Feasibility of a mobile cardiotocogram device for fetal heart rate self-monitoring in low-risk singleton pregnant women.

AIM: This study aimed to clarify the feasibility of a mobile cardiotocogram (CTG) device for self-monitoring fetal heart rate (FHR) in low-risk singleton pregnant women. METHODS: This study was conducted at six university hospitals and seven maternity clinics in Japan. Using a mobile cardiotocogram device (iCTG, Melody International Ltd., Kagawa, Japan), participants of more than 34 gestational weeks measured the FHR by themselves at least once a week until hospitalization for delivery. We evaluated the acquisition rate of evaluable FHR recordings and the frequency of abnormal FHR patterns according to the CTG classification system of the Japan Society of Obstetrics and Gynecology (JSOG). The participants also underwent a questionnaire survey after delivery to evaluate their satisfaction level of self-monitoring FHR using the mobile CTG device. RESULTS: A total of 1278 FHR recordings from 101 women were analyzed. Among them, 1276 (99.8%) were readable for more than 10 min continuously, and the median percentage of the total readable period in each recording was 98.9% (range, 51.4-100). According to the JSOG classification system, 1245 (97.6%), 9 (0.7%), 18 (1.4%), and four (0.3%) FHR patterns were classified as levels 1, 2, 3, and 4, respectively. The questionnaire survey revealed high participant satisfaction with FHR self-monitoring using the iCTG. CONCLUSION: The mobile CTG device is a feasible tool for self-monitoring FHR, with a high participant satisfaction level.

Isolation and characterization of human trophoblast side-population (SP) cells in primary villous cytotrophoblasts and HTR-8/SVneo cell line.

Recently, numerous studies have identified that immature cell populations including stem cells and progenitor cells can be found among "side-population" (SP) cells. Although SP cells isolated from some adult tissues have been reported elsewhere, isolation and characterization of human trophoblast SP remained to be reported. In this study, HTR-8/SVneo cells and human primary villous cytotrophoblasts (vCTBs) were stained with Hoechst 33342 and SP and non-SP (NSP) fractions were isolated using a cell sorter. A small population of SP cells was identified in HTR-8/SVneo cells and in vCTBs. SP cells expressed several vCTB-specific markers and failed to express syncytiotrophoblast (STB) or extravillous cytotrophopblast (EVT)-specific differentiation markers. SP cells formed colonies and proliferated on mouse embryonic fibroblast (MEF) feeder cells or in MEF conditioned medium supplemented with heparin/FGF2, and they also showed long-term repopulating property. SP cells could differentiate into both STB and EVT cell lineages and expressed several differentiation markers. Microarray analysis revealed that IL7R and IL1R2 were exclusively expressed in SP cells and not in NSP cells. vCTB cells sorted as positive for both IL7R and IL1R2 failed to express trophoblast differentiation markers and spontaneously differentiated into both STB and EVT in basal medium. These features shown by the SP cells suggested that IL7R and IL1R2 are available as markers to detect the SP cells and that vCTB progenitor cells and trophoblast stem cells were involved in the SP cell population.

Incidence of postmolar gestational trophoblastic disease in androgenetic moles and the morphological features associated with low risk postmolar gestational trophoblastic disease.

In the present study, we evaluated the incidence of postmolar gestational trophoblastic disease (GTD) in molar pregnancy. We also validated the macroscopic diagnosis based on the Japan Society of Obstetrics and Gynecology (JSOG) classification. A total of 297 samples of hydropic villi were classified according to DNA polymorphisms as androgenetic moles, dispermic triploids, or biparental diploids (hydropic abortion), clinically corresponding to complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), and hydropic abortion, respectively. These samples were also classified morphologically based on the JSOG classification. A follow-up study was performed to investigate the incidence of postmolar GTD. A subset of 267 samples eligible for testing were analyzed and diagnosed as androgenetic moles (232 cases), dispermic triploids (20 cases), and biparental diploids (15 cases). Most of the macroscopically diagnosed CHM cases were genetically androgenetic in origin. The PHM cases consisted of 30 androgenetic moles and 12 dispermic triploids. We reviewed the outcomes of 200 patients (178 cases of androgenetic mole, 13 cases of dispermic triploids, and nine cases of biparental diploids). Twenty-eight cases (16%) of androgenetic moles developed postmolar GTD. None of the patients with dispermic triploids developed postmolar GTD. Among the 28 patients who developed postmolar GTD, the shortest diameter of the largest hydropic villi was significantly longer than that of patients not developing postmolar GTD. None of the patients with androgenetic moles who had hydropic villi <2 mm in their shortest diameter developed postmolar GTD. For the patients with dispermic triploids, the risk of postmolar GTD is extremely low. The risk of postmolar GTD is also low in patients with androgenetic moles with small hydropic villi. The JSOG classification based on the morphology of hydropic villi is reliable for the diagnosis of CHM, but inaccurate for the diagnosis of PHM or "microscopic" moles.

