1 Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton OR 97006, USA and 2 Jenapharm, GmbH and Co. KG, Jena, Germany 07745
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Abstract |
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Key words: endometrium/macaque/menstruation/ovulation/progesterone antagonists
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Introduction |
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Experimental menstruation can easily be induced in both women (Critchley et al.1999) and Old World nonhuman primates (Brenner et al.1996
) by experimental progesterone withdrawal from an oestradiol plus progesterone primed endometrium. Menstruation in rhesus macaques therefore provides an experimental model that can be used to assess the ability of antiprogestins to suppress uterine bleeding.
The ability of antiprogestins to inhibit the menstrual cycle was first described in cynomologus monkeys. In that study, high doses of RU 486 (5 mg/day) administered on days 1012 of the menstrual cycle, delayed the mid-cycle LH surge and lengthened the intermenstrual interval from the normal ~30 days, to 61 days, effectively blocking one menstrual cycle (Collins and Hodgen, 1986). Similar results were reported for women treated with RU 486 during the follicular phase, where RU 486 disrupted follicle maturation and delayed progression of the menstrual cycle (Liu et al.1987
). A single high dose of RU 486 administered to women during the midluteal phase of the cycle also suppressed serum LH pulse amplitude and frequency, was luteolytic and induced menstruation (Garzo et al.1988
). The same workers also reported that corpus luteum function after a single dose of RU 486 could be rescued by exogenous HCG, but menstruation was still induced because RU 486 blocked progesterone action in the endometrium.
ZK 137 316 and ZK 230 211 are new-generation PAs with increased potency and reduced antiglucocorticoid activity. ZK 137 316, like RU 486, is a type II antagonist that can under certain circumstances exhibit agonistic activity in vitro and in vivo (Klein-Hitpass et al.1991; Chwalisz et al.2000a
). ZK 230 211 is a type III antagonist and does not display any PR agonistic activity in vitro or in vivo (Fuhrmann et al.2000
). In our previously published studies of ZK compounds we found that 0.03 mg/kg ZK 137 316 would inhibit endometrial development (Slayden et al.1998
) but still allow menstrual and ovarian cyclicity in half of the animals treated while higher doses inhibited menstrual cyclicity (Zelinski-Wooten et al.1998b
). These lower doses of ZK 137 316 were then shown to be contraceptive (Zelinski-Wooten et al.1998a
), but it was unknown if the effects of ZK 137 316 would be acutely reversible once treatment was stopped. In the current study we evaluated higher doses of these PAs to determine whether regimens could be identified that would reliably block menstrual bleeding, whether ovarian function was affected, and whether the effects of such doses would be reversible.
The current work had three aims: first, to discover the range of doses of ZK 137 316 and ZK 230 211 that would block progesterone action directly on the endometrium in artificially cycled, spayed monkeys; second, to document the histological effects of ZK 230 211 on the endometrium; and third, to determine whether such endometrial-suppressive doses administered chronically to intact cycling monkeys would cause reversible, menstrual suppression. Two dose-finding studies were carried out in the spayed animals: (i) menstrual blockade; to determine the dose of PA which, if given chronically for one cycle, would block menstruation on progesterone withdrawal, and (ii) menstrual induction; to determine the dose that would induce menstruation when given acutely at the end of an artificial cycle. The most likely doses were then selected for chronic studies of menstrual suppression in naturally cycling animals. The work was conducted over a three-year period; ZK 230 211 became available for study later in the work. Our overall goal was to obtain preclinical data useful for clinical development of a novel mode of reversible, menstrual suppression for women who may desire such suppression.
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Materials and methods |
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Animal care
Animal care was provided by the Division of Animal Resources at the Oregon Regional Primate Research Center (ORPRC) following the National Institutes of Health guidelines on the care and use of animals. Chronic treatment of cycling macaques with ZK 137 316 or ZK 230 211 had no obvious effects on general animal health. Artificial menstrual cycles were created as previously described (Slayden et al.1993). Briefly, a 3 cm Silastic capsule (0.34 cm inner diameter; 0.64 cm outer diameter; Dow Corning; Midland, MI, USA) filled with crystalline oestradiol was first inserted s.c. to induce an artificial follicular phase. After 14 days of oestradiol priming, a 6 cm Silastic capsule containing crystalline progesterone was inserted s.c. for an additional 14 days to stimulate an artificial luteal phase. Removal of the progesterone implant on day 28 completed the cycle and induced menstruation. Serum samples were collected during the artificial cycles to confirm normal concentrations of oestradiol and progesterone.
