Low-dose antiprogestin treatment prevents pregnancy in rhesus monkeys and is reversible after 1 year of treatment

S.M. Borman1, K.M. Schwinof1, C. Niemeyer2, K. Chwalisz3,5, R.L. Stouffer1,4 and M.B. Zelinski-Wooten1,4,6

Divisions of 1 Reproductive Sciences and 2 Animal Resources, Oregon National Primate Research Center, Beaverton, OR 97006 USA, 3 Fertility Control and Hormone Therapy Research, Research Laboratories of Schering AG, D13342 Berlin, Germany and 4 Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97201, USA 5 Present address: TAP Pharmaceutical Products, 675 N. Field Dr., Lake Forest, IL 60045, USA 6 To whom correspondence should be addressed at: Oregon National Primate Research Center, 505 N.W. 185th Ave, Beaverton, Oregon 97006, USA. e-mail: zelinski{at}ohsu.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Administration of low doses of an antiprogestin to rhesus monkeys permits ovarian/menstrual cyclicity, suppresses endometrial proliferation and prevents pregnancy without adverse or toxic side-effects after 5–6 months of daily treatment. The purpose of this study was to test the reversibility with respect to restoration of fertility after 1 year of low-dose antiprogestin treatment. METHODS: This experiment included a daily 1 year vehicle- or antiprogestin-treatment interval followed by a 9 month post-treatment interval for adult, female rhesus monkeys (n = 5/group) of proven fertility and exhibiting regular menstrual cycles. Co-habitation occurred with a male of proven fertility and vaginal swabs were taken to identify the presence of sperm during the treatment (antiprogestin females) and post-treatment intervals (vehicle and antiprogestin females). RESULTS: Mating and vaginal sperm were evident in all antiprogestin females during, and, in both groups, after treatment. Based on ultrasonography, none of the antiprogestin-treated females became pregnant during the treatment interval. However, upon cessation of treatment, pregnancy rates were similar between antiprogestin-treated (3/5) relative to vehicle-treated (4/5) females with live, healthy infants born in both groups. There were no differences between groups in fetal measurements, gestation lengths, live birth rates and infant weights. CONCLUSIONS: The reversal of the anti-fertility effects of chronic, low-dose antiprogestin treatment supports the clinical feasibility of potent and selective antiprogestins as potential contraceptives for women.

Key words: antiprogestin/contraception/pregnancy/reversibility/ZK 137 316


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antiprogestins represent a potential contraceptive option, which may produce alterations at several sites in the reproductive tract that individually or collectively prevent pregnancy during the menstrual cycle. Previous studies examining fertility regulation using antiprogestins in women and non-human primates have focused on the acute administration of large, single doses. RU 486 (mifepristone) disrupts the development of the dominant follicle, prevents the surge for LH, induces premature menstruation, and prevents pregnancy only when given at specific stages of the menstrual cycle (Van Look and von Hertzen, 1995Go). Large doses of antiprogestins may not be ideal for continuous contraceptive use since they disrupt ovarian and menstrual cyclicity. While once-a-month administration of high doses of antiprogestins would impair fertility during the treatment interval and can successfully prevent pregnancy in women (Gemzell-Danielsson et al., 1993Go) and non-human primates (Ghosh et al., 1997Go), the feasibility of using them as contraceptives is less practical due to the critical timing required for treatment and the added cost of accurately determining the LH surge every month.

Chronic treatment with low doses of antiprogestins may therefore comprise a new mode of regulating fertility. Previously, women given low doses of RU 486 demonstrated delayed ovulation, but normal ovarian cyclicity (Batista et al., 1992Go), prolonged follicular phase without disturbing the overall cycle length (Croxatto et al., 1993Go), suppressed endometrial maturation (Batista et al., 1992Go) and reduced levels of uterine-derived proteins (Gemzell-Danielsson et al., 1997Go), thereby supporting the possibility of using antiprogestins as contraceptives since endometrial development was delayed. Clinical studies with RU 486 suggest low-dose regimens that maintain ovarian cyclicity are less effective contraceptives and that the higher doses needed for continuous contraception which disrupt ovulation and menstrual cyclicity (Van Look and von Hertzen, 1995Go; Marions et al., 1998Go, 1999; Brown et al., 2000Go, 2002).

