Masahiro Tanikawa

Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis.

OBJECTIVE Our purpose was to investigate the relationship between the levels of interleukin-6, interleukin-6 soluble receptor, and tumor necrosis factor-alpha in peritoneal fluid and the size and number of active red endometriotic lesions. STUDY DESIGN In a university hospital 39 women of reproductive age underwent either laparoscopy for infertility workup or laparoscopic surgery for ovarian chocolate cysts. Peritoneal fluid was collected by laparoscopy. Active lesions, such as red flamelike lesions, glandlike lesions, and red vesicles, were scored according to the revised American Fertility Society classification system according to the size and number of active lesions. Peritoneal fluid levels of interleukin-6, interleukin-6 soluble receptor, and tumor necrosis factor-alpha levels were determined by enzyme-linked immunosorbent assays. The relationship between peritoneal fluid concentrations of interleukin-6 and tumor necrosis factor-alpha and the score of active endometriosis was investigated. RESULTS Peritoneal fluid levels of interleukin-6 and tumor necrosis factor-alpha were significantly higher in patients with endometriosis compared with patients without endometriosis. The concentrations in patients with active endometriosis increased as the size and the number of active lesions increased. Cyclic variations in interleukin-6 concentrations were seen in peritoneal fluid from patients with endometriosis; the concentrations in the secretary phase were significantly higher than those in the proliferative phase. CONCLUSIONS Increased peritoneal fluid levels of interleukin-6 in patients with active red endometriosis may relate to endometriosis-associated infertility and to the pathogenesis of endometriosis.

Pathogenetic Significance of Increased Levels of Interleukin-8 in the Peritoneal Fluid of Patients with Endometriosis

OBJECTIVE To investigate the role of interleukin-8 (IL-8) in peritoneal fluid of patients with endometriosis in the pathogenesis of endometriosis. DESIGN Peritoneal fluid was collected by laparoscopy. Endometrial and endometriotic stromal cells were obtained from normal endometrium and from chocolate cyst linings of the ovary. SETTING Department of Obstetrics and Gynecology of Tottori University Hospital, Yonago, Japan. PATIENT(S) Forty women who underwent either laparoscopy or laparoscopic surgery. MAIN OUTCOME MEASURE(S) The peritoneal fluid concentration of IL-8 was analyzed by enzyme-linked immunosorbent assay, and the correlation between the IL-8 concentration and the extent of active endometriosis was investigated. The effect of IL-8 on cell proliferation was examined by tetrazolium bromide and thymidine incorporation. The expression of IL-8 receptor was examined in stromal cells by reverse transcription polymerase chain reaction. RESULT(S) The level of IL-8 in peritoneal fluid was significantly higher in patients with endometriosis than in patients without endometriosis. A significant correlation was noted with the extent of active endometriosis. Interleukin-8 significantly increased the number of cells and DNA synthesis in the endometrial and endometriotic stromal cells in a dose-dependent manner. Transcripts of IL-8 receptor type A were detected in stromal cells. CONCLUSION(S) The present study suggests that IL-8 found in the peritoneal fluid of patients with endometriosis contributes to the pathogenesis of endometriosis.

Altered gene expression and secretion of interleukin-6 in stromal cells derived from endometriotic tissues

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-alpha. 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-alpha 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-alpha. 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.

Role of Cytokines in Progression of Endometriosis

Peritoneal fluid in women with endometriosis contains an increased number of activated macrophages that secrete a variety of cytokines, including interleukin (IL)-6, IL-8, vascular endothelial growth factor, and tumor necrosis factor-α (TNF-α). Cytokines may be involved in the control of implantation and the growth of endometrial cells outside the uterus. In addition, several cytokines have been implicated in or directly associated with angiogenic activity in endometriosis. There could be a relationship between the levels of cytokines in the peritoneal fluid of patients with endometriosis and the status of the lesions in such patients. Peritoneal endometriosis can be classified as having red, black, or white lesions. Red lesions are known to be an active form of early endometriosis, because vascularization and mitotic activity are shown to be most prominent in these lesions. We found that the peritoneal fluid levels of TNF-α and IL-8 were significantly higher in patients with endometriosis, and correlated with the size and number of active lesions. In addition, TNF-α and IL-8 stimulated the growth of ectopic endometrial stromal cells. These cytokines with angiogenic activity may therefore have significant roles in the pathogenesis of endometriosis.

