Objective: To investigate the factors related to clinical pregnancy outcomes of in vitro fertilization-embryo transfer(IVF-ET)in women with secondary infertility. Methods: The clinical, laboratory and follow-up data of 1129 cycles in 1099 patients with secondary infertility undergoing IVF-ET in Women's Hospital, Zhejiang University School of Medicine between July 2012 to July 2014 were retrospectively reviewed. The factors related to pregnancy outcomes were analyzed by univariate and logistic regression methods. The clinical pregnancy rates in women with different age and different number of embryos transferred were compared. The clinical outcomes of stimulation with gonadotropin releasing hormone(GnRH)agonist long protocol,GnRH agonist short protocol and GnH antagonist protocol were evaluated in secondary infertile patients aged≥40 years. Results: Among 1129 cycles, 376 cases(33.30%) had clinical pregnancy and 753 cases(66.70%)had no clinical pregnancy. There were significant differences in age, body mass index, basal follicle-stimulating hormone level, antral follicle number,paternal age and number of embryos transferred between pregnancy and no pregnancy groups(P<0.05); while only maternal age(OR=0.900,95%CI:0.873~0.928,P<0.001)and the number of embryos transferred(OR=2.248,95%CI:1.906~2.652,P<0.001)were the independent factors affecting the clinical pregnancy outcome of IVF-ET. There was no significant difference in clinical pregnancy rate between women aged 30~40 years with two embryos transferred and those aged<30 years with two or three embryos transferred(P>0.05). There were no significances in clinical pregnancy rate among women aged≥40 years using GnRH agonist long protocol,GnRH agonist short protocol and GnRH antagonist protocol for stimulation(P>0.05). Conclusion: Maternal age and number of embryos transferred have independent effect on IVF-ET clinical pregnancy outcome of secondary infertile women. We suggest that no more than two embryos should be transferred for women in their thirties to minimize the risk of multiple pregnancy while still having an acceptable pregnancy rate. The pregnancy rate of patients over 40 years decreases significantly, and there is no difference in pregnancy rate by using GnRH agonist long protocol,GnRH agonist short protocol or GnRH antagonist protocol.

Objective: To investigate the risk factors of pregnancy termination at second and third trimester in women with scarred uterus and placenta previa.Methods: Clinical data of 24 pregnant women of second and third trimester with a scarred uterus and placenta previa,who requested termination in Women's Hospital Zhejiang University School of Medicine from July 2009 to June 2014, were retrospectively analyzed. The method of mifepristone combined with ethacridine lactate was adopted for all cases. Mifepristone combined with ethacridine lactate and uterine artery embolization were routinely given for patients with complete placenta previa. Cesarean section was performed for patients who failed to delivery or underwent massive vaginal bleeding before delivery. Age, gestational weeks, gravidity and parity, times of previous cesarean section, the interval from previous operation, the position and the type of placenta previa, placenta accretet, the indication and method of termination, postpartum hemorrhage, successful rate of labor induction, placental retention ratio and uterus rupture were documented. Results: The successful rate of labor induction was 83.3%. The analysis showed that age, gestational weeks, gravidity and parity and times of previous cesarean section were not risk factors for failed labor induction, however the interval time from previous operation was related to induction failure (P<0.05). Patients with previous cesarean section ≥13 years were more likely to require cesarean section than those <13 years (P<0.05). The placenta adhered to the antetheca of the uterus or placenta accrete increased risk to have cesarean section. There were no significant differences in postpartum hemorrhage, the successful rate of labor induction, placental retention ratio and the rate of uterine rupture between patients with uterine artery embolization and those without. Conclusion: The labor induction would be feasible for women with a scarred uterus and placenta previa in second and third-trimester pregnancy. The previous operation ≥13 years, the antetheca placenta or placenta accrete might increase the incidence of labor induction, while the uterine artery embolization would rise the successful rate of labor induction.