In the search for more effective contraceptives, various ZK compounds (Schering AG, Berlin, Germany) were used as tools to further investigate the use of antiprogestins for pregnancy prevention while maintaining normal cyclicity. ZK compounds have a higher endometrial selectivity and higher affinity for the progesterone receptor; ZK 137 316 is ~3–10-fold more potent and demonstrates reduced antiglucocorticoid activity compared with RU 486 (Chwalisz et al., 2000Go). Like RU 486, ZK 98.299 (onapristone) and ZK 137 316 act as both an antiprogestin and antiproliferative agent in the reproductive tract of female rhesus monkeys (Slayden and Brenner, 1994Go; Slayden et al., 2001aGo). When low doses of ZK 98.299 (2.5 and 5.0 mg/kg) were administered to adult bonnet monkeys every 3 days during the early luteal phase for 4–7 cycles, pregnancy was prevented by inhibiting endometrial receptivity and blocking implantation without initially affecting the ovarian and menstrual cycles, but as treatment progressed cyclicity was suppressed (Katkam et al., 1995Go). However, treatment for 5–6 consecutive months with very low doses of ZK 137 316 (0.01–0.03 mg/kg) permitted continued ovarian and menstrual cyclicity in rhesus monkeys (Zelinski-Wooten et al., 1998aGo), while inducing a dose-dependent atrophy of the endometrium (Slayden et al., 1998Go) and preventing pregnancy without toxic or adverse side effects (Zelinski-Wooten et al., 1998bGo). In addition, Marions et al. (1998Go) was the initial study to observe pregnancy prevention with 5 mg RU 486 when given once a week to women. Low-dose antiprogestin treatment can prevent pregnancy, but the effects are dose-dependent and the type of antiprogestin used is important for success.

An initial study was performed to determine if female monkeys could conceive and give birth to offspring following our previous 6-month contraceptive trial (Zelinski-Wooten et al., 1998bGo). But, in order for a continual regimen of low-dose antiprogestins to be considered for clinical use, a more rigorous test of contraceptive ability following longer intervals was necessary to further investigate the interval to conception and pregnancy outcomes immediately following cessation of treatment. The objective of this study was to test the reversibility of continuous, low-dose antiprogestin treatment with respect to restoration of fertility after cessation of 1 year of daily i.m. treatment.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatments
Adult, female rhesus monkeys exhibiting normal body weights (4.5–6 kg) and regular menstrual cycles of ~28 days were kept under controlled conditions of temperature (22°C) and a standard daily 12 h light:12 h dark cycle. Each female had previously given birth to one or more live, normal, singleton infants prior to the studies. To obtain initial information on the reversibility of this regimen, animals (n = 5) from our previous trials with daily low-dose ZK 137 316 (Zelinski-Wooten et al., 1998aGo,b) were monitored post-treatment when they went into the timed mated breeding colony at ONPRC. In the current study, female monkeys were assigned randomly (n = 5 per group) to receive at 0830–0900 a single, i.m. injection of vehicle [25% ethanol/37.5% propylene glycol (Sigma, St Louis, MO, USA)/37.5% saline, v/v/v, 0.5 ml] or 0.03 mg ZK 137 316 (Schering, AG) per kg body weight for 1 year (Zelinski-Wooten et al., 1998aGo). This dose of ZK 137 316 was chosen based on previous studies from our laboratory demonstrating that 0.03 mg/kg was the lowest dose that prevented pregnancy while maintaining normal ovarian and, in some cases, menstrual cyclicity (Zelinski-Wooten et al., 1998aGo,b). The study consisted of a 1 year treatment/contraceptive interval (October–June) and a 9 month post-treatment/fertility restoration interval (October–June). During the first month of the treatment interval, individual antiprogestin-treated females were placed adjacent to a male of proven fertility in cages designed to house a breeding pair. A plexiglas barrier was positioned to physically separate the female from the male while allowing visual and olfactory exposure allowing the animals to acclimate to the co-habitation caging system (Zelinski-Wooten et al., 1998bGo). The barrier was then removed and the animals allowed to co-habitate for the duration of the treatment and post-treatment intervals. Vehicle-treated females were singly housed during treatment and paired with a male during the post-treatment interval. Animals in each group were checked daily for overt menstruation during all cycles and the duration of menstruation was recorded. To confirm that mating occurred throughout the treatment interval in the antiprogestin group as well as post-treatment in both groups, each female had vaginal swabs taken during mid-cycle for microscopic identification of sperm.

