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Effects of Subcutaneous Implantation or Intrauterine Insertion of Silastic Tube Containing Steroids on the Fertility of Rats

J. H. CASAS AND M. C. CHANG

Worcester Foundation for Experimental Biology,

Shrewsbury, Massachusetts 01545

Received October 15, 1969

Two mg of chlormadinone acetate, estradiol benzoate, ethinyl estradiol, or 6 mg of progesterone in a Silastic tube 1 cm long were subcutaneously implanted or inserted into one uterine horn of rats for 24-60 days and the absorption rate of these compounds was determined. Subcutaneous implantation of progestin did not increase body weight, nor relative oviductal and uterine weight, but increased ovarian weight slightly. Estrogen, however, increased uterine weight significantly. When inserted into one uterine horn, progestins increased the body weight while estrogen decreased body weight.

Subcutaneously implanted progestins alone were not effective in preventing pregnancy ; estrogens alone or in combination with progestins prevented pregnancy but not estrus and mating. Following the removal of implants after 45 days, the females mated and variable numbers of pregnant animals, implantation sites, and living fetuses were observed. When the Silastic tube containing progestin was inserted into one uterine horn, implantation did not occur in the treated horn but implantation sites and living fetuses were observed only in the contralateral horn as in controls. When the tube containing estrogen was inserted into one uterine horn, a systemic effect occurred and no implantation was observed in either horn. Thirty days after the removal of the tube from one uterine horn, variable numbers of pregnant animals and that of implantation sites in  either uterine horn were observed.

Histological examination revealed the enlargement of corpora lutea in the ovary, an increase of neurosecretory materials, a decrease of basophil cells, and an increase  of acidophil cells of the pituitary of estrogen-treated animals.

The antifertility activity of various steroids and related compounds administered orally or  subcutaneously at different times with various doses and their probable mode of action have been reported in different animal  species (Banik, et al., 1969;  Chang, 1966; Chang, 1967a,b;  Chang and  Yanagimachi, 1965; Schally et al., 1968; Yanagimachi and Sato, 1968) and on human beings (Mishell et al., 1968; Pincus, 1966).

Silastic tubes have been found to be useful as a subcutaneous implant because they do not cause local reactions even for prolonged periods (Folkman and Long, 1964) and because steroids placed in a silicone tube canpass through its wall in vitro and in vivo, (Chang  and  Russfield, 1968; Dziuk and Cook, 1966). The inhibition and control of estrus in ewes by subcutaneous implant of silicone rubber impregnanted with progestin (Dziuk et al., 1966; Dziuk et al., 1968) and the retention of such a preparation inserted in the uterus of rats (Doyle and Clewe, 1968) have been reported. This paper presents observations concerning the rate of absorption, the effect of estrogens and progestins as antifertility agents, and their probable mode of action when placed  into Silastic tubes, either subcutaneously implanted or inserted into the uterus.

