Hum. Reprod. Advance Access originally published online on June 28, 2006
Human Reproduction 2006 21(9):2312-2318; doi:10.1093/humrep/del182
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Mifepristone-induced amenorrhoea is associated with an increase in microvessel density and glucocorticoid receptor and a decrease in stromal vascular endothelial growth factor
1 Contraceptive Development Network, Centre for Reproductive Biology, Edinburgh, UK and 2 Shanghai Institute of Family Planning Technical Instruction, International Peace Maternity and Child Health Hospital, China Welfare Institute, Shanghai, People’s Republic of China
3 To whom correspondence should be addressed at: Centre for Reproductive Biology, Simpsons Centre for Reproductive Health, Chancellor’s Building, 51 Little France Crescent, Edinburgh EH16 4SA, UK. E-mail: dtbaird{at}ed.ac.uk
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Abstract |
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BACKGROUND: We have previously shown that the progesterone antagonist mifepristone is a contraceptive when given in a dose of 2 or 5 mg per day. The majority of women experience amenorrhoea rather than the irregular break through bleeding usually occurring with other estrogen-free contraceptive pills, such as progestogen-only pill (POP). We investigated the effects of low-dose mifepristone on endometrial parameters which may be associated with changes in endometrial function, such as microvasculature, vascular endothelial growth factor (VEGF) and glucocorticoid receptor (GR) content. METHODS AND RESULTS: Endometrial biopsies were collected from 16 women before (late proliferative phase) and 60 and 120 days after taking 2 or 5 mg mifepristone daily for 120 days. Seven of the eight women who received 2 mg mifepristone and all eight women who received 5 mg were amenorrhoeic during the study. Mean estradiol (E2) concentrations remained in the mid-proliferative range, and the majority (9/16) of women showed proliferative endometrial histology at 60 and 120 days following treatment. There was a significant increase in the density of the endometrial stroma (P < 0.05) and microvessels (P < 0.01) following 120 days of treatment. Immunocytochemistry showed that GR, hitherto localized specifically in endometrial stroma, was up-regulated in the nuclei of glands (P < 0.05) and surface (luminal) epithelium (P < 0.01) by 60 days and maintained at 120 days. There was a significant reduction in stromal VEGF protein expression by day 120 of treatment (P
0.01). CONCLUSION: The high incidence of amenorrhoea in women taking mifepristone may be related to changes in the regulation of vascular function.
Key words: contraception/endometrium/glucocorticoid receptor/mifepristone/vascular endothelial growth factor
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryAcknowledgementsReferences |
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Mifepristone is a potent antagonist of both progesterone and glucocorticoids. In daily low doses, it acts as a contraceptive by inhibiting ovulation and by altering endometrial function (Brown et al., 2002
; Baird et al., 2003a,b). Currently used estrogen-free contraceptive methods such as progestogen-only pill (POP) are often discontinued because of a high incidence of troublesome side effects, such as break through bleeding (Belsey and Farley, 1988). In contrast, the majority of women who take mifepristone have no periods. Although traditionally amenorrhoea has been classified as a disadvantage, many women now regard the absence of periods as a desirable side benefit (Glasier et al., 2003). The mechanisms underlying these disturbances in endometrial function are poorly understood and not clearly related to levels of endogenous or exogenous steroid hormones (Fraser et al., 1996).
Locally produced vasoactive substances probably play a key role in regulating endometrial angiogenesis, although these are influenced substantially by different dosage regimens and routes of administration of contraceptive steroids (Smith, 2001). Vascular endothelial growth factor (VEGF) promotes microvascular endothelial cell proliferation, migration and assembly into new vessels (Ferrara and Davis-Smyth, 1997), and estrogen has been shown to promote angiogenesis by regulating the expression of VEGF (Albrecht et al., 2003). Prostaglandins (PGs) influence contractility of endometrial vessels and their permeability (Albrecht et al., 2003). An increase in the local concentration of PGs in the endometrium is involved in the mechanism of mifepristone-induced vaginal bleeding in the luteal phase (Hapangama et al., 2002). Glucocorticoids modulate PG production in endometrial stromal cells and fibroblasts (Pakrasi et al., 1983; Schatz et al., 1986; Neulen et al., 1989; Delvin et al., 1990; Illouz et al., 2000). Glucocorticoid receptor (GR) is strongly expressed in the nuclei of endometrial cells in the stromal compartment of human endometrium during the menstrual cycle (Bamberger et al., 2001; Henderson et al., 2003). Although the exact physiological role of glucocorticoids and GR in the human endometrium is unknown, it has been suggested that they have an angiostatic role (Small et al., 2005).
