Disparate actions of mifepristone (RU 486) on glands and stroma in the primate endometrium

R.R. Greb1,3,4, L. Kiesel1, A.K. Selbmann1, M. Wehrmann2, G.D. Hodgen3, A.L. Goodman3 and D. Wallwiener1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Besides being an antiprogestin, mifepristone (RU 486) was recently shown to antagonize oestrogen-dependent growth in the endometrium. To explore the molecular mechanisms for this phenomenon, we investigated whether or not the morphological effects of mifepristone are mediated by the progesterone receptor (PR) and whether mifepristone has disparate effects on the glandular epithelium and stroma. Six groups of hypogonadal, oestrogen-primed cynomolgus monkeys were treated for 2 weeks with: vehicle only (group I); mifepristone (group II); mifepristone plus progesterone at 0.2 mg/kg (group III), 1.0 mg/kg (group IV) or 5.0 mg/kg (group V); and progesterone only (5.0 mg/kg) (group VI). Histomorphological evaluation showed strikingly compacted stroma in the mifepristone-exposed endometria (group II), which was partially reversible by additional progesterone treatment (groups III–V). Glandular proliferation (pseudostratification, glandular mitoses) in mifepristone-treated monkeys was not significantly different from that in vehicle (oestradiol)-treated monkeys, but was inhibited by progesterone-only treatment. Cells containing vacuoles were scarce in the mifepristone-exposed endometrium, but detected frequently in progesterone-exposed endometria, indicating the strong antisecretory effect of mifepristone on glands. We conclude that oestrogen-dependent oedema in the stroma is antagonized by mifepristone. The reversal of this effect by progesterone suggests a PR-mediated mechanism. In glands, mifepristone is antiprogestogenic, but not antioestrogenic. Thus, stromal cells may be the target of antiprogestin-induced inhibition of oedema and endometrial growth.

Key words: antiprogestin/endometrium/endometrial morphology/oestrogen/vascular endothelial growth factor


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the normal menstrual cycle, the primate endometrium progresses through well-known morphological changes. In the functionalis layer, proliferation is dependent on the presence of oestradiol, followed by a progesterone-dependent secretory transformation (Padykula et al., 1989Go; Okulicz et al., 1997Go).

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., 1983Go; Dukes et al., 1993Go). 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., 1985Go; Batista et al., 1992Go; Katkam et al., 1995Go; Cameron et al., 1996aGo, 1997Go; Dockery et al., 1997Go; Gemzell Danielsson et al., 1997), presumably by competitive antagonism at the PR level (Edwards et al., 1995Go).

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, 1994Go; Hodgen et al., 1994Go; Cameron et al., 1996bGo). 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., 1993Go; Katkam et al., 1995Go; Slayden et al., 1998Go).

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., 1989Go). 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., 1985Go; Berthois et al., 1991Go) as, in contrast to progestin treatment, OR and PR are upregulated during antiprogestin treatment (Neulen et al., 1990Go, 1996Go; Slayden and Brenner, 1994Go). 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., 1995Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and study design
Eighteen cynomolgus monkeys (Macaca fascicularis), each with a history of normal menstrual cycles for at least 3 months, were housed in facilities of the Division of Animal Resources at the Eastern Virginia Medical School, fully accredited by the American Association for the Accreditation of Laboratory Animal Care, as described previously (Wolf et al., 1989Go).

Hormonal treatments
The study design is shown in Figure 1Go. 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., 1989Go; Greb et al., 1997Go). 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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Figure 1. Experimental design. Each of 18 female cynomolgus monkeys received a depot preparation of the GnRH agonist leuprolide acetate (Lupron-Depot®) between cycle days 2 and 6. On study day 15, an oestradiol-releasing capsule was implanted. On study day 29, after a second injection of GnRH agonist, monkeys were assigned randomly to one of six daily i.m. treatments which continued 14 more days until study day 43. Group I, vehicle (ethanol); group II, mifepristone, 1 mg/kg bodyweight; group III, mifepristone plus progesterone, 0.2 mg/kg; group IV, mifepristone plus progesterone, 1.0 mg/kg; group V, mifepristone plus progesterone, 5.0 mg/kg; group VI, progesterone, 5.0 mg/kg. An endometrial biopsy was performed on study day 43.

