Approximately 21,550 women were diagnosed with Ovarian Cancer (OVCA) in 2009 and 14,600 died from their disease, making it the number one killer of women among gynecological cancers. Seventy percent of cases of OVCA are not detected until the disease has progressed to stage III or stage IV. Both diagnosis and aggressive treatments for late-stage OVCA can have a negative impact on women's psychosocial well-being resulting in anxiety, depression, and fear. The purpose of this integrative review of literature was to evaluate interventions designed to improve psychosocial well-being in women with OVCA. This review of literature was conducted using the CINAHL, PsycINFO, and MEDLINE databases using various key terms including "ovarian cancer or neoplasm" and "psychological and psychosocial", "well-being", "fear or anxiety or depression". Five studies met the inclusion criteria and were included in this review. These interventions include psychosocial, psychoeducational, and personal counseling interventions. The findings indicate that these interventions result in a significant improvement in participant's psychosocial well-being including decreased depressive symptoms, fear, and anxiety. This evidence supports that there is a need for psychosocial intervention in women with OVCA. It also suggests that women with OVCA benefit from tailored interventions and one-on-one educational and counseling sessions. Clinicians can use this information to anticipate potential problems in their client's psychosocial well-being and provide more education and support or refer them for counseling. More research is needed about reducing the psychosocial sequelae that results in women with OVCA.

The human ovarian surface epithelium (hOSE) is a cell monolayer that covers the surface of the ovary. Natural events like incessant ovulation, associated reproductive hormone action prior to and post-ovulation, along with the ovulationassociated inflammation, that result in injury and repair of hOSE, are considered to have a role in the development of epithelial ovarian cancer (EOC). Progesterone is apoptotic and anti-inflammatory, whereas androgens appear cytoproliferative for hOSE. Local generation of these steroid hormones is subject to 3β-hydroxysteroid dehydrogenase (3β-HSD) activity. Moreover, action of these hormones is achieved through coupling to their cognate receptors, progesterone (PR) and androgen receptors (AR). The overall aim of this thesis is to elucidate in vitro the regulation of progesterone and androgen biosynthesis and downstream signalling during the injury and repair of primary hOSE cells that were collected from pre-menopausal women who underwent surgery for benign gynaecological disorders. Injury was mimicked by treatment of cells with several pro-inflammatory cytokines, whereas repair was mimicked with T-lymphocyte, ‘anti-inflammatory’ cytokines. Immunohistochemical studies showed immunodetectable 3β-HSD in the human ovarian cell surface of whole ovary and three-week cultured hOSE cells, establishing 3β-HSD expression in vivo and in vitro. Cross-reaction of the 3β-HSD antibody with both enzyme isoforms did not allow investigation of isoform expression pattern. However, mRNA transcriptional studies with isoform specific primers and probe sets for semi-quantitative (sq) and quantitative (q) PCR revealed expression of both isoforms in hOSE cells; 3β-HSD1 mRNA was expressed at higher levels relative to 3β-HSD2 mRNA in accordance with the preference of this isoform in peripheral non-steroidogenic tissues. Of the cytokines tested, only IL-1α and IL-4 affected 3β-HSD expression. IL- 1α suppressed 3β-HSD1 mRNA, whereas it up-regulated 3β-HSD2 mRNA as assessed with qPCR, without though affecting total 3β-HSD protein and activity levels as assessed with western immunoblotting and radiometric activity assays, respectively. IL-1α did not affect AR or PR mRNA levels, suggesting a balance in androgen and progesterone biosynthesis during post-ovulatory wounding. IL-4 massively induced 3β-HSD1 and 3β-HSD2 mRNA and total 3β-HSD protein and activity. It also attenuated AR mRNA and protein, without affecting PR mRNA. Collectively, these data demonstrate that IL-4 sustains progesterone rather than androgen signalling and this may be part of the anti-inflammatory steroid action that protects hOSE from genetic damage. IL-1α effects appear to be mediated by NF-κB signalling pathway. PI-3K and p38 MAPK appeared involved in IL-1α-induced 3β- HSD2. IL-4-induced 3β-HSDs required STAT-6 and PI-3K pathways and also p38 MAPK at the case of 3β-HSD2. IL-4-attenuated AR was reversed by a p38 MAPK inhibitor. These data suggest that steroid signalling by IL-1α and IL-4 involve multiple signalling pathways. In primary EOC, 3β-HSD1 and 3β-HSD2 transcripts were attenuated relative to hOSE cells, suggestive of an acquired feature of neoplastic transformation. However, both transcripts could be restored after IL-4 treatment, attesting a therapeutic advantage of this cytokine. In conclusion, we have shown that 3β-HSD is under inflammatory control during ovarian post-ovulatory wound healing of hOSE. IL-1α- and IL-4-mediated 3β-HSD1 and 3β-HSD2 are regulated by multiple signalling pathways. Also, IL-4 was identified as an anti-inflammatory agent in hOSE with putative therapeutic benefit in malignancy.

