Differentiation of ovarian serous carcinoma from ovarian clear cell carcinoma using a 10-gene signature selected by comprehensive gene expression analysis.

AIM: Ovarian serous carcinoma (OSC) and ovarian clear cell carcinoma (OCCC) are two major histological types of epithelial ovarian carcinoma (EOC), each with different biological features and clinical behaviors. Although immunostaining is commonly used for differential diagnosis between OSC and OCCC, correct identification of EOC with mixed-type histology is sometimes a diagnostic challenge. The aim of the present study was to explore candidate genes as potential diagnostic biomarkers that distinguish OSC from OCCC. METHODS: A total of 57 surgical specimens were obtained from EOC patients who had previously undergone primary debulking surgery. Total RNAs were extracted from fresh-frozen tissues of EOC patients, and were used for comprehensive gene expression analysis using DNA microarray technology. RESULTS: Ten candidate genes, FXYD2, TMEM101, GABARAPL1, ARG2, GLRX, RBPMS, GDF15, PPP1R3B, TOB1, and GSTM3 were up-regulated in OCCC compared to OSC. All EOC patients were divided into two groups according to hierarchical clustering using a 10-gene signature. CONCLUSION: Our data suggest that the 10 candidate genes would be an excellent marker for distinguishing OSC from OCCC. Furthermore, the molecular signatures of the 10 genes may enlighten us on the differences in carcinogenesis, and provide a theoretical basis for OCCC's resistance to chemotherapy in the future.

SPON1 is an independent prognostic biomarker for ovarian cancer.

BACKGROUND: Ovarian cancer has the worst outcome among gynecological malignancies; therefore, biomarkers that could contribute to the early diagnosis and/or prognosis prediction are urgently required. In the present study, we focused on the secreted protein spondin-1 (SPON1) and clarified the prognostic relevance in ovarian cancer. METHODS: We developed a monoclonal antibody (mAb) that selectively recognizes SPON1. Using this specific mAb, we determined the expression of SPON1 protein in the normal ovary, serous tubal intraepithelial carcinoma (STIC), and ovarian cancer tissues, as well as in various normal adult tissues by immunohistochemistry, and verified its clinicopathological significance in ovarian cancer. RESULTS: The normal ovarian tissue was barely positive for SPON1, and no immunoreactive signals were detected in other healthy tissues examined, which was in good agreement with data obtained from gene expression databases. By contrast, upon semi-quantification, 22 of 242 ovarian cancer cases (9.1%) exhibited high SPON1 expression, whereas 64 (26.4%), 87 (36.0%), and 69 (28.5%) cases, which were designated as SPON1-low, possessed the moderate, weak, and negative SPON1 expression, respectively. The STIC tissues also possessed SPON1-positive signals. The 5-year recurrence-free survival (RFS) rate in the SPON1-high group (13.6%) was significantly lower than that in the SPON1-low group (51.2%). In addition, high SPON1 expression was significantly associated with several clinicopathological variables. Multivariable analysis revealed that high SPON1 was an independent prognostic factor for RFS of ovarian cancer. CONCLUSIONS: SPON1 represents a prognostic biomarker for ovarian cancer, and the anti-SPON1 mAb could be valuable as an outcome predictor.

Claudin-4-adhesion signaling drives breast cancer metabolism and progression via liver X receptor ß.

BACKGROUND: Cell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression. METHODS: By generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRß (liver X receptor ß) expression in breast cancer tissues from 187 patients. RESULTS: We uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRß. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRß, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRß- and LXRßS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRß-high" and "CLDN4-low and/or LXRß-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively. CONCLUSIONS: The identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.

Successful management of pelvic recurrence of MSI-High endometrial cancer by total pelvic exenteration followed by administration of pembrolizumab:A case report.