Menstrual blockade of progesterone-withdrawal menses by chronic administration of ZK 137 316 in artificially cycled, ovariectomized macaques
The animals were injected with ZK 137 316 daily in HPE during a complete cycle (oestradiol for 14 days and then oestradiol + progesterone for 14 days). Control animals received vehicle only during the artificial cycle (n = 4). Four doses of ZK 137 316 were tested: 0.01, 0.03, 0.05, and 0.1 mg/kg body weight (n = 3 each). At the end of the cycle, the progesterone implants were removed, and injections of ZK 137 316 were continued for 7 more days. Vaginal swabs were performed daily for 9 days after progesterone implant removal and the incidence of frank menses or minute bleeding was recorded. ZK 230 211 was not available at the time this menstrual blockade study was done. All of the animals tested for menses blockade were allowed to recover for 60 days to clear residual effects of ZK 137 316 and were then returned to the Primate Centre colony.
Menstrual induction in ovariectomized macaques
Menses induction in a progesterone-primed animal is another reliable, non-invasive indicator of antiprogestin action on the endometrium. Beginning on day 28 of an artificial menstrual cycle, the monkeys were injected daily with antiprogestin in HPE vehicle for 7 days while the progesterone implant remained in place. Control animals received vehicle only (n = 4). Five doses of ZK 137 316 were tested: 0.01 (n = 6), 0.03 (n = 6), 0.05 (n = 4), 0.1 (n = 6), and 0.15 (n = 3) mg/kg body weight. Five doses of ZK 230 211 were also tested: 0.005 (n = 2), 0.01 (n = 4), 0.03 (n = 4), 0.05 (n = 2), and 0.1 (n = 2) mg/kg. Vaginal swabs were performed daily for 9 days and the ability of the various doses of antiprogestin to induce frank, overt menses (blood detectable on the external genitalia and the cage floor, confirmed by vaginal swab) was recorded. All of the animals tested for menses induction were allowed to recover for 60 days to clear residual effects of PAs and were then returned to the Primate Centre colony.
Morphological effects of ZK 230 211 in ovariectomized hormone-treated macaques
In preliminary work, we found that treatment of artificially cycled, ovariectomized animals for 1 month with 0.1 mg/kg ZK 137 316 produced endometrial suppression essentially equivalent to the suppression we had previously observed in naturally cycling macaques treated with that dose for 5 months (Slayden et al.1998). Therefore, in this study, we focused attention on dosage effects of ZK 230 211 on histomorphometric analyses. Five groups of ovariectomized macaques were treated as described above to create artificial menstrual cycles. Beginning on day 1 of the second cycle the animals were treated with various doses as shown in Table I
. Doses were selected based on findings from the menstrual induction study. Animals treated with oestradiol alone were included to provide a baseline measure of the degree of oestradiol-dependent proliferation.
