Ovarian stem cells: From basic to clinical applications

The field of reproductive biology has undergone significant developments in the last decade. The notion that there is a fixed reserve pool of oocytes before birth was established by Zuckerman in 1951. However, in 2004, an article published in nature challenged this central dogma of mammalian reproductive biology. Tilly’s group reported the existence of ovarian germline stem cells (GSCs) in postnatal ovaries of mice and suggested that the bone marrow could be an extragonadal source of ovarian GSCs. These findings were strongly criticized; however, several independent groups have since successfully isolated and characterized ovarian GSCs in postnatal mice. The ovarian GSCs are located in the ovarian surface epithelium and express markers of undifferentiated GSCs. When transplanted into mouse ovaries, mouse ovarian GSCs could differentiate and produce embryos and offspring. Similarly, in a recent study, ovarian GSCs were found to be present in the ovaries of women of reproductive age. Conversely, there is increasing evidence that stem cells responsible for maintaining a healthy state in normal tissue may be a source of some cancers, including ovarian cancer. Cancer stem cells (CSCs) have been found in many tissues, including ovaries. Some researchers have suggested that ovarian cancer may be a result of the transformation and dysfunction of ovarian GSCs with self-renewal properties. Drug resistant and metastasis-generating CSCs are responsible for many important problems affecting ovarian cancer patients. Therefore, the identification of CSCs will provide opportunities for the development of new therapeutic strategies for treatments for infertility and ovarian cancer. In this article, we summarize the current understanding of ovarian GSCs in adult mammals, and we also discuss whether there is a relationship between GSCs and CSCs.     

Transvaginal colour flow imaging: a possible new screening technique for ovarian cancer.

OBJECTIVE--To assess whether changes in the intraovarian vasculature or blood flow impedance can be used to identify potentially malignant masses. DESIGN--Open, non-comparative prospective study. SETTING--Ovarian screening clinics at King''s College Hospital and the Hallam Medical Centre. SUBJECTS--50 Women selected on the basis of their medical history and the result of a previous transvaginal ultrasound scan. Thirty women (10 premenopausal (scan taken on days 1 to 8 of the menstrual cycle) and 20 postmenopausal) had normal ovaries, and 20 had at least one ovary with an abnormal morphology or volume, or both. INTERVENTIONS--Women with a positive result on screening were referred for laparotomy. MAIN OUTCOME MEASURES--Presence or absence of coloured areas (neovascularisation) and the pulsatility index within each ovary. The pulsatility index is a measure of the impedance to blood flow, a low value indicating decreased impedance and a high value increased impedance to blood flow. RESULTS--Two women with a positive result on screening had hydrosalpinges, 10 a benign tumour or a tumour-like condition, and eight primary ovarian cancers. No areas of neovascularisation were seen in the 30 women with morphologically normal ovaries and the two patients with hydrosalpinges; the pulsatility index ranged from 3.1 to 9.4. Similarly, nine patients (10 affected ovaries) with a non-malignant mass had no signs of neovascularisation and the pulsatility index varied from 3.2 to 7.0. One patient with bilateral dermoid cysts containing nests of thyroid-like cells had vascular changes and pulsatility index values of 0.4 and 0.8. Seven patients (eight ovaries) with primary ovarian cancer (one stage IV, four stage II, and two stage Ia) showed clear evidence of neovascularisation and pulsatility index values were from 0.3 to 1.0. One patient with an intraepithelial serous cystadenocarcinoma in a small ovary (less than 5 ml volume) had no signs of any vascular change and the pulsatility index was 5.5. CONCLUSION--Transvaginal colour flow imaging may be used to identify potentially malignant ovarian masses and help elucidate the early stages of tumorigenesis. The routine application of this technique may reduce the rate of false positive results of an ultrasonography based screening procedure.     

Advances in ovarian cancer proteomics: the quest for biomarkers and improved therapeutic interventions

Epithelial ovarian cancer is the leading cause of cancer-related death among gynecological cancers due to the asymptomatic nature of the disease, a lack of early detection markers and the development of resistance to current chemotherapeutic agents. Currently available tests (CA-125, transvaginal ultrasound or combination of both) lack the sensitivity and specificity to be useful as an efficient screening tool for surveillance of the general population. Thus, there is an urgent need for the development and validation of new molecular markers that would be both specific and sensitive indicators of disease onset, as well as progression. Proteomic profiling has emerged as a powerful tool to study ovarian cancer in an unbiased way at the molecular level, to monitor the effects of given treatment options and for the discovery of biomarkers. In this review we discuss the challenges associated with proteomics-based biomarker discovery and some recent concepts to potentially overcome these hurdles. Recent proteomics work on ovarian cancer cells and tissues will be discussed in light of obtaining new insights into fundamental biological processes, as well as their potential integration with ongoing biomarker discovery pipelines.     

