Objective To detect the expression of telomerase catalytic subunit (TERT),telomerase in human ovarian luteinized granulosa cells and to investigate the relationship between telomerase expression and the ovarian fecundity Methods Ovarian luteinized granulosa cells were recovered from 22 women with regular menses who underwent in vitro fertilization/ intracytoplasmic sperm injection programme.We carried out in situ hybridization histochemistry on luteinized granulosa cells to detect TERT mRNA expression,and telomeric repeat amplification protocol to detect telomerase activity Results TERT mRNA were present in luteinized granulosa cells.The expression of TERT mRNA and telomerase activity in ovarian luteinized granulosa cells decreased with increase of age,basal serum follicle stimulating hormone levels Conclusions These results suggest that telomerase may play an active role in ovarian function.A reduced telomerase activity of granulosa cells may be one of the important mechanisms involved in decreased ovarian function in women.

To evaluate the role of nitric oxide in local function of endometrium, the pattern of expression of endothelial and inducible nitric oxide synthases in the human endometrium at proliferative and secretory phases and decidua was studied by using immunocytochemistry. Results showed: (1) At the proliferative phase, the eNOS immunostaining was confined to vascular endothelial, whereas the iNOS was not detected in any composition of endometrium. (2) At the secretory phase, surface epithelium and grandular epithelium showed positive staining for both eNOS and iNOS. The stroma remained uniformly negative throughout the menstrual cycle. (3) In the decidua, the expression of both isoforms was increased, while moderate iNOS immunoreactivity was observed in decidualized stromal cells. It was demonstrated that the expression of eNOS and iNOS was elevated at the secretory phase and in decidua, indicating the increased production of NO at these phases. The increasing of NO might take part in implantation through dilating the vessels and relaxing the smooth muscles and in menstration through promoting apoptosis.

To evaluate the role of nitric oxide in the embryo implantation in mice, three experiments were carried out using the mice implantation model. In the experimentⅠand experimentⅡ, NG-nitro-L-arginine methyl ester (L-NAME), the non-specific NOS inhibitor, was administered intraperitoneally at doses of 0.5 mg-5 mg on the day 3 of pregnancy and at dose of 3 mg on different days of pregnancy (day 1-6) . In the experiment Ⅲ, L-aragnine, a donor of NO, was co-administered with L-NAME to evaluate the effect of L-aragnine on L-NAME, and the embryo development was assessed. In all these three experiments, the endometrium was histologically examined. Results showed compared with the control groups intraperitoneal administration of a dose of L-NAME between 1 and 5 mg on the day 3 of pregnancy led to the decrease in the number of implantation sites (P<0.05), and 4 to 5 mg of L-NAME caused inhibition of implantation completely. L-NAME resulted in failure of pregnancy when administrated at 3 mg between day 3 and 5 of pregnancy. The characteristic vascular permeability changes and decidualization in the endometrium were significantly attenuated and embryo growth was retarded. The L-NAME-mediated effects were significantly reversed when L-aragnine was co-administered with L-NAME. This study demonstrated that NO promoted the implantation in mice through regulating the permeability and decidulization of endometrium and the development of embryo.

ObjectiveTo develop a method of location of prenatal female mouse reproductive tract based on paraffin block serial sections of female fetuses, and get high quality paraffin sections of fetuses containing female reproductive tract (FRT).And to investigate the role of Wnt5a during the development of FRT of prenatal mice. Methods The sex of fetuses at gravidity 15.5 days( G15.5d), G17.5d, G19.5d) and G21.5d was identified by polymerase chain reaction(PCR),and the female fetuses were embedded in paraffin block, the specimen was sectioned serially . One of every four sections was stained by hematoxylin-eosin(HE), and the next one being used later.The images of specimen were taken with digital camera and the serial sections were obtained. The paramesonephric duct /uterus were located and recognized. Immunohistochemistry was used to determine the location and intensity of Wnt5a staining in the middle of the paramesonephric duct / uterus. Results The morphology of the paramesonephric duct /uterus was recognized first and the sections of fetuses with paramesonephric ducts were obtained. The intensity of Wnt5a immunohistochemical staining was increased from G15.5d to G21.5d in mesenchymal cells(P<0.01).Conclusion Wnt5a plays a marked role in early period of female mouse reproductive tract, and is possible to be a key factor to induce uterus differentiation and development.

Objective To evaluate the effects of biopsy methods, biopsy timing and the number of cell removed on in vitro development of embryos Methods One hundred and fifty four embryos of good morphology from in vitro fertilization patients were studied Sixty six embryos were allocated to the following three groups: chemical drilling biopsy group (26), mechanical drilling biopsy group (26) and control group (20) One cell was removed from the embryos of the two biopsy groups The remaining 88 embryos were allocated to two groups: biopsy group (44) and control group (44) Two cells were removed from biopsy group by chemical drilling method The stage of the embryo before biopsy, biopsy time, lysed blastomere, growth potential and hatching capacity of the biopsied embryos, total cell number at the blastocyst stage were recorded and evaluated Results The mean time of biopsy in the chemical drilling group (231?20) seconds was significantly shorter than that in the mechanical drilling group (262?23) seconds ( P 0 05) However, the proportion developing to the blastocyst stage was reduced after the removal of two cells from the 6 cell stage in comparison to the control (1/8 versus 5/8, P

