Successful pregnancy using immature oocytes retrieved from resected borderline ovarian tumor: a case report and literature review.

BACKGROUND: Despite the recent progress of fertility preservation technique, achievement of pregnancy in women with ovarian tumor is still challenging. Here, we report a case of OTO-IVM (ovarian tissue oocyte in-vitro maturation) resulting in a successful delivery. CASE PRESENTATION: The patient, a 33-year-old woman with a history of left borderline ovarian tumor (BOT) who underwent left salpingo-oophorectomy three years ago, presented with an enlarged right ovary during infertility treatment, indicating the recurrence of BOT. Because the patient disagreed with curative surgery and normal part-preservation surgery, we eventually performed OTO-IVM. A right salpingo-oophorectomy was first performed. Eight immature oocytes were immediately aspirated not only from visible follicles, but also from entire cortex for invisible follicles, of the removed ovary. In addition, IVM procedure generated six mature oocytes, and were subjected to intracytoplasmic sperm injection (ICSI). Accordingly, three embryos were obtained and cryopreserved. Three months after surgery, hormone replacement therapy was initiated, and a frozen-thawed embryo was transferred, resulting in a successful pregnancy. Although a cesarean section was performed at 36 weeks due to maternal ileus, the baby was delivered without complications. CONCLUSIONS: This report indicates this treatment to be an effective approach for fertility preservation in BOT patients, especially, the importance of collecting oocytes from the entire ovarian cortex was suggested.

cDNA expression library screening revealed novel functional genes involved in clear cell carcinogenesis of the ovary in vitro.

In order to identify genes involved in the pathogenesis of clear cell carcinoma of the ovary (CCC), functional screening using a cDNA expression library was performed. We extracted mRNA from a CCC cell line (RMG-1), established a cDNA library using a retroviral vector, transfected that library into mouse NIH3T3 cells and sequenced the resultant foci. The tissue-type specific expression of isolated genes and their transforming activities were evaluated. Seven genes were isolated. Of these genes, the mRNA expression of SEC61B and DVL1 is significantly stronger in CCC than in other histological types (p < .05). Immunohistochemical staining reveals the stronger expression of SEC61B and C1ORF38 than normal ovarian tissues (p < .05). Focus formation is confirmed by the transfection of SEC61B, C1ORF38, and DVL1 into NIH3T3 cells. The present study identified novel genes including SEC61B, C1ORF38, and DVL1, involved in the pathogenesis of CCC. These genes may be additional therapeutic targets for CCC.Impact statementWhat is already known on this subject? Several important genetic abnormalities, including ARID1A and PIK3CA mutations, have been reported in ovarian clear cell carcinoma (CCC).What the results of this study add? SEC61B, C1ORF38, and DVL1 were newly detected as candidate genes involved in ovarian clear cell carcinogenesis.What the implications are of these findings for clinical practice and/or further research? Functional screening using a cDNA expression library may be a useful technique to identify functional genes for pathogenesis. The information obtained using this technique may provide new therapeutic targets of CCC.

Trophoblast type-specific expression of senescence markers in the human placenta.

OBJECTIVES: Cell senescence is irreversible cell cycle arrest. The human placenta is a unique organ that grows and matures during pregnancy until 40 weeks of gestation. However, the role of senescence in placental villi, particularly in the two types of trophoblast, has not yet been elucidated in detail. Therefore, we herein investigated the expression of cell senescence-related markers in trophoblast. METHODS: Seventy normal placental tissues were used. The expression of senescence-associated beta-galactosidase (SA-ß-gal), cell senescence-related markers (p16, p21, and promyelocytic leukemia; PML), and a growth marker (MCM2) was immunohistochemically examined. The expression of these markers in BeWo cells before and after cell fusion using forskolin was also investigated. RESULTS: The expression of MCM2 is detected in cytotrophoblast (CT). The expression of SA-ß-gal in CT is strong in the first and second trimesters, but weaker in the third trimester. Syncytiotrophoblast (ST) are negative in the first and second trimesters, but become positive in the third trimester. The immunohistochemical expression of p16, p21, and PML is stronger in CT than in ST throughout pregnancy. Furthermore, the expression of these markers in ST significantly increases as pregnancy advances. The expression of SA-ß-gal, PML, and p21 in BeWo cells is stronger after than before cell fusion. CONCLUSIONS: The proliferation and senescence of CT occurred in early to mid-pregnancy in association with syncytial fusion, while senescence was observed in ST in late pregnancy. This coordinated trophoblastic senescence may be essential for maintaining placental function.

