1 Prince Henry's Institute of Medical Research, P.O.Box 5152, Clayton, Victoria, 3168, 2 Melbourne University Departments of Pathology, Obstetrics and Gynaecology, Parkville, Victoria, 3 Monash University Department of Obstetrics and Gynaecology, Clayton Victoria, 3168, Australia, 4 Human Reproduction Study Group, Department of Obstetrics and Gynaecology, University of Indonesia, Klinik Raden Salah, Jalan Raden Salah 49, Jakarta, 10330, Indonesia and 5 Monash University Department of Obstetrics and Gynaecology, Box Hill Hospital, Box Hill, Victoria, Australia
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Abstract |
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Key words: depot medroxyprogesterone acetate/leukocytes/matrix metalloproteinase-9/tissue inhibitor of metalloproteinase/uterine bleeding
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Introduction |
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Matrix metalloproteinases (MMP) are a family of zinc-dependent proteases which degrade extracellular matrix (ECM) components (Birkedal-Hansen et al., 1993). Regulation of MMP is complex and occurs at multiple levels including gene transcription, a cascade of activation in which proteases, including some MMP, are able to activate the latent zymogen and inhibition by tissue inhibitors of metalloproteinases (TIMP) with formation of 1:1 complexes with the active enzymes. MMP-9 (gelatinase B) is a 92 kDa metalloproteinase with substrate specificity for collagen IV (a major component of basement membranes), collagen V, elastin and gelatin and is unusual in that both the active and latent enzyme are inhibited by TIMP-1 (Itoh and Nagase, 1995
). 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
, 1996
; Salamonsen and Woolley, 1996
). Recent studies have also shown altered MMP (Vincent et al., 1999
, 2000
; Galant et al., 2000
; Marbaix et al., 2000
) and TIMP (Galant et al., 2000
; Marbaix et al., 2000
) expression and activation in endometrial biopsies from women using Norplant.
Leukocytes are an integral component of the endometrium and display variation in type, number, activation status and site across the menstrual cycle and are postulated to play a key role in menstruation (Salamonsen and Lathbury, 2000). Previous immunohistochemical studies have reported both increased (Song et al., 1996
; Critchley et al., 1998b
; Vincent et al., 1999
) and decreased (Clark et al., 1996
) numbers of leukocytes in the endometrium of women treated with progestin-only contraceptives. In addition to providing a source of MMP-9 (Martelli et al., 1993
; Shi et al., 1995
; Jeziorska et al., 1996
, Vincent et al., 1999
), endometrial leukocytes produce a range of bioactive molecules including cytokines and proteases which are implicated in the regulation of MMP (Salamonsen and Lathbury, 2000
).
We hypothesize that MMP, TIMP and leukocytes are involved in the pathogenesis of abnormal uterine bleeding in women using progestin-only contraceptives. Thus the aim of this study is to extend our previous studies and determine whether MMP-9, TIMP and leukocytes are present within the endometrium of women using 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 leiomyomas, endometrial polyps, endometriosis, or those who had received steroid therapy in the past 6 months were excluded. MMP 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 and embedded in paraffin. Tissue sections were cut at 5 µm, dewaxed, rehydrated and either stained with haematoxylin and eosin for histological dating according to published 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.
Menstrual diary records
Subjects recorded a daily menstrual diary from the day of 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
Immunohistochemical analysis of each antigen was performed on a single section per subject selected at random. All incubations were performed in a humid chamber. Each incubation was performed at room temperature and was followed by three Tris-buffered saline (TBS) washes over a 10 min period unless otherwise stated.
MMP-9 immunohistochemistry was performed using a monoclonal mouse anti-human MMP-9 antibody (Insight Biotechnology Ltd, Middlesex, UK) and the alkaline phosphataseanti-alkaline phosphatase (APAAP) technique as described previously (Vincent et al., 1999). Briefly, following inhibition of non-specific binding (NSB) [30 min incubation with TBS containing 10% normal goat serum (NGS)], the primary antibody (used at a concentration of 2 µg/ml in 10% NGS/TBS) was applied and the sections incubated overnight at 4°C. MMP-9 was visualized using goat anti-mouse IgG (Dako, Glostrup, Denmark) followed by mouse APAAP complex (Dako), repeated twice and with New Fuchsin (Dako) as the chromogen. Endogenous alkaline phosphatase was blocked with 1 mmol/l levamisole.
