Molecular Human Reproduction, Vol. 5, No. 4, 365-371,
April 1999
© 1999 European Society of Human Reproduction and Embryology
Progesterone analogues similarly modulate endometrial matrix metalloproteinase-1 and matrix metalloproteinase-3 and their inhibitor in a model for long-term contraceptive effects
Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia
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Abstract |
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Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in normal menstruation, while MMP-1 and MMP-3 production by human endometrial stromal cells (HESCs) is repressed in vitro by progesterone. We postulated that the repression by synthetic progestins of MMP production from HESCs may not be fully maintained in the long term, and that this may account for the disturbed uterine bleeding patterns in women using long-acting progestins. In this study, a long-term HESC culture model was established to compare the effects of natural progesterone and a number of synthetic analogues (ORG2058, medroxyprogesterone acetate, norethindrone acetate, levonorgestrel and drospirenone) on the production by these cells of MMP-1 and MMP-3 and TIMP-1. Zymographic and enzyme-linked immunosorbent analysis of culture medium after 2 weeks showed that both natural progesterone and all of the synthetic progestins tested maintained a significant inhibition of MMP-1 and MMP-3 production. Production of mRNA for MMP-1 and MMP-3 was also suppressed by all progestins, while TIMP production was increased. Thus, menstrual bleeding disturbances which occur during the use of synthetic progestins is not likely to result directly from changes in the effect of long-term progestin exposure on MMP-1 or MMP-3 or TIMP-1 production by HESCs.
endometrium/human/MMP/progestins/TIMP
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Introduction |
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Abnormal uterine bleeding is endometrial bleeding occurring outside the parameters of normal menstruation. It may affect women from the menarche to the menopause and is often associated with exogenous steroid use, either as steroid contraceptives or as menopausal hormone replacement therapy. The cellular and molecular mechanisms leading to breakthrough bleeding are not well understood although multiple endocrine factors are thought to play a role.
Special cases of abnormal bleeding arise in association with the use of progestin-only contraceptives and changes in the bleeding pattern constitute one of the most common reasons for discontinuation of their use. The incidence of breakthrough bleeding is highest during the first few months of treatment and appears related to the dose and type of progestin. A review of studies undertaken on a variety of preparations, have suggested that levonorgestrel (LNG) produces better cycle control than norethindrone acetate (NA) while the bleeding pattern with depo-medroxyprogesterone acetate (DMPA) shows the greatest aberration from the `normal' of all contraceptive methods (d'Arcangues et al., 1992
). In the episodes of erratic, unpredictable and frequent bleeding often associated with progestin-only contraceptives, it seems that synthetic progestins trigger some changes within the endometrium which cause vascular or tissue destruction (d'Arcangues et al., 1992).
Matrix metalloproteinases (MMP), the enzymes responsible for degradation of extracellular matrix components, are now recognized as being of critical importance for the tissue breakdown at menstruation (Marbaix et al., 1996; Salamonsen and Woolley, 1996), and these enzymes may also have a role in the abnormal uterine bleeding associated with the use of long-acting steroidal contraceptives (Vincent et al., 1998). Progesterone inhibits the production of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) by endometrial explants and stromal cells in culture (Marbaix et al., 1992; Schatz et al., 1994). Withdrawal of progesterone results in increased stromal cell MMP-1, 2 and –3 production but no change in expression or secretion of tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 or TIMP-3 (Salamonsen et al., 1997). Thus, we postulated that differences in the inhibitory capacity of different progestins on endometrial MMP production could account in part for the abnormal uterine bleeding seen during the use of progestogenic contraceptives. This can be demonstrated in long-term culture of endometrial stromal cells, the source of most endometrial MMP production (Salamonsen and Woolley, 1996). The objective of the present study was to determine whether natural progesterone (Nat) or any of a range of synthetic progestins mimicked the previously demonstrated inhibitory effects of the synthetic progesterone ORG2058 (Org) on endometrial stromal cell production of MMP-1 and MMP-3 (Salamonsen et al., 1997), whether these inhibitory effects were maintained during the course of long-term culture, and whether their withdrawal resulted in a similar recovery from inhibition. The relative effect of these progestins on TIMP-1 production by these cells was also determined.
