Changes in gonadal steroid receptors in the cardinal ligaments of prolapsed uteri: immunohistomorphometric data

Ayman A.A. Ewies1, John Thompson1 and Farook Al-Azzawi1,2

1 Leicester and Warwick Medical School, Leicester, UK

2 To whom correspondence should be addressed at: Gynaecology Research Unit, Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, Leicester and Warwick Medical School, Leicester LE2 7LX, UK. e-mail: fa2{at}le.ac.uk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND:The precise mechanism of uterine prolapse is poorly understood. This immunohistochemical study was performed on paraffin-embedded sections of the cardinal ligaments in an attempt to evaluate the differential expression of gonadal steroid receptors in human cardinal ligaments of prolapsed uteri compared with non-prolapsed controls. METHODS: Specimens from women with pelvic organ prolapse (POP) stage III (n = 33), together with the appropriate controls (n = 25), were stained for estrogen receptor {alpha} (ER{alpha}), ER{beta}, progesterone receptor (PR), androgen receptor (AR) and Ki-67. The control materials were samples of the cardinal ligaments obtained from pre- and post-menopausal women with no prolapse, who were not using hormonal therapy. RESULTS The prolapsed ligaments expressed 1.5–2.5 times more ER{alpha}-positive cells (statistically significant in post-menopausal women not taking HRT, P < 0.001), a 3–4 times greater percentage of AR-positive cells (P = 0.004 and P = 0.008 in pre-menopausal and post-menopausal women not taking HRT, respectively) and twice the percentage of PR-positive cells (statistically significant in the pre-menopausal group, P = 0.03) compared with the no prolapse group. Expression of ER{beta} was twice as high in the ligaments of pre-menopausal women with no prolapse compared with those with prolapse (P = 0.02), and no significant difference was found in the post-menopausal groups. The use of HRT was significantly associated with low AR and high PR expression. Ki-67 expression was not detected in these specimens. CONCLUSIONS: The clearly discernible levels of expression of ER{alpha}, ER{beta}, AR and PR in the prolapsed cardinal ligaments may suggest a relationship to the process of tissue stretch ‘trauma’, rather than an effect of the menopausal status, HRT use or cell proliferation. The use of HRT in post-menopausal women appears to offset some of the changes observed with the prolapse.

Key words: androgen receptor/cardinal ligament/estrogen receptor/progesterone receptor/prolapse


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pelvic organ prolapse (POP) is a distressing condition that compromises the quality of life and is prevalent in both developed and developing countries. In some women, prolapse begins during reproductive life and continues to be the source of symptoms for decades without much hope of relief unless surgical repair is successful (MacLennan, 2000Go). A degree of pelvic floor laxity is present in most parous women, but only 10–20% are symptomatic (Beck et al., 1991Go). The incidence of prolapse increases with age, and its prevalence among those awaiting major gynaecological surgery is 20% and rises to nearly 60% amongst elderly women (Bidmead and Cardozo, 1998Go). The true incidence of POP is difficult to estimate because many women accept the associated symptoms as inevitable consequences of childbirth and ageing (Gjorup et al., 1987Go).

The pathophysiology of POP is poorly understood, although the effect of a difficult delivery and chronically elevated intra-abdominal pressure has been widely blamed. Nonetheless, the occurrence of POP in virgins and nullipara is well recorded. The prevalence of uterine prolapse increases in the post-menopausal period, suggesting that the hypoestrogenic state may contribute to its aetiology; however, the effect of the post-menopausal hormonal milieu on pelvic supportive structures has not been assessed adequately (Mokrzycki et al., 1997Go). The precise role of estrogen on the function of the pelvic floor, and the pathogenesis, prevention or treatment of prolapse of the genital tract is not known.

The presence of estrogen receptors (ERs) and progesterone receptors (PRs) has been documented in the utero-sacral ligaments of pre- and post-menopausal women (Mokrzycki et al., 1997Go; Chen et al., 1999Go). ERs are also expressed in the levator ani muscle (Smith, 1993Go). Because the principal support of the uterus arises from the dynamic actions of the levator ani muscle and the uterosacral–cardinal ligament complex (DeLancey, 1993Go), the presence of ERs in these structures implies that they are targets for estrogen action. However, estrogen responses may be affected by the relative expression of specific receptor subtypes present in target tissues. Furthermore, the clinical development of two selective ER modulators, idoxifene (Hendrix and McNeeley, 2001Go) and levormeloxifene (Novo Nordisk, 1998Go), used in post-menopausal women to treat and prevent osteoporosis, has been discontinued because of the observed increased incidence of POP and urinary incontinence.

