Human Reproduction, Vol. 18, No. 1, 152-166,
January 2003
© 2003 European Society of Human Reproduction and Embryology
Expression of erythropoietin and erythropoietin receptor in peritoneal endometriosis
1 Department of Obstetrics & Gynecology, Tohoku University School of Medicine, 1–1, Seiryo-machi, Aoba-ku, Sendai, 980–8574, Japan and 2 Department of Gynecology, Polyclinique de l’Hotel-Dieu, 13, boulevard Charles-De-Gaulle, 63003 Clermont-Ferrand Cedex 1, France 3 Present address: Department of Gynecology, Polyclinique de l’Hotel-Dieu, 13, boulevard Charles-De-Gaulle, 63003 Clermont-Ferrand Cedex 1, France 4 To whom correspondence should be addressed at: Department of Gynecology, Polyclinique de l’Hôtel-Dieu, 13, boulevard Charles-De-Gaulle, 63003 Clermont-Ferrand Cedex 1, France. e-mail: SachikoMA{at}aol.com
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Abstract |
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BACKGROUND: Recent studies have indicated new physiological roles for erythropoietin (Epo) unrelated to erythropoiesis. We previously demonstrated that the Epo concentrations in peritoneal fluid from patients with stage I endometriosis were significantly higher than those with stages II, III and IV of the disease. Therefore, we hypothesized that Epo may play a role in the pathogenesis of endometriosis, particularly during the early stages of the disease. METHOD: We investigated the localization of Epo and the Epo receptor (Epo-R) in peritoneal endometriosis and eutopic endometrium, using immunohistochemistry. RESULTS: We detected Epo and Epo-R localized within glandular epithelial cells in both peritoneal endometriosis and eutopic endometrium. There was no significant difference in Epo expression between red and black peritoneal lesions, whereas Epo-R expression was significantly lower in black peritoneal lesions when compared to red lesions. Epo and Epo-R expression levels within red peritoneal lesions were comparable to those of eutopic endometrium from patients with endometriosis. CONCLUSION: The present findings suggest that Epo may play a role in the pathophysiology of endometriosis.
Key words: black endometriotic lesions/erythropoietin/erythropoietin-receptor/red endometriotic lesions
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Introduction |
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Erythropoietin (Epo) has a molecular weight of
30 kDa (Tilbrook and Klinken, 1999
), and is a key factor in the regulation of erythropoiesis, stimulating the proliferation of early erythroid precursors and the differentiation of late erythroid precursors via binding to the Epo receptor (Epo-R) (Krantz, 1991; Jelkman, 1992). However, recent studies have demonstrated nonclassical sites of erythropoietin and erythropoietin-receptor expression, suggesting new physiological roles unrelated to erythropoiesis (Masuda et al., 1994; Conrad et al., 1996; Yasuda et al., 1998; Masuda et al., 1999). We previously demonstrated the presence of both mRNA and protein expression of Epo and Epo-R in endometrial epithelial cells from fertile pre-menopausal women. Furthermore, we previously demonstrated that erythropoietin concentrations were elevated in the peritoneal fluid (PF) of women with endometriosis. Peritoneal endometriotic lesions are typically classified into three types: red, black, or white, according to their macroscopic appearance (American Society for Reproductive Medicine, 1997; Nisolle and Donnez, 1997). It is postulated that these three types of peritoneal lesions may represent different stages of the spontaneous evolution of endometriotic implants; the first stage characterized by red lesions, being more active than subsequent black lesions (Nisolle et al., 1993; Nisolle et al., 1997; Nisolle and Donnez, 1997; Donnez et al., 1998; Matsuzaki et al., 1998). Our observations suggested that Epo concentrations may be higher in PF from patients with red lesions only when compared to those with primarily black lesions (Matsuzaki et al., 2001a). Therefore, we hypothesized that Epo may play a role in the pathogenesis of endometriosis, particularly during the early stages of the disease (Matsuzaki et al., 2001a). In the present study we investigated the localization of Epo and Epo-R expression within peritoneal endometriosis, using immunohistochemistry. In addition, we compared the expression levels of Epo and Epo-R in red and black peritoneal lesions, in order to evaluate the pathophysiological significance of Epo in peritoneal endometriosis.
