Objective To determine the lower limit of detection (LLOD) and cut off values of K-ras mutation detection by peptide nucleic acid (PNA) clamping-PCR.Methods The genomic DNA of pancreatic cancer cell lines (PANC1 and SW1990) with codonl2,13 mutation and the genomic DNA of placenta with K-ras wild type were mixed and diluted serially into samples with different mutation rate (0,0.1％,0.2％,0.4％,0.8％,1.6％,3.1％,6.25％,12.5％,25％,50％),PANC1 cells with 1％ mutation rate and SW1990 cells with 30％ mutation rate and 4 samples with the quantity of DNA was 50,20,5,1 ng and 50,10,5,1 ng was prepared.Codon 12,13 mutation of K-ras was determined by PNA-PCR,and the mutation Ct values,overall Ct values were collected,and the △Ct values (mutation Ct values-overall Ct values) were calculated,and the tests were repeated for 10 times.ROC curve was used to analyze the △Ct values and determine the best cut off values for K-ras mutation,and the positive diagnostic rate,LLOD was evaluated.Results The mutation Ct,△Ct values of codon 12 mutation of PANC1 and codon 13 mutation of SW1990 of all the different mutation rates were statistically significantly different (P ＜ 0.05) when compared with negative control group,but the overall Ct values were not statistically significantly different from that of negative control group.For detection of K-ras codon 12 mutation by ROC curve,the relevant area of ROC curve (AUC) was 0.926,the optimum cut off value of △CT was 11,the sensitivity and specificity were 84％ and 100％,respectively,and the LLOD was 0.4 ng.For detection of K-ras codon 13 mutation by ROC curve,the relevant AUC was 0.906,the optimum cut off value of △CT was 9.5,the sensitivity and specificity were 71％ and 100％,respectively,and the LLOD was 1.5 ng.The mutation detection results of fixed rate further confirmed the LLOD.Conclusions This study successfully defines LLOD and cut off value of PNA clamping-PCR/K-ras method in detection of K-ras 12 and 13 codon mutations.This method meets the requirement of clinical application.

OBJECTIVETo determine an optimal insemination technique for unexplained infertility patients undergoing IVF or ICSI following failed intrauterine insemination (IUI).

METHODSSixty-five cycles of 61 patients with failed IUI were divided into four groups: Group A (37 cycles of IVF), Group B (19 cycles of ICSI), Group C (4 cycles of IVF and ICSI: half and half), Group D (5 cycles of ICSI after failed IVF). The conventional IVF cycles were defined as Group E (37 cycles), and the conventional ICSI cycles defined as Group F (24 cycles). The fertilization rate, completely failed fertilization rate, high quality embryo rate, clinical pregnancy rate and the implantation rate were compared among all the groups.

RESULTSThere were statistically significant differences in the fertilization rate, completely failed fertilization rate between Groups A and E (55.4% vs 72.8%, P < 0.05; 21.6% vs 0%, P < 0.005), and Groups A and B (55.4% vs 68.4%, P < 0.05; 21.6% vs 0%, P < 0.01). In Group D, there was statistically significant difference in the fertilization rate between IVF cycles and ICSI cycles (12.2% vs 74.2%, P < 0.005).

CONCLUSIONThe present study demonstrates that there may be 20% of totally failed fertilization in IVF cycles with unexplained primary infertility following failed IUI, and ICSI treatment can increase fertilization rate and avoid complete fertilization failure.

Adult ; Female ; Humans ; Infertility, Female ; etiology ; therapy ; Insemination, Artificial, Homologous ; methods ; Male ; Ovulation Induction ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic ; methods ; Treatment Failure