The layer of cells lining the outer surface of the mammalian ovary, the ovarian surface epithelium (OSE), is a constant feature throughout the dynamic tissue remodeling that occurs throughout the reproductive cycle (follicle growth, ovulation, corpora lutea formation and pregnancy). Abnormal development of these cells is responsible for 90% of all epithelial ovarian cancers in women and epidemiological studies have shown that susceptibility to ovarian cancer is negatively correlated with increasing pregnancy. Little is known about how OSE cells are affected at each stage of the cycle, so the main aim of this study was to determine how the reproductive cycle affected proliferation and degeneration of OSE cells. This study utilised three animal models each with a different type of reproductive cycle: a mono-ovular seasonal breeder (Sheep), a mono-ovular polyoestrous breeder (Cow) and a poly-ovular non human primate (marmoset) to allow comparisons to be made. Comparison of OSE proliferative activity was made in sheep and marmoset at each stage of the cycle including pregnancy and anoestrous. The bovine model was used to investigate apoptotic cell death. Proliferative activity of somatic cells within the sheep ovary was monitored throughout the reproductive cycle by detection of cell cycle markers PCNA and Ki67 using immunohistochemistry. The pattern of OSE proliferation was correlated with the pattern of follicle development at each stage (sheep and marmoset). During pregnancy cell proliferation was significantly lower in OSE and in granulosa cells, reflecting a suppression of mature follicle development during these stages whereas in cycling animals proliferation was increased. Differences in OSE proliferation were observed in relation to the local underlying tissue environment in both sheep and marmoset. Epithelial cell rupture and regeneration enhanced the hormonal mitogenic action on epithelial cells, which showed highest proliferation over corpora lutea in each animal model. To test the hypothesis that these changes are mediated by hormones or growth factors ovine OSE cells were cultured and proliferative activity monitored after treatment with several factors: fetal calf serum (FCS), follicular fluid from follicles of varying sizes, corpora lutea extracts, recombinant human IGF-1, oestradiol and progesterone. IGF alone was demonstrated to have an affect on increasing proliferation of cultured OSE cells. Levels of FSHr and LHr were monitored by quantitative real- time PCR and it was demonstrated that the concentration of gonadotrophin receptors in OSE, increased prior to and after ovulation, at which time the in vivo OSE proliferation also peaked. The in situ apoptosis index was determined in bovine tissue using TUNEL throughout the regular cycle, and at mid and late-pregnancy stages. The results showed that pregnancy induced apoptotic activity in OSE cells and up regulated the tumour suppressor gene p53. Cultured bovine OSE cells also exhibited an increased level of apoptosis following progesterone treatment. Since p53/p53 gene expression in OSE over the corpora lutea producing progesterone also increased, this progesterone-mediated apoptosis may be mediated through an up-regulation of p53 synthesis. The effect of pregnancy and low production of gonadotrophins in the regulation of OSE cell morphology and activity was further investigated in the marmoset monkey (a non-human primate) treated with GnRH antagonist and infused with BrdU to monitor proliferative activity. OSE proliferation was correlated to ovarian events (follicular growth, ovulation and luteinization) and this was suppressed during pregnancy. Inhibition of gonadotrophin secretion by treatment with a GnRH antagonist also markedly inhibited OSE proliferation. Taken together these studies support the hypothesis that pregnancy and periods of anovulation reduce proliferation of OSE cells and alter the pattern of apoptotic cell death and that this effect is independent of species and reproductive pattern. Suppression of gonadotrophins and other growth factors during pregnancy could enhance p53-mediated apoptosis of damaged and mitogenic cells arising from repeated ovulations. This effect may partly explain why increasing number of pregnancies in woman reduces the chance of epithelial ovarian cancers.

