1 Prince Henrys Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, 2 Department of Obstetrics & Gynaecology, Monash University, Clayton, Victoria 3168, Australia and 3 Department of Reproductive and Developmental Sciences, University of Edinburgh, Centre for Reproductive Biology, Edinburgh, UK
4 To whom correspondence should be addressed. E-mail: naomi.morison{at}phimr.monash.edu.au
R.L.Jones and N.B.Morison contributed equally to this work
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Abstract |
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Key words: chemokines/contraceptives/endometrium/leukocytes/progestin
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Introduction |
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P-only contraceptives have different effects on the endometrium depending on the progestin used, the dose and the route of administration. Intrauterine delivery of progestin (levonorgestrel-releasing intrauterine system; LNG-IUS; Mirena®) results in high local concentrations, causing rapid and extensive decidualization of the endometrium, epithelial atrophy and loss of cyclicity (Nilsson et al., 1978; Silverberg et al., 1986
). In contrast, subdermal implants (Norplant® and Implanon®) produce lower intrauterine concentrations of progestin and have a less dramatic and more variable effect on endometrial morphology (Marsh et al., 1995
; Mascarenhas et al., 1998
; Vincent and Salamonsen, 2000
). Norplant endometrial samples have been subclassified based on morphology as atrophic (narrow proliferative-type epithelial glands, but with low mitotic activity), shedding (areas of distinct breakdown apparent) and progestin-modified (p-modified; decidualized) (Marsh et al., 1995
; Vincent et al., 1999
).
A common feature of all p-only contraceptives is an abnormal endometrial leukocyte infiltrate. The normal endometrium possesses an active and tightly regulated immune environment, with significant immune cell populations present associated with specific endometrial events (Bulmer et al., 1991). Few leukocytes are present in the proliferative phase, but there is a dramatic accumulation of leukocytes following ovulation, associated with implantation and decidualization. The majority are a unique endometrial-specific subset of natural killer (NK) cells (uNKs), which appear to be important for endometrial remodelling associated with the establishment of pregnancy when they are highly abundant in decidua (Moffett-King, 2002
). Macrophages are present throughout the menstrual cycle but their numbers increase from the mid-secretory phase. Menstruation is associated with a dramatic influx of inflammatory leukocytes: neutrophils and eosinophils, and increased activation of resident mast cells, which provide proteases including matrix metalloproteinases and inflammatory mediators capable of initiating the tissue breakdown (Salamonsen and Lathbury, 2000
).
Leukocyte trafficking appears to be dysregulated in p-exposed endometria, with excessive numbers of inflammatory leukocytes reported (Song et al., 1996; Vincent et al., 1999
). Importantly, differences have been reported in the immune cell subpopulations in the endometria of users of different p-only contraceptives and between the different groups of Norplant-exposed endometrium; there are predominantly neutrophils and eosinophils in shedding endometria, whilst very few of these subtypes are in atrophic or p-modified endometria (Vincent et al., 1999
). In the highly decidualized endometria of women using LNG-IUS, uNKs and macrophages are abundant (Critchley et al., 1998a
).
As normal menstruation is an inflammatory-type event, with leukocyte infiltrate thought to play a major role in the focal endometrial breakdown (Salamonsen and Woolley, 1999), the abnormal leukocyte infiltrate in women using p-only contraceptives has been postulated to contribute to BTB. Leukocyte trafficking into normal endometrium is tightly regulated (Hornung et al., 1997
; Jones et al., 1997
; Akiyama et al., 1999
; Zhang et al., 2000
). Our previous studies have identified the most abundant chemokines in the endometrium associated with selective leukocyte recruitment (Jones et al., 2004
): monocyte chemoattractant protein (MCP)-3, 6Ckine, macrophage derived chemokine (MDC), hemofiltrate CC chemokine (HCC)-1, HCC-4, eotaxin, interleukin (IL)-8, and macrophage inflammatory protein (MIP)-1
. Distinct cohorts of chemokines are up-regulated coincidental with the accumulation of decidual-associated leukocytes and with the influx of menstruation-associated leukocytes. We hypothesize that the normal regulation of leukocyte entry into the endometrium is disrupted by the use of progestins, resulting in the abnormal leukocyte infiltrate that is associated with BTB. We therefore examined the production of endometrial chemokines in women using two different methods of p-only contraceptive (LNG-IUS and Norplant), and correlated these with the presence of distinct leukocyte subpopulations.