The present study was performed to evaluate the differential expression of gonadal steroid receptors in human cardinal ligaments of prolapsed uteri compared with non-prolapsed controls. Immunohistochemistry for ER{alpha}, ER{beta}, PR and androgen receptor (AR) was performed on paraffin-embedded specimens of the cardinal ligaments. The density of Ki-67-positive cells as a surrogate marker of proliferative activity was analysed in order to evaluate any potential difference between the groups.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The questions raised in our study concerned the contribution of prolapse, age and the use of gonadal steroids on the expression of the relevant cognate receptors in the cardinal ligaments. We have compared similar groups together: (i) post-menopausal women with prolapse on HRT versus no HRT; (ii) post-menopausal women with prolapse versus no prolapse; and (iii) pre-menopausal women with prolapse versus no prolapse. The samples used for this study are those derived from the cardinal ligaments, since the failure of these ligaments represents the major phenotypic change in the process of prolapse.

Thirty-three samples were obtained from women with POP; eight from pre-menopausal and 25 from post-menopausal women, 10 on HRT and 15 not taking HRT. The duration of the prolapse was not known precisely due to the insidious nature of the problem. Twenty-five control samples were taken from hysterectomy specimens with no prolapse; 15 from pre-menopausal women and 10 from post-menopausal women not on HRT. All specimens were taken from Caucasian women. All women with prolapse have had vaginal hysterectomy (except two cases in the pre-menopausal group who had abdominal hysterectomy and sacral colpopexy), while those with no prolapse have had abdominal hysterectomy. All the cases of prolapse included in this study were those with advanced POP stage III, where the uterine isthmus reached the introitus. All of the cases had a central defect, with a rectocoele and a small to moderate cystocoele. All control cases were examined pre-operatively, and no prolapse was found. None of these women used a supportive vaginal pessary or ring prior to surgery. The menopause was defined as 1 year of amenorrhoea in women over the age of 45 years. All post-menopausal women in this study met this criterion. Women in the HRT group were taking sequential combined HRT (estradiol valerate 2 mg daily and cyclic norethisterone 1 mg per day for 12 days of each 28-day treatment cycle) for at least 6 months pre-operatively.

Specimens
Slices 5 mm thick of the medial ends of the cardinal ligament were obtained from the part of the cervix above the portio vaginalis (Ewies et al., 2003Go). Tisssue samples were collected from uterine specimens of abdominal and vaginal hysterectomy. Samples were immediately fixed in 10% formol saline for a fixed period of 24 h, embedded in paraffin wax, and 5 µm sections were mounted onto silane-coated slides and allowed to dry at 37°C for 48 h. Sections were stained with haematoxylin and eosin (H&E) for histological assessment of the ligamentous tissue. The investigation protocol was approved by the local ethics committee and every patient signed a consent form pre-operatively, allowing the use of tissues removed at surgery for research purposes.

Antibodies
The primary antibodies used in this study were monoclonal mouse anti-bovine ER{alpha} antibody (clone no. 6F11, 1:50, Novacastra Laboratories Ltd, Newcastle upon Tyne, UK), polyclonal rabbit anti-rat ER{beta} antibody (catalogue no. 06-629, 1:50, Upstate Biotechnology, Lake Placid, NY), mouse monoclonal AR antibody (clone no. AR-27, 1:25, Novacastra Laboratories Ltd), mouse monoclonal PR antibody (clone no. 1A6, 1:40, Novacastra Laboratories Ltd) and mouse monoclonal Ki-67 antibody (clone no. B56, 1:150, Pharmingen, Palo Alto, CA). Biotinylated secondary antibodies; rabbit anti-mouse (ER{alpha}, AR and PR), swine anti-rabbit (ER{beta}) and goat anti-mouse (Ki-67) immunoglobulins (Dako A/S, Glostrup, Denmark) were used at a 1:400 dilution. Mouse and rabbit IgG (Vector Laboratories Inc., Burlingame, CA) at dilutions of 1:2000 and 1:1000, respectively, were used as controls for the primary antibodies.

