1 Departments of Obstetrics and Gynecology and 2 Pathology, University of Tübingen, D-72076 Tübingen, Germany and 3 The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA
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Abstract |
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Key words: antiprogestin/endometrium/endometrial morphology/oestrogen/vascular endothelial growth factor
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Introduction |
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The action of both ovarian steroids can be blocked pharmacologically by steroid receptor antagonists, which predictably disrupt the physiological development of the endometrium (Healy et al., 1983; Dukes et al., 1993
). Consequently, progesterone-dependent secretory transformation of the endometrium can be blocked by administration of progesterone receptor (PR) antagonists such as mifepristone (RU 486) or onapristone (ZK 98 299) (Schaison et al., 1985
; Batista et al., 1992
; Katkam et al., 1995
; Cameron et al., 1996a
, 1997
; Dockery et al., 1997
; Gemzell Danielsson et al., 1997), presumably by competitive antagonism at the PR level (Edwards et al., 1995
).
Surprisingly, recent studies demonstrated that besides being antiprogestins, mifepristone and onapristone also impair oestrogen-dependent endometrial proliferation in both human and non-human primates. In separate studies from different laboratories, long-term treatment with mifepristone was found to produce a thin, atrophic endometrium, even in the presence of proliferative phase concentrations of oestradiol in cynomolgus monkeys, rhesus monkeys and humans (Brenner and Slayden, 1994; Hodgen et al., 1994
; Cameron et al., 1996b
). These effects could be reproduced with the antiprogestin onapristone (ZK 98 299) and a structurally related compound (ZK 137 316) which caused endometrial atrophy and stromal compaction in monkeys (Ishwad et al., 1993
; Katkam et al., 1995
; Slayden et al., 1998
).
Since antiprogestins do not bind to the oestrogen receptor (OR), except with negligibly low affinity, this puzzling phenomenon is now called non-competitive antioestrogenic activity (Wolf et al., 1989). The mechanism mediating the non-competitive antioestrogenic activity of mifepristone is still unclear, but it is clearly different from the known intrinsic agonistic activity of these compounds (Gravanis et al., 1985
; Berthois et al., 1991
) as, in contrast to progestin treatment, OR and PR are upregulated during antiprogestin treatment (Neulen et al., 1990
, 1996
; Slayden and Brenner, 1994
). Recently, the possibility of a non-genomically mediated mechanism by mifepristone was proposed, based upon experiments in vitro demonstrating effects of the antiprogestin on macrophage functions that were attributed to antioxidant properties inherent in the drug's chemical structure (Roberts et al., 1995
).
Thus, we designed a study to investigate in vivo whether mifepristone-induced antioestrogenic effects on the morphology of primate endometrium can be antagonized by competition with progesterone at the progesterone level. Since in general, steroid receptor-induced transcriptional effects are cell-specific, we postulated that antiprogestins modulate PR-dependent gene regulation differently in the glandular epithelium and stroma in the primate endometrium. Antiprogestin treatment-induced changes in the expression of locally acting growth factors and cytokines mediating the interaction between stroma and glandular epithelium by paracrine and autocrine signalling may account for the characteristic morphological appearance described as `non-competitive antioestrogenic activity'. In order to test this hypothesis, we investigated whether mifepristone disparately affects the glandular epithelium and stroma with respect to antioestrogenic and antiprogestogenic parameters by histomorphological evaluation of endometria from steroid-treated monkeys.
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Materials and methods |
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Hormonal treatments
The study design is shown in Figure 1. Each of the 18 monkeys received a depot preparation of the gonadotrophin releasing hormone (GnRH) agonist, leuprolide acetate (Lupron-Depot®, 80 µg/kg bodyweight, s.c.; Everyready Drugs Ltd, New York, NY, USA) between cycle days 2 and 6 in order to achieve and maintain a hypo-oestrogenic state (defined as serum oestradiol concentrations <50 pg/ml). Onset of menses is considered cycle day 1; the day of the first GnRH agonist injection is defined as study day 1. On study day 15, each monkey was implanted s.c. with a 3-cm Silastic capsule (General Medical, Newport News, VA, USA) packed with crystalline oestradiol (Sigma, St Louis, MO, USA), a capsule size shown previously to maintain serum concentrations of oestradiol within the normal physiological range for macaques (Wolf et al., 1989
; Greb et al., 1997
). On study day 29 (14 days of oestradiol replacement), and after a second injection of GnRH agonist, monkeys were randomly assigned to one of six groups of daily i.m. treatments (three animals per group) which continued for 14 more days until study day 43: group I, vehicle (ethanol); group II, mifepristone (National Research Institute for Family Planning, Beijing, China, 1 mg/kg bodyweight); group III, mifepristone plus progesterone (Sigma; 0.2 mg/kg); group IV, mifepristone plus progesterone (1.0 mg/kg); group V, mifepristone plus progesterone (5.0 mg/kg); and group VI, progesterone only (5.0 mg/kg).
