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

Estrogen and progesterone receptor expression in macrophages and regulation of hepatocyte growth factor by ovarian steroids in women with endometriosis

Background: Information regarding macrophage-mediated regulation of hepatocyte growth factor (HGF) by ovarian steroid hormones in women with endometriosis is limited. Therefore, we investigated the regulation of HGF by steroid hormones in isolated macrophages and stromal cells derived from women with or without endometriosis. Methods: We isolated CD68 immunoreactive adherent macrophages in vitro from 46 women with endometriosis and 30 women without endometriosis. Estrogen receptor (ER) and progesterone receptor (PR) expression in macrophages was demonstrated by immunohistochemistry and RT-PCR. Production of HGF in the culture media of basal and ovarian steroid-stimulated macrophages was examined by enzyme-linked immunosorbent assay. Expression of mRNA for HGF and its receptor, c-Met in macrophages and stromal cells in response to ovarian steroid was investigated by RT-PCR. The single and combined effect of HGF and estrogen on the growth of macrophages and stromal cells was analysed by bromodeoxyuridine (BrdU) incorporation. Results: ER and PR were expressed in isolated macrophages and intact tissue at the protein and mRNA levels. Macrophages derived from women with endometriosis produced significantly higher concentration of HGF (352.2 ± 4.9 pg/ml) in conditioned media after treatment with estradiol (10-8 mol/l) than that of basal macrophages (221.5 ± 32.8 pg/ml, P < 0.05) or women without endometriosis (170.6 ± 2.6 pg/ml, P < 0.05). These effects were less evident after treatment with progesterone. Treatment with tamoxifen (10-6 mol/l) reversed the production of HGF and other macromolecules. Secretion of HGF in response to ovarian steroids was further enhanced after activation with lipopolysaccharide. The mRNA expressions of HGF and its receptor, c-Met, were also detected in macrophages and stroma in response to estrogen, suggesting an autocrine regulation. HGF mRNA expression was higher in cells of women with endometriosis than non-endometriosis women. Bromodeoxyuridine incorporation indicated that exogenous stimulation with HGF and estrogen, either alone or in combination, significantly increased the cell proliferation of both endometrial stroma and macrophages compared to that of non-endometriosis or non-treated cells. Conclusion: These results suggest that besides other inflammatory mediators, ovarian steroids also participate in the production of HGF by peritoneal macrophages which may be involved in the growth of endometriosis either alone or in combination with estrogen

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

Background: Information is limited regarding multifunctional role of GnRH agonist (GnRHa) therapy in reproductive diseases. We investigated 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 (Mφ) marker, and von Willebrand factor (VWF), a vessel marker, using respective antibodies. The changes in apoptosis were examined using TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay and by the immunoexpression of activated caspase-3 in tissues after GnRHa therapy. Results: The infiltration of CD68-positive M φ 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. A marked decrease in inflammatory and angiogenic responses were observed in lesions and myometria of these diseases. When compared with non-treated group, a significantly increase in apoptotic index (apoptotic cells per 10 mm2 area) and quantitative-histogram (Q-H) scores of activated caspase-3 after GnRHa therapy were observed in the eutopic endometria, lesions and myometria of these diseases. Conclusion: GnRH agonist was able to markedly reduce the inflammatory reaction and angiogenesis and 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

Effect of human seminal fluid on the growth of endometrial cells of women with endometriosis.

