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Objective: To investigate the effect of the collected oocytes by controlled ovarian stimulation (COS, long GnRHa protocol) on the clinical outcomes of intracytoplasmic sperm injection (ICSI). Methods: All couples (1 042 cycles) who underwent ICSI because of male factor were involved in this retrospective study, from January 2007 to June 2013 in our center. Those cycles were divided into 4 groups according to the number of collected oocytes: G1 as ≤4 oocytes(90 cycles), G2 as 5-10(368 cycles), G3 as 11-20 oocytes(449 cycles) and G4 as ≥21 oocytes (135 cycles). Results: In the ICSI treatment cycle, the mean age in G1 was highest among 4 groups (P<0.01), while 57.8% women were aged <35. The total fertilization failure(TFF) rate in G1, the ovarian hyperstimulation syndrome (OHSS) rate in G4 and the rates of cancelled transfer cycles in G1 and G4 were significantly higher than those in G2 and G3 (P<0.01). The rate of clinical pregnancy in G1 was the lowest in 4 groups (31.3%, P<0.05). The rate of clinical pregnancy, the implant rate and the live birth rate in G3 were the highest in 4 groups (P<0.05). Conclusions: In the ICSI treatment cycle, the number of collected oocytes lower than 4 has negative effect on fertilization; the collected oocytes between 11 to 20 have good clinical outcomes. [ABSTRACT FROM AUTHOR]

Objective: To explore the trend of changes in the parameters of the spine-pelvic sagittal plane before and after surgery in patients with double-segment lumbar spondylolisthesis (LSL) and to evaluate the value of the surgical effect., Methods: A retrospective analysis of 95 double-segment lumbar spondylolisthesis patients treated with posterior lumbar interbody fusion from October 2019 to October 2020 were analyzed, including 31 males and 64 females;age ranging from 41 to 63 years old, with an average of (52.10±4.35) years old;degree of lesion, 47 patients with gradeⅠand 48 patients with gradeⅡ. The surgical efficacy was evaluated according to the Oswestry dysfunction index(ODI) improvement rate at 3 months after operation. ODI improvement rate ≥50% was considered good, and <50% was considered bad. Ninety-five patients were divided into good curative effect group (74 cases) and poor curative effect group (21 cases) according to surgical curative effect. The clinical data, such as gender, age, body mass index, course of disease, degree of disease, operation time, intraoperative blood loss, and comorbidities were compared between two groups were compared. The parameters of spine pelvis sagittal plane were observed before and 3 months after operation, including spine sacral angle (SSA), T1 pelvic angle (TPA), lumbar lordosis (LL), pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS). Clinical symptoms were evaluated using visual analogue scale (VAS) and ODI. The correlation between the changes of spine pelvis sagittal plane parameters and the changes of VAS and ODI before and after surgery were analyzed, and the value of spine pelvis sagittal plane parameters in evaluating the surgical efficacy of patients with double level lumbar spondylolisthesis at 3 months after surgery was evaluated., Results: The courses of disease and surgical time in the group with poor efficacy were longer than those in the group with good efficacy, and the degree of lesion was higher in the group with poor efficacy ( P <0.05). The SSA, TPA, LL, and SS of the group with good efficacy were higher than those of the group with poor efficacy before and 3 months after surgery, while the PT was lower than that of the group with poor efficacy ( P <0.05). The changes in SSA, TPA, LL, PT, and SS before and after surgery in the group with good efficacy were greater than those in the group with poor efficacy ( P <0.05). The VAS and ODI of both groups were lower at 3 months after surgery than before, and the group with good efficacy was lower( P <0.05). The changes in VAS and ODI before and after surgery in the group with good efficacy were greater than those in the group with poor efficacy ( P <0.05). The changes in SSA, TPA, LL, PT, SS before and after surgery were positively correlated with the changes in VAS and ODI ( P <0.05). Three months after surgery, SSA, TPA, LL, PT, and SS were used to evaluate the surgical efficacy of patients with dual level lumbar spondylolisthesis. The area under the curve (AUC) was 0.868, 0.797, 0.875, 0.822, and 0.853, respectively. The combined evaluation of all indicators resulted in the highest AUC, 0.927, and the best sensitivity and specificity were 90.50% and 91.89%, respectively., Conclusion: The spine pelvis sagittal plane parameters SSA, TPA, LL, and SS of patients with double level lumbar spondylolisthesis before and after surgery show an upward trend;PT shows a downward trend;PI do not change significantly. And the changes of SSA, TPA, LL, SS, and PT are closely related to the patient's pain level and the improvement of lumbar function, which can be used as parameters to evaluate the surgical efficacy.

