Endocrine studies of normal and polycystic ovarian tissues in vitro.

The purpose of this study was to compare the steroidogenic potential of the granulosa, theca, and medullary tissues from polycystic and normal ovaries. These ovarian endocrine compartments were isolated from appropriate ovaries and were cultured in vitro for three days in the absence (control) and presence of follicle-stimulating hormone (FSH)/luteinizing hormone (LH) (1 lU/ml), N6,O2-dibutyryladenosine-3':5'-cyclic monophosphoric acid (Bu2cAMP) (10(-2)M), and adrenocorticotropic hormone (ACTH) (1.3 U/ml). After the incubation, steroids in the media were measured by radioimmunoassay. Granulosa cells (10(5) cells per dish) from 4 to 7 mm follicles of normal and polycystic ovaries secreted progesterone spontaneously during the culture period and the production of progesterone was markedly stimulated (between tenfold and thirtyfold) by gonadotropins and Bu2cAMP but not by ACTH. Little, if any, androgen (androstenedione, dehydroepiandrosterone, and testosterone) or estrogen (estrone and estradiol) accumulated in the media of any granulosa cell culture. The control cultures of theca tissue from normal and polycystic ovaries secreted large amounts of androstenedione and progesterone and the production of these steroids by normal and polycystic ovary theca was stimulated in most cases by LH/FSH and Bu2cAMP but not by ACTH. Both normal and polycystic ovary theca secreted some testosterone and dehydroepiandrosterone but little, if any, estrone or estradiol accumulated in any theca culture. The medullary tissue of normal and polycystic ovaries produced only trace amounts of steroids in vitro except for the results from one polycystic ovary with hyperthecosis in which case significant quantities of C19 and C18 steroids were secreted. These experiments have demonstrated that isolated granulosa and theca cells from midantral follicles of normal and polycystic ovaries have a similar capacity to secrete C21 and C19 steroids in the absence and presence of trophic agents. Therefore, it seems probable that chronic anovulation in patients with polycystic ovaries is not caused by an obvious deficiency in the de novo steroidogenic potential of the multiple midantral follicles of the polycystic ovaries or by the absence of gonadotropin receptors on the polycystic ovary follicular cells.     

Concentration of vascular endothelial growth factor released by cultured human luteinized granulosa cells is higher in women with polycystic ovaries than in women with normal ovaries

To test the hypothesis that increased serum levels of vascular endothelial growth factor (VEGF) in women with polycystic ovaries or the polycystic ovary syndrome (PCOS) result from excess release by ovarian granulosa cells.Prospective study.Academic research setting.Twenty women undergoing IVF treatment, of whom 10 had normal ovaries and 10 had polycystic ovaries.Human granulosa lutein cells were isolated from follicular fluid obtained on the day of oocyte retrieval. Release of VEGF was assessed after co-incubation of granulosa lutein cells with gonadotropins and insulin. Serum and follicular fluid concentrations of VEGF were measured.Release of VEGF from granulosa lutein cells and serum levels of VEGF.Incubation with human hCG, and luteinizing hormone increased release of VEGF into the culture medium. Insulin alone did not increase release of VEGF, but addition of insulin increased hCG-stimulated release of VEGF. Serum and follicular fluid VEGF concentrations and the amount VEGF released from granulosa lutein cells obtained from women with polycystic ovaries or PCOS and those who developed the ovarian hyperstimulation syndrome were greater than those from granulosa lutein cells obtained from women with normal ovaries and those who did not develop the ovarian hyperstimulation syndrome.The amount of VEGF released by granulosa lutein cells is gonadotropin dependent and is augmented by insulin. The increased circulating concentrations of VEGF in women with PCOS may not only be due to an increased number of actively secreting granulosa lutein cells but also due to increased secretory capacity of each granulosa cell.     

Activation of SPARC expression in reactive stroma associated with human epithelial ovarian cancer.