Immunohistochemistry
Immunostaining was carried out using standard protocols previously optimized in our laboratory (Habiba et al., 2000Go; Wahab et al., 2000Go; Taylor and Al-Azzawi, 2000Go) with some modifications. Sections were de-waxed in xylene (Genta Medical, York, UK), and rehydrated in grades of industrial methylated spirit (Genta Medical) and distilled water followed by microwave antigen retrieval in 10 mmol citrate buffer, pH 6.0, at 700 W for 15 min. For AR antigen retrieval, 1 mmol EDTA solution pH 8.0 was used. Endogenous peroxidase activity was then quenched using hydrogen peroxide (1.5% in methanol for AR detection, but 6% in distilled water for the detection of other antigens) for 10 min. Subsequently, two washes (5 min each) in de-ionized H2O and phosphate-buffered saline (PBS)–Tween-20 (0.05% v/v) were performed [Tris-buffered saline (TBS) rather than PBS was used for washing in all steps in AR runs]. Sections were blocked for 15 min with PBS containing 3% bovine serum albumin (BSA), and then further blocked for 30 min with normal rabbit serum (ER{alpha}, AR and PR), normal swine serum (ER{beta}) or normal goat serum (Ki-67) to minimize non-specific reactivity. Further blocking (except for PR) was performed with avidin–biotin blocking solution (Vector Laboratories Inc.) according to the manufacturer’s instructions. The slides were incubated overnight at 4°C with the primary antibody in a humidified chamber. Sections were incubated with the secondary antibody then with Vectastain ABC peroxidase (Elite; Vector Laboratories Ltd, Peterborough, UK) for 30 min each. Specimens were washed in PBS–Tween-20 for 20 min between steps. Bound antibodies were visualized with 0.05% diaminobenzidine (DAB) in 0.05 mol/l Tris–HCl pH 7.4, and 0.01% hydrogen peroxide, according to the manufacturer’s instructions (Vector Laboratories Inc.). Sections were then submerged in CuSO4:NaCl (16 mmol/l/123 mmol/l) solution for 5 min to enhance DAB stain. Counter-staining with haematoxylin, Gill’s formula (Vector Laboratories Inc.) for 1 min was performed to improve identification of cellular elements, followed by rewashing in tap water. Sections were dehydrated through graded alcohol, cleared with xylene and permanently mounted using XAM mounting medium (BDH, Poole, Dorset, UK). Specificity of immunostaining was confirmed using mouse non-specific IgG alongside all experiments, except for ER{beta} where rabbit IgG was used. Negative control sections were produced by omission of the primary antibody and did not show any staining, while the positive control slides were stained with the primary antibody, negative with non-specific IgG, and there was no background reaction.

Image analysis
Images of tissue sections were captured using an Axioplan microscope (Carl Zeiss, Welwyn, Herts, UK), a colour video camera (Sony CCD/RGB) and the KS 300 image analysis program (Imaging Associates Ltd, Thames, Oxfordshire, UK). From each slide, 15 randomly selected fields (x100 with oil immersion) (Hamilton, 1995Go) were captured. Within each field, the number of positive and negative nuclei was counted using the image analysis software and the data were transferred to the Microsoft Excel program for statistical analysis (Wahab et al., 1999Go). The examiner was blinded for the different groups. The number of fields per specimen was chosen to satisfy {alpha} = 0.05 and {beta} = 0.80. A preliminary evaluation of image analysis data indicated the need for a minimum of 10 specimens per group and 10–15 randomly selected fields per slide.

Power and statistical methods
Power calculations were based on early data collected on ER{alpha} and performed by simulation. Assuming 15 fields per subject, 10 subjects per group and actual mean percentage positive cells of 40% in one group and 50% in the other; if the standard deviation between subjects is 7.5%, then providing that the SD between fields on the same subject is no greater than 5%, the study will have at least 80% power when testing at the 5% level. Calculations were repeated assuming different numbers of fields per subject and it was found that there was little difference in power providing that at least 10 fields per subject were taken. The preliminary work on which the design was based had used 15 fields, and we decided to stay with that number.

