Synergistic effects of electroacupuncture and bone marrow mesenchymal stem cell transplantation on rat intrauterine adhesion： an observation. / 电针联合骨髓间å è´¨å¹²ç»†èƒžç§»æ¤æ²»ç–—å¤§é¼ å®«è ”ç²˜è¿žçš„ååŒæ•ˆåº”è§‚å¯Ÿ.

OBJECTIVES: To investigate the effects of electroacupuncture(EA) combined with bone marrow mesen-chymal stem cells(BMSCs) transplantation on the endometrium of rats with intrauterine adhesions(IUA), so as to explore the possible mechanisms underlying their combined therapeutic effects. METHODS: Forty adult female SD rats were randomly divided into control, model, cell, and combined groups. The IUA rat model was established using a dual injury method of mechanical scratching and lipopolysaccharide infection. After successful modeling, on days 1, 3, and 7, rats in the model group received tail vein injection of phosphate buffered solution, while rats in the cell group received tail vein injection of BMSCs suspension for BMSCs transplantation；and rats in the combined group received BMSCs transplantation combined with EA treatment (2 Hz/15 Hz, 1-2 mA), targeting the "Guanyuan"(CV4), bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6) for 20 min daily for 3 consecutive estrous cycles. After intervention, uterine tissue was collected from 5 rats in each group. Histological analysis was performed using hematoxylin and eosin staining to evaluate endometrial thickness and glandular number. Masson staining was used to assess endometrial fibrosis area. Immunohistochemistry was performed to detect the positive expressions of vascular endothelial growth factor(VEGF), proliferating cell nuclear antigen(PCNA), and estrogen receptor(ER). Western blot analysis was conducted to determine the protein expressions of homeobox A10(HoxA10) and leukemia inhibitory factor(LIF), both key regulators of endometrial receptivity. The remaining 5 rats in each group were co-housed with male rats, and the uterine function recovery was evaluated by assessing the number of embryo implantations. RESULTS: Compared with the control group, the model group showed thinning endometrium(P<0.001), decreased glandular number(P<0.001), increased endometrial fibrosis area(P<0.001), reduced positive expressions of VEGF, PCNA, ER, expressions of HoxA10 and LIF, and decreased embryo implantation number (P<0.001) on the injured side of the uterus. Compared with the model group, the combined group showed a reversal of the aforementioned indicators(P<0.001, P<0.01)；the cell group exhibited thicker endometrium(P<0.001) and reduced endometrial fibrosis area(P<0.001). Compared with the cell group, the combined group showed increased endometrial thickness(P<0.01), elevated glandular number(P<0.05), significantly decreased endometrial fibrosis area(P<0.05), enhanced positive expressions of VEGF, PCNA and ER, expressions of HoxA10 and LIF in the endometrium, and a significant increase in embryo implantation number (P<0.001, P<0.05, P<0.01) on the injured side of the uterus, indicating better results than the cell group. CONCLUSIONS: The combination of EA and BMSCs synergistically promotes the repair of damaged endometrium, improves endometrial morphology, reduces fibrosis levels, enhances vascular regeneration and matrix cell proliferation, improves endometrial receptivity, which ultimately facilitates embryo implantation.

Suppression of migratory/invasive ability and induction of apoptosis in adenomyosis-derived mesenchymal stem cells by cyclooxygenase-2 inhibitors.

OBJECTIVE: To investigate if stem or progenitor cells are found in adenomyosis and to characterize the role of cyclooxygenase-2 (COX-2) in adenomyosis-derived mesenchymal stem cell (AMSC)-related pathogenesis of adenomyosis. DESIGN: Experimental clinical study. SETTING: University hospital. PATIENT(S): Ten patients with adenomyosis. INTERVENTION(S): Hysterectomy. MAIN OUTCOME MEASURE(S): The gene expression of AMSCs and endometrial mesenchymal stem cells (EMSCs) were analyzed by microarray, quantitative polymerase chain reaction and Western blot. Methylthiazol tetrazolium, proliferation, apoptosis, and migration/invasion assays of AMSCs and EMSCs were evaluated after COX-2 inhibitor treatment. RESULT(S): We isolated nine EMSCs from normal endometrium (n=10) and six AMSCs (n=10) from adenomyosis. The morphology, phenotype, and potential of multilineage differentiation between EMSCs and AMSCs were not significantly different. Using complementary DNA microarrays, the expression profiles of EMSCs are related to those of bone marrow-derived mesenchymal stem cells (BMSCs), but AMSCs are different from EMSCs and BMSCs in the gene profiles. We validated the microarray results and showed that there is increased COX-2 expression in AMSCs compared with EMSCs. Treatment with a COX-2 inhibitor suppressed migration and invasion and induced apoptotic capabilities of AMSCs, but not of EMSCs. CONCLUSION(S): Overexpression of COX-2 in AMSCs may play an important role in the pathogenesis of adenomyosis. COX-2 could be a possible target for treatment and prevention of adenomyosis.