Objective To investigate the relationship between serum progesterone level at the day with human chorionic gonadotrophin (hCG) administration and pregnant outcome from in in-vitro fertilization-embryo transfer(IVF-ET). Methods From Mar. 2002 to Apr. 2007, 786 cycles with serum progesterone measurement on the day of hCG administration for final oocyte maturation in IVF were analyzed retrospectively in Reproductive Medicine Center in First Affiliated Hospital of Nanjing Medical University.All stimulations were down-regulated with gronadotrophin release hormone agonist (GnRH-a) in both long protocols and short protocols before gonadotrophin stimulation. When the thresholds of serum progesterone were set at 5.5, 6.0,6.5,7.0,7.5,8.0,8.5 and 9.0 nmol/L, respectively. If the level of progesterone was less than the thresholds, those patients were in lower progesterone group, on the contrary, more than the threshold value, those patients were in higher progesterone group. The laboratory results and the clinical outcomes between all patients at lower and higher progesterone group at different thresholds value were analyzed. Results The rate of normal fertilization, quality embryos, successful implantation, chemical pregnancy, clinical pregnancy and live birth did not exhibit remarkable difference between patients with higher and lower serum progesterone level at multiple thresholds on the day of hCG administration in the 786 cycles (P >0.05). However, when the thresholds of serum progesterone were at 8.5 and 9.0 nmol/L, early abortion rates of 27.3% (3/11) and 3/7 in higher progesterone group were significantly higher than 8.8% (26/297) and 8.6% (26/301) in lower progesterone group (P<0.05). And the total abortion rates of 3/7 in higher progesterone group were significantly higher than 11.0% (34/301) in lower progesterone group when the thresholds of serum progesterone were 9.0 nmol/L (P<0.05). Conclusions This study did not prove the correlationship between progesterone level at the clay with hCG administration and the probability of clinical pregnancy or live birth. However, early abortion rates or the total abortion rates were associated with higher progesterone level when the thresholds of serum progesterone were at 8.5 nmol/L or 9.0 nmoL/L.

Objective To evaluate the outcome of liver transplantation in patients with recurrent liver cancer after resection.Methods Data of 23 patients underwent liver transplantation for recurrent liver cancer from April 2001 to March 2008 were retrospectively collected and analyzed.Results Previous history of liver resection had little negative effect in subsequent liver transplantation in technical aspect.Liver function recovered uneventfully after transplantation in all cases.Alpha fetoprotein(AFP) recovered to normal value in 13 of 17 cases with elevated AFP before transplantation within one month after operation.Five cases(21.74%) had postoperative complications.Nineteen cases(82.61%) were followed up,average follow-up duration were 610 days.There were 5 cases(26.32%) of cancer recurrence and 6 deaths during follow-up,survival rate was 68.42%.Conclusion Liver transplantation is a reasonable treatment for recurrent liver cancer after resection.

Objective: To investigate the incidence of microvascular myocardial ischemia in diabetic patients without obstructive coronary artery disease (CAD) and its relationship with angina. Materials and Methods: Diabetic patients and an intermediate-to-high pretest probability of CAD were prospectively enrolled. Non-diabetic patients but with an intermediate-to-high pretest probability of CAD were retrospectively included as controls. The patients underwent dynamic computed tomography-myocardial perfusion imaging (CT-MPI) and coronary computed tomography angiography (CCTA) to quantify coronary stenosis, myocardial blood flow (MBF), and extracellular volume (ECV). The proportion of patients with microvascular myocardial ischemia, defined as any myocardial segment with a mean MBF ≤ of 100 mL/min/100 mL, in patients without obstructive CAD (Coronary Artery Disease–Reporting and Data System [CAD-RADS] grade 0–2 on CCTA) was determined. Various quantitative parameters of the patients with and without diabetes without obstructive CAD were compared. Multivariable analysis was used to determine the association between microvascular myocardial ischemia and angina symptoms in diabetic patients without obstructive CAD. Results: One hundred and fifty-two diabetic patients (mean age: 59.7 ± 10.7; 77 males) and 266 non-diabetic patients (62.0 ± 12.3; 167 males) were enrolled; CCTA revealed 113 and 155 patients without obstructive CAD, respectively. For patients without obstructive CAD, the mean global MBF was significantly lower for those with diabetes than for those without (152.8 mL/min/100 mL vs. 170.4 mL/min/100 mL, P < 0.001). The mean ECV was significantly higher for diabetic patients (27.2% vs. 25.8%, P = 0.009). Among the patients without obstructive CAD, the incidence of microvascular myocardial ischemia (36.3% [41/113] vs. 10.3% [16/155], P < 0.001) and interstitial fibrosis (69.9% [79/113] vs. 33.3% [8/24], P = 0.001) were significantly higher in diabetic patients than in the controls. The presence of microvascular myocardial ischemia was independently associated with angina symptoms (adjusted odds ratio = 3.439, P = 0.037) in diabetic patients but without obstructive CAD. Conclusion Dynamic CT-MPI + CCTA revealed a high incidence of microvascular myocardial ischemia in diabetic patients without obstructive CAD. Microvascular myocardial ischemia is strongly associated with angina.