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Materials and methods |
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Collection of endometrial tissue
Endometrial tissue was obtained at dilatation and curettage from women who had given informed consent, who were undergoing assessment of tubal patency and had no evidence of endometrial dysfunction. Tissue was dated initially from the patient's testimony and confirmed histologically. All tissues used were from between cycle days 10 to 22. Protocols were approved by the appropriate Institutional Human Ethics Committee.
Stromal cell isolation and culture
Human endometrial stromal cells (HESC) were prepared from tissue as described previously (Rawdanowicz et al., 1994; Salamonsen et al., 1997). Briefly, pooled tissue samples (2–3 per culture) were chopped finely and digested with bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ, USA) at a concentration of 45 IU/ml, in the presence of 3.5 µg/ml deoxyribonuclease (Boehringer Mannheim Biochemica, Mannheim, Germany) for 40 min at 37°C and filtered sequentially through 45 and 10 µm nylon filters to remove glands. Erythrocytes were removed by centrifugation on Ficoll–Paque (Pharmacia, Uppsala, Sweden). The resulting cell suspension has been shown to produce >95% pure stromal cell cultures by immunostaining for the stromal cell marker fibronectin (Marsh et al., 1994). Cells were resuspended in a 1:1 mixture of Dulbecco's minimal essential medium (DMEM) and Ham's F12 medium (Trace Biosciences) with 10% charcoal-stripped fetal calf serum (FCS) and antibiotics (penicillin, streptomycin and fungizone) and plated in 24-well dishes (2.5x105 cells/ml). After 4–5 days, when the cells were confluent (day 0), they were washed and the medium was replaced with serum-free medium containing: transferrin (10 µg/ml; Sigma Chemical Company, St Louis, MO, USA), sodium selenite (25 ng/ml, Sigma), epidermal growth factor (50 ng/ml; Sigma), linoleic acid (10 nmol/l; Sigma), insulin (10 µg/ml; human Actrapid, Novo-Nordisk Pharmaceuticals Pty Ltd, Sydney, Australia) and bovine serum albumin (BSA 0.1%; Sigma). All experiments were performed in the presence of oestradiol 17ß (10 nmol/l; Sigma), with or without (control) the synthetic progestin ORG2058 (Org, 100 nmol/l; Organon Laboratories Ltd, Oss, Holland), natural progesterone (4-pregnen-3,20-dione, Nat; Sigma), medroxyprogesterone acetate (MPA, Sigma), norethindrone acetate (NA, Sigma), levonorgestrel (Levo) or Drospirenone (Dros, kindly provided by Schering AG, Germany; all at 0.01, 0.1 or 1 nmol/l). For the progesterone withdrawal experiments, the cells were washed on day 10 and the medium was replaced with medium either with or without the progestin. Where possible, experiments were performed in triplicate wells for 15 days with medium changes every 2–3 days. Medium was collected, centrifuged to remove cellular debris and stored at –20°C prior to subsequent analysis. In some cases a protein assay was performed on cells at the end of the experiment and in others, cells from selected wells were harvested for RNA preparation.
Zymography and reverse zymography
Proteinase activity in unconcentrated culture medium was analysed by zymography on 10% sodium dodecyl sulphate (SDS)–polyacrylamide gels (all reagents from BioRad, North Ryde, Australia) containing 1 mg/ml casein (Sigma) under non-reducing conditions (Rawdanowicz et al., 1994; Salamonsen et al., 1997). Loading of samples was normalized according to the protein content of cell lysates for each culture well. Samples from multiple wells for each treatment were pooled and each complete experiment was contained on a single gel. Caseinase activity was visualized by negative staining and bands were identified by comparison with standard preparations of pure human MMPs (a gift from Dr Hideaki Nagase, Kansas City, KS) and with molecular weight markers (BioRad) (Rawdanowicz et al., 1994). MMP identity of all bands was confirmed by incubation of parallel gels in the presence of EDTA (5 mmol/l). Reverse zymography was also performed on culture medium (concentrated 2x in some cases) using 12% polyacrylamide gels containing 1% gelatin and an MMP preparation from BHK-21 cells which constitutively express proMMP-2 (Hampton et al., 1995). The presence of TIMPs was visualized by the presence of dark blue bands on a clear background. Controls were standards containing mouse TIMP-1, TIMP-2 or TIMP-3, obtained from transfected BHK cells (provided by Dr Dylan Edwards, Calgary, Canada), as previously described (Hampton et al., 1995). Relative activities of MMPs or relative concentrations of TIMPs were semi-quantified by densitometric analysis of zymograms (Kleiner and Stetler-Stevenson, 1994) using the Hewlett-Packard Scanjet IIp with Deskscan software (Hewlett-Packard, Palo Alto, CA, USA) set on a black and white photo with 256 grey shades. The areas of the bands were analysed using the NIH Image Version 1.54 equipped with gel plotting macros, by measuring the areas beneath the peaks plotted through the lane profile for the appropriate enzyme. Comparisons were made only between samples run on the same gel. Previous analyses of doubling dilutions of samples on each of the types of zymograms verified the semi-quantitative nature of these analyses, while analysis of the same samples for MMP-1 by casein zymography and enzyme-linked immunosorbent assay (ELISA), substantiated changes in MMP-1 measured by this method (Salamonsen et al., 1997).
