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Background: The standard range of D-dimer level associated with each type of gynecologic surgery is required to note the occurrence of bleeding or thromboembolism. Methods: Plasma levels of D-dimer of patients who underwent different types of gynecologic surgery were measured on the Day of Preoperative Examination (DPE) and the first postoperative day (POD-1). Patients were classified by surgery type: hysterectomy for benign diseases or cervical intraepithelial neoplasia; hysterectomy for uterine cancer; surgery for ovarian cancer; laparoscopic surgery for a benign adnexal mass; laparotomy for a benign adnexal mass; laparotomic myomectomy; cervical conization; transcervical resection of an intrauterine mass; vaginal surgery for prolapse of a pelvic organ. Results: In each type of gynecologic surgery, plasma levels of D-dimer on POD-1 were higher than those on the DPE. Prophylactic subcutaneous injection of heparin calcium for patients who underwent surgery for endometrial cancer showed no significant difference in the plasma level of D-dimer on the sixth postoperative day (POD-6) and the plasma level of D-dimer on POD-6 was in the same level as those on POD-1. Conclusions: Plasma levels of D-dimer on POD-1 were higher than those on the DPE in each type of gynecologic surgery. The D-dimer level remained high even on POD-6, and not changed by prophylactic subcutaneous injection of heparin calcium.

Background: RNA-binding motif protein 10 (RBM10), originally identified as S1-1 protein, is a nuclear protein with likely functions in transcription and RNA splicing. The RBM10 gene maps to the X chromosome and, in female cells, is inactivated in one of the two X chromosomes near the boundary with genes escaping inactivation. This study investigated the occurrence of the RBM10 gene product in complete hydatidiform mole, which is composed of cells with paternal diploid chromosomes (46, XX). Methods: Deparaffinized normal chorion or complete hydatidiform mole tissues were hybridized with a fluorescein-conjugated RBM10 gene probe in fluorescent in situ hybridization (FISH) analysis. Immunohistochemistry and immunoelectron microscopy of the tissues were performed using an anti-RBM10 antiserum. Proteins from complete hydatidiform mole tissues and those separated by anti-RBM10-linked affinity chromatography were also examined by western blotting. Results: As expected, the RBM10 gene was detected by FISH as double spots in the nuclei of complete hydatidiform mole cells. Immunohistochemistry revealed a nuclear presence of RBM10 in normal chorion and complete hydatidiform moles, and a notable cytoplasmic presence in complete hydatidiform moles. Western blotting and immunoaffinity chromatography revealed that a 50 kDa protein was predominantly found in the cytosolic fraction of complete hydatidiform moles. Conclusions: A 50 kDa protein with common antigenicity to RBM10 was found in the cytoplasm of complete hydatidiform mole cells, and could represent one of the characteristics of the disease.

Objective To investigate the possible involvement of collagen in the characteristic structure and function of human corpora lutea (CL), type V collagen expression was determined in the CL tissues during the menstrual cycle and early pregnancy. Design In vitro experiment. Setting Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama, Japan. Patient(s) Regulatory cycling women and pregnant women with ovarian tumor and ectopic pregnancy who underwent adnexectomy. Intervention(s) Composition of the various types of collagen in human CL was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Main Outcome Measure(s) Expression of type V collagen. Result(s) The ratios of type III to type I collagen and the ratios of type V to type I collagen in the CL tissues were significantly increased in early pregnancy compared with those in the menstrual cycle. Conclusion(s) These results suggest that alterations in composition of collagen might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.

Tail DNA genotyping of fetal and neonatal mice from C/EBPbeta heterozygous parents was performed to determine whether the decreased number of surviving C/EBPbeta mutants was caused by prenatal or postnatal death. Eighty-four 3-week-old mice born of heterozygous parents had significantly lower numbers of C/EBPbeta-deficient offspring than the expected Mendelian ratio (29.8%+/+, 65.5%+/-, 4.76%-/-, P0.05). The genotypes of 72 fetal mice from intercrossed heterozygotes showed approximately the expected 1:2:1 Mendelian ratio (18.1% +/+, 52.8% +/-, 29.2% -/-, P0.05). No difference in the proportions by sex could be detected in these perinates. This data indicates that C/EBPbeta-deficient mice have unknown lethal problems between the embryonic stage and weaning.

Mesothelin is a glycosylphosphatidylinositol-linked cell surface molecule expressed in the mesothelial lining of the body cavities and in many tumor cells. Based on the finding that a soluble form of mesothelin specifically binds to ovarian carcinoma cell line OVCAR-3, we isolated cDNAs encoding a mesothelin-binding protein by expression cloning. The polypeptides encoded by the two cloned cDNA fragments matched to portions of CA125, an ovarian cancer antigen and a giant mucin-like glycoprotein present at the surface of tumor cells. By flow cytometric analysis and immunoprecipitation, we demonstrate that CA125 binds to mesothelin in a specific manner. Binding of CA125 to membrane-bound mesothelin mediates heterotypic cell adhesion as anti-mesothelin antibody blocks binding of OVCAR-3 cells expressing CA125 to an endothelial-like cell line expressing mesothelin. Finally, we show that CA125 and mesothelin are co-expressed in advanced grade ovarian adenocarcinoma. Taken together, our data indicate that mesothelin is a novel CA125-binding protein and that CA125 might contribute to the metastasis of ovarian cancer to the peritoneum by initiating cell attachment to the mesothelial epithelium via binding to mesothelin.

To provide some insight into the mechanism of cervical ripening, the expression of type I collagen was investigated in human uterine cervical tissues obtained from the first (n = 4) and third (n = 3) trimesters of normal pregnancy. Indirect immunofluorescent staining was performed for type I collagen, and Northern blot analysis was done to assess expression of mRNA for the alpha1(I) chain. Collagens were also extracted from the human cervical tissues in the first and third trimesters of pregnancy. Immunohistochemical analysis revealed loose distribution of type I collagen in the uterine cervix of the first trimester compared with the third trimester of pregnancy. The relative levels of various collagens were evaluated by SDS-PAGE. The ratios of the intensity of the band of alpha1(I) to that of total collagen alpha1 chain in cervical tissues of the third trimester were significantly lower than those in cervical tissues of the first trimester of pregnancy (P0.05). In contrast, the ratios of the intensity of the band of alpha1(III) to that of total collagen alpha1 chain in cervical tissues of the third trimester were significantly higher than those in cervical tissues of the first trimester of pregnancy (P0.05). Northern blot analysis revealed that the cervical expression of mRNA for the alpha1(I) chain was significantly reduced in the third trimester compared with the first trimester of pregnancy (P0.01). These results suggest that type I collagen might play an important role in the maintenance of pregnancy and that decreased expression of this collagen could be associated with the process of uterine cervical ripening.

