Association between ovarian endometrioma and ovarian reserve.

Endometrioma is one of the main pathologies of endometriosis, though its pathogenesis still remains enigmatic. Ovarian reserve is defined as the number and quality of the follicles left in the ovary at any given time. The cause of infertility in women with endometriosis is multifactorial. Diminished ovarian reserve is major concern in women with endometriosis-associated infertility. Cystectomy for endometriomas could negatively impact on post-operative ovarian reserve. Some women had surgery for endometriomas suffer from poor ovarian response, which directly affects treatment results. In addition, endometriomas themselves may be a cause of diminished ovarian reserve. Destruction of normal histological structure in ovarian cortex may affect dormancy of primordial follicles. Therefore, determination of ovarian reserve may serve as an important role in the management of reproductive health of women with endometriosis. Although the knowledge on the physiology of follicular development and mechanism of maintenance of ovarian reserve are rapidly accumulating, results obtained by ovarian reserve testing after surgery should be carefully evaluated according to the time-points and selected test. Further investigation on this issue is warranted.

Outcome of conservative laparoscopic surgery for adnexal torsion through one-stage or two-stage operation.

AIM: We investigated the outcome on ovarian appearance and occurrence of adhesion after conservative laparoscopic surgery for adnexal torsion during reproductive age. MATERIAL AND METHODS: From April 2009 to September 2012, we treated patients with clinically suspected adnexal torsion who desired future pregnancy. We performed conservative surgery, such as cystectomy or detorsion at one-stage operation, but switched to salpingo-oophorectomy in complicated cases. We evaluated adnexal condition and pattern of adhesion by careful assessment with two-stage laparoscopy or second-look laparoscopy after first surgery. RESULTS: Mean age of patients was 25 ± 8 years. Among 37 patients with suspected adnexal torsion, 18 (49%) had adnexal torsion at first surgery. Conservative treatment was carried out in 14 of 18 cases. We obtained informed consent for second-look laparoscopy or two-stage operation in six of these 14 cases. Among these six patients, two cases were treated with only detorsion by one-stage operation and cystectomy was performed in the other four cases at first operation. At subsequent surgery, the ovary appeared normal in six cases with occurrence of mild to moderate adhesion around the adnexal lesion. Of note, two cases with para-ovarian cyst had torsion that showed complete tubal occlusions and associated severe adhesions. No major complications (peritonitis, thrombotic emboli) were observed after conservative laparoscopic surgery. CONCLUSION: Conservative laparoscopic surgery is a safe procedure to preserve ovarian function in women with adnexal torsion. Careful attention and measures should be considered during follow-up management with the fact in mind that adhesion is a common occurrence and even tubal occlusion may occur in some cases.

Involvement of hepatocyte growth factor-induced epithelial-mesenchymal transition in human adenomyosis.

Adenomyosis is commonly believed to arise from the basalis endometrium. As an estromedin growth factor, hepatocyte growth factor (HGF) exhibits multiple functions in endometriosis, a disease commonly believed to arise from the functionalis endometrium. Here, we investigated the role of HGF in the occurrence of epithelial-mesenchymal transition (EMT) in adenomyosis. Full-thickness-biopsy specimens from endometrium to myometrium were collected after hysterectomy from women with and without adenomyosis. The relationship between HGF and E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell markers) was examined at the gene and protein levels using endometrial epithelial cells (EECs) in culture and tissues by quantitative RT-PCR and immunohistochemistry. The gene and protein expressions of two transcriptional repressors of E-cadherin, SLUG and SNAIL, were examined using Ishikawa cells and in response to HGF and estrogen (E2). HGF down-regulated E-cadherin and up-regulated N-cadherin mRNA expression in EECs, and an inverse relationship in protein expression between HGF and E-cadherin was observed in basalis endometria derived from women with diffuse and focal adenomyosis. HGF induced morphological changes of EECs from a cobblestone-like appearance to spindle-shaped cells and promoted migration of EECs. Ishikawa cells exhibited up-regulation of SLUG/SNAIL gene expression in response to both HGF and E2 with an additive effect between them. HGF- and E2-promoted SLUG/SNAIL gene expression was significantly abrogated after pretreatment of cells with anti-HGF antibody or ICI 182720, an estrogen receptor antagonist. HGF may be involved in gland invagination deep into the myometrium by inducing EMT at the endo-myometrial junction in women with adenomyosis.