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Patients and methods |
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Written informed consent was obtained from all participants and ethical approval was obtained from the appropriate institutional ethics committees (96014B, LREC/2003/6/27 and LREC/2003/6/24). Stage of cycle of normal endometrial biopsies was calculated from last menstrual period and confirmed by histological assessment by a gynaecological pathologist, according to the criteria of Noyes et al. (1975). Norplant endometria were histologically stained with haematoxylin and eosin and subclassified into atrophic, shedding or p-modified groups based on morphological appearance (n = 47 per morphological group), as described previously (Marsh et al., 1995
; Vincent et al., 1999
).
Endometrial samples were fixed in 10% buffered formalin overnight prior to washing in Tris-buffered saline (TBS) and routine histological processing to paraffin blocks. A portion of each tissue from the LNG-IUS study and from the normal control tissues was immersed in RNALater solution (Ambion, Austin, TX, USA) prior to snap freezing and storage at 80°C for RNA extraction.
Leukocyte subtype immunohistochemistry
Immunohistochemistry was performed on Norplant and control endometrial biopsies using antibodies specific for leukocyte common antigen (LCA CD45; Dako, Glostrup, Denmark), macrophages (CD68; Dako) and uNK cells (CD56; Zymed; CA,USA) as described previously (Critchley et al., 1998a). Briefly, 5-µm sections of formalin-fixed, paraffin-embedded tissues were dewaxed and rehydrated. Antigen retrieval by microwaving was necessary for CD56 immunohistochemistry, whilst trypsin digestion was performed prior to CD68 immunolocalization. Endogenous hydrogen peroxidase activity was quenched using 3% H2O2 in dH2O for 5 min at room temperature. Non-specific binding was prevented by preincubation of tissue sections with a non-immune block containing 10% non-immune horse serum (Sigma-Aldrich, Sydney, Australia), 2% normal human serum (in house) in TBS-T (Tween-20; 0.1%). Primary antibodies were applied overnight (17 ± 1 h) at 4°C diluted to 1 µg/ml in non-immune block. Negative controls were included for each tissue by the substitution of the primary antibody for a matching concentration of non-immunized mouse IgG. Following stringent washing with TBS-T (0.6%), detection of positive binding was performed by the sequential application of biotinylated horse anti-mouse IgG (1:200 in non-immune block; Vector Laboratories, Burlingame, CA, USA) and avidinbiotinperoxidase conjugate (ABC-HRP; Dako), followed by the substrate diaminobenzidine (Dako) for 210 min. All samples for comparison were included in the same run to exclude inter-assay variability. Sections were counterstained with Harris haematoxylin (Sigma), dehydrated and mounted from Histosol with DPX.
Chemokine immunohistochemistry
Immunohistochemistry was performed to localize eight chemokines (MCP-3, 6Ckine, MDC, HCC-1, HCC-4, eotaxin, IL-8 and MIP-1) in formalin-fixed endometrial biopsies from users of Norplant and LNG-IUS, compared with representative normal endometrial biopsies from the times of known specific chemokine production: menstrual, proliferative and late secretory phases. Immunolocalization was performed using a monoclonal antibody specific for eotaxin (LeukoSite Inc., Cambridge, MA, USA) and polyclonal antibodies raised against human chemokine peptides (MIP-1
: R&D Systems Inc., Minneapolis, MN, USA; all other antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA, USA, and were as described previously: Zhang et al., 2000
; Jones et al., 2004
). Antibodies were applied at 0.54 µg/ml overnight at 4°C following microwave antigen retrieval. A similar protocol to that described above was used for eotaxin, but with visualization of positive localization with ABC-alkaline phosphatase (Dako), with new fushin substrate (Dako). All other antibodies were detected with sequential application of biotinylated horse anti-goat IgG and ABC-HRP. Antibodies were preabsorbed for 48 h at 4°C with a five-fold excess of specific chemokine peptide (Santa Cruz) to verify specificity. Again, all samples to be analysed were included in the same run to allow comparison of staining intensity.
Analysis of immunostaining
Abundance of leukocyte subtypes was analysed semi-quantitatively using a scoring system between 0 (no leukocytes) and 4 (highly abundant), as described previously (Vincent et al., 1999). Chemokine immunostaining was analysed semi-quantitatively by two independent observers blind to the identity of the tissue as previously described (Hannan et al., 2004
; Jones et al., 2004
). Staining intensity and heterogeneity in each of the endometrial compartments [epithelium, stroma (including decidualized stromal cells) and vasculature] was assessed and allocated a score between 0 and 3, where 0 = no stain and 3 = very intense staining. Data were statistically analysed using ANOVA with Tukeys post-hoc test. A P-value of <0.05 was deemed significantly different.