Immunolocalization of macrophages was performed using the mouse monoclonal anti-human CD68 antibody clone KP-1 (Dako) diluted 1:50 in 10% normal horse serum (NHS)/TBS. Following inhibition of endogenous peroxidase activity (0.3% H2O2 v/v in methanol for 30 min) and NSB (10% NHS/TBS for 30 min), a 2 h primary antibody incubation was performed. Incubations with biotinylated horse anti-mouse IgG (1:200 v/v in 1% fetal calf serum, 1% NHS, 5% normal human serum/TBS for 1 h) (Vector Laboratories, Burlingame, CA, USA) followed by the StreptABChorse-radish complex (Dako) (used according to the manufacturer's instructions) preceded colour development with diaminobenzidine (DAB).
T lymphocytes were identified using a polyclonal rabbit anti-human CD3 antibody (Dako) and the EnVision Peroxidase DAB kit (Dako). Immunostaining consisted of sequential applications of peroxidase blocking agent for 5 min, primary antibody (1:100 diluted in 10% NGS/TBS) for 30 min, EnVision polymer for 30 min and DAB plus kit with 5 min TBS washes between each step.
Detection of uterine natural killer (uNK) cells was performed using a microwave antigen retrieval method (Critchley et al., 1998b) adapted for our study and the APAAP method described above for MMP-9 immunohistochemistry. The tissue sections were initially dewaxed, rehydrated, microwaved at high power in 0.01 mol/l sodium citrate buffer (pH 6.0) for 15 min and then allowed to stand for 20 min. Following microwave antigen retrieval and NSB blocking (10% NGS/TBS), mouse monoclonal anti-human CD56 (diluted 1:150 in 10% NGS/TBS) (Zymed, San Francisco, CA, USA) was applied and the sections incubated overnight at 4°C. Incubations for 45 min with goat anti-mouse IgG (1:25 in 1% fetal calf serum, 1% NGS, 5% normal human serum/TBS) (Dako) and then mouse APAAP (1:50 in primary antibody diluent) (Dako) were performed and then repeated for 15 min. New Fuchsin (Dako) was used as the chromogen with colour development proceeding for 30 min.
Immunohistochemistry for TIMP-1, TIMP-2 and TIMP-3 was performed as previously described (Zhang and Salamonsen, 1997), using sheep anti-human TIMP-1 (a gift from Dr Hideaki Nagase, Kansas City, KS, USA), rabbit anti-human TIMP-2 (Triple Point, Forest Grove, OR, USA) and rabbit anti-human TIMP-3 (Triple Point) as the primary antibodies. The subsequent detection methods used were the Dako StreptABC kit (TIMP-1) and the StrAviGen (Biogenex Laboratories, San Ramon, CA, USA) supersensitive immunostaining system (TIMP-2 and TIMP-3) with New Fuchsin (Dako) as the chromogen.
For each tissue, a second section on the same slide was used as a negative control with an irrelevant -lactalbumin monoclonal IgG antibody (MMP-9, CD68 and CD56), rabbit IgG (CD3, TIMP-2 and TIMP-3; Dako) or normal sheep IgG (TIMP-1; Serotec, Oxford, UK) substituted at the same concentration as the primary antibody. Positive controls appropriate for each antibody were also included in each series of sections examined and included proliferative phase human endometrium (MMP-9), human synovium and endometrium (CD3), human endometrium and decidua (CD56; kind gift of Dr E.Wallace, Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia) and human fetal kidney (TIMP-1, TIMP-2 and TIMP-3). All tissue sections were counterstained with Harris' haematoxylin, dehydrated and cleared in xylene then mounted in DPX. Photography was performed using an Olympus BX50 microscope with filter sets for fluoroscein isothiocyanate (FITC) and Texas Red fitted with a digital camera (Videocam Fujix HC-2000; Fuji Photo Film Co., Tokyo, Japan) coupled to a Compucon Pentium PC computer using Analytical Imaging Station (Imaging Research Inc., USA) and Adobe Photoshop software.
Immunolocalization of neutrophils and eosinophils in Carnoy's fixed tissue using the antibodies, mouse monoclonal anti-neutrophil elastase and anti-eosinophilic cationic protein clone EG1 respectively, proved to be unreliable (A.J.Vincent, unpublished observations) and as DMPA samples fixed in 10% buffered formalin were not available in sufficient numbers, further immunohistochemical assessment and analysis of these leukocyte subtypes was not performed.
