C ONSIDERABLE ATTENTION has been given to the antifertility effects of

Transcription

1 Effects of Clomiphene at Different Stages of Pregnancy in the Rat Implications Regarding Possible Action Mechanisms 0. W. DAVIDSON, M.D.,"' K. WADA, M.D.,t and S. J. SEGAL, Ph.D. C ONSIDERABLE ATTENTION has been given to the antifertility effects of clomiphene and structurally related compounds. When these findings were first reported, Holtkamp et al. suggested that the compound may be a pituitary gonadotrophin inhibitor andjor may act as an antiestrogen and that these activities could account for the loss of fecundity observed in treated animals. Subsequently, Segal and Nelson found that clomiphene inhibits fertility if administered immediately postfertilization and before nidation of blastocysts. They suggested that the compound may act directly on developing zygotes to cause lysis. Further experiments on this question demonstrated that the compound interferes with the fertility of lactating inseminated females if administered during the period of nidation delay, while blastocysts are free in the uterus. 6 This work established that the period of effectiveness of the compound can be extended by prolonging the preimplantation period of the free blastocyst, thus supporting the concept of a direct action. In the present report, evidence is set forth on the effect of the compound at various stages of pregnancy in the rat. The results suggest that the compound may act in different ways, depending on dose and time of administration. MATERIAL AND METHODS Female rats of the Holtzman strain were observed through a normal pregnancy and lactation period before being used in the experiments. Animals that resumed normal cycles were placed in experimental groups. From the Laboratory of The Population Council, The Rockefeller Institute, New York, N.Y. *This work was done during the tenure of fellowships from the Conse.io Nacional de Investigaciones, Buenos Aires, Argentina, and The Population Council, New York, N.Y. ton leave from the Department of Applied Physiology, Tohoku University School of Medicine, Sendai, Japan. 195

2 196 DAVIDSON ET AL. FERTILITY & STERILITY These females were 'caged with proved fertile males and the vaginal smears examined daily for spermatozoa. The day on which sperm were found in the smear was recorded as Day 1 of pregnancy. As fertilization occurred, animals were assigned at random to experimental and control groups. In order to obtain animals with lactational delay of implantation, pregnant females on the eighteenth day of pregnancy were caged with fertile males. After delivery, vaginal smears were taken twice a day. The day on which sperm were found in the vagina was recorded as Day 1 of the postpartum pregnancy. Only rats nursing a litter of six or more were used. Clomiphene was dissolved in a small quantity of warm chloroform and the solution suspended in olive oil. The chloroform was removed under vacuum. The suspension was administered orally, by stomach tube, in all experiments. Animals received a volume of 0.1 ml.j100 gm. body weight. Controls received similar volumes of olive oil. Precoital Treatment RESULTS When clomiphene was administered daily for 8 days ( 30 p.g.j100 gm. body weight) to isolated females who were immediately exposed to males, some accepted the male immediately while others did not mate for as long as 15 days. When the interval between treatment and mating was less than 3 days, no implantations could be noted at laparotomy on Day 10 postfertilization, and there was no evidence of resorption sites. Animals which mated at least 3 days after cessation of treatment had a normal percentage of successful implantations and deliveries (Table 1). Treatment in Early Pregnancy (Before Day 10) Table 2 shows that at the dose of 30 p.g.j100 gm.jday the compound supressed fertility completely when administered during the first 4 days of pregnancy. Following treatment during postcopulatory Days 5-8, a TABLE 1. Results of Precoital Administration of Clomiphene ( 30 ftg./100 gm. for 8 Days) Interval before mating (days) No. animals No. deliveries Per cent fertile matin,q Same females, control matings