Objective: To analyze the maternal outcomes of pregnant women with pernicious placenta previa(PPP). Methods: Clinical data of 470 patients with placenta previa admitted in Women's Hospital Zhejiang University School of Medicine from August 2012 to August 2014 were collected and retrospectively analyzed. The patients were divided into pernicious group(n=101) and non-pernicious group(n=369) according to the history of cesarean section and location of placenta attached to the uterine. The general profiles, maternal outcomes of two groups were compared. Results: The age, gravidity and rate of recurrent cavity surgery of pernicious group [(32.5±4.1) y, 3.4±1.2, 28.7%] were higher than those of non-pernicious group[(30.7±4.5) y, 2.1±1.4,13.6%](P<0.05). The gestational age of pernicious group was (35.6±2.7) weeks, less than that of non-pernicious group [(36.7±2.7) weeks, P<0.001]. Rate of postpartum massive hemorrhage, rate of blood transfusion, rate of placental implantation and hysterectomy in pernicious and non-pernicious group were 29.7%, 35.6%, 27.7%, 11.9% and 8.1%, 10.8%, 5.7%, 0.8%, respectively(P<0.05). Multiple regression analysis showed that placenta accrete was significantly associated with postpartum massive hemorrhage in pernicious group(P<0.05). Conclusion: The awareness of the danger of pregnant women with PPP before operation and paying more attention to antenatal care are key measures to decrease the adverse maternal outcomes of pregnant women with placenta previa.

Objective: To investigate the risks of recurrent preeclampsia and observe the incidence and long-term prognosis of recurrent preeclampsia. Methods: One hundred and fifteen women with preeclampsia history admitted in Women's Hospital, Zhejiang University School of Medicine from January 2009 to December 2013 were enrolled in the study. The clinical data were retrospectively analyzed. Results: Among 115 women with preeclampsia, 82 cases (71.3%)had recurrent preeclampsia. The onset age, the pregnant interval time, regular prenatal check-up, weight gain during pregnancy, body mass index (BMI), hyperlipidemia, fetal growth restriction(FGR), maternal family history were closely associated with recurrent preeclampsia (P<0.05). Logistic regression analysis showed that the pregnant interval time, BMI, FGR were independent risk factors for preeclampsia recurrence. Compared with the preeclampsia in first pregnancy, the mather had earlier onset and termination of pregnancy, higher blood pressure, higher rate of urine protein≥2+ and higher rate of complications in recurrent preeclampsia. The offspring had higher rate of preterm birth, especially the time of birth for 34 weeks or earlier and lower birth weight (P<0.05). The incidence of chronic hypertension in recurrent preeclampsia group was higher than that in no recurrence group (47.5% vs 23.3%, P<0.05); the overweight rate of first pregnant offspring in recurrence group was higher than that in no recurrence group (31.25% vs 6.70%, P<0.05). Conclusion: The onset age, time interval, regular prenatal check-up, weight gain during pregnancy, BMI, hyperlipidemia, FGR, maternal family history are closely associated with recurrent preeclampsia, and the pregnant interval time, BMI, FGR are independent risk factors for preeclampsia recurrence. Recurrent preeclampsia has more serious clinical outcome and complications, and prevention need to be emphasized.

Objective: To establish a prediction model of fetal meconium-stained amniotic fluid in re-pregnant women with intrahepatic cholestasis of pregnancy(ICP). Methods: Clinical data of 180 re-pregnant women with ICP delivering in Women's Hospital, Zhejiang University School of Medicine between January 2009 to August 2014 were collected. An artificial neural network model(ANN) for risk evaluation of fetal meconium-stained fluid was established and assessed. Results: The sensitivity, specificity and accuracy of ANN for predicting fetal meconium-stained fluid were 68.0%, 85.0% and 80.3%, respectively. The risk factors with effect weight >10% were pregnancy complications, serum cholyglycine level,maternal age. Conclusion: The established ANN model can be used for predicting fetal meconium-stained amniotic fluid in re-pregnant women with ICP.