RNA extraction and purification
Total RNA was extracted from LNG-IUS (n = 6) and control endometrial samples from the menstrual, proliferative and late secretory phases of the menstrual cycle and from early pregnancy (n = 3/stage) by homogenization in Trizol reagent (Qiagen, MD, USA), according to the manufacturers instructions, with the exception of an additional chloroform extraction step to minimize carryover of phenol into the precipitated RNA. All samples were treated with RNase-free DNase (Ambion) to remove contaminating genomic DNA. RNA samples were then analysed by spectrophotometry to determine RNA concentration, yield and purity. Any samples with ratios of A260/280 <1.7 or A230/280 >1 were purified through RNeasy spin columns (Qiagen, MD, USA) according to manufacturers instructions and thereafter reanalysed by spectrophotometry. Purified RNA (0.5 µg) was then run on a 1% agarose (Roche, Castle Hill, NSW, Australia) gel to ensure integrity of rRNA subunits, or analysed using the Agilent 2100 Bioanalyzer.
RNA concentrations were also analysed by Ribogreen fluorescence RNA assay (Molecular Probes, Eugene, OR, USA). RNA samples were diluted to 40 ng/ml based on spectrophotometric readings and analysed in a 96-well plate by addition of Ribogreen fluorescent dye at a dilution of 1:500. A standard curve of serially diluted rRNA (Molecular Probes) between 0 and 80 ng/well was run on the same plate. This assay gave an intra-assay coefficient of variation (CV), as defined by the repeated analysis of a single RNA sample, of 3.3% (n = 16) and an inter-assay variation of 10.3% (n = 14).
Real time reverse transcription (RT)PCR
mRNA expression levels of MCP-3, 6Ckine, MDC, HCC-1, HCC-4, IL-8 and MIP-1 were analysed by real time PCR in LNG-IUS exposed endometrium compared with normal endometrium. Standards for real time PCR were generated by conventional PCR as described previously (Jones et al., 2004
). RNA samples were reverse transcribed (RT) using AMV-RTase (Promega, Annandale, NSW, Aust.) and 100 ng random hexanucleotide primers (Amersham Biosciences, Piscataway, NJ, USA). The efficiency and reproducibility of the reverse transcription step was then tested by real time PCR analysis of 18S expression in the triplicate RTs. The %CV was calculated and samples outside 15% variability were excluded as outliers prior to pooling of triplicates, which were thereafter used for analysis of chemokine expression. This method overcomes the inherent high variability of this technique, ensuring the RTs for subsequent analysis are representative of the original mRNA sample.
Real time RTPCR for chemokines was performed using a Roche Light Cycler (Roche) as described previously. Expression levels were quantified by comparison with standards of known concentration, using SYBR green fluorescence detection. Amplification conditions were carefully optimized for each chemokine as described previously (Jones et al., 2004). Fluorescence was monitored during cycling at the end of each elongation phase and analysis of expression was performed when amplified products were in the log-linear phase and parallel to the standards. At the end of each program, melting curve analysis was carried out to ensure specificity of the reaction products.
All samples to be compared were included within the same run (14.9% intra-assay variability). Mean expression levels were calculated from samples within each group and statistical analysis was performed using ANOVA with Tukeys post-hoc test. A P-value of <0.05 was considered statistically significant. Data were not normalized for the expression of a housekeeping gene owing to the regulated expression of all known housekeeping genes examined in the endometrium (18S, cyclophilin, GAP-DH,-actin; data not shown). Instead RNA samples were meticulously purified and quantitated by three distinct methods as described above to ensure confidence in equal RNA loading and reverse transcription efficiency and reproducibility.
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Results |
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Analysis of leukocyte subtypes revealed that generally very few CD68-positive macrophages were present in shedding and atrophic endometrium, but there were significantly higher numbers in p-modified samples (Figures 1B and 2DF). These tended to be in the subepithelial zone (Figure 2DF), with few positive cells in deeper tissue. CD56 identifies uNK cells, and these were highly abundant in p-modified tissues (P < 0.01 versus atrophic and shedding groups) (Figures 1C and 2GI), comparable to numbers seen in early pregnancy decidua (not shown). In shedding and atrophic groups there were significantly fewer uNK cells (Figure 2G and H). A summary of this data, together with existing information in the literature, is provided in Table I, clearly illustrating the distinct differences in leukocyte subpopulations between p-only groups.
