1 Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia, 2 Royal Women's Hospital, Carlton, Victoria, 3 Monash University Dept of Obstetrics and Gynaecology, Clayton, Victoria, 4 Human Reproduction Study Group, Department of Obstetrics and Gynaecology, University of Indonesia, Jakarta, Indonesia and 5 Monash University Dept of Obstetrics and Gynaecology, Box Hill Hospital, Box Hill, Victoria, Australia
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Abstract |
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Key words: abnormal uterine bleeding/depot medroxyprogesterone acetate/leukocytes/levonorgestrel/matrix metalloproteinases
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Introduction |
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The pathogenesis of abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Changes in endometrial morphology, endometrial steroid receptor profile, endometrial vascular morphology, function and haemostasis and endometrial repair mechanisms have been described (for review see Fraser et al., 1996). Bleeding is thought to arise from capillaries and venules with hysteroscopic evidence of neovascularization and increased vessel fragility in women using Norplant (Hickey et al., 1996).
Matrix metalloproteinases (MMP)s are a family of zinc-dependent proteases which degrade extracellular matrix (ECM) components. Each MMP demonstrates substrate specificity and is secreted as an inactive zymogen requiring activation, a process that is tightly regulated. For example, MMP-1 (interstitial collagenase) which degrades triple helix collagens including types I, II, III, VII and X (Birkedal-Hansen et al., 1993), is activated in vitro by MMP-3 (stromelysin-1) which is itself activated by the plasmin cascade or other proteases. MMP display spatial and temporal variation within the endometrium during the menstrual cycle, with an increase observed perimenstrually. They are postulated to be involved in the endometrial breakdown observed at menstruation (Marbaix et al., 1995
; Marbaix et al., 1996a; Salamonsen and Woolley, 1996
). Immunolocalization of MMP-9 has been reported in endometrial biopsies from women using Norplant, with increased numbers of MMP-9 positive cells observed in biopsies displaying a shedding type morphology (Vincent et al., 1999
). Thus MMPs may also play a role in the pathogenesis of abnormal uterine bleeding.
Mast cells (MC)s produce a plethora of regulatory molecules including histamine, heparin, cytokines such as tumour necrosis factor- (TNF
) and interleukin-1 (IL-1) as well as the MC specific proteases, tryptase and chymase. Thus these cells are capable of participating in many biological functions including matrix remodelling, angiogenesis, vascular permeability, leukocyte recruitment and chemotaxis (for review see Galli, 1993). Endometrial MC undergo activation and degranulation immediately prior to menstruation, as demonstrated by the extracellular immunolocalization of the MC protease, tryptase (Jeziorska et al., 1995
). Furthermore, MCs have been shown to regulate endometrial stromal cell MMP production and activation in vitro and are postulated to be involved in the regulation of MMP action at menstruation (Zhang et al., 1998
).
It is postulated that MMPs (in addition to MMP-9) and MC are involved in the pathogenesis of abnormal uterine bleeding in women using progestin-only contraceptives. Thus the aim of this study was to determine whether MMP-1, MMP-3 and MCs are present within the endometrium of women using Norplant or DMPA and the correlation with abnormal uterine bleeding.
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Materials and methods |
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To allow comparison with normal control tissues, endometrium was also obtained in Melbourne, Australia, from women who were undergoing curettage following laparoscopic sterilization or assessment of tubal patency. Patients with uterine abnormalities such as leiomyomata, endometrial polyps, endometriosis, or those who had received steroid therapy in the past 6 months were excluded. MMPs are either maximally or only present in the endometrium during the perimenstrual phase and thus the control endometrial tissues used in this study were obtained from the menstrual (day 13) or late secretory (day 2628) phases of the menstrual cycle.
All endometrial biopsies were fixed in Carnoy's fixative (Analar R®; Merck, Kilsyth, Victoria, Australia) and embedded in paraffin. Tissue sections were cut at 5 µm, deparaffinized, rehydrated and either stained with haemotoxylin and eosin for histological dating according to Noyes' criteria (Noyes et al., 1950) or subjected to immunohistochemical staining.
The project was approved by the Human Research and Ethics Committee at the Monash Medical Centre (Monash University Standing Committee on Ethics in Research on Humans), by the Family Planning Victoria Institutional Ethics Committee, by the Medical Faculty of the University of Indonesia Ethical Commission on Research on Humans and by the World Health Organization (WHO).