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Endometrial biopsies and histomorphological evaluation
Endometrial biopsies were obtained from all monkeys on study day 43 by taking a wedge section of the uterine fundus at mid-ventral laparotomy. Biopsy samples were fixed in 10% phosphate-buffered formalin for 46 h and stored in phosphate-buffered saline (PBS) until being prepared into 6-µm sections to be processed for haematoxylin and eosin (H&E) staining according to standard histological techniques. All histomorphological evaluations were performed by two independent investigators (A.K.S., M.W.), who were blinded to the hormonal exposure of the tissues.
Computer-aided image analysis (CAIA)
Stromal cellular density and the degree of glandular pseudostratification were quantified by an image analysis system. Microscope images (Leitz DMR; Leica, Wetzlar, Germany) were digitized to a 16x106 colour 640x480 pixel array via a colour video camera (Sony 3CCD, Model DXC 930P) connected to a frame grabber in a PC (Quantimet 600, Leica). All images were digitized at fixed settings of microscope and camera after at least 1 h of equipment preheating in order to obtain homogeneous and constant image illumination conditions. For each monkey, one endometrial section from the tissue block which contained the most representative full-thickness endometrial biopsy was chosen.
Stromal oedema was quantified by measuring the area covered by nuclei in the endometrial stroma similar to the procedure described by Slayden et al. (1993). From each section, several images covering the entire area beneath the surface epithelium (functionalis layer) were analysed after transformation of colour images to 256 grey level images and improving the outline of nuclei by the `delineate' function of the software. The software allowed custom definition of a certain darkness threshold above which individual pixels (equivalent to 1.04 µm2) were marked. The threshold was set to a level marking stained cell nuclei entirely, and was applied uniformly to all images after deleting the areas covered by glands. Stromal oedema (defined as reciprocal density of stromal nuclei) was calculated by the following formula: (area of the tissue inside the image area covered by glands)/area covered by stromal nuclei.
This procedure allowed good discrimination between sections judged by the pathologist as `highly oedematous' versus `non-oedematous' (see Figure 2a).
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Subjective evaluation
Endometrial sections were also evaluated by a gynaecopathologist using a method similar to that described previously by Johannisson et al. (1982), Ghosh et al. (1992) and Slayden et al. (1993). In glands, the number of mitoses, apoptotic bodies and vacuolated cells were counted and recorded per 1000 gland cells. Glandular mitosis was identified from the presence of condensed chromatin and the absence of a nuclear membrane. Apoptotic bodies in glandular cells were determined by the presence of typical cellular fragments as described by Slayden et al. (1993).
Glandular pseudostratification and stromal oedema were graded by a semiquantitative scoring system, as 0 = none; 1 = slight; 2 = moderate; and 3 = marked.
Statistical analysis
To analyse differences in means, one-way analysis of variance (ANOVA) and two-tailed t-tests with BonferroniHolm correction, if applicable, were used. A two-way ANOVA with fixed effects was used to analyse the combined influence of progesterone and mifepristone. To test for a linear dose-dependent effect of progesterone at a given dose of mifepristone, linear regression analysis was employed.
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Results |
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Glandular epithelium
The highest fraction of cells containing vacuoles (Figures 3b and 4b) was observed after progesterone treatment (group VI, 92.0 ± 3.5%), and the lowest fraction after mifepristone treatment (group II, 2.4 ± 1.2%). Progesterone-induced vacuolization was blocked substantially by mifepristone, even at the highest dose of additional progesterone treatment (Figures 3b and 4b
). The pseudostratification index in the glandular epithelium as measured by CAIA was significantly higher in group I (oestrogen exposure) than in group VI (oestrogen plus progesterone exposure, P < 0.03). In mifepristone-treated monkeys (groups IIV), intermediate pseudostratification indices were measured which were not significantly different from those in groups I and VI (Figures 3c and 4b
); in mifepristone-containing treatment groups, progesterone treatment was a marginally significant factor influencing pseudostratification (P < 0.08).
The rate of mitosis was low in all treatment groups [mean 1.9 (range 05)/1000 cells]. An ~ 18-fold higher rate in the occurrence of apoptotic bodies compared with mitoses was observed in all treatment groups [33.8 (range 0200)/1000 cells]. No statistical differences between treatment groups were observed for both parameters (data not shown).