We performed genome-wide linkage analysis in 58 patients and nine unaffected members among 28 families with no mutation in BRCA1 or BRCA2, employing a set of 410 microsatellite markers. We initially screened the whole genome, including the X chromosome, by a non-parametric method using the GENEHUNTER program. As a result, chromosome 3p22-p25 showed a suggestive score for linkage [LOD = 3.49 and non-parametric LOD (NPL) = 2.77 at D3S3611] based on a multipoint analysis. Additionally, based on a two-point analysis using dense markers, this 3p22-p25 region showed a P-value0.05 at 10 markers and there is suggestive evidence for linkage at two markers within approximately 19 cM (NPL = 2.60 and 2.49 at D3S1597 and D3S3611, respectively). To explore whether the candidate gene in this 3p22-p25 region contributed to carcinogenesis of familial ovarian cancer in a similar fashion to the tumor suppressor gene, we performed loss of heterozygosity (LOH) analysis. It was observed that the frequency of LOH at four markers in this region was50% only in tumor tissues from patients with no mutation in BRCA1 or BRCA2, not in those with a BRCA1 mutation.

To evaluate the response rate and toxicity of the combination of irinotecan (CPT-11) and cisplatin in a neoadjuvant setting, a phase II study was conducted regarding the regimen of this combination in patients with locally advanced cervical cancer. Eligibility included patients with previously untreated stage Ib2, IIb, or IIIb squamous cell carcinoma with good performance status. CPT-11 (60 mg m−2) was administered intravenously on days 1, 8 and 15, followed by cisplatin (60 mg m−2) given intravenously on day 1. Treatment was repeated every 4 weeks for a total of two or three cycles. Among 23 eligible patients (median age: 59 years), three showed complete response (13%), 15 showed partial response (65%), for an overall response rate of 78% (95% confidence interval 58–90%). Stable disease was observed in four cases (17%) and progressive disease in one (4%). The median time to failure and median survival time have not yet been reached. Of the 52 treatment cycles administered, diarrhoea and grade 3 or 4 neutropenia were observed in 10% and 75% respectively. There were no therapy-related deaths. The combination of CPT-11 with cisplatin is a promising regimen for neoadjuvant chemotherapy in locally advanced cervical cancer. The toxicities of this regimen are well tolerated. © 1999 Cancer Research Campaign

Objective: To examine the relationship between estrogen receptor (ER) and progesterone receptor (PR) content in needle biopsy specimens and the growth of uterine leiomyomata after biopsy. Design: Prospective clinical study. Setting: University teaching hospital. Patient(s): Thirty-one women with uterine leiomyomata and a normal menstrual cycle. Intervention(s): Transcervical needle biopsy of uterine leiomyomata. Main Outcome Measure(s): The relationships between histologic features (smooth muscle content, immunohistochemical expression of ER and PR) and the percent increase over a 12-month observation period in the volume of the largest myoma nodule measured by magnetic resonance imaging were analyzed. Result(s): Both the density and intensity of immunohistochemical staining of PRs in uterine leiomyoma tissue showed significant positive correlation with leiomyoma growth. Conclusion(s): The growth of uterine leiomyomata can be determined by histologic and immunohistochemical analysis of needle biopsy specimens from uterine leiomyomata.

Patients with unresectable advanced uterine leiomyosarcoma have a very poor prognosis because no effective chemotherapeutic protocols exist. There are currently few established primary human uterine leiomyosarcoma cell lines that can be used to investigate effective therapies. To overcome this problem, we carried out long-term in vitro cell culture and/or nude mouse transplantation and successfully established novel human uterine leiomyosarcoma cell lines from extrauterine and intrauterine tumors that were surgically excised from a single patient. The established cells were characterized by flow cytometry, anticancer drug-sensitivity assays and karyotyping analyses. All the established cell lines showed unstable multiple chromosome abnormalities. Since the cells can proliferate in vitro and in vivo, they will be useful for developing new therapeutic strategies for advanced uterine leiomyosarcoma patients.

We evaluated the use of 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) for the diagnosis of uterine sarcomas. FDG-PET combined with serum lactate dehydrogenase (LDH) levels were compared with FDG-PET alone for the diagnosis of leiomyosarcomas (LMS), which are the most difficult uterine sarcomas to diagnose. FDG-PET imaging of endometrial cancer (EC) was used as a reference. Immunoreactivity for glucose transporter-1 (GLUT-I) correlated with FDG uptake was evaluated in sarcomas and leiomyomas (LM), including cases not examined by FDG-PET. FDG was injected after at least 5 h fasting and standardized uptake values (SUVs) were analyzed quantitatively 50-70 min after injection. Immunohistochemical expression of GLUT-1 was studied in paraffin sections of tumors using anti-GLUT-1 antibodies and GLUT-1 expression scores were derived based on staining intensities. FDG-PET was performed preoperatively in a total of 53 patients including 10 with sarcomas, 19 with EC and 24 with LM. Immunohistochemical examination was performed in 17 sarcomas, 6 EC and 9 LM [6 usual LM, 1 uterine smooth muscle tumor of uncertain malignant potential (UMP), and 2 bizarre LM (BLM)]. SUVs for uterine sarcomas and EC were significantly higher (p=0.0001) than those for LMs. There were no significant differences in SUVs among ECs, carcino-sarcomas (CS) and LMS. Significant differences in SUVs existed between LM and LMS (p=0.003). However, the diagnostic accuracy for LMS was only 73%. The diagnostic accuracy of FDG-PET combined with serum LDH was 100%. GLUT-1 expression scores were significantly higher in sarcomas and EC than in LM (p

In vitro sensitization (IVS)を用い自己癌細胞に対し強い細胞障害性を持つcytotoxic T lymphocytes (CTL)を大量に誘導し得る条件につき検討するとともに,婦人科癌患者4例に対しその臨床応用を試みた. CTL誘導は婦人科癌患者8例を対象とし,患者単核球とMMC処理癌細胞との前培養後rIL 2を添加して行った.その際(1)培養時のrIL 2濃度の検討では0.02, 0.2, 2.0U/mlまでは細胞障害性,細胞増殖率は上昇傾向を示したが,それ以上の濃度では変化がなかった. (2)CTL感作時の癌細胞濃度は上昇させるに従い,増殖率,細胞障害性は上昇した.以上CTLの誘導条件として培養時のrIL 2濃度は2U/ml, CTL感作時の単核球:癌細胞は10:1が良好な結果となった.また臨床応用の検討では癌性腹膜炎症例に対しては腹水消失,腹水細胞診陰性化,腹水減少等,全例である程度の効果が認められたが,固形腫瘍の縮小には至らなかった.