MMP-1 ELISA
MMP-1 (including pro-, active and TIMP-bound forms) was measured in culture medium by ELISA (Amersham Australia, Baulkham Hills, NSW, Australia).
RNA extraction from cultured endometrial stromal cells
Following culture and treatment, HESCs were washed twice with phosphate-buffered saline (PBS) and stored in their culture dishes at –20°C until processing. Total cellular RNA was extracted by a modified miniprep method (Walther et al., 1994; Nie et al., 1998). Briefly, cells in the wells were incubated for 5 min at room temperature with 200 µl of 3 mol/l LiCl/6mol/l urea. The solution was transferred to a microcentrifuge tube, vortexed vigorously, heated at 50°C for 3 min and incubated for a further 5 min at room temperature. Extracted RNA was treated with DNAse because substantial amounts of DNA were often detectable. RNA was quantitated by its absorbance at 260 nm.
Quantitative RT–PCR
mRNA values for MMP-1 and MMP-3, along with the mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined by quantitative reverse transcription–polymerase chain analysis (RT–PCR) (Seibert and Larrick, 1993; Riedy et al., 1995) using a multispecific heterologous competitor which shares the same primer-binding sequences as the cellular mRNA of all three genes but yields different sized PCR products; this competitor has been fully validated (Nie et al., 1998). Briefly, a set of seven competitive RT reactions (master RTs) were set up for each sample RNA; in each of these a constant amount (300–500 ng) of total DNA-free sample RNA was combined with a serial dilution of an exact amount of synthesized competitor RNA (range 0.35–350 pg). The RNA mixture was then reverse-transcribed at 46°C for 1–1.5 h in 20 µl reaction mixture using 100 ng random hexanucleotide primers and avian myeloblastosis virus (AMV) RT (Boehringer Mannheim, Nunawading, Australia) with the cDNA synthesis buffer supplied by the company for this enzyme. The resultant cDNA mixtures were then heated at 95°C for 3 min before storage at –20°C, or immediately used for PCR amplification. PCR was carried out for GADPH, MMP-1, and MMP-3 in the same manner, using the same RT reactions. This master RT set was used in PCR for different specific genes using individual primer pairs; for each specific gene, PCR reactions corresponding to the whole series of RT reactions were performed. Each PCR (40 µl) contained 1–1.5 µl of the RT mixture, 1x PCR buffer (10 mmol/l Tris–HCl, 1.5 mmol/l MgCl2, 50 mmol/l KCl, pH 8.3), 20 µmol/l dNTPs, 10 pmol/l forward primer, 10 pmol/l reverse primer and 2.5 IU of Taq DNA polymerase (Boehringer Mannheim). The PCR was performed in three stages as follows: the first stage was one cycle of an incubation for 5 min at 95°C, 1 min at 60°C, and 2 min at 72°C; the second stage involved 28 cycles, each cycle consisting of two steps: 45 s at 95°C and 2 min at 69°C; finally the reaction mixture was incubated for 5 min at 72°C. The PCR products were separated on a 1.8% agarose TBE gel (0.045 mol/l Tris base, 0.045 mol/l boric acid, 1 mmol/l EDTA, pH 8.0), stained with ethidium bromide, and photographed using a Polaroid system. The picture was scanned using the Hewlett-Packard Scanjet IIp with Deskscan software (Hewlett-Packard, Palo Alto, CA, USA) and the band intensities were determined using NIH Image Version 1.54. To determine the competition equivalence point, the logarithm of the band intensity ratio of competitor product to the target product was graphed as a function of the logarithm of the initial amount (in mRNA copy numbers) of the competitor added. When the target and the competitor products are equal, the initial amount of the target sequence present in the RNA sample is equal to the initial amount of the competitor added to the RNA sample in the RT reaction. The exact initial amount of the target sequence in the sample was calculated by linear regression analysis of the graph. To more directly compare the differences of each mRNA species between the treatments, the expression levels of MMP-1 and MMP-3 in each treatment were normalized against GAPDH within that treatment and the data were expressed as the percentage of the control (oestradiol alone, 100%).