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Histology and immunocytochemistry
Tissue samples for morphological study were fixed in 2% glutaraldehyde and 3% paraformaldehyde, embedded in glycol methacrylate (GMA), sectioned (uterus, 2 µm; oviduct 1.5 µm) and stained with Gill's Hematoxylin (Sandow et al. 1979). Samples of fresh tissue for immunocytochemistry were microwave stabilized (Slayden et al.1995
) in an Amana Radarrange Touchmatic microwave oven (Amana, Iowa, USA) for 7 seconds in 0.5 ml Hank's Balanced Salt Solution (Gibco), then chilled on ice in 10% sucrose dissolved in 0.1 mol/l phosphate buffered saline (PBS; Sigma), mounted in Tissue Tek II OCT (Miles Inc., Elkhart, IN, USA) and frozen in liquid propane. Cryostat sections (5 µm) were thaw-mounted on Superfrost Plus (Fisher Scientific Pittsburgh, PA, USA) slides, placed on ice at 5°C, and microwaved for 2 s. Immunocytochemistry of oestrogen receptor alpha (ER
), PR and Ki-67 was carried out as recently described (Slayden et al.1998
). Briefly, the microwave-treated sections were lightly fixed (0.2% picric acid, 2% paraformaldehyde in PBS) for 10 min; and the immunocytochemistry was conducted with monoclonal anti-ER
(1D-5; Biogenex, San Ramon, CA, USA), anti-PR (JZB-39; provided by Geoffrey Greene, University of Chicago) or anti-Ki-67 antigen (Dako Corp., Carpinteria, CA, USA). In each case primary antibody was reacted with either biotinylated anti-mouse IgG (for 1D-5 and anti Ki-67) or anti-rat IgG (for JZB-39) second antibody and detected with an avidin-biotin peroxidase kit (Vector Laboratories, Burlingame, CA, USA).
Morphometrics
The abundance of mitotic cells, Ki-67 positive cells, and apoptotic cells in the endometrium was determined by a trained observer who used an ocular micrometer grid to define microscope fields and counted between 12005000 cells per animal with the aid of a mechanical tabulator. Mitotic index represented the number of mitoses per 1000 epithelial cells. Endometrial stromal cell density values (stromal compaction) were determined with the Optimas 3.0 image analysis software package. For this analysis, 10 non-overlapping fields (examined with a x25 objective) of endometrial stroma in the upper functionalis of each specimen were analysed. The number of stromal cell nuclei per 10 000 µm2 provided an index which reflected the degree to which the endometrial stroma became expanded (more oedematous) or compacted (less oedematous). Apoptotic cell counts were carried out in glycol methacrylate sections. Apoptotic bodies consist of cytoplasmic fragments containing nuclear elements and these can easily be distinguished in such sections. Percentage apoptosis was based on the number of apoptotic bodies per 5000 glandular epithelial cells.
Naturally cycling macaques: menstrual suppression
Untreated, adult macaques were first monitored for two complete menstrual cycles to document normal cycle lengths for each animal prior to treatment. Three independent protocols (see below) were conducted in naturally cycling animals. The basic design of these treatment protocols is depicted graphically in Figure 1. After treatments were complete, all of the animals were allowed to rest for a minimum of two normal menstrual cycles and were then returned to the Primate Center colony.
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Protocol 2: Long term (100 day) treatment with ZK 137 316
Starting on day 2 of menses, animals with normal menstrual cycles were injected daily for 100 days with ZK 137 316 in HPE. Three dosages (n = 4 each) were used: (i) vehicle only (control), (ii) 0.05 mg/kg ZK 137 316, and (iii) 0.1 mg/kg body weight. Daily vaginal swabs were collected during the entire treatment period. Daily blood samples were collected for analysis of oestradiol and progesterone during the last 30 days of treatment and continued until the monkeys menstruated (2 days). The monkeys were further monitored for menses during the first post-treatment cycle.
Protocol 3: Intermediate (60 day) treatment with ZK 230 211
During protocols 1 and 2 we showed that HPE vehicle injection throughout the entire treatment periods had no effect on hormone concentrations, menstrual cycles or endometrial bleeding patterns. Therefore, in protocol 3, macaques were first injected i.m. daily with HPE alone for one control, pretreatment cycle. Then beginning on the day after menses in the next cycle, the animals received ZK 230 211 in HPE (i.m.) daily for 60 days. Three dosages of ZK 230 211 (at steps of ~3-fold) were compared: (i) 0.005 mg/kg, (ii) 0.016 mg/kg and (iii) 0.05 mg/kg body weight (n = 5 each). Daily vaginal swabs and daily blood samples were collected during the pretreatment cycle, the treatment period (60 days) and the recovery period, until the monkeys menstruated for longer than 2 days. The monkeys were further monitored for menstruation by vaginal swab without blood collection during the first post-treatment cycle. Blood samples were assayed for serum concentrations of oestradiol and progesterone, and once these concentrations were known, samples that flanked the surge (or highest value) of oestradiol were assayed for bioactive LH.
