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Cell Biology International (2008) 32, 75–79 (Printed in Great Britain)
Estrogen and progesterone receptors in carpal tunnel syndrome
Amelia Toescaa*, Alessia Pagnottab, Antonella Zumboa and Raffaele Sadunb
aInstitute of Human Anatomy and Cell Biology, Catholic University, Largo Francesco Vito 1, 00168 Rome, Italy
bHand Surgery Service, Jewish Hospital, Rome, Italy


Abstract

Carpal tunnel syndrome (CTS) is a compression median nerve neuropathy common in women at menopausal age. The aim of this work was to study immunohistochemically the expression of estrogen (ER) and progesterone (PR) receptors in CTS and control specimens. Biopsies of transverse carpal ligament (TCL) and flexor tendon synovitis were collected from 23 women and from 7 men undergoing surgery for median nerve decompression at the wrist for CTS.

In TCL and synovial tissue, cells expressed ER and PR with statistically significant differences related to the age and sex of patients. Immunoreactivity was observed in fibroblasts of TCL, and in lining cells and fibroblasts of synovial tissue. In women, the number of ER-positive cells in the TCL and synovial tissue increased with the age, peaking at 55–70 years, and then decreasing. PR-immunoreactivity was observed only in fibroblasts of TCL and its expression decreased with age, while no immunolabeling was found in the synovial tissue. In TCL samples, the number of ER- and PR-positive cells in non-CTS patients was significantly lower than in CTS patients.

These results demonstrate that ER and PR are present in TCL and flexor tendon synovitis, suggesting a role for sex steroid hormones in the pathogenesis of CTS disease.


Keywords: Estrogen receptor, Progesterone receptor, Carpal tunnel syndrome, Immunohistochemistry.

*Corresponding author. Tel.: +39 0630154915; fax: +39 0630154813.


1 Introduction

Estrogens and progesterone are steroid hormones traditionally connected with female reproductive system where they affect growth, differentiation and development of reproductive tissues. Important roles of estrogens have also been reported in the male reproductive system (Hess et al., 1997) and in a number of non-reproductive systems. In bone tissue, estrogens maintain bone density and prevent osteoporosis (Syed and Khosla, 2005). They are also used as hormone therapy in treating and preventing post-menopausal symptoms and as contraceptive agents (Jerome, 2004). The biological activities of sex steroid hormones are mediated by the interaction with specific receptor proteins, namely estrogen receptor (ER) and progesterone receptor (PR) that are classified as nuclear receptors. Only through interaction with hormones and hormone-like molecules, ER and PR exert their diverse array of biological effects. In the absence of estrogen/progesterone, ER and PR are sequestrated within nuclei of target cells and maintained in an inactive state. Estrogen exerts its effects through two main isoforms of ER, ER-α, the most predominant type, and ER-β (Pavao and Traish, 2001) that share common structural and functional domains (Osborne et al., 2000). The biological response to progesterone is mediated by two distinct isoforms of progesterone receptor, PR-A and PR-B (Horwitz and Alexander, 1983) that are not functionally identical (Kastner et al., 1990; Meyer et al., 1990; Vegeto et al., 1993). The carpal tunnel syndrome (CTS) is a median nerve compression neuropathy at the wrist more common in women than in men. The carpal tunnel is an inelastic fibro-osseous canal, with its floor and lateral walls formed by carpal bones and its roof formed by the transverse carpal ligament; the median nerve pass through the tunnel under the ligament and palmarly to flexor tendons. Although most cases of CTS are idiopathic, many systemic diseases and conditions may predispose patients to develop this syndrome: diabetes (Singh et al., 2005), hypothyroidism (Palumbo et al., 2000), alcoholism (Szabo, 1999), rheumatoid arthritis (Geoghegan et al., 2004), and renal dialysis (Bicknell et al., 1991). Significant sex differences in prevalence and clinical manifestations of idiopathic CTS are female sex (Szabo, 1999), pregnancy (Szabo, 1999) and menopausal transition (Wluka et al., 2000).

Our aim was to study the expression of sex steroids receptors, ER and PR, in transverse carpal ligament and synovial tissue of idiopathic CTS and control samples to verify their possible biological role in the pathogenesis of this disease.

2 Materials and methods

Bioptic samples of transverse carpal ligament (TCL) and flexor tendon synovitis were collected from 23 women (main age 63.5, range 30–85 years) and from 7 men (mean age 66.8, range 35–80 years) undergoing surgery for median nerve decompression at the wrist for idiopathic CTS. Some of these patients suffered by concomitant diseases like diabetes (five cases), rheumatoid arthritis (two cases), dysthyroidism (eight cases) and others; three women were treated with hormonal replacement therapy. Control specimens of TCL were collected from four patients, two women (main age 55) and two men (main age 61), operated for traumatic hand pathologies and no history of CTS; no flexor tendon synovitis was detected and collected from non-CTS patients. Informed consent to specimen collection was obtained by all participating patients. After removing, tissue samples were immediately fixed in 4% paraformaldehyde at room temperature (RT) for 24h. Then they were rinsed in phosphate buffered saline (PBS, pH 7.4), dehydrated in an ascending series of alcohol and embedded in paraffin via xylene. Five-micrometer serial sections were then cut and processed for hematoxylin and eosin and immunohistochemical stainings.