Transabdominal ultrasonography (HDI 1000; ATL, Bothell, WA, USA), was performed on anaesthetized antiprogestin-treated females at various intervals during treatment and post-treatment in both groups when pregnancy was suspected based on menstrual records. Upon pregnancy confirmation, males were separated from females and pregnancies were allowed to progress until term. Ultrasonographic fetal measurements (greatest length, biparietal diameter, and femur length) were used to determine the dates of conception and delivery (Tarantal and Hendrickz, 1988Go). Fetal viability was determined by the presence of fetal heartbeat. Non-pregnant animals continued to be paired until the end of the post-treatment interval.

Body weights of each animal were obtained monthly. Blood samples were taken daily for a 2 week interval during the 4th and 9th months, and daily during the 7th month of treatment to confirm ovarian cyclicity based on patterns and levels of serum estrogen and progesterone. Based on our previous extensive observations that normal daily circulating steroid and gonadotrophin levels occurred throughout daily treatment with 0.03 mg ZK 137 316/kg for 5 consecutive months (Zelinski-Wooten et al., 1998aGo), three time-points were chosen during the treatment interval to confirm the maintenance of ovarian cyclicity. The incidence of menstruation was determined by dividing the total number of cycles a female overtly menstruated by the total possible menstrual cycles during the 12 month treatment or 9 month post-treatment interval multiplied by 100. The Clinical Pathology Laboratory at the Oregon National Primate Research Center (ONPRC) and Quest Diagnostics Incorporated (Portland, OR, USA) performed complete blood counts and serum biochemistry tests (including electrolytes, glucose, lipids, proteins, enzymes and metabolic by-products) before, during (4th and 9th months) and 10 days after treatment.

Reproductive behaviour
Female–male pairs were observed at 0800 each morning during treatment (antiprogestin) and post-treatment (antiprogestin and vehicle) for the presence of ejaculate under the cage, as well as mounting (which includes mounts with thrusting and intromission) and mating (mounting with intromission and ejaculation) behaviours. The frequencies of these reproductive behaviours were recorded for each pair. Of the five pairs in each treatment group, the number of pairs exhibiting these behaviours were compared within the antiprogestin group between months during treatment, as well as after treatment within a group and between groups.

Statistical analyses
Prior to conducting this study, we consulted a statistician at the Oregon Health & Science University, who used Ex Sample (Idea Works 3, Columbia, MO, USA) for power analysis and determining the sample size. Based on observations from our initial contraceptive study wherein the pregnancy rate in the control group was 90% (9/10 animals) (Zelinski-Wooten et al., 1998bGo) a power calculation using a one-tailed {chi}2-test (Ex Sample Idea Works, 3) was performed and determined that with five animals in each group for the present study a power of 0.08 at {alpha} = 0.05 will detect a difference in pregnancy rates of 20% assuming a pregnancy rate of 80% in the control animals. {chi}2-analysis was used to compare the number of female–male pairs that exhibited mounting and mating within and between groups and to compare the incidence of menstruation between the vehicle- and antiprogestin-treated groups with a P < 0.05 level of significance. Conception rates between control and antiprogestin-treated groups were compared by Fisher’s exact tests. Unpaired t-tests were used to analyse the interval to conception, gestation lengths and body weight of the offspring between the two treatment groups. One-way analysis of variance with one repeated measure was used to analyse blood parameters of non-pregnant females within each group over the course of treatment, followed by Neuman–Kuels test for comparison among means. Comparisons of blood parameters in all animals during the 4th, 7th and 9th months of treatment and 10 days post-treatment as well as between the antiprogestin group and the untreated females exhibiting normal menstrual cycles were made using paired and unpaired t-tests respectively.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although a detailed behavioural study (i.e. focal sampling) was not conducted, mounting with thrusting and intromission as well as mating was noted daily in both vehicle- and antiprogestin-treated pairs during and after the treatment interval. There were no differences between vehicle- and antiprogestin-treated groups with regard to the number of pairs that exhibited mounting or mating (data not shown). In addition, evidence of sperm after vaginal swabbing confirmed that mating occurred between each of the 10 pairs throughout the co-habitation period.