Effectiveness of a genetic test panel designed for gynecological cancer: an exploratory study.

To increase diagnostic efficiency and cost-effectiveness, we performed an exploratory genetic test using a newly designed panel containing 28 actionable and druggable genes, alterations in which are frequently reported in gynecological cancers (TANRE-G, Targeted variants ANalysis RElated to Gynecological cancers). Samples consisted of the formalin-fixed, paraffin-embedded tissue of endometrial (4 cases), cervical (3 cases), and ovarian (4 cases) carcinomas. The sequencing procedure was performed using Ion PGM in our institute with related sequencing kits, and data were analyzed using ClinVar. The present system achieved more than 2500 reads in all tumor samples, and enabled a copy number variation analysis. Results showed that actionable and druggable mutations were detected in 82% (9/11) and 64% (7/11) of cases, respectively, which was similar to other commercially available genetic tests. The amplification of MYC and KRAS was also detected. The analysis cost for each sample was JPY 94,000 (USD 850). These results demonstrate the potential of the TANRE-G panel as an effective tool for examining genetic alterations in gynecological cancers.

SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells.

BACKGROUND: SIRT1 is a longevity gene that forestalls aging and age-related diseases including cancer, and has recently attracted widespread attention due to its overexpression in some cancers. We previously identified the overexpression of SIRT1 in ovarian carcinoma (OvCa) as a poor prognostic factor. However, mechanistic insights into the function of SIRT1 in OvCa have yet to be elucidated. METHODS: Quantitative real-time reverse PCR (qRT-PCR) and Western blotting were employed to examine the expression of SIRT1 in a panel of human OvCa cell lines. si-RNA or sh-RNA and cDNA technologies were utilized to knockdown or overexpress SIRT1, respectively. The effects of SIRT1 on proliferation and chemoresistance were examined using a WST-1 assay, and the underlying mechanisms were confirmed using an apoptotic assay, and the quantification of glutathione (GSH), and reactive oxygen species (ROS). The aggressiveness of SIRT1 was analyzed using in vitro invasion and migration assays. RESULTS: SIRT1 was more strongly expressed in OvCa cell lines than in the immortalized ovarian epithelium at the gene and protein levels. Stress up-regulated the expression of SIRT1 in dose- and time-dependent manners. SIRT1 significantly enhanced the proliferation (P<.05), chemoresistance (P<.05), and aggressiveness of OvCa cells by up-regulating multiple antioxidant pathways to inhibit oxidative stress. Further study into the overexpression of SIRT1 demonstrated the up-regulation of several stemness-associated genes and enrichment of CD44v9 via an as-yet-unidentified pathway. CONCLUSIONS: Our results suggest that SIRT1 plays a role in the acquisition of aggressiveness and chemoresistance by OvCa, and has potential as a therapeutic target for OvCa.

Panobinostat Enhances Growth Suppressive Effects of Progestin on Endometrial Carcinoma by Increasing Progesterone Receptor and Mitogen-Inducible Gene-6.

Although progestin has been used to treat endometrial hyperplasia and endometrial carcinoma (EC), its therapeutic efficacy is limited. In order to improve this, the underlining mechanisms of the effects of progestin need to be elucidated in more detail. In the present study, we examined the involvement of mitogen-inducible gene-6 (MIG6), a negative regulator of the EGF receptor, in the progestin-mediated growth suppression of endometrial epithelia. The immunohistochemical expression of MIG6 was elevated in the early to mid-secretory phases of normal endometrium and also with endometrial hyperplasia after medroxyprogesterone acetate (MPA) therapy. The addition of progesterone (P4) to progesterone receptor (PR)-positive EC cells reduced the viability and induced MIG6 messenger RNA (mRNA) and protein expression. The silencing of MIG6 using siRNA eliminated the P4-mediated reduction of EC cell viability, indicating that MIG6 is an essential downstream component of PR-mediated growth suppression. In order to enhance PR-driven signals, we examined the effects of histone deacetylase (HDAC) inhibitors because histone acetylation has been shown to increase the expression of PR. The addition of three HDAC inhibitors (panobinostat, LBH589; trichostatin A, TSA; suberoylanilide hydroxamic acid, SAHA) decreased the viability of EC cells and up-regulated the expression of PR and MIG6, and these effects were the strongest with LBH589. The addition of LBH589 and MPA synergistically decreased the viability and increased apoptosis in EC cells. These results indicate that LBH589 has potential as an enhancer of progestin therapy via the up-regulation of PR and MIG6.