Objective: To compare the development of abnormal pronuclear zygotes after intracytoplasmic sperm injection (ICSI) and analyze their genetic polymorphism., Methods: Four hundred and ninety three abnormal pronuclear zygotes after ICSI were divided into three groups based on the number of pronuclei: 347 nonpronuclear oocytes, 71 monopronuclear zygotes and 75 multipronuclear zygotes. All of them were cultured in the medium of Vitrolife G5 series(TM). Sixteen short tandem repeats (STR) of seven blastocysts were then analyzed by ABI3100., Results: The cleavage rate of nonpronuclear group (25.4%) was lower than that of the others (P<0.01), the proportion of blocked embryos in nonpronuclear group (48.9%) was significantly higher than that of the others (P<0.05), but the blastocyst rate showed no significant difference in three groups (P>0.05). The genetic polymorphism of the 16 STRs showed that the blastocysts from the nonpronuclear and multipronuclear were diploid, and one of the blastocysts from nonpronuclear oocyte was Y-bearing., Conclusion: The zygotes with abnormal pronuclei after ICSI might have development potential, and the blastocysts from nonpronuclear oocytes and multipronuclear zygotes could be diploid.

Objective: To study the effect of patient age, the number and quality of embryo transferred on pregnancy outcome of in vitro fertilization-embryo transfer (IVF-ETs)., Methods: A retrospective study was performed in infertile women who underwent a total of 1800 cycles of IVF-ET and intracytoplasmic sperm injection (ICSI). The patients were divided into three age groups, namely<30 years group, 30-35 years group, and>or=35 years group. The clinical pregnancy rate and the multiple pregnancy rate were compared when 1, 2 or 3 embryos and 0, 1, 2 or 3 good-quality embryos were transferred., Results: In patients<30 years, no significant differences was found in the clinical pregnancy rate between 1, 2 and 3 embryos transfer groups; 2 and 3 good-quality embryos transfer resulted in similar pregnancy rate, which was significantly higher than that resulted from 0 and 1 good-quality embryo transfer. Multiple pregnancy was not found in 1 embryo transfer group. In patients aged 30-35 years, the pregnancy rate showed no significant differences not only between 1 and 2 embryos transfer groups, but also between 2 and 3 good-quality embryos transfer groups; multiple embryo transfer led to significantly increased multiple pregnancy rate. In patients aged>or=35 years, the transfer of 1, 2 and 3 embryos resulted in similar pregnancy rate; transfer of 3 good-quality embryos had obviously higher pregnancy rate than 0, 1 and 2 good-quality embryos transfer groups. Increased numbers of embryos and good-quality embryos transferred were both associated with increased multiple pregnancy rate., Conclusion: One good-quality embryo transfer in patients<30 years and 2 good-quality embryos transfer in patients>or=30 years can obtain ideal pregnancy rate and reduce the incidence of multiple pregnancy. For patients aged>or=35 years, transfer of only good-quality embryo is recommended.

Pertussis remains to be a global public health problem, even in developed countries with full coverage of universal vaccination. This has been accompanied by a change of disease epidemiology, characterized by a significant increase in reported pertussis cases among adolescents and adults. Actually, reported cases of pertussis represent only a small part of the whole pertussis cases reservoir. In response to the changing epidemiology, several countries have implemented booster programs of universal adolescent vaccine. In this paper, the anther tries to review the changing epidemiology and reveal the causes of resurgence. By comparing pertussis immunization schedules in different countries, it will be helpful for reconsidering the vaccination strategic for pertussis in our country.

