Hum. Reprod. Advance Access originally published online on November 25, 2005
Human Reproduction 2006 21(3):798-809; doi:10.1093/humrep/dei383
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Effects of mifepristone on proliferation and apoptosis of human endometrium in new users of medroxyprogesterone acetate
Department of 1 Obstetrics and Gynecology, 2 Pathology and 3 Pediatrics, The Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, The Keck School of Medicine of the University of Southern California, 1240 Mission Street, Room 8K9, Los Angeles, CA 90033, USA. E-mail: jjain{at}usc.edu
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Abstract |
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BACKGROUND: Mifepristone has been demonstrated to decrease breakthrough bleeding (BTB) in users of progestin-only contraceptives. METHODS: Endometrial biopsies were collected from 50 normal cycling women who were new users of depot medroxyprogesterone acetate (DMPA) randomized to receive either mifepristone or placebo before, during and after treatment. Proliferation, apoptosis and sex steroid receptors were evaluated by either immunohistochemistry or TUNEL assay. RESULTS: Administration of mifepristone to DMPA-exposed endometrium for 1 week significantly increased endometrial expression of Ki-67 (MKI67), estrogen receptor (ER)
and progesterone receptors A and B (PRAB) and decreased the number of TUNEL-positive and caspase-3 (CASP3)-active cells in the endometrial stroma. However, after 10 weeks of mifepristone treatment, no significant difference in proliferation, apoptosis and the expression of ER or PRAB could be detected between the endometrium treated with DMPA alone and endometrium treated with mifepristone and DMPA. CONCLUSIONS: Administration of mifepristone to DMPA users significantly increases endometrial proliferation and decreases endometrial stromal apoptosis in the short term. Prolonged exposure to mifepristone does not counteract the inhibitory effects of progestin therapy on endometrial proliferation. Estrogen and progesterone receptors may play an important role in these effects.
Key words: apoptosis/breakthrough bleeding/endometrium/mifepristone/proliferation
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Introduction |
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Progestin-only contraceptives, including depot medroxyprogesterone acetate (DMPA), are proven as effective long-term contraceptives. DMPA is an injectable contraceptive containing 150 mg of medroxyprogesterone acetate that is administered every 3 months. It has excellent contraceptive efficacy with annual failure rates of 0.3%. Breakthrough bleeding (BTB) has been reported to occur in 90% of women during the first 3 months of DMPA use (Newton et al., 1994
) and on 20–30% of days during the first 6 months of DMPA use (Tyler et al., 1970). The high rate of BTB often leads to premature discontinuation of DMPA and exposure to unintended pregnancies. The cause of irregular bleeding remains unclear. It is known that the continuous presence of progestin causes downregulation of estrogen and progestin receptors and, with time, endometrial atrophy (Lu, 1991; Lau et al., 1996; Wang et al., 1998; Zhu et al., 1999). Some authors have suggested that the decreased amount of stroma seen in atrophic endometrium may be insufficient to provide adequate support of blood vessels, thus predisposing them to fragility and bleeding (Critchley et al., 1998; Hickey et al., 2000). With time, endometrial atrophy leads to amenorrhoea.
It is well recognized that the addition of continuous ethinyl estradiol to a progestin-only contraceptive regimen reliably improves bleeding patterns (Diaz et al., 1990; Alvarez-Sanchez et al., 1996; Archer et al., 1996; Glasier et al., 2002). However, estrogen may be contraindicated in patients with conditions such as estrogen-sensitive neoplasms or predisposition to thrombosis. Recently, the progesterone receptor antagonist, mifepristone, was demonstrated to decrease the incidence of BTB in Norplant users (Cheng et al., 2000; Glasier et al., 2002) as well as in users of DMPA (Jain et al., 2003).
Although the exact mechanism by which mifepristone modulates endometrial tissue growth is poorly understood, an antiproliferative and pro-apoptotic effect has been demonstrated in studies of non-human primates (Wolf et al., 1989; Hodgen et al., 1994; Katkam et al., 1995; Zelinski-Wooten et al., 1998), women (Bagchi et al., 1992; Croxatto et al., 1993, 1998; Cameron et al., 1996) and endometrial cell lines (Schneider et al., 1998; Murphy et al., 2000; Prange-Kiel et al., 2000; Han and Sidell, 2003; Li et al., 2005b).