Correlation of objectively measured parameters with subjective evaluations
The oedema index measured by CAIA correlated significantly with the subjective oedema score (scale from 0 to 4) (R2 = 0.49, P = 0.05, Figure 5), linear regression analysis of measured and subjectively evaluated pseudostratification scores did not reach statistical significance (data not shown).
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Discussion |
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In endometrial glands, mifepristone treatment antagonized progesterone-dependent effects very potently, as would be expected from an antiprogestin: progesterone-induced vacuolization and inhibition of secretory activity were profoundly reduced in all mifepristone-containing treatment groups, indicating antagonism of progesterone-induced secretory transformation. This is in agreement with data in humans which showed that mifepristone treatment inhibits the expression of the secretory protein glycodelin in the endometrium, even at doses low enough not to disturb ovarian function (Gemzell Danielsson et al., 1997). A single dose of mifepristone administered in the early luteal phase was also shown to be sufficient to block progesterone-dependent reorganization of glandular epithelial cells at the fine structural level (Dockery et al., 1997).
Our data could not completely resolve the question as to whether all mifepristone action in the endometrium is mediated by the PR. Mifepristone-induced stromal compaction was not completely restored to levels measured in the oestrogen-only exposed endometrium, even with concomitant progesterone treatment resulting in high plasma progesterone concentrations. No clear doseresponse relationship with incremental progesterone concentrations was observed with respect to antagonism of mifepristone action. However, that progesterone was able to reverse mifepristone-induced stromal compaction is to some extent in agreement with a PR-mediated mechanism. The higher affinity of mifepristone for the PR compared with progesterone (Schreiber et al., 1983) might explain why even the highest dose of progesterone was not sufficient to antagonize mifepristone-induced stromal compaction completely. The high efficacy of mifepristone administered at a dose of 1 mg/kg could also be demonstrated in glands where progesterone-induced vacuolization was blocked completely, even in the presence of supraphysiological concentrations of progesterone in serum. Progesterone plasma concentrations were adjusted based upon the experience with previous studies (Wolf et al., 1989
; Neulen et al., 1996
; Greb et al., 1997
). The daily regimen resulted in progesterone plasma concentrations of ~ one-tenth (group III), equal (group IV) and double (groups V and VI) those of the normal luteal phase (Figure 6
) (Goodman et al., 1977
).
Our data also show that histomorphological measurements by a CAIA system and quantitative morphometry are useful tools for the evaluation of steroidal effects on the endometrium, and to discriminate between stromal and glandular effects. Similar approaches were employed previously by others (Ghosh et al., 1992; Slayden et al., 1993; Rogers et al., 1996
). Our approach was validated by rendering the anticipated results after oestradiol and progesterone treatments. Furthermore, objective measurement of stromal oedema by CAIA correlated with subjective evaluations by an experienced pathologist (Figure 5
). This implies that our measurements truly reflect hormonally induced proliferative and secretory changes of the endometrium according to established criteria (Sternberg, 1992
). However, interpretation of the data needs to take into account the small group sizes (n = 3).
The inhibitory effects of antiprogestin treatment on oestradiol-dependent growth is now well documented in non-human primates by numerous experiments performed in different laboratories (Brenner and Slayden, 1994; Hodgen et al., 1994
). Whether or not this effect occurs in humans is controversial. Endometrial morphology after daily administration of 50 mg of mifepristone over a duration of 6 months was described as being comparable with a chronic unopposed oestrogen effect (Murphy et al., 1995
). Another study confirmed the antimitogenic effects of mifepristone when administered at 2 mg/day for 30 days (Cameron et al., 1996b
). Moreover, both in humans (Kettel et al., 1996
) and monkeys (Grow et al., 1996
), the inhibitory effects of mifepristone were observed in ectopically growing endometrium (endometriosis).
The so-called `antioestrogenic action' of mifepristone and other antiprogestins seems to follow a tissue-specific pattern based upon experiments performed in rhesus monkeys by Slayden et al. (1993, 1998). Antiprogestin treatment caused inhibition of oestrogen-dependent proliferation in the endometrium, but did not affect oestrogen-dependent differentiation in the oviduct of rhesus monkeys. In a more recent paper, this group reported results comparable with our observations and employed a similar approach of histomorphological measurements after injection of another antiprogestin (ZK 137 316) during spontaneous cycles in rhesus monkeys (Slayden et al., 1998). Stromal compaction increased in all antiprogestin-treated monkeys, while contradictory observations were made in glands: ZK 137 316 treatment enhanced the expression of the proliferation marker Ki-67 dose-dependently, while the rate of mitosis was suppressed at higher doses. The authors concluded that the effects of ZK 137 316 can be explained only partly by inhibition of progesterone action. The effects of oestradiol are also inhibited by this compound.