Objective: To investigate the role of mast cells in the pathogenesis of estrogen-mediated experimental endometriosis in rats. Methods: Endometriosis model was established by transplanting autologous fragments of uterus to the inner surface of the abdominal wall in 24 unpregnant female Sprague Dawley rats. The rats were divided randomly into three groups(n=8 in each group), and were injected with different doses of estrogen: high-dose group(200 μg·kg^-1·d^-1), low-dose group(100 μg·kg^-1·d^-1) and the control group(0 μg·kg^-1·d^-1). The ovaries were surgically removed in high-dose and low-dose groups. Four rats were sacrificed in each group at 2 and 4 weeks after surgery. Their serum estradiol levels, size of lesions, total number of mast cells and degranulations, serum TNF-α levels, expression of tryptase and NGF in tissues were analyzed and compared among groups. Results: The mean levels of serum estradiol 2 weeks and 4 weeks after model established and serum TNF-α at 4 weeks in estrogen-treated groups were significantly higher than those in control group(all P<0.05). The mean size of endometriotic lesions in the estrogen-treated groups was also significantly larger than that in the control group 2 weeks and 4 weeks after model established(all P<0.05). Meanwhile, both at week 2 and week 4, the mean ratio of degranulation/total number of mast cells by toluidine blue staining in low-dose estrogen group was significantly higher than that in the control group(P<0.05). The expression of NGF in high-dose estrogen group was significantly higher than that in the control group at week 4(P<0.05). Conclusion: Estrogen can promote the growth of endometriotic lesions and may mediate the pathogenesis of endometriosis by activating mast cells, which may be associated with increasing TNF-α and NGF levels.

Objective: To investigate the effect of sodium cromoglycate on experimental endometriosis in rats. Methods: Endometriosis model was established in 36 unpregnant female SD rats by transplanting autologous fragments of endometrium to the inner surface of the abdominal wall. The endometriotic lesions were measured by a second laparotomy 2 weeks after surgery. Then the rats were randomly divided into four groups(n=8 in each group) to receive intraperitoneal injection of different doses of sodium cromoglycate for 2 weeks: high-dose group(20 mg·kg^-1·d^-1); low-dose group(10 mg·kg^-1·d^-1); the negative control group and the blank control group. The animals were sacrificed and the size of the lesions were measured. The endometriosis model of SD rats was identified by HE staining and immunohistochemical staining of keratin and vimentin. The total number of mast cells and their degranulation were measured by Toluidine blue staining; the concentrations of TNF-α in serum were measured by enzyme linked immunosorbent assay; the concentrations of estradiol in serum were measured by enzyme immunoassay; the expression of tryptase and nerve growth factor(NGF) were measured by immunohistochemical staining. Results: The number of activated mast cells(MC) by Toluidine blue staining in high-dose group was significantly lower than that in negative control group(P<0.05), and its ratio of degranulation/total number of MC was significantly lower than that in negative control group or blank control group(P<0.05). The serum TNF-α levels and tryptase expression in tissues in high-dose group were significantly lower than those in negative control group or blank control group(P<0.05). However, no significant difference in the size of endometriotic lesions and expression of NGF was found among groups(P>0.05). Conclusion: Sodium cromoglycate can stabilize mast cells from degranulation, which may relieve the clinical symptoms of endometriosis by reducing TNF-α and tryptase levels.

Objective: To investigate the effect of aquaporin 5(AQP5) on proliferation and migration of ectopic endometrial epithelial cells. Methods: AQP5 shRNA interference fragments were designed and transfected into ectopic endometrial epithelial cells stably by lentivirus technology. Fluorescence quantitative RT-PCR and Western blotting were used to detect the AQP5 mRNA and protein expression, respectively. The cell proliferation and migration were determined by using MTT method and Transwell system, respectively. Levels of phosphorylated AKT(p-AKT) and total AKT were examined by Western blotting. The nude mice model of endometriosis was constructed and the endometrial cell nodule formation was observed. Results: AQP5 shRNA transfection inhibited cell proliferation and migration compared with control group(both P<0.05). The activation of AKT in AQP5 shRNA transfected cells was lower than that in control cells(P<0.01). Compared to control group, the endometrial cells nodule formation was suppressed in mice inoculated with AQP5 shRNA-silencing ectopic endometrial epithelial cells. Conclusion: Down-regulation of AQP5 expression can suppress the proliferation and migration of ectopic endometrial epithelial cells and endometrial cell nodule formation in nude mice, in which AKT pathway may be involved.

