1 Department of Obstetrics & Gynecology, Tohoku University School of Medicine, 11, Seiryo-machi, Aoba-ku, Sendai, 9808574, Japan and 2 Department of Gynecology, Polyclinique de lHotel-Dieu, 13, boulevard Charles-De-Gaulle, 63003 Clermont-Ferrand Cedex 1, France 3 Present address: Department of Gynecology, Polyclinique de lHotel-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 lHôtel-Dieu, 13, boulevard Charles-De-Gaulle, 63003 Clermont-Ferrand Cedex 1, France. e-mail: SachikoMA{at}aol.com
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: black endometriotic lesions/erythropoietin/erythropoietin-receptor/red endometriotic lesions
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 MannWhitney U-test was applied to compare results from different groups. Statistical significance was defined as P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
American Society for Reproductive Medicine (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil. Steril., 67, 817821.[CrossRef][ISI][Medline]
Ribatti, D., Presta, M., Vacca, A., Ria, R., Giuliani, R., DellEra, P., Nico, B., Roncali, L. and Dammacco, F. (1994) Erythropoietin receptor mRNA expression in human endothelial cells. Proc. Natl Acad. Sci. USA, 91, 39743978.[Abstract]
Bikfalvi, A. and Han, Z.C. (1994) Angiogenic factors are hematopoietic growth factors and vice versa. Leukemia, 8, 523529.[ISI][Medline]
Calhaz-Jorge, C., Costa, A.P., Barata, M., Santos, M.C., Melo, A. and Palma-Carlos, M.L. (2000) Tumour necrosis factor alpha concentrations in the peritoneal fluid of infertile women with minimal or mild endometriosis are lower in patients with red lesions only than in patients without red lesions. Hum. Reprod., 15, 12561260.
Chandra, M., Clemons, G., Sahdev, I., McVicar, M. and Bluestone, P. (1993) Intraperitoneal production of erythropoietin with continuous ambulatory peritoneal dialysis. Pediatr. Nephrol., 7, 281283.[ISI][Medline]
Conrad, K.P., Benyo, D.F., Westerhausen-Larsen, A. and Miles, T.M. (1996) Expression of erythropoietin by the human placenta. F.A.S.E.B. J., 10, 760766.
Donnez, J., Smoes, P., Gillerot, S., Casanas-Roux, F. and Nisolle, M. (1998) Vascular endothelial growth factor (VEGF) in endometriosis. Hum. Reprod., 13, 16861690.[Abstract]
Fairchild Benyo, D. and Conrad, K.P. (1999) Expression of the erythropoietin receptor by trophoblast cells in the human placenta. Biol. Reprod., 60, 861870.
Halme, J., White, C., Kauma, S., Estes, J. and Haskill, S. (1988) Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro. J. Clin. Endocrinol. Metab., 66, 10441049.[Abstract]
Healy, D.L., Rogers, P.A., Hii, L. and Wingfield, M. (1998) Angiogenesis: a new theory for endometriosis. Hum. Reprod. Update, 4, 736740.
Jelkmann, W. (1992) Erythropoietin: Structure, control of production, and function. Physiol. Rev., 72, 449489.
Juul, S.E., Anderson, D.K., Li, Y. and Christensen, R.D. (1998a) Erythropoietin and erythropoietin receptor in the developing human central nervous system. Pediatr. Res., 43, 4049.[Abstract]
Juul, S.E., Yachnis, A.T. and Christensen, R.D. (1998b) Tissue distribution of erythropoietin and erythropoietin receptor in the developing human fetus. Early Hum. Dev., 52, 235249.[CrossRef][ISI][Medline]
Klein, N.A., Pergola, G.M., Rao-Tekmal, R., Dey, T.D. and Schenken, R.S. (1993) Enhanced expression of resident leukocyte interferon gamma mRNA in endometriosis. Am. J. Reprod. Immunol., 30, 7481.[ISI][Medline]
Koninckx, P.R., Kennedy, S.H. and Barlow, D.H. (1998) Endometriotic disease: the role of peritoneal fluid. Hum. Reprod. Update, 4, 741751.