OBJECTIVES: We investigated the effect of human seminal fluid on the growth of endometrial cells derived from women with and without endometriosis. STUDY DESIGN: Seminal plasma (SP) was collected from 18 healthy fertile men. Serum, peritoneal fluid (PF) and tissue specimens of eutopic and ectopic endometrium were collected from 45 women with endometriosis and 20 women without endometriosis during laparoscopic surgery. Prostaglandin (PG) E2, hepatocyte growth factor (HGF), and estradiol (E2) levels in each sample of SP, serum and PF were measured by enzyme-linked immunosorbent assay. The growth pattern of cells derived from eutopic and ectopic endometria in response to SP was examined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. RESULTS: Seminal plasma was able to significantly stimulate the growth of epithelial cells and stromal cells derived from the eutopic and ectopic endometria of women with endometriosis (2-3-fold) when compared with control media. The SP-promoted proliferation of both gland cells and stromal cells derived from eutopic endometria was also remarkably higher in women with endometriosis than that of women without endometriosis. Although levels of PGE2, HGF and E2 in SP were variable when compared with other body fluids, the levels of PGE2 and HGF in SP were significantly higher than those in either peritoneal fluid or serum of women with or without endometriosis. Pretreatment of cells with individual anti-PGE2 antibody, anti-HGF antibody and two selective estrogen receptor modulators, tamoxifen and raloxifene was unable to suppress SP-mediated growth of endometrial cells. However, pretreatment of cells with combined anti-PGE2 antibody plus anti-HGF antibody or combined anti-PGE2 antibody plus anti-HGF antibody plus tamoxifen or raloxifene was able to significantly suppress SP-promoted growth of eutopic and ectopic endometrial cells. CONCLUSION: Human seminal fluid enriched with different macromolecules may promote the growth of endometrial cells derived from women with endometriosis. Our findings may suggest some detrimental effect of unprotected sexual intercourse in women with 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

Regulation of hepatocyte growth factor by basal and stimulated macrophages in women with endometriosis

Background: The different macromolecules as secreted by macrophages (M ) in the pelvic environment are believed to enhance the growth of endometriosis. However, the possible mediator that stimulates M for the production of different growth factors is not well described. Therefore, we investigated the possible production of hepatocyte growth factor (HGF) by the basal and lipopolysaccharide (LPS)-stimulated M derived from women with or without endometriosis. Methods: Using primary culture and 4-well chamber slides, adherent M immunoreactive to CD68 were isolated from the peritoneal fluid (PF) of 20 infertile women with endometriosis and 12 women without endometriosis. The proliferation of basal and LPS-treated M was investigated by the dimethylthiazole tetrazolioum bromide (MTT) assay. The production of HGF in the culture media of basal and LPS-stimulated M was examined by enyme-linked immunosorbent assay. The expression of mRNA for HGF and its receptor, c-Met, in the M was investigated by RT-PCR. The effect of HGF on the growth of endometrial cells and M was analysed by bromodeoxyuridine (BrdU) incorporation. Results: A >100 % increase in the proliferation of peritoneal M derived from women with endometriosis, and particularly of those harbouring dominant red lesions, was observed after treatment with LPS (P < 0.05). A 4- and 3-fold increase in the production of HGF was observed by the LPS-treated M derived from women with stage I-II endometriosis and stage III-IV endometriosis, respectively, when compared with non-LPS-treated M (P < 0.001). At the transcriptional level, we found a 5-fold increase in HGF mRNA expression in LPS-treated M versus basal M in women with endometriosis (P < 0.001). The BrdU incorporation study indicates that 10-100 ng/ml of HGF enhanced the growth of endometrial epithelial cells, stroma and M (?50% increase) derived from women with endometriosis (all P < 0.05). Conclusion: LPS could be an inflammatory mediator of macrophage stimulation in the pelvic microenvironment. Besides mesenchymal cells, HGF is also produced by peritoneal M and is possibly involved in the growth of 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. Methods. Peritoneal fluid was collected from 137 women with endometriosis and 57 women without endometriosis during laparoscopy and blood sampling was collected from 37 women with endometriosis and 21 women without endometriosis before laparoscopy. The concentration of HGF in PF and serum was measured by enzyme-linked immunosorbent assay. The ability of isolated macrophages and stroma to secrete HGF in response to lipopolysaccharide (LPS) was evaluated. Results. A significantly increased concentration of HGF in PF was found in women with endometriosis (1451.75±90.7 pg/mL) than that in non-endometriosis (1120.5±77.3 pg/mL, p <0.01) without any remarkable difference in HGF levels between women with stage I-/II endometriosis and stage III-/IV endometriosis. When we distributed serum and PF levels of HGF according to different color appearances of endometriosis, we found a significantly higher serum and PF levels of HGF in women containing dominant red peritoneal lesions in pelvic cavity (740±109.3 pg/mL for serum; 1685±183.4 pg/mL for PF) than those having other pigmented lesions (649±79.5 pg/mL, p <0.05 for serum; 1224±67.8 pg/mL, p <0.05 for PF) or chocolate cysts (485±43.1 pg/mL, p <0.05 for serum; 1118±83.1 pg/mL, p <0.01 for PF). Exogenous stimulation with LPS significantly increased the production of HGF in the culture media by macrophages and stroma derived from women with endometriosis than that in women without endometriosis. Conclusions. These results suggest that women with early or advanced endometriosis as measured by r-ASRM scoring system are not associated with an increase in either serum or PF concentrations of HGF. Rather HGF levels in serum and PF were significantly increased in women harboring blood-filled red peritoneal lesions and may be clinically useful to predict the activity of pelvic endometriosis