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Chemokines in Norplant endometrium
All chemokines examined (Table II) were detected in endometrium from women using Norplant, although significant differences were seen in chemokine production between different Norplant groups and in comparison with normal endometrium.
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In non-pregnant endometrium, the luminal and glandular epithelia are the predominant sites of chemokine synthesis. MDC, HCC-1, MCP-3 and eotaxin were highly expressed in epithelial cells in endometria from all women using Norplant, with levels equivalent to maximal immunostaining levels seen during the normal menstrual cycle (Figures 3 and Figures 4A). Very intense staining was identified for MDC and HCC-1 in epithelial cells of p-modified endometrium (Figure 3). In contrast, 6Ckine, MIP-1 and HCC-4 were present in lower levels in epithelial cells from Norplant tissues than normal control endometrial samples, with a similar pattern in all three groups (Figure 3). IL-8 followed a distinct pattern, with elevated epithelial immunostaining in the shedding Norplant group comparable to levels seen in normal endometrium perimenstrually (Figure 3). Immunostaining was low or absent in atrophic and p-modified endometria. Representative photomicrographs of chemokines are shown in Figure 4, illustrating the faint immunostaining for most chemokines (eoxtain is a notable exception) in atrophic endometrium (Figure 4B); intense staining for MCP-3 and IL-8 in areas of breakdown in shedding endometrium (Figure 4C); and reduced staining for HCC-4 and MIP-1
compared with proliferative controls in p-modified endometrium, whilst 6Ckine, MCP-3 and MDC are more highly expressed, consistent with secretory levels (Figure 4D).
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Individual stromal cells, exhibiting histological characteristics of leukocytes, stained intensely for a number of the chemokines: MDC, HCC-4, eotaxin, MIP-1, 6Ckine and IL-8. MDC- and MCP-3-positive cells were particularly abundant in p-modified endometrium (Figure 4D), whilst those producing IL-8 were abundant in breakdown areas in shedding tissues (Figure 4C, arrowheads). Occasional immunoreactivity for chemokines was detected in vascular endothelial cells of Norplant users, as illustrated for eoxtain (Figure 4C).
Chemokines in LNG-IUS endometrium
Chemokine immunostaining after insertion of LNG-IUS was consistent with the highly decidualized nature of the tissue and closely resembled that seen in the p-modified Norplant endometrium. Most marked was the strong up-regulation of many chemokines (HCC-1, 6Ckine, MCP-3 and MDC) in the decidualized stroma (Figures 4E and 5). However, HCC-4 and eotaxin exhibited faint immunostaining (Figure 5), whilst MIP-1 was markedly absent (data not shown). Epithelial staining remained high post-insertion for MDC, HCC-1 and MCP-3, whilst again 6Ckine and HCC-4 production by epithelial glands was reduced with continued exposure to intrauterine levonorgestrel, although this failed to reach significance (Figure 5). No significant changes were seen with continued exposure to levonorgestrel, except that stromal chemokine immunostaining was limited to those cells that had undergone decidualization, and thus the staining became more homogeneous as the degree of decidualization increased. It is important to note that while some samples were collected during times of bleeding there was no correlation with bleeding and chemokine expression compared with those samples collected during non-bleeding episodes.
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Real time RTPCR
mRNA expression levels of seven of the chemokines were analysed by real time RT-PCR in endometrium from LNG-IUS users, and compared with normal non-pregnant and pregnant endometrial levels. Most of the chemokines examined were expressed at elevated levels after insertion of LNG-IUS compared with normal cycling endometrium (Figure 6) and were generally consistent with those seen in the decidua of early pregnancy, although HCC-1 was slightly reduced (in keeping with the lower staining intensity in glandular epithelium seen in these tissues; Figure 5). MCP-3 was highly elevated in the LNG-IUS users, consistent with the intense immunostaining seen after levonorgestrel exposure (Figure 5). IL-8 mRNA was only detected in menstrual phase tissue, with a low degree of expression in endometrium from LNG-IUS users. These expression patterns are consistent with and support the protein expression data from immunohistochemical studies.