For immunohistochemical experiments, CTS patients were classified into four groups: (1) women under 50 years (n=9, mean age 42.1), (2) women 50–70 age old (n=11, mean age 61.6), (3) women over 70 years (n=3, mean age 82), (4) men (n=7, mean age 66.8).

Endogenous peroxidase activity was quenched with 0.5% H2O2 in absolute methanol for 15min at RT. The preparations were permeabilized with 0.3% Triton X-100 in PBS for 15min at RT and incubated with 3% normal goat serum in PBS for 30min at RT to block non-specific bindings. Sections were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-estrogen receptor-α (ER-α) (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit polyclonal anti-progesterone receptor (PR) (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Control sections were treated with normal rabbit immunoglobulins at the same concentration used for primary antibodies. Reactions were revealed by standard avidin–biotin peroxidase complex procedure (Vectastain Elite ABC, Vector, CA, USA) using 3,3′-diaminobenzidine tetrahydrochloride as chromogen. Sections were examined under a Zeiss Axiophot (Germany) light microscope.

The immunoreactivity of sex steroid receptors was evaluated separately in three components, i.e. fibroblasts of TCL, lining cells and fibroblasts of synovial tissue. For quantitative analysis, positive cells that fell into 15 randomly selected 50μm squares for each slides (lens: 20×) were counted using a computerized system. The percentage of immunoreactivity of positive vs. total number of counted cells in each field, i.e. the labelling index, was determined. For this purpose, images from immunostained sections were captured using a Sony XC77CCD camera (Sony Corp, Cypress, CA) coupled to a Macintosh IIvX computer (Apple Computer, Cupertino, CA) by means of a Scion LG3 frame grabber (Scion Corp, Frederick, MD). The number of marked cells was then evaluated on the captured images using the public domain software NIH Image 1.59 (developed at the US National Institutes of Health, Bethesda, MD, available from the Internet).

Statistical analysis was performed using one-way analysis of variance (ANOVA) and P<0.05 was considered significant.

3 Results

Hematoxylin and eosin stained TCL sections of CTS patients showed a dense connective tissue with aligned fibroblasts intermingled between collagen fibers and few areas of fibrosis (Fig. 1A). Histology of synovial tissue exhibited a superficial layer of synoviocytes lining a fibrous matrix constituted of spared fibroblasts and collagen fibers and rich of vessels (Fig. 1B).


Fig. 1

Transverse carpal ligament (A) and synovial tissue (B) of CTS women 50–70 age old (Hematoxylin – eosin stain) (80×).


ER-α and PR immunoreactivities were always localized intranuclearly in both CTS and non-CTS samples. In particular, in the TCL of CTS patients the ER-α-immunoreaction was detected in fibroblasts (Fig. 2A) and in vascular walls. In synovial tissue, expression was observed in lining cells and fibroblasts of the fibrous matrix (Fig. 2B). ER-α labelling index was higher in the TCL than in the synovial tissue (Table 1). In CTS women, the number of ER-positive cells in the TCL increased with the age (<50 years, 27.5±12.1), peaked in women ranging from 50 to 70 years (63.2±22.6), and then decreased (>70 years, 18.6±11.2). These differences were statistically significant (P<0.005). Similarly, in the synovial fibroblasts and lining cells, the ER-α labelled index was higher in the 50–70 years old women group as compared with male group and other female groups. With the exception of 50–70 years old women group (P<0.005), there were no statistically significant differences in the ER-α labelling index in the TCL between men and women.


Fig. 2

ER-α-immunoreactivity in transverse carpal ligament (A) and synovial tissue (B) of a CTS women 50–70 age old. The immunoreactivity is localized in the nuclei (*) of fibroblasts in the TCL (A) and in the nuclei (*) of lining cells and fibroblasts of synovial tissue (B) (80×).


Table 1.

