Xanthine oxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis

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1 FERTILITY AND STERILITY VOL. 75, NO. 4, APRIL 2001 Copyright 2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Xanthine oxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis Hirotaka Ota, M.D., a Shinichi Igarashi, M.D., b and Toshinobu Tanaka, M.D. a Akita University School of Medicine, Akita, and Koto General Hospital, Hachirogata-machi, Japan Received July 3, 2000; revised and accepted October 16, Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Japan (no ). Reprint requests: Hirotaka Ota, M.D., Department of Obstetrics and Gynecology, Akita University School of Medicine, Hondo, Akita, Akita-ken , Japan (FAX: : otah@obgyn.med.akitau.ac.jp). a Department of Obstetrics and Gynecology, Akita University School of Medicine. b Department of Obstetrics and Gynecology, Koto General Hospital /01/$20.00 PII S (01) Objective: To investigate the expression of xanthine oxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis. Design: Immunohistochemical identification of xanthine oxidase in endometrial tissues by using polyclonal antibody. Setting: University hospital. Patient(s): Thirty-four women with endometriosis, 34 women with adenomyosis, and 44 fertile control women. Intervention(s): Biopsy samples were obtained from the endometrium throughout the menstrual cycle. Main Outcome Measure(s): Semiquantitative immunostaining (evaluation nomogram) score of endometrial cells. Result(s): The level of xanthine oxidase expression in the glandular epithelium of control varied according to menstrual phase, but no such variation in expression was seen in endometriosis. Variation in xanthine oxidase expression was observed during the menstrual cycle in patients with adenomyosis; this variation differed completely from that in controls. Xanthine oxidase expression was found in ectopic endometrial tissue in all cases. The mean evaluation nomogram levels in the glandular epithelium in adenomyosis tissue were as high as those in the early secretory phase in the eutopic endometrium. Conclusion(s): Aberrant expression of xanthine oxidase in eutopic and ectopic endometrium appears to play a pathologic role in endometriosis and adenomyosis. (Fertil Steril 2001;75: by American Society for Reproductive Medicine.) Key Words: Xanthine oxidase, free radical, endometrium, endometrial tissue, uterus, endometriosis, adenomyosis Free radicals are intimately involved in the physiology of reproduction. Enzymes that produce and eliminate various free radicals are distributed throughout the body. These enzymes are believed to modulate concentrations of free radicals at an optimal level and maintain the homeostasis of the body. Some enzymes that produce or eliminate free radicals are xanthine oxidase, superoxide dismutase (SOD), glutathione peroxidase, and nitric oxide synthase (NOS). Xanthine oxidase produces superoxide, whereas SOD eliminates superoxide by converting it to hydrogen peroxide. Hydrogen peroxide is then converted to water and oxygen by glutathione while simultaneously producing hydroxyl radicals, which are powerful free radicals. At this point, glutathione peroxidase eliminates hydroxyl radicals. Meanwhile, nitric oxide is generated when L-arginine is catalyzed to citrulline by NOS. Enzymes associated with free radicals are present in the glandular epithelium of the endometrium in humans, and their level varies dynamically depending on the menstrual phase. It has been found that in normal women, levels of SOD and NOS in the endometrium are low during the proliferative phase and increase during the early and midsecretory phases (1, 2). In addition, levels of SOD and NOS remain constant throughout the menstrual cycle, and their expression is pronounced in endometriosis and adenomyosis (3, 4). Furthermore, expression of glutathione peroxidase ceases to vary during the menstrual cycle in endometriosis (5). Levels of this substance are lower than those in normal women during the early secretory 785

