From Skin Cells to Ovarian Follicles?

Generating oocytes from cells derived from skin in vitro may provide a valuable modelfor identifying factors involved in germ cell formation and oocyte differentiation. In addition, the“oocytes” produced could potentially be useful for therapeutic cloning, and thus offer newpossibilities for tissue therapy. We recently reported the differentiation of cells derived fromporcine fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cellsexpressed germ cell markers and formed aggregate like oocyte-cumulus complexes that secretedovarian steroid hormones and responded to gonadotropin stimulation. Some of these aggregatesextruded large oocyte-like cells that expressed markers appropriate to oocytes. We now showfurther evidence of germ cell marker expression during differentiation. We have also comparedthe oocyte-like cells with natural oocytes for their expression levels of Oct4, growthdifferentiation factor-9b (GDF9b), the deleted in azoospermia -like (DAZL) gene, vasa, zonapellucida (ZP), and the meiosis marker synaptonemal complex protein 3 (SCP3), and haverevealed interesting similarities and differences.     

Effect of Prolactin on Gonadotropin Regulation of Mouse Ovarian Function

We have demonstrated that prolactin inhibits gonadotropin-induced ovulation in PMSG-primed mice.The number of ova in oviducts considerably decreased in the group of hCG plus prolactin (PRL)(19.7 + 4.9) as compared with that of hCG alone (31.7 + 6.7). PRL inhibition of hCG-inducedovulation in mice may be through decreasing the ovarian plasminogen activator (PA) activity on onehand, and inhibiting the preovulatory increase in estrogen (E) secretion on the other.     

The Contribution of Germline BRCA1 and BRCA2 Mutations to Familial Ovarian Cancer: No Evidence for Other Ovarian Cancer–Susceptibility Genes

To establish the contribution of germline BRCA1 and BRCA2 mutations to familial ovarian cancer, we have analyzed both genes in DNA samples obtained from an affected individual in each of 112 families containing at least two cases of epithelial ovarian cancer. Germline mutations were found in 43% of the families; BRCA1 mutations were approximately four times more common than BRCA2 mutations. The extent of family history of ovarian and breast cancers was strongly predictive of BRCA1-mutation status. Segregation analysis suggests that a combination of chance clustering of sporadic cases and insensitivity of mutation detection may account for the remaining families; however, the contribution of other genes cannot be excluded. We discuss the implications for genetic testing and clinical management of familial ovarian cancer arising from the data presented in these studies.     

Ovarian cancer and ovulation induction drugs--is there a link?

Screening for ovarian cancer in a group of women with induced ovulations was encouraged by recently reported controversies about a possible association between the use of ovulation induction drugs and the increased risk of ovarian carcinoma. Transvaginal color Doppler ultrasonography was applied in screening for early stage ovarian malignoma in 110 asymptomatic women who received an ovulation induction therapy for infertility. Already reported standard parameters for discriminating malignant from benign flows, such as resistance index RI < 0.40, pulsatility index PI < 1 and morphological score (borders, cyst quality, septate areas, papilla and ovarian tissue echogenicity) were used. Screening included 110 women and was carried out from April 1, 1198 to March 31, 1999. Seven examinees had abnormal ovarian findings. The finding spontaneously regressed in five of them, one underwent surgery for a persistent cyst with a benign pathohistologic diagnosis, and one was diagnosed with early stage ovarian malignoma. RI < 0.40 was reported in one patient (0.9%) with a morphologically suspect finding and a pathohistologically confirmed malignoma, PI < 1 was found in 40 subjects or 36.4%, while malignoma was demonstrated in one case alone. The results showed the advantage of RI over PI in discriminating malignant from benign structures. The association between the use of ovulation stimulation drugs and the increased risk of ovarian carcinoma remains unproved and also challenges new dilemmas. The paper cautions against undesirable, potentially serious long-term effects of the use of ovulation induction agents. Additional trials should therefore be performed including a long-term prospective follow-up of women with induced ovulations.     

Intra-Peritoneal Hyperthermia Combining α-Galactosylceramide in the Treatment of Ovarian Cancer