We have previously reported the effects of daily low-dose mifepristone on the histology of the endometrium, sex hormone receptors and various proliferation markers (Baird et al., 2003a,b; Narvekar et al., 2004). Mifepristone treatment is associated with a striking up-regulation of the expression of androgen receptors (ARs) in the glands and with an increase in stromal density. The aim of this study was to look at the effects of low-dose mifepristone on endometrial microvessel density, VEGF and GR protein expression and correlate with endometrial histology and bleeding patterns.
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Materials and methods |
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Human endometrial samples were obtained from 16 women with regular menstrual cycles (25–35 days), who were a subset of 58 volunteer women from Edinburgh, as previously reported (Brown et al., 2002
; Baird et al., 2003a; Narvekar et al., 2004). The local ethics committees (Institutional Review Board) approved the studies, and all the women provided written informed consent. Subjects were randomly allocated to receive 2 (n = 8) or 5 mg (n = 8) of mifepristone daily for the 120 treatment days. Subjects had a mean age of 30.5 years (range 24–40) and a mean BMI of 24.5 kg/m2 (range 21–34). Endometrial biopsies were collected using a Pipelle endometrial sampling device (Prodimed, Neuilly-en-Thelle, France) in the late follicular phase of the pretreatment cycle (day 12), after 60 days of mifepristone treatment and after 120 days of treatment. Specimens were fixed in normal buffered formalin, processed and embedded in paraffin wax. Endocrine and endometrial findings have been reported previously (Brown et al., 2002; Baird et al., 2003a; Narvekar et al., 2004).
Immunocytochemistry
Immunocytochemistry was performed for the immunolocalization of VEGF, CD31 (endothelial marker) and GR. Immunostaining procedures followed those previously published (Nayak et al., 2000; Bamberger et al., 2001). All antibodies were tested individually at a range of dilutions and different antigen retrieval conditions to determine the protocol which gave the least background and highest specific staining (Table I). Positive and negative controls were included. All tissue sections were initially prepared in a similar manner. Five-micrometre paraffin-embedded tissue sections were dewaxed in Histoclear (National Diagnostics, UK) and rehydrated in descending grades of alcohol to distilled water. The tissue sections were subjected to antigen retrieval in a pressure cooker (5 min) or microwave (10 min) using 0.01 M sodium citrate at pH 6 (Table I) and were then allowed to cool for 20 min. Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide (BDH, Poole, UK) in methanol for 30 min at room temperature. Non-specific binding of the primary antibody was blocked by incubating the sections for 20 min at room temperature in a 1:5 dilution of normal immune serum (Autogen Bioclear, Holly Ditch Farm, Wilts, UK) in buffer containing 5% bovine serum albumin. Sections were incubated at 4°C with primary and control antibodies overnight. An avidin–biotin–peroxidase system was used as the detection system. The sections were incubated with biotinylated secondary antibody (Vector Laboratories, Peterborough, UK) in normal immune serum followed by avidin–biotin–peroxidase complex (Vectastain horse-radish peroxidase and Vectastain Elite PK 6101, Vector Laboratories) for 30–60 min each at room temperature. All tissue sections underwent an identical epitope visualization step. The peroxidase substrate 3,3'-diaminobenzidine (DAKO) was used as chromogen. Sections were then counterstained with haematoxylin, dehydrated in ascending grades of alcohol to xylene and mounted using Pertex (Cellpath plc., Hemel Hempstead, UK).