Advances in ovarian cancer proteomics: the quest for biomarkers and improved therapeutic interventions

Epithelial ovarian cancer is the leading cause of cancer-related death among gynecological cancers due to the asymptomatic nature of the disease, a lack of early detection markers and the development of resistance to current chemotherapeutic agents. Currently available tests (CA-125, transvaginal ultrasound or combination of both) lack the sensitivity and specificity to be useful as an efficient screening tool for surveillance of the general population. Thus, there is an urgent need for the development and validation of new molecular markers that would be both specific and sensitive indicators of disease onset, as well as progression. Proteomic profiling has emerged as a powerful tool to study ovarian cancer in an unbiased way at the molecular level, to monitor the effects of given treatment options and for the discovery of biomarkers. In this review we discuss the challenges associated with proteomics-based biomarker discovery and some recent concepts to potentially overcome these hurdles. Recent proteomics work on ovarian cancer cells and tissues will be discussed in light of obtaining new insights into fundamental biological processes, as well as their potential integration with ongoing biomarker discovery pipelines.     

Pattern of arterial supply to the ovaries in Nili-Ravi buffaloes: Relationship with the distribution of ovarian structures

Reproductive tracts of 12 heifers and 18 adult buffaloes were collected from a slaughter house to study gross ovarian morphology and the distribution of follicles and corpora lutea on the surface of the ovary relative to the pattern of the arterial supply. Mean weight and volume of the ovaries were 3.8 ± 0.3 g and 4.08 ± 0.07 cm³, respectively, and were higher in adult buffaloes, but the difference was a function of body weight. Seventy percent of the ovaries were supplied by a single artery while the remaining 30% received two branches of the ovarian artery. Regardless of the position of entry of a single branch, ovarian follicles were located more frequently in the central (41.1%) than in medial (30.7%) or lateral portions of the ovary (28.2%). In ovaries supplied by two branches of the ovarian artery, follicles were distributed more uniformly on the ovarian surface. Number of follicles on the ovarian surface tended to increase when one of these branches was medial in position. Corpora lutea were observed more frequently in the part(s) of the ovary adjacent to the point of arterial entry regardless of the number of branches. Presence of a corpus luteum on the ovary had a negative effect on follicular development. The data suggested a possible relationship of the ovarian activity of the buffaloes with the pattern of arterial supply to the ovaries. Pattern of arterial supply might be contributing to the growth of the follicles, possibly by either saving them from atresia or by stimulating their growth.     

Prediction of ovarian tumor malignancy

Ovarian cancer is the leading cause of mortality among gynecological cancers. The aim of the study was to form the decision rules for distinguishing benign from malignant ovary lesions. The research was conducted on 201 women with ovary tumor. Commonly used specific markers for ovarian cancer (biochemical marker Ca 125, ultrasound and vascular markers) were used. The significant difference in the presence of an ultrasound and vascular markers between benign and malignant ovary changes along with the significantly different level of Ca 125 is confirmed. To a specific marker certain score number was appointed and the scoring system was formed. The incidence of benign/malignant ovary changes was observed in the researched group regarding anthropometric parameters (age, marital and menopausal status and number of deliveries). There is also significant difference in the incidence of benign/malignant ovary tumor regarding these parameters. Based on combination of the scoring system and anthropometric parameters the decision rules for distinguishing benign from malignant ovary tumors were formed. The logistic regression method was used. We proved that this method has higher accuracy in prediction of malignancy in women with ovary tumors than using morphological, Doppler or anthropometric parameters separately.     

Screening for early familial ovarian cancer with transvaginal ultrasonography and colour blood flow imaging.

