Proliferation and maturation of intratumoral blood vessels in women with malignant ovarian tumors assessed with cancer stem cells marker nestin and platelet derived growth factor PDGF-B.

OBJECTIVES: Platelet-derived growth factor B (PDGF-B) and nestin have been suggested to be useful in the assessment of neoangiogenesis in malignant ovarian masses. We aimed to investigate a possible association of these markers with newly formed microcapillaries and perivascular cells in ovarian tumors. MATERIAL AND METHODS: Microvessel density (MVD) and pericytes were studied in 82 women with ovarian neoplasms, including 7 benign cysts, 7 borderline masses, 64 epithelial ovarian cancers and 4 other malignant ovarian tumors. Immunohistochemical staining included antibodies to CD34, PDGF-B and nestin. RESULTS: Median values of CD34-positive and nestin-positive MVD were: 24,5 (range:17-32) and 21 (range: 12-31), respectively. No significant correlation between intratumoral CD-34 positive MVD and nestin-positive MVD was found. Benign and borderline lesions more frequently than malignant tumors displayed low or medium values of nestin-positive MVD (p = 0.01). Histological grading of malignant tumors was associated with nestin-positive MVD (p = 0.01). Nestin expression in tumor cells was not correlated with tumor grade or histological subtype. PDGF-B expression was found in tumor microves-sels in 72% of cases (59/82). High expression of PDGF in pericapillary cells was strongly associated with high expression of this marker in cancer cells (p = 0.007). Significant correlations between PDGF-B and nestin expression in malignant tumor microvessels were also found (p = 0.04). Nestin and PDGF-B expressions were strongly associated with high grade tumors when compared to low grade or benign masses. CONCLUSIONS: We conclude that the assessment of PDGF-B and nestin-positive MVD could be used to identify only highly active, angiogenic malignant ovarian masses, where tumor vasculature is formed.

Decreased expression of pigment epithelium-derived factor and increased microvascular density in ovarian endometriotic lesions in women with endometriosis.

OBJECTIVES: To determine whether women with endometriosis have altered expression of pigment epithelium-derived factor (PEDF) in ovarian endometriotic lesions as compared to women without endometriosis. STUDY DESIGN: Ectopic and eutopic and normal endometrial tissues were sampled from 40 women with ovarian endometriosis and 20 control women, respectively. Endometrial PEDF expression and microvascular density (MVD) using an antibody to von Willebrand factor (vWF) and alpha-smooth muscle actin (α-SMA) were evaluated by using immunohistochemical staining. RESULTS: We detected decreased PEDF expression and increased MVD using anti-vWF and -α-SMA in ovarian endometriotic lesions in women with endometriosis compared with the control group. In women with endometriosis, the MVD using anti-vWF and -α-SMA but not PEDF expression in ovarian endometriotic lesions correlated with the size of ovarian endometriotic cysts and the severity of the disease. Moreover, the MVD using anti-vWF was negatively correlated with PEDF expression in control endometrium but not in ovarian endometriotic lesions. CONCLUSIONS: Our results suggest that decreased PEDF expression and increased MVD in ovarian endometriotic lesions might play an important role in the pathogenesis of ovarian endometriosis.

Three-dimensional power Doppler vascular sonographic sampling for predicting ovarian cancer in cystic-solid and solid vascularized masses.