Menstrual diary records
Subjects recorded a daily menstrual diary from the day of insertion of Norplant or DMPA administration to the day of endometrial biopsy. The total duration of implant use was recorded for each subject. Menstrual bleeding charts were analysed by calculating the total number of bleeding days (any bleeding or spotting) in the 90 day reference period prior to endometrial biopsy. Where endometrial biopsy was performed before 90 days of treatment had elapsed, the ratio of number of bleeding + spotting days/total duration was calculated and multiplied by 90 to obtain an equivalent bleeding/spotting days per 90 day reference period (Hourihan et al., 1991).
Immunohistochemical staining
MMP-1 immunohistochemistry was performed using a polyclonal mouse anti-human MMP-1 antibody (kind gift of Dr. Woolley, Manchester, UK), used at a concentration of 1.5 µg/ml in Tris-buffered saline (TBS) containing 10% normal goat serum (NGS; Prince Henry's Institute of Medical Research Animal Facility, Werribee, Victoria, Australia) with incubation at room temperature for 2 h, followed by incubation with a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) and the biotinyl tyramide TSA indirect amplification kit (NEN Life Sciences, Boston, MA, USA) as per manufacturer's instructions with diaminobenzamine (DAB) as the chromagen.
MMP-3 was demonstrated using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique (Vincent et al., 1999) and a mouse monoclonal anti-human MMP-3 (Calbiochem, Cambridge, MA, USA). The primary antibody (used at a concentration of 2 µg/ml, diluted in TBS/10% NGS) was applied and the sections incubated overnight at 4°C. MMP-3 was visualized using goat anti-mouse immunoglobulin G (IgG) (Dako, Glostrup, Denmark) followed by mouse APAAP complex (Dako), repeated twice and with New Fuchsin (Dako) as the chromagen. Endogenous alkaline phosphatase was blocked with 1 mmol/l levamisole.
MCs were immunolocalized using a monoclonal mouse anti-human MC tryptase (MC tryptase) (Dako) and the Dako streptABCalkaline phosphatase kit. The primary antibody [1 µg/ml diluted in 10% normal horse serum (Sigma, St Louis, MO, USA)/TBS] was then applied and incubated for 2 h at room temperature. After washing, slides were incubated with biotinylated horse anti-mouse IgG (Vector) for 30 min followed by the detection system used according to the manufacturer's instructions with New Fuchsin (Dako) as the chromagen. Endogenous alkaline phosphatase was blocked with 1 mmol/l levamisole.
Dual immunolocalization of MC tryptase and MC chymase was performed on selected samples using mouse monoclonal antibodies to MC tryptase (Dako) and MC chymase (Chemicon, Temecula, CA, USA) at a concentration of 4 µg/ml and 6.75 µg/ml respectively and the Envision Double stain detection kit (Dako) used according to the manufacturer's instructions with New Fuchsin (Dako) and 5 bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT) (Dako) as the respective chromagens.
Negative and positive controls appropriate for each antibody were included in each series of sections examined. The negative control was an irrelevant -lactalbumin monoclonal mouse IgG antibody (gift from Dr E. Thean, Prince Henry's Institute of Medical Research) or normal rabbit IgG (Dako) at the same concentration as the primary antibody. Positive controls included HT1080 cell cytospins (gift from Dr P. Hertzog, Clayton, Victoria, Australia) and proliferative phase human endometrium (from laboratory tissue collection) (MMP-1), human synovium (MMP-3; gift from Dr D. Woolley, Manchester, UK) and human endometrium and myometrium (MC tryptase and MC chymase). Tissue sections were counterstained with 1/10 Harris' haemotoxylin. Photography was performed using an Olympus BH2® photomicroscope.
Histological assessment
Histological assessment of the biopsies was performed by an experienced gynaecological pathologist (AO) with no knowledge of the patient characteristics. The samples were assessed as to the morphological appearance (atrophic, progestin-modified or shedding (Vincent et al., 1999) and the degree of pseudo-decidualization observed in the progestin-modified samples was graded from 0 (none) to 3 (complete).