Our initial observations to determine the reversibility of contraception after 5–6 months of continuous, low-dose ZK 137 316 treatment in macaques was successful. Animals (n = 5) from the dose-ranging (Zelinski-Wooten et al., 1998aGo) and contraceptive (Zelinski-Wooten et al., 1998bGo) trials were subsequently reassigned to the timed mated breeding colony at ONPRC within 5–8 months after cessation of treatment. All monkeys became pregnant within 1–3 months of reassignment and delivered live, healthy infants (Table I).


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Table I. Initial observations of reversibility in female rhesus monkeys receiving daily administration of antiprogestin for 5–6 consecutive cycles
 
Table II shows the number of animals in the present study that demonstrated ovarian and menstrual cyclicity as well as number of conceptions during the year of daily treatment with either vehicle or antiprogestin (Table II). All animals in both groups demonstrated ovarian cyclicity (5/5 per group) based on estradiol and progesterone profiles. Peak levels of estradiol measured at mid-cycle did not differ between animals treated with vehicle (245 ± 80 pg/ml) or antiprogestin (223 ± 38 pg/ml). Likewise, peak levels of progesterone observed at mid-luteal phase were similar between vehicle- (3 ± 1.2 ng/ml) and antiprogestin-treated (5.5 ± 2.3 ng/ml) animals. There were no differences in the duration of menstruation or cycle length among animals in each group during the first 6 months of the treatment interval and in non-pregnant animals during the post-treatment interval (data not shown). However, over the year of treatment timely menstruation occurred at regular intervals in all vehicle animals throughout the ovulatory rhesus macaque season (September–May), but as the monkey anovulatory season (June–August) approached, 2/5 vehicle animals did not mense for two consecutive cycles and in the antiprogestin group 3/5 females did not mense for four consecutive cycles. Therefore, the percentage of females displaying consistent overt menstruation in the antiprogestin-treated group (18 ± 2%) was significantly less (P < 0.05) compared with vehicle-treated females (88 ± 5%) and by the 6th month of treatment all antiprogestin-treated females were amenorrhoeic. There were no conceptions and/or pregnancies during the consecutive 12 month treatment interval in antiprogestin-treated females.


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Table II. Cyclicity and conceptions during the treatment interval in female rhesus monkeys receiving daily administration of vehicle or antiprogestin for 12 consecutive cycles
 
Figure 1 illustrates the interval to conception during the post-treatment cycles. The animals conceived during the 9 month post-treatment interval before the summer hiatus of ovulatory activity typical of rhesus monkeys. Within the vehicle group, 4/5 conceptions occurred during the first paired cycle post-treatment with an average interval to conception of 20 ± 5.3 days (Figure 1) and a cumulative conception rate of 80% (Table III). The remaining vehicle animal never conceived before the end of the experiment. Within the antiprogestin group, 3/5 animals conceived after cessation of treatment (Figure 1), two rapidly (21 and 55 days) and one during the 8th post-treatment cycle. Demonstrating the limitations of paired breeding studies, mating became inconsistent or stopped altogether during cycles 3–7 post-treatment with the remaining three antiprogestin-treated females. Therefore, males previously paired with pregnant females were removed and reintroduced with non-pregnant females (vehicle or antiprogestin) during the third post-treatment cycle. Male-to-female aggressive tendencies and cessation of mating were again noted, so males were placed with unfamiliar females during the 5th and 7th cycles (Figure 1). During the 8th post-treatment cycle, a third conception occurred in an antiprogestin-treated female within 15 days of reintroduction to a new male (Figure 1). Therefore, in the antiprogestin-treated group, 3/5 total pregnancies resulted in a cumulative conception rate of 60% (Table III). An estimated average interval to conception was 38 ± 17 days, including only the first two conceptions; when the third conception, calculated from time of re-introduction of a new male, is included this value becomes 30 ± 12 days. There was no significant difference in pregnancy rate between antiprogestin- and vehicle-treated groups (Table III).



