregnanediol and total estrogens. Acta ndocrinol (Kbh) 64:38, 197 16. Thompson, Arfania J, Taymor ML: ffects of estrogen and progesterone on pituitary response to stimulation by luteinizing hormone-releasing factor. J in ndocrinol Metab 37:152, 1973 17. Netter A, Gorins A, Thomas K, Cohen M, Jotinaux J: Blocage du pic d'ovulation de LH et de FSH par la progesterone a faibles doses chez la femme. Ann ndocrinol (Paris) 34:43, 1973 18. Weiner, Johansson DB: Plasma levels ofd-norgestrel, estradiol and progesterone during treatment with Silastic implants containing d-norgestrel. Contraception 14:81, 1976 19. Weiner, Johansson DB: Contraception with d norgestrel Silastic rods. Plasma levels of d-norgestrel and influence on the ovarian function. Contraception 14:551, 1976 2. Spies HG, Niswender GD: Blockade on the surge of preovulatory serum luteinizing hormone and ovulation with exogenous progesterone in cycling rhesus (Macaca mulatta) monkeys. J in ndocrinol Metab 32:39, 1971