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ObjectiveTo identify the differential expressed proteins,and to investigate the relationship between altered expression of annexin A4 during window of implantation [ WOI ( at day-6 after ovulatory day )] in infertile patients with endometriosis and endometrial receptivity.MethodsTwo-dimensional fluorescence differential in-gel electrophoresis (2D-DIGE) and matrix-assist laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were used to detect protein expression in endometrial WOI in 10 infertile cases with endometriosis as endometriosis group and 10 infertile cases with tubal factors as control group.The semi-quantitative validation of annexin A4 in the eutopic endometrial tissue during WOI was analyzed by western blot.Results By comparing protein profiles,there were 7 meaningful differential proteins during WOI in infertile patients with endometriosis.One protein with an isoelectric point of 5.84 and relative molecular weight of 36 100 were down regulated 348％ in samples of endometriosis group.It was identified as annexin A4 by mass spectrometry.By western blot,relative intensity of annexin A4 in endometriosis group was 7.2 ±0.9,which was lower than 17.8 ± 2.6 in control group significantly (t =7.654,P =0.002 ).ConclusionLower expresssion of annexin A4 during WOI in infertile patients with endometriosis might be associated with the decrease of endometrial receptivity.
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OBJECTIVE@#To investigate the role of focal adhesion kinase (FAK) in extracellular signal-regulated kinase (ERK) signaling pathway mediated invadsion of trophoblasts.@*METHODS@#We established a human extravillous cytotrophoblasts in vitro invasion model. Different concentrations of herbimycin A(FAK inhibitor)and PD98059 (ERK inhibitor) were given to observe the influence on the growth of trophoblast cells, FAK, ERK phosphorylation, and trophoblast invasion abilities.@*RESULTS@#The expression of phosphorylated FAK in the extravillous cytotrophoblasts (EVCT) was inhibited by herbimycin A in a concentration-dependent manner and expression of phosphorylated ERK1/2 was also partially reduced. PD98059 had no effect on the expression of phosphorylated FAK. Herbimycin A and PD98059 suppressed the in vitro invasion of EVCT to various degrees.@*CONCLUSION@#ERK signaling pathway may be the common pathway for many invasive signals,and play a key role in the regulation of trophoblast invasion.
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Humans , Benzoquinones , Pharmacology , Cell Division , Physiology , Cell Movement , Physiology , Extracellular Signal-Regulated MAP Kinases , Metabolism , Flavonoids , Pharmacology , Focal Adhesion Protein-Tyrosine Kinases , Metabolism , Lactams, Macrocyclic , Pharmacology , Phosphorylation , Rifabutin , Signal Transduction , Physiology , Trophoblasts , Cell Biology , PhysiologyABSTRACT
Objective To investigate the value of ratio of luteinizing hormone (LH) to folliclestimulating hormone (FSH) in diagnosis of polycystic ovarian syndrome (PCOS) among women with ploycystic ovary (PCO) and to compare the difference of the diagnostic criteria between the Rotterdam Consensus and the Committee for Reproductive and Endocrine in Japan Society of Obstetrics and Gynecology.Methods By means of transvaginal Doppler ultrasound, 195 women with PCO were diagnosed in Nanfang Hospital of Reproductive Medicine Center and compare difference of multiple clinical indexes according to Rotterdam consensus and Japan consensus respectively. In the mean time, the ratio of LH/FSH, the level of LH, testosterone (T) and recevier operating characteristic (ROC) curve were explored to on the value of diagnosis of PCOS. Results By Rotterdam consensus, 144 women were diagnosed with PCOS and 51 women were non-PCOS, while 111 were identified as PCOS and 84 were non-PCOS according to Japan consensus. LH/FSH in PCOS and non-PCOS were 1.59 ±0. 84 and 0. 85 ±0. 47 respectively when based on Rotterdam consensus, and this ratio were 1.87 ± 0. 76 in PCOS and 0. 78 ± 0. 39 in non-PCOS based on Japan consensus. When using LH/FSH to diagnosis PCOS by Rotterdam consensus and Japan consensus,areas under ROC curve are 0. 786 and 0. 942, respectively. Conclusions The ratio of LH/FSH ≥ 1 provide the significant value in the diagnosis of PCOS. The criteria of the Committee for Reproductive and Endocrine in Japan Society of Obstetrics and Gynecology is more suitable for Chinese patients.