We previously reported that activin A, not inhibin, was localized to endometrial tissues, and that the endometrium might be a major source of activin A during the menstrual cycle, using an immunohistochemical method. However, there are few detailed reports concerning the expression of inhibin subunits, activin receptors and Smad proteins in the ectopic endometrial tissues of endometriosis. In this study, our purpose was to evaluate the immunohistochemical localization of inhibin alpha-, betaA-subunits, activin A, activin receptor, and Smad proteins in ovarian endometriosis. Tissue samples from ovarian endometriosis were obtained from 13 women. Normal endometrial tissues were obtained during the proliferative phase from 5 premenopausal women without endometriosis who were undergoing a hysterectomy for the treatment of uterine cervical intraepithelial neoplasia 3. We examined the immunohistochemical localization of inhibin/activin alpha-, betaA-subunit, activin A, activin receptors types IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 using an avidin-biotin-peroxidase complex technique. No immunostaining for the alpha-subunit of inhibin was observed in ovarian endometriosis and the normal endometrium. Positive immunostaining for the betaA-subunit of inhibin, activin A, activin receptors types IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 was observed in ovarian endometriosis and the normal endometrium. In conclusion, these results suggest that activin A, but not inhibins, is produced by ovarian endometriosis and the normal endometrium, and that the activin signal transduction system exists in both ovarian endometriosis and the normal endometrium.

Although death-associated protein kinase (DAPK) is a Ca 2+ /calmodulin-regulated serine/threonine kinase that plays important roles in various types of apoptotic cell death, there have been no reports of its tissue distributions and functions in female reproductive organs. By comparing C57BL/6 wild-type mice with DAPK-mutant mice lacking the 74-amino acid catalytic kinase domain of DAPK, the cellular distributions and biological functions of DAPK in murine ovaries were investigated. In situ hybridization analyses with sense and antisense riboprobes revealed that DAPK mRNA was selectively and highly expressed in granulosa cells in the ovaries of both types of mice. There were no significant differences in the body weights, ovarian weights and unstimulated ovarian follicular numbers between the wild-type and DAPK-mutant mice. Intraperitoneal injection of CPT-11, an anticancer topoisomerase I inhibitor that causes granulosa cell-specific apoptosis partly through Fas-Fas ligand (FasL) interactions in MCH mice, induced follicular apoptosis in both the wild-type and DAPK-mutant mice. However, the numbers of apoptotic follicles were significantly reduced in the DAPK-mutant mice. The Fas and FasL expression levels in the CPT-11-injected mice did not differ significantly between the wild-type and DAPK-mutant mice. These results indicate that DAPK positively regulates intracellular signaling pathways for CPT-11-induced granulosa cell apoptosis.

The aim of this study (JGOG1063) was to determine the recommended dose (RD) for combination chemotherapy with irinotecan hydrochloride (CPT-11) and nedaplatin (NDP) for advanced cervical squamous cell carcinoma. CPT-11 was given intravenously in fixed doses of 60 mg/m 2 on days 1 and 8 and NDP, in escalating doses, on day 1, every 4 weeks. A total of 15 patients were enrolled in the study. At level 1 (NDP: 50 mg/m 2 ), one of the 3 patients developed grade 3 diarrhea, so 3 additional patients were enrolled at this level. As none of the 3 additional patients exhibited dose-limiting toxicity, level 1 was elevated to level 2 (NDP: 60 mg/m 2 ). The maximum tolerated dose was not reached, even at the highest dose level (level 4; NDP: 80 mg/m 2 ). No further dose escalation was carried out, and level 4 (CPT-11: 60 mg/m 2 , NDP: 80 mg/m 2 ) was determined as the RD.

This study aimed to clarify the mechanism by which apoptosis and Fas ligand (FasL) expression are induced in the ovarian granulosa cells of mice injected with irinotecan HCl (CPT-11). To this end, the direct effects of CPT-11 and its active metabolite, SN38, on granulosa cells were investigated. Normal ovarian tissue fragments obtained from 8-week-old female MCH mice were cultured in vitro with CPT-11 or SN38 and paraffin-embedded. After sectioning, the ovarian fragments were analyzed by TUNEL staining to detect apoptotic cells and by immunohistochemistry with an anti-FasL antibody to detect FasL expression. The results revealed no increase in TUNEL-positive granulosa cells in the ovarian tissue fragments cultured with CPT-11 or SN38. Furthermore, CPT-11 and SN38 did not induce FasL expression in the ovarian fragments. In conclusion, apoptosis and FasL expression induced in the ovarian granulosa cells of mice injected with CPT-11 is not caused by direct stimulation with CPT-11 or SN38. Therefore, systemic CPT-11 administration appears to induce apoptosis and FasL expression in granulosa cells via currently unknown endogenous FasL-inducing factors or by active metabolites of CPT-11 other than SN38.

母体にとってSemiallograftである胎児を維持する免疫学的機序を知るべく,脱落膜組織に浸潤している単核球(MNC)を取り出し,その細胞膜表面の性格をモノクローナル抗体を用い検討するとともに,それらの細胞の存在部位を免疫組織学的に検討した.さらにその機能についても検討した. MNCは初期妊娠中絶により得られた脱落膜組織より得た.細胞膜表面の性格の検討では末梢血MNC (P-MNC)に比し, CD 8+細胞が多く, CD 4+/CD 8+ ratioが著明に低下していた.またこのCD 8+細胞はCD11b-細胞でcytotoxic T-cellと判断された.これらの細胞の占拠部位は脱落膜緻密層にはマクロファージ(Mφ)のみ存在し,リンパ球はみられなかった.一方,脱落膜海綿層ではリンパ球およびMφがみられ,その他NK細胞も少数ながらみられた.これらの多くの細胞は, HLA-DR+細胞であった.しかしIL-2 receptor陽性細胞はみられなかった.またCD 8+細胞はCD 8+, CD11b-のcytotoxic T-cellであった.そこで次にこれらの細胞の機能をP-MNCと比較検討した.PHA反応性は低値を示した.しかしIL-2に対する反応性は高値を示した.またNK活性も認められず,逆にsuppressor活性が高かった.以上,脱落膜組織のT-cellは膜表面の性格はcytotoxic T-cellで,また活性化されているが,膜表面にIL-2 receptorをもたず,また全体の細胞の機能として抑制機能が有意で,妊娠維持に好環境が形成されているものと判断された.