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Discussion |
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P-only contraceptive methods induce differential effects on endometrial morphology and the different morphologies contain different populations of immune cells. Norplant-treated endometrium with an atrophic appearance contains very few leukocytes. However, there is a large leukocyte infiltrate evident in the shedding group, which previous studies show to be predominantly neutrophils and eosinophils (Vincent et al., 1999), and also in the p-modified tissues with an increase in macrophages and uNK cells, as we have shown here. In LNG-IUS users where morphology is highly decidualized (p-modified), macrophages and uNK cells also predominate (Critchley et al., 1998a), as seen normally in early pregnancy decidual samples, where uNK cells comprise 40% of all cells in the stromal compartment (Moffett-King, 2002). Consistent with these leukocyte patterns, we present evidence for different repertoires of chemokines in each of the three different groups of Norplant users, with LNG-IUS-exposed endometria closely resembling that of the p-modified Norplant endometrium.
In normal endometrium chemokines are produced predominantly by epithelial cells, with cyclical variations in expression levels. We have previously identified groups of chemokines that are up-regulated in specific stages of the cycle: menstrual-associated (IL-8), proliferative phase-associated (MIP-1 and HCC-4), decidual-associated (MDC and 6Ckine) and constantly present (MCP3, FKN and HCC-1) (Jones et al., 2004
). Expression of most chemokines examined in this study (HCC-1, MCP-3, eotaxin and 6Ckine) was similar in the shedding and atrophic groups and was consistent in localization and expression levels with normal non-decidualized endometrium. IL-8 was strikingly elevated in the shedding Norplant group, consistent with the high levels seen perimenstrually, supporting a role for IL-8 in the recruitment of the large neutrophil infiltrate peculiar to this Norplant group and absent from the p-modified tissue of some Norplant and all LNG-IUS users (Jones and Critchley, 2000
). The marked reduction of the proliferative-associated chemokines, HCC-4 and MIP-1
(Jones et al., 2004
), in the atrophic quiescent endometrium compared with proliferative endometrium reinforces a role for these chemokines in the regeneration and proliferation of the normal endometrium.
In the p-modified Norplant and LNG-IUS endometrium, epithelial immunostaining levels were consistent with those seen in the mid-late secretory phase of the cycle, with very strong staining for HCC-1, MDC, MCP-3 and eotaxin, but low expression of HCC-4 and MIP-1. The former group are all chemoattractants for the leukocyte subsets abundant in p-modified Norplant and LNG-IUS tissues (macrophages, uNKS) (Table I and II), and confirms an important role for the glands in positioning and activation of leukocytes in the decidualized endometrium. However, in marked contrast to normal decidualized endometrium, 6Ckine and IL-8 were selectively down-regulated in epithelial cells in p-modified endometria from women using both Norplant and LNG-IUS. Such down-regulation has been reported previously for fractalkine (Hannan et al., 2004
) and indicates either differential regulation of these three chemokines in artificially induced or extensive decidualization, or differential effects of levonorgestrel and natural progesterone. One possible explanation is that levonorgestrel is a highly androgenic progestin (Kloosterboer et al., 1988
) and may indirectly influence chemokine expression through interactions with stromal androgen receptor (Mertens et al., 2001
).
The most marked finding in these studies relates to stromal production of chemokines, and again we confirm that stromal cells are not a source of chemokines until they undergo decidualization, when they become the predominant production site in the endometrium (Jones et al., 2004). This is entirely consistent with the abundance of uNKs and macrophages in the decidua and in the highly decidualized LNG-IUS and p-modified Norplant endometrium, and with the strong expression of their chemoattractants (MDC, HCC-1, MCP-3, MIP-1
) (Bacon et al., 2002
; Rabin, 2003
). Decidual-derived chemokines have been implicated in the recruitment of uNKs to the implantation site (Jones et al., 2004
), and also for specific targeted migration of a cytotrophoblast to decidual vessels (Sato et al., 2003
; Drake et al., 2004
). The high chemokine production and uNK cell abundance in these groups of p-only contraceptive users is likely to be unrelated to BTB, but rather, a consequence of extensive decidualization.