Objective: To investigate the influence of gelatin particle(GP) size and gelatin/calcium phosphate cement(GP/CPC) ratio on repairing potency of comparison artificial bone material. Methods: Composite GP/CPC materials with different GP size(100~200 μm vs. 200~300 μm) and ratio(5% vs. 10%) were prepared. Physiochemical and biological properties, including porosity, resistance to compression, ultrastructure and biocompatibility were compared among 4 groups of GP/CPC materials. Different GP/CPC materials were used to repair the critical-size cranial bone defect in rabbit model, and the histology and newly formed bone inside scaffolds(nBIS) were examined and compared among different groups. Results: GP/CPC with GP of 200~300 μm possessed larger micropores than that with GP of 100~200μm(P<0.05). The GP/CPC containing 10% GP had higher porosity than that containing 5% GP(P<0.05). The animal model study showed that more new bone formed in those defects filled with GP/CPC containing 10% GP with 200~300 μm in size compared with GP/CPC containing 5% GP with 100~200 μm in size(P<0.05). While GP/CPC containing 5%GP with 100~200 μm in size showed a higher level of resistance to compression. Conclusion: Both the particle size of GP and its ratio in the GP/CPC affect the properties of the composite biomaterials and their role in bone repairing. In particular, the GP/CPC containing 10% GP with 200~300 μm in size is most suitable for repairing critical-size cranial bone defect in animal model.

Objective: To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. Methods: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1×10^-4, 1×10^-5, 1×10^-6 and 1×10^-7 mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2(BMP-2), Runt-related transcription factor 2(RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. Results: ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1×10^-5, 1×10^-6 and 1×10^-7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1×10^-6 mol/L was the optimal concentration. Besides,icariin(1×10^-6 mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group. Conclusion: Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.

Objective: To establish a model of chondrocyte degeneration in vitro. Methods: Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR. Results: The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group. Conclusion: The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.

Objective: To investigate whether the waveform of electrical stimulus affects the antiepileptic effect of focal low-frequency stimulation (LFS). Methods: The antiepileptic effects of the LFS in sine, monophase square and biphase square waves were investigated in hippocampal kindled mice, respectively. Results: Compared to the control group, sine wave focal LFS(30 s) inhibited seizure stages(2.85±0.27 vs 4.75±0.12,P<0.05), lowered incidence of generalized seizures(53.6% vs 96.5%,P<0.01) and reduced afterdischarge durations[(16.22±1.69)s vs (30.29±1.12)s,P<0.01] in hippocampal kindled mice, while monophase or biphase square wave LFS (30 s) showed no antiepileptic effect. Monophase square LFS(15 min) inhibited seizure stages(3.58 ± 0.16,P<0.05) and incidence of generalized seizures(66.7%,P<0.01), but had weaker inhibitory effect on hippocampal afterdischarge durations than sine wave LFS. In addition, pre-treatment and 3 s but not 10 s post-treatment with sine wave LFS resulted in suppression of evoked seizures(P<0.05 or P<0.01). Conclusion: The antiepileptic effect of LFS is dependent on its waveform. Sine wave may be optimal for closed-loop LFS treatment of epilepsy.