Apoptosis plays a pivotal role in the reproductive physiology of the endometrium (Kokawa et al., 1996; Nakano and Shikone, 1996; Shikone et al., 1997; Vaskivuo et al., 2000, 2002; Castro et al., 2002) and is highly regulated by estrogens and progesterone (Critchley et al., 1999; Vaskivuo et al., 2000; Castro et al., 2002). Moreover, sex steroids can modify the expression of proteins related to apoptosis in endometrial cells during the menstrual cycle (Koh et al., 1995; Critchley et al., 1999). In a recent study, the levonorgestrel-releasing intrauterine system (LNg-IUS) was shown to cause a decrease in endometrial proliferationand an increase in apoptosis in endometrial glands and stroma (Maruo et al., 2001). In this study, there were no differences in the levels of expression of Bcl-2, Fas or caspase-3 (CASP3) in the endometrium of levonorgestrelimplant (Norplant) users with and without BTB (Rogers et al., 2000). Fewer data are available regarding the effect of mifepristone on the regulation of endometrial cell proliferation and survival in the endometrium of women using DMPA as a contraceptive.
Thus, the present study was conducted to determine the changes in the proliferative activity and apoptosis in the endometrium of women using DMPA as a contraceptive with or without the addition of mifepristone.
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Materials and methods |
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This was a prospective, randomized, double-blind, placebo-controlled study approved by the Institutional Review Board of the University of Southern California and Los Angeles County Medical Center. Written informed consent was obtained from all subjects prior to enrolment.
Tissue collection
Endometrial biopsies were taken with a Pipell (Prodimed, Neuilly-en-Thelie, France) instrument from a total of 50 regularly menstruating healthy women. Subjects were equally randomized in a double-blinded fashion to receive 50 mg of mifepristone or placebo every 14 days for six cycles beginning on day 14 following the first injection of DMPA (150 mg). As shown in Figure 1, four biopsies were obtained from each patient, one prior to treatment, one after administration of DMPA and an additional two biopsies to evaluate early and late effects of mifepristone within one cycle of DMPA treatment (the first 3 months). Since there are no clear data on when the receptors undergo induction and suppression after steroidal contraceptives, we arbitrarily chose the time points of 2 weeks post-DMPA, and 1 week and 10 weeks after mifepristone. Altogether, a total of 96 placebo biopsies and 94 mifepristone biopsies were taken. Part of each biopsy of the 190 biopsies was processed for routine histological evaluation with haematoxyalin and eosin. All endometrial samples were also evaluated for determination of phase using the criteria of Noyes et al. (1975).
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Immunohistochemistry
Endometrial biopsies obtained at the end of the 3 month study contained scant endometrium. In many instances, the amount of tissue and the absence of functionalis precluded the performance of immunohistochemistry. Immunohistochemistry was performed in only 80 endometrial biopsies sampled at four different treatment periods of each woman selected from patients with sufficient and adequate tissue to perform all assays in both the placebo (n = 8) and mifepristone group (n = 12). Proliferation was assessed immunocytochemically with MKI67, a monoclonal antibody against a nuclear antigen present only in proliferating cells (Gerdes et al., 1984). The proliferation marker MKI67 is expressed during all phases of the cell cycle with the exception of the early G1 phase of resting cells, or differentiated cells (in G0 phase) (Landberg et al., 1990). Because of its short half-life (Schluter et al., 1993), MKI67 indicates the growth fraction of the examined tissue (Gown et al., 1996). Apoptosis was identified by detecting CASP3 activity using an anti-cleaved CASP3 antibody, which is immunolocalized to the cytoplasm of apoptotic cells (Jain et al., 2005b). Immunolocalization of estrogen receptor subtypes and 
(ER and ER) and progesterone receptors A and B (PRAB) was evaluated as described previously with a few modifications (Jain et al., 2005a,b; Li et al., 2005a). Tissue sections were de-waxed, rehydrated and immersed in 10 mmol/l (pH 6) sodium citrate buffer and heated in a microwave oven for 8 min at 700 W for antigen recovery. They were then allowed to cool to room temperature and immersed in phosphate-buffered saline (PBS). Endogenous peroxidase activity was quenched using 3% hydrogen peroxide for 10 min at room temperature. After normal serum blocking, an avidin/biotin blocking kit (Vector Laboratories, Inc., Burlingame, CA) was used to ensure that all endogenous biotin, biotin receptors or avidin-binding sites present in tissues are blocked prior to the addition of the labelled avidin reagent. The sections were then incubated with either an antihuman MKI67 mouse monoclonal antibody, anti-human ER rabbit polyclonal antibody (1:100, BioGenex, San Ramon, CA), anti-human cleaved CASP3 rabbit polyclonal antibody (1:50, Oncogene, San Diego, CA), anti-human ER or antihuman PRAB mouse monoclonal antibodies (1:100, Novocastra Laboratories Ltd, Burlingame, CA) at 4°C overnight. After washing with PBS, biotinylated anti-mouse or anti-rabbit IgG was applied for 30 min at room temperature. Following washing of PBS, avidin/biotin/peroxidase solution was applied for 50 min and visualized by Peroxidase Substrate Kit DAB (Vector Laboratories, Inc.). Counterstaining was performed lightly with haematoxylin. The sections were then dehydrated and coverslipped with mounting medium (Richard-Allan Scientific, Kalamazoo, MI). Examination and photography were performed usingan Olympus BX-50 light microscope equipped with a Canon EOS D30 digital camera (Tokyo, Japan).
