Search

Problem How is the tumor necrosis factor (TNF) α-induced inhibitor of apoptosis (IAP) protein expression in endometriotic stromal cells (ESCs) involved in cell viability and signaling pathways? Method of study Endometriotic stromal cells were isolated from ovarian chocolate cysts in 20 patients who underwent laparoscopic surgery. IAP protein expression and IκB phosphorylation were evaluated by Western blot analysis. Interleukin (IL)-8 protein expression and cell proliferation were assessed by ELISA. Results Cellular IAP (cIAP)-2 protein expression in endometriotic tissue was higher than that of endometrium. TNFα markedly enhanced cIAP-2 protein expression in ESCs. Pretreatment with a nuclear factor (NF)-κB inhibitor attenuated TNFα-induced cIAP-2 expression. An antagonist of IAPs abrogated TNFα-induced cIAP-2 protein expression and showed a decrease in TNFα-induced IL-8 protein expression and BrdU incorporation in ESCs. Conclusions TNFα and its downstream NFκB pathway have proven to be critical regulators of highly expressed cIAP-2 in ESCs. cIAP-2 may be a novel therapeutic target for endometriosis.

Objective To evaluate the effects of parthenolide on human endometriotic cells and murine endometriotic lesions. Design Experimental study. Setting University hospital and laboratory of animal science. Patient(s) and Animal(s) Twenty women with ovarian endometrioma and 30 mice. Intervention(s) Ectopic endometrial tissue from the endometrioma was collected. Main Outcome Measure(s) Human endometriotic stromal cells (ESCs) were pretreated with parthenolide and exposed to tumor necrosis factor (TNF)-α. Interleukin 8 ( IL-8 ) and COX-2 gene expressions were evaluated by real-time reverse transcription–polymerase chain reaction. Interleukin-8 protein, prostaglandin E 2 (PGE 2 ) level, and intranuclear p65 protein concentration were determined by ELISA. Cell proliferation was assessed by 5-bromo-2′-deoxyuridine–ELISA. Phosphorylation of signaling pathways in ESCs was evaluated by Western blotting. Gene expression and proliferative activity in murine endometriosis-like lesions were assessed by real-time reverse transcription–polymerase chain reaction and Ki67 staining, respectively. Result(s) With parthenolide pretreatment, TNF-α–induced IL-8 gene and protein expression in ESCs were diminished. Tumor necrosis factor α–induced COX-2 expression and PGE 2 synthesis were also inhibited. Adding parthenolide repressed TNF-α–induced 5-bromo-2′-deoxyuridine incorporation and IκB phosphorylation in ESCs. As in vivo experiments, administering parthenolide reduced the number, surface area, and weight, the level of Vegf , Il-6 , Mcp-1 , and Lif gene expression, and the percentage of Ki67-positive cells in murine endometriosis-like lesions. Conclusion(s) Parthenolide repressed the development of endometriosis by suppressing the inflammatory peritoneal environment through the nuclear factor κB pathway.

Aims: We investigated the potential of gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists to inhibit cell proliferation in endometriotic and endometrial stromal cells. Methods: Twenty patients with ovarian endometriomas and 18 patients with uterine fibromas were recruited. Endometriotic and endometrial stromal cells were obtained from the ovarian chocolate cyst linings and the eutopic endometria of premenopausal women with uterine fibromas, respectively. Results: GnRH agonist or antagonist treatment attenuated tumor necrosis factor (TNF)-α-induced cell proliferation in the endometrial stromal cells, whereas endometriotic stromal cells did not respond to treatment. The endometriotic stromal cells exhibited a decreased expression of the type I GnRH receptor compared with the endometrial stromal cells. GnRH agonists or antagonists did not repress TNF-α-induced IL-8 production in endometriotic stromal cells. Conclusion: GnRH agonists and antagonists have similar effects in slowing the growth of endometrial stromal cells. Endometriotic stromal cells resist the antiproliferative effect of GnRH agonists and antagonists.

Objective To search for the demethylated cytosine-phosphate-guanine (CpG) islands within the aromatase gene in stromal cells derived from endometriotic chocolate cysts. Design Prospective study. Setting Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago, Japan. Patient(s) Twenty-eight women who underwent laparoscopy (n = 14) and laparotomy (n = 14). Intervention(s) Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary. Main Outcome Measure(s) We searched for the CpG island and examined methylation profile and the association of methyl-binding proteins with the CpG island. Result(s) Up-regulation of aromatase messenger RNA (mRNA) expression was demonstrated in endometriotic cells. Three proximal promoters drove the mRNA expression. In endometrial cells, a marginal level of aromatase mRNA expression was observed. Treating endometrial cells with the demethylating agent 5-aza-2′-deoxycytidine markedly enhanced aromatase mRNA expression. The same promoters as in the endometriotic cells were used. To identify the unmethylated CpGs in endometriotic cells, we searched for CpG islands within the aromatase gene and subsequently examined the methylation profiles. Sequence analysis of bisulfite-treated genomic DNA demonstrated a stretch of CpG demethylation within a nonpromoter CpG island of the aromatase gene in endometriotic cells. In endometrial cells, the CpG sequences were heavily methylated and associated with methyl-CpG-binding proteins. Conclusion(s) The up-regulation of the aromatase gene in endometriosis may be ascribed to the epigenetic disorder associated with aberrant DNA demethylation in a nonpromoter CpG island.

Objective To determine whether serum interleukin (IL)-8 concentration can be measured in patients with ovarian endometrioma and whether this measurement is a useful tool in diagnosing this disease. Design A controlled clinical study and an in vitro study. Setting Department of Obstetrics and Gynecology, Tottori University, Japan. Patient(s) Seventy patients with ovarian endometrioma and 21 patients with benign ovarian cyst. Intervention(s) Laparoscopic surgery or laparotomy for ovarian endometriomas or benign ovarian cyst was performed. Preoperative blood samples were obtained. Endometriotic stromal cells obtained from nine patients with endometrioma were cultured. Main Outcome Measure(s) Interleukin-8 concentration in the serum or supernatant of the cell culture was measured with use of ELISA. Result(s) The serum concentration of IL-8 in patients with endometrioma was significantly higher than in patients with benign ovarian cyst. The serum IL-8 threshold (25 pg/mL) had a higher sensitivity (71.4%) for diagnosing ovarian endometrioma than did serum CA-125 level. The increased rates of IL-8 concentration in the culture supernatants after adding tumor necrosis factor α were significantly higher in patients whose serum IL-8 levels were ≥25 pg/mL than in those with levels Conclusion(s) Measuring of serum IL-8 concentration may be a valuable tool in diagnosing endometriosis.