Preoperative diagnosis of pelvic actinomycosis by clinical cytology

Background: The purpose of this work was to investigate whether clinical cytology could be useful in the preoperative diagnosis of pelvic actinomycosis. Methods: This study involved the prospective collection of samples derived from the endometrium and the uterine cervix, and retrospective data analysis. Nine patients with clinically diagnosed pelvic actinomycosis were enrolled. The clinical and hematological characteristics of patients were recorded, and detection of actinomyces was performed by cytology, pathology, and bacteriological culture of samples and by imprint intrauterine contraceptive device (IUD) cytology. Results: The detection rate of actinomyces was 77.7% by combined cervical and endometrial cytology, 50.0% by pathology, and 11.1% by bacterial culture. Conclusion: The higher detection rate of actinomyces by cytology than by pathology or bacteriology suggests that careful cytological examination may be clinically useful in the preoperative diagnosis of pelvic actinomycosis

Differential infiltration of macrophages and prostaglandin production by different uterine leiomyomas

Background: The association between uterine myoma and infertility is still controversial. The anatomical defect of endometrium by uterine fibroids could be a factor for reducing pregnancy rates and increasing miscarriage rates. However, pregnancy and implantation rates were found to be significantly lower in women with intramural myomas (IMMs), when there was no deformity of uterine cavity. This could be due to other biological factors such as increased accumulation of inflammatory cells within fibroid tissue and corresponding endometrium that might impair fertility. Therefore, we tried to investigate the pattern of macrophage (Mψ) accumulation in different uterine fibroids and the production of chemokine and prostaglandin (PG) by these tissues. Methods: The selection criteria of uterine fibroids were based on the classification of European Society of Hysteroscopy. Biopsy specimens were collected from respective nodules and autologous endometrium of 20 women with submucosal myoma (SMM), 29 women with IMM and 18 women with subserosal myoma (SSM). CD68 immunoreactive Mφs were identified in these tissues by immunohistochemistry. A fraction of corresponding tissues were homogenized, and levels of monocyte chemotactic protein-1 (MCP-1) and PGF 2α were measured by enzyme-linked immunosorbent assay (ELISA). Results: Mφ infiltration in the myoma nodule and corresponding endometrium of women with SMM and IMM was significantly higher than that of women with SSM or control women (P < 0.01 and P < 0.05, respectively). This tissue accumulation of inflammatory cells was independent of the sizes of the myoma nodules and phases of menstrual cycle. The tissue concentration of MCP-1 corresponded to increased Mφ infiltration and was significantly higher in women with SMM and IMM than that in women with SSM (P < 0.05 for each). A positive correlation was observed between MCP-1 concentration and accumulated Mψ numbers in the endometrium of women with SMM and IMM but not in women with SSM. The tissue levels of PGF2α were also significantly higher in the nodule and corresponding endometrium of women with SMM and IMM than that in SSM or control women (P < 0.05 for each). Conclusions: Higher production of MCP-1 could be responsible for the increased accumulation of Mψ in women with SMM and IMM. The augmented inflammatory reaction in endometrium and increased PGF 2α levels might be detrimental to reproductive outcome in women with SMM or IMM

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