Krantz, S.B. (1991) Erythropoietin. Blood, 77, 419434.[ISI][Medline]
Mandal, M., Bandyopadhyay, D., Goepfert, T.M. and Kumar, R. (1998) Interferon-induces expression of cyclin-dependent kinase-inhibitors p21WAF1 and p27Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating kinase (CAK). Oncogene, 16, 217225.[CrossRef][ISI][Medline]
Matsuzaki, S., Murakami, T., Uehara, S., Yokomizo, R., Noda, T., Kimura, Y. and Okamura, K. (2001a) Erythropoietin concentrations are elevated in the peritoneal fluid of women with endometriosis. Hum. Reprod., 16, 945948.
Matsuzaki, S., Canis, M., Murakami T., Dechelotte, P., Bruhat, M.A. and Okamura, K. (2001b) Expression of the cyclin-dependent kinase inhibitor p27Kip1 in eutopic endometrium and peritoneal endometriosis. Fertil. Steril., 75, 956960.[CrossRef][ISI][Medline]
Masuda, S., Okano, M., Yamagishi, K., Nagao, M., Ueda, M. and Sasaki, R. (1994) A novel site of erythropoietin production. J. Biol. Chem., 269, 1948819493.
Masuda, S, Nagao, M. and Sasaki, R. (1999) Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production. Int. J. Hematol., 70, 16.[ISI][Medline]
Nisolle, M., Casanas-Roux, F., Anaf, V., Mine, J.M. and Donnez, J. (1993) Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil. Steril., 59, 681684.[ISI][Medline]
Nisolle, M. and Donnez, J. (1997) Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil. Steril., 68, 585596.[CrossRef][ISI][Medline]
Nisolle, M., Casanas-Roux, F. and Donnez, J. (1997) Immunohistochemical analysis of proliferative activity and steroid receptor expression in peritoneal and ovarian endometriosis. Fertil. Steril., 68, 912919.[CrossRef][ISI][Medline]
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 35.[ISI][Medline]
Tabibzadeh, S.S., Satyaswaroop, P.G. and Rao, P.N. (1988) Antiproliferative effect of interferon-gamma in human endometrial epithelial cells in vitro: potential local growth modulatory role in endometrium. J. Clin. Endocrinol. Metab., 67, 131138.[Abstract]
Taniguchi, S., Dai, C.H., Price, J.O. and Krantz, S.B. (1997) Interferon gamma downregulates stem cell factor and erythropoietin receptors but not insulin-like growth factor-I receptors in human erythroid colony-forming cells. Blood, 90, 22442252.
Tilbrook, P.A. and Klinken, S.P. (1999) Erythropoietin and erythropoietin receptor. Growth Factors, 17, 2535.[ISI][Medline]
Yasuda, Y., Musha, T., Tanaka, H., Fujita, Y., Fujita, H., Utsumi, H., Matsuo, T., Masuda, S., Nagao, M., Sasaki, R. et al. (2001) Inhibition of erythropoietin signalling destroys xenografts of ovarian and uterine cancers in nude mice. Br. J. Cancer, 84, 836843.[CrossRef][ISI][Medline]
Yasuda, Y., Masuda, S., Chikuma, M., Inoue, K., Nagao, M. and Sasaki, R. (1998) Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis. J. Biol. Chem., 273, 2538125387.
Yokomizo, R., Matsuzaki, S., Uehara, S., Murakami, T., Yaegashi, N. and Okamura, K. (2002) Erythropoietin and erythropoietin receptor expression in human endometrium throughout the menstrual cycle. Mol. Hum. Reprod., 8, 441446.
Submitted on April 18, 2002; resubmitted on June 27, 2002; accepted on September 5, 2002