17ß-estradiol and lipopolysaccharide additively promote pelvic inflammation and growth of endometriosis.

Endometriosis is a multifactorial disease mostly affecting women of reproductive age. An additive effect between inflammation and stress reaction on the growth of endometriosis has been demonstrated. Here we investigated the combined effect between 17ß-estradiol (E2) and lipopolysaccharide (LPS) on pelvic inflammation and growth of endometriotic cells. Peritoneal fluid was collected from 46 women with endometriosis and 30 control women during laparoscopy. Peritoneal macrophages (Mφ) and stromal cells from eutopic/ectopic endometrial stromal cells (ESCs) were isolated from 10 women each with and without endometriosis in primary culture. Changes in cytokine secretion (interleukin 6 [IL-6] and tumor necrosis factor α [TNF-α]) by Mφ and proliferation of ESCs in response to single and combined treatment with E2 and LPS were measured by enzyme-linked immunosorbent assay and by bromodeoxyuridine incorporation assay, respectively. A significantly increased secretion of IL-6 and TNF-α in Mφ culture media was found in response to E2 (10(-8) mol/L) compared to nontreated Mφ. This effect of E2 was abrogated after pretreatment of cells with ICI 182720 (10(-6) mol/L; an estrogen receptor [ER] antagonist). Combined treatment with E2 and LPS (10 ng/mL) additively promoted IL-6 and TNF-α secretion by peritoneal Mφ and growth of eutopic/ectopic ESCs. The additive effects of E2 + LPS on cytokine secretion and growth of ESCs were effectively suppressed after combined blocking of ER and Toll-like receptor 4. An additive effect was observed between E2 and LPS on promoting proinflammatory response in pelvis and growth of 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.

Evaluation of obstetrical factors related to Sheehan syndrome.

AIM: To evaluate factors related to the occurrence of Sheehan syndrome. METHODS: The obstetrical disseminated intravascular coagulation score, total volume of hemorrhage, shock index, level of consciousness at the time of shock occurrence and pituitary magnetic resonance imaging findings were evaluated in nine women who showed massive hemorrhage during delivery. These clinical outcomes were analyzed in all these patients who were prospectively followed-up to identify any possible occurrence of Sheehan syndrome. RESULTS: Compared to six women with non-Sheehan syndrome, three women who were diagnosed with Sheehan syndrome showed significant elevation of the obstetrical disseminated intravascular coagulation score, decrease in the level of consciousness during shock and remarkable pituitary gland atrophic change with an empty sella turcica detected by pituitary magnetic resonance imaging. The volume of hemorrhage during delivery and shock index were not significantly different between these two groups of women. CONCLUSION: Careful attention and follow-up should be paid to women with post-partum massive hemorrhage for early detection and management of women with Sheehan syndrome.

Occult microscopic endometriosis: undetectable by laparoscopy in normal peritoneum.