Surgery can be curative treatment for pelvic locoregional recurrence of endometrial cancer; however, a cure is contingent on complete resection. Here, we report the case of a patient in whom recurrent endometrial tumor remained in the pelvis after resection; long-term control was achieved with postoperative administration of pembrolizumab.The patient had recurrent endometrial cancer of stage IA and was treated with chemotherapy and radiation, but tumor persisted in the pelvic cavity. We therefore attempted total pelvic exenteration, but the tumor was adherent to the pelvic wall and complete resection could not be achieved. However, postoperative administration of pembrolizumab controlled the residual tumor for more than two years without regrowth. We believe that since the resected tumor was MSI-High, the residual tumor responded well to pembrolizumab. It is not known whether cytoreductive surgery contributes to a long-term response to pembrolizumab, but at least in our patient, pembrolizumab appeared to be a very effective drug therapy for MSI-High endometrial cancer that was refractory to chemotherapy and radiotherapy.

Claudin­9 is a novel prognostic biomarker for endometrial cancer.

The tight­junction protein claudin­9 (CLDN9) is barely distributed in normal adult tissues but is ectopically expressed in various cancer types. Although multiple databases indicated upregulation of CLDN9 in endometrial cancers at the mRNA level, its protein expression and biological roles remain obscure. In the present study, the prognostic significance of CLDN9 expression in endometrial cancer was evaluated by immunohistochemical staining and semi­quantification using formalin­fixed paraffin­embedded specimens obtained from 248 endometrial carcinoma cases. A total of 43 cases (17.3%) had high CLDN9 expression, whereas 205 cases (82.7%) exhibited low CLDN9 expression. The 5­year disease­specific survival rates in the high and low CLDN9 expression groups were 62.8 and 87.8% (P<0.001), respectively. In addition, multivariate analysis revealed that high CLDN9 expression was an independent prognostic factor (hazard ratio, 4.99; 95% CI, 1.96­12.70; P<0.001). Furthermore, CLDN9 expression was significantly correlated with the expression of CLDN6 (P<0.001), which is the closest CLDN member to CLDN9 and a poor prognostic factor for endometrial carcinoma. The 5­year disease­specific survival rate of cases with CLDN6­high/CLDN9­high, CLDN6­high/CLDN9­low and CLDN6­low/CLDN9­high status was 30.0, 37.5 and 72.7%, respectively, whereas that of CLDN6­low/CLDN9­low was 89.8% (P=0.004). In conclusion, aberrant CLDN9 expression is a predictor of poor prognosis for endometrial cancer and may be utilized in combination with CLDN6 to achieve higher sensitivity.

Rare Hereditary Gynecological Cancer Syndromes.

Hereditary cancer syndromes, which are characterized by onset at an early age and an increased risk of developing certain tumors, are caused by germline pathogenic variants in tumor suppressor genes and are mostly inherited in an autosomal dominant manner. Therefore, hereditary cancer syndromes have been used as powerful models to identify and characterize susceptibility genes associated with cancer. Furthermore, clarification of the association between genotypes and phenotypes in one disease has provided insights into the etiology of other seemingly different diseases. Molecular genetic discoveries from the study of hereditary cancer syndrome have not only changed the methods of diagnosis and management, but have also shed light on the molecular regulatory pathways that are important in the development and treatment of sporadic tumors. The main cancer susceptibility syndromes that involve gynecologic cancers include hereditary breast and ovarian cancer syndrome as well as Lynch syndrome. However, in addition to these two hereditary cancer syndromes, there are several other hereditary syndromes associated with gynecologic cancers. In the present review, we provide an overview of the clinical features, and discuss the molecular genetics, of four rare hereditary gynecological cancer syndromes; Cowden syndrome, Peutz-Jeghers syndrome, DICER1 syndrome and rhabdoid tumor predisposition syndrome 2.