 
Plasma concentrations of oestradiol and progesterone
Blood samples (3 ml) were collected on alternate days throughout the study period by femoral venepuncture under ketamine anaesthesia (10 mg/kg, i.m.) and analysed by commercially available oestradiol and progesterone radioimmunoassays (ICN Biomedicals, Costa Mesa, CA, USA). The intra- and interassay coefficients of variation (CV) were 11 and 18% for oestradiol, and 10 and 15% for progesterone, respectively.

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 4–6 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 2aGo).



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Figure 2. Histomorphometric measurements performed by computer-assisted image analysis on digitized images. Examples of (a) a dense (left) and highly oedematous (right) stromal area and (b) a pseudostratified (right) and non-pseudostratified (left) gland are shown. Discriminative indices were obtained by applying the image analysis system.

 
Glandular pseudostratification was calculated based upon measurements of individual cross-sectioned glands in the functionalis layer (3–14 glands/animal, average number of glands/animal, 9.4). For better discrimination of glandular nuclei, all images were subjected to the `white sharpen' function of the software. The luminal and abluminal sides of the glandular cell nuclei were manually encircled using the computer mouse and marking the area covered by glandular nuclei. The average distance between the inner and outer circles was calculated by the software and divided by the average diameter of individual nuclei in a particular gland. Thus, one layer of columnar epithelium yielded a value of one, a double layer columnar epithelium resulted in a value of two, etc. (see Figure 2bGo).

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 Bonferroni–Holm 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometrial stroma
The highest density of stromal cell nuclei was observed in treatment group II, where oestrogen-primed endometrium was exposed to mifepristone-only treatment. This stromal compaction is reflected in the low oedema index as measured by CAIA which was approximately one-half of the oedema index measured in group I (oestrogen exposure only) (P < 0.003, ANOVA). Comparable indices of stromal oedema were observed in groups I and VI (oestrogen plus progesterone exposure) (Figures 3a and 4aGoGo).



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Figure 3. Stromal and glandular morphometry in endometrial biopsies. Means of particular treatment groups (n = 3) are shown as squares ({blacksquare}); means of different treatment combinations are shown as bars. (a) Stromal oedema index measured by computer-assisted image analysis (CAIA) in mifepristone-treated monkeys (group II) was significantly lower than in vehicle (oestradiol only)-treated monkeys (group I). (b) Progesterone treatment induced vacuolization in most glandular cells (group VI). This effect was blocked substantially in all mifepristone-containing treatment groups (II–V). (c) Glandular pseudostratification index measured by CAIA was inhibited significantly by progesterone treatment compared with oestradiol-only exposure (group VI versus group I).

 


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Figure 4. Photomicrographs of representative endometrial sections from each hormonal treatment group. Stromal oedema was quantified at low-power (a), and pseudostratification at high-power (b) magnification. Scale bar = 100 µm. See Results for description.

 
In groups III–V, where progesterone was added at three different dose levels to oestrogen/mifepristone treatment, mifepristone-induced stromal compaction was partially reversed by progesterone. Progesterone influenced stromal oedema significantly at a given dose of mifepristone (Figure 3aGo, groups II–V, P < 0.01). However, no linear dose–response relationship was observed (P = 0.27).

Glandular epithelium
The highest fraction of cells containing vacuoles (Figures 3b and 4bGoGo) 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 4bGoGo). 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 II–V), intermediate pseudostratification indices were measured which were not significantly different from those in groups I and VI (Figures 3c and 4bGoGo); 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 0–5)/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 0–200)/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 5Go), linear regression analysis of measured and subjectively evaluated pseudostratification scores did not reach statistical significance (data not shown).



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Figure 5. Regression analysis of objective measurements by computer-assisted image analysis (CAIA) and subjective evaluation of tissue sections.