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Figure 1. Interval to conception during the post-treatment cycles. Individual vehicle- (n = 4/5) and antiprogestin-treated (n = 3/5) female rhesus monkeys conceived upon cessation of treatment. The average interval to conception is represented in days as mean ± SEM for each treatment group. During cycles 3–7, mating declined (see text). Solid arrows represent the cycle during which males were re-introduced to novel non-pregnant females. Open arrow represents the beginning of the summer hiatus/rhesus monkey anovulatory season.

 

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Table III. Pregnancy outcome of animals that conceived after vehicle or antiprogestin treatment
 
Table III summarizes the pregnancy outcomes of the animals that conceived after the cessation of vehicle or antiprogestin treatment. In the vehicle group, 3/4 (75%) pregnancies resulted in live, singleton births (one male, two females); the remaining pregnant animal miscarried at gestational day 90. In the antiprogestin group, 2/3 (67%) pregnant females had a normal delivery of a single live infant (two females). The remaining pregnant female delivered a stillborn infant at term (165 days), and upon pathological evaluation the placenta and infant appeared normal. There were no differences in the fetal characteristics at ultrasound (data not shown), length of gestation, number of live births and infant weights between vehicle and antiprogestin-treated animals or between 10 pregnant animals from the timed mated breeding colony at ONPRC (Table III). Delivery dates estimated from fetal measurements taken at ultrasonography in both groups were within 3–11 days of the actual delivery date. Birth weights of all infants in both treatment groups (Table III) were within the normal range for male and female rhesus infants at ONPRC. All infants appeared normal at birth and to date have not exhibited any physical abnormalities. There were no differences in body weights between groups prior to or throughout treatment with vehicle or antiprogestin in all non-pregnant animals (data not shown). Pregnant animals in both groups typically began to gain weight between the first and second trimester.

Table IV lists the number of non-pregnant animals exhibiting menstruation during the post-treatment interval (9 consecutive months). There were no differences in the length of menstrual cycles or duration of menses among non-pregnant animals in each group after the cessation of treatment. Antiprogestin-treated females recovered overt menstruation within 2 months of treatment cessation, unlike the single control animal that demonstrated consistent incidence of overt menstruation during the entire post-treatment interval. Although each antiprogestin-treated female did not demonstrate consistent incidence of overt menstruation every month, each mensed three to five times throughout the 9 month post-treatment interval. The two remaining non-pregnant females in the antiprogestin-treated group did demonstrate mense (50% of the time) during the first 7 months of post-treatment (October–April); however, they did not menstruate during the final 8th and 9th months post-treatment (May and June), possibly because of the normal impending anovulatory summer hiatus (June–August) that occurs in rhesus monkeys.


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Table IV. Incidence of menstruation after cessation of treatment in non-pregnant animals
 
Table V lists a sample of the salient values from serum biochemistry and haematological determinations in untreated females exhibiting normal menstrual cycles selected randomly from the colony, as well as those collected from animals daily for a 2 week interval during the 4th and 9th months of treatment with vehicle or antiprogestin, and once 10 days post-treatment. Despite the following changes, all values remained within normal limits for rhesus monkeys (Fernie et al., 1994Go; Buchl and Howard, 1997Go) and other macaques (Durand et al., 1998Go; Takenaka et al., 2000Go). Antiprogestin-treatment increased (P < 0.05) aspartate aminotransferase (AST), an indicator of muscle function, during the 4th and 9th months of treatment, as well as triglycerides and very-low-density lipoprotein (VLDL) during treatment and 10 days post-treatment relative to vehicle-treated values within the month. Additionally, antiprogestin treatment decreased (P < 0.05) high-density lipoprotein (HDL) during treatment and low-density lipoprotein (LDL) during and after treatment relative to vehicle-treated values within each month. When comparing values within the antiprogestin group, AST and LDL were significantly lower (P < 0.05) after treatment than during treatment, whereas triglycerides, HDL and VLDL were significantly higher (P < 0.05) after treatment than during treatment. Finally, there was no significant change in cholesterol values between groups during or post-treatment. General indices of circulating glucose and ions, renal function (blood urea nitrogen, creatinine), liver function (e.g. total protein, albumin/globulin, {gamma}-glutamyl transferase, alanine aminotransferase), muscle function (e.g. lactate dehydrogenase) and blood cell constituents (e.g. erythrocytes) or any other parameters typically measured revealed no significant difference between vehicle- or antiprogestin-treated females and non-pregnant females (data not shown).