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Objective To establish an animal model of PCOS and to detect the DNA methylation states of LHR, INSR and AR genes. Methods 24-days-old female Sprague-Dawley (SD) rats were randomly divided into two groups. The rats in the experimental group were given subcutaneous implanting of levonorgestrel silica gel staff and injected 1.5 IU hCG twice daily for 9 days from the 4th day. The rats in the control group were injected with normal saline at the same time. Ovarian morphologic changes, sex hormone levels, fasting serum insulin and glucose were detected. The LHR, INSR and AR genes' DNA methylation patterns were checked by methylation specific PCR in modeling group and control group. Results The ovarian weight and volume in modeling group were higher than those in control group. The ovaries in modeling group showed multiple follicular cysts, and the number of theca cells and interstitial cells increased. Less developed follicles and corpus lutea were seem. The serum level of progesterone, testosterone, luteinizing hormone, fasting insulin and glucose were significantly higher in experimental group than those in control group, so as the LH/FSH ratio and HOMA index. No methylation of LHR and AR genes were found in both groups. The methylation frequency of INSR gene (76.7%) was significantly higher in modeling group than that in control group (P<0. 001). Conclusion The depression of INSR gene's transcriptional induced by DNA methylation is important in the development of insulin resistance in PCOS.
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Objective To establish an animal model of PCOS and to detect the DNA methylation states of LHR,INSR and AR genes. Methods 24-days-old female Sprague-Dawley (SD) rats were randomly divided into two groups. The rats in the experimental group were given subcutaneous implanting of levonorgestrel silica gel staff and injected 1.5 IU hCG twice daily for 9 days from the 4th day. The rats in the control group were injected with normal saline at the same time. Ovarian morphologic changes,sex hormone levels,fasting serum insulin and glucose were detected. The LHR,INSR and AR genes' DNA methylation patterns were checked by methylation specific PCR in modeling group and control group.Results The ovarian weight and volume in modeling group were higher than those in control group. The ovaries in modeling group showed multiple follicular cysts,and the number of theca cells and interstitial cells increased. Less developed follicles and corpus lutea were seem. The serum level of progesterone,testosterone,luteinizing hormone,fasting insulin and glucose were significantly higher in experimental group than those in control group,so as the LH/FSH ratio and HOMA index. No methylation of LHR and AR genes were found in both groups. The methylation frequency of INSR gene (76.7%) was significantly higher in modelinggroup than that in control group (P
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8 oocytes) according to the number of oocytes collected in one ovary. On day of human chorionic gonadotrophin injection, PSV, EDV, PI, RI, S/D of ovarian stromal artery in the ovarian were detected and perifollicular vascularity were graded with color Doppler ultrasonography. Results Not only PSV and EDV of stromal artery but also perifollicular vascularity in low-response group was significantly lower than that of normal-response and high-response groups. PSV and EDV of ovarian stromal artery and perifollicular vascularity were highly interrelated with ovarian response. Conclusions The increase of PSV and EDV of ovarian stromal artery and perifollicular vascilarity indicate the improvement of perfusion in ovary and ovarian response.
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Objective To observe the role of calcitonin (CT) during the implantation. Methods Human endometrial epithelial cells were cultured. After stimulated with various concentrations of CT, intracellular calcium(Ca 2+)in the epithelial and stroma cells and pre-embryos were measured by the laser scanning confocal microscope. Resutls When stimulated with different concentrations of CT, mean fluorescence levels in the epithelial cells were similar to that of the control. However CT can improve intracellar Ca 2+ of preimplantation embryos and stroma cells in a dose-dependent manner and significantly higher than those of controls. When 10 nmol/L CT was added to the culture medium, the intracellular Ca 2+ concentration in 8-cell embryos rose immediately. Embryo exposure to CT was followed by a series of Ca 2+ bursts that persisted for at least 2 hours. No change in Ca 2+ was observed when culture medium alone was added to the embryos. Pre-loading embryos and stoma cells with the Ca 2+ chelator,prevented the increased fluorescence after CT addition. Conclusions CT play an important role during the procesess of implantation. It maybe improve intracellar of preembryos and accerate the development of preembryos.
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Objective To study the expression of HOXA10 gene in the endometrium of normal fertile women and patients with unexplained infertility during different phases of menstrual cycle. Methods Endometrium samples were obtained by curettage in 52 normal fertile women and 38 patients with unexplained infertility during different phases of menstrual cycle, HOXA10 mRNA expression were detected by in situ hybridization and reverse polymerase chain reaction (RT-PCR). Results (1) HOXA10 mRNA were detected in the glandular and stromal cells of endometrium of fertile women during the menstrual cycle. By in situ hybridization (positive unite, PU), HOXA10 mRNA levels were significantly higher in the mid-secretory phase [glandular cells (G) 5.69?0.57,stromal cells (S) 7.48?0.67] and late-secretory phase(G 5.99?0.40,S 7.98?1.08) than those in late proliferative phase (G 3.35?0.20,S 3.20?0.37) and early secretory phase (G 3.07?0.26,S 3.18?0.27)(P
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Objective:To explore whether human chorioic gonadotrophin(hCG) might change the invasiveness of trophoblast by regulating the prodution of TGF-?3. Methods: The effect of hCG on TGF-?3 mRNA expression in JEG-3 cells was investigated by employing the semi-quantiative method of reverse transcription polymerase chain reaction(RT-TCR). Results:TGF-?3 was shown to be expressed in JEG-3 cells. After incubated with 0,50,500,5 000,25 000 mU/ml hCG for 48 hours respectively, the expression of TGF-?3 in JEG-3 cells increased gradually with the concentration of hCG increased. Additionally,TGF-?3 mRNA expression in JEG-3 cells incubated with 25 000 mU/ml hCG experienced a significant induction at 30 h point which was followed by a less significant decrease.Conclusion:The autocrine of TGF-?3 might be involved in the effect of hCG on trophoblast or trophoblast-derived choriocarcinoma cell invasiveness.