An endometrial section in the phase of proliferation was used as a positive control for MKI67. A rat mammary gland specimen (CHEMICON, Temecula, CA) was used as a positive control for cleaved CASP3 antigen. A breast cancer specimen was used as a positive control for PR and ER. The specificity of the immunohistochemical reaction was verified by the omission of the primary antibody as well as using non-relevant primary antibody mouse or rabbit IgG (1:1000, Vector Laboratories, Inc.)
TUNEL
To determine late-stage apoptosis in endometrium from placebo- or mifepristone-treated groups, deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) analysis was performed to identify cells involved in the apoptotic fragmentation of DNA. The DeadEndTM Colorimetric TUNEL System (Promega, Madison, WI) was used to detect apoptotic cells in 44 biopsies sampled at four different treatment periods of each woman randomly selected from both the placebo (n = 6) and the mifepristone group (n = 6). Sections were deparaffinized in fresh xylene and washed twice in 100% ethanol for 5 min. Then rehydration was done with decreasing concentrations of ethanol (100, 95, 85, 70 and 50%) for 3 min each time, followed by immersion in 0.85% NaCl and PBS for 5 min each. The slides were then fixed in 4% paraformaldehyde in PBS for 15 min and washed in PBS twice for 5 min each time. The permeabilization was then performed in 100 µl of a 20 µg/ml proteinase K solution for 15 min. After a 5 min wash in PBS, the slides were repeatedly fixed in 4% paraformaldehyde in PBS for 5 min. Equilibration in 100 µl of equilibration buffer was done for 5 min following another PBS wash. Labelling was performed by adding 100 µl of TdT reaction mix to the tissue sections on the slides. Plastic coverslips were used to ensure even distribution, and incubation was for 60 min in a 37°C humidified chamber. The reaction was stopped by immersing slides in 2x SSC for 15 min followed by three consecutive 5 min PBS washes. Immersion in 3% hydrogen peroxide for 3 min was done as the blocking step followed by another triple wash in PBS. A 100 µl aliquot of streptavidin–horseradish peroxidase (HRP) diluted 1:500 in PBS was then added followed by 30 min of incubation. A triple wash was then performed followed by the addition of 100 µl of Peroxidase Substrate Kit diaminobenzidine (DAB) (Vector Laboratories, Inc.) that was prepared immediately before, and allowed to incubate until a light brown background developed. Finally the slides were washed by immersing several times in deionized water and counterstained lightly with haematoxylin. The sections were then mounted and photographed as described above. The positive control sections were treated with DNase I (Promega) prior to TUNEL staining, and the TdT was replaced by PBS for the negative controls.
Quantification of proliferating and apoptotic cells
The number of MKI67-, TUNEL- and activated CASP3-positive cells was quantified by counting the number of labelled cells per 1000 total cells manually by two trained observers blinded to the experiment. Two consecutive, randomly selected 400x microscopic fields were selected for each specimen processed. Fields were evaluated separately for glandular and stromal cells.
Semi-quantification of ER- and PR-positive cells
Semi-quantification of immunoreactivity for the expression of ER and PR was performed separately for glands and stroma in each biopsy by evaluating the percentage of stained cells and staining intensity. The proportion of stained cells was graded as 0, 1, 2 or 3 (positive staining signal in 0, <10, 10–50 or >50 of cells, respectively). The intensity of staining was graded as: 1 (weak), 2 (moderate) or 3 (strong). The staining index was calculated according to the following equation: percentage of stained cells multiplied by the staining intensity, allowing for a minimum score of 0 and a maximum score of 9 (Nap et al., 2004). The average of the scores of two independent observers was calculated and taken as the staining index. All samples were evaluated at a 400x magnification.