Further analysis of antiprogestin action in the endometrium revealed that oestrogen-dependent pathways seem to be only partially antagonized. Even in the presence of high plasma concentrations of oestradiol, tissue growth was inhibited although other oestrogen receptor-mediated effects, e.g. upregulation of OR and PR levels, were not disrupted by antiprogestins (Neulen et al., 1990, 1996
; Slayden and Brenner, 1994
). The antiproliferative action of mifepristone does not require competition with progestins at the PR level. In fact, this effect was observed paradoxically, even in the absence of its cognate agonist, progesterone (Wolf et al., 1989
; Chwalisz et al., 1991
; Slayden and Brenner, 1994
; Cameron et al., 1996b
; Grow et al., 1996
; Neulen et al., 1996
).
As a potential molecular mechanism to explain the tissue-specific antioestrogenic activities of antiprogestins, it was suggestedbased upon experiments in transfected mammalian cellsthat the A-isoform of the human PR can act as a dominant inhibitor of OR-dependent transcriptional activity in the presence of antiprogestins (McDonnell and Goldman, 1994). In-vivo studies investigating OR-dependent signalling showed that the pathways regulating cell proliferation are not completely impaired during mifepristone treatment. Studies in human (Cameron et al., 1996b
) and non-human (Heikinheimo et al., 1996
; Slayden et al., 1998
) primate endometrium demonstrated a divergence between lack of mitosis and an increase of cell cycle-promoting factors involved in mitosis such as PCNA, Ki-67 and cyclin-B during mifepristone treatment. Based on these data, it was proposed by Heikinheimo et al. (1996) that mifepristone treatment induces a cell cycle block in the G2M interphase, preventing the cell from undergoing mitosis at the stage where cell cycle-promoting factors are already expressed.
The results obtained in the present study are in agreement with the studies referenced above by showing that, after 2 weeks of mifepristone treatment, stromal compaction rather than antiproliferative action in glands is associated with its growth inhibitory effect. Our data suggest that a disturbance of normal stromal function may explain why antiprogestin treatment ultimately leads to endometrial atrophy. The vasculature represents an important component of the stroma, and formation of new blood vessels (angiogenesis) in a menstrual cycle-dependent pattern was described as a distinctive process during cyclic development of the endometrium (Markee, 1940; Findlay, 1986
). It was also shown that menstrual cycle-dependent changes in blood vessel permeability occur which most likely cause stromal oedema (Torry et al., 1996
). Indeed, studies investigating the ultrastructure of the endometrium after mifepristone exposure could identify blood vessels as a target of antiprogestin action (Johannisson et al., 1989
), but as yet it is unknown what is mediating these effects. We and others recently demonstrated that mifepristone treatment was associated with reduced expression levels of the specific angiogenesis and vasopermeability factor (vascular endothelial growth factor/vascular permeability factor; VEGF/VPF) in primate endometrium (Greb et al., 1995
, 1997
; Ghosh et al., 1998
). Preliminary data from endometrial cells in culture suggest an upregulation of VEGF-expression levels by progestins (Shifren et al., 1996
; Sharkey et al., 1997
). Since VEGF is a very potent stimulator of vasopermeability (Senger et al., 1993
), the distinctive compaction of the stroma in our study would suggest an inhibition in the production of VEGF/VPF as a potential mechanism for the morphological effects associated with chronic mifepristone treatment. If we presume that stromal compaction is due to a lack of interstitial fluid possibly caused by decreased vasopermeability, mifepristone treatment-induced reduction in the production of VEGF/VPF would be in agreement with the observed high stromal cell density (Yuan et al., 1996
). Impairment of angiogenesis and vascular permeability may be a mechanism whereby mifepristone inhibits oestrogen-dependent growth without affecting other oestrogen-dependent pathways.
Thus, several mechanisms of action of antiprogestins might contribute to their potential usefulness as contraceptive agents preventing implantation: besides their antiprogestogenic potency blocking differentiation, endometrial growth is inhibited by a disturbance of normal stromal function. Additionally, mifepristone was shown to inhibit the growth and viability of preimplantation-stage embryos in the rhesus monkey (Ghosh et al., 1997).
In conclusion, we have demonstrated that the antiprogestin, mifepristone, affects stromal and glandular morphology in primate endometrium in a disparate fashion. In the stroma, mifepristone antagonizes the action of oestradiol, and increases cellular density by an as yet unidentified mechanism. In glands, mifepristone antagonizes the action of progesterone and blocks secretory transformation. Both actions appear to be mediated by the PR. These findings might be important in designing contraceptive strategies which prevent implantation.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 10, 1998; accepted on October 1, 1998.