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CD31
Sections were incubated at 4°C either with mouse anti-human CD31 (Novacastra Laboratories, Newcastle upon Tyne, UK) overnight at a 1:800 dilution in normal horse serum (NHS) or similarly with a control mouse immunoglobulin (Ig)G1 antibody at 1:8000 dilution in NHS.
VEGF
Sections were incubated overnight at 4°C either with polyclonal rabbit anti-human VEGF (SantaCruz Biotechnology, CA, USA) antibody at 1:600 dilution in normal goat serum (NGS) or similarly with a control VEGF preabsorbed antibody at 1:60 dilution in NGS.
GR
Non-specific binding of avidin and biotin was blocked by incubating with avidin and biotin for 15 min each followed by non-immune rabbit serum (NRS) for 20–30 min at room temperature. Sections were incubated at 4°C either with monoclonal mouse anti-human GR IgG2a (Novocastra Laboratories, Newcastle upon Tyne, UK) at a dilution of 1:40 in NRS or similarly with a control mouse IgG2a antibody at 1:320 dilution in NRS.
Semi-quantitative immunocytochemistry score
Location and intensity of immunostaining were measured for VEGF and GR using a semi-quantitative scoring system. Sections were scored blind by two observers (blind to study groups and to other’s results). This scoring system is a standard method used in previous studies (Wang et al., 1998; Critchley et al., 2001; Narvekar et al., 2004), and a high correlation has been demonstrated between objectively measured immunoreactivity (image analysis) and subjective semi-quantitative scoring of immunostaining patterns (Wang et al., 1998). Immunostaining intensity and distribution of epitopes in all tissue sections were assessed on an arbitrary four-point scale: 0 = no staining, 1 = mild staining, 2 = moderate staining and 3 = intense staining.
Vessel counts and stromal density
Vessel counts and stromal density were measured using image analysis. The system used a Carl Zeiss Axistop 2 microscope (x40 objective) connected to a MacIntosh G3 computer, using Openlab 2.08 image analysis software (Improvision, Coventry, UK). At least 12 fields of view were selected at random from each tissue section at a magnification of x 40. The glands and stroma from each digitized image were interactively dissected.
CD31 immunoexpression was used to identify endothelial cells (Rees et al., 1993). All immunostained (brown) structures were considered positive and counted for each digitized image, even if lumen was not identified. A similar methodology has been described to study changes in vessel density following Norplant use (Hickey et al., 1999). The results were averaged and expressed as vessels per square millimetre. Endometrial stromal density appears to be increased following daily low-dose mifepristone (Baird et al., 2003a). To compensate for this, the total number of cells not expressing CD31 (blue haematoxylin) was measured separately for each digitized image using Openlab colour discrimination software, and the results are also expressed as vessels per 1000 stromal nuclei. This method of image analysis has previously been described and validated in our laboratories (Critchley et al., 1996; Wang et al., 1998).
Stromal density was expressed as the total number of stromal nuclei (brown CD31 and blue haematoxylin) per square millimetre of endometrial tissue.
Statistical methods
Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA) and Excel 2002 (Microsoft Corporation). Continuous data are expressed as mean with SE and categorical data as median with range. Non-parametric tests (Friedman test, Wilcoxon-signed rank test and Mann–Whitney test), with and without Bonferroni correction, were used to compare study variables at various time points.
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Results |
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Endometrial histology, serum estradiol and bleeding patterns
All 16 pretreatment endometria were sampled in the proliferative phase. Following treatment, 9/16 endometria were still proliferative at 60 and 120 days. Four endometria showed cystic dilatation of the glands and five inactive epithelium at 60 and 120 days of treatment, respectively. Serum estradiol (E2) levels remained in the mid-proliferative range following treatment (511 ± 70.21 pmol/l, pretreatment; 459 ± 90.94 pmol/l, mifepristone 60 days; 290 ± 63.39 pmol/l, mifepristone 120 days). There was no significant change in the level of E2 following treatment (P > 0.05, Bonferroni correction). Seven of the eight women on 2 mg mifepristone and all eight women on 5 mg of mifepristone were completely amenorrhoeic during treatment. Detailed description of menstrual physiology, endocrinology and endometrial histology has been reported previously (Brown et al., 2002
; Baird et al., 2003a). There was no significant difference (P > 0.05, Mann–Whitney test) between women treated with 2 or 5 mg mifepristone for any parameters studied except for the degree of GR protein expression in the vascular endothelium at 60 days of treatment [median (range) at 60 days = 3 (3), 2-mg group; 2 (2–3), 5-mg group; P = 0.012, Bonferroni correction, Mann–Whitney test]. As the GR expression was identical by 120 days in the two groups and there were no differences in any of the other 36 parameters tested, it was considered that this difference could have occurred by chance. Therefore, the 2- and 5-mg data sets were combined, and results reported are for all 16 subjects.