OBJECTIVE: The purpose of this study was to explore the role of 3-dimensional (3D) power Doppler (PD) sonography to discriminate between benign and malignant cystic-solid and solid vascularized adnexal masses and to define cutoff values for 3D PD indices to be used in a clinical setting. METHODS: A total of 143 consecutive women (mean age, 50.4 years; range, 17-82 years) with diagnoses of cystic-solid or solid vascularized adnexal masses on B-mode and 2-dimensional PD sonography were evaluated by 3D PD sonography before surgery. Three-dimensional PD sonography was used to assess vascularization within papillary projections and solid areas with a virtual organ computer-aided analysis program. Three-dimensional PD vascular indices (vascularization index [VI], flow index [FI], and vascularization-flow index [VFI]) were automatically calculated. A definitive histologic diagnosis was obtained in each case. RESULTS: A total of 113 masses (74%) were malignant, and 39 (26%) were benign. Morphologic evaluation revealed 30 unilocular solid masses (19.7%), 43 multilocular solid masses (28.3%), and 79 mostly solid masses (52%). The mean VI (9.365% versus 3.3%; P< .001), FI (34.318 versus 28.794; P< .001), and VFI (3.233 versus 1.15; P<0.01) were significantly higher in malignant tumors. No differences were found in the resistive index, pulsatility index, and peak systolic velocity. Receiver operating characteristic analysis revealed an area under the curve of 0.77 (95% confidence interval, 0.69-0.85), 0.71 (0.60-0.81), and 0.75 (0.66-0.83) for the VI, FI and VFI, respectively. For reducing the false-positive rate by almost one-third, sensitivity values for the VI (cutoff, 1.556%), FI (25.212), and VFI (0.323) were 92%, 95%, and 93%, respectively. CONCLUSIONS: Three-dimensional PD vascular indices could be helpful for reducing the false-positive rate in cystic-solid and solid vascularized adnexal masses.

Relationship between microvascular density and expression of vascular endothelial growth factor in patients with ovarian endometriosis.

OBJECTIVE: The aim of this investigation was to assess whether a correlation exists among microvascular density (MVD), expression of endothelial growth factor, and pelvic pain in patients with ovarian endometriosis. METHODS: Sixty-five patients (mean age 33.3 years, range 20-49 years) were diagnosed as having suspected cystic ovarian endometriosis and were scheduled for surgery. Patients were classified into two groups according to clinical complaints: group A, asymptomatic patients or patients with mild dysmenorrhea, and group B patients with severe dysmenorrhea and/or chronic pelvic pain and/or dispareunia. Immunohistochemical staining for CD34 and vascular endothelial growth factor (VEGF) in histological specimens for MVD and VEGF cellular expression assessment were performed. RESULTS: Five patients were excluded after surgery because no ovarian endometriosis was found in histological analysis. Thirty women were included in each group. MVD was higher in the symptomatic group. No differences were found in VEGF cellular expression. CONCLUSIONS: We conclude that pain symptoms in ovarian endometriosis are directly correlated with MVD but not with VEGF cellular expression. Based on our results, it appears that in endometriotic cysts, the angiogenic processes are present but not completely mediated by VEGF.

Expression of vascular endothelial growth factor receptors in bovine cystic follicles.

Cystic follicles have excess fluid derived from blood flow in the theca interna of the follicle; therefore, the vasculature network is related to cystic follicle formation. Vascular endothelial growth factor (VEGF) is a potent stimulator of blood vessel permeability and angiogenesis. The aim of this study was to examine the expression of VEGF receptors proteins and mRNA in cystic follicles to elucidate the VEGF system in cystic follicles. The expression of protein for VEGF receptors; fms-like-tyrosine kinase-1 (Flt-1) and foetal liver kinase-1 (Flk-1) was detected by the immunohistochemical method. The mRNA expression of Flt-1 and Flk-1 in cystic follicles was determined by RT-PCR. Concentration of oestradiol-17beta and progesterone in the follicular fluid of cystic follicles was determined using ELISA. Flt-1- and Flk-1 proteins were localized in granulosa and theca interna cells and endothelial cells of theca layers. The intensity of Flt-1 and Flk-1 immunoreaction was similar among cystic follicles with various ratios of oestradiol-17beta/progesterone concentrations. The expression of Flt-1 and Flk-1 mRNA was similar, regardless of the ratio of oestradiol-17beta to progesterone in follicular fluid. These results demonstrate that cystic follicles have both VEGF receptors in the granulosa and theca interna layers, which may be responsible for the increased permeability of microvessels, causing the accumulation of follicular fluid in cystic follicles.