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Materials and methods |
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Experimental subjects
Patients undergoing laparoscopic surgery for infertility or investigation of pelvic pain, or a pelvic mass, were recruited for the study. None of the patients received hormonal treatments, such as GnRH agonist or sex steroids, during a minimum 6 month period preceding surgery. All patients had regular menstrual cycles, confirmed by their menstrual history and measurement of their serum 17ß-estradiol and progesterone levels. During laparoscopic surgery, paired samples of peritoneal endometriotic lesions (2–5 mm in size, n = 44) and eutopic endometrium were obtained from a total of 23 patients (23–38 years of age) at Tohoku University Hospital, Sendai, Japan and Polyclinique de l’Hôtel-Dieu, Clermont-Ferrand, France. Peritoneal endometriotic lesions were further categorized as red, black or white lesions as proposed in the latest revision of the American Society for Reproductive Medicine classification (American Society for Reproductive Medicine, 1997). Finally, 21 samples were categorized as red lesions (including red, red-pink and clear lesions, 10 in the proliferative phase and 11 in the secretory phase) and the remaining 23 samples were of black lesions (including black and blue lesions, 10 in the proliferative phase and 13 in the secretory phase). Eutopic endometrial tissues were obtained from 23 patients (10 in the proliferative phase and 13 in the secretory phase as determined using established criteria) (Noyes et al., 1950
). Tissues were fixed immediately after collection in 4% paraformaldehyde (pH 7.4) and embedded in paraffin for routine histopathological examinations and immunohistochemical analysis. All samples contained a glandular epithelium surrounded by stromal tissue. The presence of these features was sufficient to meet the criteria for histopathological diagnosis of endometriosis. Sections of 3 µm thickness were routinely prepared, mounted on glass slides and dried overnight at 37°C before being used for immunohistochemistry. All tissue samples were obtained with the patients’ fully informed consent. The research protocol was approved by the Human Research Board of the Ethical Committee of the Tohoku University School of Medicine, Sendai, Japan and the Polyclinique de l’Hotel-Dieu, Clermont-Ferrand, France.
Immununohistochemical staining
Immunohistochemical staining was performed on paraffin sections using a goat polyclonal antibody against human erythropoietin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a rabbit polyclonal antibody against the human erythropoietin receptor (Santa Cruz Biotechnology). Immunohistochemistry for Epo and Epo-R was performed using a commercial streptavidin-biotin system (Histofine kit; Nichirei, Tokyo, Japan) and has been previously described in detail (Yokomizo et al., 2002). The primary antibody, either anti-human Epo or anti-human Epo-R, was diluted 1:100 in PBS with 3% bovine serum albumin. Specific immunolocalization of Epo and Epo-R has been reported previously in human placenta and fetal kidney; therefore, these tissues were chosen as positive controls (Conrad et al., 1996; Juul et al., 1998a; Juul et al., 1998b; Fairchild Benyo and Conrad, 1999; Yokomizo et al., 2002). Negative controls were performed by replacing the primary antibody with normal goat IgG (for Epo) or rabbit IgG (for Epo-R) diluted at the same concentration as the primary antibody.
Quantitation of Epo and Epo-R immunostained cells
To quantify immunopositivity, positively stained cells were counted as previously reported (Yokomizo et al., 2002). For each section, the number of positive cells was counted, regardless of the staining intensity. In all samples of eutopic endometrium, 10 non-overlapped fields were analysed. In peritoneal endometriosis, because of the tissue heterogeneity, we analysed all glandular and stromal cells within each sample. The number of areas analysed in peritoneal endometriosis varied from five to 13 areas per sample. The percentage of positive cells for Epo or Epo-R was calculated for each sample.
Statistical analysis
Statistical analysis was performed with the Stat View 4.5 program (Abacus concepts, Inc., Berkeley, CA, USA). The Kruskal-Wallis test or Mann–Whitney U-test was applied to compare results from different groups. Statistical significance was defined as P < 0.05.
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Results |
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Erythropoietin expression
The results are shown in Figure 1 and Table I. Homogeneous cytoplasmic immunoreactivity to erythropoietin was detected in glandular epithelial cells, while stromal cells stained heterogeneously for eutopic endometrium and peritoneal endometriosis samples at each stage of the menstrual cycle. The percentage of positive glandular epithelial cells was significantly increased in secretory eutopic endometrium when compared to proliferative eutopic endometrium (P < 0.01). No significant change was detected in peritoneal endometriotic tissues between each phase of the menstrual cycle. During the proliferative phase, there was no significant difference in the percentage of positive glandular epithelial cells among eutopic endometrium, black or red peritoneal lesions. During the secretory phase, the percentage of positive glandular epithelial cells found in red lesions was similar to that of eutopic endometrium, whereas that in black lesions was significantly lower in number than in eutopic endometrium (P < 0.02). However, there was no significant difference in the percentage of positive glandular epithelial cells between red and black peritoneal lesions. The percentage of positive stromal cells was <1% in both eutopic endometrium and peritoneal endometriosis at each phase of the menstrual cycle.