Stromal density
An average of 0.5 mm2 (SE = 0.05) of endometrial stroma was examined in the pretreatment samples and 0.37 mm2 (SE = 0.04) in the treatment samples. Stromal density, expressed as nuclei per square millimetre of endometrial tissue, increased significantly (24%) by day 60 of treatment (P < 0.001), and the increase (18%) was maintained at 120 days (P < 0.05, Figure 1a; 8057 ± 355, pretreatment; 10 003 ± 355, day 60; 9510 ± 351, day 120).
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Microvessel density
Vessel density per square millimetre of endometrial tissue increased (47%) by day 60 of treatment (P
0.01, Figures 1b and 2). This increase was highly significant and was maintained (49%) at 120 days (P < 0.01, Figure 1b; 267 ± 18, pretreatment; 392 ± 32, day 60; 398 ± 37, day 120). To compensate for an increase in stromal density, microvessel density was calculated per total nuclei in each endometrial sample. There was a modest (P = 0.08, ns) increase (15%) by day 60 and a significant (P < 0.01) increase (34%) by day 120 of treatment (Figure 1c; and Figure 2b and c; 33 ± 1.9, pretreatment; 38 ± 2.1, day 60; 42 ± 2.75, day 120).
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VEGF
VEGF protein was strongly expressed in the cytoplasm of endometrial glands and surface epithelium. Expression in the stroma was patchy and faint (Table II, Figure 2e). VEGF expression remained unchanged in the glandular [median (range) = 2 (1–3) pretreatment, 1 (1–3) day 60 and 1 (0–2) day 120], surface epithelium [median (range) = 1 (0–3) pretreatment, 1 (0–3) day 60 and 1 (0–2) day 120] and endothelial cells [median (range) = 1 (0–2) pretreatment, 0.5 (0–2) day 60 and 1 (0–1) day 120] following treatment with mifepristone. There was a decrease (P
0.01) in the stromal expression [median (range) = 1 (0–2) pretreatment, 0 (0–2) day 60 and 0 (0–1) day 120] of VEGF following 120 days of treatment (Table II, Figure 2f and g).
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GR
The pretreatment proliferative phase endometrial samples showed strong GR immunoexpression in the nuclei of stromal [median (range) = 2 (0–3)] and endothelial [median (range) = 3 (1–3); Table III, Figure 2p] cells and a complete absence of expression in the surface epithelium [median (range) = 0 (0–0); Figure 2i] and glands [median (range) = 0 (0–0); Table III, Figure 2i and j]. Following treatment with mifepristone, nuclear immunoexpression was induced in glands [median (range) = 0 (0–2) day 60 and 1 (0–2) day 120, P < 0.05] and surface epithelium [median (range) = 1 (0–3) day 60 and 1 (1–3) day 120, P < 0.01]. This was evident by 60 days and maintained at 120 days (Table III, Figure 2k–n). Immunoexpression in the stroma [median (range) = 2 (1–3) day 60 and 2 (1–3) day 120] and endothelial cells [median (range) = 3 (2–3) day 60 and 3 (2–3) day 120; Figure 2q and r] was unchanged.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryAcknowledgementsReferences |
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This study extends our previous report of the effects of daily low-dose mifepristone on endometrial development (Brown et al., 2002
; Baird et al., 2003a,b; Narvekar et al., 2004). Here, we show that mifepristone-induced amenorrhoea is associated with an increase in glandular GR and microvessel density and a decrease in stromal VEGF.