 
Plasma hormone concentrations
GnRH agonist application suppressed plasma concentrations of oestradiol to <50 pg/ml in all six groups from study day 9 onward after an initial flare up. The insertion of the oestradiol-containing capsule on study day 15 resulted in peak oestradiol concentrations of 200–400 pg/ml in all groups, which stabilized at a plateau between 80–150 pg/ml for the duration of the study. Plasma progesterone concentrations were <0.3 ng/ml in groups I and II. In progesterone-treated animals, the average plasma progesterone concentrations during i.m. treatments between study days 29 and 43 were 1.6 ± 0.3 ng/ml, 23.0 ± 3.9 ng/ml, 33.6 ± 3.8 ng/ml and 38.2 ± 3.5 ng/ml in groups III, IV, V and VI, respectively (Figure 6Go).



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Figure 6. Plasma concentrations of oestradiol and progesterone in different treatment groups (n = 3).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our data indicate that the antiprogestin, mifepristone, antagonizes the action of oestradiol in the endometrial stroma. In the stroma, cellular density was increased significantly by mifepristone when compared with oestrogen exposure only. Along with the inhibitory effect on oestrogen-dependent growth, stromal compaction could be identified as one of the key morphological features of the antioestrogenic action of mifepristone. In contrast, mifepristone treatment did not significantly change morphological parameters associated with oestrogen stimulation in the glandular epithelium. The pseudostratification index as a parameter of glandular proliferation was only marginally reduced in response to mifepristone exposure, but was significantly suppressed in progesterone-treated monkeys, in agreement with the known antiproliferative action of progesterone in glands. Hence, with respect to the described `non-competitive antioestrogenic action', mifepristone treatment affects glandular and stromal morphology disparately in oestrogen-primed primate endometrium.

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., 1997Go).

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 dose–response 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., 1983Go) 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., 1989Go; Neulen et al., 1996Go; Greb et al., 1997Go). 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 6Go) (Goodman et al., 1977Go).

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., 1993Go; Rogers et al., 1996Go). 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 5Go). This implies that our measurements truly reflect hormonally induced proliferative and secretory changes of the endometrium according to established criteria (Sternberg, 1992Go). 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, 1994Go; Hodgen et al., 1994Go). 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., 1995Go). Another study confirmed the antimitogenic effects of mifepristone when administered at 2 mg/day for 30 days (Cameron et al., 1996bGo). Moreover, both in humans (Kettel et al., 1996Go) and monkeys (Grow et al., 1996Go), 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., 1998Go). 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., 1990Go, 1996Go; Slayden and Brenner, 1994Go). 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., 1989Go; Chwalisz et al., 1991Go; Slayden and Brenner, 1994Go; Cameron et al., 1996bGo; Grow et al., 1996Go; Neulen et al., 1996Go).

As a potential molecular mechanism to explain the tissue-specific antioestrogenic activities of antiprogestins, it was suggested—based upon experiments in transfected mammalian cells—that 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, 1994Go). 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., 1996bGo) and non-human (Heikinheimo et al., 1996Go; Slayden et al., 1998Go) 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 G2–M 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, 1940Go; Findlay, 1986Go). It was also shown that menstrual cycle-dependent changes in blood vessel permeability occur which most likely cause stromal oedema (Torry et al., 1996Go). Indeed, studies investigating the ultrastructure of the endometrium after mifepristone exposure could identify blood vessels as a target of antiprogestin action (Johannisson et al., 1989Go), 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., 1995Go, 1997Go; Ghosh et al., 1998Go). Preliminary data from endometrial cells in culture suggest an upregulation of VEGF-expression levels by progestins (Shifren et al., 1996Go; Sharkey et al., 1997Go). Since VEGF is a very potent stimulator of vasopermeability (Senger et al., 1993Go), 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., 1996Go). 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., 1997Go).

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.


    Acknowledgments
 
The authors wish to thank the following members of the Jones Institute for Reproductive Medicine: Dr Keith Gordon, Bruce Lamar, Wendy Babbitt, Alice Holland and Victoria Prince for assistance in monkey surgeries. Gratefully acknowledged is the expert histological assistance of Susan Downing. We also thank Prof. H.-K.Selbmann of the Institut für Medizinische Informationsverarbeitung at the University of Tübingen for his helpful advice on the data analysis and Ms Schröder of the Department of Surgery at the University of Tübingen for instruction on the image analysis system.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 10, 1998; accepted on October 1, 1998.