The biological roles of laminin and type IV collagen in human endometrial stromal tissues were investigated by the evaluation of the expression levels in human endometrial tissues using immunohistochemistry. In addition, normal human endometrial stromal cells were cultured in vitro on laminin- or type IV collagen-coated plates and subjected to cytological analyses. Cyclic production of laminin and type IV collagen were detected and the two productions were significantly increased in late proliferative and late secretory endometrial stromal cells. Unstimulated endometrial stromal cells proliferated with specific growth structures that varied depending on the extracellular matrix component coated on the culture plates. The expression levels of integrin subunits on endometrial stromal cells were sufficiently enhanced by 8Br-cAMP treatment to mask any differences in the growth structures induced by the extracellular matrix components. 8Br-cAMP-stimulating stromal cells exhibited significant survival on laminin-coated plates, while 8Br-cAMP-deprived stromal cells, after 8Br-cAMP stimulation, showed significant survival on type IV collagen-coated plates. In conclusion, human endometrial stromal cells produce laminin and type IV collagen, and these productions are possibly regulated by ovarian estrogen and progesterone. Human endometrial stromal cells specifically bind to laminin and type IV collagen via integrins, and regulate endometrial stromal cell structures, viability and differentiation. Thus, laminin and type IV collagen may autoregulate human endometrial stromal remodeling during the menstrual cycle in an autocrine and paracrine fashion.

Minimal optimal surgery without chemotherapy is often performed for patients with ovarian immature teratoma, which frequently occurs in young women who hope for future pregnancies. If tumors recur after the operation, anticancer drug chemotherapy is often administered, although few studies have highlighted differences between the recurrent and the primary tumor cells. Therefore, we have established experimental animal models of recurrent ovarian immature teratoma cells after optimal surgery and characterized the anticancer drug sensitivity and antigenicity of the recurrent tumors. Surgically-excised tumor cells of a grade II ovarian immature teratoma were cultured in vitro and transplanted into nude mice to establish stable cell lines. Differential drug sensitivity and antigenicity of the tumor cells were compared between the primary and the nude mouse tumors. Nude mouse tumor cells showed a normal 46XX karyotype. Cultured primary cells showed a remarkably high sensitivity to paclitaxel, docetaxel, adriamycin and pirarubicin, compared to peritoneal cancer cells obtained from a patient with ovarian adenocarcinomatous peritonitis. The drug sensitivity of teratoma cells to 5-fluorouracil, bleomycin or peplomycin was also significantly higher. However, there was no significant difference in sensitivity to platinum drugs between the primary teratoma and the peritoneal adenocarcinoma cells. As for nude mouse tumor cells, sensitivity to 12 anticancer drugs was significantly lower than that of the primary tumor cells, while there was little difference in sensitivity to carboplatin or peplomycin between the primary and nude mouse tumor cells. Flow cytometry showed that the expression of smooth muscle actin (SMA) significantly decreased in nude mouse tumor cells when compared to cultured primary cells. In conclusion, ovarian immature teratomas with normal karyotypes have a malignant potential to recur after minimal surgery. During nude mouse transplantation, SMA-overexpressing cells appeared to be selectively excluded and nude mouse tumor cells were less sensitive to the majority of anticancer drugs than the primary tumor cells. These results indicate that after optimal surgery for ovarian immature teratoma, recurrent cells can be more resistant to anticancer drugs than the primary tumors. Therefore, it is likely that adjuvant chemotherapy lowers the risk of ovarian immature teratomas recurring after optimal surgery. BEP and PBV regimens are frequently given to teratoma patients. However, paclitaxel/carboplatin or docetaxel/carboplatin, which are the most effective chemotherapy treatments for epithelial ovarian cancer patients, are considered to be an alternative regimen, especially in the prevention of reproductive toxicity.

To better understand the molecular mechanisms of carcinogenesis induced in uterine endometrium by therapeutic anti-estrogenic Tamoxifen (TAM) exposure, 27 uterine tumors (4 benign endometrial polyps and 23 carcinomas) associated with TAM exposure were analyzed for the presence and spectrum of p53 and K-ras mutations. Although there was no significant difference between TAM-associated endometrial carcinomas and sporadic endometrial tumors in the frequency of these mutations, the spectrum of p53 mutations was characteristically unique to the TAM-associated tumors. The median duration of TAM exposure was significantly longer in patients with p53 mutations than those without p53 mutations (62 vs. 30 months, p=0.028). Our observation suggests that prolonged TAM exposure may directly inactivate the p53 gene by acting as a mutagen in a significant fraction of TAM-associated endometrial carcinomas.

The effects of irinotecan HCl (CPT-11) combination chemotherapies on the hypothalamus-pituitary-ovary endocrine system were examined clinically. The incidences of typical menopausal malaises and/or endocrinological findings were investigated in 32 gynecological cancer patients treated by CPT-11 combination chemotherapies. Patients who complained of menopausal malaises or had been treated by hormone replacement therapy before chemotherapy were excluded from the study. Menopausal malaise-like symptoms (MMLS) appeared in 6 of 32 patients (18.8%) during CPT-11 combination chemotherapy, and these symptoms were completely cured within a few days by administration of conjugated estrogen tablets (0.625 mg/day). All the MMLS cases were perimenopausal patients (47-57 years of age), and MMLS were not found in any of the postmenopausal patients who had exceeded 3 years since endocrinological menopause or patients who had recurrent cancer after pelvic radiotherapy. After exclusion of these 3-year-postmenopausal patients and postirradiation patients, 6 of 7 patients aged 45-59 years complained of MMLS during CPT-11 combination chemotherapy. The incidence of CPT-11-induced MMLS showed no relationships with the anticancer drugs combined with CPT-11, mean total CPT-11 dose, mean number of CPT-11 injections, mean individual CPT-11 dose, grade of CPT-11-specific diarrhea or anticancer effects of each CPT-11 combination chemotherapy. The perimenopausal cancer patients with CPT-11-induced MMLS showed decreased serum estradiol and increased serum FSH and LH levels accompanying the CPT-11 injections. A young patient with CPT-11-induced secondary amenorrhea showed decreased serum estradiol and increased serum FSH and LH levels accompanying the CPT-11 injections. None of the postmenopausal patients with high FSH and LH levels showed any significant differences in their serum FSH, LH, PRL and TSH levels during CPT-11 combination chemotherapy. No differences in the results of LHRH and TRH tests during chemotherapy were found for postmenopausal patients. Histopathological examinations of normal ovarian tissues surgically removed from 4 young cervical cancer patients treated with preoperative CPT-11 combination chemotherapies revealed no growing ovarian follicles in the ovarian tissues. CPT-11 injections can induce estrogen-rescued MMLS in cancer patients aged approximately 50 years at a very high rate and may induce secondary amenorrhea in young women. The endocrinological and histopathological studies revealed that CPT-11 causes ovarian follicular loss and ovarian failure within a short time without affecting hypothalamic and pituitary hormone secretion. These clinical results indicate that CPT-11 has strong ovarian toxicity and that repeated CPT-11 administrations may frequently induce ovarian follicular loss and premature ovarian failure, even in young women.