Hormone assays
All blood samples were analysed for concentrations of oestradiol and progesterone by routine radioimmunoassay and LH determinations were made by bioassay (Zelinski-Wooten et al.1998b). All assays were performed by the ORPRC Hormone Assay Core.
Statistical analysis
Quantitative data were compared by analysis of variance (ANOVA). Significant differences among means were determined by Fisher's protected least significant difference test (Petersen, 1985). These data included lengths of the pre-treatment menstrual cycles, treatment-induced intermenses interval, length of the recovery period (time to return to menses) after the last injection, post-treatment menstrual cycle length, tissue weights, percentage fimbrial ciliation, mitotic index, percentage Ki-67 positive epithelial cells, apoptotic index and degree of stromal compaction.
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Results |
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ZK 230 211:
As Figure 3 shows, the first significant increase in mean days of bleeding was induced by 0.03 mg/kg, and increasing the dose to 0.05 mg/kg and 0.1mg/kg induced significantly more days of bleeding.
Histological effects of ZK 230 211:
Animals treated with oestradiol + progesterone alone displayed an hypertrophied, progestational (secretory) endometrium (Figure 4a) with sacculated glands and expanded stroma in both the functionalis and basalis zones (Figures 4a and e
), and well developed spiral arteries (Figure 4i
). In the basalis zone, the glandular epithelium was tall, columnar and mitotically active. Under this hormonal condition, the epithelium of the oviductal fimbriae was deciliated and cuboidal (Figure 4m
), indicative of the normal suppressive action of progesterone on the oviduct (Brenner and Slayden, 1994
).
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Immunocytochemical effects of ZK 230 211:
The effect of each dose of ZK 230 211 on endometrial ER, PR and Ki-67 is presented in Figure 5
. In oestradiol + progesterone-treated control animals, oestrogen, progesterone and Ki-67 staining in the functionalis was generally low because of the suppressive action of progesterone (Brenner et al.1990
; Hild-Petito et al.1992
; Okulicz et al.1993
). Treatment with ZK 230 211 at 0.005 mg/kg had no effect but 0.016 and 0.032 mg/kg blocked progesterone suppression and increased ER
(compare Figure 5 ad
) PR (Figure 5 eh
) and Ki-67 (Figure 5 il
) staining in glands and stroma.
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In the basalis zone of the macaque, mitosis is progesterone-dependent, not oestradiol-dependent (Brenner and Slayden, 1994). Consequently there was considerable Ki-67 and mitotic activity in this zone in the animals treated with oestradiol + progesterone alone. ZK 230 211 treatment with both the 0.016 and 0.032 mg/kg doses dramatically suppressed both the Ki-67 and mitotic indices in the basalis.
In the oestradiol + progesterone treated animals, the apoptotic index was significantly lower than that seen after oestradiol treatment because progesterone treatment tends to suppress endometrial apoptosis. ZK 230 211 at doses 0.016 mg/kg, blocked the action of progesterone on apoptosis and raised the level to that seen under oestradiol alone (Table III
).
Naturally cycling macaques
Based on the above dosage information, we selected two doses of ZK 137 316 (0.05 and 0.1 mg/kg) and three doses of ZK 230 211 (0.005, 0.016 and 0.05 mg/kg) for study of menstrual suppression in naturally cycling monkeys.
Table IV summarizes the effects of both PAs on (i) treatment-induced intermenstrual intervals, (ii) the lengths of the recovery periods, and (iii) the lengths of the post-treatment cycles. Figures 611
graphically display the serum oestradiol and progesterone concentrations associated with these treatments.
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Oestradiol and progesterone concentrations in macaques treated with ZK 137 316 for 40 days (shaded area) are shown in Figure 7. In the 0.05 mg/kg group, ovulation was unaffected in four animals and suppressed in five animals; of the latter, two had relatively normal oestradiol surges. In all animals, whether or not ovulation was affected, non-surge oestradiol concentrations were normal (~30100 pg/ml). Similar results were seen in the 0.1 mg group in that three ovulated and six failed to ovulate. At both doses, in the monkeys that ovulated, there was no menstruation, but some animals showed minute bleeding, detectable only by vaginal swab, around the time of progesterone decline. After the recovery period, normal menstrual cycles resumed, and Figure 8
shows oestradiol and progesterone concentrations during the second post-treatment menstrual cycle.