MATERIALS AND METHODS

The compounds used in the present study were progesterone, 6 mg per tube; chlormadinone acetate, 2 mg per tube; estradiol benzoate, 2 mg per tube; in Bouin’s Holland fixative and stained according to ethinyl estradiol, 2 mg per tube; or the combination of estrogen and progestin placed in a Silastic tube and an empty Silastic tube  as control, 1 cm  in length and closed with medical adhesive (Type A, No. 891, Dow  Corning  Corporation). For subcJtaneous  implantation, 0.192 in. o.d. and 0.104 in. i.d. tubes (No. 601-321) were used. For intrauterine insertion, 0.125 in. o.d. and 0.078 i.d. tubes (No. 602-301) were used. Fema’e  rats,  weighing  between 180  and 220 g obtained frcm Charles River Breeding Co. were used.  Vaginal smears were taken for 5  days. On the 5th day  the  tube  was  subcutaneously  implanted  under  the skin in the neck region or inserted into one uterine horn. Smears were taken for an additional 5 days. For the determination of absorption  rates, 2  animals of each group of 10 were killed daily on 24th to 28th day after subcutaneous implantation, or 36-60  days  after  the  insertion  of the tube into the utens. The tubes were removed and the remaining compound was measured by spectrophotometric determination and corrected according to Allen (1950). The  body  weight, ovarian, oviductal, and uterine weights were recorded and the presence or absence of ovulation was ascertained by the presence of corpora lutea on the ovaries or that of eggs in the tube. For the  determination  of  fertility, 15 days after the  subcutaneous implantation or  intrauterine insertion  of the tube, two sexually  mature !flees of proven  fertility  were introduced into each  cage of Five females. Vaginal smears were taken daily. Day 1  was the day on which sperm were found in the smear.  Two  anima’s  from  each  group  were killed on day 1 for the determination of fertilization. On day 8 laparotomies were done in order to determine the  number  of  implantation  sites.  On  day 18 all pregnant animals were killed. The number of eggs, the  implantation  sites  and  living  fetuses  were  recorded. The  animals  that  did  not  become  pregnant  as proved by laparotomy on day 8 were placed with males on day 13 after the first mating. They were laparotomized again on day 8 after the second time of introduction  of males. The capsules  were  taken out 45 days after subcutaneous implantation or 36-60 days after insertion into the uterus and vaginal smears were taken daily. After the animals showed two apparently normal cycles, approximately 30 days after the removal of the tube, they were placed with males for the third time. At autopsy the number of pregnant  animals, site  of  implantation,  and  living fetuses were recorded. For histological study  the  ovaries,  oviducts, and uteri were fixed in Bouin’s fixative and stained with hematoxylin and eosin. Pituitary glands were fixed PAS orange G procedure. For statistical analysis  of  results  the  Duncan’s multiple range  test at the 5%  level of significance was employed.

RESULTS

Absorption of the Steroids

Following  subcutaneous  implantation  of the tube the average absorption rate in micrograms  as determined  on  days 24-28 was progesterone 84.32 per animal/day,  chlormadinone acetate 10.76 per animal/day, estradiol benzoate 13.91 per animal/day, and ethinyl estradiol 21.52 per animal/day (Table 1). When the tubes containing estrogen or progestin were inserted into one of the uterine horns the absorption rate in micrograms was progesterone 31.80 per animal/day, chlormadinone acetate 9.90 per animal/day, estradiol benzoate 8.90 per animal/day, and ethinyl estradiol 12.05 per animal/day (Table 2). From  the  results  presented  in Tables 1 and 2 it appears that the absorption rate was slightly higher when the tube was implanted under the skin rather than inserted into  the  uterus.  Moreover,  the  absorption rate of progesterone was much higher in both cases. This is not due to a large amount of progesterone (6 mg instead of 2 mg) placed  into each tube because the absorption rate averaged 81  tg when 2 mg progesterone was placed into each tube.

Effects on Body and Organ Weight

Following subcutaneous implantation, the body weights and the relative uterine and oviductal  weights to body weights  were neither increased nor reduced by implantation of progesterone or chlormadinone acetate as compared  with  control  animals. Following subcutaneous implantation of ethinyl estradiol, a decrease of body weight was observed (Table 1). Following insertion of estrogens into the uterus, the body weight was neither increased nor decreased significantly as compared with control. Insertion of tubes containing progestins however, induced a significant increase of body weight (Table 2). As shown in Table 3, the ovarian weight was significantly increased after subcutaneous implantation of progestins but significantly decreased after subcutaneous implantation of estrogens. There were no significant changes of oviductal and uterine weights following subcutaneous implantation of progestin. Subcutaneous implantation of estrogen, however caused a significant increase of uterine weight.

