Akira Fujishita

Changes in tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma after GnRH agonist therapy

BACKGROUND Information is limited regarding the multifunctional role of GnRH agonist (GnRHa) therapy in reproductive diseases. We investigated the pattern of changes in inflammatory reaction, micro-vessel density and apoptosis in the tissues collected from women with endometriosis, adenomyosis and uterine myoma who were treated with or without GnRHa therapy. METHODS Biopsy specimens were collected from lesions, myometria and corresponding endometria of 45 women with ovarian endometrioma, 35 women with adenomyosis and 56 women with uterine myoma. A fraction of these women were treated with GnRHa therapy for a variable period of 3-6 months before surgery. We performed immunohistochemical analysis of CD68, a macrophage (Mvarphi) marker and von Willebrand factor (VWF), a vessel marker, using respective antibodies. Changes in apoptosis were examined using TdT-mediated dUTP-biotin nick end-labeling assay and by the immunoexpression of activated caspase-3 in tissues after GnRHa therapy. RESULTS The infiltration of CD68-positive Mvarphi and VWF-positive micro-vessel density were significantly decreased in the endometria of women with endometriosis, adenomyosis and uterine myoma in the GnRHa-treated group when compared with that in the non-treated group. Marked decreases in inflammatory and angiogenic responses were observed in lesions and myometria of these diseases. When compared with the non-treated group, a significant increase in apoptotic index (apoptotic cells per 10 mm(2) area) and quantitative-histogram scores of activated caspase-3 after GnRHa therapy were observed in the eutopic endometria, lesions and myometria of these diseases. CONCLUSIONS GnRHa was able to markedly reduce the inflammatory reaction and angiogenesis and to significantly induce apoptosis in tissues derived from women with endometriosis, adenomyosis and uterine myoma. These multiple biological effects at the tissue level may be involved in the regression of these reproductive diseases.

Expression of estrogen and progesterone receptors in endometrium and peritoneal endometriosis: an immunohistochemical and in situ hybridization study

OBJECTIVE To clarify the role of ovarian steroids in the development and progression of endometriosis, estrogen receptors (ERs) and progesterone receptors (PRs) were localized by immunohistochemistry, and ER messenger RNA (mRNA) was detected by in situ hybridization in the uterine endometrium and in normal and altered pelvic peritoneum. DESIGN Retrospective and prospective study. SETTING Nagasaki University School of Medicine, Nagasaki, Japan. PATIENT(S) A retrospective study of 61 formalin-fixed uterine endometria and normal and altered pelvic peritonea from patients suffering from various gynecologic diseases was conducted. In addition, in 22 fresh frozen tissue specimens, ER mRNA expression was evaluated prospectively. MAIN OUTCOME MEASURE(S) In formalin-fixed tissues, ER and PR were localized immunohistochemically. The results of immunohistochemical staining were scored from 0 to 4, depending on the signal intensity and frequency of positive cells. In fresh frozen specimens, ER mRNA expression was assessed by nonradioactive in situ hybridization using thymine-thymine dimerized oligonucleotide probes. RESULTS The highest score of ERs and PRs was observed in the epithelial and stromal cells of the normal uterine endometrium at the early proliferative phase of the menstrual cycle. The ER and PR scores declined throughout the secretory phase. In typical endometriotic lesions, the ER and PR scores were constantly high independent of the menstrual cycle. The expression pattern of ER mRNA was mostly in parallel with that of ERs. In typical endometriosis, ERs and PRs were found in both glandular epithelial cells and their surrounding stromal cells. Expression of ER mRNA was found in typical endometriotic peritonea and in pelvic peritoneum with columnar epithelial cells, but not in normal pelvic peritoneum (mesothelium). Estrogen receptors and PRs were negative in mesothelium, but were positive in the nuclei of fibroblasts in the connective tissue. CONCLUSION(S) We demonstrated the expression of ERs, ER mRNA, and PRs in the columnar cells in pelvic peritonea and typical endometriosis, but not in normal mesothelium. These results suggest that endometriosis may originate from the columnar cells with ERs and PRs in the pelvic peritoneal lining.