Quantitative analysis of ER- and PR-immunoreactivity (labelling index) evaluated separately in three components, i.e. fibroblasts of transverse carpal ligament (TCL), lining cells and fibroblasts of synovial tissue

<50 years (n = 9)Women 50–70 years (n = 11)>70 years (n = 3)Men (n = 7)Controls (n = 4)
ER
TCL27.5 ± 12.163.2 ± 22.618.6 ± 13.218.7 ± 11.20.8 ± 0.2
Fibroblasts12.1 ± 1.218.5 ± 5.36.8 ± 7.110.5 ± 3.2
Lining cells2.8 ± 25.9 ± 41.3 ± 1.14.3 ± 3.1

PR
TCL29.3 ± 15.125.9 ± 17.32.6 ± 2.419.2 ± 9.30.7 ± 0.4
Fibroblasts0000
Lining cells0000



PR-immunoreactivity was mainly detected in fibroblasts and in the wall of vessels in the transverse carpal ligament (Fig. 3A), while in the synovial tissue no immunoreactivity was found (Fig. 3B). In the TCL of CTS samples, the number of PR-labelled cells decreased with age (Table 1). PR labelling index was significantly lower in women >70 years old (P<0.05) in respect to the other female and male groups. In non-CTS patients, very few nuclei appeared immunoreactive in both ER- and PR-labelled sections (Fig. 4) with no statistically significant differences between receptors labelling indices (Table 1). In the TCL ligament from specimens of CTS, the number of ER- and PR-positive cells in each age and gender group was significantly higher than the controls (P<0.005; Table 1).


Fig. 3

PR-immunoreactivity in transverse carpal ligament (A) and synovial tissue (B) of a CTS women 50–70 age old. The immunoreactivity is localized in the nuclei (*) of fibroblasts in the TCL (A); in the synovial tissue no PR-immunoreactivity is detected (B) (80×).


Fig. 4

ER-α-immunoreactivity (A) and PR-immunoreactivity (B) in transverse carpal ligament of a non-CTS women 55 age old; the immunoreactivity is localized in the nuclei (*) of fibroblasts (80×).



4 Discussions

The pathogenesis of idiopathic CTS is still under debate. The hypothesis that chronic tenosynovitis as result of repeated mechanical stress (Armstrong et al., 1984) is the underlining cause of the syndrome has been challenged. Many authors believe in a non-inflammatory ischemia–reperfusion etiology, as demonstrated by the low incidence of synovial hyperplasia (Fuchs et al., 1991) and normal levels of inflammatory cytokines (Freeland et al., 2002). For the first time, our immunohistochemical findings suggest a sex steroid involvement in the pathogenesis of this disease.

All samples of TCL and synovial tissue from CTS patients expressed ER-α. ER-labelling index was higher in the TCL than in the synovial tissue, suggesting a major role of TCL as target tissue for hormonal action. In women, this expression increased with age showing a peak in menopausal age, then decreased in the elderly group. The peak of ER expression in women between 50 and 70 years could correlate with clinical features of CTS, disease affecting typically peri- and post-menopausal women.

PR-immunoreactivity was observed only in TCL samples and its expression decreased with age; differently to data obtained for ER no peak of PR-immunoreactivity was found in women ranging 50–70 years. The absence of PR-immunoreaction in synovial tissue and the lower number of PR-positive cells in TCL could suggest a minor role for progesterone activity in the pathogenesis of the CTS disease.

This study has also demonstrated that there were no statistically significant differences between men and women groups in the number of ER- and PR-positive cells with the exception of the 50–70 years age group. These data correlate with a minor sex hormonal involvement and a minor incidence of the disease in the male population.

These results have shown that in TCL samples, the number of ER- and PR-positive cells in non-CTS patients was significantly lower than in CTS patients. The localization of the ER and PR proteins in the normal TCL suggests that female sex hormones may influence the physiological modulation of TCL metabolism.

The presence of the ER and PR in target tissues is a prerequisite for direct estrogen and progesterone action via their receptors. These sex steroid receptors have been already described in the human anterior cruciate ligament (Liu et al., 1996), where they may have an effect on the structure of the ligament. It has also been demonstrated that sex hormones fluctuations could change the composition of the ligament predisposing it to the higher injury rate in female athletes (Liu et al., 1997; Yu et al., 2001). In fact, estrogens influence the structure and the composition of anterior cruciate ligament, regulating collagen types I and III synthesis (Lee et al., 2004) and fibroblast proliferation (Yu et al., 2001). Similarly, fluctuation of estrogens and progesterone could affect TCL growth and development, acting on fibroblast proliferation and collagen synthesis. Some possible estrogen actions on synovial tissue are the immunomodulation of synovitis and synovial hyperplasia (Cutolo et al., 2004), and the regulation of pro-inflammatory cytokines (Kawasaki et al., 2000).

The role of hormone replacement therapy (HRT) in post-menopausal women is controversial. Some authors report an improvement of carpal tunnel symptoms (Hall et al., 1992), while others associated HRT with higher risk of requiring carpal tunnel surgery (80% increase) (Solomon et al., 1999).

The presence of sex hormone receptors in transverse carpal ligament and in synovial tissue supports strong clinical features of CTS like female sex, pregnancy, and menopause.

Idiopathic CTS appears to be affected by sex hormone status, but the role of estrogen and progesterone in its etiology or modulation of symptoms is still undefined.

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Received 1 February 2007/30 April 2007; accepted 29 August 2007

doi:10.1016/j.cellbi.2007.08.014


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)