The statistical analysis was performed using Stata (StataCorp 2001 Stata Statistical Software Release 7.0, Stata Corporation College Station, TX). Each receptor type was analysed separately. The data on the proportion of positively stained nuclei were analysed using logistic regression with adjustment of the standard errors to allow for clustering of measurements made on different fields within the same slide. Results are presented as percentages of positively stained nuclei and their 95% confidence intervals (CIs) obtained from the logistic regression. Patient groups were compared using the corresponding Wald tests. Each comparison between patient groups was made with and without adjustment for age. In general, the age adjustment made no material difference, and unadjusted results are given unless otherwise stated.


    Results
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 Materials and methods
 Results
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The demographic data of the five groups of women described in this study are summarized in Table I.


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Table I. The demographic attributes of patients included in the study
 
ER{alpha}
The average percentage of ER{alpha}-positive cells for each patient group together with their corresponding 95% CIs are shown in Figure 1a. In women not on HRT, the ER{alpha} expression in prolapsed ligaments was 1.5–2.5 times greater than in non-prolapsed ligaments for pre-menopausal (P = 0.09) and post-menopausal (P < 0.001) groups. The test of interaction of prolapse versus menopausal status (P = 0.04) suggests that the difference in ER{alpha} expression with prolapse may be larger in the post-menopausal women (74 versus 32%) than in the pre-menopausal women (60 versus 43%). In the post-menopausal women with prolapse, there was no significant difference between those on HRT and those not on HRT (P = 0.38). There was no material change in these comparisons when they were adjusted for age (Figure 2a).






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Figure 1. The percentage expression (mean and 95% CI) of gonadal steroid receptors in tissue sections of the cardinal ligaments. (a) ER{alpha}: a significantly higher expression is seen in the prolapse group when compared with the no prolapse group in post-menopausal women not taking HRT. (b) ER{beta}: a significantly higher expression is seen in the no prolapse group when compared with the prolapse group in the pre-menopausal women. (c) AR: a significantly higher expression is seen in the prolapse group when compared with the no prolapse group in both pre- and post-menopausal women not taking HRT. The expression was significantly lower in the post-menopausal women taking HRT when compared with non-HRT takers. (d) PR: a significantly higher expression is seen in the prolapse group when compared with the no prolapse group in the pre-menopausal women. The expression was significantly higher in the post-menopausal women taking HRT when compared with non-HRT users. PrM = pre-menopausal; PM = post-menopausal; P = prolapse; NP = no prolapse *P < 0.05; **P < 0.001.

 


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Figure 2. Immunohistochemical staining of the cardinal ligament sections for gonadal steroid receptors (x1000). (a–c) ER{alpha}, (d–f) ER{beta}, (g–i) AR and (j–l) PR. The group effect is demonstrated by marked expression of ER{alpha} protein (b) and of AR (h) in cases of prolapse with a suppressed expression in the group treated with sequential combined HRT (a and g, respectively). ER{beta} protein, on the other hand, is strongly expressed (nuclear and cytoplasmic staining) in the pre-menopausal woman with no prolapse (e). This is markedly contrasted with pre-menopausal women with prolapse (f) and minimal expression in post-menopausal women with prolapse who were using HRT until the time of surgery (d). PrM = pre-menopausal; PM = post-menopausal; P = prolapse; NP = no prolapse.

 
ER{beta}
The average percentage of ER{beta}-positive cells for each patient group together with their corresponding 95% CIs are shown in Figure 1b. The expression of ER{beta} in the pre-menopausal women with no prolapse was significantly higher (P = 0.02) than for those with prolapse. However, the expression was nearly similar in the different groups of post-menopausal women. The test of interaction of prolapse versus menopausal status (P = 0.03) suggests that the difference in ER{beta} expression with prolapse exists only in the pre-menopausal women (32 versus 67%), but not in post-menopausal women (53 versus 45%). In the post-menopausal women with a prolapse, there was no significant difference between those on HRT and those not on HRT (P = 0.52). There was no material change in these comparisons when they were adjusted for age (Figure 2b).

AR
The average percentage of AR-positive cells for each patient group together with their corresponding 95% CIs are shown in Figure 1c. In women not on HRT, the AR expression in prolapsed ligaments was 3–4 times greater than in non-prolapsed ligaments for pre-menopausal (P = 0.004) and post-menopausal (P = 0.008) groups. The test of interaction of prolapse versus menopausal status (P = 0.62) suggests that the difference in AR is related to the prolapse regardless of the menopausal status. Women with prolapse on HRT showed significantly lower expression (21%) of AR in comparison with those not taking HRT (44%). There was no clear evidence for an effect of age (Figure 2c).