Protein assay
Cells were lysed in culture wells by sonication in 0.1% sodium dodecyl sulphate solution, and the protein content was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA).
Statistical analyses
Data from individual cell cultures were expressed as percentage of control (100%), and from combined cultures as mean percentage of control ± SEM. Where possible all treatments were in triplicate wells. Data in Figures 1 and 6 were analysed by unpaired Student's t-test. P < 0.05 was considered to be statistically significant.
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) and MMP-3 (
) production by long-term human endometrial stromal cell cultures. Culture medium was analysed by casein zymography (A) (representative one of three cultures) followed by densitometry (B). After an initial settling period of 4–5 days, cells were maintained in culture for 14–15 days during which time oestradiol (E; 10 nmol/l) treatment was maintained with or without the appropriate progestin (100 nmol/l). Lane 1, oestradiol alone (control); lane 2, natural progesterone (NAT); lane 3, ORG2058 (Org); lane 4, medroxyprogesterone acetate (MPA); lane 5, levonorgestrel (LNG); lane 6, norethindrone acetate (NA); and lane 7, drospirenone (Dros). Data are presented as the relative densitometric intensity expressed as the percentage of control values within each culture ± SEM for combined data from three separate cell cultures. *P < 0.01 compared with control for MMP-1; **P < 0.01, ***P < 0.05 compared with control for MMP-3. E = oestradiol. |
Results |
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Comparison of effect of natural and different synthetic progestins on MMP-1 and MMP-3 production by endometrial stromal cells in long-term culture
Analysis by casein zymography demonstrated that all progestins tested, both natural and synthetic, decreased the production of both MMP-1 and MMP-3 by HESCs in long-term culture compared with the oestradiol-only control. Figure 1A
shows a zymogram of conditioned medium from culture 1 (representative of three cultures performed). Although the relative degree of inhibition varied between the three cultures performed and between the progestins in each of the cultures, in each case inhibition by all progestins compared with the oestradiol-only control was substantial. Statistical analysis could not be performed for each separate culture due to the limited number of wells which could be included on each gel and the necessity to contain complete experiments within one gel. Figure 1B shows the combined data from the three cultures, after quantification of zymography gels by densitometry. These combined results yielded statistically significant differences between all progestins compared with the oestradiol-alone control for both MMP-1 and MMP-3. There were no significant differences between any of the individual progestins. Inhibition of MMP-1 by all of the progestins was confirmed by an ELISA performed on the same conditioned media samples (Figure 2). These results also show that for all of the progestins this inhibition occurred in a dose-responsive manner between 0.01 and 1 µmol/l, with the exception of natural progesterone in culture 1. No differences in relative cell number or metabolic viability were observed throughout the culture period between cells treated with the different progestins or with oestradiol alone, as measured by protein assay of cell lysates at the end of the culture period and by the rate of Phenol Red colour change in the medium (data not shown).
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); and 1 µmol/l (Effect of progestin withdrawal on MMP-1 and MMP-3 production
Assay of total MMP-1 in culture media by ELISA demonstrated that withdrawal of most of the progestins on day 10 of culture resulted in an increased value of MMP-1 production on day 15 compared with cultures in which progestin was maintained (Figure 3
). The exceptions were that of Nat, in which MMP-1 production was restored almost to control values in culture 1 (Figure 3A), but not in culture 2 (Figure 3B), and LNG, where MMP-1 production was not restored in either culture following withdrawal. For all but Nat, although the relative degree of MMP-1 production and restoration differed between the two cultures assayed, a similar trend was observed. No differences in relative cell number or metabolic viability were observed throughout the culture period between culture wells in which progestin had been either maintained or withdrawn.