Long-term (100 day) suppression of menses with ZK 137 316:
The mean (± SEM) intermenstrual intervals in the control, 0.05 and 0.1 groups were 28.1 ± 0.83, 131.1 ± 1 0.1 and 134 ± 8.7 days, respectively (P < 0.001); the recovery period after treatment stopped was ~approximately 3135 days, and post-treatment cycles were of normal length (Table IV). In this group, blood samples were collected only for the last 30 days of treatment. During this 30 day sampling period, control cycles remained normal. All the monkeys in the 0.05 mg/kg group had ovulated (n = 4), as indicated by normal luteal phase concentrations of progesterone and a subsequent progesterone decline during days 7080 of treatment, but there was no menstrual bleeding when progesterone declined on treatment day 78. A subsequent oestradiol surge around day 85 of treatment was not followed by ovulation (Figure 9
). At the 0.1 mg/kg dose none of the monkeys ovulated during the last 30 days of treatment nor showed any menstrual bleeding.
All the treated monkeys showed normal non-surge concentrations of oestradiol (~30100 pg/ml). When treatment ended (at 100 days) most of the animals in both groups developed an oestradiol surge, ovulated, expressed a normal luteal phase and menstruated when progesterone declined. These data indicate that the 100 day treatment was fully reversible.
Intermediate term (60 day) suppression of menses with ZK 230 211:
Treatment with 0.005 mg/kg ZK 230 211 had no effect on ovulation or menstruation, but the 0.016 and 0.05 mg/kg doses suppressed all ovulation and menstruation, significantly extending the intermenstrual interval to ~100 days (P < 0.01; Table IV).
The recovery period was around 40 days (see Table IV). Both 0.016 mg and 0.05 mg/kg doses also increased the length of the first post-treatment menstrual cycle to ~50 days (Table IV
P, < 0.05); menstrual cycles thereafter were of normal length.
In this protocol we bled the animals daily throughout the entire study period until the first menstruation occurred. Macaques treated with 0.005 mg/kg had normal oestradiol surges and luteal phase progesterone concentrations. The 0.016 mg/kg dose resulted in follicular phase oestradiol surges of normal amplitude, but normal luteal phase concentrations of progesterone did not develop (Figure 10, and see Figure 11
). Monkeys treated with 0.05 mg/kg failed to develop either a normal oestradiol surge or normal luteal phase concentrations of progesterone (Figures 10 and 11
). All ZK 230 211 treated monkeys showed normal non-surge concentrations of oestradiol (~30100 pg/ml). Approximately 10 days after treatment ended, there was a rise in serum progesterone and a normal length luteal phase for both higher dose groups. Menses occurred following progesterone decline at the end of this luteal phase (Figure 10
).
To determine whether ZK 230 211 could suppress LH, serum samples flanking the highest concentrations of oestradiol in each group were re-assayed for LH by bioassay (bLH, Figure 11). Samples from the pretreatment period, the recovery period, and those treated with 0.005 mg ZK 230 211 all showed rises of bLH associated with peak concentrations of oestradiol. However, bLH concentrations remained low in animals treated with 0.016 and 0.05 mg/kg. This effect on the pituitary was fully reversible, as the post-treatment cycles showed normal LH surges.
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Discussion |
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Histological and morphometric effects
ZK 230 211 dramatically suppressed the endometrium in oestradiol plus progesterone-primed animals at 0.032 mg/kg over a 1 month treatment period. This dose is around 3-fold lower than the 0.1mg/kg dose of ZK 137 316 that blocked the endometrium in naturally cycling monkeys over a 5 month period (Slayden et al.1998). As stated in Materials and methods, we also found that 0.1 mg/kg of ZK 137 316 severely suppressed the endometrium within a 1 month treatment period (data not shown).