OBJECTIVE: SPARC (secreted protein, acidic, rich in cysteine) is a calcium-binding counteradhesive glycoprotein that has the potential to play an important role in promoting tumor progression and invasiveness. SPARC has been reported to be markedly down-regulated in ovarian carcinomas relative to the normal surface epithelium and has been suggested to act as a tumor suppressor in ovarian cancer. To more precisely define potential changes in SPARC expression associated with malignant transformation of the ovary, we compared the distribution of SPARC mRNA and protein expression in patient specimens of malignant and nonmalignant ovaries. METHOD: SPARC mRNA and protein expression was examined in 24 human invasive ovarian cancers, 5 tumors of low malignant potential (LMP), and 8 nonmalignant ovaries by in situ hybridization and immunohistochemistry. RESULTS: In nonmalignant ovaries, SPARC mRNA expression was restricted to thecal and granulosa cells of vessiculated follicles. Cytoplasmic SPARC immunoreactivity was observed in these compartments, whereas variable SPARC immunostaining was observed in normal surface epithelial cells. In contrast, high-level expression of SPARC mRNA and protein was detected in stroma of ovaries containing malignant tumor cells, particularly at the tumor-stromal interface of the invading tumors. Lower levels and a more diffuse pattern of SPARC mRNA expression were associated with LMP specimens. SPARC mRNA was not expressed by ovarian adenocarcinoma or by surface epithelial cells. Consistent with the in situ hybridization data, SPARC immunoreactivity was found throughout the reactive stroma of specimens containing ovarian carcinoma. However, despite the lack of detectable SPARC mRNA, SPARC immunoreactivity was consistently observed within the cytoplasm of cancer cells. CONCLUSION: The pattern of SPARC expression shown in this study indicates that SPARC is up-regulated in reactive stroma associated with invasive ovarian cancer. Moreover, these results raise the possibility that SPARC secreted from the stroma is internalized by ovarian cancer cells and may exert important intracellular effects upon these cells.     

Ovarian steroid production in a woman with polycystic ovary syndrome associated with endometrial cancer

A young woman with typical polycystic ovary syndrome (PCO) underwent laparotomy for moderately differentiated endometrial cancer. Specimens from the hyperplastic thecal and stromal tissue of the ovaries were incubated for 2 hours in the presence or absence of hCG, 100 IU/ml. Following incubation the tissue content of cyclic AMP and the amounts of progesterone (P), androstenedione (A), testosterone (T) and estradiol-17 beta (E2) in the incubation medium were analysed. For comparison, thecal cells from normal ovaries of regularly menstruating women were incubated under identical conditions. In vivo, the PCO ovaries secreted several-fold greater amounts of T than normal ovaries. In vitro, the thecal cells were much more active, steroidogenically, than the stromal cells of the PCO ovary. Furthermore, the hyperplastic thecal cells of the PCO ovary produced several-fold greater amounts of androgens, and appeared more sensitive to stimulation with hCG, as compared with thecal cells from normal ovaries. The results indicate that in women with PCO associated with endometrial cancer the hyperplastic thecal cells are a significant site of abnormal androgen production and abnormal sensitivity to gonadotropin.     

热休克蛋白10、27和47在多囊卵巢综合征患者卵巢中的表达

目的:探索热休克蛋白(heat shock protein,HSP)10、27和47在多囊卵巢综合征(PCOS)患者卵巢组织中的表达及其意义。方法:采用荧光实时定量PCR(SYBR GREEN Iassay)、Western blot和免疫组织化学技术分别对3例正常妇女(对照组)、3例无排卵PCOS患者(PCOS组)卵巢组织的HSP10、HSP27和HSP47的mRNA及蛋白进行测定。结果:①HSP10 mRNA在PCOS患者卵巢组织的表达与对照组相比,差异无显著性(P>0.05);HSP10蛋白在PCOS患者卵巢组织的表达与对照组相比,表达降低,差异有显著性(P<0.05);免疫组化结果显示HSP10表达于对照组卵巢的所有细胞,而在PCOS组仅在卵泡膜细胞表达水平较高。②HSP27 mRNA和蛋白在PCOS患者卵巢组织的表达降低与对照组相比,差异有显著性(P<0.05);免疫组化结果显示HSP27高表达于对照组卵巢原始卵泡的卵母细胞。③HSP47 mRNA和蛋白在PCOS患者卵巢组织的表达高于对照组,PCOS组卵巢,差异有显著性(P<0.05);免疫组化结果显示HSP47高表达于PCOS组卵巢的卵母细胞、颗粒细胞和间质细胞。结论:HSP10、HSP27和HSP47在PCOS患者卵巢和正常人卵巢中存在差异表达。     