Epithelial Ovarian Cancer (EOC) kills more women annually in the United Kingdom than any other gynaecological cancer. Survival rates for women diagnosed with EOC have not improved over the past 30 years, due to the often advanced stage at presentation, where widespread intra-peritoneal dissemination has occurred. The natural history of the disease remains uncertain but the ovarian surface epithelium (OSE) is a strong candidate for the tissue of origin. The OSE undergoes cyclical damage and repair in women of reproductive age following ovulation, which can be considered an acute inflammatory event. Factors that prevent ovulation (pregnancy, breastfeeding and contraceptive pill use) also protect against the development of EOC. Previously published data show that the OSE is able to upregulate the enzyme 11-beta hydroxysteroid dehydrogenase type 1 (11βHSD1) in response to inflammation, the enzyme responsible for converting inactive cortisone to anti-inflammatory cortisol. This thesis hypothesises that 11βHSD isozymes are deregulated in ovarian cancer; that the peritoneal surface epithelium (PSE) is indistinguishable from the OSE in its response to inflammation and should be considered a potential source of some “ovarian cancers”; and finally that the expression of the tumour suppressor gene OPCML (OPioid binding Cell adhesion Molecule-Like) is altered by inflammation. These hypotheses were examined at three levels. Firstly, primary cultures of EOC were established, and glucocorticoid metabolism and the response to inflammation was compared to normal OSE. Results from these investigations reveal that the11βHSD1 response to IL-1α stimulation is impaired in EOC compared to normal OSE at the mRNA level but there is no significant difference when 11βHSD1 enzyme activity is measured in these tissues. When basal levels of 11βHSD1, 11βHSD2 and COX2 are compared amongst untreated samples of EOC and OSE, there was a significant correlation between 11βHSD1 and COX2 mRNA expression (P<0.001). 11βHSD2 mRNA expression was significantly higher in the EOC specimens compared to OSE (P<0.05). Secondly the response to inflammation was compared in primary cultures of human peritoneal surface epithelial (PSE) cells and OSE. The data suggest that the mRNA response to inflammation was similar in OSE and PSE, but that the 11βHSD1 enzyme activity was reduced in PSE (P<0.05), which may result in differences in tissue healing. Finally, the effect of inflammation on the expression of the ovarian cancer associated tumour suppressor gene (TSG), OPCML (OPioid binding Cell adhesion Molecule-Like) and the other members of the IgLON family, was examined in OSE. These results suggest that OPCML mRNA expression can be induced by IL-1α, an effect that is inhibited by cortisol.

O apelo pela conservação ambiental e o significativo aumento no número de organismos cultivados de alto valor genético demandam tecnologias que permitam conservar sua genética, mesmo após a morte do animal. A criopreservação de gametas possibilita a preservação da genética de espécies ameaçadas e de interesse comercial, prolongando sua vida reprodutiva evitando assim a perda de material genético por doenças, catástrofes, transferência de animais ou perda do habitat natural. A criopreservação tem sido aplicada à conservação de ovários e tecido ovariano, no entanto, há muitas controvérsias acerca de qual seria o melhor protocolo a ser utilizado. Tendo isso em vista, o presente estudo teve como objetivo avaliar a morfologia do tecido ovariano de zebrafish criopreservado em cápsula de metal com o uso de diferentes soluções crioprotetoras. As soluções crioprotetoras utilizadas foram: 1,5 M metanol + 4,5 M propileno glicol (SC1); 1,5 M metanol + 5,5 M Me2SO (SC2); 1,5 M metanol + 4,5 M propileno glicol + 0,5 M sacarose (SC3); 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose (SC4). Após o descongelamento a integridade de cinco estágios de desenvolvimento folicular foi avaliada em cada grupo. A morfologia celular foi observada através de análise histológica. A análise dos dados mostrou que os folículos em estágio I e II foram os melhores criopreservados em todos os grupos experimentais. Sendo que, os grupos SC4 e SC2 foram os que apresentaram os melhores resultados, respectivamente com 88,26% e 84,2% de folículos sem alterações morfológicas. Já os estágios foliculares mais avançados de desenvolvimento (estágios IV e V) apresentaram-se com alterações em todos os grupos. Portanto, apesar do sucesso na criopreservação dos estágios foliculares mais iniciais (I e II) foi possível identificar alterações morfológicas em todos os grupos avaliados. Dentre as principais alterações identificadas estão a aglutinação do citoplasma e enrugamento e ruptura da membrana do envelope celular. Ao avaliar os resultados pode-se concluir que apesar do uso da capsula de metal em associação com as soluções SC4 e SC2 apresentarem os melhores resultados, as soluções SC1 e SC3 também foram eficientes na manutenção da integridade morfológica de folículos imaturos, e portanto essa metodologia pode ser utilizada com sucesso na criopreservação de folículos imaturos. / The appeal for environmental conservation and the significant increase in the number of farmed organisms with high genetic value demand technologies to allow preserving their genetics, even after the death. Cryopreservation of gametes allows the preservation of genetics of the endangered and commercial