Objective: To investigate the effects of extremely low frequency electromagnetic field (ELF-EMF) on human osteosarcoma cells and its mechanisms.Methods: Human osteosarcoma MG-63 cells were exposed to 50 Hz,1 mT ELF-EMF for 1, 2 and 3 h in vitro, with or without pretreatment by reactive oxygen species (ROS) inhibitor N acetylcysteine (NAC) or p38MAPK inhibitor SB203580. The proliferation of MG-63 cells was determined by MTT method; the apoptosis rate and ROS level in MG-63 cells were detected by flow cytometry. The expression of p38MAPK in MG-63 cells was determined by Western blotting.Results: ELF-EMF decreased the viability of MG-63 cells, inhibited cell growth, induced cell apoptosis and increased the level of ROS significantly. The apoptosis rate declined significantly after treatment with ROS inhibitor NAC or p38MAPK inhibitor SB203580. After exposure to ELF-EMF, p38MAPK in MG-63 cells was activated, and the phosphorylation level was also inhibited after treatment with NAC. Conclusion: ELF-EMF can induce the apoptosis of MG-63 cells. Increased ROS and p38MAPK activation may be involved in the mechanism.

Objective: To investigate the mRNA and protein expression levels of S100A8 and S100A9 in giant cell tumor(GCT) of bone,and its relation with radiological findings and biological behavior. Methods: Forty three patient with GCT of bone admitted in Ruijin Hospital Shanghai Jiaotong University School of Medicine from January 2009 to June 2012 were enrolled in the study. The expression levels of S100A8 and S100A9 mRNA and protein were detected by using semiquantitative RT-PCR and Western blotting in 43 specimens of GCT and 6 specimens of normal bone marrow. The CT and MRI findings of patients were retrospectively reviewed, its relation with tissue expression of S100A8 and S100A9 was analyzed. Results: Among 43 GCT cases 40 showed positive expression of S100A8 and S100A9 mRNA and protein, and the expression levels were significantly higher than those in normal bone marrow(P<0.05). The expression level of S100A8 protein was significantly different in bone GCT with different composition ratio on MRI(P<0.05).The expression level of S100A9 protein was significantly different in GCT with different degree of bone destruction on CT scan(P<0.05). Conclusion: The expression of S100A8 and S100A9 mRNA and protein is up-regulated in GCT of bone. The expression of S100A8 and S100A9 is associated with the real composition ratio and the degree of bone destruction, respectively, indicating that S100A8 and S100A9 may be involved in the biological behavior of bone GCT.

Silver-Russell syndrome (SRS) is a rare genetic disorder with non-specific manifestations and severity, so that the clinical diagnosis of SRS remains difficult. We reported a 23-year-old female patient with SRS characterized with short body stature, asymmetry, obesity, fifth finger clinodactyly and dislocation of hip. The patient had a past history of lengthening operation on the right lower limb at the age of 10. Chromosome analysis revealed (46, XX). The patient was admitted due to severe asymmetry in low extremities caused by right-side obesity. After successful orthopedic surgery in the right hips and thighs the symptoms of patient were relieved.

The routine prenatal maternal serum testing is widely used for screening of birth defects, including Down syndrome, trisomy 18 syndrome and neural tube defects. The testing results are also associated with other adverse pregnant outcomes such as fetal surface structural abnormalities, gestational hypertension disease, intrahepatic cholestasis of pregnancy, premature rupture of membranes, abortion, stillbirth, intrauterine growth restriction and macrosomia; therefore the abnormal levels of serum markers might be used for predicting these adverse pregnant outcomes.

Vertical transmission is the major route of HCV infection in children and draws much attention recently. With the development of assisted reproductive technology (ART), more and more HCV-serodiscordant infertile couples seek assisted reproduction treatment. Vertical transmission of HCV in ART cannot be avoided. Understanding the state of HCV infection of oocyte and embryo is helpful to solve the fertility problem and to control mother-to-child transmission.

Epidemiological and experimental studies have demonstrated that the risk of cardiovascular disease(CVD) in adulthood is influenced by the environment in early life. Intrauterine and early postnatal malnutrition and the following catch-up growth have a long-term effect on blood pressure and endothelial function in adulthood. Well-established prenatal or/and postnatal animal models are used to study the impact of different nutritional intervention on CVD in adulthood. This article reviews the early original cause of chronic CVD in adulthood with the hypothesis of DOHaD(the developmental origins of health and disease), and proposes possible preventions in early life on the basis of this theory.