Detection of ovarian tumors in chicken by sonography: a step toward early diagnosis in humans?

OBJECTIVE: Animal models of spontaneous ovarian cancer are important for understanding early tumor development. Ovarian imaging may play an important role in following changes in tumor development. Laying hens are the only animals that develop spontaneous ovarian cancer similar to humans. The aim of this study was to determine the feasibility of detecting ovarian tumors in laying hens using sonography. METHODS: Ovaries of commercial strains of White Leghorn laying hens (n = 29, 2.5-3.0 years old) were examined by transvaginal 2-dimensional gray scale and color Doppler sonography. Sonographic evaluations were compared with ovarian anatomy and histologic features. RESULTS: Results of in vivo sonography and ovarian anatomic and histologic examinations were consistent. The presence of gross ovarian tumors was correctly detected in all hens by sonography. The resistive and pulsatility index values associated with ovarian tumors were lower than for normal ovaries (P < .001) suggesting that blood flow velocity was increased in ovarian tumors. Values associated with abnormal ovarian histologic findings but no gross tumors were intermediate. CONCLUSIONS: Transvaginal sonography can be used to determine ovarian status in hens. It offers the ability to make repeated examinations on the same hen to monitor early changes in the ovary associated with ovarian cancer.

Endothelial cell survivin is involved in the growth of ovarian endometriotic cysts.

BACKGROUND: The aim of the present study was to evaluate microvessel density (MVD) in the cellular layers of ovarian endometriomata, with particular interest in the relationship with VEGF and survivin expressions by endothelial cells and with the diameter of the cysts. MATERIALS AND METHODS: MVD and VEGF and survivin endothelial cell expressions were evaluated in 26 ovarian endometriotic cysts and correlated with the cyst diameter. RESULTS: The mean MVD was higher in the inner specialized stroma of ectopic endometrium than in the outer fibrous capsule, but only in the fibrous capsule was MVD correlated with endothelial cell VEGF and survivin expressions as well as with the cyst diameter. CONCLUSION: The diameter of ovarian endometriotic cysts seems to be related to the angiogenic process involving the outer fibrous capsule, and not the inner specialized stroma of ectopic endometrium, since only in the capsule are vessels stimulated to proliferate by VEGF and protected from apoptosis by survivin, and their density is correlated to cyst diameter.

Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles.

The aim of this study was to examine the distribution of microvessels in the theca and the expression of vascular endothelial growth factor (VEGF) in the theca and granulosa of cystic follicles. Paraffin sections of cystic follicles were stained with Bandeiraea simplicifolia-I (BS-I) to visualize the endothelial cells of microvessels. The other sections were immunostained with anti-VEGF antibody. The mRNA expression of VEGF in the theca interna of cystic and healthy follicle was determined by RT-PCR. In the theca interna, cystic follicles with granulosa cells had significantly greater microvessel number density (the number of microvessels per given field) and area (area occupied by microvessels per given area) than healthy follicles in various sizes (<3, 4-8, >9 mm). Loss of granulosa cells from cystic follicles resulted in a similar number density, but significantly smaller area of microvessels in the theca interna. There was no significant difference in the microvessel number density and area of the theca externa between the types of follicle. VEGF protein was expressed in the granulosa and theca interna of healthy and cystic follicles. These results demonstrate that cystic follicles have a highly developed vasculature network in the theca interna, especially in cystic follicles containing granulosa cells. It is also suggested that VEGF is highly expressed in the cystic follicle as well as healthy follicle, which may be associated with advanced vasculature and the accumulation of follicular fluid in cystic follicles.

[Three dimensional sonography and 3D power angiography in differentiation of adnexal tumors]. / Wspólczesna ultrasonografia w diagnostyce guzów jajnika: porównanie wartosci predykcyjnych oceny morfologicznej, badania przeplywu dopplerowskiego i obrazowania trójwymiarowego 3D.