Objective To evaluate the influence of peroxisome proliferator-activated receptor-γ (PPARγ) ligand (pioglitazone) on tumor necrosis factor-α (TNF-α)-induced interleukin-8 (IL-8) expression in endometriotic stromal cells (ESCs) and on proliferation of ESCs. Design Prospective study. Setting Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. Patient(s) Twenty-seven patients who underwent laparoscopic surgery. Intervention(s) The ESCs were obtained from the chocolate cyst linings of ovaries. Main Outcome Measure(s) The expression of PPARγ gene and protein was determined by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunocytochemistry. We determined the effect of pioglitazone on the production of TNF-α-induced IL-8 protein in culture supernatant of ESCs using ELISA. The effect of pioglitazone on TNF-α-induced proliferation of ESCs was evaluated by 5-bromo-2′-deoxyuridine proliferation assay. The activation of nuclear factor (NF)-κB in ESCs was evaluated by Western blot analyses and NF-κB transcription factor assays. Result(s) Immunocytochemistry and RT-PCR revealed the expression of PPARγ gene and protein in ESCs. The PPARγ protein was predominantly located in the cell nucleus. Measurement of IL-8 protein by ELISA showed that adding TNF-α (100 pg/mL) significantly increased IL-8 protein. Treating ESCs with 0.1–10 μM of pioglitazone significantly reduced the TNF-α-induced IL-8 production. The presence of 0.1–10 μM of pioglitazone significantly suppressed growth of ESCs. The TNF-α increased the expression of phosphorylation of inhibitor κB (IκB). Adding pioglitazone (10 μM) did not influence the expression of phosphorylated inhibitor κB (IκB). The TNF-α markedly increased the intranuclear concentration of p65, and adding pioglitazone (10 μM) significantly reduced the concentration of p65. Conclusion(s) The present study demonstrates for the first time that PPARγ is expressed in ESCs, and that pioglitazone reduced IL-8 secretion and the proliferation of ESCs. The PPARγ ligand may be an attractive therapeutic agent for endometriosis.

Objective To investigate whether and how P, dienogest (synthetic progestin), and danazol affected tumor necrosis factor α (TNFα)-induced interleukin-8 (IL-8) expression in endometriotic stromal cells. Design Prospective study. Setting Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. Patient(s) Ten patients who underwent laparoscopic surgery. Intervention(s) Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary. Main Outcome Measure(s) In the presence of TNFα (0.1 ng/mL) and E 2 (10 −7 mol/L), the cells were cultured in medium with P (10 −6 mol/L), danazol (10 −6 mol/L), or dienogest (10 −7 mol/L). The expression of the IL-8 gene and protein was determined by Northern blotting and ELISA, respectively. Activation of nuclear factor (NF)-κB was evaluated by electrophoretic mobility shift assay. Result(s) Adding TNFα (0.1 ng/mL) together with E 2 markedly enhanced gene and protein expression of IL-8. The up-regulation of the IL-8 gene and protein expression by TNFα and E 2 was significantly reduced by the addition of P, dienogest, or danazol. Electrophoretic mobility shift assay revealed that incubation with TNFα and E 2 induced NF-κB activation. Adding P, dienogest, or danazol attenuated NF-κB activation. Conclusion(s) The present study demonstrates for the first time that P and progestational compounds attenuate the expression of IL-8 by reducing TNFα-induced NF-κB activation in endometriotic stromal cells, suggesting a possible molecular mechanism of hormone therapy for controlling the growth of endometriosis.

Objective To examine the involvement of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) in the induction of interleukin-6 (IL-6) by tumor necrosis factor-α (TNF-α) in endometriotic stromal cells (ESC). Design Prospective study. Setting Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. Patient(s) Twelve patients who underwent laparoscopic surgery. Intervention(s) Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary. Main outcome measure(s) We determined the effect of TNF-α on the production of IL-6 and the effect of inhibitors for NF-κB and the MAPK pathway on IL-6 production using ELISA. Western blottings and electrophoretic mobility shift assays were used to detect activation of NF-κB and extracellular signal-regulated kinase 1/2 (ERK1/2). Result(s) The addition of TNF-α (0.1 ng/mL) significantly increased IL-6 protein in endometriotic stromal cells. Western blottings and electrophoretic mobility shift assays revealed that incubation with TNF-α induced degradation of inhibitor κB (I κB) and expression of phosphorylated ERK1/2. The NF-κB inhibitor (TPCK) and MAPK inhibitor (U0126) blocked the TNF-α-induced IL-6 expression. Electrophoretic mobility shift assay revealed that U0126 attenuated activator protein-1 (AP-1) activation induced by TNF-α. Conclusion(s) These findings demonstrate that NF-κB and AP-1 activation is critical for TNF-α-induced IL-6 expression in endometriotic stromal cells. Novel therapeutic modalities targeting these molecules may be possible in the near future.

Problem The cytokine, interleukin-6 (IL-6), stimulates the production of human chorionic gonadotropine (hCG) in chorionic cells. The purpose of this study was to examine the role of nuclear factor-kappaB (NF-kappaB) during the induction of IL-6 by IL-1beta in human trophoblast cells. Method of study Reverse transcription-polymerase chain reaction was used to determine the gene expressions of IL-1, IL-1 receptor (IL-1R), IL-6R and gp130 in a human choriocarcinoma cell line, BeWo. The BeWo cells were cultured for 24 hr with IL-1beta (0-10 ng/mL), IL-1beta (10 ng/mL) together with IL-1 receptor antagonist (IL-1Ra) or NF-kappaB inhibitor, N-tocyl-l-phenylalanine chloromethyl ketone (TPCK). The concentrations of IL-6 in culture supernatants were measured by enzyme-linked immunosorbent assay. Results The gene expressions of IL-1R, IL-6R and gp130, but not IL-1beta, were observed. The concentration of IL-6 was enhanced by IL-1beta in a dose-dependent fashion. The addition of IL-1Ra neutralized the effects of IL-1beta on IL-6 secretion. IL-1beta induced expression of phosphorylated IkappaB and the activation of NF-kappaB. The addition of TPCK reduced the IL-1beta-induced IL-6 expression. Adding IL-1beta increased beta-hCG production in a dose-dependent manner, and the addition of TPCK neutralized the effect of IL-1beta. Conclusion IL-1beta may induce the IL-6 expression and hCG production in human BeWo cells via NF-kappaB.

Hepatocyte growth factor (HGF) is a mesenchymal-derived paracrine factor that acts through a c-met receptor. The activated c-met receptor recruits various signal proteins. We used a steroidogenic human granulosa-like tumor cell line (KGN cells) to analyze the biological function of HGF in human ovary cells. First, we designed a method to analyze local production and action of HGF in the human ovary. Although c-met mRNA is expressed in KGN cells, granulosa lutein, theca, and ovarian stroma cells, we observed HGF mRNA only in theca and stroma cells. Adding HGF to the medium enhanced mitogenic activity in KGN cells. We next examined the activation of intracellular signal transduction molecules induced by HGF in KGN cells. Here, we showed that HGF activated the distinct phosphorylation of Raf-1, MEK1/2, and ERK1/2, but did not induce phosphorylation of Akt. HGF enhanced the phosphorylation of Elk-1 and c-Jun as nuclear transcription factors. U0126, a MEK1/2 inhibitor, completely abrogated the phosphorylation of ERK1/2 and the cell proliferation in response to HGF. In contrast, H-89, a protein kinase A inhibitor, further enhanced the HGF-induced phosphorylation of ERK1/2 and cell proliferation. In addition, we revealed that HGF suppressed progesterone synthesis in KGN cells. Adding HGF suppressed the forskolin-induced steroidogenic acute regulatory protein (StAR) expression, which is a key regulator in progesterone synthesis. Crosstalk signals between PKA and the mitogen-activated protein kinase (MAPK) pathway were mutually inhibitory. These results demonstrated for the first time that theca cell-derived HGF may be capable of stimulating the proliferation of granulosa cells and suppressing progesterone synthesis via an activating MAPK pathway.