2 phase, and it is overexpressed in each phase of the menstrual cycle in adenomyosis. These findings suggest that the overall free radical metabolism is abnormal in endometriosis and adenomyosis (6). Xanthine oxidase metabolizes hypoxanthine and xanthine into uric acid and generates superoxide under high concentrations of oxygen (7). Xanthine oxidase is widely distributed in the liver, kidney, and other organs (8) and is present in ovarian follicular or luteal tissues (9). In addition, xanthine oxidase in follicles may be involved in the ovulation mechanism (10). It has been reported that xanthine oxidase is present in the uterus (11), but details are lacking about its distribution in the endometrium and its variation during the menstrual cycle. We therefore investigated the expression of xanthine oxidase in eutopic and ectopic endometrium to learn more about the role of xanthine oxidase in endometriosis and adenomyosis. MATERIALS AND METHODS Patients We studied 112 women who were treated at the Department of Obstetrics and Gynecology at Akita University Hospital, Akita, Japan. The women were divided into three groups: fertile controls (n 44), women with laparoscopically diagnosed endometriosis (n 34), and women with histologically diagnosed adenomyosis (n 34). Patients with endometriosis associated with adenomyosis and those with adenomyosis associated with endometriosis were excluded. All patients with adenomyosis underwent hysterectomy because of severe dysmenorrhea or iron deficiency anemia. Of the 44 fertile controls, 14 were in the proliferative phase and 30 were in the secretory phase. None of the controls had identifiable endometriosis confirmed by laparoscopy or adenomyosis according to serum CA-125 measurement, ultrasonography, or magnetic resonance imaging. Controls were parous women with definite male factor infertility (mild oligospermia or azoospermia). These women conceived after artificial insemination with their husband s semen or a donor s semen within three treatment cycles and delivered full-term babies. Before controls underwent this procedure and any hormonal treatment, endometrial tissue was obtained during each optional phase. Ectopic endometrial tissues in ovarian chocolate cysts in patients with endometriosis (n 10) or in the myometrium in patients with adenomyosis (n 8) were also studied in some patients. The menstrual cycle of the patients was estimated by endometrial dating by using the method of Noyes et al. (12). The study was approved by the ethical committee of the Medical Center (Institutional Review Board), and all patients gave informed consent. Reagents Rabbit polyclonal antibody for bovine xanthine oxidase (AB1242) was obtained from Chemicon International, Inc. (Temecula, CA). The second antibody (goat [Fab ]anti-rabbit Ig (H L) horseradish peroxidase conjugate; 458) was obtained from Medical and Biological Laboratories, Inc. (Nagoya, Japan). Staining Tissues were fixed in neutral buffered 10% formalin solution. The tissue samples were cut into blocks of approximately 1 cm 3. Serial 4- m sections of tissue were cut and stained with hematoxylin and eosin. The sections were deparaffinized and rehydrated through ethanol, as is routine. The sections were stained by using the peroxidase antiperoxidase method (3). First, the sections were incubated in 1% hydrogen peroxide in phosphate-buffered saline (0.1 mol/l) for 15 minutes to block endogenous peroxidase activity. Nonspecific background staining was reduced by treating the sections with nonimmune 10% swine serum (809-10; Cosin Bio, Sakato, Japan) in phosphate-buffered saline. The polyclonal antibody (x200 dilution) was added and the sections were incubated for 1 hour at 37 C. After washing the sections with phosphate-buffered saline, the second antibody ( 1500 dilution) was added. They were incubated for 1 hour at 37 C and the peroxidase antiperoxidase complex was layered on the slides. After washing the sections with phosphate-buffered saline, they were stained with diaminobenzidine and hydrogen peroxide. Finally, the sections were counterstained with 2% methyl green. In each run, a section of hepatic tissue with strong xanthine oxidase staining was routinely included as a positive control. Negative controls for immunostaining were prepared by substituting the first antibody with nonimmune rabbit serum IgG. Evaluation of Staining Ten nonoverlapping fields of view per biopsy were examined in a systematic random sampling pattern (magnification, 400). Surface and glandular epithelia and stromal cells in eutopic and ectopic endometria were examined for xanthine oxidase staining. Staining was evaluated by using a nomogram reported elsewhere (13). In brief, each section was graded according to the frequency of positive cells and intensity of staining in endometrium. Frequency was defined as 1, 2, or 3 when the number of positive cells in the endometria in each section was 10%, 10% 50% 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 1 to 3. 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 to the source of the specimen. Sections were 786 Ota et al. Xanthine oxidase in endometriosis Vol. 75, No. 4, April 2001

3 FIGURE 1 Evaluation nomogram score of xanthine oxidase in the glandular surface epithelium during the menstrual cycle in fertile controls and women with adenomyosis. TABLE 1 Evaluation nomogram scores for xanthine oxidase in the glandular epithelium during the menstrual cycle in women with endometriosis or adenomyosis. Phase of menstrual cycle Study group Control Endometriosis Adenomyosis a assigned a score by a first observer that was confirmed by a second observer. Statistical Analyses Results are expressed as the mean SE where applicable. The nomogram score for the antigens among the six phases in each group and among the three groups was compared by using the Kruskal Wallis test. The nomogram score for antigens between the two patient groups or the two phases was compared using the Mann Whitney test. Differences were considered statistically significant at P.05. RESULTS Changes in Xanthine Oxidase Expression in the Eutopic Endometrium During the Menstrual Cycle The level of xanthine oxidase expression in the glandular epithelium of controls varied according to menstrual phase; it was low during the early and midproliferative phases and high from the ovulatory phase to the secretory phase (Fig. 1, Table 1). In women with endometriosis, expression of xanthine oxidase tended to be higher than that in controls. It was expressed throughout the menstrual cycle, but no variation in expression was seen. Finally, in women with adenomyosis, xanthine oxidase expression varied during the menstrual cycle, but this variation differed completely from that in controls: It was high in every menstrual phase and was higher than that in controls and in women with endometriosis at every phase except the late proliferative phase. In the surface epithelium of controls, changes in the level Proliferative phase Early (3) (3) (3) Middle b (5) (8) (6) Late (6) (4) (8) Secretory phase Early b (12) (8) (5) Middle c (10) (6) (6) Late b (8) (5) (6) Note: Values are means SE. Values in parentheses are the number of patients examined for the antigen. a P.05 among the six phases in the adenomyosis group (Kruskal Wallis test). b P.01 among the three groups (Kruskal Wallis test). c P.05 compared with the midproliferative phase in the control group (Mann Whitney test). of xanthine oxidase expression were similar to that in the glandular epithelium. The level of expression was low in the early and midproliferative phases, increased in the late proliferative phase, and was high in the secretory phase, which continued until the menstrual phase (Table 2). In contrast, women with endometriosis did not experience changes in the level of xanthine oxidase expression during the menstrual cycle. In women with adenomyosis, expression of xanthine oxidase was lowest in the late proliferative phase of the menstrual cycle and peaked in the late secretory phase. Furthermore, it was the highest of the three groups at every phase except for the late proliferative phase. Expression of xanthine oxidase was lower in stromal cells than in the glandular and surface epithelia. It was low in the early and midproliferative phases and was on the high side in the late proliferative phase (Table 3). In women with endometriosis and adenomyosis, no variation in xanthine oxidase expression was seen during the menstrual cycle, similar to that in the glandular and surface epithelia. Xanthine Oxidase Expression in the Ectopic Endometrium Xanthine oxidase expression was found in ectopic endometrial tissue in all participants. The mean levels on the evaluation nomograms of the glandular epithelium and stromal cells from the walls of ovarian chocolate cysts were FERTILITY & STERILITY 787