The purpose of this study was to investigate the anti-tumor effect and potential mechanisms of i.p. hyperthermia in combination with α-galactosylceramide (α-GalCer) for the treatment of ovarian cancer. In this study, immuno-competent tumor models were established using murine ovarian cancer cell lines and treated with i.p. hyperthermia combining α-GalCer. Th1/Th2 cytokine expression profiles in the serum, NK cell cytotoxicity and phagocytic activities of dendritic cells (DCs) were assayed. We also analyzed the number of CD8⁺/IFN-γ⁺ tumor specific cytotoxic T cells, as well as the tumor growth based on depletion of lymphocyte sub-population. Therapeutic effect on those ovarian tumors was monitored by a non-invasive luminescent imaging system. Intra-peritoneal hyperthermia induced significant pro-inflammatory cytokines expression, and sustained the response of NK and DCs induced by α-GalCer treatment. The combination treatment enhanced the cytotoxic T lymphocyte (CTL) immune response in two mouse ovarian cancer models. This novel treatment modality by combination of hyperthermia and glycolipid provides a pronounced anti-tumor immune response and better survival. In conclusion, intra-peritoneal hyperthermia enhanced the pro-inflammatory cytokine secretion and phagocytic activity of DCs stimulated by α-GalCer. The subsequent CTL immune response induced by α-GalCer was further strengthened by combining with i.p. hyperthermia. Both innate and adaptive immunities were involved and resulted in a superior therapeutic effect in treating the ovarian cancer.     

Ovarian follicular response to different hormonal stimulation treatments in Canindé goats

The objective of the present study was to evaluate the effect of different hormonal stimulation treatments on the antral follicular population of naturalized Canindé goats. Adult goats (n=17) having estrous cycles at regular intervals were treated with intra-vaginal sponges containing 60 mg medroxyprogesterone acetate for 11 days, combined with an application of 50 μg d-cloprostenol on the Day 8 of treatment. For ovarian stimulation, goats were distributed into the following experimental groups: (a) multiple doses (MD), with a total of 120 mg NIH-FSH P1 in five intramuscular injections (30/30; 20/20 and 20 mg) at 12-h intervals; (b) three doses (TD), with a total of 120 mg NIH-FSH P1 in three intramuscular injections (60; 40 and 20 mg) at 24 h intervals; (c) one dose (OD), which consisted of the use of 70 mg NIH-FSH P1 combined with 200 IU eCG administered intramuscularly 36 h before sponge removal. In the MD and TD groups, FSH injections were begun on the Day 8 of progestagen treatment. The ovaries of all animals were observed by transrectal real time ultrasonography (TRU) during the follicular stimulation protocols. All follicles ≥2 mm were counted, measured and classified according to greatest diameter. The ultrasonographic assessment of the ovaries provided for well-defined ovarian structures. At the time of insertion of the sponges (Day 0), significant differences were observed (P<0.05) for the mean number of large follicles between the treated groups. Meanwhile, on Day 11, the three treatments did not differ (P<0.05), regardless of the follicular category. The diameter of small follicles was similar in MD, TD and OD during the whole period of the study. In the TD group, diameter of the large follicles was less (P<0.05) on Day 10 when compared to MD and OD. However, these differences were not observed on Day 11. In conclusion, the three treatments produced comparable distribution of the follicular populations. However, the single dose treatment can be preferred because of its simplicity and efficacious follicular response.     

Adrenomedullin in Ovarian Cancer: Foe In Vitro and Friend In Vivo?

Stromal elements within a tumor interact with cancer cells to create a microenvironment that supports tumor growth and survival. Adrenomedullin (ADM) is an autocrine/paracrine factor produced by both stromal cells and cancer cells to create such a microenvironment. During differentiation of macrophages, ADM is produced in response to pro-inflammatory stimuli and hypoxia. In this study we investigated the role of ADM as a growth factor for ovarian cancer cells and as a modulator of macrophages. We also analyzed ADM expression levels in a retrospective clinical study using nanofluidic technology and assessment of ADM at the gene level in 220 ovarian cancer patients. To study the effects of ADM, ovarian cancer cell lines A2780, OVCAR-3, and HEY and their drug-resistant counterparts were used for proliferation assays, while monocytes from healthy donors were differentiated in vitro. ADM was a weak growth factor, as revealed by proliferation assays and cell cycle analysis. After culturing cancer cells under stressing conditions, such as serum starvation and/or hypoxia, ADM was found to be a survival factor in HEY but not in other cell lines. In macrophages, ADM showed activity on proliferation/differentiation, primarily in type 2 macrophages (M2). Unexpectedly, the clinical study revealed that high expression of ADM was linked to positive outcome and to cancer with low Ca125. In conclusion, although in vitro ADM was a potential factor in biological aggressiveness, this possibility was not confirmed in patients. Therefore, use of an ADM antagonist would be inappropriate in managing ovarian cancer patients.     

