1 Department of Obstetrics and Gynecology, Akita University School of Medicine, Akita-city, Akita-ken 010-8543, and 2 Koto General Hospital, Hachirogata-town, Akita-ken 018-1605, Japan
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Abstract |
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Key words: adenomyosis/cyclooxygenase/endometriosis/endometrium/prostaglandin
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Introduction |
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It is known that human endometrium produces PGE2 and PGF2 (Smith and Kelly, 1988
). It has been reported (Jones et al., 1997
) that COX-2 is distributed in endometrial glandular epithelia and vascular endothelia and that it varies during the menstrual cycle. Other studies have also found that COX-2 is distributed in the human placenta (Wetzka et al., 1997
) and decidual tissue (Ishihara et al., 1995
). A significant amount of prostaglandins are produced from the endometrium and endometriotic tissues (Lumsden et al., 1984
). It is interesting to note that low concentrations of arachidonic acid are only metabolized by COX-2 (Morita et al., 1995
). Accordingly, COX-2 expression may be abnormal in endometriosis, but there appear to be no reports of investigations of COX-2 kinetics in this disorder. Therefore, in this study, we investigated the expression of COX-2 in eutopic and ectopic endometria in endometriosis.
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Materials and methods |
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These women conceived after artificial insemination using husband's or donor's semen within three treatment cycles and delivered full-term babies. The mean ages in the fertile control group, the endometriosis group, and the adenomyosis group were 28.9 years (range 2236 years), 32.1 years (range 2341 years), and 43.1 years (range 3449 years) respectively.
Before starting any medication in the control group, or just after laparoscopy in the patients with endometriosis or after hysterectomy in the patients with adenomyosis, endometrial tissue was obtained when it became available during any phase of the menstrual cycle. Ectopic endometrial tissues in ovarian chocolate cysts in endometriosis (n = 10) or in the myometrium in adenomyosis (n = 9) were studied in the identical patients at the same time. In the control and the endometriosis group, endometrial specimens were obtained by curettage. The tissues were fixed in neutral-buffered 10% formalin solution. The menstrual cycle of the patients was estimated by endometrial dating according to previously described criteria (Noyes et al., 1950). Informed consent was obtained in each case, and approval for the study was granted by the Institutional Review Board.
Reagents
The goat polyclonal antibody for human COX-2 (sc-1745) raised against a peptide mapping at the carboxy terminus of COX-2 of human origin was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The second antibody [rabbit anti-sheep immunoglobulins (Ig) H+L horseradish peroxidase conjugate; 615504] was obtained from Southern Biotechnology Associates Inc. (Birmingham, AL, USA).
Staining
The endometrial tissue samples were cut into blocks (~1 cm3). Serial 3 mm sections of tissue were cut, deparaffinized, and rehydrated through ethanol, as in routine histology. The sections were stained using the indirect method. Before the staining, a microwave antigen retrieval technique was utilized, whereby the sections were heated in sodium citrate buffer (pH 6.0) for 5 min at high power (500 W), and this was repeated four times. First, the sections were incubated in 3% hydrogen peroxide in phosphate-buffered saline solution (PBS; 0.1 mol/l) for 15 min to block endogenous peroxidase activity. Non-specific background staining was reduced by treating the sections with non-immune 10% swine serum (80910; Cosin Bio, Sakato, Japan) in PBS. Then, the polyclonal antibody (x50 dilution) was added and the sections were incubated overnight at 4°C. After washing them with PBS, the second antibody (x400 dilution) was added and they were incubated for 1 h at 37°C. After washing the sections with PBS, they were stained with 0.2 mg/ml 3,3'-diaminobenzidine (DAB) tetrahydrochloride containing 0.005% hydrogen peroxide in PBS. Finally, the sections were counterstained with Carazzi's haematoxylin. Negative controls for immunostaining were prepared by substituting the first antibody with non-immune rabbit serum IgG. In each run, a section of placenta with strong COX-2 staining was routinely included as a positive control.
Evaluation of staining
Ten non-overlapping fields of view were examined per biopsy in a systematic random sampling pattern (magnification x400). Surface and glandular epithelia and stromal cells in eutopic and ectopic endometria were evaluated for COX-2 staining. Staining was evaluated using an evaluation nomogram as previously reported (Ota and Igarashi, 1993). Briefly, each section was graded according to the frequency of positive cells and intensity of staining in endometrium. The frequency was defined as 1+, 2+ or 3+ when the number of positive cells in the endometrium in each section was <10%, 1050% or >50% respectively. Intensity was defined as 3+ when staining of the cells was as strong as that observed in the positive controls, as 1+ when staining was weakly positive but distinct from the negative controls, and as 2+ when the staining was between 1+ and 3+. The sections were ranked from 1 to 5 according to the evaluation nomogram. Sampling and grading of each specimen were done by two different observers blinded as to the specimen source. Sections were assigned a score by a first observer, and confirmed by a second observer.