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Effect on Fertility

Results presented in Table 4 reveal that neither progesterone nor chlormadinone acetate alone was effective in preventing pregnancy during a period of 45 days. The number of eggs, implantation sites, and living fetuses were not significantly different from those of control animals. However, estrogens alone or in combination with progestins were effective in preventing pregnancy for a period of 45 days. Fifteen days after the removal of the tube apparently  normal  cycles appeared in the animals and by day 30 all the animals had at least two normal cycles. When the females were  caged  with  males,  mating  occurred within 1-5 days. The numbers of pregnant animals varied, however, according to different treatments (Table 5). A significant reduction of implantation sites when examined on day 8, however, was found in the rats previously treated with ethinyl estradiol alone, ethinyl estradiol with progesterone or  with chlormadinone acetate, and estradiol benzoate with chlormadinone acetate. The number of ilving fetuses as examined on day 18 were also significantly lower in the rats previously implanted with estrogen alone or in combination of progestin, except in the rats implanted with  Silastic tube containing progesterone and estradiol benzoate. When Silastic tubes containing estrogens or progestins were inserted into one uterine horn for 15 days and the rats were mated implantation sites or living fetuses were not found in the uterine horn where the Silastic tube was inserted. Exception in the control animals however was observed (Table 6). When a Silastic tube containing progestin was inserted into one horn, implantation and living fetuses were obtained in the other horn.

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When a Silastic tube containing estradiol benzoate  or ethinyl  estradiol  was inserted into one horn no implantation was obtained in the other horn. This shows that an empty Silastic tube or a tube containing either estrogen or progestin inserted in one uterine horn prevents the implantation in that horn by virtue of its action as an intrauterine device.

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When a Silastic tube containing estrogen, not progestin, was inserted into one uterine horn a systemic effect occurred as implantation was prevented also in the control uterine horn. When the Silastic tube was removed from one uterine horn 30 days later and the animals were mated again, fetuses were found in the uterine horn where the empty tube was inserted and in the uterine horn where the tube was not inserted in the control rats. In the rats in which estradiol benzoate in the tube had been inserted into one uterine horn, a small number of fetuses was found in the control horn but not in the experimental horn due to infection. In the rats in which ethinyl estradiol in the tube had been inserted into one uterine horn, the number of fetuses was significantly higher in the experimental than in the control horn (Table 7).

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Histological Studies

Inhibition of  follicular  development  and enlargement of corpora lutea were noticed in estrogen-treated animals 24-60 days after the implantation  of  tube. Their  corpora lutea were enlarged and similar to corpora lutea of pregnancy (Figs. 5, 6)  although some of them showed signs of regression. There was stimulation of interstitial  tissue in some places and their cells  showed  vacuolated cytoplasm. The effect on the ovary of estradiol  benzoate appeared less  than  that of ethinyl estradiol. In the progestin-treated animals the surface epithelium  of uterine lumen had low columnar cells and showed subnuclear vacuoles. The uterine glands were tortuous and their  epithelium  cells  had  vacuolar  base. Stroma were increased in interstitial structures with edema in the subepithelial layer in the endometrium, indicating general characteristics of progestational stimulation. The surface epithelium of the uterus had high columnar cells and showed invaginations in the estrogen-treated animals. Their uterine glands were straight, dilated, some of them being  tortuous.  The epithelium had high columnar cells with nucleus lying on the base of the cells and with vacuolated cytoplasm. The stroma showed  edema  and  increased cellular components characteristic of estrogenic  stimulation.  The  effect  of  estradiol benzoate appeared to be less than that of ethinyl estradiol. In the pituitary gland an increase  of neurosecretory material was seen in the pars nervosa of rats treated with progestins. Pars intermedia were slightly enlarged and the cells also showed strong basophilia reaction in  the  progestin-treated animals. Acidophil cells in the pars distalis were apparently normal in number, some of them showing mitosis. Basophil cells of different types with PAS-positive  material,  probably  LH and  FSH secretory cells, were increased. The sinusoids showed an increased amount of PAS-positive material in some places. The effect on the pituitary of progesterone was less than that of chlormadinone acetate. Following the treatment with estrogen the pars nervosa contained a large amount of neurosecretory material, the pars intermedia were slightly enlarged with basophil cells, which  showed a granular cytoplasm with PAS-positive material. An increase of acidophil cells was seen in the pars distalis, (Figs. 11, 12) indicating probably prolactin type cells, but some had pycnotic nuclei  and  others showed mitoses. Basophil cells were scarce. The effect on the pituitary of estradiol benzoate was less than that of ethinyl estradiol.