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Natural and synthetic progestins modulate MMP-1 and MMP-3 by inhibiting mRNA production
Quantitative RT–PCR of mRNA extracted from cell lysates at the termination of the culture period showed inhibition of both MMP-1 and MMP-3 mRNA by all the progestins tested compared to the oestradiol-only control (Figure 4
). Cells from a number of similar treatment wells were pooled for the analysis.
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Effect of natural and different synthetic progestins on TIMP production
The relative effects of natural and synthetic progestins on TIMP-1, TIMP-2 and TIMP-3 production after 2 weeks of culture varied between the three cultures performed, and variation between TIMPs within cultures was also observed. However, a consistent increase in TIMP-1 was observed when cells were treated with all progestins compared with the oestradiol-alone control. Figure 5A
shows a reverse zymogram of one representative culture while Figure 5B shows combined data for TIMP-1 from three cultures after conversion of results to relative densitometric units following scanning densitometry. TIMP-2 and TIMP-3 could not be quantified as their concentrations were too low. The combined data in Figure 5B shows that the observed increase in TIMP-1 production was significant for Nat, LNG, NA (P < 0.01) and Dros (P < 0.05), and that the increase observed for Nat compared with the control was significantly higher (P < 0.05) than for Dros.
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Discussion |
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The major finding of this study is that all the progestins tested, including natural progesterone and a variety of synthetic formulations, similarly were able to continuously inhibit the production of MMP-1 and MMP-3 by HESCs in a long-term culture system, designed to mimic the endometrium under the control of long-acting progestogenic contraceptives. We had postulated that, although progesterone has the potential to inhibit MMP production by stromal cells within the short term (of the order of the time it is present in the normal menstrual cycle), when administered continuously in the longer term, some or all progestins may lose the capacity to maintain this inhibition. This postulate was not substantiated by the experimental findings. Thus a reduction in the direct inhibitory effect of one or more of these progestins with time, on MMP-1 and MMP-3 production by HESCs is not likely to account for the breakdown of endometrial tissue and increased fragility of blood vessels commonly found in women using long-acting progestogens as contraceptives. All the synthetic progestins tested, over a range of concentrations, mimicked the effects of natural progesterone by inhibiting the production of MMP-1 and MMP-3 and increasing the production of TIMP-1 by HESCs in long-term culture. Both of these effects would act to favour tissue integrity over tissue breakdown. Furthermore, following withdrawal of the progestins, similar increases in MMP production were seen over a 5 day time frame, for all the progestins with the exception of levonorgestrel. The effect of all the progestins was directly on the expression of MMP mRNA.
The degree of variability between separate cultures in these studies is common with experiments using human endometrium, and has been experienced not only by our laboratory but also by others. It emphasizes the difficulty of working with such tissue and the variability between the endometria of different women. The time of the cycle of the tissue from which cells are cultured can produce considerable variability in short-term cultures, but, that such variation was retained even after 15 days of cell culture under the same hormonal conditions was somewhat surprising. However, this is probably an accurate reflection of the clinical situation in which some women will display disturbed menstrual bleeding patterns when exposed to certain progestin-only formulations while others will not. Another possible cause of variability between cultures with respect to patterns of MMP-1 and MMP-3 and TIMP expression could be that individual progestins have different effects on disparate subpopulations of stromal/decidual cells within the cultures, and that the proportions of these subpopulations vary between cultures established from different patients. Whether such subpopulations exist has not been formally tested and indeed would be difficult to define. However, immunostaining of cultures of endometrial stromal cells for a number of peptides, including endothelin-1 and TIMPs, has demonstrated that in any culture at any one time, positive immunostaining for such secreted peptides will be apparent in some but not all cells of similar morphological appearance (M.M.Marsh, J.Zhang and L.A.Salamonsen, unpublished observations). There is a possibility that a low level of contamination by macrophages could have contributed to the MMP production in the cultures. These are the only adherent cells which separate from endometrial tissue with the fibroblasts (L.J.Lathbury, personal communication). However, their ready attachment to laboratory glassware used during the cell isolation procedure and their lack of proliferation in culture, suggests that their contribution to the total MMPs in these experiments will be minimal. In addition, previous work in our laboratory using this cell separation method has demonstrated no contamination by macrophages or leukocytes in stromal cell cultures after 48 h in culture (Marsh et al., 1994).