Statistical analyses
The results are expressed as the mean ± SEM where applicable. Statistical analysis was performed by the KruskalWallis test. P < 0.05 was considered to be statistically significant.
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Results |
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Histological findings
COX-2 formed a granular pattern or texture in the cytoplasm of glandular cells and cells were stained almost diffusely (Figure 3). Pronounced localization was not found in cell membranes. Polarity in the basal and apical sides was not particularly pronounced. No localization was seen in nuclei. Cytoplasm of interstitial cells was slightly positive.
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Discussion |
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This is the first study to look at the expression of COX-2 in eutopic and ectopic endometria in endometriosis. Expression of COX-2 in eutopic endometrium varied significantly during the menstrual cycle. Expression was higher in the secretory phase than in the proliferative phase, but it was higher than that in the control throughout the menstrual cycle. Over-expression of COX-2 leads to increased production of prostaglandins. The mechanism of over-expression of COX-2 in eutopic endometrium in endometriosis is unclear. It has been indicated that an increased autoimmune response occurs in the endometrium in endometriosis. For instance, T cells and B cells are increased (Witz et al., 1994; Ota et al., 1996a
), macrophage is activated (Haney et al., 1981
), and immunoglobulin and complements are deposited in endometrial glandular epithelial cells (Weed and Arquembourg, 1980
; Ota and Maki, 1990
). Furthermore, human leukocyte antigen expression in glandular epithelial cells is increased (Ota and Igarashi, 1993
) and adhesion molecules are abnormally expressed (Lessey et al., 1992
; Ota et al., 1996b
). In addition, expression of free radical-related enzymes such as nitric oxide synthase (Ota et al., 1998
), superoxide dismutase (Ota et al., 1999a
), and glutathione peroxidase is increased (Ota et al., 1999b
; Ota et al., 2000
). Consequently, a variety of cytokines are secreted from these immune cells and macrophages, including IL-1 and IL-2. These cytokines may very well initiate COX-2 expression. Over-expression of COX-2 and the accompanying increase in prostaglandin production likely initiate an abnormal state in the uterus. Endometriosis is often accompanied by dysmenorrhoea. Abnormal uterine contraction is also observed in endometriosis. Myometrial movement in patients with endometriosis was observed using ultrasonography (Leyendecker et al., 1996
) and they found that hyperperistalsis and dysperistalsis are frequently observed in patients with endometriosis, and speculated that abnormal peristalsis may inhibit normal sperm transport in the uterus. Secondly, excessive prostaglandins may be impairing implantation of fertilized oocytes. The implantation rate was said to have decreased significantly in an experimental endometriotic model using rabbits that were subsequently artificially inseminated (Hahn et al., 1986
).
COX-2 expression was found in endometriotic tissues of the ovarian chocolate cyst wall in all cases, and expression of COX-2 in glandular epithelial cells was almost as pronounced as that in the secretory phase in the eutopic endometrium. In fact, there have been many reports that large amounts of PGs are produced in ascites and endometrial tissues in endometriosis (Badawy et al., 1985; De Leon et al., 1986
; Moon et al., 1981
).
Over-expression of COX-2 in ectopic endometrial tissue, and prostaglandins that is produced as a result, may be involved in the proliferation and differentiation of cells and malignant transformation. For example, it was reported that when COX-2 was over-expressed in epithelial cells of mouse small intestine, expression of adhesion molecules such as laminin increased, while apoptosis was suppressed (Tsujii and DeBois, 1995). It has also been reported that the incidence of colon cancer and the number and size of colorectal polyps decreased in people who took aspirin for an extended length of time (Giovannucci et al., 1995
; Marcus, 1995
). It is said that apoptosis is reduced in the eutopic and ectopic endometria in endometriosis compared with normal women (Dmowski et al., 1998
; Imai et al., 2000
). Furthermore, apoptosis is further reduced in the ectopic endometrium compared with the eutopic endometrium (Gebel et al., 1998
). In fact, loss of heterozygosity was frequently observed on chromosomes in endometriotic tissue from ovarian chocolate cysts (Jiang et al., 1996
). It is concluded that COX-2 expression in endometriosis appears to be intimately involved in its pathology. Further investigations into the involvement of COX-2 are required.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 4, 2000; accepted on November 17, 2000.