3 VoL. 16, No. 2, 1965 CLOMIPHENE IN PREGNANCY 197 TABLE 2. Results of Postcoital Administration of Clomiphene (30,ug./100 gm./ day) to Nonlactating Females Treatment days No. animals of pregnancy No. animals implanted No. deliveries Control normal percentage of females had implantations on Day 12 (as observed by laparotomy), but only 50% maintained pregnancy to parturition. Daily treatment on Days 7-10 postcoitus influenced neither implantation nor postimplantation pregnancy. Single-day treatment (30 /Lg.j100 gm. three times a day) on Day 1, 2, or 3 prevented maintenance of pregnancy. Although fertilized ova could be recovered from the fallopian tubes up to Day 4, at laparotomy on Day 10 no evidence of nidation sites was seen. As shown in Table 3, 11 of 14 animals had no implantation sites following treatment on Day 4. The remaining animals had a reduced number of implantations, and only 45% of these survived the complete pregnancy. Single-day treatment on Day 5 or 6 did not interfere with the normal number of implantations observed on Day 10, but there was a slight reduction of survival rate of implanted embryos. Treatment after Implantation Daily treatment from Day 8 postcoitus through Day 22, the expected day of parturition, caused a slight reduction in the percentage of developing TABLE 3. Results of Single-day Postcoital Administration of Clomiphene ( 90,ug./100 gm.) Survival of Av. No. nidations Av. No. viable nidated embryos Day after coitus No. females on Day 10 newborn ( o/o) Control

4 198 DAVIDSON ET AL. FERTILITY & STERILITY TABLE 4. Results of Daily Administration of Clomiphene (30 ftg./100 gm.) on Days 8-22 Group* Av. No.- 'implantations on Day 10 Av. No. fetu. es on Day 22 Av. No. viable newborn Control ( 28) Treated ( 33) *Control group was composed of 8 females laparotomized on Day 10 and sacrificed on Day 22, and 20 females allowed to deliver normally at term. Treated group was composed of 11 females laparotomized on Day 10 and sacrificed on day 22, and 22 females allowed to deliver normally at term. fetuses between Day 10 and Day 22 (as observed by laparotomy in both instances). The drastic effect of this treatment was the loss of full-term fetuses due to fetal distress during parturition. An average of 1.8 viable newborns were delivered, compared with 10.0 average number of viable preparturition fetuses (Table 4). The most frequent cause of fetal death was incomplete labor; in other cases following prolonged labor the mother delivered the full litter, but all were dead. When the treatment was confined to 4 days before expected day of delivery, the dose of 30 1Lg.j100 gm. did not affect fetus viability or parturition. However, larger doses reduced by almost 50% the number of viable newborn as compared with the number of implantations observed at laparotomy on Day 10 (Table 5). Treatment of Lactating Females During Postpartum Pregnancy Postpartum estrus and ovulation in the rat lead to pregnancy in lactating rats if newly delivered females are exposed to fertile males. In our colony 80% of postpartum matings result in full-term pregnancies. The pregnancies range in length from 23 to 34 days. Almost always there is a delay in implantation of from 5 to 12 days while blastocysts remain free and viable in the uterine cavity. Hence, the preimplantation period for lactating pregnant females can be as long as 18 days, while the preimplantation period TABLE 5. Results of Preparturition Administration of Various Doses of Clomiphene on Days Dose (J-Lg./100 gm.) No. animals Av. No. 'implantations A v. No. living young

5 VoL. 16, No.2, 1965 CLOMIPHENE IN PREGNANCY 199 TABLE 6. Results of Postcoital Administration of Clomiphene ( 30 p.g./100 gm./ day) to Lactating Females Treatment days Length of pregnane]! post coitus No. animals No. deliveries (days) Control" *Controls were pregnant lactating females, given olive oil orally for 4 days during expected period of delay of implantation. for nonlactating pregnant females is no longer than 6 days. It may be recalled that clomiphene treatment in nonlactating pregnant females was ineffective if started after Day 6 (Table 2). Of 53 lactating pregnant females treated with 30 flg.j100 gm. from Days 7-10, 9-12, or 10-13, only 20% delivered litters (Table 6). In these deliveries, it can be calculated from the length of pregnancy that there was a very brief period of implantation delay, so that treatment was administered after implantation had occurred. DISCUSSION Previous work with clomiphene and its analogue ethamoxytriphetol led to the suggestion that these compounds possess a specific antifertility action, with the time of action limited to the period between fertilization and blastocyst implantation. The present results confirm these findings but, at the same time, indicate that the concept of time-specific activity may be an oversimplification. Precoital treatment with clomiphene has no effect on fertility if the interval between treatment and mating is longer than 2 days. However, treatment that is extended closer to the time of mating is as effective in preventing pregnancy as is postfertilization treatment. Since such animals achieve sexual estrus, accept males, and ovulate, as evidenced by vaginal-smear records, it may be assumed that precoital treatment does not prevent normal ovarian cyclicity. This finding leads us to conclude that a dose and treatment schedule that does not interfere with normal gonadotrophic stimulation for follicle development and ovulation is capable of complete antifertility activity so that at least under these circumstances the reported antigonadotrophic activity of clomiphene need not be involved in the antifertility effect. Since similarly treated animals are capable of forming deciduomata if the uteri are traumatized on Day 5 postfertilization, it appears that normal ovarian steroid production is maintained when the antifertility effect is absolute.