Statistical analysis
The Wilcoxon rank sum test was used to compare the numbers of MKI67- and activated CASP3-possitive cells and TUNEL-labelled cells per 1000 total cells at various time points between the mifepristone and placebo groups. The data are expressed as median and range. Differences were considered significant at a level of P < 0.05. The differences of staining index of ER and PR immunoreactivity between the mifepristone and placebo groups were also compared using Wilcoxon rank sum test and paired t-test.
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Histological assessment
Adequate tissue for histological assessment from all four endometrial biopsies was available for 20 out of 25 women in each of the treatment groups. Histological analysis of these tissues revealed that most baseline biopsies contained secretory endometrium post-ovulation day 2–11 (Table I). All samples obtained 14 days after the first dose of DMPA displayed a progestogenic effect, although the degree of pseudo-decidualization varied. At the time of the third biopsy, 7 days after commencement of mifepristone or placebo, 15 out of 20 samples (75%) in the mifepristone group showed weakly proliferative or proliferative endometrium with frequent mitosis, whereas two out of 20 (10%) women showed weakly proliferative endometrium in the placebo group (Table I and Figure 2). The majority of samples at the time of the fourth biopsy, 10 weeks of commencing mifepristone or placebo, displayed atrophic endometrium in both groups. However, eight out of 20 (40%) endometrial samples at the time of the fourth biopsy showed a mild to strong progestogenic effect in the placebo group, while only three out of 20 (10.5%) showed a progestogenic effect in the mifepristone group.
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Proliferation
The proliferation rate assessed by means of the monoclonal antibody MKI67 is shown in Figure 3 and Tables II and III. Pre-treatment secretory phase endometrium (first biopsy) exhibited abundant glandular expression of MKI67 in post-ovulation day (POD) 2–4 endometrium (Figure 3A and E) but minimal or absent glandular expression at POD5–9 (Figure 3I and M). In contrast, expression of MKI67 in stroma showed less variability between different periods of the secretory phase. Generally, staining of stroma was equally distributed in all areas, whereas staining of glands showed a marked heterogeneity.
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Fourteen days following exposure to DMPA (second biopsy), endometrium when treated at POD2–4 displayed a significant reduction in the number of endometrial cells expressing MKI67 when compared with untreated endometrium with a median of 4 per 1000 cells versus 257 per 1000 cells (glands) and a median of 51 per 1000 cells versus 210 per 1000 cells (stroma) (P < 0.05, Figure 3B and F). There were no significant changes in the number of cells expressing MKI67 in the endometrium when treated at POD5–9 versus those of the second biopsy (P > 0.05, Figure 3J and N and Table II). One week following the administration of mifepristone (Figure 3G and O), both glandular and stromal cells demonstrated a significant increase in MKI67 immunoreactivity when compared with endometrium treated with DMPA alone (placebo group) during the same time period (251 per 1000 cells versus 5 per 1000 cells and 125 per 1000 cells versus 28 per 1000 cells, respectively). Ten weeks after mifepristone treatment (Figure 3H and P), the number of MKI67-positive cells decreased significantly when compared with the third biopsy of the same group in both glands (2.5 per 1000 cells versus 251 per 1000 cells) and stroma (23 per 1000 cells versus 125 per 1000 cells) (P < 0.05). There was no statistical difference between the endometrium of the fourth biopsy in women of the mifepristone group and placebo group (P > 0.05, Table III).
As shown in Figure 4A–H and Table III, pre-treatment secretory phase endometrium contained rare apoptotic cells that were scattered among ordinary cells in both glands and stroma. Fourteen days after administration of DMPA, there was a significant increase in the number of apoptotic cells seen in the stroma of the DMPA-treated endometrium with a median of 7 per 1000 cells versus 1 per 1000 cells in the stroma of the pre-treated endometrium (P < 0.05). Apoptotic cells in glands did not change significantly upon DMPA treatment (0.5 per 1000 cells versus 0 per 1000 cells) (P > 0.05, Table III). At the time of the third biopsy, the median of TUNEL-positive cells in the stroma of the mifepristone-treated women (4 per 1000 cells) was significantly lower than in the stroma of the women treated with DMPA alone (11.5 per 1000 cells, P = 0.03). The median of TUNEL-positive cells in the glands and stroma of the mifepristone-treated women remained low, without significant changes after 10 weeks of mifepristone exposure (Table III). Because endometrial breakdown is significantly associated with apoptosis in the endometrium, we evaluated the bleeding status of each woman at the time of the endometrial biopsy. Clinical bleeding at the time of each biopsy in women whose biopsies were used for immunohistochemistry is shown in Table IV. There was no significant difference in the bleeding patterns between placebo and mifepristone groups except in the third biopsy (1 week following the administration of mifepristone), where more women in the mifepristone group were bleeding than women in the placebo group.