Objective To evaluate the efficacy of an abdominal wall sealing device (the LAP DISK) used during laparoscopically assisted myomectomy (LAM). Design Retrospective study. Setting Tottori University Hospital, Yonago, Japan. Patient(s) All 43 patients who underwent LAM using the LAP DISK. Intervention(s) Ultrasonography and magnetic resonance imaging. Main outcome measure(s) Treatment strategy, operative outcome, and postoperative pregnancy rate. Result(s) Weight and size of the myomas removed ranged from 40–700 g (mean: 208.0 g) and 2–10 cm (mean: 5.4 cm). Mean blood loss was 42.3 mL. Half of the 18 patients who had been diagnosed with primary infertility for >2 years became pregnant without postoperative assisted reproductive techniques. Conclusion(s) The LAP DISK, a useful device for LAM, allows surgeons to remove myomas safely and repair uterine defects effectively while minimizing blood loss and trauma.

Objective To determine whether serum interleukin (IL)-6 can be measured in patients with ovarian endometriomas and whether these measurements are useful in managing this disease. Design A controlled clinical study and an in vitro study. Setting Department of Obstetrics and Gynecology, Tottori University, Japan. Patient(s) Twenty-two patients with ovarian endometriomas. Intervention(s) Laparoscopic cystectomy for ovarian endometriomas was performed. Gonadotropin-releasing hormone (GnRH) agonist was administered for 3 months in nine patients before laparoscopic surgery. Endometriotic stromal cells obtained from patients with endometriomas with or without GnRH agonist treatment were cultured. Main outcome measure(s) IL-6 concentrations in serum or supernatant of the cell culture were measured using ELISA. Result(s) The serum concentration of IL-6 in patients with endometriomas was higher at the time of diagnosis than in those without endometriomas. Laparoscopic surgery significantly reduced serum levels of IL-6. Serum IL-6 concentrations also decreased after treatment with GnRH agonist. IL-6 production was attenuated in the endometriotic stromal cells obtained from patients with GnRH agonist treatment compared with patients without such treatment. Conclusion(s) GnRH agonist treatment may decrease IL-6 production in endometriotic cells. Measurement of serum IL-6 concentrations may be of value in managing patients with endometriomas.

Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. We previously reported that TNFalpha promoted proliferation of endometriotic stromal cells by inducing IL-8 gene and protein expression. We hypothesize that TNFalpha may induce IL-8 production in endometriotic cells through nuclear factor-kappa B (NF-kappa B) activation. Western blot analyses and electrophoretic mobility shift assays revealed that incubation with TNF alpha induced the expression of phosphorylated inhibitor kappa B (p-I kappa B) and activation of NF-kappa B in endometriotic stromal cells. The NF-kappa B inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone, reduced TNFalpha-induced IL-8 gene and protein expression. The medical treatment of endometriosis with GnRH agonist (GnRHa) has been shown to induce hypoestrogenemia and reduce the observable number of endometriotic implants. We compare the expression of IL-8 gene and protein in endometriotic stromal cells of patients treated with GnRHa and those of patients without treatment before laparoscopic cystectomy for endometrioma. The addition of TNFalpha (0.1 ng/ml) significantly increased protein and gene expression of IL-8 in the cells of patients without GnRHa treatment, but this expression was not observed in the cells of patients with GnRHa. The addition of estradiol (E2; 10(-7) M) enhanced the expression of IL-8. However, in the cells of patients who received GnRHa treatment, TNFalpha and E2 did not show any significant effect. In endometriotic stromal cells without GnRHa treatment, TNFalpha and E2 increased the expression of p-I kappa B. In contrast, TNFalpha and E2 had no significant effect on the expression of p-I kappa B in cells that received GnRHa treatment. These findings demonstrate that NF-kappa B activation is critical for TNFalpha-induced IL-8 expression in endometriotic stromal cells. The current study showed for the first time that GnRHa treatment attenuated the expression of IL-8 by reducing TNFalpha-induced NF-kappa B activation.

Toll-like receptor (TLR) can activate dendritic cells (DC) through common signaling pathways requiring a cytoplasmic adapter, MyD88. However, the signaling is differentially regulated among TLR family members. TLR4 can activate MyD88-deficient bone marrow-derived DC (BMDC), and lead to induction of IFN-inducible genes and up-regulation of co-stimulatory molecules such as CD40, implying that the MyD88-independent signaling pathway functions downstream of TLR4. Because these effects can also be induced by type I IFN, we have analyzed whether type I IFN is involved in TLR4-induced responses. In response to lipopolysaccharide (LPS), IFN-beta gene expression was augmented in both wild-type and MyD88-deficient BMDC. Expression of all IFN-inducible genes except immune-responsive gene 1 (IRG1) was abolished and CD40 up-regulation was decreased in LPS-stimulated BMDC lacking either IFN-alpha/beta receptor (IFN-alpha/betaR) or signal transducer and activator of transcription 1 (STAT-1). Similar to the LPS response, TLR9 signaling can also induce expression of IFN-beta and IFN-inducible genes, and up-regulation of CD40. However, all these effects were MyD88 dependent. Thus, in TLR4 signaling, IFN-beta expression can be induced either by the MyD88-dependent or -independent pathway, whereas, in TLR9 signaling, it is dependent on MyD88. In CpG DNA-stimulated DC, expression of IFN-inducible genes except IRG1 was dependent on type I IFN signaling as in LPS-stimulated DC. However, in contrast to TLR4 signaling, TLR9 signaling requires type I IFN signaling for CD40 up-regulation. Taken together, this study demonstrates differential involvement of type I IFN in TLR4- and TLR9-induced effects on DC.

Tumor progression is often regulated through interactions between carcinoma cells and host stromal cells. In this study of endometrial cancer, we investigated one mechanism potentially involved in hepatocyte growth factor (HGF)-mediated cancer-stromal interactions. Endometrial cancer cells (HEC-1 and ISHIKAWA) expressed the c-met receptor, but HGF did not. HGF, however, did stimulate the proliferation and invasion of these cells. The HGF gene was expressed in stromal cells, which had been separated from primary cultures of endometrial cancers, 6.4 times more than in isolated normal endometrial stromal cells. Immunohistochemical staining revealed immunoreactive HGF in cancer stromal cells, the staining intensity being more pronounced in cancer tissue than in normal endometrium. The conditioned medium from normal epithelial cells and cancer cell lines induced HGF production in normal stromal cells. We identified basic fibroblast growth factor as an HGF inducer derived from endometrial cancer cell lines. Basic fibroblast growth factor derived from tumor cells may induce HGF in endometrial stromal cells, whereas stromal cell-derived HGF leads to the invasive growth of carcinoma cells. These interactions, mediated by HGF and HGF inducers, may play a significant role in the progression of endometrial cancer.

Endometriosis, which is common in women of reproductive age, may affect fertility. It is also clear that mechanical disruption of the pelvic anatomy may cause infertility. However, our understanding of the association between the early stage of endometriosis and infertility remains incomplete. Bloody peritoneal fluid (PF) is frequently observed in the cul-de-sac of endometriosis patients and contains various biologically active factors. We found that the concentrations of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in PF from patients with endometriosis were significantly higher than that of patients with endometriosis. There were significantly positive correlations between the levels of TNF-α and IL-6. We compared the levels of these cytokines with regard to the R-AFS stages and scores, but no differences were observed. In contrast, these cytokines correlate with the number and extent of red color peritoneal endometriosis. TNF-α increased the expression of IL-6 messenger RNA and protein in endometriotic stromal cells derived from chocolate cyst in a dose-dependent manner. The addition of IL-6 inhibited the development of mouse preimplantation embryo and impaired sperm function. We concluded that increased levels of IL-6 in peritoneal fluid of patients with active red endometriosis might be related to endometriosis-associated fertility.