Assessment of immunostaining
Quantitative analysis of positive MMP-1 and -3 immunostaining was performed using an Olympus BX-50® microscope and a x40 objective. The image was captured using a Pulinex TMC-6 video camera coupled to a Pentium PC computer using a Screen Machine II FAST multimedia video adaptor (FAST Multimedia AG, Munich, Germany). A software package (Olympus DK CASTGRID V1.10®; Olympus, Denmark) was used to generate a nominated field of points directly on to the video screen. Fields to be counted were selected using a systematic uniform sampling scheme generated by the CASTGRID V1.10® computer program with the aid of a motorized stage (Multicontrol 2000®; ITK, Ahornweg, Germany). The percentage ratio of positively stained stromal area to total stromal area was calculated in at least 10 fields of each section. Quantitative analysis was performed by the same observer, with no knowledge of the clinical characteristics of the patient donor.
Quantitative analysis of the number of MC (expressed as number of positive cells per 1000 stromal cells) and assessment of the distribution of MC tryptase positive cells in various endometrial compartments, graded from 0 (no cells) to 3 (many cells), was performed using the method previously described (Vincent et al., 1999).
Statistical analysis
Comparisons between groups were made using either the MannWhitney test (clinical characteristics) or the KruskalWallis test (immunostaining results). Multiple comparisons were made after statistical significance was found at the 5% level using the method described by Conover (Conover, 1980). Correlations between variables were calculated via Spearman's rank correlation.
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Results |
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Clinical characteristics
The clinical characteristics of the patients are shown in Table I. There was no significant difference between the two treatment groups with respect to age, body mass index (BMI), duration of contraceptive use and number of bleeding days reported. Significantly fewer bleeding days were observed with increasing duration of progestin-only contraceptive use (P = 0.04)(data not shown).
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MC immunohistochemical staining of endometrial biopsies
Immunolocalization of MC tryptase was observed in 9/10 control, 16/19 Norplant biopsies and all DMPA samples. Quantitative analysis of the numbers of positive cells revealed no significant difference between control subjects, Norplant or DMPA users (Figure 3A). Extracellular MC tryptase, indicating MC activation, was observed in association with greater than 80% of positive cells identified in patients treated with progestin-only contraceptives and menstrual and premenstrual controls (Figures 1 and 3B
). MCs were distributed in the endometrial stroma and mainly observed adjacent to glands, blood vessels and intervening stroma (Figure 3C
). However, in endometrial samples from women using progestin-only contraceptives, MCs could be seen adjacent to the subluminal epithelium and, in one case, between adjacent luminal epithelial cells. Intravascular MCs were not seen and MC were not specifically associated within sites of tissue breakdown although they could be observed at the periphery of such sites. There was no correlation between MC numbers and bleeding patterns reported by women.
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Discussion |
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MMP-1 cleaves fibrillar collagens including collagen types I and III, which are major components of endometrial interstitial ECM thereby initiating ECM breakdown. MMP-1 mRNA and protein in endometrium are expressed exclusively during the late secretory and menstrual phase of the normal cycle (Hampton and Salamonsen, 1994; Rodgers et al., 1994
) and are localized to the stromal components of the endometrium; primarily stromal cells but also observed perivascularly (Rodgers, 1994; Kokorine et al., 1996
). In the present study, small foci of MMP-1 positively stained stromal cells and adjacent stroma were distributed throughout the endometrium of progestin-treated women and not confined to areas of tissue breakdown as was observed in the perimenstrual control samples. In women using progestin-only contraceptives, subtle disruption of endometrial matrix components only visible at electron microscopy, as has been reported in perimenstrual samples previously (Roberts et al., 1992
), may account for the MMP-1 immunolocalization pattern. An alternative explanation is that the MMP-1 immunolocalization observed constitutes proMMP-1 (the antibody used detects both inactive proMMP-1 and active MMP-1) and thus would not necessarily be associated with areas of tissue breakdown. In a study of the normal menstrual cycle, perivascular MMP-1 immunostaining was only observed in 5/13 perimenstrual samples (Korkorine et al., 1996) and thus the lack of perivascular MMP-1 immunolocalization in the endometrial samples from women using progestin-only contraceptives may reflect sampling variability, differing immunohistological techniques or an alteration in MMP-1 expression. Alternatively, as the expression and activation of MMPs is tightly regulated, perivascular MMP-1 may only be apparent in samples collected at bleeding sites or from women experiencing bleeding. The observed pattern of MMP-1 immunostaining is consistent with the hypothesis that MMP-1 may be involved in tissue fragility and breakdown observed in women with abnormal uterine bleeding; however, different patterns of expression are associated with different progestin-only contraceptives. Differences in the pattern of MMP-9 immunolocalization in endometrial biopsies obtained from women treated with levonorgestrel administered through either Norplant or the levonorgestrel-releasing intrauterine device (IUD) have also been described (Vincent et al., 1999
; Skinner et al., 1999
) suggesting that the delivery system, resultant local progestin concentration and micro-environment are important determinants of MMP expression in the endometrium.