Identification and clinical significance of somatic oncogenic mutations in epithelial ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer (EOC) is a heterogeneous disease with diverse clinicopathological features and behaviors, and its heterogeneity may be concerned with the accumulation of multiple somatic oncogenic mutations. The major goals of this study are to systematically perform the comprehensive mutational profiling in EOC patients, and investigate the associations between somatic mutations and clinicopathological characteristics. METHODS: A total of 80 surgical specimens were obtained from EOC patients who had previously undergone primary debulking surgery, and genomic DNAs were extracted from fresh-frozen tissues. We investigated mutational status in hot spot regions of 50 cancer-related genes by targeted next-generation sequencing using an Ion AmpliSeq Cancer Hotspot Panel v2 Kit. RESULTS: Validated mutations were detected in 66 of the 80 tumors (82.5%). The five most frequently mutated genes were TP53 (43.8%), PIK3CA (27.5%), KRAS (23.8%), PTEN (10%) and CTNNB1 (10%). PTEN and CTNNB1 mutations were associated with younger age. PIK3CA1, KRAS and CTNNB1 mutations were observed in early-stage, whereas TP53 mutations were more common in advanced stage. Significant associations were observed between TP53 mutation and serous carcinoma, and between KRAS mutation and mucinous carcinoma. Both PIK3CA mutation and CTNNB1 mutation were also significantly associated with endometrioid and clear cell carcinoma. The patients with PIK3CA and KRAS mutations were significantly associated with favorable progression free survival (PFS). In particular, PIK3CA mutations had more significant associations with favorable PFS than PIK3CA wild-type in the endometrioid subtype (P = 0.012). Patients with mutations only in TP53 were significantly associated with worse PFS. CONCLUSION: EOCs were heterogeneous at the genomic level and harbored somatic oncogenic mutations. Our molecular profiling may have the potential for becoming a novel stratification within histological subtypes of EOC. Further studies are needed to define molecular classification for improved clinical outcomes and treatment of EOC patients in future.

Reduced Claudin-12 Expression Predicts Poor Prognosis in Cervical Cancer.

BACKGROUND: Within the claudin (CLDN) family, CLDN12 mRNA expression is altered in various types of cancer, but its clinicopathological relevance has yet to be established due to the absence of specific antibodies (Abs) with broad applications. METHODS: We generated a monoclonal Ab (mAb) against human/mouse CLDN12 and verified its specificity. By performing immunohistochemical staining and semiquantification, we evaluated the relationship between CLDN12 expression and clinicopathological parameters in tissues from 138 cases of cervical cancer. RESULTS: Western blot and immunohistochemical analyses revealed that the established mAb selectively recognized the CLDN12 protein. Twenty six of the 138 cases (18.8%) showed low CLDN12 expression, and the disease-specific survival (DSS) and recurrence-free survival rates were significantly decreased compared with those in the high CLDN12 expression group. We also demonstrated, via univariable and multivariable analyses, that the low CLDN12 expression represents a significant prognostic factor for the DSS of cervical cancer patients (HR 3.412, p = 0.002 and HR 2.615, p = 0.029, respectively). CONCLUSIONS: It can be concluded that a reduced CLDN12 expression predicts a poor outcome for cervical cancer. The novel anti-CLDN12 mAb could be a valuable tool to evaluate the biological relevance of the CLDN12 expression in diverse cancer types and other diseases.

Aberrant Claudin-6-Adhesion Signaling Promotes Endometrial Cancer Progression via Estrogen Receptor α.

Cell adhesion proteins not only maintain tissue integrity, but also possess signaling abilities to organize diverse cellular events in a variety of physiologic and pathologic processes; however, the underlying mechanism remains obscure. Among cell adhesion molecules, the claudin (CLDN) family is often aberrantly expressed in various cancers, but the biological relevance and molecular basis for this observation have not yet been established. Here, we show that high CLDN6 expression accelerates cellular proliferation and migration in two distinct human endometrial cancer cell lines in vitro. Using a xenograft model, we also revealed that aberrant CLDN6 expression promotes tumor growth and invasion in endometrial cancer tissues. The second extracellular domain and Y196/200 of CLDN6 were required to recruit and activate Src-family kinases (SFK) and to stimulate malignant phenotypes. Knockout and overexpression of ESR1 in endometrial carcinoma cells showed that the CLDN6-adhesion signal links to estrogen receptor α (ERα) to advance tumor progression. In particular, aberrant CLDN6-ERα signaling contributed to collective cell behaviors in the leading front of endometrial cancer cells. Importantly, we demonstrate that CLDN6/SFK/PI3K-dependent AKT and SGK (serum- and glucocorticoid-regulated kinase) signaling in endometrial cancer cells targets Ser518 in the human ERα to activate ERα transcriptional activity in a ligand-independent manner, thereby promoting tumor progression. Furthermore, CLDN6, at least in part, also regulated gene expression in an ERα-independent manner. IMPLICATIONS: The identification of this machinery highlights regulation of the transcription factors by cell adhesion to advance tumor progression.