Studies on steroidogenesis of polycystic ovary syndrome--endocrinological analysis of follicular fluid and progesterone biosynthesis of cultured granulosa cells

In order to determine the difference between steroidogenesis in normal and polycystic ovaries (PCO), endocrinological analysis of follicular fluid and monolayer cell culture of human granulosa cells were carried out, and human LH and dibutyryl cyclic AMP were added to the cultures. The concentrations of LH and androstenedione in the follicular fluid from PCO ovaries were significantly higher than those found in normal ovaries in the early and mid-follicular phases. The cultured granulosa cells from the mid-follicular phase of normal women responded significantly to LH, showing an 8 to 10 fold increase in progesterone production. In contrast, exogenous LH caused only a slight stimulation (2 to 4 fold increase) in progesterone production by granulosa cells from PCO follicles. The cultured granulosa cells from normal women and PCO ovaries responded significantly to dibutyryl cyclic AMP, showing an 8 to 10 fold increase in progesterone production. These results strongly suggest that there is no alteration in the steroidogenic pathway after the cyclic AMP stage and that the persistent elevation of LH in serum and follicular fluid may lead to a disturbance of progesterone production in response to exogenous LH in the cultured granulosa cells from PCO ovaries.     

Ovarian enlargement as a possible marker of androgen activity in polycystic ovary syndrome.

This study has been carried out in an attempt to analyze clinical, hormonal and ultrasonography data and to evaluate their possible interrelationships in a group of 72 women with polycystic ovary syndrome (PCOS). Seventeen (23.6%) PCO women were found to have ovarian volume within normal range, while 72.2% had enlarged ovaries. Serum testosterone, androstenedione and DHEAS levels were higher in PCO women with enlarged ovaries when compared to patients who had ovarian volume within normal range, although the difference was not significant. However, when PCO patients were divided into subgroups according to the degree of ovarian enlargement, it was found that patients with the most enlarged ovaries had significantly higher serum androstenedione levels than those with normal ovarian volume (p = 0.039). Significant positive correlation was established between serum androstenedione concentration and ovarian volume (r = + 0.23, p less than 0.05). Hirsutism was found to be equally present in patients with normal ovarian volume (70.6%) and in patients with enlarged ovaries (71.2%) while oligomenorrhea was present more frequently among PCO women who had ovarian volume within normal range (64.7% vs. 40.4%). The results of our study allow us to suggest ovarian enlargement as a marker of excessive androgen production and disturbances of menstrual cycle in polycystic ovary syndrome.     

Alterations in SPARC and VEGF Immunoreactivity in Epithelial Ovarian Cancer

Objective. Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular protein that modulates cell adhesion and growth. It is thought to play a decisive role in tissue remodeling and angiogenesis. Alterations in SPARC expression have been observed in a variety of solid tumors; however, no consistent pattern of deregulation has been characterized. Vascular endothelial growth factor (VEGF) has emerged as an important regulator of tumor neovascularization. Recent work has shown that SPARC modulates the mitogenic activity of VEGF in normal endothelium. While its role in malignant transformation remains elusive, SPARC may contribute to tumor propagation and invasion. This study examines the immunoreactivity of SPARC and VEGF associated with neoplastic transformation of the ovary.Methods. Immunostaining for VEGF and SPARC protein was performed on 62 archival specimens.Results. Fourteen normal ovaries and 48 ovarian carcinomas were evaluated. SPARC was detected in the stroma of 63% of ovarian carcinomas. In contrast, SPARC was observed in the stroma of only 29% of normal ovaries (P = 0.02). Furthermore, SPARC was limited in normal ovaries to premenopausal patients, juxtaposed either with vesiculated follicles or within the corpus luteum. VEGF was observed in 42% of ovarian carcinomas with immunoreactivity confined to tumor cells. The level of VEGF immunoreactivity was significantly higher in ovarian carcinoma compared to normal ovary epithelium (42 vs 7%, P = 0.02).Conclusions. Immunoreactivity of SPARC and VEGF is heightened in association with ovarian carcinoma, with a distinct distribution of SPARC in the stroma of neoplastic ovaries and VEGF within tumor cells. No obvious pattern of coincident SPARC and VEGF immunoreactivity was detected. These results indicate the possibility of an aberration in the interaction that has been described in normal endothelium between SPARC and VEGF in association with malignant transformation.     

