Koichi Hiraki

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.

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.

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.

Cell proliferation effect of GnRH agonist on pathological lesions of women with endometriosis, adenomyosis and uterine myoma

BACKGROUND We recently demonstrated the effect of gonadotrophin-releasing hormone agonist (GnRHa) on tissue inflammation, angiogenesis and apoptosis in endometriosis, adenomyosis and uterine myoma. Here, we investigated expression of GnRH receptors (GnRHRs) and effect of GnRHa on the proliferation of cells derived from endometria and pathological lesions of women with these reproductive diseases. METHODS Biopsy specimens were collected from lesions and corresponding endometria of 35 women with pelvic endometriosis, 45 women with ovarian endometrioma, 35 women with adenomyosis and 56 women with uterine myoma during laparoscopy or laparotomy. The gene and protein expressions of GnRHR in eutopic/ectopic cells and tissues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The immunoreactivity of GnRHR in tissue was analysed by quantitative-histogram (Q-H) scores. The exogenous effect of GnRHa on cell proliferation was examined by 5-bromo-2-deoxyuridine incorporation assay. The Ki-67-immunoreactive cell proliferation index was analysed in biopsy specimens derived from GnRHa-treated and -non-treated women. RESULTS Types I and II GnRHRs mRNA and proteins were expressed in eutopic endometria and pathological lesions derived from women with endometriosis, adenomyosis and uterine myoma. GnRHR expression was the highest in the menstrual phase when compared with other phases of the menstrual cycle. Higher Q-H scores of GnRHR immunoreaction were found in blood-filled opaque red lesions than in other peritoneal lesions. Exogenous treatment with GnRHa significantly suppressed the proliferation of cells derived from respective endometria and pathological lesions when compared with GnRHa-non-treated cells. CONCLUSIONS Local tissue expression of GnRHR was detected in endometriosis, adenomyosis and uterine myoma. In addition to a hypo-estrogenic effect, a direct anti-proliferative effect of GnRHa may be involved in the regression of these reproductive diseases with consequent remission of clinical symptoms.

Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70

BACKGROUND We investigated the role of human heat-shock protein 70 (Hsp70) in Toll-like receptor 4 (TLR4)-mediated growth of endometriosis. METHODS TLR4 expression was examined in macrophages (M) isolated in primary culture from the peritoneal fluid of women with and without endometriosis. The production of a number of macromolecules by non-treated M, Hsp70-treated M and after treatment with anti-TLR4 antibody was examined by enzyme linked immunosorbent assay (ELISA). The single and combined effects of Hsp70 and lipopolysaccharide (LPS) on the growth of endometrial stromal cells were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation study. Hsp70 levels in eutopic and ectopic endometria were measured by ELISA. RESULTS TLR4 was detected in isolated M at protein and gene level. Hsp70 (10 microg/ml) significantly stimulated the production of hepatocyte growth factor, vascular endothelial cell growth factor, interleukin-6 and tumor necrosis factor alpha by M derived from women with endometriosis compared with M derived from women with no endometriosis (P < 0.05 for each). This effect of Hsp70 was abrogated after pretreatment of M with anti-TLR4 antibody. BrdU incorporation indicated that Hsp70 significantly enhanced the growth of endometrial stromal cells ( approximately 50% increase) from women with endometriosis compared to non-treated cells. A synergistic effect on cell proliferation was observed between exogenous Hsp70 and LPS and this was significantly suppressed by pretreatment of cells with anti-TLR4 antibody (P < 0.05). Tissue levels of Hsp70 were significantly higher in the eutopic endometria (P < 0.05) and opaque red lesions (P < 0.01) derived from women with endometriosis than from other peritoneal lesions or from women with no endometriosis. CONCLUSIONS A prominent stress reaction was observed in blood-filled opaque red peritoneal lesions. Human Hsp70 induces pelvic inflammation and may be involved in TLR4-mediated growth of endometrial cells derived from women with endometriosis.

Peritoneal fluid and serum levels of hepatocyte growth factor may predict the activity of endometriosis

Background. The suitable parameter in PF as well as in serum that may predict the activity of endometriosis is not well described. Therefore, we tried to examine the peritoneal fluid (PF) and serum concentrations of hepatocyte growth factor (HGF) in different revised American Society of Reproductive Medicine (r‐ASRM) staging and morphologic appearances of endometriosis in an attempt to determine whether HGF can be clinically useful to predict the activity of pelvic endometriosis.

Intra-uterine microbial colonization and occurrence of endometritis in women with endometriosis