Clinical relevance of oncogenic driver mutations identified in endometrial carcinoma.

PURPOSE: Endometrial carcinoma (EC) is a clinically heterogeneous disease characterized by a number of different histological subtypes, and its heterogeneity may be involved in the accumulation of multiple genetic alterations. The aim of this work was to investigate the comprehensive mutational profile of EC tumors, and examine the associations between somatic mutations and clinicopathological features or survival in EC patients. METHODS: A total of 100 surgical tumors were obtained from EC patients who had previously undergone surgery. Genomic DNA samples extracted from fresh-frozen tissues were analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit, covering 50 tumor-related genes. RESULTS: Validated mutations were detected in 91 of the 100 tumors (91%) and identified in eight of the most frequently mutated genes, namely PTEN (57%), PIK3CA (51%), TP53 (30%), KRAS (23%), CTNNB1 (21%), FBFR2 (13%), FBXW7(10%) and RB1 (9%). PTEN mutations were found to associated with young age (< 60), early-stage, endometrioid histology, non-recurrence and better overall survival (OS). CTNNB1 mutations were associated with young age, endometrioid histology and better OS. On the other hands, TP53 mutations were associated with late-stage, non-endometrioid histology, high-grade, recurrence and worse OS. FBWX7 mutations were associated with late-stage, vascular invasion and lymph node metastasis. FGFR2 mutations correlated with deep (≥ 1/2) myometrial invasion. CONCLUSION: Our comprehensive mutational profile will be useful for understanding and evaluating the molecular characteristics of EC tumors, and may lead to the establishment of novel treatment strategies that improve the survival of patients with EC in the future.

Clinical implication of oncogenic somatic mutations in early-stage cervical cancer with radical hysterectomy.

It is well known that tumour initiation and progression are primarily an accumulation of genetic mutations. The mutation status of a tumour may predict prognosis and enable better selection of targeted therapies. In the current study, we analysed a total of 55 surgical tumours from stage IB-IIB cervical cancer (CC) patients who had undergone radical hysterectomy including pelvic lymphadenectomy, using a cancer panel covering 50 highly mutated tumorigenesis-related genes. In 35 patients (63.6%), a total 52 mutations were detected (58.3% in squamous cell carcinoma, 73.7% in adenocarcinoma), mostly in PIK3CA (34.5%) and KRAS and TP53 (9.1%). Being mutation-positive was significantly correlated with pelvic lymph node (PLN) metastasis (P = 0.035) and tended to have a worse overall survival (P = 0.076). In particular, in the patients with squamous cell carcinoma, there was a significant association between being mutation-positive and relapse-free survival (P = 0.041). The patients with PLN metastasis had a significantly worse overall survival than those without (P = 0.006). These results indicate that somatic mutation status is a predictive biomarker for PLN metastasis in early-stage CC, and is consequently related to poor prognosis. Therefore, comprehensive genetic mutations, rather than a single genetic mutation, should be examined widely in order to identify novel genetic indicators with clinical usefulness.

Prognostic Significance of Aberrant Claudin-6 Expression in Endometrial Cancer.