OBJECTIVE--To assess the value of transvaginal ultrasonography with colour blood flow imaging in detecting early ovarian cancer in women with a family history of the disease. DESIGN--Study of self referred symptomless women with a close relative who had developed the disease. Each woman was screened to detect persistent lesions and defined changes in ovarian volume. Morphological score and pulsatility index were recorded. SETTING--Ovarian screening clinic. SUBJECTS--1601 self referred women. INTERVENTIONS--Women with a positive screening result were recommended to have further investigations. MAIN OUTCOME MEASURES--Findings at surgery and histology of abnormal ovaries. Morphological score > or = 5 and pulsatility index < 1.0 at last scan. RESULTS--Women were aged 17 to 79 (mean 47) years; 959 (60%) were premenopausal, 469 (29%) were naturally postmenopausal, and 173 (11%) had had a hysterectomy. 157 women had a pedigree suggestive of the site specific ovarian cancer syndrome and 288 of multiple site cancers. 61 women had a positive screening result (3.8%, 95% confidence interval 2.9 to 4.9%), six of whom had primary ovarian cancer detected at surgery (five stage Ia, one stage III). Use of a high morphological score or a low pulsatility index increased the odds of finding ovarian cancer from 1:9 to about 2:5 (1:1 in the highest risk groups). Five interval cancers were reported (three ovarian and two peritoneal). Eight of the 11 cancers developed in women with pedigrees suggestive of inherited cancer. CONCLUSIONS--Transvaginal ultrasonography with colour flow imaging can effectively detect early ovarian cancer in women with a family history of the disease. The screening interval should be less than two years.     

Ovarian contents of immunoreactive beta-endorphin and alpha-N-acetylated opioid peptides in rats

beta-Endorphin was measured by radioimmunoassay in homogenates of ovaries from immature Sprague-Dawley rats (21-29 days of age) and found to be present at levels of about 0.6-0.7 ng/ovary. After administration of PMSG there was approximately a 4-fold increase (2-3 ng/ovary) in total ovarian immunoreactive (ir) beta-endorphin 48 h after injection. Analysis of follicular fluid from similarly treated rats indicated about the same amount of ovarian ir-beta-endorphin (2-3 ng/ovary) as in ovarian homogenates, suggesting that most of the ir-beta-endorphin is localized in follicular fluid of PMSG-primed immature rats. Immature rats were made pseudopregnant by administration of hCG 48 h after PMSG, and at 24 h after injection of hCG there was a slight, but significant and reproducible, increase in the ovarian content of ir-beta-endorphin. The serum concentration of ir-beta-endorphin was in the range of 1-3 ng/ml and was unaffected by PMSG and PMSG/hCG; likewise, the pituitary content of ir-beta-endorphin did not change following administration of gonadotrophins to immature rats. In mature cyclic animals, levels of 2-4 ng ir-beta-endorphin/ovary were found, comparable to those in the ovaries of PMSG-primed immature rats, and there were only small changes during the oestrous cycle. In addition to ir-beta-endorphin, we also obtained evidence for the presence of alpha-N-acetylated opioid peptides (endorphins or enkephalins) in the ovaries of PMSG-primed immature and mature rats. The physiological role of the opioid peptides in reproductive tissue is unknown, but they are presumably acting in an autocrine or paracrine fashion.     

Radiation dose and second cancer risk in patients treated for cancer of the cervix

The risk of cancer associated with a broad range of organ doses was estimated in an international study of women with cervical cancer. Among 150,000 patients reported to one of 19 population-based cancer registries or treated in any of 20 oncology clinics, 4188 women with second cancers and 6880 matched controls were selected for detailed study. Radiation doses for selected organs were reconstructed for each patient on the basis of her original radiotherapy records. Very high doses, on the order of several hundred gray, were found to increase the risk of cancers of the bladder [relative risk (RR) = 4.0], rectum (RR = 1.8), vagina (RR = 2.7), and possibly bone (RR = 1.3), uterine corpus (RR = 1.3), cecum (RR = 1.5), and non-Hodgkin's lymphoma (RR = 2.5). For all female genital cancers taken together, a sharp dose-response gradient was observed, reaching fivefold for doses more than 150 Gy. Several gray increased the risk of stomach cancer (RR = 2.1) and leukemia (RR = 2.0). Although cancer of the pancreas was elevated, there was no evidence of a dose-dependent risk. Cancer of the kidney was significantly increased among 15-year survivors. A nonsignificant twofold risk of radiogenic thyroid cancer was observed following an average dose of only 0.11 Gy. Breast cancer was not increased overall, despite an average dose of 0.31 Gy and 953 cases available for evaluation (RR = 0.9); there was, however, a weak suggestion of a dose response among women whose ovaries had been surgically removed. Doses greater than 6 Gy to the ovaries reduced breast cancer risk by 44%. A significant deficit of ovarian cancer was observed within 5 years of radiotherapy; in contrast, a dose response was suggested among 10-year survivors. Radiation was not found to increase the overall risk of cancers of the small intestine, colon, ovary, vulva, connective tissue, breast, Hodgkin's disease, multiple myeloma, or chronic lymphocytic leukemia. For most cancers associated with radiation, risks were highest among long-term survivors and appeared concentrated among women irradiated at relatively younger ages.     