STUDY QUESTION Is there any risk of intra-uterine bacterial colonization and concurrent occurrence of endometritis in women with endometriosis? SUMMARY ANSWER An increase in intra-uterine microbial colonization and concurrent endometritis occurred in women with endometriosis that was further increased after GnRH agonist (GnRHa) treatment. WHAT IS KNOWN ALREADY Higher bacterial contamination of menstrual blood and increased endotoxin level in menstrual and peritoneal fluids have been found in women with endometriosis than in control women. However, information on intra-uterine microbial colonization across the phases of the menstrual cycle and possible occurrence of endometritis in women with endometriosis is still lacking. STUDY DESIGN, SIZE AND DURATION This is a case-controlled study with prospective collection of vaginal smears/endometrial samples from women with and without endometriosis and retrospective evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODS Vaginal smears and endometrial smears were collected from 73 women with endometriosis and 55 control women. Twenty of the women with endometriosis and 19 controls had received GnRHa therapy for a period of 4-6 months. Vaginal pH was measured by intra-vaginal insertion of a pH paper strip. The bacterial vaginosis (BV) score was analyzed by Gram-staining of vaginal smears and based on a modified Nugent-BV scoring system. A panel of bacteria was analyzed by culture of endometrial samples from women treated with GnRHa or not treated. Immunohistochemcial analysis was performed using antibody against Syndecan-1 (CD138) and myeloperoxidase in endometrial biopsy specimens from women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE A significant shifting of intra-vaginal pH to ≥4.5 was observed in women with endometriosis compared with control women (79.3 versus 58.4%, P < 0.03). Compared with untreated women, use of GnRHa therapy also shifted vaginal pH to ≥4.5 in both control women (P = 0.004) and in women with endometriosis (P = 0.03). A higher risk of increasing intermediate flora (total score, 4-6) (P = 0.05) was observed in women with endometriosis who had GnRHa treatment versus untreated women. The number of colony forming units (CFU/ml) of Gardnerella, α-Streptococcus, Enterococci and Escherichia coli was significantly higher in endometrial samples from women with endometriosis than control women (P < 0.05 for each bacteria). GnRHa-treated women also showed significantly higher colony formation for some of these bacteria in endometrial samples than in untreated women (Gardnerella and E. coli for controls; Gardnerella, Enterococci and E. coli for women with endometriosis, P < 0.05 for all). Although there was no significant difference in the occurrence of acute endometritis between women with and without endometriosis, both GnRHa-treated controls and women with endometriosis had a significantly higher occurrence of acute endometritis (P = 0.003 for controls, P = 0.001 for endometriosis versus untreated women). Multiple analysis of covariance analysis revealed that an intra-vaginal pH of ≥4.5 (P = 0.03) and use of GnRHa (P = 0.04) were potential factors that were significantly and independently associated with intra-uterine microbial colonization and occurrence of endometritis in women with endometriosis. These findings indicated the occurrence of sub-clinical uterine infection and endometritis in women with endometriosis after GnRHa treatment. LIMITATIONS, REASONS FOR CAUTION We cannot exclude the introduction of bias from unknown previous treatment with immunosuppressing or anti-microbial agents. We have studied a limited range of bacterial species and used only culture-based methods. More sensitive molecular approaches would further delineate the similarities/differences between the vaginal cavity and uterine environment. WIDER IMPLICATIONS OF THE FINDINGS Our current findings may have epidemiological and biological implications and help in understanding the pathogenesis of endometriosis and related disease burden. The worsening of intra-uterine microbial colonization and higher occurrence of endometritis in women with endometriosis who were treated with GnRHa identifies some future therapeutic avenues for the management, as well as prevention of recurrence, of endometriosis. Further studies are needed to examine intra-uterine colonization of a broad range of common bacteria as well as different viruses and their role in the occurrence of endometritis. STUDY FUNDING/COMPETING INTERESTS This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan. There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER Not applicable.

Pelvic pain in women with ovarian endometrioma is mostly associated with coexisting peritoneal lesions

STUDY QUESTION Is the occurrence of pelvic pain in women with ovarian endometrioma associated with coexisting peritoneal lesions (PLs)? SUMMARY ANSWER Pelvic pain in women with ovarian endometrioma is usually associated with coexisting PLs. An increased tissue inflammatory reaction with elevated prostaglandin (PG) production may be responsible for the generation of pain. WHAT IS KNOWN ALREADY Severe pelvic pain in women with ovarian endometrioma is reported to be associated with deeply infiltrating endometriosis. However, information on pelvic pain in women with ovarian endometriosis with and without coexistent peritoneal superficial lesions is limited. STUDY DESIGN, SIZE AND DURATION Retrospective clinical study with case-controlled biological research using prospectively collected tissue samples derived from women with and without endometriosis and their retrospective evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODS We performed a retrospective cohort study conducted in 2988 cases who had laparoscopic surgery for indications of ectopic pregnancy, tubal infertility and other benign gynecologic diseases. We analyzed the occurrence of pelvic pain in the cases with ovarian endometrioma according to the distribution of coexisting PLs and pattern of intrapelvic adhesions. Inflammatory reaction of eutopic and ectopic endometria was measured by immunoreaction to macrophage marker, CD68. The tissue expression of cyclooxygenase (COX) 2 was examined by immunohistochemistry and tissue concentrations of PG F2α were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE Among the 2988 surgical cases, 350 (11.7%) were found to have ovarian endometrioma at laparoscopy. Coexisting PLs were present in 269 of these women and in this group 85.4% of cases experienced pelvic pain and 14.6% had no pain. In contrast, among the 81 women with ovarian endometrioma only, 38.3% cases experienced pelvic pain and 61.7% cases had no pain and the difference between the groups was statistically significant (P < 0.01). The infiltration of CD68-immunoreactive macrophages was significantly higher in the eutopic and ectopic endometria of women with peritoneal endometriosis than in ovarian endometrioma. The tissue expression of COX2 and levels of PGF2α were significantly higher in both the eutopic and ectopic endometria derived from women with peritoneal endometriosis than in similar tissues derived from women with ovarian endometrioma. LIMITATIONS, REASONS FOR CAUTIONS Lack of evaluation in the detection of general or disseminated deeply infiltrating endometriosis in the pelvic cavity could be a bias or limitation in this study. Further multicenter prospective studies are needed to strengthen our current findings. WIDER IMPLICATIONS OF THE FINDINGS Our findings may provide some new insights to understand the physiopathology of pelvic pain in women with ovarian cystic endometriosis and may hint at proper surgical manipulation to prevent the recurrence of pelvic pain in these women. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology of Japan. There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER Not applicable.