species, prolonging their reproductive life. Furthermore, this technology prevents the loss of genetic material caused by diseases, disasters, transfer of animals or loss of natural habitat. Cryopreservation has been applied to the conservation of ovaries and ovarian tissue, however, there are many controversies regarding what would be the best protocol to use. Thus, the aim of this study was to evaluate the morphology of cryopreserved zebrafish ovarian tissue using a metal capsule with four different cryoprotectant solutions. The cryoprotectant solutions used were: 1.5M methanol + 4.5 M propylene glycol (CS1); 1.5M methanol + 5.5 M Me2SO (CS2); 1.5M methanol + 4.5 M propylene glycol + 0.5 M sucrose (CS3); Methanol + 1.5 M 5.5 M 0.5 M sucrose + Me2SO (CS4). After heating the integrity of the five stages of follicular development was assessed in each group. Cell morphology was observed by histological analysis. The thermal gradient inside the capsule and the sample was verified by a thermistor Pt500, model Keithley 2001A. The data analysis shows that the follicles at stage I and II were better cryopreserved among all experimental groups. The treatments CS4 and CS2 showed the best results, respectively with 88.26% and 84.2% of follicles without morphological changes in stage I. The most advanced follicular development stages (stages IV and V) showed changes in all treatments. Therefore, despite the successful cryopreservation of earlier follicular stages (I and II), it was possible to identify morphological changes in all the groups. Among the main changes identified, agglutination of cytoplasm and rupture of cell and wrinkling of the egg envelope could be observed. Despite the use of the metal capsule in association with the CS4 and CS2 solutions showed the best results, CS1 and CS3 solutions were also effective in maintaining the morphological integrity of immature follicles, therefore this method can be successfully used in the cryopreservation of immature follicles.

While the etiology of ovarian cancer (OC) is not completely understood, evidence suggests that chronic inflammation may promote malignant transformation. However, familial history remains the strongest risk factor for developing OC and is associated with germline BRCA1 mutations, such as the 185delAG mutation. Normal human ovarian surface epithelial cells expressing the 185delAG mutant, BRAT, exhibit molecular and pathological changes that may contribute to OC oncogenesis. In the current study, I sought to determine whether BRAT could promote an inflammatory phenotype by investigating BRAT's impact on the expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Using a culture model system of normal human ovarian surface epithelial (OSE) cells with and without the BRCA1 185delAG frameshift mutation, BRAT, I investigated BRAT's role in IL-1β expression. OSE cells stably expressing the 185delAG mutation and ovarian surface epithelial cells with endogenous 185delAG were analyzed for differential target gene expression by real time PCR, western blot, ELISA, luciferase reporter and siRNA assays. Normal and malignant breast epithelial cell lines transiently expressing BRAT were also evaluated by real time PCR to determine whether BRAT-induced IL-1β expression is tissue specific. BRAT-expressing OSE cells exhibited enhanced IL-1β mRNA and protein expression. However, expression of BRAT in all breast cell lines failed to significantly alter IL-1β expression levels so that BRAT-mediated IL-1β expression promoting a chronic inflammatory phenotype conducive to malignant transformation may be limited to the ovary. Secondly, since OSE cells expressing the BRCA1 185delAG mutation have increased levels of IL-1β that may contribute to malignant transformation, in a pilot study, I sought to assess whether elevated urinary levels of IL-1β are associated with OC as well as compare urinary IL-1β levels with clinical parameters. Urinary and serum levels of IL-1β were analyzed by ELISA and biostatistical analysis from a patient cohort consisting of healthy women (N=10), women with ovarian benign disease (N=23), women with OC (N=32), women with other benign gynecological conditions (N=22), and women with other gynecological cancers (N=6). Urinary IL-1β levels were elevated in patients with ovarian benign disease and a first degree family history of ovarian and/or breast cancer. Urinary IL-1β levels were also correlated with increased body mass index. Urinary and serum IL-1β levels were increased in ovarian benign and OC patient samples supporting the theory of elevated urinary IL-1β being associated with cancer progression. Lastly, I sought to begin early molecular characterization of BRCA1 185delAG to better understand its role in ovarian transformation. I isolated 185delAG protein expressed in E. coli and utilized web tools to analyze the amino acid sequence to determine the molecular and structural characteristics. The study results showed the predicted BRCA1 185delAG protein product is an ordered, self-aggregating, alpha helical protein structurally and molecularly distinct from wild-type BRCA1. The BRCA1 185delAG amino acid sequence contained domains with resemblance to the Peptidase M20 family. Isolation of the BRCA1 185delAG protein product will allow for further protein analysis to better understand its' oncogeneic functions; as well as, elucidate the mechanism of tissue-specific BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility.