Histological appearance of the endometrium does not correlate well with menstrual patterns, except with the development of extreme histological atrophy, which typically predicts amenorrhoea. In the primate model, continuous mifepristone induces marked endometrial atrophy, and the spiral arteries are the primary targets that are damaged or inhibited by progesterone antagonists such as mifepristone (Chwalisz et al., 2000). Microvessel density increases in conditions of spontaneous (post-menopausal) and induced (Norplant, danazol and goserelin) endometrial atrophy, and the mechanisms involved may vary according to the nature of the atrophic stimulus (Hickey et al., 1996, 1999). In women exposed to high- and medium-dose progestogens and long-term users of levonorgestrel-releasing intrauterine system (Mirena), a decrease in endometrial vascular density has been observed (Song et al., 1995; Oliveira-Ribeiro et al., 2004). In this study, over 90% women were amenorrhoeic. The endometrium was proliferative, and there was a significant increase in microvessel density. We used CD31 to identify the vessels because preliminary studies with the alternative immunohistochemical marker CD34 were unsatisfactory. The use of CD31 may explain the discrepancy in the numerical count of vessels per area of endometrium between our study and that by other investigators (Hickey et al., 1996; Lau et al., 1999; Schindl et al., 2001). A study that used CD31 to identify vessels in endometrial samples of 16 normally menstruating premenopausal women showed wide individual variation in vessel density and no clear trend or difference over the menstrual cycle (Moller et al., 2001).
VEGF is a major regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and capillary hyperpermeability (Ferrara and Davis-Smyth, 1997; Ferrara, 1999a,b; Smith, 2001). The presence of VEGF mRNA, protein and its receptors has been demonstrated in the human and primate endometrium throughout the menstrual cycle (Torry et al., 1996; Ferrara and Davis-Smyth, 1997; Meduri et al., 2000; Nayak et al., 2000; Moller et al., 2002; Nayak and Brenner, 2002; Sugino et al., 2002), and proliferative endometrium demonstrates prominent glandular immunoreactivity and faint, inconsistent staining in stromal cells, similar to observations in this study (Sugino et al., 2002). Mifepristone abolished VEGF expression in the endometrial glandular epithelium of cynomolgous monkeys (Greb et al., 1997), and this might represent a mechanism for the suppression of angiogenesis and severe endometrial atrophy observed following mifepristone treatment in the primate model. In this study, VEGF immunoexpression was significantly reduced in the stroma following mifepristone treatment. There was a non-significant reduction in glandular VEGF immunoexpression, whereas microvessel density was significantly increased following treatment. It has been suggested that VEGF may not be the primary regulator of endothelial cell proliferation in the human endometrium (Gargett et al., 1999). Besides VEGF, other factors such as angiopoetin and fibroblast growth factor also regulate endometrial angiogenesis (Smith, 2001). VEGF regulates vascular permeability, and the decrease in stromal VEGF may explain the increase in stromal density following treatment with mifepristone (Ferrara, 1999a,b).
The most striking observation in this study was the change in the expression of GR protein and its location. In the normal menstrual cycle, GR is located only in the nuclei of endometrial stromal and endovascular cells and is absent in the glands (Bamberger et al., 2001). In this study, the pretreatment proliferative phase samples showed a strong nuclear receptor expression in the stroma and a complete absence in the glands and surface epithelium. Following treatment with low-dose mifepristone, there was a significant induction of nuclear GR protein expression in both glands and surface epithelium. Although the presence of GR has been reported in the luminal (surface) epithelium of rat uterus (Korgun et al., 2003), this is the first study to report the presence of nuclear GRs in endometrial gland cells. Mifepristone binds strongly to GR and progesterone receptor but more weakly to AR (Spitz and Bardin, 1993), and we have previously reported that chronic treatment with low-dose mifepristone up-regulates AR and down-regulates progesterone receptor in the endometrium (Narvekar et al., 2004). The underlying cellular mechanisms are poorly understood, but the striking temporo-spatial up-regulation of AR and GR in the glands and surface epithelium suggests that they share common mechanistic pathways. However, it is unlikely that the changes described in this study are the result of a change in the levels of cortisol because this dose of mifepristone has no demonstrable effect on the pituitary–adrenal axis (Brown et al., 2002).