4 TABLE 2 Evaluation nomogram scores for xanthine oxidase in the surface epithelium during the menstrual cycle in women with endometriosis or adenomyosis. Phase of menstrual cycle Study group Control Endometriosis Adenomyosis a Proliferative phase Early (3) (3) (3) Middle b (5) (8) (6) Late (6) (4) (8) Secretory phase Early c (12) (8) (5) Middle d (10) (6) (6) Late e (8) (5) (6) Note: Values are means SE. Values in parentheses are the number of patients examined for the antigen. a P.01 among the six phases in the adenomyosis group (Kruskal Wallis test). b P.01 among the three groups (Kruskal Wallis test). c P.05 among the three groups (Kruskal Wallis test). d P.05 compared with the midproliferative phase in the control group (Mann Whitney test). e P.05 compared with the control group (Mann Whitney test). similar to that of SOD (4). It did not necessarily peak at the early secretory phase, and that level of expression was maintained until the late secretory phase. Superoxide dismutase is expressed even after a woman becomes pregnant (14). Thus, xanthine oxidase expression may be involved in development of the fertilized egg after nidation, as is SOD. In endometriosis, expression of NOS and SOD was pronounced but that of glutathione peroxidase and xanthine oxidase was not very enhanced. On the contrary, no variation was observed during the menstrual cycle. This lack of variation was not limited to xanthine oxidase; it is also seen in the other three enzymes. Therefore, abnormal expression of xanthine oxidase may be closely linked with abnormalities in these enzymes. Xanthine oxidase expression in ectopic endometrial tissues from ovarian chocolate cysts may be clinically significant. Expression of xanthine oxidase in the ovaries may lower the quality of eggs by generating an excess amount of superoxide. In one report, egg quality was low when an egg was harvested for in vitro insemination in a woman with endometriosis (15), although data are conflicting (16). Low concentrations of free radicals are believed to create an ideal environment for embryonic development during a specified period in which fertilized eggs divide (17). Second, excessive free radicals in ovaries may initiate a chromosomal aberration. The addition of xanthine oxidase under hypoxanthine in an ovarian cell strain culture system derived from Chinese hamsters induced a chromosomal aberration (18). In fact, and , respectively; those in the glandular epithelium and stromal cells of adenomyosis tissue were and Levels were almost as high as that in the early secretory phase in the eutopic endometrium. Histologic Findings Localization of xanthine oxidase was found in the endometrial surface epithelium and glands and endometrial stromal cytoplasm (Fig. 2). Staining intensity and incidence of positive cells was slightly high in women with adenomyosis. The positive site was stained diffusely in the cytoplasm of glandular cells, but no localization was seen in the nuclei. No clear difference was seen between the basal area and the apical side in the endometrial glands. There was no intracellular polarity of note, and no pronounced localization was seen in cell membranes. Almost no individual cells had isolated pronounced staining. DISCUSSION This is the first study to report that xanthine oxidase is specifically distributed in the glandular and surface epithelia, the functional layers of the human endometrium. In normal women, expression of xanthine oxidase showed variation TABLE 3 Evaluation nomogram scores for xanthine oxidase in the stromal cells during the menstrual cycle in women with endometriosis or adenomyosis. Phase of menstrual cycle Study group Control Endometriosis Adenomyosis Proliferative phase Early (3) (3) (3) Middle (5) (8) (6) Late (6) (4) (8) Secretory phase Early (12) (8) (5) Middle (10) (6) (6) Late (8) (5) (6) Note: Values are means SE. Values in parentheses are the number of patients examined for the antigen. 788 Ota et al. Xanthine oxidase in endometriosis Vol. 75, No. 4, April 2001