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Objective To perfect gene profile expressed in pre-implantation embryos. Methods Using nested RT-PCR to investigate the expression of seven imprinted genes: P57~ KIP2, LIT1, TSSC3, GRB10, PEG3, ARHI, and ZAC1 in human oocytes and pre-implantation embryos. Results Transcripts of P57~ KIP2 and ZAC1 were detected in human oocytes and at all stages of pre-implantation; LIT1 was expressed only in stages of 8-cell and blastocyst; transcripts of TSSC3 could not be detected; GRB10 mRNA could be detected in oocytes and pre-implantation embryos except for 2-cell embryo; ARHI was expressed in oocytes and 2,8-cell embryos and blastocyst; Peg3 mRNA existed in 4,8-cell embryos and blastocyst. Conclusion Except for TSSC3, transcripts of the other six imprinted genes are detected in human pre-implantation development, which are helpful for pre-implantation diagnosis of imprinted diseases, and provide the theoretical basis for understanding the correlation among assisted reproductive technology, genetic imprinted diseases and tumor.
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To show the protective effect of macrophage colony-stimulating factor (M-CSF) on macrophages under oxidative stress,we investigated the effect of M-CSF on RAW264.7 cells incubated with tert-butyl hydroperoxide (tbOOH) using L929 cell conditioned medium (L929-CM) as the source of M-CSF.The results showed that M-CSF could alleviate the tbOOH- induced oxidative injury to RAW264.7 cells.We also found that M-CSF could improve superoxide dismutase (SOD) activity in the cells.MnSOD mRNA expression was also shown to be increased by RT-PCR technique,and the induction could be blocked by actinomycin D.So,we concluded that M-CSF could protect RAW264.7 cells from oxidative injury through inducing MnSOD expression.
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Objective To investigate the effects of leptin on steroidogenesis of human luteinized granulosa cell in vitro. Methods Human luteinized granulosa cells were isolated from follicular fluid obtained during oocyte retrieval of in vitro fertilization-embryo transfer program and were cultured with M199 medium plus various concentration of leptin (0, 10, 30, 100, 300 ng/ml)，human menopausal gonadotropin (hMG, 0, 0.1, 0.2, 0.5, 1, 2, 5, 10 IU/ml) and testosterone 100 μg/ml. For 2 days the media were collected for estradiol and progesterone measurements. Results Addition of leptin alone did not alter estradiol and progesterone production (P>0.05) by human luteinized granulosa cells. Leptin of 10～30 ng/ml concentrations caused a dose-dependent inhibition of estradiol production (P<0.05) while greater than 0.5 IU/ml of hMG were added. There was no effect of leptin on hMG -stimulated progesterone production (P>0.05). Conclusion Leptin can directly inhibit hMG-stimulated estradiol production by human luteinized granulosa cells in vitro, but has no effect on progesterone production. Leptin may play an important role in follicle development and luteinization.
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Objective To evaluate the function of vascular endothelial growth factor (VEGF) and other clinical indexes to forcast ovarian hyperstimulation syndrome (OHSS). Method Collecting the serum and follicular fluid (FF) of 42 cases in the in vitro fertilization and embryo transfer (IVF-ET) cycles and then analysis the relationship of the clinical and detected indexes between the OHSS (10 cases) and the non-OHSS (32 cases). Results The concentration of VEGF in FF?E 2 on the day of human chorionic gonadotropin (hCG) injection, basal luteinizing hormone (LH) and the number of ovum retried much higher in the OHSS than in the non-OHSS. Conclusions The concentration of VEGF in FF of OHSS cases is higher than that of controls, supporting the role of VEGF as a mediator of OHSS. Therefore VEGF in FF is a forcast index of OHSS.