Prognostic implications of the nuclear localization of Y-box-binding protein-1 and CXCR4 expression in ovarian cancer: their correlation with activated Akt, LRP/MVP and P-glycoprotein expression.

The nuclear localization of Y-box-binding protein-1 (YB-1) is known to be a poor prognostic factor in several human malignancies, including ovarian carcinoma. Following on from our basic study dealing with microarray analyses of YB-1-associated gene expression in ovarian cancer cells, we examined whether nuclear localization of YB-1 is associated with the expression of CXCR4, a vault protein named lung resistance-related vault protein (LRP/MVP), phosphorylated Akt (p-Akt) or P-glycoprotein (P-gp) in human ovarian carcinoma. Fifty-three surgically resected ovarian carcinomas treated with paclitaxel and carboplatin were examined immunohistochemically for nuclear YB-1 expression and intrinsic expression of p-Akt, P-gp, LRP/MVP and CXCR4. Nuclear expression of YB-1 demonstrated significant correlation with p-Akt, P-gp and LRP expression, but no relationship with CXCR4 expression. By multivariate analysis, only YB-1 nuclear expression and CXCR4 expression were independent prognostic factors with regard to overall survival. These results indicate that YB-1 nuclear expression and CXCR4 expression are important prognostic factors in ovarian carcinoma.

Complete mole coexistent with a twin fetus.

We report two cases of a complete hydatidiform mole coexistent with a live fetus diagnosed by DNA polymorphism analysis. A 27-year-old woman revealed symptoms of pre-eclampsia and ultrasound showed multicystic tumor and placenta coexistent with a live fetus at 16 weeks' gestation. The placenta with partly hydropic change and the fetus without anomaly were consequently evacuated. Another 30-year-old woman had a multicystic mass attached to a normal placenta with a 20-week live fetus on ultrasound examination. A hysterotomy was carried out because of persistent bleeding due to placenta previa. In both cases, DNA was extracted from the placental tissue and the tumor, as well as from maternal and paternal blood. Genetic analysis demonstrated that the placental tumor consisted of only paternal origin, which is consistent with the diagnosis of complete hydatidiform mole.

Adenovirus-mediated calponin h1 gene therapy directed against peritoneal dissemination of ovarian cancer: bifunctional therapeutic effects on peritoneal cell layer and cancer cells.

PURPOSE: Calponin h1 (CNh1), one of the family of actin-binding proteins, stabilizes the filaments of actin and modulates various cellular biological phenotypes. Recent studies revealed the close correlation between the invasive tumor spread and the reduced expression of CNh1 and alpha-smooth muscle actin in the surrounding stromal cells. The purpose of this study is to evaluate the efficacy of i.p. CNh1 gene therapy against peritoneal dissemination of ovarian cancer. EXPERIMENTAL DESIGN: We used an adenoviral vector to induce the CNh1 gene into peritoneal cells and ovarian cancer cells as a means of enhancing or inducing the expression of alpha-smooth muscle actin as well as CNh1. The efficacy of gene transfer was examined by in vitro cell culture and in vivo animal experiments. RESULTS: The formation of longer and thicker actin fibers was observed in each transfected cell line, and the localization of these fibers coincided with that of externally transducted CNh1. With respect to changes in cell behavior, the CNh1-transfected peritoneal cells acquired an ability to resist ovarian cancer-induced shrinkage in cell shape; thus, cancer cell invasion through the monolayer of peritoneal cells was inhibited. In addition, CNh1-transfected ovarian cancer cells showed suppressed anchorage-independent growth and invasiveness, the latter of which accompanied impaired cell motility. The concomitant CNh1 transfection into both peritoneal cells and ovarian cancer cells produced an additive inhibitory effect with respect to cancer cell invasion through the peritoneal cell monolayer. By in vivo experiments designed to treat nude mice that had been i.p. inoculated with ovarian cancer cells, we found that the i.p. injected CNh1 adenovirus successfully blocked cancer-induced morphologic changes in peritoneal cell surface and significantly prolonged the survival time of tumor-bearing mice. Moreover, CNh1 adenovirus could successfully enhance the therapeutic effect of an anticancer drug without increase in side effects. CONCLUSIONS: Thus, CNh1 gene therapy against peritoneal dissemination of ovarian cancer is bifunctionally effective (i.e., through inhibitory effects on the infected peritoneal cell layers that suppress cancer invasion and through direct antitumor effects against invasion and growth properties of cancer cells).