Objective: To determine whether there is a major difference in protein levels between isoniazid-resistant and susceptible M. tuberculosis strains., Methods: The proteins extracted from 9 isoniazid-resistant M. tuberculosis strains and 7 isoniazid-susceptible M. tuberculosis strains were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Protein spots remarkly increased in isoniazid-resistant M. tuberculosis strains were digested in gel by enzyme and the mass of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in protein database search. These genes of all strains were sequenced., Result: In all protein spot differences, 5 protein spots were upregulated in isoniazid-resistant strains and identified as Rv1446c, Rv3028c, Rv0491, Rv2971, and Rv2145 by (MALDI-TOF-MS)., Conclusion: These results suggest that the differentially expressed proteins from isoniazid-resistant strains might be used as potential immuno-diagnostic antigens and candidate novel drug targets against drug resistant tuberculosis.

Aim: To prepare specific single-epitope antibody using immunomagnetic beads coated with multiple antigen peptides(MAP)., Methods: Eight-branch MAP is synthesized by Fmoc SPPS. After immunizing the BALB/c mice with the MAP, polyclonal antiserum was obtained. Then, the immunomagnetic beads (IB) were prepared by coating with the MAP in covalent bond form and were used to purify the specific single-epitope antibody from the antiserum of immunized animals., Results: The titer of antiserum was 1:12,800, which showed the strong immunogenicity of MAP. The best coating concentration of MAP was 100 mg/L and the max coating efficiency was 79%. After purifying specific antibody from polyclonal antiserum using the IB, the obtained antibodies only interacted with one epitope rather than other epitopes. The purity of the antibody reached 95% and the mean recovery rate was 5.8%., Conclusion: The beads coated with MAP can be used to quickly isolate specific single-epitope antibody which is similar to monoclonal antibody. This method provide a convenient shortcut in preparing small amount of antibody with high specificity.

Objective: To establish a protocol for culturing of human embryonic stem cells (HUES4) without any animal-derived feeder cells and to investigate the karyotype stabilities of HUES4 cells after long-term cultivation., Methods: HUES4 cells were cultured on mitomycin C treated MEFs or human foreskin fibroblast feeder cells. The pluripotency of the ES cells was analyzed by immunocytochemistry staining to detect the expression of pluripotent marker, karyotype of the ES cells at passage 27, 34, 41, 44 and short tandem repeat (STR) at passage 27 were analyzed., Results: The HUES4 cells cultured on human feeder cells were positive for alkaline phosphatase activity, SSEA-4, TRA-1-60 and TRA-1-81 staining, but negative for SSEA-1. Analysis of karyotype at different passages suggested an abnormal karyotype 46, XY, t(9;15)(q22;q26) mosaicism occurred in HUES4, and the ratios of abnormal increased with passage., Conclusion: HUES4 could be cultured without animal-derived feeder cells and the incidence of abnormal karyotype might be increased with long-term culture.

Objective: To set up and evaluate the method of phage amplified biologically assay (PhaB) in rapid detection of pyrazinamide (PZA) resistance., Methods: The PhaB assay was developed and applied in detecting PZA resistance in 108 clinical isolates of Mycobacterium tuberculosis and the results were compared with those of the absolute concentration method. The minimum inhibitory concentration (MIC) was detected for all discrepancy isolates., Results: The results showed that the optimal detecting condition was pH 5.5, PZA 200 microg/ml and 48 h. Of the 108 strains of Mycobacterium tuberculosis, 28 strains were PZA-susceptible and 80 strains were PZA-resistant detected by PhaB; while 32 strains were PZA-susceptible and 76 strains were PZA-resistant by absolute concentration method. Twenty-eight of the 108 strains were PZA-susceptible and 71 were PZA-resistant by the two methods. The concordant isolates of determination of PZA resistance were 99 by the two methods and the concordance rates was 91.7%. There were 9 strains in discordant isolates, of which 7 were the same with MIC method and gene chip in drug susceptibility. If the results of absolute concentration method was the gold standard, the sensitivity, specificity, positive and negative predictive values, as well as accuracy of PhaB assay was 94.7%, 84.8%, 93.4%, 87.5% and 91.7% respectively., Conclusion: The PhaB assay can be used as a rapid screening method for detection of drug susceptibility of PZA in clinical isolates of Mycobacterium tuberculosis.