Ultrasonographic and laparoscopic evaluation of the reproductive tract of the captive female African lion (Panthera leo)

The use of transabdominal ultrasonography to assess the oestrous cycle has not been previously described in the African lion (Panthera leo). Twelve sexually mature lionesses and five female cubs had their reproductive organs assessed by transabdominal ultrasound. Ovarian findings were compared to laparoscopic findings while performing laparoscopic ovariectomy or salpingectomy. Vaginal cytology was performed and serum progesterone levels were determined. By combining all data the oestrous cycle stage of each lion was determined. One lion was far pregnant and was not operated on. In adults a uterine body could be seen ultrasonographically in 67% of lions while mural structures could be distinguished in 44% of lions. Five uterine horns could be seen in 3 lions. In 12 adults 10 ovaries were found of which eight had discernable follicles or luteal structures. During laparoscopy 12 active ovaries were seen with luteal structures seen in 11 ovaries and follicles in 2 ovaries. Using laparoscopy as the gold standard, ultrasonography had a sensitivity of 66% and specificity of 83% to detect ovarian reproductive activity. Two uterine cysts and a cluster of periovarian cysts were seen in three different lions. Three lions were pregnant, two were in oestrus, three in a luteal phase (dioestrus), and four were in anoestrus. Transabdominal ultrasound in combination with serum progesterone levels and vaginal cytology can be used to assess ovarian cyclical activity with reasonable accuracy in captive bred lions.     

Cell-nonautonomous induction of ovarian and uterine serous cystadenomas in mice lacking a functional Brca1 in ovarian granulosa cells

Women with germline mutations in BRCA1 have a 40% risk of developing ovarian cancer by age 70 and are also predisposed to cancers of the fallopian tubes. Given that ovulatory activity is a strong risk factor for sporadic ovarian cancer, we hypothesized that reduced BRCA1 expression might predispose to gynecological cancers indirectly, by influencing ovarian granulosa cells. These cells secrete sex steroids that control the ovulatory cycle and influence the growth of ovarian epithelial tumors. Granulosa cells also secrete mullerian inhibiting substance (MIS), a hormone that inhibits both the formation of female reproductive organs in male embryos and the proliferation of ovarian epithelial tumor cells. We tested this hypothesis by using the Cre-lox system to inactivate the Brca1 gene in mouse ovarian granulosa cells. A truncated form of the Fsh receptor promoter served as the Cre driver. Here, we show that indeed, inactivation of the Brca1 gene in granulosa cells led to the development of cystic tumors in the ovaries and uterine horns. These tumors carried normal Brca1 alleles, supporting the view that Brca1 may influence tumor development indirectly, possibly through an effector secreted by granulosa cells.     

Long interspersed element-1 open reading frame 1 protein expression profiles in ovarian cancers

Long interspersed element-1 (LINE-1) retrotransposons are autonomous mobile DNA elements with unique activity that account for about one-fifth of the human genome. Recently, it has been reported that the expression of LINE-1 is closely related to cancer prognosis, and LINE-1 hypomethylation might contribute to the acquisition of aggressive tumor behavior. Despite the importance of LINE-1 expression in cancers, research on the expression of LINE-1 open reading frame (ORF) proteins is very limited. Here, we investigated the expression profiles of LINE-1 ORF1p in ovarian cancer tissue microarrays containing 100 surgical specimens including adjacent normal ovary tissue, primary ovarian cancers, and metastatic ovarian cancers in lymph node. The tissue microarray was stained with mouse monoclonal antibody to LINE-1 ORFp1 for immunofluorescence analysis, and expression levels were evaluated by image analysis. LINE-1 ORFp was significantly overexpressed in ovarian cancers compared with normal tissues and especially upregulated in metastatic ovarian cancers. In addition, the expression of LINE-1 ORF1p was significantly higher in older ovarian cancer patients compared with young patients. These results indicate that expression of LINE-1 ORF1p is related to the progression of ovarian cancers and, in particular, to the age of the patient and the metastatic potential of the cancer.     

P-3THE VALUE OF PERITONEAL WASHING CYTOLOGY IN THE STAGING OF GYNAECOLOGICAL MALIGNANCY

Introduction:  Current protocols for staging gynaecological cancers include cytopathological examination of peritoneal washings taken at the time of definitive surgery. We investigated the clinical usefulness of this procedure.
Methods:  During 2004 and 2005, 140 peritoneal washings were submitted for cytopathological examination in our institutions for staging of 36 ovarian, 101 endometrial and 3 synchronous ovarian/endometrial cancers.
Results:  The washings contained malignant cells in 39 cases (28%). 35 of these cases had high stage disease – not confined to the organ of origin (i.e. stage 2 or more for ovary and stage 3 or more for endometrial). The other 4 were stage 1C ovarian cancers where there was either rupture or tumour involvement of the capsule. In only 2 of the 39 positive cases the cancer was marginally upstaged by the positive washings – these were ovarian cancers upstaged from 2A /B to 2C.
Discussion:  These findings suggest that peritoneal washing cytology as a routine procedure for staging ovarian and endometrial cancer is of limited clinical value. A larger study is needed to determine whether this procedure should continue to be included in staging protocols for gynaecological cancer.     