Dual immunofluorescence
Dual immunofluorescent staining was used on selected specimens to identify the cellular source of the MMP-9 using the amplification technique described previously (Vincent et al., 1999). MMP-9 antiserum, used at a concentration of 0.2 µg/ml, was visualized using the Renaissance TSA Indirect Amplification kit (NEN Life Sciences, Boston, MA, USA) with FITC-conjugated streptavidin as the detection system. A subsequent conventional fluorescent staining with the second primary antibody and visualization using a sheep anti-mouse or donkey anti-rabbit second antibody conjugated to Texas Red (Amersham Life Science, Little Chalfont, Buckinghamshire, UK) was then performed. The second primary antibodies used were (i) mouse monoclonal anti-mast cell tryptase (Dako), (ii) mouse monoclonal anti-CD56 (Zymed), (iii) mouse monoclonal anti-CD68 (clone KP1) (Dako), (iv) rabbit polyclonal anti-CD3 (Dako), (v) mouse monoclonal anti-neutrophil elastase (Dako) and (vi) mouse monoclonal anti-eosinophilic cationic protein clone EG1 (Pharmacia Ltd, Milton Keynes, Bucks, UK). No detectable signal was observed with conventional immunofluorescent staining using the same MMP-9 antibody concentration as used in the amplification technique. Other controls included omission of the primary antibody with resultant minimal background staining. The tissue sections were mounted with immunomount (Dako) and photographed using the Olympus photomicroscope described above with filter sets for FITC and Texas Red. As the antibodies, mouse monoclonal anti-neutrophil elastase and anti-eosinophilic cationic protein clone EG1 do not reliably detect antigen in Carnoy's fixed tissue (A.J.Vincent, unpublished observations), 10% buffered formalin-fixed biopsies obtained from two Australian DMPA users were included in this experiment.
Assessment of immunostaining
Quantitative analysis of positive MMP-9 and leukocyte immunostaining was performed as described previously (Vincent et al., 1999) 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, Birkeroed, Denmark) was used to generate a counting frame (14565 µm2) 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 number of positive cells (excluding intravascular and glandular lumen cells) in
15 random fields was counted for each section. Stromal cell density was also assessed by counting the number of stromal cells in eight of the random fields above, which contained only stroma (i.e. excluding glands and large blood vessels). The number of positive cells was expressed as per 1000 stromal cells. Quantitative analysis was performed by the same observer, with no knowledge of the clinical characteristics of the patient donor.
TIMP immunostaining intensity in each endometrial compartment (including luminal and glandular epithelium, stroma, endothelium, vascular smooth muscle cells, and decidual cells) was assessed using a published method (Zhang and Salamonsen, 1997). All endometrial samples were compared with the fetal kidney positive control (included in each immunostaining run) and the negative control section (included on each endometrial sample slide). Immunostaining was scored on a scale from 0 (no staining) to 4 (equivalent to the maximal staining intensity observed on the fetal kidney positive control).
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 a published method (Conover, 1980). Correlations between variables were calculated via Spearman's rank correlation.
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Results |
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Clinical characteristics
As described previously, the duration of DMPA use varied from 19 to 376 days (Vincent et al., 2000). Dividing the subjects into three groups according to duration of DMPA use: (I) duration <31 days (n = 7), (II) duration 80110 days (n = 4) and (III) duration >300 days (n = 5) revealed that the shedding morphology was only observed in association with a shorter duration of use and the atrophic morphology associated with a longer duration of use. The progestin-modified morphological appearance was observed in all groups. The median number of bleeding days ratio in the 90 day reference period prior to biopsy was greatest in group I (36 days, range 2050) compared with group II (median 17 days, range 928) and group III (median 26 days, range 2130). However, there were no statistically significant differences in the bleeding ratios between the three groups.
Immunohistochemical staining of endometrial biopsies
No specific positive staining was observed in any of the sections where the primary antibody was substituted with the appropriate negative control antibody.
MMP-9
Immunoreactive MMP-9 was observed in all DMPA biopsies (Figure 1A) and in those from normal cycling women during the perimenstrual period. In endometrial samples from women using DMPA, MMP-9 immunolocalization was mainly confined to either intravascular leukocytes where intracellular staining was observed or in association with leukocytes in areas of tissue lysis where both intracellular and adjacent extracellular immunostaining was noted. There was no glandular epithelial, endothelial or stromal cell expression of MMP-9.
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Quantitative analysis demonstrated that MMP-9 immunostaining was similar in DMPA users and premenstrual and menstrual controls (Figure 2). There was no correlation between the number of MMP-9 immunopositive cells and the number of leukocytes or bleeding patterns as recorded in menstrual diaries.