Clinical significance of RCAS1 as a biomarker of uterine cancer.

OBJECTIVES: Expression of RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is associated with prognosis of various malignancies including uterine cancer. Proteolytic cleavage of RCAS1 at extracellular domains (ectodomain shedding) yields soluble RCAS1. Although RCAS1 can induce apoptosis in normal peripheral lymphocytes, its biologic function in cancer patients is unclear. Here, we evaluated serum RCAS1 concentrations to clarify its biologic activity in uterine cancer. METHODS: Via ELISA, we measured serum RCAS1 concentrations in samples from 54 healthy blood donors and 113 patients-63 with cervical cancer and 50 with endometrial cancer. We also counted the peripheral lymphocyte number. We correlated via statistical means the RCAS1 values with patients' clinicopathologic variables. We assessed inhibition of growth of K562 cells, which express the putative RCAS1 receptor, via WST-1 assay of serum samples to clarify RCAS1's biologic activity. RESULTS: Uterine cancer patients had significantly higher serum RCAS1 concentrations than did healthy blood donors (P<0.05). Patients with adenocarcinoma had significantly higher RCAS1 concentrations than did those with squamous cell carcinoma (P=0.0340). RCAS1 values were also significantly associated with response to treatment (P<0.001). FasL and TNF-alpha serum concentrations were not significantly different for the different groups, however. The WST-1 assay showed that patients' serum induced K562 cell growth inhibition, but this effect partially recovered after immunodepletion of RCAS1. Peripheral lymphocyte number and serum RCAS1 concentration were inversely related (P=0.0310). CONCLUSION: RCAS1 may be a biomarker of uterine cancer because of its potential to predict results of uterine cancer treatment and inhibit growth of immune cells.

Prophylactic effect of pemirolast, an antiallergic agent, against hypersensitivity reactions to paclitaxel in patients with ovarian cancer.

We have previously shown that sensory nerve peptides contribute to the pathogenesis of pulmonary hypersensitivity reactions (HSRs) to paclitaxel in rats. Moreover, pemirolast, an antiallergic agent, reverses the HSRs to paclitaxel, although the mechanism is considered to result from the blockade of paclitaxel-induced release of sensory peptides, rather than the inhibition of histamine release. In the present study, we investigated the preventive effect of pemirolast against acute HSRs in a total of 84 patients who undertook postoperative paclitaxel plus carboplatin chemotherapy every 4 weeks for ovarian cancer. Patients were assigned to receive oral lactose (placebo) or pemirolast (10 mg), 2 hr before paclitaxel infusion. All patients received conventional premedication, including oral diphenhydramine, intravenous ranitidine and intravenous dexamethasone, 30 min before paclitaxel infusion. The HSRs that led to the discontinuance of paclitaxel infusion (grade>or=2) occurred in 5 of 42 patients in placebo group, whereas none of pemirolast-treated 42 patients showed any signs of HSRs. Plasma histamine concentrations were not changed after paclitaxel infusion in either group. Our present findings suggest that pemirolast is potentially useful for prophylaxis of paclitaxel-induced HSRs. In this respect, the use of pemirolast as premedication is expected to be beneficial to the safety management in patients who undergo chemotherapy containing paclitaxel.

Genome-wide definitive haplotypes determined using a collection of complete hydatidiform moles.