Clinical use of CPT-11 combination chemotherapy frequently induces ovarian dysfunction in premenopausal and perimenopausal cancer patients, but its mechanism remains unclear. Mouse experiments were performed to clarify the molecular mechanism of CPT-11-induced ovarian dysfunction. Clinically therapeutic doses of CPT-11 were injected intraperitoneally into 8-week-old female MCH mice, and their ovaries were examined by the TUNEL assay to detect dead cells. Immunohistochemical examinations were simultaneously performed to detect the expression of activated caspase 3, Fas antigen and Fas ligand (FasL). Furthermore, normal murine ovarian tissue fragments were incubated with recombinant soluble FasL in organ cultures and stained by the TUNEL assay to detect apoptotic cells. Intraperitoneal CPT-11 injections induced specific TUNEL-positive cells and cell death with cleaved caspase 3 expression among large ovarian follicular granulosa cells. Apoptotic follicles (follicles containing >/=10 TUNEL-positive cells per ovarian section) were only found among large follicles. The final apoptotic follicle ratios were approximately 30% of the total follicles independent of the CPT-11 dose, while CPT-11 dose-dependently enhanced apoptotic processes in murine ovarian follicles. Fas antigen was expressed in most ovarian cells, with extremely high expression levels detected in luteal cells. CPT-11 injections did not significantly increase the Fas expression levels in ovarian cells. Although no FasL expression was detected in normal ovarian tissues, CPT-11 injections significantly induced specific FasL expression in granulosa cells. Incubation of organ-cultured normal murine ovarian tissue fragments with recombinant mouse soluble FasL significantly increased the numbers of TUNEL-positive granulosa and luteal cells. In conclusion, CPT-11 dose-dependently induced specific FasL expression in granulosa cells of developing ovarian follicles. The induced FasL reacted with the Fas antigen constitutively expressed on granulosa cells, such that apoptosis can only be enhanced and induced in granulosa cells in an autocrine and/or paracrine manner. This cell lineage-specific and differentiation stage-specific apoptosis in granulosa cells is thought to be the main molecular mechanism of the ovarian dysfunction induced by CPT-11 combination chemotherapy.

妊娠の維持機構を明らかとすべく,妊婦の免疫系による胎児抗原の認識ならびに免疫応答を最もよく反映すると考えられる子宮近傍リンパ節(regional lymph nodes draining the uterus: DLN)の免疫動態について検討を行った.まず,各種のmononuclear cell (MNC)を識別するモノクローナル抗体およびFACS analyzerを用い,細胞の膜表面抗原を解析し,リンパ節での免疫動態の特徴について検討した.その結果,妊婦のDLNでは, HLA-DRおよびCD11b陽性細胞の増加が認められた.さらに,免疫組織学的観察を加えたところ, HLA-DR陽性細胞は, DLNのB cell領域とともに, T cell領域にも多く認められ, T cellは活性化されているものと考えられ, DLNでの細胞免疫能が亢進しているものと推察された.そこで, DLNよりMNCを分離し, mitogenに対する反応性を測定したところ,末梢血MNCとの間には差は認められなかった.しかし, controlとして用いた子宮筋腫患者のDLNより分離したMNCの反応性より上昇していた.さらに瞬帯血のMNCをstimulatorとしmixed lymphocyte reaction系で検討してみたところ,末梢血と比較して反応性の充進が認められた.これらの結果は,母体の免疫系が胎児に反応していると理解された.次にこの反応が胎児を許容すべく作用するか,それとも傷害的に作用するかsuppressor活性およびcytolytic activityについて検討してみた.その結果, suppressor活性は認められなかったが, cytolytic activityが認められた.このことは, DLNのMNCが胎児に対して傷害的に作用している可能性を推察させた.

The efficacy and toxicity of combined therapy with irinotecan (CPT-11) and mitomycin-C (MMC) in a neoadjuvant setting were evaluated in patients with locally advanced squamous cell carcinoma (SCC) of the uterine cervix.Eligibility included patients with previously untreated cervical carcinoma. CPT-11 (100 mg/m2) was administered on days 1, 8 and 15 intravenously (i.v.), while MMC (10 mg/m2 i.v.) was given on day 1. Survival curves were generated using the Kaplan-Meier method.Among 35 eligible patients, 3 showed a complete response and 27 a partial response, with an overall response rate of 85.7%. No patient showed progressive disease. Thirty-three patients were able to undergo radical surgery after neoadjuvant chemotherapy and only 2 patients (stage IIIb) received radiotherapy without the optimal surgery. The median disease-free survival (DFS) period was 42 months (range 5-73). The median overall survival (OAS) period was 44 months (range 17-74). Two-year DFS and OAS rates were 74.3% and 91.4%, respectively. Of the 58 treatment cycles administered, grade 3 or 4 neutropenia and thrombocytopenia were observed in 50% and 9% of the treatment cycles, respectively. Grade 3 or 4 diarrhea was observed in 6%.Neoadjuvant chemotherapy with CPT-11 and MMC can be effective and well-tolerated against locally advanced SCC of the uterine cervix.

The functions of activin, a member of TGF-beta superfamily, in ovarian clear cell adenocarcinoma remain unsolved, although we recently found that inhibin betaA-subunit, activin A, activin receptor type IA, type IB, type IIA, type IIB, Smad2, Smad3 and Smad4 were localized in tumor cells of the ovarian clear cell adenocarcinoma tissue by immunohistochemistry. In the present study, in order to investigate the role of activin concerning cell growth in ovarian clear cell adenocarcinoma cells, we determined the production of activin A and inhibin A, and the expression of activin receptors and Smads using the human ovarian clear cell adenocarcinoma cell line JHOC-5. Moreover, we examined the effects of activin A on the activation of activin signaling pathway and on the proliferation in JHOC-5 cells. We detected a measurable amount of activin A in the culture medium of JHOC-5 cells, although inhibin A was not detected. The expression of activin receptor type IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 was observed in JHOC-5 cells. Activin A induced a significant increase in proliferation of JHOC-5 cells compared with the untreated control. On the other hand, activin A did not affect the growth of JHOC-5 cells and no statistically significant difference was observed in the presence of follistatin which is a specific binding protein of activin. Phosphorylated Smad2, an activated form of Smad2, was detected both in treated JHOC-5 cells and in untreated cells by activin A. Activin A significantly increased the expression of phosphorylated Smad2 in JHOC-5 cells. Therefore, it is possible that activin has autocrine roles in tumor growth of ovarian clear cell adenocarcinoma cells.