STUDY QUESTION: Is there any occurrence of hidden (occult) endometriotic lesions in normal peritoneum of women with and without visible endometriosis? SUMMARY ANSWER: We detected a slightly higher occurrence of occult microscopic endometriosis (OME) in normal peritoneum of women with visible endometriosis than in control women. WHAT IS KNOWN ALREADY: Based on a small number of cases, the concept of invisible microscopic endometriosis in visually normal peritoneum has been reported for more than a decade but there is controversy regarding their tissue activity and clinical significance. STUDY DESIGN, SIZE, DURATION: This case-controlled research study was conducted with prospectively collected normal peritoneal samples from 151 women with and 62 women without visible endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Normal peritoneal biopsy specimens from different pelvic sites of were collected during laparoscopy. A histological search of all peritoneal biopsy specimens for the detection of invisible endometriosis was done by immunoreaction to Ber-EP4 (epithelial cell marker), CD10 (stromal cell marker) and Calretinin (mesothelial cell marker). Tissue expression of estrogen/progesterone receptors (ER/PR) and cell proliferation marker, Ki-67, was performed by immunohistochemistry to identify tissue activity. MAIN RESULTS AND THE ROLE OF CHANCE: Three different patterns of OME were detected based on (I) the presence of typical gland/stroma, (II) reactive hyperplastic change of endometrioid epithelial cells with surrounding stroma and (III) single-layered epithelium-lined cystic lesions with surrounding stroma. A higher tendency toward the occurrence of OME was found in women with visible endometriosis (15.2%, 23/151) compared with control women (6.4%, 4/62) (P = 0.06, χ(2) test). The epithelial cells and/or stromal cells of OME lesions were immunoreactive to Ber-EP4 and CD10 but not reactive to Calretinin. ER and PR expression was observed in all patterns of OME lesions. Ki-67 index was significantly higher in pattern I/II OME lesions than in pattern III OME lesions (P< 0.05 for each). LIMITATIONS, REASONS FOR CAUTION: Bias in the incidence rate of OME lesions in this study cannot be ignored, because we could not analyze biopsy specimens from the Pouch of Douglas of women with revised classification of the American Society of Reproductive Medicine Stage III-IV endometriosis due to the presence of adhesions in the pelvis. WIDER IMPLICATIONS OF THE FINDINGS: We re-confirmed a decade long old concept of invisible (occult) endometriosis in visually normal peritoneum of women with visible endometriosis. The existence of a variable amount of tissue activity in these occult lesions may contribute to the recurrence/occurrence of endometriosis or persistence/recurrence of pain manifestation in women even after successful ablation or excision of visible lesions by laparoscopy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science. There is no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: Not applicable.

Uterine preservation surgery for placental polyp.

AIM: To examine clinical and surgical performances of cases with placental polyps in which uterine preservation surgery was conducted. METHODS: During the period September 2002 to April 2009, we examined eight cases (hysteroscopic resection, six cases; laparotomy, one case; dilatation and curettage, one case) diagnosed with placental polyp that had been treated with polyp extraction surgery. Imaging evaluation was done using magnetic resonance imaging and 2-D ultrasound. RESULTS: Three of the eight cases (37.5%) had been first-time pregnancies. Most of our cases experienced minimal surgical manipulation after medical abortion. Among them, six cases (75%) were mid-term medical abortions, one case (12.5%) received no treatment after spontaneous abortion, and one case (12.5%) had postsurgical abortion (dilatation and curettage). All cases showed variable amount of blood flow in the internal mass and myometrium by color Doppler ultrasound. Magnetic resonance imaging angiography showed contrast effects in the intrauterine cavity and myometrium in selected cases. The average duration from diagnosis to surgery was 32 days (range, 11-105). Color Doppler revealed a reduction in blood flow in five cases during the waiting period until surgery with an average blood loss of 10 g (range, 0-20) during surgery. CONCLUSION: Use of color Doppler ultrasound may be useful in diagnosing placental polyp. Although hysteroscopic resection of placental polyp is effective in patients hoping for uterine preservation, delaying timing of surgery may reduce blood loss during operative procedure.

Toll-like receptor system and endometriosis.

Endometriosis is an estrogen-dependent chronic inflammatory condition associated with variable degrees of pelvic pain and infertility. Studies have 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 innate immune system, increased infiltration of macrophages has been described in the intact tissue and peritoneal fluid of women with endometriosis. Different immune cells and dendritic cells express Toll-like receptors (TLR) and exhibit functional activity in response to microbial products. In this review article, we discuss the role of the TLR system in endometrium and endometriosis and outline the involvement of cytokines/endotoxin in causing adverse reproductive outcome. In the first part of this review article, the fundamentals of innate immune system, functional characteristics of TLR and signaling pathways of TLR4 are discussed for easy understanding by the readers.

Additive effects of inflammation and stress reaction on Toll-like receptor 4-mediated growth of endometriotic stromal cells.