We present genome-wide definitive haplotypes, determined using a collection of 74 Japanese complete hydatidiform moles, each carrying a genome derived from a single sperm. The haplotypes incorporate 281,439 common SNPs, genotyped with a high throughput array-based oligonucleotide hybridization technique. Comparison of haplotypes inferred from pseudoindividuals (constructed from randomized mole pairs) with those of moles showed some switch errors in resolution of phases by the computational inference method. The effects of these errors on local haplotype structure and selection of tag SNPs are discussed. We also show that definitive haplotypes of moles may be useful for elucidation of long-range haplotype structure, and should be more effective for detecting extended haplotype homozygosity indicative of positive selection.

Expression of RCAS1 in female genital organs.

Receptor-binding antigen expressed on a human uterine adenocarcinoma cell line, SiSo (RCAS1), has been reported to be a prognostic factor of various malignant tumors, and it has also been proven to induce apoptosis of lymphoid cells. However, its normal distribution and function have not yet been elucidated. The purpose of this study was to disclose the distribution of RCAS1 expression in normal female genital organs. Immunohistochemical staining using anti-RCAS1 and anti-MIB-1 antibodies was performed on 123 surgical specimens of a histologically normal uterus, ovary, or fallopian tube from 66 patients, and the apoptotic index was determined. In uterine cervical glands, the expression of RCAS1 was seen in 93% of the cases, and it was mainly localized in the superficial cervical glands. Near the areas of squamous metaplasia, RCAS1 was strongly expressed in all samples. In the uterine cervical squamous epithelium, RCAS1 was seen in 84% of cases. In the uterine corpus, RCAS1 was seen in 87% of all cases, and it was mainly expressed in the endometrial glands of basalis layer. There was significant positive correlation between age and RCAS1 expression, but no significant difference was found regarding the endometrial status and RCAS1 expression in endometrium. No significant correlation was found between RCAS1 expression and MIB-1 index/apoptotic index. RCAS1 may affect these metaplastic processes and tumor progression.

Invasive potency related to RCAS1 expression in uterine cervical cancer.

OBJECTIVES: RCAS1 expression is significantly associated with clinical prognosis in various human cancers, which suggests that RCAS1 may be involved in acquisition of malignant phenotypes. To investigate the relationship between RCAS1 and one such characteristic, tumor invasiveness, we examined RCAS1 expression in cervical neoplasms ranging from the precancerous state to invasive cancer. METHODS: RCAS1 expression was studied retrospectively via immunohistochemical methods. Samples consisted of biopsy tissue from 90 patients with intraepithelial neoplasia and resected tumor tissue from 154 patients with invasive cancer. Statistical analysis was done to correlate RCAS1 expression and clinicopathologic variables in patients with a depth of cancer cell invasion into stromal tissue of >5 mm. RESULTS: RCAS1 expression was detected in patients with intraepithelial cancer and invasive cancer but not in patients with dysplasia. The occurrence and degree of RCAS1 expression increased with the depth of invasion. In patients with invasive cancer, RCAS1 overexpression was significantly correlated with invasion of the lymph-vascular space, lymph node metastasis in two or more sites, and tumor volume; RCAS1 expression was not associated with histologic subtype. Overall survival rates for patients with RCAS1 overexpression were significantly shorter than those for patients without RCAS1 overexpression. In connective tissue surrounding tumor cells, the number of cells expressing vimentin significantly decreased in relation to RCAS1 expression level. Moreover, significant associations between expression levels of RCAS1 and those of MMP-1 and laminin-5 were found. CONCLUSION: RCAS1 may contribute to acquisition of malignant uterine cervical phenotypic characteristics including invasion, metastasis, and tumor growth via connective tissue remodeling.

Differential diagnosis between complete and partial mole by TSSC3 antibody completely correlates to DNA diagnosis.