Ovarian and placental expression of 20α-hydroxysteroid dehydrogenase during pregnancy in deer

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme has been shown to play a critical role in the regulation of luteal function in experimental animals. In this study, we cloned and expressed the gene encoding elk deer 20α-HSD from reproductive placental and ovarian tissues. PCR, 3'- and 5'-RACE, and northern blot analysis were performed for the cloning and characterization of deer 20α-HSD gene. We expressed recombinant deer 20α-HSD protein and used western blot analysis to determine protein expression levels in the placenta and ovary during pregnancy. The full cDNA sequence of 20α-HSD was used to clone an open reading frame encoding 323 amino acids and consisting of 1142 bp. The nucleotide sequence of deer 20α-HSD showed high homology with the sequences of the bovine (96%), goat (96%), and human (83%) 20α-HSD genes. 20α-HSD mRNA was strongly expressed in the placenta on days 30, 60, and 70 of pregnancy. A high level of the protein was also detected in the placenta but not in fetal skin tissue. The recombinant 20α-HSD protein produced in mammalian cells and bacterial systems had a molecular weight of approximately 37-kDa. The deer 20α-HSD protein signal was specifically localized in the basal part of the primary chorionic villi and chorionic stem villus of the placenta during early pregnancy. The 20α-HSD protein was also intensively localized in the larger luteal cells of the corpus luteum during pregnancy.     

Preoperative Anti-Class III β-Tubulin Antibodies As Relevant Clinical Biomarkers in Ovarian Cancer

Class III β-tubulin (TUBB3) overexpression in ovarian cancer (OC) associates with poor prognosis. We investigated whether TUBB3 overexpression elicited anti-TUBB3 antibody production in OC patients and whether these antibodies may have diagnostic and prognostic impact. The presence of serum anti-TUBB3 antibodies was investigated in 49 untreated OC patients and 44 healthy individuals by an in-house developed ELISA that used recombinant TUBB3 as the antigen. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of the assay. Anti-TUBB3 antibodies discriminated OC patients and healthy individuals with excellent sensitivity and specificity (91.8% and 90.9%, respectively). In multivariate analysis, anti-TUBB3 antibody level emerged as an independent prognostic factor for progression free and overall survival. The ELISA was then optimized using a biotin-labeled TUBB3 C-terminal peptide_424-450 instead of recombinant TUBB3 as the antigen and streptavidin-coated plates. The diagnostic role of the anti-TUBB3 antibodies was studied in an independent series of 99 OC patients and 80 gynecological benign disease patients. ROC-curve analysis showed a valuable diagnostic potential for serum anti-TUBB3 antibodies to identify OC patients with higher sensitivity and specificity (95.3% and 97.6%, respectively). Overall, our results provide evidence that preoperative anti-TUBB3 antibody level is a promising diagnostic and prognostic biomarker for the management of OC patients.     

Are Early Relapses in Advanced-Stage Ovarian Cancer Doomed to a Poor Prognosis?

Objective

Early recurrence (ER) after completion of therapeutic regimen in advanced-stage ovarian cancer is a challenging clinical situation. Patients are perceived as invariably having a poor prognosis. We investigated the possibility of defining different prognostic subgroups and the parameters implicated in prognosis of ER patients.

Study Design

We analyzed a multi-centric database of 527 FIGO stage IIIC and IV ovarian cancer patients. We defined patients relapsing within 12 months as ER and investigated using Cox logistic regression the prognostic factors in ER group. We subsequently divided ER patients into good and poor prognosis groups according to a lower or higher overall survival (OS) at 12 months after relapse and determined parameters associated to poor prognosis.

Results

The median follow up was 49 months. One hundred and thirty eight patients recurred within 12 months. OS and Disease Free Survival (DFS) were 24.6 and 8.6 months, respectively, in this group of patients. Among the ER patients, 73 had a poor prognosis with an OS after relapse below 12 months (mean OS = 5.2 months) and 65 survived after one year (mean OS = 26.9 months). Residual disease (RD) after debulking surgery and mucinous histological subtype negatively impacted prognosis (HR = 1.758, p = 0.017 and HR = 8.641, p = 0.001 respectively). The relative risk of death within 12 months following relapse in ER patients was 1.61 according to RD status. However, RD did not affect DFS (HR = 0.889, p = 0.5).

Conclusion

ER in advanced-stage ovarian cancer does not inevitably portend a short-term poor prognosis. RD status after initial cytoreduction strongly modulates OS, that gives additional support to the concept of maximum surgical effort even in patients who will experience early recurrence. The heterogeneity in outcomes within the ER group suggests a role for tumor biology in addition to classical clinical parameters.     