This study was designed to investigate the relationship between apoptosis and Bcl-2 and Bax expressions in uterine cervical cancer after balloon-occluded arterial infusion (BOAI).Twenty-four specimens were obtained before and after BOAI. The occurrence of apoptosis was examined with molecular biochemical techniques. The expressions of Bcl-2 and Bax proteins were investigated by immunohistochemical staining.Labelling of DNA in situ indicated that apoptotic cells were sporadically seen before BOAI (6.1 +/- 1.9). Apoptotic cells apparently increased at 5 days (25.1 +/- 6.4) after BOAI The autoradiographic analysis revealed that the DNA-ladder was identified at 5 days after BOAI. Although Bcl-2 immuno-reactivity was faintly detected, the expression of Bax increased at 3 days (49.4 +/- 10.4%) after BOAI.The results indicated that treatment with BOAI resulted in transient increases of apoptosis in cervical cancer in association with the increased expression of Bax.

Patients with unresectable advanced carcinoma of the uterine cervix are usually treated with chemotherapy or chemoradiotherapy. In the present study, the optimal administration protocol for etoposide in chemotherapy and chemoradiotherapy for advanced cervical cancer patients was investigated in vitro using the radio-sensitive and anticancer drug-sensitive human cervical squamous cell carcinoma cell line ME180. Therapeutic doses of concurrent irradiation reduced the cellular etoposide sensitivity in a dose-dependent manner, while postirradiation-surviving subclones established from repeatedly irradiated ME180 cells showed significantly higher etoposide sensitivities than the non-irradiated parent cells. Of the 6 monoclonal etoposide-resistant subclones established from ME180 cells, 5 were significantly radio-resistant. Although the etoposide-resistant subclones were also significantly resistant to other anticancer drugs, such as cisplatin, carboplatin, nedaplatin, pirarubicin, paclitaxel and docetaxel, they were more sensitive to 5-fluorouracil, mitomycin C and SN38 than the parent cells. Flow cytometric analyses revealed that the etoposide-resistant subclones showed significantly increased cell surface expression of CD40 compared to the parent cells, which expressed undetectable levels of CD40. However, the expression of some integrin receptor subunits, such as CD29, CD49a and CD49f, was apparently reduced in the etoposide-resistant subclones. These results indicate that etoposide should be administered to advanced cervical squamous cancer patients after the completion of radiotherapy, rather than as a concurrent chemoradiotherapy. In order to kill surviving etoposide-resistant cancer cells more effectively, 5-fluorouracil, mitomycin C and irinotecan may be candidate combination drugs for use with etoposide. Differential expression of integrin receptors and CD40 may be involved in the acquisition of etoposide resistance by cervical squamous cancer cells.

The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.

Our purpose was to examine the immunolocalization of inhibin, activin and the activin signaling system in ovarian clear cell adenocarcinoma tissue. Tissue samples of ovarian clear cell adenocarcinoma were collected from ten women. The patients' ages ranged from 41 to 71 years (mean +/- standard deviation; 53.4+/-9.9). Their surgicopathological staging was based on the criteria of the International Federation of Gynecology and Obstetrics (FIGO); there were 5 patients with stage I, 3 with stage II, and 2 with stage III. We examined the immunohistochemical localization of inhibin/activin alpha-subunit, betaA-subunit, activin A, and activin receptor types IA, IB, IIA, IIB, Smad2, Smad3 and Smad4 using an avidin-biotin peroxidase complex technique. With the antibodies against the betaA-subunit, and activin A, we observed positive immunoreactive staining in the cytoplasm of clear cell adenocarcinoma, whereas we did not observe any staining for the alpha-subunit of inhibin. We observed positive immunoreactive staining in the cell cytoplasm and surface with the antibodies against the activin receptors, types IA, IB, IIA and IIB. We observed positive immunoreactive staining in the cytoplasm and nucleus with the antibodies against the Smad2, Smad3 and Smad4. It is suggested that activin A, not inhibins, might be secreted by ovarian clear cell adenocarcinoma, and that an activin signal transduction pathway might exist in ovarian clear cell adenocarcinoma.

Objective To investigate the characteristic structure and function of human corpora lutea (CL), various types of collagen expression were determined in the CL tissues during the menstrual cycle and early pregnancy. Design In vitro experiment. Setting Department of obstetrics and gynecology at a medical university. Patient(s) Regulatory cycling women and pregnant women with ovarian tumor and ectopic pregnancy who underwent adnexectomy. Intervention(s) Immunohistochemistry for human type I, III, and IV collagen with specific monoclonal antibodies was used for analysis. Main Outcome Measure(s) Expression of type I, III, and IV collagen. Result(s) Immunohistochemical staining for type I and III collagen revealed intense staining of the CL stroma during early pregnancy, as compared with those in the menstrual cycle. Moreover, pericellular intense immunostaining for type IV collagen was observed around the luteal cells, especially luteal granulosa cells, of early pregnancy. Conclusion(s) These results suggest that alterations in distribution of collagen might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.

Death-associated protein kinase (DAP kinase) is a Ca2+/calmodulin-dependent serine/threonine kinase implicated as a positive apoptosis mediator. However, little is known about DAP kinase involvement with apoptosis in renal diseases.In order to determine whether DAP kinase has a role in renal cell apoptosis in kidney diseases, we performed an immunohistochemical study using a monoclonal antibody to DAP kinase. Firstly, by examining the cellular distribution of DAP kinase in normal human renal tissues and cultured proximal tubule cells. We then used western blotting and immunohistochemical analysis to examine directly whether DAP kinase protein levels could be modulated in rat kidneys with chronic obstructive uropathy created by unilateral ureteric ligation.Immunohistochemistry of normal human kidney tissues showed that DAP kinase was exclusively localized in the cytoplasm of renal tubule cells. Expression analysis of DAP kinase using cultured cells confirmed DAP kinase mRNA and protein presence in human renal tubule cells. Immunocytochemical analysis directly visualized DAP kinase in the cytoplasm of the renal tubule cells in culture. Finally, DAP kinase was found up-regulated in renal tubule cells of rat kidneys with chronic obstructive uropathy.Our study demonstrates that DAP kinase is localized to renal tubule cells, implying a crucial role for DAP kinase in renal tubular cell apoptosis in progressive renal diseases.