The current findings, that all progestins similarly inhibit MMP production from HESC, are not necessarily surprising, as evidence from studies in normal endometrium, has suggested that progesterone, while being important overall in regulating the function of the endometrium, is not the primary regulator of endometrial MMPs, and hence tissue destruction at menstruation (Salamonsen and Woolley, 1996). This hypothesis is strongly supported by the fact that endometrial tissue breakdown at menstruation is focal, rather than generalized and thus local, rather than endocrine, regulation must ultimately be responsible. It should be stressed that the present findings relate only to the direct effects of the progestins on MMP production by HESCs and not to the possible indirect effects of these progestins on MMP production in vivo (for review see Salamonsen and Woolley, 1996). For example, endothelin-1 (whose production is markedly reduced in women using slow-release levonorgestrel, (Marsh et al., 1995), tumour necrosis factor 
and interleukin-1 (IL-1) all increase MMP-1 and MMp-3 production by HESCs in short-term culture (Rawdanowicz et al., 1994, L.A. Salamonsen, unpublished observations). Paracrine actions of both IL-1 (Singer et al., 1997) and transforming growth factor ß (Bruner et al., 1995) within the endometrium modify production of certain endometrial MMPs while mediators derived from activated mast cells modulate both MMP production and activation by HESCs (Zhang et al., 1998). Importantly, these latter effects occur even in the presence of progesterone, thus emphasizing that even in the progestin-dominated endometrium, it would be possible for the inhibitory effect of the progestins on MMP production to be overridden by other local factors. In endometrium from women using the contraceptive Norplant® (slow release levonorgestrel), increased numbers of macrophages (Clark et al., 1996) neutrophils and eosinophils have been detected (Vincent et al., 1998), particularly at sites where tissue fragility is observed. Products of these cells are likely stimuli for local MMP expression, overriding the overall inhibitory effect of the levonorgestrel. This hypothesis is currently being tested experimentally in our laboratory.
Changing responses to progestins could be accounted for by differences in both progesterone receptor (PR) and androgen receptor (AR) subtypes in the endometrium of normal cycling women, compared with those using progestogenic steroids. Surprisingly, endometrium from women receiving Norplant has increased total immunoreactive PR (Critchley et al., 1993) and PR mRNA (Lau et al., 1996) although the receptor subtype was not determined. PR B is a stronger activator of target genes while PR A can act as a dominant repressor of PR B and other hormone receptors. Both subtypes are present in normal human endometrium with some cyclical changes (Mangal et al., 1997; Wang et al., 1998). Likewise as some of the synthetic progestins also have androgenic activity (for example, levonorgestrel and norethindrone are highly androgenic while medroxyprogesterone acetate is not) and as AR activation also regulates MMP expression (Schneikert et al., 1996), the presence or absence of AR on endometrial cells could also be relevant in the presence of synthetic progestins. AR have recently been shown to be cycle dependent in normal endometrium (Mertens et al., 1996) while, in a single sample, the AR B:A ratio was 1.8:0.2 (Wilson and McPhaul, 1996). Identification of these receptor subtypes and their cellular location in endometrium of women using a variety of progestins could contribute important information relating to the regulation of endometrial MMPs by progestins.
There are a number of advantages associated with the use of progestin-only contraceptives. These include administration in long-acting formulations (Norplant and Depo-Provera), the frequent need for only low doses and the avoidance of oestrogenic side effects. It is, therefore, of significant clinical importance to continue to work towards a greater understanding of the abnormal uterine bleeding associated with their use, as this will ultimately result in new effective therapies, and assist in the development of more acceptable contraceptive and HRT formulations.
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Acknowledgments |
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The authors wish to thank Professor Gabor Kovacs and Dr Jason Clarke for providing the endometrial tissue used in this study, Sister Cathy Canny for collecting the tissue, and the patients who agreed to participate in the study. We wish also to thank Drs Hideaki Nagase, Kristof Chwalisz, and Dylan Edwards for providing reagents, Sue Pankridge for assistance in the preparation of the figures, and Professor Jock Findlay for helpful discussion. This work was supported by the National Health and Medical Research Council of Australia (Grant 971292) and the NIH (Grant HD-33233–02).
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Notes |
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1 To whom correspondence should be addressed
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References |
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Submitted on August 19, 1998; accepted on January 7, 1999.
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