BACKGROUND: Among the claudin (CLDN) family, CLDN6 exhibits aberrant expression in various cancers, but its biological relevance has not yet been established. We generated a monoclonal antibody (mAb) against human CLDN6 and verified its specificity. By immunohistochemical staining and semi-quantification, we evaluated the relationship between CLDN6 expression and clinicopathological parameters in tissues from 173 cases of endometrial cancer. RESULTS: The established mAb selectively recognized CLDN6 protein. Ten of the 173 cases (5.8%) showed high CLDN6 expression (score 3+), whereas 19 (11.0%), 18 (10.4%) and 126 (72.4%) cases revealed low CLDN6 expression (score 2+, 1+ and 0, respectively). In addition, intratumor heterogeneity of CLDN6 expression was observed even in the cases with high CLDN6 expression. The 5-year survival rates in the high and low CLDN6 groups was approximately 30% and 90%, respectively. Among the clinicopathological factors, the high CLDN6 expression was significantly associated with surgical stage III/IV, histological grade 3, lymphovascular space involvement, lymph node metastasis and distant metastasis. Furthermore, the high CLDN6 expression was an independent prognostic marker for overall survival of endometrial cancer patients (hazard ratio 3.50, p = 0.014). CONCLUSIONS: It can be concluded that aberrant CLDN6 expression is useful to predict poor outcome for endometrial cancer and might be a promising therapeutic target.

Pelvic Exenteration as Potential Cure and Symptom Relief in Advanced and Recurrent Gynaecological Cancer.

BACKGROUND/AIM: Pelvic exenteration is a radical procedure for certain advanced or recurrent gynaecological cancers, performed with curative or palliative intent. Its validity has evolved as operative mortality and morbidity have improved. This surgery was evaluated to determine the validity of these claims. PATIENTS AND METHODS: The details of surgery and outcomes of 13 patients who underwent pelvic exenteration (6 curative intent, 7 palliative intent) for advanced or recurrent gynaecological cancers in our Department were retrospectively evaluated. RESULTS: There were no significant differences in blood loss, surgical time, hospital stay, and complications between curative pelvic exenteration and palliative pelvic exenteration. The curative intent group had a good prognosis; the palliative-intent group showed a trend to a worse prognosis. All patients' symptoms were relieved, but in patients with short survival, symptom relief lasted for up to 3 months. CONCLUSION: Pelvic exenteration is an acceptable and valuable procedure for gynaecological cancers.

Prediction of lymphovascular space invasion in endometrial cancer using the 55-gene signature selected by DNA microarray analysis.

Lymphovascular space invasion (LVSI) is considered to be the beginning of lymphogenous and hematogenous metastases. It is strongly related to dissemination, and therefore could be a valuable predictive sign of lymph node metastases and distant spread. Recently, the presence of LVSI in endometrial cancer (EC) has been shown to be an independent prognostic factor. The preoperative diagnosis of LVSI by pathological examination is difficult and LVSI is detected after surgery. The aim of the current study was to explore candidate genes as potential diagnostic biomarkers and determine whether they are predictors of LVSI in patients with EC. A total of 88 surgical specimens obtained from EC patients who had undergone surgical resection at Fukushima Medical University Hospital between 2010 and 2015 were analyzed using DNA microarray. LVSI was significantly associated with poor prognostic factors in EC such as higher tumor grade, lymph node metastasis, deep myometrium invasion, advanced stage and recurrence. Fifty-five candidate genes were significantly differentially expressed between 26 LVSI-positive and 62 LVSI-negative samples. All 88 samples were divided into two groups according to hierarchical clustering of 55 genes. Regarding diagnostic accuracy, sensitivity and negative predictive value were both high (92% and 95%, respectively); further, specificity and positive predictive value were both moderate (63% and 71%, respectively). Our data suggests that the 55-gene signature could contribute to predicting LVSI in EC, and provide clinically important information for better management. The molecular signatures of 55 genes may be also useful for understanding the underlying mechanism of LVSI.