Objective Using of cumulative live birth rate(CLBR)per oocytes retrieved cycle,to assess the clinical outcomes of in vitro fertilization or intracytoplasmic sperm injection(IVF/ICSI),and to explore impact factors on CLBR following utilization of all fresh and frozen embryos in one complete IVF/ICSI cycle using gonadotropin-releasing hormone(GnRH)agonist, GnRH-antagonist and clomiphene mild stimulation protocols. Methods Of the patients who underwent IVF/ICSI from January 1st, 2014 to December 31st, 2015 in the First Affiliated Hospital, Nanjing Medical University, a total of 6 142 oocytes retrieved cycles were included. The clinical and laboratory parameters of different ovarian stimulation protocols, and the effects of the age, number of oocytes retrieved and number of embryos available on the CLBR of each oocytes retrieved cycle were analyzed.Results The CLBR was 69.0%(2 004/2 906)in the GnRH-agonist protocol versus 67.4%(644/955)in the GnRH-antagonist protocol (P>0.05); the CLBR of clomiphene mild stimulation protocol was 53.2%(1 215/2 281),significantly lower than those of the other two protocols (all P<0.05). The CLBR significantly decreased with age increased. When divided into four groups according to the patients′ age, we found that CLBR were not statistically significant using three different protocols in the 20-25 years old group(all P>0.05).There was a strong association between the number of oocytes retrieved and embryos available on CLBR. CLBR rose significantly with an increasing number of oocytes up to 6, then the rising trend slowed down. Patients were categorized into four groups according to the number of oocytes retrieved,CLBR was significantly higher using GnRH-antagonist protocol (50.0%)than mild stimulation protocol(37.0%)in low ovarian responder(0-4 oocytes)group(P<0.05). The CLBR were no significant difference among three protocols in normal(10-15 oocytes)and high responders(≥15 oocytes)group(all P>0.05).The incidence rate of ovarian hyperstimulation syndrome in GnRH-agonist protocols(5.2%,152/2 906)were significantly higher than those of GnRH-antagonist(4.4%, 42/955)and clomiphene mild stimulation protocols(1.5%,34/2 281;all P<0.05).Conclusions CLBR is an important index to assess the clinical outcomes of IVF/ICSI. Age, number of oocytes retrieved and embryos available could affect CLBR obviously. According to the different age and ovarian response of patients, we should design ovarian stimulation protocols based on target oocytes number in order to get higher CLBR and reduce complications.

Objective To investigate the impact of dyslipidemia on in-vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) pregnancy outcome in patients with polycystic ovary syndrome (PCOS).Methods From July 2013 to March 2016,468 PCOS patients with antagonist protocol in IVF/ICSI of First Affiliated Hospital of Nanjing Medical University,cycles were divided into dyslipidemia group (108 cases) and normol blood lipids group (360 cases) according to the serum cholesterol,triglyceride (TG),high-density lipoprotein,low density lipoprotein levels.The general condition and clinical outcomes of the two groups were analyzed retrospectively,including the implantation rate,clinical pregnancy rate,live birth rate and the incidence of moderate to severe ovarian hyperstimulation syndrome (OHSS),etc.Besides,stratified analysis and multivariate logistic regression analysis were used to correct the impact of body mass index (BMI).Results (1) Comparing the based data of dyslipidemia group and normal blood lipids group:age,years of infertility,basic FSH,basic LH,basic estradiol and other indexes had no significant differences (all P＞0.05),but BMI of dyslipidemia group was significantly higher than normal blood lipids group [(25.0±3.0) versus (23.1±3.0) kg/m2],difference had statistical significance (P＜0.01).(2) The high score embryo rate,endometrial thickness on the day of hCG injection,progesterone and LH levels on the day of hCG injection,moderate to severe OHSS rate and miscarriage rate in the two groups did not exhibit remarkable differences (all P＞0.05).However,the number of dominant follicle,retrieved oocyte number,estrogen level on the day of hCG injection,implantation rate,biochemical pregnancy rate,clinical pregnancy rate and the live birth rate in dyslipidemia group were significantly less than those of normal blood lipids group (all P＜0.05),the dose of gonadotropin (Gn) and days of stimulation were significantly higher compared with the normal blood lipids group,there were significant differences statistically (all P＜0.05).(3) Stratified analysis showed that no matter in BMI＜24 or BMI≥24 kg/m2 group,the dose of Gn and days of stimulation were significantly higher in the dyslipidemia group than those of the normal blood lipids group,the difference was statistically significant (P＜0.05).However,the number of oocytes retrieved,estrogen level on the day of hCG injection had obvious downtrend,and the difference was statistically significant (P＜0.05) in BMI≥24 kg/m2 group.Multivariate logistic regression analysis found that,even after the correction of BMI,dyslipidemia still had negative impact on implantation rate,biochemical pregnancy rate,clinical pregnancy rate and the live birth rate (P＜0.05).(4) Further analysis of the different components of blood lipids in the clinical pregnancy group and unobtained pregnancy group revealed that the level of triglyceride (TG) in the unobtained pregnancy group was significantly higher than that in the pregnancy group,and the difference was statistically significant (P＜0.05);logistic regression analysis also showed that the increase of TG levels was negatively correlated with the clinical pregnancy rate of PCOS patients (P＜0.05).Conclusions PCOS patients combined with dyslipidemia have a higher BMI,and dyslipidemia increases the dosage of Gn,reduces the implantation rate,clinical pregnancy rate and live birth rate,especially the increase of TG level,which has adverse effects on IVF/ICSI outcome in patients with PCOS.