Complete hydatidiform moles (CHMs) are a type of androgenetic fertilization without an ovum. Cases of CHM exhibit a generalized swelling of the villi and are known to be highly associated with persistent disease or carcinoma. In contrast, partial hydatidiform moles (PHMs) also show characteristic hydropic changes among the villi, but the incidence of secondary disease is relatively low. Because PHMs are fertilized by one ovum and two sperm and CHMs are fertilized by one or two sperm alone, we considered whether or not maternally imprinted genes might be useful for achieving a differential diagnosis. The validity of the imprinted genes in CHMs was assessed by implementation of a microarray technique. Among the genes examined, TSSC3, SLC22A1L, KCNQ1, and Decorin were shown to be down-regulated, and TSSC3 was the most markedly suppressed of these genes. In this study, 20 cases of CHM, the diagnosis of which was confirmed by DNA polymorphism, were investigated. In all of these cases, the expression of TSSC3 was completely absent, as determined by Western blot analysis. Conversely, 12 cases of PHM, also diagnosed by DNA polymorphism, were examined here; in all of these 12 cases, TSSC3 was found to be expressed normally. Immunohistochemical (IHC) analysis also produced the same results. The complete silencing of TSSC3 in cases of CHM will provide a novel, convenient strategy for the diagnosis of molar lesions in the placenta.

Association between RCAS1 expression and microenvironmental immune cell death in uterine cervical cancer.

OBJECTIVE: : The presence of regional lymph node metastasis is one of the prognostic factors for uterine cervical cancer. The development of metastasis requires that cancer cells avoid lymphocyte attack. Impaired lymphocyte function is mediated by apoptotic factors including receptor-binding cancer antigen expressed on SiSo cells (RCAS1), Fas ligand (FasL), and tumor necrosis factor-alpha (TNF-alpha). Our aim was to evaluate the association between expression of these factors and microenvironmental lymphocyte apoptosis in this disease. METHODS: : Immunohistochemical methods were used to determine the relationship between the expression of RCAS1, FasL, and TNF-alpha, and the number of apoptotic lymphocytes in primary lesions and metastatic lymph nodes in patients with cervical cancer. RESULTS: : Expression of these apoptosis-inducing molecules was quite different in primary tumors and metastatic lymph nodes: RCAS1 expression in lymph nodes was significantly higher than that in primary lesions (P < 0.0001), whereas FasL and TNF-alpha expressions at these two locations were not significantly different. The number of cells with positive expression of RCAS1, but not of FasL or TNF-alpha, was significantly correlated with the number of apoptotic lymphocytes in uterine cervix and metastatic lymph nodes (P < 0.0001 for both). CONCLUSION: : RCAS1 expression may be related to tumor cell evasion of immune surveillance via induction of lymphocyte apoptosis in primary lesions and metastatic lymph nodes in uterine cervical cancer.

Incidence and risk factors for paclitaxel hypersensitivity during ovarian cancer chemotherapy.

Hypersensitivity reaction (HSR) is still a major concern during cancer chemotherapy with paclitaxel. In the present study, we investigated retrospectively the incidence of HSRs to paclitaxel and the risk factors in 105 patients (553 courses) who received adjuvant chemotherapy (paclitaxel and carboplatin) for ovarian cancer. Moderate to severe HSRs that led to cessation or discontinuation of the chemotherapy, including respiratory distress and hypotension, were observed in 14 patients (13.3%) and 16 courses (2.9%), regardless of the use of conventional premedication with glucocorticoid, and histamine H(1) and H(2) antagonists. The incidence of HSRs to paclitaxel in patients with ovarian cancer seemed to be considerably higher than those reported by other investigators in patients with other carcinomas such as non-small-cell lung cancer and breast cancer. Four risk factors were identified: (1) history of mild dermal reactions such as facial flushing and urticaria in previous courses, (2) presence of respiratory dysfunction, (3) obesity (body mass index >25), and (4) postmenopausal at the time of ovariectomy. The incidence of hypersensitivity increased linearly as the number of risk factors increased (r=0.992, P=0.008). It is likely that disappearance of the estrous cycle facilitates the occurrence of HSRs to paclitaxel.

Frequent microsatellite instability in synchronous ovarian and endometrial adenocarcinoma and its usefulness for differential diagnosis.