BACKGROUND: Three-dimensional sonography is a novel diagnostic method proposed to be an additional non-invasive tool in the assessment of ovarian tumors. OBJECTIVE: To study diagnostic potential of 3D sonography and power Doppler angiography in the preoperative differentiation of adnexal masses. MATERIAL AND METHODS: One hundred twenty-eight women with tumors thought to be of adnexal origin were examined preoperatively. Following morphological (papillae, septae, tumor size and volume) and color Doppler (PI, RI, Vmax and TAMX) assessment, 3D ultrasound of adnexal tumors according to Kurjak et al. (2000) was performed. Various scanners were used and included: ATL 5000 HDI (Phillips, USA) and Combison 530 and Voluson 730 (Kretztechnik, Austria) machines. Following variables were studied: inner wall structure, presence of papillae, thickening > 3 mm of septa as well as vascular branching pattern, number and localization of small blood vessels and the presence of vascular anastomoses. RESULTS: Twenty-one tumors were malignant (3 FIGO stage I) and 101 masses were benign. Power Doppler combined with 3D sonography predicted malignancy with a sensitivity of 92.6% (25 of 27 patients). Commonly used morphological and Doppler criteria produced lower sensitivity, the values being in range of 45% to 87.5%. Negative predictive value of 97.2% was the highest for 3D sonography (> 7 points in Kurjak's scale). CONCLUSIONS: Selective use of 3D ultrasound and power Doppler angiography could be used to better characterize adnexal tumors. Detailed 3D sonography may help to identify women who, if needed, may have less invasive surgical procedure such as laparoscopy or be referred to gynecological oncologist.

Three-dimensional power Doppler sonography: imaging and quantifying blood flow and vascularization.

OBJECTIVES: To assess the feasibility of imaging low-velocity blood flow in adnexal masses by transvaginal three-dimensional power Doppler sonography, to analyze three-dimensional power Doppler sonography data sets with a new computer-assisted method and to test the reproducibility of the technique. METHODS: A commercially available 5-MHz Combison 530 ultrasound system was used to perform three-dimensional power Doppler sonography transvaginally. A cube (= volume of interest) was defined enclosing the vessels of the cyst and the Cartesian characteristics were stored on a hard disk. This cube was analyzed using specially designed software. Five indices representing vascularization (the vascularization index (VI) or blood flow (the flow index (FI)) or both (the vascularization-flow index (VFI)) were calculated. The intraobserver repeatability of cube definition and scan repetition was assessed using Hartley's test for homogeneous variances. Interobserver agreement was assessed by the Pearson correlation coefficient. RESULTS: Imaging of vessels with low-velocity blood flow by three-dimensional power Doppler sonography and cube definition was possible in all adnexal massed studied. In some cases even induced non-vascular flow related to endometriosis was detected. The calculated F value with intraobserver repeated Cartesian file-saving ranged from 0 to 18.8, with intraobserver scan repetition from 4.74 to 24.8 for VI, FI 1, FI 2 and VFI 1; for VFI 2 the calculated F value was 64. The interobserver correlation coefficient ranged between 0.83 and 0.92 for VI, FI 1, FI 2 and VFI 1; for VFI 2 the correlation coefficient was less than 0.75. CONCLUSION: Vessels with low-velocity blood flow can be imaged using three-dimensional power Doppler sonography. Induced non-vascular flow was detected in endometriotic cyst fluid. Three-dimensional power Doppler sonography combined with the cube method gave reproducible information for all indices except VFI 2. These indices might prove to be a new predictor in all fields of neoangiogenesis. The clinical relevance remains to be determined.

Blood flow in functional cysts and benign ovarian neoplasms in premenopausal women.