Previous studies have identified MMP-3 (Hampton and Salamonsen, 1994; Rodgers et al., 1994
; Jeziorska et al., 1995
) mRNA and protein consistently within menstrual endometrium and variably in late secretory endometrium. All studies agree that the cellular source of MMP-3 is endometrial stromal cells. The absence of positive MMP-3 immunostaining in the majority of biopsies examined in this study was unexpected. However, this may reflect the limitations of sampling and/or inter-individual variation. For example, MMP-3 mRNA detected by Northern analysis of a large number of samples from across the menstrual cycle (Hampton and Salamonsen, 1994
) was only observed in menstrual or late secretory endometrium. Importantly, a proportion of the biopsies collected at these phases of the menstrual cycle were MMP-3 negative. MMP-3 is an important enzyme as it plays a central role in establishing a cascade of MMP activation (Salamonsen and Woolley, 1996
). However, in its absence, stromelysin-2 (MMP-10) may perform the same function. MMP-10 has the same substrate specificity as MMP-3 (stromelysin-1) and MMP-10 mRNA has been localized to focal areas of endometrial stromal cells only during the menstrual and late secretory phase (Rodgers et al., 1994
). Activation of proMMP-9, proMMP-1, proMMP-7 and proMMP-8 by MMP-10 has been observed in vitro (Nakamura et al., 1998
) and this profile is similar to that activated by MMP-3. Thus MMP-10 may provide an alternative to MMP-3 in the endometrium.
This study also demonstrates that endometrial MCs of varying phenotype are present in the endometrium of Norplant and DMPA users and that they display a state of activation which is similar to that seen at menstruation. In a previous immunohistochemical study of MC distribution throughout the normal menstrual cycle (Jeziorska et al., 1995) it was demonstrated that although there was little change in MC numbers throughout the cycle, the state of activation/degranulation varied (as judged by the presence of extracellular tryptase) with extensive activation/degranulation seen 2 days prior to and during menstruation. The authors postulated that the release of various soluble mediators by activated MCs is likely to contribute to endometrial remodelling. Variation in MC phenotype in the normal menstrual cycle, as determined by the pattern of tryptase/chymase expression, has been observed in the different layers of the endometrium (Jeziorska et al., 1995
) with MCs of the functionalis having a MC tryptase phenotype and that of the basalis a MC tryptasechymase phenotype. The results of this study suggest that alteration in MC phenotype associated with the use of progestin-only contraceptives has occurred with a change from the MC tryptase phenotype to the presence of tryptase, tryptasechymase and chymase positive MCs in the superficial endometrial samples obtained by Pipelle suction curette or microhysteroscopy. This is likely to represent a change in function (enzyme secretion patterns) of the resident MCs rather than migration of a new population of MCs into the superficial endometrium as the number of MCs did not differ between the control and progestin-treated women. Phenotypic changes in MC populations in response to alteration in the micro-environment have been observed in mice (Galli et al., 1993). It is possible that the sampling technique resulted in activation of the MCs although it is unlikely as endometrial biopsy samples obtained by similar techniques during the proliferative and secretory phases of the normal cycle did not show evidence of MC activation (Jeziorska et al., 1995; Vincent, unpublished observations).