The purpose of this study was to evaluate the effects of laparoscopic surgery on the symptoms associated with ovarian endometrioma. We also examined serum IL-6 concentrations in patients with endometrioma. Ninety-two patients who underwent laparoscopic surgery for ovarian endometrioma were enrolled in this study. The mean duration of follow-up was 27.6 months. Transvaginal ultrasound examinations revealed a recurrence of endometrioma in 13% of the cases. We evaluated the severity of dysmenorrhea using a 0–3-point verbal rating scale, and found that the dysmenorrhea score was statistically improved after the operation. Follicular growth was preserved in 94%, and the pregnancy rate was 43%. We measured serum IL-6 concentrations in 14 patients with ovarian endometrioma and 4 patients with benign gynecologic disease without endometriosis. IL-6 was significantly higher in patients with endometrioma than in those without endometriosis at the time of diagnosis. The mean serum IL-6 concentration significantly decreased after the operation. In conclusion, laparoscopic surgery is effective for alleviating pain and preserving fertility in patients with endometrioma. Measurements of serum IL-6 concentrations may be useful for the management of endometrioma.

Endometriosis, a common disease among women of reproductive age, is characterized by the presence and growth of endometrial tissue (glands and stroma) outside the uterus. Dysmenorrhea and infertility common in endometriosis compromise the quality of life of reproductive age women. Despite a long history of clinical experience and experimental research, endometriosis remains an enigma, and its pathogenesis is still controversial. The peritoneal fluid (PF) of women with endometriosis contains an increased number of activated macrophages that secrete a variety of local products, such as growth factors and cytokines. In this chapter, we review the current understanding of the role of cytokines in the pathogenesis of endometriosis.

We have investigated the possible roles of oncostatin M (OSM), which is a member of the interleukin-6 family of cytokines, in endometrial and endometriotic stromal cell growth. Endometrial and endometriotic stromal cells were collected from the uterus or ovarian chocolate cysts. We observed the expression of mRNA transcripts for OSM, OSM receptor subunit beta, leukaemia inhibitory factor receptor subunit (LIFR), and glycoprotein 130 in endometrial and endometriotic stromal cells. We also examined the effects of OSM (0-50 ng/ml) and LIF (0-10 ng/ml) on endometrial and endometriotic stromal cell proliferation and evaluated the effects of OSM on endometrial stromal cell differentiation. The presence of 10-50 ng/ml OSM significantly suppressed endometrial stromal cell growth in secretory phase tissue but not in proliferative phase tissue. In contrast, stromal cells in endometriotic tissues were resistant to the inhibitory effects of OSM. Addition of LIF did not influence the growth of endometrial stromal cells. We also showed that 10 ng/ml OSM stimulated markers of differentiation causing increased prolactin secretion and cyclooxygenase-2 gene expression in endometrial stromal cells from the secretory phase. These results suggest that OSM may play a pivotal role in regulating the growth and differentiation of endometrial cells. Endometriotic cells may behave differently from normal endometrial cells in terms of the inhibitory response to OSM.

Objective: Several growth factors and cytokines appear to participate in the proliferation or differentiation of trophoblast cells. The purpose of this study was to investigate the extent to which keratinocyte growth factor participates in the development of human embryonic and trophoblast cells at the maternal-fetal interface. Study Design: The reverse transcriptase–polymerase chain reaction method was used to determine the gene expression of keratinocyte growth factor and keratinocyte growth factor receptor in human choriocarcinoma cells (BeWo), human teratocarcinoma cells (PA-1), and human endometrial stromal cells. We also examined the effects of keratinocyte growth factor on cell proliferation and production of human chorionic gonadotropin in BeWo and PA-1 cells. Results: Keratinocyte growth factor gene was expressed in all cell types. The expression was pronounced in stromal cells of the endometrium collected during the secretory phase and early pregnancy. The keratinocyte growth factor expression was also enhanced in the differentiated BeWo cells. The expression of keratinocyte growth factor receptor gene was observed only in the BeWo cells. The addition of keratinocyte growth factor to the medium did not affect cell proliferation of the BeWo and PA-1 cells. On the other hand, keratinocyte growth factor (100 ng/mL) significantly enhanced human chorionic gonadotropin production in the BeWo cells. Stimulatory action of keratinocyte growth factor on human chorionic gonadotropin production in the BeWo cells was markedly enhanced after forskolin-induced differentiation. Conclusions: We conclude that keratinocyte growth factor may play an important role in promotion of human chorionic gonadotropin production in the trophoblast cells. (Am J Obstet Gynecol 2000;182:692-8.)

Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. We and others showed that several cytokine levels, including interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFalpha), are elevated in the peritoneal fluid of women with endometriosis compared with those in women without endometriosis. We also demonstrated that the addition of IL-8 to the culture medium stimulated the proliferation of cultured endometriotic stromal cells. TNFalpha is a multipotent cytokine that induces IL-8 production in various cell types. Therefore, we hypothesized that TNFalpha may also contribute to the pathogenesis of endometriosis by inducing the production of IL-8. To test this hypothesis, we analyzed the peritoneal fluid concentrations of IL-8 and TNFalpha using enzyme-linked immunosorbent assay (ELISA). We observed a significant correlation between the levels of TNFalpha and IL-8 in the peritoneal fluid of endometriosis patients. We also obtained the endometriotic stromal cells from chocolate cyst linings of the ovary. The expression of the receptors for TNFalpha (TNFR) was examined by RT-PCR. We observed the expression of both TNFR-I and TNFR-II genes in endometriotic stromal cells. The expression of IL-8 gene and protein was analyzed by Northern blot hybridization and enzyme-linked immunosorbent assay, respectively. TNFalpha induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. The addition of TNFalpha promoted the proliferation of the endometriotic stromal cells, and the stimulatory effects of TNFalpha were abolished by adding anti-IL-8 antibody. We demonstrated for the first time that TNFalpha stimulated proliferation of endometriotic stromal cells through induction of IL-8 gene and protein expression. We concluded that the TNFalpha may be one of the essential factors for the pathogenesis of endometriosis.

Objective: To compare the expression of interleukin-6 (IL-6) in endometrial and endometriotic cells. Design: Prospective study. Setting: Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. Patient(s): Twenty patients who underwent either hysterectomy or laparoscopic surgery. Intervention(s): Endometrial and endometriotic stromal cells were obtained from normal endometrium and from chocolate cyst linings of the ovary. Peritoneal macrophages were isolated from peritoneal fluids. Cells were cultured in the presence or absence of tumor necrosis factor-α. Main Outcome Measure(s): Gene expression of IL-6 was examined by Northern blot analysis. Interleukin-6 protein production was examined by immunocytochemical staining and ELISA. Result(s): A single IL-6 messenger RNA band of approximately 1.3 kilobases was detected in endometriotic stromal cells. Tumor necrosis factor-α increased the expression of IL-6 messenger RNA in endometriotic cells in a dose-dependent manner. In endometrial stromal cells, IL-6 messenger RNA signals were much weaker. Endometriotic stromal cells produced significantly larger amounts of IL-6 compared with endometrial stromal cells under basal conditions and after stimulation with tumor necrosis factor-α. Interleukin-6 protein was detected in cells isolated from endometriotic tissues by immunocytochemical staining. Interleukin-6 production by cultured macrophages from patients with endometriosis and endometriotic stromal cells was comparable. Conclusion(s): Altered gene expression and protein secretion of IL-6 in patients with endometriosis may contribute to the pathogenesis of the disease and/or to endometriosis-associated infertility.

Embryo implantation is a complex process that requires the interaction of embryo and endometrium. Several growth factors and cytokines appear to be involved in this process. Stem cell factor (SCF) and its receptor c-kit regulate the proliferation and survival of germ cells and play an important role in follicular development. However, little information is available on the role of SCF and c-kit in the process of blastocyst implantation. In the present study, we examined the expression of SCF and c-kit mRNA in mouse embryos and in the stromal and epithelial cells of the uterine endometrium by reverse transcription-polymerase chain reaction (RT-PCR). SCF mRNA was expressed in the spreading blastocysts and endometrial cells, with especially strong expression occurring in the stromal cells. Expression of c-kit mRNA was detected in the blastocysts and spreading blastocysts, as well as in the endometrial cells. By immunocytochemical studies, staining for c-kit protein was observed in the in-vitro spreading trophoblasts. We found that 50-100 ng/ml SCF significantly promoted the expansion of the surface area of the spreading blastocysts (P < 0.01). These results are consistent with the hypothesis that SCF derived from endometrial cells and the implanting embryo exerts paracrine and/or autocrine action on the process of implantation by stimulating trophoblast outgrowth through its receptor c-kit.

Objective: This study investigated the possible roles of interleukin 6 and soluble interleukin 6 receptor in the growth of endometrial and endometriotic cells. Study Design: Endometrial and endometriotic stromal cells were collected from the uterus or from ovarian chocolate cysts. We examined the effects of interleukin 6, soluble interleukin 6 receptor, and a combination of both factors on the proliferation of endometrial and endometriotic stromal cells. The action of sex steroids on the interleukin 6 regulation of the growth of stromal cells was also evaluated. The gene expressions of interleukin 6 receptor and glycoprotein 130 were examined in endometrial and endometriotic cells by reverse transcription-polymerase chain reaction. Results: Interleukin 6 had no effect on the growth of stromal cells in tissue from the proliferative phase. In contrast, the addition of concentrations of ≥100 pg/mL interleukin 6 induced significant inhibition of stromal cell proliferation in tissue from the secretory phase. Similarly, the addition of soluble interleukin 6 receptor caused significant suppression in the growth of endometrial stromal cells in tissue from the secretory phase but not the proliferative phase. On the other hand, stromal cells of endometriotic tissues were resistant to interleukin 6, showing no inhibitory response. Although the combination treatment did not affect the proliferation of stromal cells of the proliferative phase and of endometriotic tissues, 10 pg/mL interleukin 6 inhibited proliferation of stromal cells of the secretory phase in the presence of 1 ng/mL soluble interleukin 6 receptor. Treatment with estradiol and progesterone for 10 days newly induced the inhibitory response to interleukin 6 in the endometrial cells from the proliferative phase. Expressions of transcripts of interleukin 6 receptor and glycoprotein 130 were observed in the endometrial cells from the proliferative and secretory phases and in endometriotic cells. Conclusions: Interleukin 6 may play a central role in regulation of the growth of endometrial cells as a mediator of endocrine action. Endometriotic cells may behave differently from their normal counterparts in terms of the inhibitory regulation exerted by interleukin 6. (Am J Obstet Gynecol 1999;180:1088-94.)

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor alpha (TGF-alpha) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100-500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1-100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors.

Purpose: We wished to explore the role of transforming growth factor (TGF)-α in mouse embryonic development.

To evaluate the effects of parthenolide on human endometriotic cells and murine endometriotic lesions.Experimental study.University hospital and laboratory of animal science.Twenty women with ovarian endometrioma and 30 mice.Ectopic endometrial tissue from the endometrioma was collected.Human endometriotic stromal cells (ESCs) were pretreated with parthenolide and exposed to tumor necrosis factor (TNF)-α. Interleukin 8 (IL-8) and COX-2 gene expressions were evaluated by real-time reverse transcription-polymerase chain reaction. Interleukin-8 protein, prostaglandin E₂ (PGE₂) level, and intranuclear p65 protein concentration were determined by ELISA. Cell proliferation was assessed by 5-bromo-2'-deoxyuridine-ELISA. Phosphorylation of signaling pathways in ESCs was evaluated by Western blotting. Gene expression and proliferative activity in murine endometriosis-like lesions were assessed by real-time reverse transcription-polymerase chain reaction and Ki67 staining, respectively.With parthenolide pretreatment, TNF-α-induced IL-8 gene and protein expression in ESCs were diminished. Tumor necrosis factor α-induced COX-2 expression and PGE2 synthesis were also inhibited. Adding parthenolide repressed TNF-α-induced 5-bromo-2'-deoxyuridine incorporation and IκB phosphorylation in ESCs. As in vivo experiments, administering parthenolide reduced the number, surface area, and weight, the level of Vegf, Il-6, Mcp-1, and Lif gene expression, and the percentage of Ki67-positive cells in murine endometriosis-like lesions.Parthenolide repressed the development of endometriosis by suppressing the inflammatory peritoneal environment through the nuclear factor κB pathway.

The management of growth-restricted fetuses requires accurate diagnosis to optimize the timing of delivery. Doppler velocimetry is the only noninvasive method for assessing the fetoplacental hemodynamic status. This review will give a critical overview of the current knowledge on fetal venous blood flow in pregnancies complicated by in-trauterine growth-restricted fetuses. Adaptation of the circulation in intrauterine growth-restricted fetuses is described. Normal and abnormal venous Doppler waveforms are presented. Correlations of abnormal waveforms with the presence of acidemia and perinatal outcomes are emphasized. Limitations of venous Doppler velocimetry for optimizing the time for delivery and the perinatal outcome are also presented.

Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. Accumulated evidence suggest that apoptosis helps to maintain cellular homeostasis during the menstrual cycle by eliminating senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phase of the cycle. BCL-2 family and Fas/FasL system have been extensively studied in human endometrium and endometriotic tissues. Eutopic endometrium from women with endometriosis reportedly has some fundamental differences compared with normal endometrium of women without endometriosis. The differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and the development of endometriosis. One mechanism that recently gained a lot of interest is the finding that apoptosis appeared in eutopic and ectopic endometrium of patients with endometriosis. This study is a current review of the literature focused on the physiological role of apoptosis in normal endometrium and the alterations in regulation of apoptosis in eutopic and ectopic endometrium from women with endometriosis. Finally, role of apoptosis in the treatment of endometriosis is reviewed to link the basic research findings into clinical applications.

To assay DNA and RNA synthesis by developing mouse embryos in vitro and in vivo, we measured the uptake and incorporation of 3H-thymidine and 3H-uridine by morulae and blastocysts. We also evaluated the effect of adding EDTA to the culture medium on the uptake and incorporation of nucleic acid precursors by blastocysts.Thymidine and uridine incorporation increased after morulae developed into early blastocysts both in vitro and in vivo. However, the rates of uptake and incorporation were significantly lower by embryos grown in vitro than by those grown in vivo. The ratios of incorporation to total uptake were similar in embryos grown in vitro and in vivo. EDTA (100 microM) added to the culture medium significantly increased the incorporation of uridine into RNA by blastocysts grown in vitro (P0.01) but did not increase the total uptake of uridine.These observations showed that both DNA and RNA synthesis increased during the early development of preimplantation embryos and that those activities were reduced in embryos undergoing development in vitro. The results also suggested that through the mechanism of EDTA effect in embryo culture remains unknown, it appeared to reduce the retardation of RNA synthesis by embryos cultured in vitro through a selective stimulation of uridine incorporation.

The aim of our study was to determine the efficacy of postponing administration of human chorionic gonadotropin while continuing daily gonadotropin-releasing hormone agonist therapy (‘coasting’) to prevent the occurrence of severe ovarian hyperstimulation syndrome (OHSS) for patients with polycystic ovary (PCO) syndrome. Five patients with PCO who had been hospitalized due to severe OHSS in previous in vitro fertilization and embryo transfer or intrauterine insemination cycles at the Tottori University Hospital were included in the study. The rates of mature oocytes and fertilization were comparable between the cycles. A singleton pregnancy was achieved in a patient during the coasting cycle, and none of the women developed severe OHSS in coasting cycles. The results suggest that coasting may be an alternative method for reducing the severity of OHSS in patients with PCO.

Endometriosis causes pelvic pain and infertility in women of reproductive age. We explored TNFalpha-induced specific signaling pathways and gene expressions in endometriotic stromal cells (ESCs). Based on the data of the pathway specific cDNA array, we analyzed the role of TAK1, which is believed to work as a common mediator for NF-kappaB and MAPK pathways. Using the NF-kappaB pathway array, we found that TNFalpha upregulated ICAM-3, IL-6, IL-8, TAK1, JNK2, RelA, and TLR4 expressions. TNFalpha augmented the phosphorylation of TAK1. By transfection of TAK1 siRNA, TNFalpha-induced phosphorylation of IkappaBalpha, JNK1/2, and p38MAPK, as well as IL-6 or IL-8 expression, were repressed. TAK1 silencing in TNFalpha-pretreated ESCs caused a decrease in the proportion of cells in S-phase, and reduced TNFalpha-promoted BrdU incorporation. We provide the first evidence that TNFalpha and its downstream TAK1, which are key mediators for NF-kappaB and MAPK pathways, may be involved in the pathogenesis of endometriosis.

Aims: To evaluate fetal disorders using detailed quantitative values from the actocardiogram (ACG) involving simultaneous tracing of ultrasonic Doppler fetal movement bursts and fetal heart rate (FHR). Methods: Duration of FHR accelerations and fetal movement bursts were measured manually in 20 common fetal disorders. The severity of the fetal disorder was estimated using the FHR acceleration duration to movement burst ratio (A/B ratio) and 10–0 clinical severity ranks derived from the A/B ratio. The correlation of the A/B ratio and 1 and 5 min Apgar scores, as well as numerically expressed long-term outcomes were studied. Results: A/B ratios were significantly correlated with the 1 and 5 min Apgar scores and the numerically evaluated long-term outcomes. Controversial cases of FHR pattern were more easily understood using the A/B ratio. The 10–0 severity derived from the A/B ratio was useful in clinical fetal studies. Conclusion: Common fetal disorders were evaluated quantitatively and in more detail using the A/B ratio from the actocardiogram than when using common binary good or bad evaluation. The A/B ratio was useful in outcome estimation, where the prognostic capability of the A/B ratio was confirmed by significant correlation with 1 and 5 min Apgar scores and long-term outcomes of fetal disorders.

Chorioamnionitis is implicated in the pathogenesis of preterm delivery. However, the detailed mechanisms by which infection induces preterm labor are not well understood. This study has assessed the involvement of mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-induced pro- and anti-inflammatory cytokine and prostaglandin (PG) production in human choriodecidua. Samples of choriodecidua were collected before the onset of labor from women undergoing elective cesarean sections at term for breech presentation, previous cesarean delivery or cephalopelvic disproportion. Concentrations of TNFalpha, IL-10, PGE(2) and PGF(2)alpha in culture supernatants were measured by ELISA. Expression of COX-2 protein was analyzed by Western blotting. In human choriodecidual explants, LPS induced TNFalpha and IL-10 production in a dose- and time-dependent manner. LPS also up-regulated COX-2 expression and PG synthesis. Phosphorylations of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal kinases (JNK) were also confirmed by Western blotting. Furthermore, the effect of MAPK inhibitors was examined on LPS-induced pro- and anti-inflammatory cytokines and PG synthesis. Among the MAPK inhibitors examined, the p38 MAPK inhibitor, SB202190, significantly suppressed LPS-induced cytokine and PG production. SB202190 most profoundly suppressed the TNFalpha to IL-10 ratio, demonstrating that p38 MAPK inhibitor reduced predominantly TNFalpha other than IL-10 production. Phospho-p38 MAPK immunostaining was intense in extravillous trophoblast cells. The p38 MAPK seems to be most involved in signaling mechanisms when infection and inflammation cause preterm labor through PG synthesis. Novel therapeutic modalities targeting p38 MAPK may prevent to arrest preterm labor.

Ovarian surface epithelial cells (OSEs) are considered to be the common source of endometrioma and epithelial ovarian cancer. The present study reveals that keratinocyte growth factor receptor (KGFR) messenger RNA was expressed in OSEs of endometriomas but not in those of normal ovaries, suggesting that autocrine KGF/KGFR and paracrine fibroblast growth factor 10/KGFR signaling loops may be involved with the proliferation in OSEs of endometrioma.

BACKGROUND: The survival of endometriotic cells in the ectopic site has been investigated from the aspect of susceptibility of endometriotic tissues to apoptosis. In order to investigate the nature of abnormal survival of endometriotic cells in ectopic locations, we compared drug-induced apoptosis in endometrial and endometriotic cells. METHODS: Endometrial stromal cells were obtained from normal endometrium in 11 patients who underwent hysterectomy for leiomyoma without endometriosis. Endometriotic cells were isolated from the chocolate cyst linings of the ovary in 13 patients who underwent laparoscopic surgery. Cells were cultured in the presence or absence of staurosporine. Apoptotic cell death was evaluated by staining nuclei with propidium iodide and phosphatidylserine (a marker of early apoptotic events) with Annexin V as well as by DNA fragmentation assay. The number of viable cells was estimated by modified MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide WST-8] assay. RESULTS: After 3 h of exposure to staurosporine, >50% of the endometrial stromal cells became Annexin V positive. In contrast, >30% of the endometriotic cells were Annexin V positive. DNA fragmentation was not clearly induced in the endometriotic cells. Less than 20% of the endometrial cells survived after staurosporine exposure, while >40% of the endometriotic cells survived. Cell death induced by staurosporine was partially blocked by incubation with the caspase inhibitor, N-benzyoxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl-ketone (ZVAD-fmk), suggesting that a caspase cascade may play a role in the cell death process. CONCLUSIONS: Attenuated susceptibility to apoptosis in endometriotic stromal cells may be associated with abnormal survival in ectopic sites in an environment that is probably unfavourable. These results may be implicated in the pathophysiology of endometriosis.