PR
The average percentage of PR-positive cells for each patient group together with their corresponding 95% CIs are shown in Figure 1d. In women not on HRT, the PR expression in prolapsed ligaments was double that in the non-prolapsed ligaments for both pre-menopausal (P = 0.03) and post-menopausal (P = 38) groups. The test of interaction of prolapse versus menopausal status (P = 0.64) suggests that the difference in PR is related to the prolapse regardless of the menopausal status. PR expression in the ligaments of post-menopausal women with prolapse and taking HRT was double the average expression in those not on HRT (P = 0.02). There was no material change in these comparisons when they were adjusted for age (Figure 2d).

Ki-67
No Ki-67 expression was detected in any of the ligaments tested.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our knowledge, this is the first study that evaluates the difference in the pattern of expression of gonadal steroid receptors in the cardinal ligament of women with POP compared with those with no prolapse. The discovery reported in this article refers to the fact that the event of prolapse per se is the determinant of finding significantly altered gonadal steroid receptor protein expression. This means that this clinical state of tissue failure is associated with an alteration of nuclear activation factors, as a direct result of injury or as a part of a repair process. This type of tissue injury is associated with raised levels of steroid receptor proteins, possibly to help heighten tissue sensitivity to the respective ligands.

In this study, we have elected to investigate advanced stages of POP only, for two reasons. First, given the nature of immunohistochemistry of gonadal steroid receptors, a POP stage-dependent discrimination will not be possible with current technology. Secondly, the clinical classification is not precise, with insidious progression of the prolapse which makes it hardly noticeable by the woman in its early stages, and therefore this tissue material is not generally available. When these factors are combined with the subjects’ heterogeneity, the differential expression of the protein may not be apparent.

Our data showed that, apart from ER{beta} expression, the expression of the three steroid receptors investigated was consistently higher in the prolapsed ligaments compared with non-prolapsed ligaments, suggesting a prolapse effect rather than a menopausal status or HRT effect. We report that prolapsed ligaments express 1.5–2.5 times more ER{alpha}-positive cells, 3–4 times more AR-positive cells and twice as many PR-positive cells compared with the controls irrespective of the menopausal status. Nonetheless, the expression of ER{beta}-positive cells is twice as high in pre-menopausal women with no prolapse compared with those with prolapse, and no significant difference is found between the post-menopausal groups. The use of HRT in post-menopausal women with prolapse appears to ameliorate some of the changes in gonadal steroid receptors observed with the prolapse. It is associated with a statistically significant reduction in the expression of AR and increase in the expression of PR.

It is to be emphasized that the above descriptions are average effects of the groups prolapse, age and HRT usage on tissue expression of gonadal steroid receptors and not the direct relationship of expression among different receptors in a particular tissue sample.

Since one of the possibilities of estrogen’s mode of action could be ascribed to its proliferating effect on fibroblasts, we stained all the sections for Ki-67 antigen to determine the prevalence of such cells. We found that Ki-67 was not expressed in the human cardinal ligaments from all groups, suggesting that the vast majority, if not all, constituting cells are either at G0 or early G1 of the cell cycle where Ki-67 antigen is not expressed (Scotti et al., 2000Go). This also suggests that the detected changes in the steroid receptor expression are not the result of an increased proliferative activity of the fibroblasts, but are a prolapse-related phenomenon. The evaluation of Ki-467 staining has been widely used to detect the proliferative activity of many tissues including uterine epithelium (Dahmoun et al., 1999Go) and endometriotic lesions (Matsuzaki et al., 2000Go; Scotti et al., 2000Go), but has not been reported previously in the assessment of proliferative activity of pelvic ligaments. Furthermore, the smooth muscle content of the vaginal muscularis in the upper anterior wall was significantly decreased in women with prolapse of the anterior vaginal wall compared with normal control subjects (Boreham et al., 2002Go). However, increased Ki-67-positive cells and observation of mitotic figures were prevalent in the fibroblasts of the degenerating cranial cruciate ligaments of beagle dogs, which attests to the proliferating potential of ligamentous fibroblasts (Narama et al., 1996Go).