Objective: To investigate the hepatic safety of itraconazole intravenous solution in the treatment of invasive fungal infection., Methods: Forty-nine patients with invasive fungal infection, such as pneumonia, meningitis, endocarditis, and blood stream infection, caused by Aspergillus spp. Cryptococcus neoformans, Candida spp. Penicillium marneffei, and Prototheca wickerhamii, 50 of which had underlying diseases, including hepatic diseases, such as hepatitis, cirrhosis, fatty liver etc. were treated with intravenous itraconazole for 12.6 days (2 - 42 days). Liver function test was conducted before and after the treatment. The relationship between liver adverse reaction and itraconazole treatment was evaluated., Results: Liver function test was abnormal in 20 patients. The patients with liver enzyme abnormalities before itraconazole treatment were easier to suffer from hepatic damage during itraconazole treatment (chi(2) = 11.54, P = 0.001). However, only in 2 patients with mild liver function test abnormality the liver adverse reaction could be associated with itraconazole treatment., Conclusion: Itraconazole treatment for invasive fungal infection is rather safe, even in the patients with underlying hepatic diseases. Liver function test is recommended for all patients suggestive of liver dysfunction and protective therapy for liver is needed.

Objective: To develop a new multiplex allele-specific polymerase chain reaction (MAS-PCR) assay to detect the main mutations in the rifampin resistance dependent region, which has been reported to account for the majority of clinic Mycobacterium tuberculosis resistant to rifampin., Methods: Based on the sequence of rpoB gene, three specific primers were designed for the MAS-PCR to detect the most common mutations in codons 531, 526, 516 of rpoB gene., Results: The purified DNA preparations of 91 clinical strains of Mycobacterium tuberculosis were used to optimize the PCR. The mutations in codon 531, 526, 516 were detected by the MAS-PCR. Compared with the results of direct sequencing of rpoB gene, no mutation was detected in the sensitive strains. For rifampin-resistant strains, the total sensitivity was 81.5% (66/81)., Conclusions: MAS-PCR is a new molecular method with a high sensitivity and specificity, which can be used to detect the 3 main mutations of rpoB gene rapidly and economically. It can be used in clinical laboratories to detect the rifampin-resistant strains of Mycobacterium tuberculosis.

Aim: To express ESP30, a novel serine protease, in Escherichia coli and to prepare anti-ESP30 antibody., Methods: ESP30 gene was amplified by PCR from the genome of aeromonas hydrophila, and cloned into the expression vector pDH2. The ESP30 expression was carried out under thermal induction. The antiserum was prepared by immunizing rabbit with ESP30. The titer and specificity of the antibody were detected by ELISA and Western blot respectively., Results: The ESP30 non-fusion protein with relative molecular mass (M(r)) of 66,000 was highly expressed in E. coli. The rabbit antibody against ESP30 was obtained. The ELISA titer of antiserum against ESP30 was about 1:128,000. Western blot analysis showed that the antiserum could bind to the expressed ESP30 specifically., Conclusion: The rabbit antibody against ESP30 has been successfully prepared, which lays the foundation for further studying the structure and function of the novel protease ESP30.

Aim: To develop a new method for antibody detection based on fluorescence polarization (FP) technique., Methods: BALB/c mice were immunized with single epitope synthetic 8 branches peptide antigen (SVEVGKVADL)8 of Helicobacter pylori (Hp) UreB protein. FP values of the corresponding linear peptide antigen labeled with FITC in different concentration were measured to determine the optimal concentration of the antigen. The antigenicity of the synthetic linear peptide was identified by FP assay. Then, the antibody-positive and negative murine sera were used in FP assay to determine the optimal dilution factor of serum samples. To apply FP technique in Hp antibody detection, 126 human serum samples were detected either by FPIA (fluorescence polarized immunoassay) using the single epitope linear synthetic peptide as antigen or by a commercial ELISA kit. Then, receiver operating characteristic (ROC) curve analysis was performed on the FPIA results by MedCalc software., Results: The UreB single epitope peptide had strong antigenicity. 1.0 nmol/L of the synthetic linear peptide labeled with FITC was the optimal concentration of the antigen and the optimal dilution factor of serum sample was 1:25. In 77 Hp antibody-positive serum samples detected by ELISA, 66 samples were positive by FP detection method. The sensitivity and specificity of the FPIA assay was 85.7% and 98%, respectively., Conclusion: The single epitope synthetic peptide antigen can be used in FP method for rapid detection of serum antibody against Hp UreB. The established FP assay for antibody detection may be used in clinical diagnosis in the future.