Ovarian hormone level alterations during rat post-reproductive life-span influence CD8?+?T-cell homeostasis

The study examined the putative role of ovarian hormones in shaping of rat peripheral T-cell compartment during post-reproductive period. In 20-month-old rats ovariectomized (Ox) at the very end of reproductive period, thymic output, cellularity and composition of major TCRαβ + peripheral blood lymphocyte and splenocyte subsets were analyzed. Ovariectomy led to the enlargement of CD8 + peripheral blood lymphocyte and splenocyte subpopulations. This reflected: (i) a more efficient thymic generation of CD8 + cells as indicated by increased number of CD4+CD8 + double positive and the most mature CD4-CD8+TCRαβ^high thymocytes and CD8 + recent thymic emigrants (RTEs) in peripheral blood, but not in the spleen of Ox rats, and (ii) the expansion of CD8 + memory/activated peripheral blood lymphocytes and splenocytes. The latter was consistent with a greater frequency of proliferating cells among freshly isolated memory/activated CD8 + peripheral blood lymphocytes and splenocytes and increased proliferative response of CD8 + splenocytes to stimulation with plate-bound anti-CD3 antibody. The former could be related to the rise in splenic IL-7 and IL-15 mRNA expression. Although ovariectomy affected the overall number of CD4 + T cells in none of the examined compartments, it increased CD4+FoxP3 + peripheral blood lymphocyte and splenocyte counts by enhancing their generation in periphery. Collectively, the results suggest that ovariectomy-induced long-lasting disturbances in ovarian hormone levels (mirrored in diminished progesterone serum level in 20-month-old rats) affects both thymic CD8 + cell generation and peripheral homeostasis and leads to the expansion of CD4+FoxP3 + cells in the periphery, thereby enhancing autoreactive cell control on account of immune system efficacy to combat infections and tumors.     

Immunohistochemical Analysis of DNA ‘mismatch-repair’ Enzyme Human Mut-S-Homologon-2 in Ovarian Carcinomas

The human Mut-S-Homologon-2 (hMSH-2) gene product is a member of a highly conserved family of proteins involved in postreplication mismatch repair. We have analysed hMSH-2 expression in normal ovarian tissue (n=15) and ovarian carcinomas (n=40). hMSH-2 protein was investigated immunohistochemically on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). A hMSH-2-immunoreactivity score (hMSH-2-IRS) for semiquantitative analysis of hMSH-2 expression is presented. In normal ovarian tissue, we only found weak nuclear immunoreactivity for hMSH-2 in 60%, while the remaining 40% were hMSH-2 negative (mean hMSH-2-IRS: 0.73; SD: ±0.70). All ovarian carcinomas analysed revealed moderate to strong nuclear immunoreactivity (mean hMSH-2-IRS: 8.05; SD: ±3.65). hMSH-2 staining was heterogeneous, with visual differences between individual tumour cells. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of ovarian carcinomas as compared to normal ovarian tissue. No statistically significant correlation in comparing the labelling patterns for hMSH-2 with the labelling patterns for Ki-67 (mean percentage of Ki-67 positive tumour cells: 25.88%; SD: ±18.43) was observed in ovarian carcinomas. Furthermore, no statistical significant correlations between hMSH-2-IRS and histological grading (p=0.47), histological type of carcinoma (p=0.706) or FIGO-classification (p=0.054) were found. Our findings indicate that (a) hMSH-2 is expressed in normal human ovarian tissue, (b) expression of hMSH-2 is increased in ovarian carcinomas, (c) expression of hMSH-2 may be of importance for the genetic stability of ovarian carcinomas in vivo, (d) hMSH-2 mutations may not cause microsatellite instability in ovarian carcinomas, (e) hMSH-2 may contribute to mechanisms responsible for resistance to anticancer drugs.     

Ovarian cancer stem cells: Can targeted therapy lead to improved progression-free survival?

Despite significant effort and research funds, epithelial ovarian cancer remains a very deadly disease. There are no effective screening methods that discover early stage disease; the majority of patients are diagnosed with advanced disease. Treatment modalities consist primarily of radical debulking surgery followed by taxane and platinum-based chemotherapy. Newer therapies including limited targeted agents and intraperitoneal delivery of chemotherapeutic drugs have improved disease-free intervals, but failed to yield longlasting cures in most patients. Chemotherapeutic resistance, particularly in the recurrent setting, plagues the disease. Targeting the pathways and mechanisms behind the development of chemoresistance in ovarian cancer could lead to significant improvement in patient outcomes. In many malignancies, including blood and other solid tumors, there is a subgroup of tumor cells, separate from the bulk population, called cancer stem cells(CSCs). These CSCs are thought to be the cause of metastasis, recurrence and resistance. However, todate, ovarian CSCs have been difficult to identify, isolate, and target. It is felt by many investigators that finding a putative ovarian CSC and a chemotherapeutic agent to target it could be the key to a cure for this deadly disease. This review will focus on recent advances in this arena and discuss some of the controversies surrounding the concept.