Unique Learning System for Uterine Artery Embolization for Symptomatic Myoma and Adenomyosis for Obstetrician-Gynecologists in Cooperation with Interventional Radiologists: Evaluation of UAE From the Point of View of Gynecologists Who Perform UAE.

STUDY OBJECTIVE: To evaluate a unique learning system for uterine artery embolization (UAE) and examine its feasibility and clinical outcomes for the treatment of symptomatic uterine leiomyomas and adenomyosis when performed by obstetrician-gynecologists in cooperation with interventional radiologists (IVRs). DESIGN: Retrospective study (Canadian Task Force classification II-2). SETTING: University hospital. PATIENTS: One hundred seventy-three patients who underwent UAE for symptomatic leiomyomas and adenomyosis. INTERVENTIONS: We examined the medical records of patients who underwent UAE for symptomatic uterine leiomyomas and adenomyosis at our department between 2003 and 2012 using our learning system for UAE for obstetrician-gynecologists in cooperation with IVRs. The charts of all patients were reviewed, and data on etiologic factors, past medical history of leiomyomas and adenomyosis, symptoms, details of UAE, and clinical outcomes after UAE were extracted. MEASUREMENTS AND MAIN RESULTS: A total of 173 patients who underwent 177 UAEs were identified, including 4 patients who underwent embolization twice because of primary treatment failure or symptom recurrence. During the study period, 2 gynecologists successfully acquired endovascular skills. The technical success rate was 97.7% (174 of 177). The duration of fluoroscopy in procedures performed by obstetrician-gynecologists who acquired endovascular skills was not significantly different from that in procedures performed by IVRs at our institution; however, this duration was significantly longer in procedures performed by obstetrician-gynecologists who did not have sufficient experience with our learning protocol for UAE because of inadequate live observation of UAEs performed by skilled IVRs. Complications that necessitated discontinuation of the procedure occurred in 2.3% of cases (4 of 177). The clinical outcomes were similar to those reported in previous studies. Adverse events after UAE included myeloid passages in 7.0% (11 of 158), infections in 2.5% (4 of 158), vaginal discharge in 2.5% of patients with leiomyomas (4 of 158), and vaginal discharge in 7.1% of patients with adenomyosis (1 of 14). All the adverse events were adequately treated by the obstetrician-gynecologists themselves. The timing of hysterectomy due to complications or recurrence of symptoms after UAE varied widely. CONCLUSION: UAE performed by obstetrician-gynecologists in cooperation with radiologists can be achieved safely and successfully with acceptable clinical outcomes. Live observation of the procedure performed by skilled IVRs is essential to improving the skills and reducing the fluoroscopic time of obstetrician-gynecologists.

Sclerosing stromal tumor of the ovary treated with 2-incision total laparoscopic cystectomy.

Sclerosing stromal tumor (SST) is an extremely rare benign tumor of the ovary that is derived from the sex cord stroma [1,2] and occurs in young women [3-11]. Preoperative diagnosis of the tumor is difficult as it often mimics a malignant tumor. Oophorectomy is usually performed in many cases of young women [3], and diagnosis of SST is made based on post-operative pathological examination. Laparoscopic surgery is seldom performed in SST cases. Here, we report a case of SST of the ovary in an 18-year-old girl who was diagnosed by preoperative imaging and underwent laparoscopic cystectomy. Accurate preoperative imaging helped to perform minimally invasive surgery for SST.

Prospective evaluation of the incidence of uterine vascular malformations developing after abortion or delivery.