This work, and much other research in the literature, is driven by the hypothesis that immune cells (particularly inflammatory neutrophils) are key effector cells initiating menstruation and BTB. Their absence in the decidualized endometrial groups of p-only users therefore seems to vilify this hypothesis. However, most studies examining bleeding patterns in Norplant users have not distinguished between the different morphological groups. One study from our laboratory reported that women with a p-modified endometria experience almost half the number of bleeding days (24 days) than the shedding or atrophic groups (37 and 40, respectively), and more than double the number of non-bleeding days (16 versus 5.8 and 6.4) in the 90-day period prior to endometrial biopsy (Vincent et al., 1999). This finding, however, failed to reach significance when the statistical analysis was corrected for body mass index (BMI), as the women with a p-modified endometrium had an overall higher BMI (although still in the normal weight range). This trend towards less BTB in women with a decidualized endometrium than in those with an atrophic or shedding endometrium, is consistent with the leukocyte profile of this tissue, and raises the suggestion that achieving a decidualized endometrium, complete with non-inflammatory chemokines and leukocytes, is desirable to reduce BTB. Women using LNG-IUS do experience BTB, although there is a notable improvement in bleeding patterns after the first 6 months of use. There are likely to be different mechanisms involved in endometrial fragility between Norplant and LNG-IUS users owing to the considerable difference in endometrial exposure to progestin, leading to distinct imbalances in the endometrial microenvironment. Indeed, local delivery of progestin results in suppression of progesterone receptors in the first 6 months of use (Critchley et al., 1998a
), whilst Norplant endometrium remains strongly responsive to steroids (Critchley et al., 1993
), indicating major differences in the functional downstream effects of progestin in these groups of women.
Certain chemokines are known to be progesterone regulated (Kelly et al., 1994; 1997
), and generally inflammatory chemokines are inhibited by glucocorticoids (Miyamasu et al., 1998
). From the differential expression of chemokines between the three groups of Norplant endometrium it seems unlikely that levonorgestrel is exerting direct effects on chemokine expression. This is supported by the fact that in women using LNG-IUS there is an almost total down-regulation of nuclear progesterone receptor (PR) in epithelial cells (Critchley et al., 1998b
), although this could be explained by the presence of membrane bound PR (Zhu et al., 2003
). Alternatively, the progestin effect on epithelial cells could be mediated through paracrine interactions with underlying stromal cells, which also possess glucocorticoid and androgen receptors (Bamberger et al., 2001
; Mertens et al., 2001
), with which the high concentrations of levonorgestrel may interact. Epithelial and decidual chemokine production during the normal secretory phase and in women using p-only contraceptives is therefore more likely to be stimulated indirectly by progesterone-regulated locally produced factors.
In this study, we have not identified a single chemokine that is responsible for BTB, leading to the conclusion that BTB is most likely caused by very focal perturbations in the endometrial environment leading to loss of tissue integrity in discrete focal areas. This is supported by the hysteroscopic visualization of dilated bleeding vessels (Hickey et al., 2000), and histologically by the focal clusters of leukocyte infiltrate in breaking-down tissue, surrounded by unaffected atrophic endometrium (Clark et al., 1996
; Song et al., 1996
; Vincent et al., 1999
). Selective leukocyte accumulation and activation in breakdown foci must be caused by focal up-regulation of specific chemokines. The chemokines examined in this study were selected on the basis of their abundant expression in normal endometrium, from global gene array studies (Jones et al., 2004
), which would not identify chemokines that are lowly and focally expressed. Nevertheless, closer examination of normal endometrium using immunohistochemistry identified local up-regulation of neutrophil and macrophage chemokines (IL-8, FKN, MDC, 6Ckine) in vascular endothelium, only in the immediate premenstrual phase (Jones et al., 1997
; 2004
; Hannan et al., 2004
). Identification of the critical effector cells and mediators involved in BTB is likely to be possible only by detailed analysis of actual bleeding sites, as described by Lockwood et al. (2000)
, correlating leukocyte infiltrate, localized chemokine expression, blood vessel abnormalities and progesterone receptor content in the areas known to be breaking down.
To summarize, the current study verifies that leukocytes are abundant in the endometrium of women using p-only contraceptives, but that the leukocyte profile of the endometrium differs significantly between subgroups of contraceptive users. In keeping with the elevated numbers of leukocytes present, we show that chemokine production is overall elevated and comparable to levels seen during maximal leukocyte recruitment in normal endometrium. Furthermore, we see significant differences in chemokine expression patterns in the different groups of p-only users, consistent with endometrial morphology and leukocyte content. Importantly, these findings reinforce that these contraceptives not only differentially affect endometrial morphology, but also endometrial physiology. Overall, these data demonstrate chemokine expression is dysregulated in p-exposed endometria, reinforcing a critical role for chemokines in abnormal leukocyte recruitment in women using p-only contraceptives.
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Acknowledgements |
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Submitted on January 25, 2005; resubmitted on March 28, 2005; accepted on May 11, 2005.