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Biopsies obtained from women using DMPA and perimenstrual control subjects revealed positive TIMP-3 immunostaining in the same endometrial components as TIMP-1 and TIMP-2 (Figure 1G). As demonstrated with TIMP-1 and TIMP-2, significantly less immunoreactive TIMP-3 was observed in luminal epithelial (P = 0.01) and stromal (P = 0.02) endometrial compartments of DMPA users compared with perimenstrual control subjects (Figure 3
). However, in contrast to TIMP-1 and TIMP-2, TIMP-3 immunostaining intensity, although low, was significantly higher in endometrial endothelium of women using DMPA compared with perimenstrual control subjects (P = 0.007). No correlation between stromal TIMP-3 immunostaining intensity and bleeding parameters was observed (data not shown).
Leukocytes
T lymphocytes, macrophages and uNK cells were identified in all endometrial biopsies from DMPA users (Figure 1L, M and N respectively) and control women (not shown). CD3+ T lymphocytes were widely distributed throughout the stroma including areas of tissue breakdown, with occasional cells infiltrating the luminal and glandular epithelium and present intravascularly. Periglandular aggregates of T lymphocytes were commonly observed. CD56+ uNK cells displayed a similar pattern to T cells. Macrophages were also dispersed throughout the stroma and areas of tissue lysis. Infiltration of luminal and glandular epithelium was prominent with CD68+ cells observed in the gland lumens. Macrophages were occasionally noted in association with the lymphoid aggregates. Quantitative analysis of the numbers of positive cells revealed significantly more (P = 0.01) CD3+ T cells in endometrial biopsies from DMPA users compared with premenstrual or menstrual controls (Figure 2
). No significant difference was observed between the number of uNK cells and macrophages in control endometria compared with endometrial samples obtained from women using DMPA (Figure 2
). However, the number of macrophages was positively correlated (P = 0.01) with the morphological grade of decidualization of the progestin-modified histological group (data not shown). There was no correlation between leukocyte numbers and duration of DMPA use or bleeding patterns reported by women.
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Discussion |
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In agreement with previous investigations of the normal menstrual cycle (Hampton and Salamonsen, 1994; Rodgers et al., 1994
; Zhang and Salamonsen, 1997
), immunoreactive TIMP-1, TIMP-2 and TIMP-3 were observed in all endometrial compartments except leukocytes in both progestin contraceptive users and perimenstrual controls. However, differences in endometrial TIMP immunostaining intensity were observed between DMPA users and control subjects. In comparison with perimenstrual controls, the extent of TIMP-1 and TIMP-2 immunostaining intensity was decreased in the endometrium of DMPA users. Importantly, the endometrial compartments in which altered TIMP-1 and TIMP-2 immunoreactivity was observed included endothelium, stroma and luminal epithelium which are important for the maintenance of endometrial integrity. MMP are inhibited by TIMP with a 1:1 stoichiometry (Birkedal-Hansen et al., 1993
) with ECM integrity/degradation dependent upon the MMP/TIMP balance. Thus the significant decrease in immunoreactive TIMP-1 observed in multiple endometrial compartments concomitant with the influx and localization of MMP-9 immunopositive leukocytes to areas of tissue lysis and the perimenstrual-like extent and widespread distribution of immunoreactive MMP-1 (Vincent et al., 2000
) in biopsies obtained from DMPA users, suggests that the MMP/TIMP ratio may be altered in favour of MMP action predisposing to tissue breakdown. Indeed, the increased foci of stromal oedema reported in the endometrium of DMPA users (Ludwig, 1982
) is consistent with increased MMP activity leading to ECM degradation. Decreased endothelial and perivascular TIMP-1 could also contribute to vascular fragility, which has been noted in women using Norplant (Hickey et al., 1996
) but not assessed in DMPA users. In accordance with these findings, endometrial biopsies obtained from Norplant users at the start of a bleeding episode (compared with non-bleeding intervals) demonstrated immunopositive MMP-1, MMP-2, MMP-3 at sites of tissue breakdown, activation of MMP-1, MMP-2, MMP-3 and MMP-9 with decreased production of TIMP-1 (Galant et al., 2000
; Marbaix et al., 2000
). In comparison with the findings of the current study, endometrial MMP-9 immunostaining was increased in women using Norplant whereas TIMP-1 immunoreactivity was similar to perimenstrual controls (Vincent et al., 1999
). Such differences in the pattern of MMP and TIMP immunostaining observed between the different progestins may relate to the differences in bleeding patterns observed between the different agents. However, despite a heterogeneous response observed in the endometrium following treatment with different progestins, alteration in the MMP/TIMP balance in favour of MMP activity and subsequent ECM degradation is a common theme.