5 FIGURE 2 Cells stained for xanthine oxidase antigen. (A, B), Eutopic endometrium in the midproliferative phase (A: 100, evaluation nomogram score 1) and the early secretory phase in the control group (B: 100, evaluation nomogram score 2). (C, D), Eutopic endometrium in the midproliferative phase in the adenomyosis group (C: 100, evaluation nomogram score 3) and surface epithelium in the early secretory phase in the endometriosis group (D: 100, evaluation nomogram score 3), (E, F), E, Ectopic endometrial tissue in the early proliferative phase in the endometriosis group (E: 50, evaluation nomogram score 2) and in the late proliferative phase in the adenomyosis group (F: 50, evaluation nomogram score 3). chromosomal analysis of endometriotic tissue in the ovarian chocolate cyst has revealed loss of heterozygosity on various chromosomes (19). Furthermore, free radicals may increase fetal aberrations. Jenkinson et al. (20) reported that when they added xanthine oxidase under the presence of xanthine and exposed rats with a fetal age of 9.5 days to excessive active oxygen, the incidence of neural suture deformities increased in a dose-dependent manner. Fetal deformities increase even if nitric oxide is increased by adding nitric oxide donor (21). FERTILITY & STERILITY 789

6 Abnormal expression of xanthine oxidase is seen in disorders other than endometriosis. As a result of xanthine oxidase activation when reperfusion occurs after vascular ischemia (in, for instance, myocardial infarction), an excess amount of superoxide is generated locally, leading to destruction of tissue (22). Xanthine oxidase is strongly expressed in the articular cavities of patients with rheumatoid arthritis, resulting in generation of superoxide, which resolves hyaluronic acid and lowers its viscosity, exacerbating the disease (23). In adenomyosis, xanthine oxidase was overexpressed throughout the menstrual cycle. In this disorder, expression of NOS, SOD, and glutathione peroxidase was more enhanced than that in normal women. Thus, the data strongly suggest that generation of nitric oxide, superoxide, and other free radicals is heightened in women with adenomyosis compared with normal women. This raises the question, What is the mechanism that leads to overexpression of xanthine oxidase in adenomyosis? Menstrual pain is intense in women with adenomyosis, and hysterectomy is often performed because of it. Pain in adenomyosis may result from an imbalance in contraction of the uterus, but almost nothing is known of the contraction mechanism. Several factors have been implicated as being involved in contractions. Levels of calcium, prostaglandin, or oxytocin, which are contractive factors, may be increased; substances that may promote relaxation include magnesium, nitric oxide, progesterone, relaxin, and -adrenergic agents (24). In fact, prostaglandin and nitric oxide are two major substances that are known to be directly involved in uterine contraction during menstruation. Accordingly, it is tempting to suggest that contraction of the uterus is basically regulated by prostaglandin and nitric oxide. Expression of NOS is pronounced in adenomyosis, and large amounts of nitric oxide appear to be generated (3). Meanwhile, it has been reported that large amounts of prostaglandin are generated in adenomyosis (25). Consequently, because abnormal constriction of the myometrium occurs through an imbalance in prostaglandin and nitric oxide, xanthine oxidase is strongly expressed in the eutopic and ectopic endometrial cells, and free radicals are regenerated. How excessive superoxide caused by xanthine oxidase is actually involved in the pathology of adenomyosis needs to be investigated further. References 1. Telfer JF, Lyall F, Norman JE, Cameron IT. Identification of nitric oxide synthase in human uterus. Hum Reprod 1995;10: Narimoto K, Noda Y, Shiotani M, Tokura T, Goto Y, Takakura K, et al. Immunohistochemical assessment of superoxide dismutase expression in the human endometrium throughout the menstrual cycle. Acta Histochem Cytochem 1990;23: Ota H, Igarashi S, Hatazawa J, Tanaka T. 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Am J Physiol 1992;262:E Gatzuli E, Aten RF, Behrman HR. Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells. Endocrinology 1991;128: Gossrau R, Frederiks WM, Van Noorden CJF. Histochemistry of reactive oxygen-species (ROS)-generating oxidases in cutaneous and mucous epithelia of laboratory rodents with special reference to xanthine oxidase. Histochemistry 1990;94: Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1: Ota H, Igarashi S. HLA-DR expression in endometriotic tissue in patients with endometriosis and adenomyosis. Fertil Steril 1993;60: Telfer JF, Thomson AJ, Cameron IT, Greer LA, Norman JE. Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition. Hum Reprod 1997;12; Wardle PG, Mitchell JD, McLaughlin EA, Ray BD, McDermott A, Hull MG. Endometriosis and IVF: effect of prior therapy. 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