The growth hormone-releasing hormone (GHRH) antagonist JV-1-36 inhibits proliferation and survival of human ectopic endometriotic stromal cells (ESCs) and the T HESC cell line.

OBJECTIVE: To determine the effect of the GHRH antagonist JV-1-36 on proliferation and survival of primary ectopic human endometriotic stromal cells (ESCs) and the T HESC cell line. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENT(S): 22 women with endometriosis (aged 34.8+/-5.7 years) undergoing therapeutic laparoscopy. INTERVENTION(S): Eutopic (n=10) and ectopic (n=22) endometrial tissues were collected from women who underwent therapeutic laparoscopic surgery for endometriosis (stage III/IV). MAIN OUTCOME MEASURE(S): Expression of GHRH, GHRH receptor (GHRH-R) and GHRH-R splice variant (SV) 1 mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR). The ESC proliferation was assessed by 5-bromo-2-deoxyuridine incorporation, cell survival by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Trypan blue assay. The T HESC survival was evaluated by MTT, cyclic adenosine monophosphate (cAMP) levels by ELISA, extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation by Western blot, and insulin-like growth factor (IGF)-2 mRNA by real-time PCR. RESULT(S): The ESCs and T HESCs, but not normal endometrial tissues, expressed GHRH-R mRNA; SV1 mRNA was determined in normal endometrial tissues, ESCs, and T HESCs; GHRH mRNAwas found in T HESCs; JV-1-36 inhibited ESC proliferation and ESC and T HESC survival. In T HESCs, JV-1-36 reduced cAMP production and ERK1/2 phosphorylation but had no effect on IGF-2 mRNA expression. CONCLUSION(S): The GHRH antagonist JV-1-36 inhibits endometriotic cell proliferation and survival, suggesting that GHRH antagonist may represent promising tools for treatment of endometriosis.

Effects of the special extract ERr 731 from Rheum rhaponticum on estrogen-regulated targets in the uterotrophy model of ovariectomized rats.

A recent clinical study with a two-year application of the extract ERr 731 from Rheum rhaponticum demonstrated its efficacy and potentially suggested it safety regarding unwanted endometrial side effects. The aim of the present study is to provide experimental proof for the latter observation in a preclinical experimental animal model by assessing dose-dependent effects of ERr 731 - either alone or in combination with estradiol (E2) - on growth and proliferation in the uterus of ovariectomized (ovx) rats. ERr 731 was given in a dose corresponding to human therapeutic application and additionally in three pharmacologically relevant doses. In addition to uterine wet weight, this study examines the effects of ERr 731 on the uterine mRNA expression of the proliferation marker Ki67, proliferating cell nuclear antigen (PCNA), insulin-like growth factor-1 (IGF-1), type 1 IGF receptor (IGF-1R), the two estrogen receptor (ER) subtypes alpha and beta (ERalpha and ERbeta) and the estrogen-responsive gene complement C3 (C3). ERr 731 did neither stimulate an uterotrophic response in the uterotrophic assay with ovx rats nor stimulate or modulate the expression of genes associated with proliferation. In combination with E2, ERr 731 reduced the E2-induced uterine growth stimulation. These observations were further substantiated by the expression pattern of genes related to proliferation control, in view of the fact that the E2-induced elevation of Ki67 mRNA and PCNA protein levels in the uterus were counteracted by simultaneous treatment of the animals with ERr 731. In conclusion, the experimental findings presented here provide further evidence for the safety of ERr 731 towards unwanted uterine and endometrial proliferative events in response to ERr 731 and support observations from recent clinical trials.

Effects of Yiweining Recipe on expressions of metalloproteinase-2 and cyclooxygenase-2 mRNAs in ectopic endometrium of rats with endometriosis.