Objective: To determine the changed expression levels, biological roles and underlying mechanism of LncSox4 in non-small cell lung cancer (NSCLC), providing novel biomarkers for NSCLC diagnosis and therapy. Methods: QRT-PCR was used to detect the expression of LncSox4 in the tumor tissues of NSCLC patients. Colony formation, cell growth curve, Transwell migration and invasion assays were used to determine the effects of LncSox4 knockdown on A549 cell function, respectively. Flow cytometry was used to determine the effects of LncSox4 on the progression of A549 cell cycle. QRT-PCR and western blot were used to explore the expressions of genes and proteins in epithelial-mesenchymal transition (EMT). Results: The expression of LncSox4 was upregulated significantly in carcinoma tissues of NSCLC compared to the para-carcinoma tissues (t=7.109，P＜0.01). The growth rate of A549 cells slowed down in LncSox4 knockdown group and the number of formed cell colonies was less than that in control group(P＜0.01). LncSox4 knockdown reduced the migration and invasion abilities of A549 cells (P＜0.01) and induced cell cycle arrest at G1 phase(P＜0.01). LncSox4 knockdown downregulated the protein expressions of Cyclin D1, c-Myc, N-cadherin, and Vimentin, while upregulated the expression of E-cadherin in A549 cells. LncSox4 knockdown also decreased the expressions of EMT-related transcription factors including snail, slug and twist. Conclusion The high expression of LncSox4 in NSCLC may promote malignant progression of NSCLC by enhancing cell proliferation, migration and invasion, suggesting that it should be a promising target for diagnosis and therapy of NSCLC.

Objective: To investigate the expression change, biological role and action mechanism of long non-coding RNA (lncRNA) LINC00978 in non-small cell lung cancer (NSCLC). Methods: The expression levels of LINC00978 in tumor tissues and serum samples of NSCLC patients were detected by the qRT-PCR. The effects of knockdown and overexpression of LINC00978 on the biological function of A549 cells were determined by the CCK-8, colony formation, Transwell migration and invasion assays. The action mechanisms of LINC00978 in NSCLC were investigated by the flow cytometry, qRT-PCR and western blot, respectively. Results: The expression levels of LINC00978 in the tissues ( t =2.465, P ＜0.05) and serum samples ( t =8.781, P ＜0.01) of NSCLC patients increased. The knockdown of LINC00978 inhibited the proliferation, migration and invasion of A549 cells ( P ＜0.01) and induced cell cycle arrest at G1 phase and apoptosis of A549 cells ( P ＜0.01). The knockdown of LINC00978 downregulated the expression of Cyclin D1 and Bcl-2 , and upregulated the expression of Bax ( P ＜0.05). In addition, the knockdown of LINC00978 inhibited the expression of N-cadherin, Vimentin, Snail, Slug and Twist, and promoted the expression of E-cadherin ( P ＜0.05). The overexpression of LINC00978 had the opposite effect. Conclusion LINC00978 is highly expressed in NSCLC and can promote the occurrence and progression of NSCLC, which may serve as a potential target for the diagnosis and therapy of NSCLC.