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Activated CASP3
Subsequently, we investigated the CASP3 activity in endometrium from mifepristone- or placebo-treated groups by immunohistochemistry to confirm the apoptosis patterns seen in the TUNEL assay. As shown in Figure 4I–P and Table III, the expression pattern of cleaved CASP3 in response to the DMPA and DMPA plus mifepristone treatment is identical to that seen with the TUNEL assay.
ER and PR expression
To determine whether the changes of proliferation and apoptosis in response to the treatment are correlated with the expression of steroid receptors, we also evaluated the expression of endometrial ER, ER and PRAB in these samples by immunohistochemistry. As shown in Figure 5 and Table V, treatment with mifepristone for 1 week significantly increased ER and PRAB immunoreactivity in both glandular and stromal cells. However, 10 weeks after mifepristone treatment, the expression of ER and PRAB was reduced to the baseline (second biopsy, DMPA treatment) in both glands and stroma. No significant difference in ER expression was seen throughout all treatment periods and groups (data not shown).
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Discussion |
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We have shown that the anti-progestin, mifepristone at 50 mg significantly increases endometrial proliferation in new users of DMPA, as demonstrated by histology and by increased expression of the proliferation marker MKI67. We also demonstrate a decrease in stromal apoptosis as demonstrated by decreased TUNEL-positive cells and cleaved CASP3 activity. Mifepristone also increased the immunoreactivity of ER
and PRAB. These effects were only seen after 7 days of exposure to mifepristone but returned to baseline by 10 weeks of treatment. We have confirmed previous findings in the literature demonstrating a decline in the expression of MKI67 in endometrium, and an increase of apoptotic cells in stroma after DMPA exposure. We believe that the increased proliferation and decreased apoptosis associated with increased ER and PRAB seen with the addition of mifepristone may play an important role in decreasing breakthrough bleeding of the DMPA-treated endometrium at an earlier stage.
In our study, we show a relatively short-lived estrogen-like effect as demonstrated by increased proliferation in the endometrium of mifepristone- and DMPA-treated women when compared with that of women treated with DMPA alone. This effect, however, disappeared after 10 weeks of combined mifepristone and DMPA therapy. We theorize that the short-lived proliferative effect seen with mifepristone exposure is due to a direct anti-progestogenic effect of mifepristone that blocks the inhibitory effects of DMPA. As such, we note a transient increase in the number of bleeding days after 1 week of mifepristone (Table IV). This increase in bleeding seen shortly after mifepristone administration probably represents ‘progesterone withdrawal’ caused by the antagonistic effect of mifepristone on the PR. Our data also demonstrate that the exposure of DMPA-treated endometrial tissue to mifepristone for 1 week led to a significant increase of ER and PRAB, also consistent with an anti-progestin effect. This is consistent with previous studies showing that short-term treatment with a PR antagonist leads to elevations of the two main uterine steroid receptors, ER and PR (Neulen et al., 1990; Slayden and Brenner, 1994; Cameron et al., 1996). The increase of endometrial ER and PR is likely to contribute to early effects of mifepristone on endometrial proliferation leading to cessation of bleeding and the improvement of BTB in the long term. This long-term effect can be gleaned from our data as evidenced by the return of bleeding to a baseline level by the third month (fourth biopsy) after the initiation of mifepristone therapy.
Another explanation for the short-term increase in proliferation may be that the MKI67 detects cells throughout all stages of the cell cycle, e.g. G1, S, G2 and M. It is possible that mifepristone treatment blocks completion of the cell cycle at G2–M interphase (Heikinheimo et al., 1996). MKI67 protein can persist for sometime after cell division has been arrested (Gerdes et al., 1984; Endl and Gerdes, 2000). The concept that mifepristone may cause cells to enter the cell cycle but somehow prevent entry of cells into the mitotic phase of the cell cycle is consistent with findings showing a prevention of endometrial proliferation with resulting endometrial atrophy. This hypothesis has been postulated previously in a study demonstrating a decrease in the mitoticindex after 21 days of treatment with mifepristone although the MKI67 immunostaining was increased (Cameron et al., 1996). However, it is also possible that the transient pro-proliferative effect is due to the low dose and intermittent administration of mifepristone used in our study.