The specific histological effects induced by ZK 230 211 included overall shrinkage, stromal compaction, glandular atrophy and hyalinization of the spiral artery walls. In addition, the suppressive effects of progesterone on endometrial ER and PR were blocked, as were the antagonistic effects of progesterone on oestradiol action in the oviduct. The data in Table III
indicate that ZK 230 211 suppressed endometrial mass, endometrial thickness and the functionalis mitotic index significantly below the level in animals treated with oestradiol alone, which is a clear antiproliferative effect. Because ZK 230 211 treatment elevated both ER
and Ki-67 concentrations, the findings suggest that oestradiol acts through ER
(directly or indirectly) to stimulate cells to produce Ki-67 and enter the cell cycle, but some unknown aspect of ZK 230 211 action blocks the cells from entering mitosis. The exact mechanism underlying this mitotic suppressive effect remains to be discovered.
In addition, ZK 230 211 elevated apoptotic counts in the functionalis to the level seen during oestradiol treatment alone (Table III). Because ZK 230 211 treatment suppressed the mitotic rate but raised the apoptotic index, cell death would greatly outpace cell birth in the functionalis zone, and over time this difference would contribute to the decrease in endometrial cell mass and thickness induced by these doses. In the basalis, which grows under progesterone influence in macaques, the blockade of progesterone action by ZK 230 211 would block the growth of this zone as well. Suppression of growth of both zones contributes to the overall shrinkage of the endometrium. Clearly, moderate doses of ZK 230 211 can block the effects of both oestradiol and progesterone on endometrial growth and development. This is the first report to show that low doses of ZK 230 211 can induce these effects directly on the endometrium and oviduct within 28 days in ovariectomized, artificially cycled macaques.
Studies in naturally cycling animals
ZK 137 316:
The combined results of the 40 and 100 day studies with ZK 137 316 suggest that treatment with 0.05 mg/kg, long or short term, is near but generally below the threshold dose that blocks ovulation, while the 0.1 mg/kg dose generally blocked ovulation. There was no obvious residual effect of antiprogestin treatment and effects appeared completely reversible, as post-treatment cycles were normal in all respects. The two treatment periods, one for 40 and one for 100 days, were designed to mimic those cases where women might want to block either just one or several menstrual periods. During the 40 day study ovulation was blocked in 5/9 animals at 0.05 mg/kg and 6/9 animals at 0.1 mg/kg. In those animals in which ovulation occurred, the luteal phase length, the serum concentrations of progesterone and oestradiol, and the time of decline of progesterone were normal, but there was no frank menstrual bleeding. Undoubtedly this was due to the combined antagonistic effects of ZK 137 316 on both oestradiol-dependent growth, (Wolf et al.1989; ,Slayden et al.1994, 1998
) and progesterone-dependent endometrial progestational development (Brenner and Slayden, 1994
).
In the 40 day study, the ovulatory animals showed minute bleeding, detectable only by vaginal swab, around the time of progesterone decline. These positive vaginal swabs detected bleeding that was far less in quantity than the so-called spotting or breakthrough bleeding which occurs in women on continuous progestin treatment (e.g. Norplant, or Depo Provera). However, in the 100 day study, animals that ovulated were completely amenorrheic with no minute bleeding. Apparently such minute bleeding can disappear with continued treatment.
ZK 230 211:
At 0.005 mg/kg, ZK 230 211 failed to suppress either menstruation or ovulation, but the higher doses of 0.016 and 0.05 mg/kg completely suppressed both ovulation and menstruation. The suppression of the LH surges induced by these doses clearly indicates that this compound had central inhibitory effects. We previously noted that the antiovulatory effects of ZK 137 316 were accompanied by suppression of LH surges, implicating the hypothalamic-pituitary axis as the site of the antiovulatory action for both these antiprogestins (Zelinski-Wooten et al.1998b). Of great interest were the animals treated with the 0.016 mg/kg dose that showed oestradiol surges but no LH surges (Figure 11
), which suggests that ZK 230 211 has separate, dose related antagonistic effects at the ovarian and pituitary levels. These divergent effects deserve additional attention in a separate research study.