The differential expression of ER subtypes in human cells has been reported. Chen et al. (1999Go), using RT–PCR, reported differential expression of ER subtypes in the uterosacral ligament. It was found that mRNA transcripts for ER{alpha} were present in all the 16 samples examined, but ER{beta} mRNA was found in most samples from pre-menopausal women (10 out of 12) and in some (two out of four) samples from post-menopausal women. Similar results were reported in prolapsed human vaginal tissues (Gebhart et al., 2001Go) using the same techniques. Such differences in ER or AR expression were not observed in an earlier immunohistochemical study of the vagina and vulva of pre- or post-menopausal women with no prolapse (Hodgins et al., 1998Go). The distribution of the ER{beta} isoform appears to be closely related to the expression of ER{alpha} in most tissues. Taylor and Al-Azzawi (2000Go) found that some human ER{alpha}-positive cells lack ER{beta}, and vice versa, suggesting that estrogen action in some human tissues may be mediated via the activation of one subtype rather than the other or both together. This raises the possibility that there are distinct ER{alpha}- and ER{beta}-dependent transcriptional pathways. Estrogen induces pleiotropic responses in target tissues, acting through classic steroid receptor-mediated pathways and through transactivation of fos/jun (AP1) mechanisms. While both ER{alpha} and ER{beta} bind estrogen, the specific receptor subtypes may mediate different functions. The tissue- and ligand-specific functions of ER{alpha} appear to differ from those mediated by ER{beta}. The tissue responses could be modulated by the extent to which the ERs form homodimers ({alpha}{alpha} and {beta}{beta}) and heterodimers ({alpha}{beta}), and by the affinity of dimers for a specific ligand (Kuiper et al., 1998Go).

The clearly discernible raised levels of expression of ER{alpha}, AR and PR in the cardinal ligaments of the uterine prolapse group may indicate a relationship to the process of tissue stretch ‘trauma’. Nonetheless, it is not clear whether the changes in steroid receptor expression are the cause or the effect of the prolapse. Interestingly, the expression of ER{alpha} and PR was elevated in the varicose segments compared with the normal parts of the veins obtained from men and women. However, the increase was much more pronounced in women (Mashiah et al., 1999Go). It may well be that changes in the extracellular matrix composition trigger the overexpression of steroid receptors in the prolapsed cardinal ligaments. Supportive evidence from in vitro studies cited the inhibition of ER-mediated transcriptional activity, including proliferation and PR expression, when MCF-7 and T47D breast cancer cell lines were grown on laminin, but estrogen treatment induced DNA synthesis and restored other ER-mediated transcriptional activity including PR expression when these cancer cell lines were grown on collagen 1 or fibronectin. Furthermore, the laminin effect seemed to be more specific against ER-mediated actions, since the mitogenic responses of these cell lines to insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) were unaffected (Woodward et al., 2000Go). We also found the expression of both collagen III and tenascin to be significantly higher and the expression of elastin was significantly lower in prolapsed when compared with non-prolapsed ligaments regardless of the menopausal status (Ewies et al., 2003Go). The use of HRT significantly reduced collagen III expression to levels similar to those of the non-prolapsed ligaments. The pattern of change may fit a picture of the healing phase of traumatized tissue as evidenced by the raised tenascin expression. The trauma itself may have been initiated by events such as childbirth, and post-menopausal estrogen deficiency results in decompensation. The absence of a reliable animal model limits our ability to understand the cellular and extracellular matrix changes alongside the progression of clinical stages of pelvic organ prolapse, although the rhesus macaque recently has been reported as a potential model (Otto et al., 2002Go). The reported number in that study was 11 divided among three different treatment groups. If this model is validated, a mechanism for this pattern of tissue failure might be elucidated.

This report establishes the pattern of change in gonadal steroid receptors in prolapsed tissue. It also documents that post-menopausal hormonal supplementation with gonadal steroids partly reverses some of the changes associated with prolapsed cardinal ligaments, since such regimens lack the myriad steroids manufactured by the pre-menopausal ovaries.

The changes observed with ER{beta} are completely different from ER{alpha} expression and suggest a different role for these two proteins in tissue homeostasis. Tissue responses to trauma, or chronic stretch, may be responsible for our observations, and the understanding of the underlying mechanisms may form the basis to design agents that strengthen tissue architecture and enhance tissue healing.


    Acknowledgements
 
We would like to thank Miss Michelle Duffus and Miss Eryl Roberts, for their technical support.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 19, 2003; accepted on March 31, 2004.