To determine the (tttta)n repeat polymorphisms at the promoter region of CYP11alpha gene, and study its linkage to hyperandrogenism of polycystic ovary syndrome (PCOS) in Chinese women, a case-control study was conducted in the Reproductive Medical Center of the Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). 96 PCOS patients and 78 healthy control women were included. CYP11alpha (tttta)n repeat-polymorphism genotyping analysis was performed by using polymerase chain reaction (PCR). Serum pituitary hormone and total testosterone levels were measured by ELISA. 4 different CYP11alpha (tttta)n allelles were identified, corresponding to 4-, 6-, 8-, and 9-repeat-unit alleles. The frequency and distribution of these alleles are 0.16, 0.33, 0.38, and 0.13 respectively in PCOS patients, as compared with 0.20, 0.34, 0.35, and 0.11 respectively in healthy controls. There were no significant differences between these two groups. Moreover, no correlation between the polymorphism of CYP11alpha gene and serum testosterone level of patients with PCOS and controls was observed. It is concluded that microsatellite polymorphism (tttta)n of gene CYP11alpha exists in Chinese women and the polymorphism of CYP11alpha gene does not play an important role in the pathogenesis of Chinese patients with PCOS, especially in patients with hyperandrogenism.
Cholesterol Side-Chain Cleavage Enzyme/*genetics ; Hyperandrogenism/complications ; Hyperandrogenism/*genetics ; Microsatellite Repeats ; Polycystic Ovary Syndrome/complications ; Polycystic Ovary Syndrome/*genetics ; *Polymorphism, Genetic/genetics

This study examined the expression and distribution of angiopoietin-1/-2 (Ang-1/-2) in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohisto-chemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups: control group, D2 group (2 days after pregnancy), D4 group (4 days after pregnancy), D6 group (6 days after pregnancy) and D8 group (8 days after pregnancy), each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups (P<0.01). Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy (day 2), and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice (P<0.01). In situ hybridization showed Ang-1 mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 (P<0.01). It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.

In order to study the angiogenesis in endometriosis, the samples of eutopic and ectopic endometria from patients with endometriosis were quantitatively analyzed by color morphometric image system (CMIS) for vascular surface area, and by examining endometrial blood vessel for microvessel density (MVD). The results showed that within each menstrual phase the vascular surface area and MVD were significantly higher in ectopic endometria with endometriosis than those in eutopic endometria with endometriosis or normal endometrium (P < 0.05). It is concluded that angiogenesis might be involved in the development of endometriosis.
Endometriosis/etiology ; Endometriosis/*pathology ; Endometrium/*blood supply ; Menstrual Cycle ; *Neovascularization, Pathologic ; *Pelvis

HOXA10 gene plays an essential role in differentiation of the endometrium and in human reproduction. The aim of this study was to investigate the regulatory effect of sex steroids and HB-EGF on HOXA10 gene in Ishikawa cells. Ishikawa cells were incubated with 17-beta estradiol (10(-8) mol/L), medroxyprogesterone acetate (MPA) (10(-6) mol/L), RU486 (10(-5) mol/L) or HB-EGF (10 ng/mL) for 48 h respectively. The expression of HOXA10 gene was detected by immunofluorescence, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Our results showed that either estrogen alone, progestin alone or progestin combined with estrogen could significantly increase the expression of HOXA10 gene 48 h after the treatment (P<0.05). But estrogen combined with progestin and RU486 could inhibit the up-regulation by estrogen and progestin. HB-EGF could elevate the expression of HOXA10 gene 48 h after the treatment (P<0.05). It is concluded that both estrogen and progestin can up-regulate the expression of HOXA10 gene in Ishikawa cells, but RU486 can inhibit the effect and HB-EGF can elevate the expression level of HOXA10 gene.
Cell Line, Tumor ; Endometrial Neoplasms/*pathology ; Estradiol/*pharmacology ; Genes, Homeobox ; Homeodomain Proteins/genetics ; Homeodomain Proteins/*metabolism ; Intercellular Signaling Peptides and Proteins/*pharmacology ; Medroxyprogesterone Acetate/*pharmacology ; RNA, Messenger/genetics ; RNA, Messenger/metabolism

ObjectiveTo study microRNA (miRNA) expression and role of cell cycle regulation in decidualized endometrial stormal cells (ESC) in vitro.MethodsESC was induced decasualization in vitro and matched with non-decidualized cells as controls.The expression repertoire of miRNA was measured by microarray chip and was validated by real-time PCR.Flow cytometry was used to identify ESC cycle during decidual reaction in vitro and after miRNA222 inhibitor was transfected into it.Results (1) Between decidualized and undecidualized stromal cells,there were 49 miRNAs significantly different expression by microarray chip,including 16 miRNA up-regulation and 33 miRNA down-regulation.hsa-miR-27b,30c,143,101,181 b,29b,30d,507,23 a,222,221 exhibited significantly differential expression between decicualized and undecidualized stromal cells by real-time PCR (P ＜0.05).(2) After miRNA222 inhibitor (NC-FAM) transfection to decidual ESC,ESC were cultured by FBS medium for 24 hours,the rate of transfection was 70％.ESC were transfected with miRNA 222 inhibitor and cultured for 48 hours,the percentage of ESC at Sphase of (6.2 ± 0.7 ) ％ were significantly lower than ( 10.9 ± 0.8 ) ％ in control group ( P ＜ 0.05 ) ; the percentage of ESC at G0/G1 phase increased at transfection group [ (77.5 ± 1.3 ) ％ vs.(73.0 ± 1.6) ％ at control group ],but there was no significant difference (P ＞ 0.05 ).Decasualization ESC were transfected with miRNA 222 inhibitor and cultured for 48 h,the percentage of ESC at S-phase was ( 3.3 ± 0.6) ％ in transfection group,which were significantly lower than (7.8 ± 0.9 ) ％ in control group ( P ＜ 0.05 ).The percentage of ESC at G0/G1 phase was ( 80.7 ± 1.6 ) ％ in transfection group and ( 74.9 ± 1.1 ) ％.In control group,which did not reached statistical difference ( P ＞ 0.05).ConclusionmiRNA was involved in ESC decidual process in vitro by regulating cell cycle.