Synchronous tumors of the ovary and endometrium are a well-known phenomenon. There are histological criteria for defining double primary tumors or metastasis from one organ to another, but in some cases a precise diagnosis is difficult. In this study we reviewed 17 cases of synchronous ovarian and endometrial adenocarcinoma by previously reported histological criteria and performed a microsatellite analysis, combined with X-linked clonality analysis. We also analyzed 8 cases of endometrial adenocarcinoma with pelvic lymph node metastasis as a control. Five dinucleotide microsatellite markers were selected, and microsatellite analysis was performed by a high-resolution method using fluorescence-labeled polymerase chain reaction and laser scanning. In synchronous tumors, 11 ovarian carcinomas (65%) and 10 endometrial carcinomas (59%)demonstrated microsatellite instability (MSI). In total, 13 of the 17 patients demonstrated MSI in the ovarian tumor, the endometrial tumor, or both. Four cases of endometrial carcinoma with pelvic lymph nodes metastases displayed MSI, and MSI findings of the endometrial tumor and lymph node metastasis were same in all cases. Based on these findings, we considered that similar MSI findings indicate metastatic tumors. According to the MSI findings, 13 of the 17 patients (76%) had single or double clonal tumors, 11 (67%) with double primary tumors and 2 (13%) with metastatic tumors. Using X-linked clonality analysis, 3 patients were diagnosed with double primary tumors. The molecular diagnosis corresponded with the histological criteria in all but 1 case. In conclusion, using both MSI and X-linked clonality analysis, most patients (82%) could be diagnosed as having single or double clonal tumors. The histological criteria are accurate and useful in most cases; however, in some cases where the relationship between the 2 tumors is difficult to determine, high-resolution MSI analysis may be helpful.

Inhibin-producing ovarian granulosa cell tumor as a cause of secondary amenorrhea: case report and review of the literature.

We report the case of 31-year-old patient with an inhibin B-secreting granulosa cell tumor of the left ovary who presented with secondary amenorrhea. Preoperative serum hormonal levels were as follows: follicle-stimulating hormone (FSH) 0.3 mIU/mL, luteinizing hormone (LH) 9.81 mIU/mL, estradiol 142.0 pg/mL and inhibin B 2429 pg/mL. Gonadotropin-releasing hormone (GnRH) test revealed no FSH response and a normal LH response. After removal of the tumor, the levels of FSH and inhibin B returned to within the normal range, and regular menses resumed 27 days postoperatively. In premenopausal women, secondary amenorrhea may be the initial manifestation of granulosa cell tumor. A low FSH level coupled with normal levels of E2 and LH, the inhibition of the FSH response to GnRH and an elevated inhibin level suggest the presence of an inhibin-secreting ovarian tumor and also rule out the possibility of isolated FSH deficiency.

Uterine cervical cancer: a holistic approach to mental health and it's socio-psychological implications.

This correlation study investigated outpatients with early stage uterine cervical cancer. The subjects' mental health and its' relationship with demographic characteristics, clinical characteristics, and quality of life were examined. One hundred and seventy six patients from three major hospitals in the Fukuoka area were surveyed with a structured questionnaire. The status of mental health measured by CES-D (Center for Epidemiologic Studies Depression Scale) indicated an average score of 13+/-8 (mean+/-SD). No clinical parameters were found to have significant correlation to CES-D. However, increased pain (p< 0.001) and the absence of a husband or a partner (p < 0 .01) had greater CES-D score which indicated worse mental health outcome. The QOL (Quality of Life) scale developed for this study consists of the four domains: "Feel satisfied with life" (r = -.526, p < 0. 01), "Find life worth living" (r= -.485, p < 0.01), "Feel no hindrance in daily life" (r= -.319, p<0.01), and "Feel no anxiety with illness" (r= -. 578, p < 0.01) all which have statistically significant correlations with CES-D scores respectively. Upon examination using the multi-regression model, a strong relationship between CES-D scores and "Feel no anxiety with illness" (r= -.331, p<0.001) was evident. This showed to be the strongest indicator affecting the depression outcome, followed by "Strong pain" (r= .231, p<0.01). Clinical parameters, such as performance status, clinical stage, and medical treatment did not show any correlation to CES-D scores. The research suggests that the mental health of outpatients with uterine cervical cancer was influenced by pain and quality of life, rather than the clinical parameters. The presence of a husband or a partner played the role as social support to reduce the level of depression. In order to provide complete care of patients, pain management, anxiety management, and spousal involvement are crucial to patients' mental health, especially in the ongoing care of uterine cervical cancer.

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation.

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.