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Conclusions |
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Of great importance, however, circulating oestradiol concentrations were never suppressed below normal, follicular phase concentrations by either PA at any dose. Therefore, if women used these compounds to block menses, and if ovulation were also blocked, it is unlikely there would be any associated symptoms of oestrogen deprivation, such as hot flushes, vaginal atrophy or loss of bone density, though we did not address these issues in this study. Moreover, the antiovulatory effects of PAs would be accompanied by secretion of naturally occurring oestrogens, an important difference from the current mode of menstrual inhibition, namely blockade of ovulation by combined synthetic oestrogens and progestins in a continuous contraceptive tablet regimen. Synthetic progestins are usually administered to prevent unopposed actions of synthetic oestrogens on the endometrium, but PA therapy, through its endometrial antiproliferative effects, may obviate this need.
Chronic low dose PA therapy is therefore a powerful method to reversibly suppress menstrual bleeding, and it can act in several ways depending on dose and PA type: (i) it can allow ovulation, but block the effects of both oestradiol and progesterone on endometrial growth and development, suppress menstruation upon progesterone withdrawal and induce a state of amenorrhea, (ii) it can inhibit ovulation so that progesterone concentrations remain low, resulting in amenorrhea, (iii) it can allow normal follicular phase concentrations of oestradiol so that the effects of oestrogen on bone and other peripheral target tissues should be normal and (iv) it can block any unopposed oestrogenic effects in the endometrium through the endometrial antiproliferative effect. The potential also exists for suppression of various sorts of abnormal uterine bleeding, though long term studies in women are needed to validate these various possibilities.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed. E-mail: brennerr{at}ohsu.edu
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References |
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Brenner, R.M., West, N.B., and McClellan, M.C. (1990) Estrogen and progestin receptors in the reproductive tract of male and female primates. Biol. Reprod., 42, 1119.[Abstract]
Brenner, R.M., Rudolph, L., Matrisian, L. et al. (1996) Non-human primate models: artificial menstrual cycles, endometrial matrix metalloproteinases and s.c. endometrial grafts. Hum. Reprod., 11, 150164.[ISI][Medline]
Chwalisz, K., Brenner, R.M., Fuhrmann, U. et al. (2000a) Antiproliferative effects of progesterone antagonists and progesterone receptor modulators (PRMs) on the endometrium. Steroids, 65, 741752.[ISI][Medline]
Chwalisz, K., Brenner, R.M., Nayak, N. et al. (2000b) A comparison of the endometrial effects of a mesoprogestin (J1042) with the antiprogestins ZK 137 316 and ZK 230 211 in cynomolgus monkeys. In Society for Gynecologic Investigation Program and Abstracts: 47th Annual Meeting (held in Chicago, IL, March 2225, 2000), abstract 221A.
Collins, R.L. and Hodgen, G.D. (1986) Blockade of the spontaneous midcycle gonadotropin surge in monkeys by RU 486: A progesterone antagonist or agonist? J. Clin. Endocrinol. Metab., 63, 12701276.[Abstract]
Coutinho, E.M. and Segal, S.J. (1999) Is menstruation obsolete? Oxford University Press, Oxford, 190 pp.
Critchley, H.O.D., Jones, R.L., Lea, R.G. et al. (1999) Role of inflammatory mediators in human endometrium during progesterone withdrawal and early pregnancy. J. Clin. Endocrinol. Metab., 84, 240248.
Elger, W., Bartley, J., Schneider, B. et al. (2000) Endocrine pharmacological characterization of antiprogestins and progesterone receptor modulators (PRMs) with respect to PR-agonistic and -antagonistic activity. Steroids, 65, 713723.[ISI][Medline]
Fuhrmann, U., Hess-Stumpp, H., Cleve, A. et al. (2000) Synthesis and biological activity of a novel, highly potent progesterone receptor antagonist. J. Med. Chem., 43, 50105016.[ISI][Medline]
Garzo, V.G., Liu, J., Ulmann, A. et al. (1988) Effect of an antiprogesterone (RU486) on the hypothalamic- hypophyseal-ovarian-endometrial axis during the luteal phase of the menstrual cycle. J. Clin. Endocrinol. Metab., 66, 508517.[Abstract]
Goodman, A.L. and Hodgen, G.D. (1996) Progesterone Receptor Antagonists. In Adashi, E.Y., Rock, J.A. and Rosenwaks, Z. (eds), Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers, Philadelphia, pp. 548558.