Concern has been expressed previously that long-term use of PR antagonists may lead to endometrial hyperplasia and possible malignancy due to exposure of the endometrium to the effects of unopposed estrogen (Murphy et al., 1995; Newfield et al., 2001; Eisinger et al., 2003). Evidence of estrogenic stimulation of the endometrium has been observed in rats receiving long-term PR antagonist treatment (Rumpel et al., 1993; Bofinger et al., 2001). However, in non-human primate and human endometrium, treatment with mifepristone has shown endometrial atrophy, suggesting anti-estrogenic activity (Chillik et al., 1986; Koering et al., 1986; Wolf et al., 1989; Ishwad et al., 1993; Cameron et al., 1996; Neulen et al., 1996; Slayden et al., 1998; Brown et al., 2002; Baird et al., 2003; Narvekar et al., 2004). In addition, Slayden et al. (1993) reported a significant decrease of proliferation in the endometrial stroma in rhesus monkeys with mifepristone at a dose of 1mg/kg, daily, during a 2 week time period. Our findings confirm that although proliferation was increased by 1 week of mifepristone and DMPA, there is no evidence of persistent endometrial proliferation after 10 weeks of mifepristone and DMPA. To support these findings, we further demonstrated that the exposure of DMPA-treated endometrial tissue to mifepristone for 10 weeks led to a downregulation of ER and PRAB in parallel with endometrial atrophy. Therefore, the transient nature of the effect of mifepristone is probably due to this subsequent decrease in ER and PR content after prolonged exposure to mifepristone and MPA.
We have demonstrated an increase in stromal cell apoptosis following treatment with DMPA, a finding seen in several other studies (Maruo et al., 2001). Importantly, we demonstrate a significant decrease in stromal apoptosis in the endometrium of women in the mifepristone group (mifepristone and DMPA) when compared with women in the placebo group (DMPA alone) after 1 week treatment. These data are different from what was reported previously by Slayden et al. (1993) for rhesus monkeys treated with higher doses of mifepristone. In their studies, a marked increase in the rate of apoptotic cell death in the glandular epithelium occurred in both the absence and presence of progesterone in the endometrium of the mifepristone-treated animals. The higher dosages and continuous delivery of mifepristone used by Slayden et al. may account for the differences observed.
The increase in endometrial proliferation and prevention of stromal apoptosis may be an instrumental early effect in the decrease of BTB that we have shown in DMPA users by the addition of mifepristone (Cheng et al., 2000; Glasier et al., 2002; Jain et al., 2003). Although neither the increase in proliferation nor the decrease in apoptosis is sustained for a prolonged period of time (not seen after 10 weeks of treatment), the temporary changes may be sufficient to decrease bleeding in the transition of the endometrium from functional to atrophy. The administration of mifepristone to DMPA-exposed endometrium early on appears to antagonize the inhibitory action of the progestin as evidenced by the observed upregulation of ER and PR and a transient increase in endometrial bleeding. We postulate that this upregulation causes an induction of factors responsible for endometrial proliferation that mimics the physiological process observed during the proliferative phase of the normal menstrual cycle leading to the eventual, longer term decrease of bleeding and a total increase in bleeding-free days. Similarly, we postulate that since apoptosis in endometrial stromal cells is maximally observed in menstrual or ‘bleeding’ endometrium (Dahmoun et al., 1999), the decrease in apoptosis seen after the addition of mifepristone results from a downregulation of factors that decrease stromal breakdown.
In summary, we have shown that the exposure to mifepristone reversed several effects of DMPA alone on the endometrium in the short term by inducing a transient increase in endometrial proliferation and decrease in stromal apoptosis causing cessation of bleeding. Prolonged exposure to mifepristone and DMPA led to endometrial atrophy where nothing was shed, suggesting no increased risk in long-term proliferation or proliferation-associated diseases.
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Acknowledgements |
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These studies were supported by NIH grants NIH RO-1 HD 43189 and NIH-1 RO3 HDO 47322 to J.K.J.
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Submitted on July 11, 2005; resubmitted on October 5, 2005; accepted on October 6, 2005.
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