To assess the value of transvaginal color Doppler sonography in the differentiation of functional cysts from benign ovarian neoplasms in premenopausal women, 100 premenopausal women with the diagnosis of adnexal mass were enrolled in a prospective study. All patients underwent transvaginal color Doppler sonography during the follicular phase. We evaluated 107 masses. Tumor volume and morphology were assessed, as were tumor blood flow location, the number of vessels, the resistive and pulsatility indices, and the peak systolic velocity. Patients were followed up after 8 to 10 weeks by transvaginal sonography. Functional cysts were considered when spontaneous resolution occurred. Surgery was performed if a tumor enlarged or persisted after two scans. Thirty-nine (36.5%) cysts regressed spontaneously and 68 (63.5%) were removed surgically. Seven of the latter were follicular or luteal cysts and were considered to be functional cysts. No carcinoma was found. Arterial blood flow was detected in 28 (60.8%) functional cysts and in 42 (68.8%) benign neoplasms (P = 0.3446). The vessels were located peripherally in 27 (94.6%) functional cysts and in 37 (88.1%) benign neoplasms (P = 0.2226). No differences were found between functional cysts and benign neoplasms in mean resistive index (0.65, 95% confidence interval: 0.59 to 0.71 versus 0.64, 95% confidence interval: 0.60 to 0.69), mean pulsatility index (1.47, 95% confidence interval: 1.17 to 1.84 versus 1.57, 95% confidence interval: 1.26 to 1.86), number of vessels (1.1, 95% confidence interval: 0.7 to 1.3 versus 1.4, 95% confidence interval: 1.1 to 1.8), and peak systolic velocity (28.6 cm/s, 95% confidence interval: 24.7 to 34.2 versus 24.9 cm/s, 95% confidence interval: 21.6 to 28.3). We concluded that transvaginal color Doppler sonography is not useful to discriminate between functional ovarian cysts and benign ovarian neoplasms in premenopausal women.

Visualization of blood flow in small ovarian tumor vessels by transvaginal color Doppler sonography after echo enhancement with injection of Levovist.

The echo-enhancing effect of an ultrasound contrast medium (Levovist) in color Doppler sonography of small vessels in ovarian tumors was studied in a group of 30 women. The vascularity of adnexal masses with a suspicion of primary carcinoma in 3 and benign lesions in 27 cases was investigated before and after injection of 300 mg/ml of Levovist. In 17 cases color flow mapping showed increased vascularity either in the cystic wall or in solid structures within the tumor. In all patients we observed enhancement of Doppler signal intensity. Our results show that Levovist facilitates waveform analysis in very small vessels with poor velocity patterns.

Color Doppler sonography of ovarian masses: a multiparameter analysis.

This study analyzed vessel location, maximum systolic velocity, impedance, and waveform shape in 25 benign and 25 malignant surgically excised and pathologically examined ovarian masses as depicted by transvaginal or transabdominal color Doppler sonography, or both. Those parameters that achieved statistical significance (P > 0.05) between the two types of masses included vessel location, impedance, and waveform shape. Malignant masses typically were characterized by centrally located vessels that had low impedance without a diastolic "notch" in the waveform. Maximum systolic velocities are statistically similar in benign and malignant lesions. Multiparameter analysis may improve the diagnostic specificity and sensitivity of this technique in distinguishing benign from malignant ovarian masses.

Transvaginal color Doppler for the assessment of pelvic circulation.

Transvaginal color Doppler was used to analyse a group of 56 patients including 41 with proven pelvic masses and 15 infertility patients with normal pelvic anatomy. All patients were examined by ultrasound before laparoscopy or/and laparotomy. The ultrasonographer had not been informed of other clinical findings and indications for operative treatment. Comparison of blood flow characteristics within uterine fibromas and ovarian malignancies revealed less impedance and higher blood velocity in cases of malignancy. When predicting the malignant nature of pelvic tumors, no false-negative results were encountered, and only one false positive diagnosis of malignancy.

Angiography of the ovarian artery in adnexal lesions.

Selective catheterization of the ovarian artery was performed in 39 women with pelvic tumor. Previously the combination of ultrasound and bilateral selective internal iliac angiography was proven to be valuable in the evaluation of gynecologic lesions. The present series demonstrates that selective ovarian angiography still enhances the possibilities for a correct preoperative diagnosis.