Diffuse luteinization of the ovaries associated with the masculinization syndrome

The syndrome of masculinization in the female, attributed to pituitary basophilism or adrenal cortical lesions, usually includes regressive changes in the ovaries. At times, however, as evidenced by the two cases described in this report, there may be bilateral ovarian enlargement, due to excessive perifollicular proliferation and luteinization of the theca cells and diffusely scattered luteinized cells within the ovarian parenchyma.The ovrian effects are suggestive of increased gonadotropic stimulation and are probably secondary. It does not appear that they are responsible for the production of the masculinization syndrome.In the clinical investigation of virilism the presence of an ovarian enlargement has been and should be regarded as suspicious evidence of a possible arrhenoblastoma or adrenal rest neoplasm of the ovary. In view of this report, a third possibility exists, namely, enlargement of the ovary due to diffuse luteinization. It may be possible to decide, at operation, by incision of the ovaries, whether or not tumor is present. If, in young patients, tumor can unequivocally be ruled out by this device, one or both ovaries may be left in situ.     

Immunohistochemical detection of plasminogen activator inhibitor-1 in polycystic ovaries.

Anovulation in women with polycystic ovary syndrome (PCOS) is incompletely understood. The concentration of the glycoprotein plasminogen activator inhibitor-1 (PAI-1) is raised in insulin resistance. This has been described in the granulosa and theca cell layers of the animal but not the human ovary. This study was performed to investigate the location of PAI-1 in the human ovary and investigate whether it may contribute to anovulation in PCOS. PAI-1 was localized immunohistochemically and quantitated using computer image analysis in 17 ovarian follicles from five women with a diagnosis of PCOS and compared with 15 follicles from six normal ovaries. PAI-1 was predominantly found in the granulosa and theca cells in both polycystic and normal ovaries. Image analysis did not reveal a difference in the PAI-1 signal from polycystic compared with normal ovaries. This study shows that PAI-1 plays a role in human ovulation, but its role in PCOS requires further research.     

Gherlin expression in women with polycystic ovary syndrome--a preliminary study

The etiology and pathogenesis of polycystic ovary syndrome (PCOS) is still unknown. Using real-time PCR, we detected that polycystic ovaries showed almost ten times lower expression of ghrelin mRNA than normal ovaries, whereas the mRNA levels in blood cells were similar in both study groups. This suggests that the presence of ghrelin in PCOS and normal ovaries may have an autocrine/paracrine modulatory effect on ovary functions and local significance in the etiology of PCOS.     

The effect of short-interval laparoscopic lysis of adhesions on pregnancy rates following Nd-YAG laser photocoagulation of polycystic ovaries.

Laparoscopic Nd-YAG laser photocoagulation of the ovaries was performed in 40 anovulatory women with clomiphene citrate-resistant polycystic ovary disease. Following this procedure, the subjects were randomly assigned to have either a second-look laparoscopy with lysis of adhesions within 3-4 weeks of the initial laparoscopy (N = 19) or expectant management (N = 20). One patient assigned to the laparoscopy group refused the procedure. Minimal and mild adhesions that did not distort the normal tubo-ovarian relationship were encountered in 13 patients (68%) in the second-look laparoscopy group; these adhesions were easily lysed using sharp or blunt dissection. The pregnancy rates over 6 months were similar in the two groups (47% in the second-look group and 55% in the expectant-management group; P greater than .05). These data suggest that early laparoscopic lysis of adhesions does not improve short-term conception rates following laparoscopic Nd-YAG laser photocoagulation of polycystic ovaries.     