The exact physiological role of GR and glucocorticoids in the human endometrium is not clear. The expression pattern points to a functional role in the complex process of decidualization (Bamberger et al., 2001). Several effects of glucocorticoids on endometrial cells have been reported. Uterine events such as menstruation, implantation, cervical softening and parturition share similarity with non-reproductive inflammatory situations (Kelly, 1996). At high concentrations, glucocorticoids inhibit most immunological responses and are well-known anti-inflammatory agents. PGs have a pivotal role in menstruation and endometrial bleeding (Baird et al., 1996), and glucocorticoids have been shown to suppress PGF2 alpha production (Schatz et al., 1986; Neulen et al., 1989; Delvin et al., 1990; Illouz et al., 2000) and phospholipase A2 (Pakrasi et al., 1983), one of the enzymes considered to be rate-limiting in generating free arachidonic acid for PG synthesis. An increase in the local concentration of PGs in the endometrium has been postulated in the mechanism of bleeding observed following administration of mifepristone in the mid-luteal phase (Hapangama et al., 2002). The expression of GR in endothelial cells suggests a role for glucocorticoids in modulation of angiogenesis in the endometrium as has been reported in the rat aorta (Small et al., 2005). Endogenous and exogenous glucocorticoids exert tonic inhibition, whereas treatment with a GR antagonist enhances angiogenesis in the mouse model (Small et al., 2005). An increase in GR expression could potentiate the effects of circulating glucocorticoids on the endometrium, thereby suppressing local PG concentrations and inhibiting angiogenesis. Contrary to theoretical expectations, we have demonstrated a small increase in microvessel density. This increase may represent a counting artefact in endometrial sections following stromal compaction and gland dilatation. A better understanding of the role of glucocorticoids and androgens and their receptors in the endometrium is needed to reconcile the different endometrial effects into a working hypothesis.
Glucocorticoids may also modulate human fertility. Elevated levels of glucocorticoids disrupt normal uterine development and implantation (Campbell, 1978; Monheit and Resnik, 1981; Bigsby, 1993; Hicks et al., 1994). Mechanisms by which glucocorticoids may influence implantation include their known effects on actin polymerization, lysosomal activity, PG synthase, PGE nitric oxide synthase and matrix metalloproteinases (Salamonsen, 1996), all of which have known roles in implantation. Excess glucocorticoid exposure can disturb the normal pattern of growth and differentiation of the primate fetus (Arcuri et al., 1997). On the other hand, cortisol may modulate local immunosuppressive activity within the decidua by inhibiting the production of anti-inflammatory cytokines, such as interleukin-1 (Snyder and Unanue, 1982), thus protecting the developing blastocyst from maternal immune rejection (Ricketts et al., 1998). Although low-dose mifepristone acts as a contraceptive by inhibiting ovulation in up to 90% subjects (Brown et al., 2002), an increase in surface (luminal) and glandular GR expression may play a role in the endometrial anti-fertility effects of mifepristone.
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Summary |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSummaryAcknowledgementsReferences |
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The most striking observation in this study was the change in the expression of GR protein and its location. Glucocorticoids and GR modulate angiogenesis. We have demonstrated an increase in endometrial vessel density and VEGF. The high incidence of amenorrhoea in women taking mifepristone may be related to the regulation of vascular function.
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Acknowledgements |
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We are grateful to Mrs Ann Mayo and Dr Karen Smith in Edinburgh for their help in running and coordinating this study, Dr Alistair Williams for expert gynaecological pathology, Mrs Teresa Henderson for expert technical assistance and Dr Rob Elton for statistical support. The work was supported by a grant to the Contraceptive Development Network from the Department for International Development and the Medical Research Council, UK (G9523250). The mifepristone was supplied through WHO Special Programme of Research, Development and Research Training in Human Reproduction (project no. 96503).
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Submitted on October 20, 2005; resubmitted on January 26, 2006; resubmitted on March 8, 2006; accepted on March 14, 2006.
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