BRCA1 and BRCA2 pathways and the risk of cancers other than breast or ovarian

OBJECTIVE: Germline mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose women to breast and ovarian cancer. Female carriers of BRCA1 or BRCA2 gene mutations have very high lifetime risks for breast and ovarian cancers. Genetic abnormalities occur in all cancers, so BRCA-related pathways are critical because they serve to safeguard genetic content. Although protecting genetic information is a general function, BRCA-related pathways seem largely specific to preventing breast and ovarian cancer. The objective of this study was to resolve this difference between the theoretical functions of BRCA genes and their specific clinical effects. DATA SOURCES, DATA EXTRACTION, DATA SYNTHESIS: The author collected data published in > 30 epidemiologic studies on the incidence of cancers other than breast or ovarian in mutation carriers and in large populations eligible for mutation testing. Data were extracted and used directly as published whenever possible with a minimum of statistical manipulation. CONCLUSIONS: Although mutations target breast and ovary, a broader spectrum of cancers also occur with statistically significant elevated frequencies. Risks for "all cancers except breast or ovary" are elevated, with some population subgroups differing with regard to how frequently elevated risks were found at individual sites. Additional sites at risk included stomach, pancreas, prostate, and colon. The increased risk ranged from about 20% to 60%, with the greatest increases in risk in stomach and pancreas. The collected data show BRCA-pathway functions are probably required at multiple sites, not just in breast or ovary. Known interactions and relationships among BRCA-related pathways strongly support the idea that their inactivation provides growth or survival advantages for a variety of cancers. The data suggest applying an increased level of clinical alertness to those with defects in BRCA-related pathways. Identifying molecules that confer growth or survival advantages to BRCA-related cancers may provide broadly useful targets for chemotherapy or chemoprevention.     

Changes in the cellular localization of estrogen receptor alpha in the growing and regressing ovaries of Gallus domesticus during development

In this work, the presence of estrogen receptor alpha (ER-α) was determined in different cell subpopulations in the left growing and right regressing ovaries of Gallus domesticus from 13-day-old chicken embryos to one-month-old chickens by immunohistochemistry. Results revealed positive ER-α immunostaining in both ovaries during development, but the percentage, staining intensity, and cellular distribution of ER-α immunostaining changes according to whether it is the left or right ovary and with the animal’s age. In the left ovary, the ER-α was localized in the nuclei of the germinal epithelium and in germ cells of the ovarian cortex, as well as in the interstitial cells, undifferentiated cells, and epithelial cells of the lacunar channels of the ovarian medulla in all ages. In contrast, in the right ovary from 13-day-old chicken embryos to one-week-old chickens, only the epithelial cells of lacunar channels were ER-α immunoreactive, but in the right ovary of one-month-old chickens both the epithelial cells of lacunar channels and the interstitial cells presented ER-α. These results demonstrate differential expression of ER-α in both chicken ovaries during development in a cell type-specific distribution, suggesting that these differences may be regarded as an important cause in the process of asymmetric ovarian development in the chicken.     

Animal models of ovarian cancer

Ovarian cancer is the most lethal of all of the gynecological cancers and can arise from any cell type of the ovary, including germ cells, granulosa or stromal cells. However, the majority of ovarian cancers arise from the surface epithelium, a single layer of cells that covers the surface of the ovary. The lack of a reliable and specific method for the early detection of epithelial ovarian cancer results in diagnosis occurring most commonly at late clinical stages, when treatment is less effective. In part, the deficiency in diagnostic tools is due to the lack of markers for the detection of preneoplastic or early neoplastic changes in the epithelial cells, which reflects our rather poor understanding of this process. Animal models which accurately represent the cellular and molecular changes associated with the initiation and progression of human ovarian cancer have significant potential to facilitate the development of better methods for the early detection and treatment of ovarian cancer. This review describes some of the experimental animal models of ovarian tumorigenesis that have been reported, including those involving specific reproductive factors and environmental toxins. Consideration has also been given to the recent progress in modeling ovarian cancer using genetically engineered mice.