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

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 α (TGF‐α) 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. Mol. Reprod. Dev. 50:54–62, 1998.

Hepatocyte growth factor and stem cell factor involvement in paracrine interplays of theca and granulosa cells in the human ovary.

OBJECTIVE To examine gene expression of hepatocyte growth factor (HGF), the receptor for HGF, c-met, and the receptor for stem cell factor (SCF), c-kit, in the human ovary and to investigate the effects of HGF and SCF on the proliferation and function of granulosa and theca cells. DESIGN Prospective study. SETTING University hospital. PATIENT(S) Six premenopausal women. INTERVENTION(S) Follicular fluid and granulosa cells were collected during IVF cycles. Ovarian tissues were obtained from women who underwent surgery. MAIN OUTCOME MEASURE(S) Gene expression of HGF, c-met, and c-kit in the human ovary was determined. RESULT(S) Reverse-transcription polymerase chain reaction showed the presence of HGF and c-kit mRNA in the theca and stroma cells of the ovary, whereas c-met mRNA was observed in the granulosa, theca, and stroma cells. HGF increased the expression of SCF gene in granulosa cells, and SCF reciprocally increased the expression of HGF gene in theca cells. SCF stimulated the proliferation of theca cells. HGF stimulated progesterone production in granulosa cells. CONCLUSION(S) A positive feedback loop between theca cells and granulosa cells was identified that is mediated by HGF and SCF. HGF and SCF modulate the interplay between theca and granulosa cells by promoting cell proliferation and steroid hormone production.

Menstrual cycle–specific inhibition of the proliferation of endometrial stromal cells by interleukin 6 and its soluble receptor ☆ ☆☆

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.

Transvaginal ultrasound-guided follicular aspiration in the management of anovulatory infertility associated with polycystic ovaries *

STUDY OBJECTIVE To investigate whether or not transvaginal ultrasound (US)-guided follicular aspiration can effectively induce ovulation and facilitate pregnancy in anovulatory patients with polycystic ovaries (PCO). DESIGN Eight patients with polycystic ovarian disease (PCOD) and 10 patients with PCO were participants who failed to ovulate by the medical therapies. Most of persistent follicles were punctured, and their contents were thoroughly aspirated during the midluteal phase. The same ovarian stimulation regimen as used in the previous cycles were administered in the cycles after the aspiration. MAIN OUTCOME MEASURES Evidence of ovulation and a subsequent pregnancy was ultrasonically monitored after the aspiration, and the responsiveness of pituitary gonadotropins to gonadotropin-releasing hormone was tested in these patients. RESULTS The ovulation rates were 87.5% per patient, 52.6% per cycle monitored in PCOD patients and 100% per patient, 63.3% per cycle monitored in PCO patients, respectively. Half of the patients both with PCOD and PCO achieved pregnancy after the aspiration. A significant decrease (P less than 0.05) of the basal and peak levels of serum luteinizing hormone was observed after the aspiration. CONCLUSIONS The US-guided follicular aspiration seems to be a new surgical method for treating anovulatory patients with PCO.