The increased endothelial TIMP-3 immunostaining observed in women using progestin-only contraceptives was unexpected and may relate to (i) sampling at non-bleeding endometrial sites, (ii) subject amenorrhoea or (iii) alternative functions of TIMP-3 such as mitogenesis or induction of apoptosis, which have been described in other tissues (Woessner and Nagase, 2000) but not as yet described in the endometrium. Apoptosis has been proposed as a mechanism involved in the regulation of endometrial endothelial cell numbers (Rogers, 1996
).
As reported previously in Norplant users (Vincent et al., 1999), this study demonstrates that MMP-9 is present within endometrial leukocytes in women with abnormal bleeding associated with the use of DMPA. These MMP-9 positive cells were identified as CD3+ T cells and neutrophils, although not all cells of any one type were MMP-9 positive. Prior immunohistochemical (Jeziorska et al., 1996
; Vincent et al., 1999
) and zymography studies (Shi et al., 1995
) have also identified endometrial macrophages, eosinophils and uNK cells as a source of MMP-9, although human endometrial mast cells do not appear to produce MMP-9 (Jeziorska et al., 1996
; Zhang et al., 1998
). The observed differences in this study may relate to the small sample size available for experimentation (e.g. eosinophils) and phenotypic variation where only a subset of each type of leukocyte is MMP-9 positive. Both of these are particularly relevant for those cells in very low numbers. Variation in leukocyte phenotype associated with different clinical settings, as observed with endometrial mast cells in control women compared with progestin-only contraceptive users (Vincent et al., 2000
), may be an alternative explanation. We therefore cannot exclude endometrial macrophages, eosinophils and uNK cells as a potential source of MMP-9 in women treated with DMPA. Immunoreactive MMP-9 has also been localized to endometrial glandular, stromal, endothelial and perivascular cells in women using the levonorgestrel-releasing intrauterine system (LNG-IUS) (Skinner et al., 1999
) and these differences may relate to immunohistochemical technique or the different clinical context.
Progesterone regulation of endometrial MMP production in vitro is well recognized (Salamonsen and Woolley, 1996). Alteration in endometrial progesterone receptor (PR) expression has been observed in response to exposure to progestin-only contraceptives (Critchley et al., 1993
, 1998a
; Lau et al., 1996
; Mangal et al., 1997
) with differences demonstrated in relation to PR isoform, type and duration of the contraceptive agent, potentially contributing to the observed changes in endometrial MMP expression in women using these agents. One group (Critchley et al., 1998a
) reported decreased prostaglandin dehydrogenase immunostaining and activity as well as diminished endometrial PR (PR-B more suppressed than PR-A) following the insertion of the LNG-IUS, concluding that the down-regulation of endometrial PR was associated with a functional response. PR-B expression, identified using Western analysis, was reduced in the endometrium of two subjects using DMPA (Mangal et al., 1997
).
The pattern of leukocyte response to the use of exogenous progestins varies with the type of progestin used, duration and pattern of administration (cyclical or continuous), route of administration, presence of bleeding and endometrial morphological appearance (Vincent and Salamonsen, 2000). Consistent with the endometrial leukocyte influx observed in DMPA users in this study, focal and diffuse infiltration of leukocytes, predominately lymphocytes and monocytes, were observed in a light and electron microscopic study of endometrial biopsies obtained from women using long-term, low dose oral progestins or DMPA (Ludwig, 1982
). In the current study, the
6-fold increase in CD3+ T lymphocytes observed is consistent with an immunohistochemical study (Song et al., 1996
) which reported increased numbers of leukocytes in the endometrium of women treated with high dose oral progestins compared with secretory phase controls. These included CD3+ T cells, leukocyte common antigen positive cells, neutrophils, CD68+ macrophages and phloxine-positive uNK cells. In the present study, endometrial macrophage and uNK cells numbers were similar between DMPA users and perimenstrual controls, the time of the normal menstrual cycle when leukocyte numbers are maximal (Salamonsen and Lathbury, 2000
). These results also concur with the observations of Song et al. (1996) above and those of another study (Critchley et al., 1998b
) which reported the presence of CD56+ uNK cells and CD68+ macrophages in the endometrium of women using the LNG-IUS. Interestingly, in the current study a positive correlation was observed between macrophage numbers and the grade of pseudo-decidualization. In this context, increased macrophage numbers and immunoreactive stromal granulocyte-macrophage colony stimulating factor (GM-CSF), a macrophage chemokine, were observed in the endometrium of LNG-IUS users (in whom significant stromal pseudo-decidualized change occurs) (Critchley et al., 1998b
). In contrast, decreased macrophage numbers were demonstrated in morphologically atrophic endometrium in Norplant users (Clark et al., 1996
). Chemokines, including monocyte chemotactic protein-1 (MCP-1) (Arici et al., 1995
; Hampton et al., 2000
), MCP-2 (Hampton et al., 2000
), GM-CSF (Critchley et al., 1998b
), interleukin-8 (Jones and Critchley, 2000
) have been identified in the endometrium of women using progestin-only contraceptives; however, their contribution to leukocyte infiltration or other functions remains unclear.