Objective: To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput., Methods: Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated., Results: The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD., Conclusion: TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.

Objective: To study the relationship between KatG 315 mutation and isoniazid (INH) resistance in M. tuberculosis by template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP), and to develop a new method that can detect INH-resistant M. tuberculosis precisely and quickly., Methods: Isolates of M. tuberculosis resistant to INH from 82 tuberculosis patients were cultured on culture medium with different INH concentrations. DNA of all M. tuberculosis samples was extracted and amplification of 271 bp KatG gene fragment was carried out by polymerase chain reaction (PCR). After digestion of the excess primers and dNTPs in PCR products by clean-up reagents, template-directed dye-terminator incorporation reaction was performed, and the Acyclo-terminators labeled Rhodamine 110 (R110) or 6-carboxytetramethylrhodamine (TAMRA) were incorporated into the detection primers specifically. Then fluorescence polarization value was measured using Victor2 multi-label counter and the genotypes of the mutation site in 315 condon of KatG gene of all samples were investigated., Results: There were 15 out of the 29 isolates of M. tuberculosis with high INH-resistance showed G-->C mutation in 315 condon of KatG gene, including 4 isolates with both mutated and non-mutated trains, the mutation frequency being 52%. In 32 isolates of M. tuberculosis with low INH-resistance, 15 were mixed infection, the mutation frequency being 47%. No mutation in 315 condon of KatG gene was found in 21 susceptible isolates of M. tuberculosis., Conclusions: The G-->C mutation in 315 condon of KatG gene was associated with INH-resistance of M. tuberculosis. TDI-FP technology was a reliable, easy to use and high-through method to detect mutation of KatG gene. It may be a useful technique for the diagnosis of INH-resistance in the future.

Aim: To explore the induction of HepG2 cells by NDRG2 gene., Methods: Human NDRG2 gene was obtained by RT-PCR. Sequence analysis proved that sequence of NDRG2 gene was correct. Then the gene was inserted into the eukaryotic expression vector pIRES2-EGFP and transfected into NDRG2 gene-negative HepG2 cells. The changes of cell morphology and structure were observed under light, fluorescence and transmission electron microscope, respectively. The variation of cell cycle was detected by flow cytometry., Results: The NDRG2 gene had been obtained and its expression vector was constructed successfully. The NDRG2 gene-transfected HepG2 cells were shown a bad condition, structure damage, and a great number of died cells. The expression of target gene in cytoplasm of HepG2 cells was seen under fluorescence microscope. Flow cytometry analysis showed G1 phase arrest and apoptosis peak appeared.The typical manifestation of cell apoptosis could be observed under transmission electron microscope., Conclusion: NDRG2 gene can arrest HepG2 cell proliferation and induce their apoptosis.

For studying the expression and distribution of angiotensinogen (AGT), the C-teminus of rat AGT gene was expressed in E.coli. Rabbits were immunized with expressed AGT protein and sera from different rabbits were raised. ELISA showed a high titre (1:25600) of the antiserum. With the antiserum, Western blotting recognized not only the prokaryotic expressed AGT, but also the endogenous AGT protein in liver tissue of both rats and humans. Using this antiserum, immunohistochemistry showed the expression of AGT protein in islet cells of human pancreas as well as in epithelium of human bile duct. These results suggest that the prokaryotic expressed AGT protein is an effective immunogen for the preparation of anti-AGT antiserum. Our present work provides an important tool for study of the pathophysiological role of AGT as well as local renin-angiotensin system.