Expression of transforming growth factor alpha (TGF-α) gene in mouse embryonic development

Purpose: The expression of genes for TGF-α, epidermal growth factor (EGF), and the EGF receptor (EGFR) in mouse blastocysts was evaluated by the reverse transcription-polymerase chain reaction (RT-PCR). We evaluated the effects of TGF-α and EGF on the development of mouse embryo prior to implantation.Results: The results revealed the presence of transcripts of TGF-α and EGFR. However, EGF mRNA was not observed in repeated experiments. None of these growth factors influenced the rate of development from the two-cell stage to the blastocyst stage when added to the culture medium. These effects were further examined on measuring the incorporation of tritiated thymidine and leucine, providing indices of the synthesis of DNA and protein, respectively. A concentration of only 0.1 ng/ml of TGF-α, which shares a cell surface receptor with EGF, stimulated the synthesis of both DNA and protein. EGF at a concentration of 10 ng/ml stimulated the synthesis of DNA and protein by blastocysts. To explore autocrine effects of TGF-α on the rate of blastocoel expansion, TGF-α antisense oligodeoxynucleotides was used to reduce expression of the TGF-α gene. TGF-α at a concentration of 0.1 ng/ml stimulates the rate of blastocoel expansion in early cavitating mouse blastocysts. In contrast, TGF-α antisense oligonucleotides significantly reduced the rate of expansion.Conclusions: Our present observations suggest that TGF-α/EGF and the EGFR may be involved in regulating embryonic development. In particular, TGF-α may serve as an autocrine factor in the regulation of embryonic development.

Presence of stem cell factor in follicular fluid and its expression in the human ovary

Stem cell factor (SCF), a pleiotropic glycoprotein with a molecular weight of 25‐36 kd, is a ligand for the tyrosine kinase receptor encoded by the c-kit protooncogene. The SCF and c-kit are essential for germ cell development, melanogenesis, and hematopoiesis. Although several lines of evidence in experimental animals suggest that SCF and c-kit may also play essential roles in human ovaries, little information is available. We previously reported that c-kit receptor messenger RNA (mRNA) was expressed in human oocytes (1). In the present study, we examined the expressions of SCF, a ligand for c-kit, in human oocytes, granulosa, theca, and ovarian stromal cells. We also investigated the relationship between SCF and steroid hormone concentrations in follicular fluid. Fifty-five samples of follicular fluid were collected from nine women (mean age 6 SD, 37.0 6 5.1 years; range, 29 ‐ 43 years) who were undergoing oocyte pick up for IVF-ET. The patients undergoing IVF-ET were stimulated with a combination of a GnRH analog (Suprecur, Hoechst Marrion Roussel, Tokyo, Japan) and hMG; clinical pregnancy was achieved in two of the nine women. Human preovulatory granulosa cells were collected from each of the nine women who underwent IVF-ET. Ovarian tissue was obtained from three additional women with regular menstrual cycles who were undergoing benign gynecological surgery unrelated to an ovarian condition; theca internal layers were microdissected from the follicle wall. A patient who underwent IVF-ET for research purposes donated seven oocytes used in this study. All of the oocytes used in this study were metaphase II. The total RNA was extracted from the oocytes as well as from cultured granulosa, theca, and stromal cells by the guanidium thiocyanate method. Informed consent was obtained from all subjects. We performed a reverse transcription polymerase chain reaction (RT-PCR) study with a Gene Amp RNA PCR Core Kit (Perkin, Elmer, NJ). The specific primers for SCF were 59-CCAGAACCCAGGCTCTTTAC-3 9 (sense), and 59-AGGCTCCAAAAGCAAAGC-3 9 (antisense). The SCF primers were designed to span exon 6, producing a 316-base pair fragment for the secretable form of SCF and a 233-base pair fragment for the membrane-bound form that lacks exon 6. The specificity of the PCR product was confirmed by a Southern blot analysis using the biotinylated oligonucleotide internal probe. The concentration of soluble SCF in the follicular fluid was determined by means of ELISA kit (R & D Systems Inc., Minneapolis, MN). The concentrations of E2 and P in the follicular fluid were measured with the use of an enzyme immunosorbent assay kit (Johnson & Johnson, Clinical Diagnostics, Amersham, UK). The concentration of T was measured by use of a radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA).