[Culture and identification of fibroblast from human endometriosis].

OBJECTIVE: To establish the model of cultivating and identifying fibroblast from human endometriosis (HEFC) in vitro. METHODS: The tissues of human endometriotic cysts of ovary were digested by collagenases I, II and IV The resulting cells were purified by centrifugation and differential adhesion. HEFC was identified by observing the morphologic changes under an inverted microscope and the expressions of vimentin, α-SMA (α-smooth muscle actin) and keratin were detected by immunocytochemistry. RESULTS: Immunohistochemical staining of vimentin was positive, α-SMA rarely positive and keratin completely negative in cultured fibroblasts. HEFC grew as a confluent monolayer of short fat fusiform, triangular, star-shaped and polygonal fiber-like cells. Furthermore HEFC could be well sub-cultured. CONCLUSION: Acquired fibroblast can be cultured in vitro stably. It is quite important to study the specificities of HEFC. Sufficient and reliable target cells may be obtained for studying the mechanisms of fibrosis and adhesion in endometriosis at the molecular level.

Prevention of osteoporosis in mice after ovariectomy via allograft of microencapsulated ovarian cells.

It is believed that estrogen deficiency is one of the major risk factors associated with osteoporosis. To investigate the effects of the transplantation of microencapsulated ovarian cells in estrogen-deficient mice, ovarian cells from female Kunming (KM) mice (6-weeks old) were separated, cultured, and microencapsulated with alginic acid-polylysine-alginic acid. Female KM mice (8-weeks old) were randomly separated into three groups: intact (normal), ovariectomized (OVX), and treatment (OVX+ implantation). Microencapsulated ovarian cells were found to secrete estrogen at normal levels in vitro. Ninety days after transplantation, serum estradiol levels in the OVX group were significantly lower, and the trabecular bone amount and volume were decreased when compared with the normal group. The expression of alkaline phosphatase in chondrocytes appeared lower, while the expression of matrix metalloproteinase 9 (MMP-9) in the bone matrix was higher. The ratio of MMP-9-positive chondrocytes and osteoblasts to osteoclasts was significantly lower than that of the normal group. The concentrations of hydroxyproline (Hyp), Ca, and P in the left femurs of the OVX group were lower than those of the normal group. However, the aforementioned changes were not seen in the treatment group. In conclusion, microencapsulated ovarian cells survive well after transplantation and secrete estrogen in vivo, and they can prevent in some degree osteoporosis caused by ovariectomy.

Gonadotropin stimulation as a challenge to calibrate cisplatin induced ovarian damage in the female rat.

Cisplatin administration for treatment of cancer causes damage to the ovaries, potentially leading to ovarian failure. To extend our previous work, we tested the hypothesis that serum anti-Mullerian hormone (AMH) levels after ovarian stimulation can be used as a biomarker to calibrate the degree of ovarian damage following cisplatin. Female rats were injected with two weekly doses of cisplatin followed by an injection of pregnant mare serum gonadotropin to stimulate the ovaries. Sera and ovaries were collected 54 h after gonadotropin challenge. Analysis of serum and ovarian AMH by Western blot and ELISA revealed that increasing doses of cisplatin caused dose-dependent decrease in AMH after gonadotropin stimulation. Immunohistochemical analysis demonstrated that the AMH positive follicles declined in a dose-dependent manner after cisplatin, resulting in AMH levels that are lower after cisplatin exposure. Cisplatin damage to the ovaries can be identified by the serum AMH levels following exogenous ovarian stimulation.

Baseline and stimulated serum inhibin levels as biomarkers of cisplatin-induced ovarian damage in female rats.

OBJECTIVE: Chemotherapeutic agents, such as cisplatin, injure the ovary. It was hypothesized that serum inhibins can serve as biomarkers of ovarian damage from cisplatin. STUDY DESIGN: Adult female rats were administered cisplatin and afterward pregnant mare serum gonadotropin (PMSG) was given to stimulate ovarian inhibin production. RESULTS: At baseline, the serum inhibin A levels in the cisplatin-treated groups were slightly lower than saline. However, after PMSG stimulation, the cisplatin groups had dose-related and significantly blunted serum inhibin A and B responses. Results of Western blot and enzyme-linked immunosorbent assay inhibin analysis of ovarian lysate were consistent with the serum levels. Investigation of the ovaries showed that cisplatin groups at baseline had higher percentages of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive follicles in inhibin positive follicles, suggesting that cisplatin induced apoptosis in the inhibin follicles. CONCLUSION: We conclude that serum inhibin A and B after PMSG ovarian challenge can be used as biomarkers to define rat ovarian damage after exposure to cisplatin.

Serum and ovarian Müllerian inhibiting substance, and their decline in reproductive aging.

The objectives of this study were to identify whether there is a decline in Müllerian inhibiting substance (MIS) in the female rat during chronological aging, and to define the physiological basis of aging-related changes in MIS. The results demonstrate that there is an exponential decline in both serum and ovarian levels of MIS with increasing female age, and that the histologic origin for the reduction in serum levels of MIS is a decline in the number of small ovarian follicles expressing MIS.

Müllerian inhibiting substance as a novel biomarker of cisplatin-induced ovarian damage.

Müllerian inhibiting substance (MIS) has been investigated as a possible serum biomarker in human aging to estimate the number of female germ cells remaining. Cisplatin is an effective chemotherapeutic agent that is associated with ovarian injury. In this study, we tested the hypothesis that decreasing serum MIS can serve as a biomarker of ovarian damage after cisplatin. Adult female rats were treated with saline, 4.5, or 6.0 mg/kg cisplatin. The serum MIS levels were lower in both cisplatin groups, in a dose-related fashion. The ovarian lysates of both cisplatin groups had less MIS than control. Immunofluorescence analysis showed that the percentage of MIS-positive follicles was lower in the 6.0 mg/kg group. TUNEL assays showed that there was a dose related increase in the number of apoptotic follicles in the cisplatin groups. In summary, a decrease in serum MIS could serve as a biomarker to discriminate the degree of ovarian damage after cisplatin. These data are the first to establish in the rat that ovarian injury due to a chemotherapeutic agent could be monitored with the non-invasive serum biomarker MIS.