STUDY QUESTION: Is there any combined effect between inflammation and stress reaction on Toll-like receptor 4 (TLR4)-mediated growth of endometriotic cells? SUMMARY ANSWER: A combined effect of local inflammation and stress reaction in the pelvic environment may be involved in TLR4-mediated growth of endometriotic stromal cells. WHAT IS KNOWN ALREADY: In endometriosis, higher endotoxin levels in the menstrual fluid (MF) and peritoneal fluid (PF) and higher tissue concentrations of human heat shock protein 70 (HSP70) in the eutopic and ectopic endometria promote TLR4-mediated growth of endometriotic cells. STUDY DESIGN, SIZE AND DURATION: This is a case-controlled research study with prospective collection and retrospective evaluation of sera, MF, PF and endometrial tissues from 43 women with and 20 women without endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: PF was collected from 43 women with endometriosis and 20 control women during laparoscopy. Sera and endometrial biopsy specimens were collected from a proportion of these women. MF was collected from a separate population of 20 women with endometriosis and 15 control women. HSP70 concentrations in sera, MF, PF and in culture media were measured by ELISA. Gene expression of HSP70 by endometrial cells in response to lipopolysaccharide (LPS) was examined by qRT-PCR. The individual and combined effects of LPS and HSP70 on the secretion of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by PF-derived macrophages (M[Symbol: see text]) were examined by ELISA, while their effects on endometrial cell proliferation were examined by bromodeoxyuridine and [(3)H]-thymidine incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Concentrations of HSP70 were maximal in MF, intermediate in PF and the lowest in sera. In MF and PF, HSP70 levels were higher in women with endometriosis than in controls. LPS stimulated gene expression and secretion of HSP70 by eutopic endometrial stromal cells (ESCs) and this effect was abrogated after pretreatment of cells with an anti-TLR4 antibody. This effect was significantly higher for ESCs derived from women with endometriosis than for ESCs from control women. Exogenous treatment with either HSP70 or LPS significantly stimulated the production of IL-6 and TNFα by M[Symbol: see text] and promoted the proliferation of ESCs, and a significant additive effect between LPS and HSP70 was observed. While individual treatment with either polymyxin B, an LPS antagonist, or anti-HSP70 antibody was unable to suppress the combined effects of LPS and HSP70 on cytokine secretion or ESC proliferation, pretreatment of cells with the anti-TLR4 antibody was able to significantly suppress their combined effects. LIMITATIONS, REASONS FOR CAUTIONS: Further studies are needed to examine the mutual role between other secondary inflammatory mediators and endogenous stress proteins in promoting pelvic inflammation and growth of endometriotic stromal cells. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that endotoxin and HSP70 are mutually involved in a stress reaction and in inflammation. A combined effect between local inflammation and a stress reaction in pelvic environment may be involved in TLR4-mediated growth of endometriotic cells. Since endometriosis is a multi-factorial disease, it is difficult to explain uniformly its growth regulation by a single factor. Our findings may provide some new insights in understanding the physiopathology or pathogenesis of endometriosis and may hold new therapeutic potential. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grants-in-Aid for Scientific Research (grant no. 16591671 and 18591837) from the Ministry of Education, Sports, Culture, Science and Technology of Japan (to K.N.K.). 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.

Role of prostaglandin E2 in bacterial growth in women with endometriosis.