Immunopathogenesis of Pelvic Endometriosis: Role of Hepatocyte Growth Factor, Macrophages and Ovarian Steroids

Endometriosis, a chronic disease characterized by endometrial tissue located outside the uterine cavity is associated with chronic pelvic pain and infertility. However, an in‐depth understanding of the pathophysiology of endometriosis is still elusive. It is generally believed that besides ovarian steroid hormones, the growth of endometriosis can be regulated by innate immune system in pelvic microenvironment by their interaction with endometrial cells and immune cells. We conducted a series of studies in perspectives of pelvic inflammation that is triggered primarily by bacterial endotoxin (lipopolysacccharide) and is mediated by toll‐like receptor 4 and showed their involvement in the development of pelvic endometriosis. As a cellular component of innate immune system, macrophages were found to play a central role in inducing pelvic inflammatory reaction. We further report here that peritoneal macrophages retain receptors encoding for estrogen and progesterone and ovarian steroids also participate in producing an inflammatory response in pelvic cavity and are involved in the growth of endometriosis either alone or in combination with hepatocyte growth factor (HGF). As a pleiotropic growth factor, HGF retains multifunctional role in endometriosis. We describe here the individual and step‐wise role of HGF, macrophages and ovarian steroid hormones and their orchestrated involvement in the immunopathogenesis of pelvic endometriosis.

Changes in serum anti-Müllerian hormone levels may predict damage to residual normal ovarian tissue after laparoscopic surgery for women with ovarian endometrioma.

We measured serum anti-Müllerian hormone levels before and after surgery in women undergoing unilateral and monolocular cystectomy for benign ovarian diseases. Comparing to control benign cysts, we found a significant decline in serum anti-Müllerian hormone levels with consequent depletion of follicles in tissue specimens after surgery for women with ovarian endometrioma.

Immunoexpression of hepatocyte growth factor and c-Met receptor in the eutopic endometrium predicts the activity of ectopic endometrium

OBJECTIVE To investigate the mitogenic and angiogenic activity of the eutopic and ectopic endometrium throughout the menstrual cycle and to examine whether the activity of the eutopic endometrium is useful to predict greater activity of the ectopic endometrium. DESIGN Controlled clinicopathologic study using intact tissue. SETTING Nagasaki University School of Medicine, Nagasaki, Japan. PATIENT(S) Fifteen infertile women with pelvic endometriosis and 10 women without endometriosis undergoing laparoscopy. INTERVENTION(S) Biopsies from the ectopic endometrium and the corresponding eutopic endometrium were collected. Immunohistochemical staining was performed using respective antibodies, and a computer analyzed modified quantitative-histogram (Q-H) score was used to quantify immunostaining. MAIN OUTCOME MEASURE(S) The immunoreactions of hepatocyte growth factor (HGF), its receptor, c-Met, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), and von Willebrand factor (VWF) in eutopic and ectopic endometrium were examined, and their relation with different revised American Society for Reproductive Medicine (r-ASRM) stages and the morphology of endometriosis was evaluated. RESULT(S) The immunoexpressions of HGF and c-Met were significantly higher in the eutopic endometrium of patients with endometriosis than in that of controls. The Q-H scores of HGF, c-Met, VEGF, PCNA, and microvessel density (MVD) were markedly higher in red peritoneal lesions when compared with other lesions. The Q-H scores did not reveal r-ASRM stage-dependent variation in any of these markers. We observed a significant correlation between the immunoexpressions of HGF, c-Met, and PCNA or microvessel counts. When we combined the Q-H scores of the glandular epithelium and stroma, we found that increased activity of the eutopic endometrium as measured by the immunoreaction of HGF, c-Met, VEGF, PCNA, and MVD was similar to highly active red lesions and was significantly higher than that of controls and other lesions. CONCLUSION(S) Immunoexpression of HGF and c-Met in the eutopic endometrium of patients with pelvic endometrioisis is possibly useful to predict greater activity of the ectopic endometrium.