Hild-Petito, S., Verhage, H.G. and Fazleabas, A.T. (1992) Immunocytochemical localization of estrogen and progestin receptors in the baboon (Papio anubis) uterus during implantation and pregnancy. Endocrinology, 130, 23432353.[Abstract]
Klein-Hitpass, L., Cato, A.C.B., Henderson, D. et al. (1991) Two types of antiprogestins identified by their differential action in transcriptionally active extracts from T47D cells. Nucleic Acids Res., 19, 12271234.[Abstract]
Liu, J.H., Garzo, G., Morris, S. et al. (1987) Disruption of follicular maturation and delay of ovulation after administration of the antiprogesterone RU 486. J. Clin. Endocrinol. Metab., 65, 11351140.[Abstract]
Okulicz, W.C., Balsamo, M., and Tast, J. (1993) Progesterone regulation of endometrial estrogen receptor and cell proliferation during the late proliferative and secretory phase in artificial menstrual cycles in the rhesus monkey. Biol. Reprod., 49, 2432.[Abstract]
Petersen, R.G. (1985) In Owen, D.B. (ed), Design and Analysis of Experiments. Marcel Dekker, Inc., New York, pp. 72111.
Sandow, B.A., West, N.B., Norman, R.L. et al. (1979) Hormonal control of apoptosis in hamster uterine luminal epithelium. Am. J. Anat., 156, 1536.[ISI][Medline]
Slayden, O.D. and Brenner, R.M. (1994) RU 486 action after estrogen priming in the endometrium and oviducts of rhesus monkeys (Macaca mulatta). J. Clin. Endocrinol. Metab., 78, 440448.[Abstract]
Slayden, O.D., Hirst, J.J., and Brenner, R.M. (1993) Estrogen action in the reproductive tract of rhesus monkeys during antiprogestin treatment. Endocrinology, 132, 18451856.[Abstract]
Slayden, O.D., Koji, T., and Brenner, R.M. (1995) Microwave stabilization enhances immunocytochemical detection of estrogen receptor in frozen sections of macaque oviduct. Endocrinology, 136, 40124021.[Abstract]
Slayden, O.D., Zelinski-Wooten, M.B., Chwalisz, K. et al. (1998) Chronic treatment of cycling rhesus monkeys with low doses of the antiprogestin ZK 137 316: morphometric assessment of the uterus and oviduct. Hum. Reprod., 13, 269277.[Medline]
Spitz, I., Croxatto, H.B., and Robbins, A. (1996) Antiprogestin: mechanism of action and contraceptive potential. Annual Review of Pharmacolologic Toxicology, 36, 4781.[ISI][Medline]
Thomas, S.L. and Ellertson, C. (2000) Nuisance or natural and healthy: should monthly menstruation be optional for women? Lancet, 355, 922924.[ISI][Medline]
Wolf, J.P., Hsiu, J.G., Anderson, T.L. et al. (1989) Noncompetitive antiestrogenic effect of RU 486 in blocking the estrogen- stimulated luteinizing hormone surge and the proliferative action of estradiol on endometrium in castrate monkeys. Fertil. Steril., 52, 10551060.[ISI][Medline]
Zelinski-Wooten, M.B., Chwalisz, K., Iliff, S.A. et al. (1998a) A chronic, low dose regimen of the antiprogestin ZK 137 316 prevents pregnancy in rhesus monkeys. Hum. Reprod., 13, 21322138.[Abstract]
Zelinski-Wooten, M.B., Slayden, O.D., Chwalisz, K. et al. (1998b) Chronic treatment of female rhesus monkeys with low doses of the antiprogestin ZK 137 316: establishment of a regimen that permits normal menstrual cyclicity. Hum. Reprod., 13, 259267.[Medline]
Submitted on September 18, 2000; accepted on March 23, 2001.