The correlation of MC numbers with bleeding patterns is variable (Mehra et al., 1970; Houihan et al., 1991; Yin et al., 1993
) depending on the study and the timing of the endometrial biopsy. Increased endometrial MCs were reported in women using an IUD with abnormal uterine bleeding who were biopsied during a bleeding phase compared with women biopsied during a bleed-free interval or women using an IUD without abnormal uterine bleeding (Mehra et al., 1970
). It is important to note that many of these earlier studies were performed using histochemical granule staining techniques (e.g. toluidine blue) which does not identify either MC phenotype or immature/degranulated MCs (thus underestimating the number of MCs present). These immunohistochemical stains also provide no information regarding the state of activation of the MCs. Indeed, Jeziorska and co-workers (Jeziorska et al., 1995
) demonstrated that MC tryptase immunolocalization was able to detect approximately twice as many MC as toluidine blue staining.
Endometrial MCs may contribute to the endometrial breakdown and thus the pathogenesis of abnormal uterine bleeding via a role in the regulation of MMP production and activation, by their production of heparin, histamine and other proteases and as a source of cytokines and chemokines. MC chymase is capable of activating proMMP-1 in vitro (Lees et al., 1994) and MC tryptase activates proMMP-3 in vitro (Zhang et al., 1998
). MCs produce IL-1, which has been shown to overcome progesterone-mediated suppression of MMP-1 production from stromal cells in vitro (Singer et al., 1997
; Zhang et al., 1998
). MCs also secrete chemokines such as IL-8, a neutrophil chemotactic factor (McNeil, 1996
). Neutrophils are a source of MMP-9 and elevated numbers have been reported in the endometrium of Norplant users (Vincent et al., 1999
). Neutrophil elastase, an enzyme secreted by neutrophils, is capable of activating pro-MMP-3 and 9 and inactivating tissue inhibitor of metalloproteinase-1 (TIMP-1) (Salamonsen and Lathbury, 2000
). The observation of an altered MC phenotype in the endometria of progestin-treated women is likely to both reflect and contribute to functional changes leading to endometrial breakdown.
Comparison between Indonesian women treated with Norplant and Australian controls (predominantly Caucasian women) was a potential source of error in this study as differences in bleeding patterns between women of different ethnicity have been described (Gao et al., 1987). However, it was not possible to obtain control tissue from Indonesian women for this study. No difference in endometrial endothelin or neutral endopeptidase immunostaining was observed between Australian and Indonesian control subjects (Marsh et al., 1995
).
The lack of correlation of MMP-1, MMP-3 or MCs with bleeding patterns may reflect the complexity of the system and the likelihood that the pathogenesis of abnormal uterine bleeding is multifactorial and not dependent upon a single variable. It may also be related to the difficulty in obtaining tissue at the time and site of bleeding. Indeed, it is difficult in many subjects to obtain sufficient biopsy sample for histological and immunohistochemical analysis; in this study approximately half the Norplant and DMPA samples collected could not be used for analysis. Similar findings were reported by Hadisputra and co-workers (Hadisputra et al., 1996) who also observed an increased number of bleeding days reported by women in whom a successful biopsy was obtained. Thus the results of this study may only apply to a subgroup of Norplant users. Even had sampling at times of bleeding been possible, it is extremely difficult to sample bleeding sites as compared with non-bleeding sites, even at hysteroscopy (M. Hickey, Imperial College School of Medicine, London, UK, personal communication). It is likely that analysis of bleeding sites would have provided better correlations between bleeding and the parameters studied here. Histological assessment of the samples revealed that the majority displayed a morphological appearance consistent with the effect of exogenous progestins. However, an interesting finding was the variability in the degree of stromal cell pseudo-decidualization observed. Decidualized stromal cells display a quite different phenotype to non-decidualized stromal cells and differences in the degree of pseudo-decidualization may translate to functional differences observed in the endometrium. No correlation between bleeding patterns and histological appearance of biopsies from women using Norplant has been observed in previous studies (Rogers, 1996
) although this factor was not specifically assessed.
In conclusion, this study demonstrates that in women using progestin-only contraceptives, MC phenotype alteration and activation occurs and amounts of MMP-1 and -3 are similar to or higher than those seen at menstruation, a time of tissue breakdown. These changes, which vary according to the clinical context, may contribute to abnormal uterine bleeding seen in women using these contraceptives.
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Acknowledgments |
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Part of this work was presented at the WHO and NIH sponsored meeting on Steroids and Endometrial Breakthrough Bleeding held on 45 May 1999 at Monash Medical Centre, Clayton, Victoria, Australia.
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Notes |
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References |
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Submitted on June 28, 1999; accepted on October 6, 1999.