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Table V. Concentrations of blood constituents in female monkeys during the 4th and 9th months of treatment with vehicle or antiprogestin (n = 5/group) and 10 days post-treatment
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A chronic daily regimen of the low-dose antiprogestin treatment is contraceptive after 6 months of daily treatment (Zelinski-Wooten et al., 1998bGo). While antiprogestin treatment permits continuous ovarian/menstrual cyclicity (Zelinski-Wooten et al., 1998aGo) and induces a dramatic antiproliferative effect in the endometrium of non-human primates after 5–6 months of treatment (Slayden et al., 1998Go), it was unknown if the pregnancy prevention was reversible. This study identifies for the first time that a chronic low-dose antiprogestin regimen may be considered as a contraceptive to be used clinically since a rigorous test of its ability to restore subsequent fertility following a longer treatment interval was successful.

None of the antiprogestin females became pregnant during the treatment interval, and no adverse effects were observed, supporting previous studies after a shorter treatment interval (Zelinski-Wooten et al., 1998bGo). Ovarian cyclicity was evident in both vehicle- and antiprogestin-treated females based on steroid measurements taken during the 4th, 7th and 9th cycles of treatment, even though antiprogestin-treated animals were amenorrhoeic by the 6th month of treatment. Timely menstruation at regular intervals occurred during the treatment interval in all vehicle animals throughout the ovulatory rhesus macaque season (September–May), but, as the monkey anovulatory season (June–August) approached, 2/5 animals did not mense for two consecutive cycles, accounting for a cumulative 88% menses rate over the course of the year of treatment compared with the 18% cumulative mense rate for the antiprogestin-treated females. Menstruation did recover within 2–3 months after cessation of antiprogestin treatment. Although the remaining three non-pregnant antiprogestin-treated females did not resume consistent menstruation, each female did menstruate three to five cycles of the nine post-treatment cycles (October–June) before the summer anovulatory monkey season (June–August). The ongoing amenorrhoea in the antiprogestin group is most likely due to the drug itself during the year of treatment. However, recovery of mense to pretreatment conditions never occurred near the end of the post-treatment cycles. Therefore, it is difficult to determine if more females would have returned to normal menstrual cyclicity had the study ended at a different point of the season. There was only one non-pregnant vehicle female and she mensed every post-treatment cycle.

The suppressive effects on the endometrium after administration of antiprogestins (Slayden et al., 1998Go) most likely contributed to the increased prevalence of amenorrhoea in these animals. Nevertheless, our extensive observations that normal circulating steroid and gonadotrophin concentrations occur throughout the treatment interval suggests that lack of overt menstruation does not equate with cessation of ovarian cyclicity during this antiprogestin regimen (Zelinski-Wooten et al., 1998aGo). Chronic low doses of antiprogestin treatment may also provide a new option for women who wish to suppress their menstrual periods (Slayden et al., 2001aGo). The antiprogestin used in the current study, ZK 137 316 (a type II antiprogestin), and ZK 230 211 (a type III antiprogestin) suppress menstruation during the treatment interval in non-human primates, while maintaining normal follicular phase hormonal profiles. Timely menstrual cyclicity was returned to normal within 60 days post-treatment in macaques (Slayden et al., 2001aGo). Reversible amenorrhoea can therefore be achieved with low-dose antiprogestin treatment and may also protect the endometrium from the effects of unopposed estrogen (Slayden et al., 2001aGo).