OBJECTIVE: To explore the effects of Yiweining Recipe (YWNR), a compound Chinese herbal medicine, on expressions of metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) mRNAs in rats with endometriosis (EM). METHODS: Operational self-transplantation was applied in establishing the rat models. Detection of MMP-2 and COX-2 mRNAs was conducted with hybridization in situ. RESULTS: There were significant differences in the expressions of MMP-2 and COX-2 mRNAs between the untreated group and the high-dose YWNR-treated group. YWNR could reduce the expressions of MMP-2 and COX-2 mRNAs. CONCLUSION: YWNR can treat EM through reducing the positive expressions of MMP-2 and COX-2 mRNAs.

Expression of leptin receptor in human endometrium and fluctuation during the menstrual cycle.

Leptin is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors (OB-R). Accumulated evidence shows that leptin is involved in the stimulation of reproductive functions and that local expression of leptin and OB-R in the ovary, oocyte, embryo, and placenta plays a role in early development. To investigate the role of leptin in implantation, we examined the expression of OB-R and leptin in the human endometrium. Northern and Western blot analyses and RT-PCR showed that the long form of OB-R (OB-R(L)) messenger ribonucleic acid (mRNA) and protein were expressed. In contrast, leptin mRNA or protein was not detected. All of the splice variants of OB-R (OB-R(T)) and OB-R(L) transcripts were expressed in 90% and 84% of the cases, respectively. OB-R mRNA expression peaked in the early secretory phase. Decidual tissue of early gestation also expressed OB-R(T) and OB-R(L). Their incidence and abundance were comparable among endometria with benign uterine diseases and disease-free endometria and were not related to a body mass index within the normal range. The present results indicate that OB-R, but not leptin, is expressed in the human endometrium.

Endocrine defects in mice carrying a null mutation for the progesterone receptor gene.

Mice carrying a null mutation of the progesterone receptor gene exhibit several reproductive abnormalities, including anovulation, attenuated lordotic behavior, uterine hyperplasia, and lack of mammary gland development. The hormonal correlates of these abnormalities are unknown, however, and were the focus of these studies. Serum samples from female wild-type (WT) and progesterone receptor knockout (PRKO) mice were obtained and analyzed by RIA for LH, FSH, PRL, estrogen (E2), and progesterone. Hypothalamic tissues were also processed for measurement of LHRH by RIA. Serum LH levels in PRKO mice were found to be elevated by approximately 2-fold over basal (metestrus) values in WT mice. By contrast, basal FSH levels were not different in PRKO and WT mice. Basal levels of E2 and progesterone in serum were likewise similar in the two groups, as were hypothalamic LHRH concentrations. Basal PRL levels were slightly higher in PRKO vs. WT mice. Ovariectomy of both groups of mice was accompanied by significant increases in both LH and FSH. At 5 days following ovariectomy, LH levels were elevated in both groups by 2-fold over PRKO basal and 4-fold over WT basal levels; however, by 10 days postovariectomy LH levels had continued to rise to a greater extent in PRKO mice than in WT animals. The FSH response to ovariectomy was greater for the PRKO mice at 5 days, but was no different from WT at 10 days. Of seven PRKO mice that were exposed to male odor, none exhibited preovulatory surges 3 days later, on the day of presumptive proestrus; this was in marked contrast with WT females, in which 100% exhibited robust LH surges. These results confirm the essential role of progesterone receptors in the regulation of hypothalamic and/or pituitary processes that govern gonadotropin secretion. The finding that basal LH levels are elevated in PRKO mice confirms that circulating progesterone normally conveys a significant portion of the total ovarian negative feedback control of the gonadotropin. That gonadotropin responses to ovariectomy are slightly enhanced in PRKO mice suggests that adrenal progesterone may contribute to the imposition of negative feedback control. The apparent inability of PRKO mice to respond to male odor suggests that anovulation in these mice may not be solely due to reproductive abnormalities within the ovary itself; rather, PRKO mice additionally harbor neuroendocrine defects that render them incapable of mounting normal preovulatory gonadotropin surges. It remains to be determined how the absence of PR in brain and pituitary of PRKO mice may produce this hormonal acyclicity and, conversely, how the presence of PR in brain and pituitary of WT mice may be obligatory in the generation of gonadotropin surges.