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Erythropoietin receptor expression
The results are shown in Figure 1 and Table I. Cytoplasmic immunoreactivity to Epo-R was observed to be homogenous in glandular epithelial cells and heterogeneous in stromal cells within eutopic endometrium and peritoneal endometriosis tissue at each stage of the menstrual cycle. No significant cyclical change in Epo-R immunoreactivity was detected in glandular epithelial or stromal cells either in eutopic endometrium or peritoneal endometriosis samples. Black lesions possessed the lowest percentage of positive glandular epithelial cells when compared to eutopic endometrium tissue (proliferative phase; P < 0.02, secretory phase; P < 0.001) or red lesions (proliferative phase; P < 0.04, secretory phase; P < 0.004). There was no significant difference in the percentage of positive glandular epithelial cells between eutopic endometrium and red peritoneal lesions. The percentage of positive stromal cells was <1% in both eutopic endometrium and peritoneal endometriosis tissue at each phase of the menstrual cycle.
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Discussion |
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In the present study we detected Epo and Epo-R localization in glandular epithelial cells in peritoneal endometriosis as well as in eutopic endometrium. The present study and our previous studies (Matsuzaki et al., 2001a
; Yokomizo et al., 2002) suggested that a potential source of Epo in PF from women with endometriosis may include endometriotic tissues as well as eutopic endometrial tissues. We previously reported that Epo concentrations in PF from patients with only red lesions may be higher than those with only black lesions (Matsuzaki et al., 2001a), suggesting that red endometriotic lesions may produce more Epo than black ones. Another source of Epo that should be considered is from activated macrophages, which are increased in the peritoneal fluid of patients with endometriosis (Halme et al., 1988; Koninckx et al., 1998). Peritoneal macrophages from continuous ambulatory peritoneal dialysis (CAPD) patients produce Epo (Chandra et al., 1993). A recent study suggested an impaired function of peritoneal macrophages in those patients with only red lesions (Calhaz-Jorge et al., 2000). Erythropoietin is a soluble factor and thus the present findings do not indicate the local production of Epo in peritoneal endometriosis.
The functional role of Epo and Epo-R in endometriosis remains to be determined. A recent study demonstrated high expression levels of Epo and Epo-R in human solid tumours and in vitro stimulation of cell proliferation of breast cancer cells by Epo (Acs et al., 2001). Inhibition of Epo signalling induces tumour regression of xenografts of ovarian and uterine cancers in nude mice (Yasuda et al., 2001). Our recent study suggests that Epo may be involved in cyclic proliferation and differentiation of endometrial epithelial cells, acting in an autocrine mechanism (Yokomizo et al., 2002). The present study showed prominent expression of Epo in glandular epithelial cells in peritoneal endometriosis and no significant difference between red and black peritoneal lesions. However, we detected that Epo-R immunoreactivity was significantly lower in black peritoneal lesions when compared to red ones. Black peritoneal lesions have lower cell-proliferating activity compared with red ones (Nisolle and Donnez, 1997; Matsuzaki et al., 2001b). A recent study demonstrated that downregulation of Epo-R by interferon (IFN)-
led to a reduced effect of Epo on erythroid cell proliferation and differentiation (Taniguchi et al., 1997). Studies demonstrated that IFN- inhibited the proliferation of endometrial and endometriotic epithelial cells (Tabibzadeh et al., 1988; Klein et al., 1993). Furthermore, we previously reported increased expression of cell cycle inhibitor p27Kip1 in black lesions compared with red lesions, suggesting that expression of p27Kip1 is involved in progression of peritoneal endometriosis ( Matsuzaki et al., 2001b). p27Kip1 has a role in mediating IFN--induced terminal growth arrest (Mandal et al., 1998). The present findings suggest that Epo may play a role in the pathophysiology of endometriosis. We hypothesize that local production of Epo in endometrial, endometriotic and activated macrophages in PF may be involved in the development and growth of peritoneal endometriosis and that the down-regulation of Epo-R expression may possibly lead to a reduced effect of Epo on cell proliferation in black peritoneal lesions. However, comparisons between red and black lesions by a morphometric approach have some unavoidable limitations. The different morphological appearance of red and black lesions makes it easy to identify the laparoscopic appearance from each section (Nisolle et al., 1993; Nisolle and Donnez, 1997). Further studies are required to clarify roles of Epo and Epo-R in the pathogenesis of peritoneal endometriosis.
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Acknowledgements |
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We are grateful to Ms Keiko Abe and Mr Satoshi Okamoto for their expert technical assistance.
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Submitted on April 18, 2002; resubmitted on June 27, 2002; accepted on September 5, 2002
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