The cytokine, interleukin-6 (IL-6), stimulates the production of human chorionic gonadotropine (hCG) in chorionic cells. The purpose of this study was to examine the role of nuclear factor-kappaB (NF-kappaB) during the induction of IL-6 by IL-1beta in human trophoblast cells.Reverse transcription-polymerase chain reaction was used to determine the gene expressions of IL-1, IL-1 receptor (IL-1R), IL-6R and gp130 in a human choriocarcinoma cell line, BeWo. The BeWo cells were cultured for 24 hr with IL-1beta (0-10 ng/mL), IL-1beta (10 ng/mL) together with IL-1 receptor antagonist (IL-1Ra) or NF-kappaB inhibitor, N-tocyl-l-phenylalanine chloromethyl ketone (TPCK). The concentrations of IL-6 in culture supernatants were measured by enzyme-linked immunosorbent assay.The gene expressions of IL-1R, IL-6R and gp130, but not IL-1beta, were observed. The concentration of IL-6 was enhanced by IL-1beta in a dose-dependent fashion. The addition of IL-1Ra neutralized the effects of IL-1beta on IL-6 secretion. IL-1beta induced expression of phosphorylated IkappaB and the activation of NF-kappaB. The addition of TPCK reduced the IL-1beta-induced IL-6 expression. Adding IL-1beta increased beta-hCG production in a dose-dependent manner, and the addition of TPCK neutralized the effect of IL-1beta.IL-1beta may induce the IL-6 expression and hCG production in human BeWo cells via NF-kappaB.

Objective To examine the effect of interleukin-6 (IL-6) on estrogen production and aromatase activity using a human granulosa tumor cell line (KGN cells). The involvement of the mitogen-activated protein kinase (MAPK) cascade in the inhibitory effects of IL-6 on estrogen production was also evaluated. Design Molecular and biological studies of KGN cells. Setting Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. Main Outcome Measure(s) Gene expression of IL-6 and the IL-6 receptor was analyzed by reverse transcription–polymerase chain reaction and Southern blot analysis. KGN cells were cultured for 48 hours with IL-6 (0.1–10 ng/mL) or IL-6 (10 ng/mL) plus a mitogen activated protein kinase-extracellular signal regulated kinase kinase 1/2 (MEK1/2) inhibitor U0126 (10 μM). Estradiol concentration in the culture supernatants was measured by means of enzyme immunoassay, [1β- 3 H] androstenedione was added to the cell lysate supernatant, and aromatase activity was determined by measuring the amount of [ 3 H] H 2 O released upon the conversion of [1β- 3 H] androstenedione to estrone. To examine the activation of intracellular signal transduction molecules induced by IL-6, the phosphorylation of Stat3, p38 MAPK, and extracellular signal-regulated kinase 1/2 (ERK1/2) was examined by Western blotting. Result(s) Gene expression of IL-6 and its receptor was detected in KGN cells. Estradiol secretion was significantly inhibited by adding IL-6, which also suppressed aromatase activity to 50% of the control. In addition, pretreatment with U0126 restored the IL-6–induced suppression of aromatase activity. IL-6 markedly enhanced the phosphorylation of ERK1/2, but not Stat3 and p38 MAPK. U0126 markedly reduced the level of the IL-6–induced phosphorylation of ERK1/2. Conclusion(s) These findings demonstrate that IL-6 may reduce estrogen production via the MAPK signal pathway in human granulosa cells. The results may support the notion that IL-6 is related to impaired estrogen biosynthesis in patients with endometriosis.

Background We previously reported that the level of interleukin (IL)-6 is increased in the peritoneal fluid of women with endometriosis. This study was undertaken to assess the effects of IL-6 and soluble IL-6 receptor (sIL-6R) on in vitro sperm motility. Methods Sperm (n = 20) were cultured with IL-6 or sIL-6R, or with a combination of both. After 24 h cultures, sperm motility was evaluated using a computer-assisted semen analysis system. Gene and protein expressions of IL-6, IL-6 receptor (IL-6R), and glycoprotein 130 (gp130) were examined in sperm by RT-PCR analysis and western blot analysis. Results Addition of IL-6 or sIL-6R individually to the culture media had no affect on sperm motion. However, adding a combination of IL-6 and sIL-6R dose-dependently reduced the percentage of motile and rapidly moving sperm. Adding anti-IL-6R antibody abolished these adverse effects. Sperm expressed the gp130 gene and protein, but not IL-6 or IL-6R. Conclusions A combination of IL-6 and sIL-6R may be associated with gp130 expressed in the sperm and reduce sperm motility. IL-6 and sIL-6R may contribute to the pathogenesis of endometriosis-associated infertility.

Hepatocyte growth factor (HGF) is a mesenchymal-derived paracrine factor that acts through a c-met receptor. The activated c-met receptor recruits various signal proteins. We used a steroidogenic human granulosa-like tumor cell line (KGN cells) to analyze the biological function of HGF in human ovary cells. First, we designed a method to analyze local production and action of HGF in the human ovary. Although c-met mRNA is expressed in KGN cells, granulosa lutein, theca, and ovarian stroma cells, we observed HGF mRNA only in theca and stroma cells. Adding HGF to the medium enhanced mitogenic activity in KGN cells. We next examined the activation of intracellular signal transduction molecules induced by HGF in KGN cells. Here, we showed that HGF activated the distinct phosphorylation of Raf-1, MEK1/2, and ERK1/2, but did not induce phosphorylation of Akt. HGF enhanced the phosphorylation of Elk-1 and c-Jun as nuclear transcription factors. U0126, a MEK1/2 inhibitor, completely abrogated the phosphorylation of ERK1/2 and the cell proliferation in response to HGF. In contrast, H-89, a protein kinase A inhibitor, further enhanced the HGF-induced phosphorylation of ERK1/2 and cell proliferation. In addition, we revealed that HGF suppressed progesterone synthesis in KGN cells. Adding HGF suppressed the forskolin-induced steroidogenic acute regulatory protein (StAR) expression, which is a key regulator in progesterone synthesis. Crosstalk signals between PKA and the mitogen-activated protein kinase (MAPK) pathway were mutually inhibitory. These results demonstrated for the first time that theca cell-derived HGF may be capable of stimulating the proliferation of granulosa cells and suppressing progesterone synthesis via an activating MAPK pathway.

Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. Accumulated evidence suggests that apoptosis helps to maintain cellular homeostasis during the menstrual cycle by eliminating senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phase of the cycle. The BCL-2 family and Fas/FasL system have been extensively studied in human endometrium and endometriotic tissues. Eutopic endometrium from women with endometriosis reportedly has some fundamental differences compared with normal endometrium of women without endometriosis. The differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and the development of endometriosis. One mechanism that recently gained a lot of interest is the finding that apoptosis appeared in eutopic and ectopic endometrium of patients with endometriosis. This study is a current review of the literature focused on the physiological role of apoptosis in normal endometrium and the alterations in regulation of apoptosis in eutopic and ectopic endometrium from women with endometriosis. Similarities in characteristics of endometriosis at a molecular level with gynaecological tumours are also discussed. Finally, the role of apoptosis in the treatment of endometriosis is reviewed to link the basic research findings into clinical applications. Hum Reprod Update

Endometrial stromal cells reportedly have a role in the initial invasion of endometrial tissue into the peritoneum. Hepatocyte growth factor (HGF), which is a ligand for the c-met protooncogene product (Met), stimulates proliferation and invasion of a large number of cells. In this study we investigated the role of the HGF/Met system in the pathogenesis of endometriosis. HGF concentrations in the peritoneal fluid of patients with endometriosis were significantly higher than in those without endometriosis and correlated positively with revised American Society of Reproductive Medicine scores. We showed that the peritoneum and endometriotic stromal cells may be major sources of HGF in peritoneal fluid. Endometrial and endometriotic stromal cells expressed the Met receptor, which was activated by endogenous and exogenous HGF. HGF enhanced stromal cell proliferation and invasion. We also demonstrated that the HGF-stimulated stromal cell invasion was due in part to the induction of urokinase-type plasminogen activator, a member of the extracellular proteolysis system. In conclusion, the HGF/Met system is involved in the pathogenesis of endometriosis by promoting stromal cell proliferation and invasion of shed endometria and endometrial lesions via autocrine and paracrine pathways.