Antiprogestins bind the progesterone receptor (PR) and inhibit progesterone initiated gene transcription and thereby progesterone actions. Chronic low-dose treatment with antiprogestins in women and non-human primates either during their menstrual cycle or with a combined estrogen therapy will inhibit endometrial proliferation. There are several proposed mechanisms. For example, during the proliferative phase, suppression of estrogen-dependent mitotic activity by antiprogestins is accompanied by inhibition of glandular, stromal and arterial growth (Chwalisz et al., 2000Go). A decrease in vascular support is hypothesized to involve changes in nitric oxide synthetase resulting in a decrease in the nutritional and oxygenated state of the endometrium (Chwalisz et al., 2000Go). Additionally, over-expression of the androgen receptor occurs in the stroma and glands following antiprogestin treatment (Slayden et al., 2001bGo). Since androgens can block estradiol-stimulated endometrial androgen receptors the result is antagonistic leading to growth inhibition of the primate endometrium (Slayden et al., 2001bGo). Another proposed mechanism of the antiproliferative effects of low-dose antiprogestins on the endometrium may be due to the differential binding of the PR isoforms, PR-A or PR-B. PR-A acts as a negative repressor of estrogen, androgen and glucocorticoid receptors (McDonnell, 1995Go). Antiprogestins can induce the inhibitory activity of PR-A in vitro, which may allow them to function as potent antiestrogens without directly interacting with the estrogen receptor (McDonnell, 1995Go). Whether these mechanisms are acting alone or in combination to prevent pregnancy remains to be determined.

While our acute studies with continuous, low-dose ZK 137 316 demonstrated complete contraceptive efficacy while permitting ovarian cyclicity (Zelinski-Wooten et al., 1998bGo), recent reports in women taking low doses of RU 486 indicate that the overall success rate of pregnancy prevention was poor. In women that received daily low doses (0.5 mg/day or 5 mg/1x week) of RU 486 for 6 months, a 16–30% failure rate was reported (Marions et al., 1998Go, 1999). Likewise, women given 2 or 5 mg RU 486 once a week for only 2 months exhibited a 6% contraceptive failure rate (Bygdeman et al., 1999Go). Additionally, women given 2 or 5 mg RU 486 daily for 4 months did not get pregnant, but they demonstrated complete suppression of ovulation and significant amenorrhoea (Brown et al., 2000Go, 2002). Therefore, clinical studies with RU 486 suggest that low-dose regimens that maintain ovarian cyclicity are less effective contraceptives whereas higher doses needed for continuous contraception disrupt ovulation and menstrual cyclicity (Van Look and von Hertzen, 1995Go; Marions et al., 1998Go, 1999; Brown et al., 2000Go, 2002). Mifepristone (RU 486) treatment is best when used as emergency contraception post-coitally, but it is not effective as a long-term, low-dose contraceptive. The ZK compounds on the other hand are more potent, requiring smaller doses to achieve normal ovarian cyclicity (estrogen and progesterone profiles), menstrual cyclicity, and they prevent pregnancy without the added expense of determining the mid-cycle gonadotrophin surge for every cycle. In contrast to RU 486, perhaps the in-vivo half-life, metabolism and potency of ZK 137 316 contributed to its success in preventing pregnancy in non-human primates during continual exposure to low doses that allow normal ovarian cyclicity and when the treatment regimen is discontinued fertility is restored and conception and live births can result as shown in the present study.

This study for the first time demonstrates the rapid reversibility with respect to subsequent fertility after antiprogestin treatment. There was no difference in the conception or pregnancy rates between vehicle- or antiprogestin-treated females in either the 6 month (Zelinski-Wooten et al., 1998bGo) or 1 year (present study) contraceptive trials. While difficulties due to animal behaviour preclude an accurate assessment of the interval to conception after 1 year of antiprogestin treatment, the fact that two animals conceived during the first two post-treatment cycles supports a rapid return to fertility. Thus, fertility can be restored after 6 months to 1 year of consecutive, low-dose antiprogestin administration. The current study also demonstrates for the first time that normal, live healthy infants can develop following daily maternal treatment with antiprogestin and upon cessation of treatment; this regimen did not adversely affect the subsequent progression of fetal development to term. Gestation lengths, live birth rates and infant weights were similar between vehicle- and antiprogestin-treated animals and both groups were within normal values as seen in the ONPRC colony.