Overexpression of SIRT1 is Associated With Poor Outcomes in Patients With Ovarian Carcinoma.

Sirtuin 1 (SIRT1), originally identified as a longevity gene, regulates DNA repair and metabolism by deacetylating target proteins such as p53. SIRT1 plays a key role in the pathophysiology of metabolic diseases and neurodegenerative disorders, and is considered to protect against age-related diseases including cancer. In contrast, SIRT1 may be oncogenic because its overexpression has been detected in many cancers. The aim of the present study was to clarify the expression and the role of SIRT1 in ovarian carcinoma (OvCa). The expression of SIRT1 was evaluated immunohistochemically in 16 cases of normal ovaries, 35 cases of endometriosis with/without carcinoma, and 68 cases of OvCa (endometrioid, 16; clear cell, 20; mucinous, 16; serous, 16). Staining results were evaluated semiquantitatively by the Immunoreactive Scoring System, and the relationships with clinicopathologic features and outcomes of patients were analyzed. The expression of SIRT1 was higher in endometrioid, mucinous, and clear-cell carcinomas than in the inclusion cysts of normal ovaries, but not in serous carcinoma (P=0.038). The expression of SIRT1 on OvCa did not correlate with age, stage, location of metastasis, or capsular penetration. However, elevated SIRT1 expression was a significant predictor of shorter survival in univariate (P=0.038) and multivariate (P=0.037) survival analyses, regardless of the tumor stage. Results of the present study suggest a positive role for SIRT1 in the development of OvCa and its potential as a novel therapeutic target.

Lipocalin 2 Enhances Migration and Resistance against Cisplatin in Endometrial Carcinoma Cells.

PURPOSE: Lipocalin 2 (LCN2) is a secretory protein that is involved in various physiological processes including iron transport. We previously identified LCN2 as an up-regulated gene in endometrial carcinoma, and found that the overexpression of LCN2 and its receptor, SLC22A17, was associated with a poor prognosis. However, the functions and mechanism of action of LCN2 currently remain unclear. METHODS: The LCN2-overexpressing endometrial carcinoma cell lines, HHUA and RL95-2, and LCN2-low-expressing one, HEC1B, were used. The effects of LCN2 on cell migration, cell viability, and apoptosis under various stresses, including ultraviolet (UV) irradiation and cisplatin treatment, were examined using the scratch wound healing assay, WST-1 assay, and Apostrand assay, respectively. RESULTS: LCN2-silencing using shRNA method significantly reduced the migration ability of cells (p<0.05). Cytotoxic stresses significantly decreased the viability of LCN2-silenced cells more than that of control cells. In contrast, LCN2 overexpression was significantly increased cisplatin resistance. These effects were canceled by the addition of the iron chelator, deferoxamine. After UV irradiation, the expression of phosphorylated Akt (pAkt) was decreased in LCN2-silenced cells, and the PI3K inhibitor canceled the difference induced in UV sensitivity by LCN2. The cisplatin-induced expression of pAkt was not affected by LCN2; however, the expression of p53 and p21 was increased by LCN2-silencing. CONCLUSIONS: These results indicated that LCN2 was involved in the migration and survival of endometrial carcinoma cells under various stresses in an iron-dependent manner. The survival function of LCN2 may be exerted through the PI3K pathway and suppression of the p53-p21 pathway. These functions of LCN2 may increase the malignant potential of endometrial carcinoma cells.

Lipocalin 2 attenuates iron-related oxidative stress and prolongs the survival of ovarian clear cell carcinoma cells by up-regulating the CD44 variant.

Ovarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.