Surface ultrastructure of polycystic ovaries as viewed by electron microscopy

Scanning electron microscopy was used to observe surface fine structure of germinal epithelium and granulosa cells of human ovaries from 24 patients with polycystic ovaries and primary infertility. Synthetic LH-RH was administered intravenously and serum LH was measured at frequent intervals before and after LH-RH injection. The PCO patients were arbitrarily classified into two groups on the basis of ovarian morphology: typical PCO (Type I) with greater LH response than the lower LH response of a typical PCO (Type II). The germinal epithelium which did not completely cover the surface of the normal ovary was characterized by patchy areas of cells with and without dense microvilli. In normal preovulatory follicles, most granulosa cells were polyhedral in shape with smooth surfaces, whereas those facing the follicular cavity were elongated and flattened and were covered with material having a filamentous/reticular texture. In PCO the germinal epithelium surrounding the whole surface possessed dense microvilli, solitary cilia, and blebs, resembling the fetal ovary. In normal ovaries, the follicular cells were uniform in size and shape with microvilli and evaginations. The PCO cells have irregular size and shape with few microvilli.     

A comparison of bone mineral density in oligomenorrhoeic adolescents with polycystic ovaries and normal ovaries

We evaluated whether the presence of polycystic ovaries in adolescent girls as a cause of oligomenorrhoea and amenorrhoea would pose any protective effect against osteoporosis or low bone mineral density (BMD) compared with girls having similar menstrual dysfunction but normal ovaries. A cross-sectional observational study was done in consecutive girls, aged between 16 and 19 years, presenting to the adolescent gynaecology clinic with oligomenorrhoea or amenorrhoea. All patients underwent full hormonal profile assessment, pelvic ultrasound for ovarian morphology, bio-impedance estimation of body fat, and dual-energy X-ray absorptiometry and quantitative peripheral computed tomography scans to determine BMD in axial and appendicular skeletal sites. Polycystic ovaries were diagnosed according to ultrasound morphology. These were then compared with an age-matched eumenorrhoeic control group that had undergone the same evaluation. Of 45 patients with oligomenorrhoea or amenorrhoea, 14 (31%) were diagnosed to have polycystic ovaries, while the other 31 had normal ovaries. The control group consisted of 45 age-matched eumenorrhoeic girls. The group with normal ovaries had lower BMD at the lumbar spine and hip, as well as lower total tibial volumetric BMD, than the eumenorrhoeic controls, but there were no significant differences between the group with polycystic ovaries and eumenorrhoeic controls. We conclude that adolescents with oligomenorrhoea and amenorrhoea with normal ovaries had lower BMD than eumenorrhoeic ones, but those with polycystic ovaries had BMD values comparable to those of eumenorrhoeic controls despite their menstrual dysfunction.     

Immunocytochemical localization of angiotensin II immunoreactivity and demonstration of angiotensin II binding in the rat ovary

The cellular localization of angiotensin II immunoreactivity and the presence of angiotensin II binding in rat ovaries were studied. Angiotensin II immunohistochemical staining was demonstrated throughout the corpora lutea of gonadotropin-stimulated immature rats and pseudopregnant adult rats, as well as in some stromal and thecal cells surrounding large antral follicles. No immunostaining was observed in granulosa cells of preantral or antral rat follicles or in ovaries from unstimulated immature rats. With in vitro autoradiography, specific, saralasin-suppressible 125I-angiotensin II binding was demonstrated in normal cycling rat ovaries: diestrus greater than proestrus greater than estrus. The combined findings of angiotensin II immunostaining in ovarian follicles and corpora lutea and of cycle-related angiotensin II binding support the hypothesis of a functional role for the ovarian renin-angiotensin system.     

Ovarian response after laparoscopic ovarian cystectomy for endometriotic cysts in 132 monitored cycles.