STUDY OBJECTIVE: To describe the incidence of uterine vascular malformations (UVMs) including uterine arteriovenous malformations (AVMs) in patients after abortion or delivery and in outpatients. DESIGN: Prospective study (Canadian Task Force classification II-3). SETTING: Fukushima Red Cross Hospital. PATIENTS: Six patients with a UVM including 1 with an AVM. INTERVENTIONS: Clinical screening of patients using transvaginal color Doppler ultrasonography between April 2010 and March 2012. MEASUREMENTS AND MAIN RESULTS: The incidence of UVM developing after abortion or delivery or in outpatients was prospectively evaluated using transvaginal color Doppler ultrasonography. From 959 patients, we identified 6 (0.63%) with UVMs, including 1 (0.10%) with a uterine AVM. Specifically, we detected UVMs in 4 of 77 patients (5.2%) after abortion, 1 of 458 patients (0.22%) after delivery, and 1 of 424 outpatients (0.24%). Four patients after abortion and 1 after delivery reported mild symptoms, which were treated conservatively; however, the outpatient had a severe uterine AVM, which was confirmed via 3-dimensional computed tomography angiography. CONCLUSION: The incidence of UVMs was relatively higher, in particular in the patients after abortion, and was significantly higher than that in postpartum or outpatient groups. Therefore, it is important to consider the possibility of UVMs in any patient with episodes of unexplained uterine bleeding and to perform follow-up analysis using color Doppler ultrasonography. Such an approach will facilitate accurate diagnosis and lead to appropriate clinical management to prevent unnecessary dangerous repeat curettage, which might induce profuse uterine bleeding.

Acceleration of the Cleavage in Sea Urchin Eggs by Treatmentswith Local Anesthetics (I) Procaine Treatment*: (acceleration of cleavage/local anesthetics/procaine/sea urchin eggs).

Unfertilized sea urchin eggs were pre-treated with 2-10 mM procaine sea water for 10 min and then fertilized 5 min later. These eggs hatched about 1-1.5 hr earlier than the control eggs. This acceleration of hatching seems to be the result of a reduction in the individual cleavage cycles. In the second series of experiments, eggs were inseminated at 5, 30 and 60 min after the procaine treatments. In this case, the rate of accelerated cleavage decreased gradually, as the intervals between the treatments and insemination were extended. Next, the minimum time-length of procaine treatment to accelerate cleavage was determined. When eggs were immersed in procaine for 1 min, cleavage was accelerated as in the case of 10 min-treatment, although acceleration to the same degree required higher concentrations of procaine solution. When fertilized eggs were post-treated with procaine, cleavage was also accelerated, but both the rate of acceleration and the effective concentrations decreased with time after fertilization.

Acceleration of the Cleavage in Sea Urchin Eggs by Treatments with Local Anesthetics (II) Treatments with Some Other Local Anesthetics than Procaine*: (acceleration of cleavage/local anesthetics/caffeine/sea urchin eggs).

Unfertilized sea urchin eggs were exposed to sea water solutions of local anesthetics, such as caffeine, tetracaine and ethyl urethane, and the herbicide, isopropyl N-phenyl carbamate (IPC) for 10min and returned to normal sea water. Then they were inseminated 5min later. When eggs were pre-treated with 1-2 mM caffeine, 0.02-0.05 mM tetracaine, 50-100 mM ethyl urethane and 2% saturated sea water of IPC, respectively, they could cleave and hatch earlier than the control eggs. However, when fertilized eggs were continuously post-treated with solutions of the agents except IPC at the same concentrations as those in the case of the pre-treatments, the fertilized eggs could not cleave or were retarded in development. The possible mechanisms of the cleavage acceleration by pre-treatments with local anesthetics were discussed.

Effects of D2 O on Parthenogenetic Activation and Cleavage in the Sea Urchin Egg.

When unfertilized sea urchin eggs are treated with D2 O-sea water, fertilization membranes are raised, cytasters appear in the cytoplasm and cleavages occur. Such observations are in agreement with those reported by Gross and his collaborators. However, D2 O-treated eggs can develop at least to the gastrula stage under appropriate conditions, in contradiction to descriptions by Grosset al. This is the case in eggs of three species of Japanese sea urchins used here. The double treatment with D2 O is more effective in inducing parthenogenetic development than the single treatment and, further, double treatments, such as the combinations of D2 O and hypertonic sea water or butyric acid and D2 O, also, can effectively induce the development. In addition, centrifugally enucleated egg-halves can be activated and developed to a blastulalike stage by the double treatment with D2 O.