DISCUSSION

From the results of the present study, it appears that chlormadinone acetate, progesterone, ethinyl estradiol, and estradiol benzoate passed through the wall of the Silastic tube at a rather constant rate in vivo. This confirms the observation of Dziuk and Cook (1966)  who  reported  that  steroids passed through Silastic tube at a quite constant rate in vivo and in vitro. Chang and Russfield (1968) reported that the absorption rates of norethynodrel and mestranol in mice were not constant and showed exponential curves for a period of up to 3 months, and the diffusion rate was less variable in vitro. Since the same length but different grade of Silastic tube was used in our experiments the difference in rate of absorption between various steroids might be affected by the wall thickness, the polarity, and the presence of side chains on the steroid’s nucleus, as had been suggested by Dziuk and Cook (1966). Thus, in addition to the weight of steroid placed in the tube, these other factors may affect the absorption rate. Subcutaneous implant  of silicone rubber impregnated with a progestogen can be used to inhibit and control ovulation in the ewes (Dziuk et al., 1966; Dziuk et al., 1968) and it has been reported that progestin prevents pregnancy  in  the  rat (Doyle  and  Clewe, 1968).

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The  present  study  revealed  that progestin had no antifertility activities under our  experimental conditions. The minimum effective daily doses of chlormadinone acetate and progesterone by subcutaneous injection to produce significant antifertility effects has been reported to be 5000 to 15,000 lig, re spectively in rats by Kind  and  Dorfman (1965).  The lack of antifertility effect by implantation of progesterone  and  chlormadinone acetate in Silastic tube in the present study may be due to an insufficient amount of progestin absorbed by the animal. However, estrogen alone or in combination with progestin can be used as antifertility agents. The principal site of action by steroids may be in the central nervous system and in the pituitary. Minaguchi and Meites (1967) and Schally et al. (1968) suggested that estrogens might inhibit the release of LH-RF and FSH-RF through the hypothalamus. Moreover, Dorfman and Dorfman (1963), McCann and Taleisnik (1961), suggested  that  estrogens could inhibit the release of both FSH and LH from the pituitary and thus prevent ovulation. Watnick et al., (1964) suggested that progesterone produced by the corpora lutea may act independently or together with estrogens to inhibit the release of LH. In view of the fact that estrus and mating occurred in all the treated animals as revealed in the present investigation,  inhibition  of  ovulation with persistent estrus may be the major cause of estrogen antifertility activity. It is known that the life span of the corpus luteum could be prolonged by direct effect of estrogens as suggested by Banik et al., (1969). The antifertility activity of estrogen may be due also to a certain disturbance of the existence of active corpora lutea. Our results suggest that estrogens could maintain the life span of the corpus  luteum  either  by  direct  effect  or through the hypothalamus and pituitary and thus cause certain disturbances in ovulation, fertilization, egg transport, and implantation. Progestin, however, had no antifertility effects  under  our  experimental   conditions. Moreover,  since  accurate  equilibrium  between estrogen and progesterone is required for ovum  implantation (Psychoyos, 1966), it is more likely that antifertility effects of estrogen  as  revealed  in  the present study may have affected the mechanism of implantation. The long period necessary (30 days) of recovery of a normal estrous cycle after the removal of the implant may indicate a long potent effect by such treatment.

ACKNOWLEDGMENTS

This study was supported from a grant from the Ford Foundation and a grant (HD-03003)  from the National Institute of Child Health and Development of the U.S. Public Health Service.

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