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AIM:To investigate the influence of F10 on the activities of transcription factor NF-?B and AP1 in A549 cells.METHODS:The luciferase report plasmids of NF-?B-Luc,AP1-Luc and F10 gene were introduced into A549 cells and the luciferase activity was detected.The DNA binding activities of AP1 and NF-?B in the cells were measured by the electrophoretic mobility shift assay(EMSA).RESULTS:The luciferase activity in F10+ transfection group decreased 30% and increased 2-fold respectively 48 h after transfected with the luciferase report plasmid of NF-?B-Luc and AP1-Luc in A549 cells.The DNA binding activity decreased 49% and increased 23%,respectively.CONCLUSION:F10 gene up-regulates the transcription activity of AP1 and down-regulates the NF-?B in A549 cells.
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AIM: To study the change of some metabolism-associated genes expression in preeclamptic placenta. METHODS: The mRNA expression levels of metabolism-associated genes in preeclamptic or normal placenta were detected by employing complementary DNA microarray representing over 220 human hormone-associated genes. RESULTS: Many redox metabolism-related genes (GenBank: U78168, X16699, D32143, AL035079, AF069668, X53463, J03746, X02317, X91247, etc) were highly expressed in preeclamptic placenta compared with normal placenta. Additionally, the mRNA expression levels of some genes which were related to other metabolism such as hormone (GenBank: NM -001718, Z22535, M38180, M76665, X75252, J03258, U56725), were also highly expressed in preeclamptic placenta.CONCLUSION: The change of genes expression in placenta is associated with the change of metabolism in patients sufferring from preeclampsia.
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AIM: To uncover the effect of human chorionic gonadotrophin (hCG) on vascular epithelial growth factor (VEGF) expression in trophoblasts. METHODS: The semi-quantitative method of reversely transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of VEGF in trophoblast-derived JEG-3 cell line. RESULTS: VEGF 121 , VEGF 165 and VEGF 189 was shown to express in JEG-3 cells. After incubated with 0, 50, 500, 5 000 , 25 000 U/L hCG for 48 hours respectively, the expression of VEGF (including its 3 isoforms: VEGF 121 , VEGF 165 , VEGF 189 ) was induced gradually with the concentration of hCG increased. Furthermore, VEGF mRNA expression in JEG-3 cells incubated with 25 000 U/L hCG experienced a transient increase before a less significant decrease. CONCLUSION: The autocrine of VEGF might be involved in the effect of hCG on trophoblast or trophoblast-derived choriocarcinoma cell proliferation, migration and invasiveness.
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Hoxa gene plays an essential role in the generation and development of the human female reproductive system. This gene is expressed in the Mullerian duct and the adult female reproductive system. Expression of Hoxa10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation. Female mice lacking Hoxa10 have a uterine factor defect that results in death of the preimplantation embryo and failure of implantation. These results suggest Hoxa10 may have an important function in implantation. Hoxa10 - deficient males exhibit cryptorchidism that lead to male infertility.
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Objective:To investigate the influences of laparoscopic surgery on Th1/Th2 balance in patients with uterine myomas.Methods:In a prospective study,the number of the Th subsets,Th1/Th2 ratio,the serum level of IL-18 and IL-10 in 20 patients submitted to laparoscopic operation and 20 patients undergoing conventional open operation were evaluated preoperatively as well as 2,24,48 h postoperatively.Results:The level of Th1 cell,IL-18,and the Th1/Th2 ratio decreased significantly (laparoscopic group: P
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Objective To investigate the expression of human leucocyte antigen-G (HLA-G) in the endometrium of patients with endometriosis (EM). Methods The expression of HLA-G protein was detected in ovarian endometriosis (OEM) and adenomyosis (AM) using immunohistochemistry method, and the expression of HLA-G mRNA was detected using in situ hybridization technique (ISH), to compare with that in the endometrium of hysteromyoma as the control group. Results The rate of positive HLA-G expression in EM (including AM and OEM) was significantly higher than that in control group (P0.05). The expression of HLA-G protein showed no relation with different clinical phase or different endometrial cycle. Conclusion HLA-G is overexpressed in EM, which may play certain role in the pathogenesis of the disease.
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Objective To study the expression of ARHI mRNA,an imprinted suppressor gene,in ovarian serous carcinoma and the methylation status of its three CpG islands,and to determine the possible role of aberrant methylation of ARHI gene in pathogenesis of ovarian serous carcinoma.Methods Twenty normal ovarian specimens and 20 ovarian serous carcinoma specimens were analyzed.Total RNA was extracted and semi-quantitative RT-PCR was employed to detect the mRNA expression of ARHI gene.Methylation status of three CpG islands were examined by DNA bisulfite treatment,PCR amplification and methylation specific restriction enzymes,TagI for CpG I and III,BstUI for CpG II.Results The average of ARHI/G3PDH was 0.73?0.09 in normal ovarian specimens,and it was 0.43?0.37 in ovarian carcinoma specimens(P