Recent reports have demonstrated that the peritoneal fluid and serum concentrations of leptin are increased in women with endometriosis. However, the pathophysiological roles of leptin in endometriosis have not been well characterized. In this study, we examined the direct effects of leptin on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. No effects of leptin on cell viability and prolactin secretion were found in unstimulated endometrial stromal cells. Leptin dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and leptin while PRL secretion from the cells was significantly inhibited in a dose-dependent manner. As for 8-Br-cAMP-stimulated cells, leptin significantly enhanced their cell viability in a dose-dependent manner but not their PRL secretion. These results indicate that leptin enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that it inhibits the decidualization process of endometrial stromal cells. Increased leptin in endometriotic patients might play an antiapoptotic role in some activated ESCs in the peritoneal cavity to stimulate endometrial cell implantation, and might cause infertility by inhibiting stromal decidualization.

Decreased numbers of eutopic endometrial apoptotic cells have been reported in endometriotic patients, indicating the possibility of antiapoptotic effects of their sera. In this preliminary study, we examined the effects of serum from 8 endometriotic patients on cell proliferation and viability of eutopic human endometrial stromal cells obtained from 3 non-endometriotic women. Endometrial stromal cell proliferation was stimulated in a dose-dependent manner by all sera examined, including those of healthy women. Compared with the sera from healthy women, all sera from 8 endometriotic patients significantly enhanced stromal cell proliferation, and 5 of the 8 sera significantly inhibited 8-Br-cAMP-activation-induced cell death in endometrial stromal cells. Considering these results, we suggest that the stromal cell growth and surviving factor(s) from the serum of most endometriotic patients have a higher activity, which may stimulate ectopic implantation and proliferation of endometrial stromal cells.

Fas antigen (CD95) is expressed on almost all types of human cells and is believed to mediate receptor-specific apoptotic signals. In human endometrial tissues, high Fas and Fas ligand expressions and Fas-mediated apoptosis in endometrial epithelial cells have been discussed in many reports but no study has examined Fas-mediated signals in endometrial stromal cells. In this study we investigated Fas expression and Fas-mediated signals of normal human endometrial stromal cells. Flow cytometric analysis revealed Fas antigen expression on the stromal cells and their Fas expression was enhanced by 8-Br-cAMP, a strong inducer of decidualization. Neither short-term nor long-term cultures with anti-Fas IgM affected proliferation or viability of the stromal cells. Anti-Fas IgM alone affected neither viable cell numbers nor PRL release of unstimulated stromal cells. However, anti-Fas IgM dose-dependently stimulated viable cell numbers of stromal cells co-stimulated with 8-Br-cAMP and anti-Fas IgM, whereas PRL secretion of the co-stimulated cells was not affected. Anti-Fas IgM dose-dependently stimulated viable cell numbers of 8-Br-cAMP-stimulated cells but did not affect PRL secretion of 8-Br-cAMP-induced decidualized cells. These results indicate that Fas antigen on human endometrial stromal cells cannot mediate receptor-specific apoptotic signals, and that Fas-mediated signals stimulate survival of 8-Br-cAMP-stimulated non-decidualized stromal cells. Thus, stimulation of Fas-antigen on the endometrial stromal cells enhances anti-apoptotic/survival signals in certain stromal cells, autoregulating functional cellular subpopulations of human endometrial stromal cells in a paracrine manner.

Normal human endometrial stromal cells (ESCs) stimulated with 8-Br-cAMP secrete significantly large amounts of PRL and granulocyte colony-stimulating factor (G-CSF), whereas unstimulated stromal cells secreted little. However, there is no relation between PRL and G-CSF levels secreted from the stimulated cells, which suggests that PRL-secreting stromal cells may not completely coincide with the G-CSF-producing stromal cells. Recently we found that macrophage colony-stimulating factor (M-CSF) inhibits 8-Br-cAMP-induced decidualization, differentiation to prolactin (PRL)-secreting cells, by suppressing viable decidualizing cells. Therefore, we investigated the effects of M-CSF on G-CSF secretion from normal human endometrial stromal cells using an in vitro decidualization activity assay. M-CSF did not show any significant effects on viable cell numbers of unstimulated ESCs, while M-CSF dose-dependently enhanced G-CSF release from the non-decidualized stromal cells. A high concentration of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells co-stimulated with 8-Br-cAMP and M-CSF, although G-CSF release from the co-stimulated stromal cells was not affected by M-CSF. Moreover, M-CSF did not affect viable cell numbers, PRL secretion or G-CSF secretion of 8-Br-cAMP-stimulated cells. These results indicate that M-CSF enhances G-CSF secretion from 8-Br-cAMP-unstimulated human endometrial stromal cells but not from 8-Br-cAMP-stimulated stromal cells, thus suggesting that there exists a functional subpopulation of G-CSF-secreting stromal cells that are different from the predecidualized ESCs that differentiate into PRL-secreting ESCs under stimuli with 8-Br-cAMP. Hence, M-CSF may autoregulate functional cellular subpopulations of human endometrial stromal cells in an autocrine or a paracrine manner.

It is well known that oncostatin M binds and activates leukemia inhibitory factor-specific receptors while leukemia inhibitory factor cannot bind oncostatin M-specific receptors. In this study, the differential effects of oncostatin M and leukemia inhibitory factor on cell survival and decidualization of normal human endometrial stromal cells were investigated by using 8-Br-cAMP-induced decidualization assay. Oncostatin M did not affect the viable cell numbers or the prolactin release of unstimulated stromal cells. However, oncostatin M dose-dependently suppressed prolactin releases from 8-Br-cAMP-stimulating stromal cells but enhanced their viable cell numbers. Although oncostatin M significantly enhanced viable cell numbers of 8-Br-cAMP-stimulated stromal cells, it did not affect prolactin secretion from 8-Br-cAMP-induced decidualized cells. Leukemia inhibitory factor significantly enhanced viable cell numbers of 8-Br-cAMP-stimulating cells in a dose-dependent manner as did oncostatin M, while leukemia inhibitory factor did not show any significant suppression of PRL secretion from 8-Br-cAMP-stimulating cells. These results indicate that oncostatin M inhibits the 8-Br-cAMP-induced decidualization process via oncostatin M-specific receptors and that oncostatin M and leukemia inhibitory factor enhance cell survival of 8-Br-cAMP-stimulated prolactin-non-secreting stromal cells mainly via leukemia inhibitory factor receptors. Thus, oncostatin M may autoregulate human endometrial stromal cell survival and decidualization in a paracrine manner.