OBJECTIVE: To investigate the follicular response of ovaries after laparoscopic ovarian cystectomy for endometriotic cysts. DESIGN: A retrospective, controlled study. SETTING: Obstetrics and Gynaecology Department of a university hospital. PATIENT(S): Patients with history of infertility who had laparoscopic ovarian cystectomy for endometriotic cysts. INTERVENTION(S): Laparoscopic ovarian cystectomy, transvaginal ultrasound monitoring of follicles in natural cycles, stimulated cycles using clomiphene citrate (CC), chronic low-dose FSH, and "flare down" regimen. MAIN OUTCOME MEASURE(S): Follicular response of postcystectomy and normal ovaries. RESULT(S): For women < 35 years of age, the mean follicular response of postcystectomy ovaries was reduced significantly when compared with normal ovaries in natural cycles (0.30 versus 1.00 follicle per cycle) and in CC-stimulated cycles (0.87 versus 1.27 follicles per cycle). The mean follicular response was not statistically significantly different when the ovaries were stimulated with chronic low-dose FSH or with the flare down regimen. For women > or = 35 years, postcystectomy ovaries responded with a comparable number of follicles as the normal ovaries in natural cycles and the three different ovarian stimulation regimens. CONCLUSION(S): Postcystectomy ovaries showed reduced follicular response in natural and CC-stimulated cycles for women < 35 years of age. Postcystectomy ovaries produced a comparable number of follicles as normal ovaries when stimulated with gonadotropins.     

Ultrasound diagnosis of polycystic ovaries in women who have no symptoms of polycystic ovary syndrome is not associated with subfecundity or subfertility

OBJECTIVE: To evaluate the effect on fertility of the appearance of polycystic ovaries in women who have no symptoms of polycystic ovary syndrome. DESIGN: Case-control study. SETTING: Teaching hospitals in Hull, United Kingdom. PATIENT(S): Women with the appearance of polycystic ovaries on ultrasound and women with normal ovaries. INTERVENTION(S): A questionnaire about previous subfertility, pregnancies, menstrual pattern, features of polycystic ovary syndrome, gynecological history, and individual lifestyle factors. MAIN OUTCOME MEASURE(S): Time to pregnancy (TTP) and relative risk (RR) of subfertility in symptomatic and asymptomatic subgroups of both groups. RESULT(S): Women with PCOs took longer TTP and were significantly less fertile if they were obese (RR = 2.6), had menstrual disturbances (RR = 4.6), hirsutism (RR = 2.5), and/or acne (RR = 2.7). Further reductions in fecundity occurred with an increasing number of symptoms (threefold, sevenfold, and 10-fold longer TTP with two, three, and four symptoms, respectively). The TTP of women with no symptoms was not significantly longer and they were not more likely to be subfertile than women with normal ovaries. These symptoms were not associated with significantly reduced fecundity in women with normal ovaries. CONCLUSION(S): The appearance of polycystic ovaries has been shown to have no significant impact on fertility in women with no symptoms. Appearances alone do not reflect the pathological features of polycystic ovary syndrome, and additional diagnostic criteria should be considered. Obesity, menstrual disturbances, and/or hyperandrogenism are factors associated with subfertility in women with polycystic ovaries.     

A comparison of bone mineral density in oligomenorrhoeic adolescents with polycystic ovaries and normal ovaries.

We evaluated whether the presence of polycystic ovaries in adolescent girls as a cause of oligomenorrhoea and amenorrhoea would pose any protective effect against osteoporosis or low bone mineral density (BMD) compared with girls having similar menstrual dysfunction but normal ovaries. A cross-sectional observational study was done in consecutive girls, aged between 16 and 19 years, presenting to the adolescent gynaecology clinic with oligomenorrhoea or amenorrhoea. All patients underwent full hormonal profile assessment, pelvic ultrasound for ovarian morphology, bio-impedance estimation of body fat, and dual-energy X-ray absorptiometry and quantitative peripheral computed tomography scans to determine BMD in axial and appendicular skeletal sites. Polycystic ovaries were diagnosed according to ultrasound morphology. These were then compared with an age-matched eumenorrhoeic control group that had undergone the same evaluation. Of 45 patients with oligomenorrhoea or amenorrhoea, 14 (31%) were diagnosed to have polycystic ovaries, while the other 31 had normal ovaries. The control group consisted of 45 age-matched eumenorrhoeic girls. The group with normal ovaries had lower BMD at the lumbar spine and hip, as well as lower total tibial volumetric BMD, than the eumenorrhoeic controls, but there were no significant differences between the group with polycystic ovaries and eumenorrhoeic controls. We conclude that adolescents with oligomenorrhoea and amenorrhoea with normal ovaries had lower BMD than eumenorrhoeic ones, but those with polycystic ovaries had BMD values comparable to those of eumenorrhoeic controls despite their menstrual dysfunction.