Objective To evaluate the effect of lipopolysaccharide (LPS) on the expression of tumor necrosis factor α (TNFα) and interleukin-8 (IL-8) protein in endometriotic stromal cells (ESC) and their effect on the proliferation of ESC. Design Prospective study. Setting Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan. Patient(s) Seventeen patients who underwent laparoscopic surgery. Intervention(s) Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary. Main outcome measure(s) We determined the effect of LPS on the production of TNFα and IL-8 and the effect of IL-8 antisense oligonucleotide and nuclear factor-κB (NF-κB) inhibitor on IL-8 production using ELISA. TNFα production was examined by immunocytochemical staining. We determined the effect of LPS and the effect of IL-8 antisense oligonucleotide and NF-κB inhibitor on LPS-promoted ESC proliferation. Result(s) LPS-stimulated ESC produced significant amounts of TNFα and IL-8 in a dose- and time-dependent fashion. Adding LPS promoted ESC proliferation. Anti-TNFα antibody and anti-IL-8 antibody inhibited the stimulatory effects of LPS. IL-8 antisense oligonucleotide and NF-κB inhibitor significantly decreased LPS-induced IL-8 protein production and LPS-induced ESC proliferation. Conclusion(s) Pelvic inflammation may promote the progression of endometriosis.

Fibroblast growth factors (FGFs) exert diverse effects resulting from their interaction with cognate receptors on target cells. Our current study was designed to examine the local production and action of two specific stromal-epithelial cell mediatory factors, keratinocyte growth factor (KGF) and FGF-10, in human endometrial carcinoma cells. The RT-PCR method was used to determine gene expression of KGF, FGF-10, and KGF receptor in human endometrial carcinoma cells (HEC-1) and human endometrial stromal cells. KGF mRNAs were expressed in both of these cell types. On the other hand, FGF-10 mRNA was detected only in the endometrial stromal cells, and KGF receptor mRNA was observed in the HEC-1 cells. The novel finding of the present study is that KGF is expressed in carcinoma cells and FGF-10 is expressed in human endometrial stromal cells. The distinct phosphorylation of ERK-1 and -2 (ERK1/2), which are members of the MAPK family, was observed when HEC-1 cells were treated with KGF or FGF-10. KGF and FGF-10 could induce the prompt phosphorylation of ERK1/2 and consequently stimulate DNA synthesis. KGF and FGF-10 did not activate the phosphorylation of Akt, protein kinase C, or signal transducer and activator of transcription-3. Blocking the MAPK pathway with the specific methyl ethyl ketone 1/2 inhibitor (U0126) completely neutralized the enhancement of cell proliferation induced by KGF and FGF-10. In addition, KGF and FGF-10 activated expressions of downstream nuclear transcription factors, such as Elk-1 and c-myc, but not c-fos. These results demonstrate for the first time that KGF and FGF-10 are capable of stimulating the growth of endometrial carcinoma cells via activating MAPK pathway through autocrine/paracrine fashion.

Objective To evaluate the expression, regulation, and role of interleukin (IL)-8 in human ovary. Design Prospective study. Setting University hospital. Patient(s) Sixteen premenopausal women. Intervention(s) Follicular fluid and granulosa lutein cells (GLCs) were collected during IVF cycles. Ovarian stromal and theca cells were obtained from women underwent surgery. KGN cells, the human granulosa cell tumor cell line, were also used. Main outcome measure(s) The levels of IL-8 and IL-1β in follicular fluid and IL-8 protein production were determined using ELISA. Interleukin-8 and IL-8 receptor gene expression in ovarian cells and the effect of IL-8 on the proliferation of stromal cells were determined. The expression of pIκB was evaluated by Western blot, and the effect of NF-κB inhibitor APDC was examined by Northern blot analysis and ELISA in KGN cells. Result(s) The levels of IL-8 and IL-1β in follicular fluid; each concentration and the volume showed a positive correlation. Reverse transcription polymerase chain reaction showed the presence of IL-8 mRNA in all ovarian cells. In contrast, IL-8 receptor mRNA was only detected in stromal cells. The expression of IL-8 in GLCs and KGN cells was increased by addition of IL-1β and TNFα. Interleukin-8 increased the proliferation of ovarian stromal cells. The expression of pIκB in KGN cells was induced by IL-1β, and the effects were reduced by APDC. Conclusion(s) Interleukin 8 induced by IL-1β via activation of NF-κB in granulosa cells may have a role in the periovulatory period of follicular maturation.

Toll-like receptor (TLR) signaling activates dendritic cells (DC) to secrete proinflammatory cytokines and up-regulate co-stimulatory molecule expression, thereby linking innate and adaptive immunity. A TLR-associated adapter protein, MyD88, is essential for cytokine production induced by TLR. However, in response to a TLR4 ligand, lipopolysaccharide (LPS), MyD88-deficient (MyD88(-/-)) DC can up-regulate co-stimulatory molecule expression and enhance their T cell stimulatory activity, indicating that the MyD88-independent pathway through TLR4 can induce some features of DC maturation. In this study, we have further characterized function of LPS-stimulated, MyD88(-/-) DC. In response to LPS, wild-type DC could enhance their ability to induce IFN-gamma production in allogeneic mixed lymphocyte reaction (alloMLR). In contrast, in response to LPS, MyD88(-/-) DC augmented their ability to induce IL-4 instead of IFN-gamma in alloMLR. Impaired production of T(h)1-inducing cytokines in MyD88(-/-) DC cannot fully account for their increased T(h)2 cell-supporting ability, because absence of T(h)1-inducing cytokines in DC caused impairment of IFN-gamma, but did not lead to augmentation of IL-4 production in alloMLR. In vivo experiments with adjuvants also revealed T(h)2-skewed immune responses in MyD88(-/-) mice. These results demonstrate that the MyD88-independent pathway through TLR4 can confer on DC the ability to support T(h)2 immune responses.

Objective: To review the literature on the role of cytokines in the pathogenesis of endometriosis and endometriosis-associated infertility. Design: Pertinent studies were identified by a computer search of MEDLINE. References of selected articles were hand-searched for additional citations. Result(s): Recent studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages that secrete various local products, such as growth factors and cytokines. Levels of several cytokines were reported to be elevated in the peritoneal fluid of women with endometriosis. Because the peritoneal environment may be controlled by locally regulated factors, cytokines are believed to play a role in the development and progression of endometriosis and endometriosis-associated infertility. A possible pathogenic mechanism links cytokines with endometriosis. Conclusion(s): Cytokines, which are produced by many cell types including endometriotic tissues, play diverse roles in the pathogenesis of endometriosis and endometriosis-associated infertility. More studies about the specific role of these cells and soluble factors are needed to improve understanding of endometriosis and to develop novel therapies.