Ovarian cancer is a fatal disease because of the lack of symptoms and markers for early detection. Most ovarian neoplasms resemble and are classified according to the complex characteristics of Mullerian duct epithelia. We tested the hypothesis that Mullerian epithelial characteristics influence early ovarian neoplastic progression. The most common type of ovarian cancer is the serous carcinoma which resembles Mullerian-derived oviductal epithelium. We discovered that oviduct-specific glycoprotein (OVGP1), a tubal differentiation marker, was present in inclusion cysts, which are the preferential sites for malignant transformation, and in most low grade serous tumors, but absent in ovarian surface epithelium and most high grade carcinomas. OVGP1 was almost entirely limited to ovarian neoplasms with the notable exception of endometrial hyperplasia and carcinoma. A new antibody against OVGP1 detected elevated serum levels from most women with low grade ovarian cancers compared to normal controls. OVGP1 also identified a subset of patients with high grade serous carcinomas who had a more favorable outcome. To examine whether the differentiated phenotype of early ovarian neoplasms alters invasiveness, we established the first permanent cell line for serous borderline ovarian tumors (SBOT), which are differentiated but noninvasive. The results revealed a striking phenotypic similarity between two lines regardless of their cytogenetic diversity. They retained Mullerian epithelial characteristics in vitro, as demonstrated by their morphologic appearance and the differentiation markers keratin, E-cadherin, CA125 and OVGP1. Neither disruption of the growth pattern nor manipulations of the cadherin profile induced invasivenesss. Induction of invasiveness by SV40 early genes was associated with a loss in morphologic differentiation and of differentiation markers but increased motility. MMP secretion was independent of the invasion status. Our findings indicate that OVGP1 is an indicator of early ovarian epithelial neoplasia. It can be detected in the sera from women with early ovarian cancer, and thus, may be a new promising diagnostic marker for the early detection of ovarian cancer. In addition, the results show that Mullerian differentiation does not directly prevent invasiveness, but it diminishes in parallel with invasion caused by other factors. The lack of invasiveness by SBOT cells may depend on factors that regulate motility.

Ovarian cancer is a significant concern for women’s health as it is the most lethal of all gynaecological malignancies. One of the reasons for the high mortality of this disease is that traditionally used chemotherapeutic treatments tend to have poor initial or sustained efficacy against ovarian tumours. Resistance to such treatments may in part be mediated by autophagy, a cell survival process in which unnecessary or damaged components of the cytoplasm are engulfed within a double-membraned vesicle known as an autophagosome and ultimately degraded upon fusion of the autophagosome with a lysosome. Autophagy has been shown to be employed by cells to aid in their survival under stresses such as nutrient deprivation, hypoxia, chemotherapy treatment, and growth factor withdrawal. As these stresses are commonly encountered by ovarian cancer cells, it is possible that autophagy promotes ovarian cancer cell survival. This thesis aims to investigate which stimuli induce autophagy in ovarian cancer cells and whether or not this induction can promote cell survival. In addition, there is a particular focus on the comparison of autophagy utilization between subtypes of ovarian cancer, as the subtypes are in fact considered different diseases and may vary in their usage of autophagy. The first chapter of this thesis provides relevant background information on autophagy as well as ovarian cancer and its subtypes. In the second chapter, I describe studies in which tumours from a large cohort of patients with ovarian cancer are assessed for LC3A, a marker of autophagy, in addition to markers of other cellular processes including hypoxia. Here I found that LC3A was significantly associated with poor patient survival in patients with the clear cell subtype of ovarian cancer, but not other subtypes. I also found that LC3A expression was associated with markers of hypoxia in the clear cell patient tumours and that clear cell carcinoma cell lines preferentially induced autophagy in response to hypoxia in vitro as compared to cell lines of the high-grade serous subtype. These results indicate that clear cell ovarian tumours are uniquely dependent upon autophagy in response to hypoxia. In the third chapter, I investigated the autophagic response to treatment with the standard ovarian cancer chemotherapy drugs carboplatin and paclitaxel in a syngeneic mouse model of ovarian cancer. I found that these drugs did indeed induce autophagy and that the cancer cells utilized autophagy to promote resistance to these chemotherapeutics. In addition, when the tumour cells were grown in syngeneic mice, treatment with the autophagy inhibitor hydroxychloroquine resulted in a significant suppression of tumour growth. Together, my findings indicate that further investigation into the use of autophagy inhibitors in ovarian cancer patients is warranted and that different specific rational drug combinations for each subtype will likely yield optimal results. / Graduate

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.