Female macaques tolerated the contraceptive dosing regimen with no detrimental effects noted in biochemical and haematological determinations throughout 1 year of treatment. Although in the current study antiprogestin treatment increased triglycerides, HDL, VLDL and LDL compared with vehicle-treated females, the levels were still within normal parameters of female rhesus macaques (Takenaka et al., 2000Go). In rats, ZK 137 316 has weak androgenic activity, which would induce slight changes in lipids and could possibly explain the effects on HDL in the present study (K.Chwalisz, unpublished data). Likewise, no concerns arose following the toxicological and pathological studies after the previous 6 month treatment interval (Zelinski-Wooten et al., 1998bGo). Various blood constituents were analysed from untreated females exhibiting normal menstrual cycles selected randomly from our colony, as well as from non-pregnant animals during the final cycle of treatment with vehicle, 0.01 and 0.03 (dose used in present study) mg/kg ZK 137 316. General indices of circulating ions, liver function, muscle function and blood cell constituents revealed no significant differences in these or any other parameters between antiprogestin-treated and untreated females.

The endometrial antiproliferative effect following a chronic, low-dose regimen of the antiprogestin ZK 137 316 in macaques (Slayden et al., 1998Go; Zelinski-Wooten et al., 1998bGo) and mifepristone in women (Batista et al., 1992Go; Croxatto et al., 1993Go; Cameron et al., 1996Go; Gemzell-Danielsson et al., 1996Go, 1997) alleviates concern for unopposed estrogenic effects in the endometrium (Chwalisz et al., 2000Go). However, consideration must be given to possible effects of unopposed estrogen in tissues where classical inhibition of progesterone action in the presence of circulating estrogen concentrations typical of the follicular phase allows manifestation of estrogen-dependent effects. Previous studies following treatment with low-dose antiprogestins in macaques demonstrated that the oviducts were devoid of histological abnormalities and that the endometrium was protected from the effects of unopposed estrogen (Slayden et al., 1998Go, 2001a). The long-term effects of chronic low-dose antiprogestin on other estrogen-dependent tissues such as the cervix, vagina or mammary glands will require further consideration.

Our study supports the concept of a daily low-dose antiprogestin regimen as a potential contraceptive strategy as proposed previously by others (Batista et al., 1992Go; Kettel et al., 1992Go; Croxatto et al., 1993Go; Cameron et al., 1995Go, 1996; Gemzell-Danielsson et al., 1996Go; Slayden et al., 1998Go; Zelinski-Wooten et al., 1998aGo,b). Continual administration of low-dose ZK 137 316 permits continued ovarian cyclicity, timely overt menses initially followed by amenorrhoea, and prevents pregnancy in rhesus monkeys without adverse side-effects. The rapid reversal of the anti-fertility effects and the live births observed after 1 year of antiprogestin administration in macaques increases confidence in considering the clinical feasibility of potent and selective antiprogestins as a potential contraceptive for women. Whether the contraceptive action of chronic low-dose antiprogestin is due to prevention of implantation and/or other actions in the embryo (Ghosh et al., 1997Go) or reproductive tract remains to be tested.


    Acknowledgements
 
Schering AG (Berlin, Germany), generously provided the ZK 137 316 used in the present study. The authors are grateful to the dedicated and conscientious staff of the Division of Animal Resources for their enthusiastic participation in this study. We also thank Julie White for aid in preparation of the manuscript. These studies were supported by National Institute of Health grants HD31633 (to M.Z.W.) and 2T32 HDO7133 (Department of Physiology, Oregon Health & Science University to S.M.B.) and RR00163.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 12, 2001; resubmitted on August 29, 2002. accepted on September 10, 2002