Escherichia coli contamination of menstrual blood and effect of bacterial endotoxin on endometriosis

To test the hypothesis that bacterial contamination of menstrual blood could be a local biologic event in the development of endometriosis, menstrual blood was cultured and bacterial endotoxin was measured in menstrual blood and peritoneal fluid. Our results suggest that compared with control women, higher colony formation of Escherichia coli in menstrual blood and endotoxin levels in menstrual fluid and peritoneal fluid in women with endometriosis may promote Toll-like receptor 4-mediated growth of endometriosis.

Toll-Like Receptors in Innate Immunity: Role of Bacterial Endotoxin and Toll-Like Receptor 4 in Endometrium and Endometriosis

Macrophages, dendritic cells, and Toll-like receptors (TLRs) are integral components of the innate immune system. This rapidly reactive system responds immediately to infectious or other non-self agents, thereby inducing an inflammatory response to protect the host until the activation of the slower adaptive immune system. The fundamentals of the innate immune system, functional characteristics of TLRs, and signaling pathways of TLR4 are discussed for the easy understanding by readers. Studies showed that the growth and progression of endometriosis continue even in ovariectomized animals. This indicates that besides ovarian steroid hormones, the growth of endometriosis can be regulated by the innate immune system in the pelvic environment. As a component of the innate immune system, increased infiltration of macrophages has been described in the intact tissue and peritoneal fluid of women with endometriosis. In this review article, we discuss the role of bacterial endotoxin and TLR4 in endometrium and endometriosis and outline the involvement of endotoxin in causing adverse reproductive outcome.

Immunohistochemical Study of Angiogenic Factors in Endometrium and Endometriosis

The proliferative activity and angiogenic factors of pelvic endometriosis and uterine endometrium and their correlation with different pigmented lesions are important throughout the menstrual cycle. The proliferative activity and/or expression of angiogenic factors appears to be different between endometrium and endometriosis, and also different in each pigmented lesion. Immunohistochemical studies using computerized image analysis have shown that in normal endometrium, the PCNA index shows cyclic variation, but no cyclic change is observed in endometriosis. In the pelvic peritoneum, the PCNA index is higher in red lesions compared to black and white lesions. There is no difference in VEGF expression among different pigmented lesions. However, a cyclic variation of VEGF concentration was found in the peritoneal fluid of patients with endometriosis. As a newly determined angiogenic factor, the number of endoglin-positive cells and the mean endothelial area were higher in red lesions than in black or white lesions. These results suggest that the pathogenesis and activity of endometriosis possibly depends on internal angiogenesis. The cellular activity of endometriotic lesions may not necessarily coincide with the regulation of angiogenesis, and may not be synchronized in each pigmented pelvic lesion of endometriosis.

Higher activity by opaque endometriotic lesions than nonopaque lesions

Background.  Higher activity by early endometriosis than advanced endometriosis has been reported. However, the pattern of activity in individual colored endometriotic lesions in pelvic cavity is unknown. We investigated the variation in activity of the different colored morphologic lesions as proposed by the current revised American Society of Reproductive Medicine (ASRM) classification in women with endometriosis.

Modified Reduction Surgery for Adenomyosis

We describe the preliminary clinical results of a modified method of reduction surgery for easy approach and effective removal of lesions in women with adenomyosis. Old classical reduction surgery was performed in 5 women with imaging diagnosis of adenomyosis who were selected retrospectively among 104 patients undergoing conservative surgery. A transverse H incision method in the reduction surgery was applied to 6 of 83 patients wishing to preserve fertility. Benefit in operative procedure, complications, patients’ compliance, and pregnancy outcome were analyzed and compared between 5 women with the classical method and 6 women with the H incision method for adenomyosis. No apparent difference was observed in the operation time, blood loss and volume of excised specimens between these two groups. The major complication of perforation during surgery occurred in 2 patients (40%) by the classical method and in only 1 patient (17%) by the H incision technique. The subjective relief of pain was relatively more evident in the modified than in the classical group. There was no case of pregnancy in the classical group; however, 1 patient conceived spontaneously 4 months after operation by this H incision procedure. Our H incision technique may be considered a useful method for an easy surgical approach and satisfactory removal of adenomyotic lesions and may be better than the old classical method of reduction surgery.