Endometrial leukocytes do not express PR (King et al., 1996; Salamonsen et al., 2002
); however, alteration in endometrial epithelial and stromal cell PR expression (as described above) may result in changes in the endometrial chemokine and cytokine milieu leading to leukocyte influx and activation. Leukocytes, as well as providing a source of MMP-9, produce a variety of enzymes, cytokines and other bioactive mediators. These molecules, in addition to involvement in MMP and TIMP regulation (Salamonsen and Lathbury, 2000
), may further influence the endometrial micro-environment thereby contributing to endometrial breakdown.
The association between bleeding patterns and leukocytes remains unclear. Endometrial leukocyte infiltrations reported by Ludwig were observed more frequently closer to the time of last reported bleeding episode and were seen less frequently in morphologically atrophic endometrial samples or those from amenorrhoeic patients (Ludwig, 1982). It was reported that the number of CD68+ macrophages was significantly increased in women using Norplant who reported irregular bleeding compared with non-bleeders (Clark et al., 1996
). More recently, endometrial mast cells were increased in women using the LNG-IUS who reported breakthrough bleeding compared with non-bleeders (Milne et al., 2001
). In contrast, as observed in the current study, no correlation was observed between bleeding patterns and endometrial MMP-9 or leukocyte immunostaining in Norplant users (Vincent et al., 1999
) or following the insertion of the LNG-IUS (Critchley et al., 1998b
). This lack of correlation may reflect the complexity of the endometrial micro-environment and the likelihood that the pathogenesis of menstrual bleeding disturbance associated with progestin-only contraceptives 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. It is often difficult to obtain sufficient biopsy sample for analysis; as reported previously (Vincent et al., 2000
) approximately half the DMPA samples collected could not be used for analysis. Similar findings have been reported together with an increased number of bleeding days reported by women in whom a successful biopsy was obtained (Hadisputra et al., 1996
). Thus the results of the current study may only apply to a subgroup of DMPA users.
Previous studies have demonstrated temporal variation in endometrial vascular membrane components following Norplant exposure (Hickey et al., 1999a,b
) and leukocyte distribution following LNG-IUS insertion (Critchley et al., 1998b
) indicating the importance of the timing of sampling in relation to the administration of progestin. No difference in MMP-9, TIMP or leukocytes was observed according to long (>300 days) or short (<31 days) duration of DMPA use; however, the sample size of each group was small.
Comparison between the Indonesian women treated with DMPA and Australian controls (predominately 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. Furthermore, no difference was observed between the four DMPA endometrial biopsies obtained from Australian women and the Indonesian DMPA samples. No difference in endometrial endothelin or neutral endopeptidase immunostaining was observed between Australian and Indonesian control subjects (Marsh et al., 1995
).
The findings of this study provide further evidence linking MMP to endometrial breakdown associated with abnormal uterine bleeding, although a causal relationship remains to be established. These investigations also emphasize the variation in endometrial response to different progestins and indicate the importance of the MMP/TIMP balance in determining potential loss or maintenance of endometrial integrity. Such variability may relate to the different patterns of bleeding disturbance reported with different agents (Odlind and Fraser, 1990) and indicates similarities and differences in the potential mechanisms underlying breakthrough bleeding and the process of normal menstruation (Fraser et al., 1996
).
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Acknowledgements |
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Notes |
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References |
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Submitted on August 17, 2001; accepted on December 11, 2001.