STUDY QUESTION: Can prostaglandin E(2) (PGE(2)) in menstrual and peritoneal fluid (PF) promote bacterial growth in women with endometriosis? SUMMARY ANSWER: PGE(2) promotes bacterial growth in women with endometriosis. WHAT IS KNOWN ALREADY: Menstrual blood of women with endometriosis is highly contaminated with Escherichia coli (E. coli) compared with that of non-endometriotic women: E. coli-derived lipopolysaccharide (LPS) promotes the growth of endometriosis. STUDY DESIGN, SIZE AND DURATION: Case-controlled biological research with a prospective collection of body fluids and endometrial tissues from women with and without endometriosis with retrospective evaluation. PARTICIPANTS/MATERIALS, SETTING AND METHODS: PF and sera were collected from 58 women with endometriosis and 28 women without endometriosis in an academic research laboratory. Menstrual blood was collected from a proportion of these women. Macrophages (Mφ) from PF and stromal cells from eutopic endometria were isolated in primary culture. The exogenous effect of PGE(2) on the replication of E. coli was examined in a bacterial culture system. Levels of PGE(2) in different body fluids and in the culture media of Mφ and stromal cells were measured by ELISA. The effect of PGE(2) on the growth of peripheral blood lymphocytes (PBLs) was examined. MAIN RESULTS AND THE ROLE OF CHANCE: The PGE(2) level was 2-3 times higher in the menstrual fluid (MF) than in either sera or in PF. A significantly higher level of PGE(2) was found in the MF and PF of women with endometriosis than in control women (P < 0.05 for each). Exogenous treatment with PGE(2) dose dependently increased E. coli colony formation when compared with non-treated bacteria. PGE(2)-enriched MF was able to stimulate the growth of E. coli in a dilution-dependent manner; this effect was more significantly enhanced in women with endometriosis than in control women (P < 0.05). PGE(2) levels in the culture media of LPS-treated Mφ/stromal cells were significantly higher in women with endometriosis than in non-endometriosis (P < 0.05 for each). Direct application of PGE(2) and culture media derived from endometrial Mφ or stromal cells significantly suppressed phytohemagglutinin-stimulated growth of PBLs. LIMITATIONS AND REASONS FOR CAUTION: Further studies are needed to examine the association between PGE(2)-stimulated growth of E. coli and endotoxin level and to investigate the possible occurrence of sub-clinical infection within vaginal cavity. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may provide some new insights to understand the physiopathology or pathogenesis of the mysterious disease endometriosis and may hold new therapeutic potential. 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.

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.

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.

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.

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.

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.

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.

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 (lipopolysaccharide) 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 ometriosis. 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.

Re-evaluation of the indication for and limitation of laparoscopic salpingotomy for tubal pregnancy.

OBJECTIVE: We investigated the outcome of laparoscopic salpingotomy for tubal pregnancy by follow-up hysterosalpingography (HSG) or second-look laparoscopy (SLL) and reexamined the indication for and limitation of this conservative surgery. STUDY DESIGN: From April 1991 to December 2003, we treated 181 cases of tubal pregnancy using laparoscopic salpingotomy. The tubal patency was assessed by either HSG or SLL performed at 3 months post-surgery. The patients with a successful initial operation and confirmed ipsilateral patent tubes at follow-up were classified as truly successful cases (group I). Even after successful operation, if the treated tubes were found to be occluded, they were considered as unsuccessful cases. Therefore, those cases that were unsuccessful at initial surgery as well as at follow-up were categorized as group II. RESULTS: One hundred and thirty-four cases (74%) were successfully treated by salpingotomy at initial laparoscopy and 85 of them (63.4%) were found to be truly successful at follow-up (group I). The remaining 47 cases (26.0%) were unsuccessful at initial surgery and 18 (13.4%) cases at follow-up (group II). Thirty-one other patients refused to accept a tubal patency test or were not examined for personal reasons or were lost to follow-up. No difference in surgical outcome was observed between these two groups of patients with regard to gestational age, intra-operative hemorrhage, size or anatomic location of the pregnancy mass, and pre-operative adhesions of the fallopian tube. However, pre-operative serum levels of hCG were significantly higher in group II than in group I. In addition, the unsuccessful cases were more frequently associated with positive fetal heart beat (FHB), tubal rupture, and pre-operative serum levels of hCG of more than 10,000 IU/l (p<0.05, chi2 test). The log-rank test indicated a higher pregnancy success rate in group I (p<0.05) than in group II in those who desired future pregnancy. CONCLUSION: Laparoscopic salpingotomy may be practised as conservative surgery for proximal ectopic pregnancy, and gestational mass size is not as important and is not a relative contraindication for conservative laparoscopic surgery, as previously reported. Low pre-operative HCG levels, absence of FHB, absence of tubal rupture initially or minimal rupture may be considered suitable parameters for successful surgery and for achieving future pregnancy.