The purpose of this study was to evaluate PET using (18)F-FDG for gynecologic lesions with continuous bladder irrigation to eliminate artifacts from the (18)F-FDG activity in the bladder.Forty-one patients were studied. They had 23 cervical uterine lesions (15 cases of cancer, 5 recurrences, 3 nonrecurrences); 8 cases of uterine corpus cancer, including 2 recurrences; and 10 ovarian masses (6 malignant, 4 nonmalignant). All cases of cancer were histologically proven; however, 2 cases of nonrecurrent uterine cervical carcinomas were diagnosed by clinical course. Continuous bladder irrigation was performed 35-55 min after intravenous administration of 185-370 MBq (18)F-FDG, and an emission scan was obtained 40-55 min after intravenous administration. Standardized uptake value (SUV) was used to estimate the degree of (18)F-FDG uptake quantitatively.After bladder irrigation, the (18)F-FDG activity in the urinary tract was eliminated in 33 patients, so that detection of tumor (18)F-FDG accumulation was easy. Two patients showed residual activity in the urinary bladder, and 6 patients showed activity in the ureter. An artifact was seen in 1 patient with residual activity in the urinary bladder caused by insufficient irrigation. However, these residual activities had no influence on detecting (18)F-FDG accumulation in tumor. The mean (+/-SD) of SUVs of malignant lesions was 6.04 +/- 3.22, that of nonmalignant lesions was 1.71 +/- 1.12, and the difference was significant (P = 0.0002). SUVs of all malignant lesions were greater than 2.0, and SUVs of all nonmalignant lesions, except the 1 case of ovarian fibroma, were less than 2.0.(18)F-FDG PET with continuous bladder irrigation is useful for eliminating (18)F-FDG activity in the bladder and for differentiating between malignant and nonmalignant uterine or ovarian masses.

Leukemia inhibitory factor (LIF) is produced locally in decidual tissues, but its direct effects on endometrial stromal cells have not been well characterized. In this study, we examined the direct effects of LIF on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. We found no effects of LIF on cell viability and prolactin secretion of unstimulated endometrial stromal cells. LIF dose-dependently enhanced cell viability, but not prolactin secretion of 8-Br-cAMP-stimulated cells. LIF dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and LIF without any significant effect on PRL secretion from the cells. Further, the extracellular matrix did not affect these stimulatory effects of LIF on cell viability of 8-Br-cAMP-stimulated endometrial stromal cells. These results indicate that LIF enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that the cell survival signals generated by LIF are independent of those generated by the extracellular matrix. LIF produced locally in decidual tissues may enhance cell viability in certain activated endometrial stromal cells in an autocrine or paracrine manner, and possibly protect against cell damage during embryo implantation and trophoblastic invasion.

Local danazol therapy reduces the signs and symptoms of endometriosis without inhibition of regular ovulation and menstruation and without atrophic changes to the endometrium or vaginal wall. It has been suggested that danazol has possible non-cytotoxic direct actions on eutopic endometrial cells and endometriotic cells. We have investigated the direct effects of danazol on a human endometrial epithelial cell line, HHUA, which is believed to retain many normal intracellular signaling pathways. A thoroughly dissolved solution of danazol enhanced Fas-mediated apoptosis in HHUA cells without inhibiting cell proliferation. Semi-quantitative flow cytometric analysis revealed that danazol did not enhance cell surface expression of Fas antigens. The enhancement of Fas-mediated apoptosis by endometrial cytokines such as EGF, IL-1beta and interferon-gamma was not additively enhanced by danazol; nor did danazol enhance growth suppression by anticancer drugs such as paclitaxel, carboplatin and 5-fluorouracil. Moreover, danazol did not enhance the irradiation-induced cell growth suppression of radiation-sensitive human cervical squamous cancer ME180 cells. These results indicate that danazol may regulate endometrial epithelial cell proliferation and apoptosis within normal physiology.

We investigated the expression and function of laminin and type IV collagen (COL-4) in the human endometrial epithelium throughout the menstrual cycle. Their expression was demonstrated by immunohistochemistry, while cytological analysis of the human endometrial epithelial cell line HHUA cultured on laminin- or COL-4-coated plates provided information on their roles in cell proliferation, structure and apoptosis. Laminin and COL-4 were detected in subepithelial basement membrane-like structures and their expression levels varied throughout the menstrual cycle. Laminin expression was significantly decreased in secretory phase endometrial surface epithelium, while COL-4 expression was significantly decreased in late proliferative phase endometrial epithelium and in vascular endothelium. The morphology of proliferating HHUA cells varied depending on the extracellular matrix component coated on the culture plates. COL-4 strongly inhibited cell proliferation of HHUA cells and enhanced Fas antigen (CD95)-mediated apoptosis, while laminin inhibited Fas-mediated apoptosis. These results indicate that the cyclic expression of laminin and COL-4 in basement membrane-like structures of endometrial epithelium may regulate the endometrial epithelial remodeling and embryo implantation during the menstrual cycle.

Objective : To analyze histopathologic features related to myoma volume reduction after treatment with a GnRH agonist, buserelin acetate, in needle biopsy specimens obtained before treatment. Design : Prospective clinical study. Setting : University teaching hospital. Patient(s) : Twenty women with normal menstrual cycles and symptomatic uterine myomas. Intervention(s) : Buserelin acetate was administered intranasally (900μg/d) for at least 16 weeks; transcervical needle biopsy of the uterine myoma was done before this treatment. Main Outcome Measure(s) : The relation between histopathologic features (degree of cellularity, hyaline change, and collagen content) and the percent decrease in the volume of the largest myoma at 16 weeks measured by magnetic resonance imaging. Result(s) : The correlation between the degree of hyaline change and the percent decrease in the myoma volume was significant, as was that with the proportion of collagenous tissue in the specimens. Conclusion(s) : We predicted myoma volume reduction after treatment with a GnRH agonist by histologic analysis of the uterine leiomyoma before treatment.

It is generally recognized that an endometrial thickness of 4-8 mm is considered a definite cutoff point for detection of endometrial malignancy by transvaginal sonography. We report on 2 patients with endometrial cancer and on 1 with endometrial hyperplasia, in each of whom endometrial thickness was < 4 mm.