6 200 DAVIDSON ET AL. FERTILITY & STERILITY Barnes and Meyer found that animals treated throughout the preimplantation stage of pregnancy with a high dose of clomiphene ( 'g.jlOO gm.) had no implantation sites on Day 8 and that no ova were recoverable. This observation supports the suggestion that nonimplanted zygotes are susceptible to lysis under clomiphene treatment. In order to test this hypothesis further, we obtained data on the effect of the compound on blastocysts in an extended period of delay of implantation. (Preliminary results have been reported earlier). 6 The condition of lactational delay, used in these experiments, has several limitations in that the period of delay can be variable and the hormonal conditions of lactation and pregnancy cannot be placed under experimental control. Nevertheless, the results show that clomiphene treatment during the period of delay prevents subsequent implantation of blastocysts. It is not apparent from these results whether the effect is directly on the delayed blastocysts or on the hormonal status of the lactating pregnant female. Since clomiphene has mild estrogenic and antiestrogenic activity, it cannot be discounted that interference with normal estrogen levels at the endometrium could prevent implantation subsequent to treatment. Further observations relating to this question are reported elsewhere. 4 The interpretation that the effect is, indeed, on the blastocysts is supported by the observation of Davidson et al. that a 50% reduction in recoverable blastocysts occurs within 48 hr. after clomiphene treatment during lactational delay. Daily administration of 30 1-'g.jlOO gm. clomiphene from the time that implantations have been established until the expected day of delivery has no significant effect on the number of viable fetuses that develop, but only a small percentage of these are delivered normally by the mother. The remaining fetuses die in utero as a result of incomplete or prolonged labor. This effect can be observed also if the treatment is confined to the last 4 days of pregnancies, but with this limited period of treatment, higher dosages are required. This confirms the observation of Barnes and Meyer. The compound's activity at this late stage of pregnancy appears to be on the mechanism controlling parturition, most likely the hormonal mechanism. The nature of the interference suggests that the compound prevents normal and complete uterine contraction; this could be a result of an antiestrogen influence. The general term antifertility action covers a broad spectrum of possible interferences. Clomiphene appears to exert this effect in different manners at different points in the reproductive sequence of events, and by different mechanisms. lnstituto de Anatomia General Y Embriologia Facultad de Medicina Paraguay 2155 Buenos Aires, Argentina

7 VoL. 16, No.2, 1965 CLOMIPHENE IN PREGNANCY 201 REFERENCES I. BARNES, L. E., and MEYER, R. K. Effects of ethamoxytriphetol, MRL-37, and clomiphene on reproduction in rats. Fertil. & Steril. 13:412, DAVIDSON, 0. W., ScHUCHNER, E. B., and WADA, K. Effects of clomiphene on rat zygotes. Anat. Rec. 148:214, HoLTKAMP, D. E., GRESLIN, S. C., RooT, C. A., and LERNER, L. J. Gonadotrophin inhibiting and anti-fecundity effects of clomiphene. Proc. Soc. Exper. Biol. & Med. 105:191, PRAsAD, M. R. N., KALRA, S. P., and SEGAL, S. J. Effect of clomiphene on blastocysts during delayed implantation in the rat. Fertil. & Steril. 16:101, SEGAL, S. J., and NELSON, W. 0. Anti-fertility action of chloramiphene. Anat. Rec. 139:213, SEGAL, S. J., and DAVIDSON, 0. W. Prolonged anti-fertility action of clomiphene in delayed implantation. Anat. Rec. 142:218, Conference on Gamete Transport, Fertilization, and Preimplantation A conference on Gamete Transport, Fertilization, and Preimplantation Mechanisms, sponsored jointly by The Population Council, Inc., and the Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, will be held on the Vanderbilt Campus May 19-21, Twentysix papers will be presented by authorities from nine countries. The conference is open to physicians and scientists on application to the Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tenn