In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further development capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cytoplasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hybridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chromosomal diseases.

To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID(50), 10 TCID(50) and 1 TCID(50)). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID(50) of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.
Blastocyst ; Cells, Cultured ; Cleavage Stage, Ovum ; Cytomegalovirus Infections ; Fertilization ; Muromegalovirus/*pathogenicity ; Oocytes/cytology ; Oocytes/growth & development ; Oocytes/*virology

Background and purpose: AMP-activated protein kinase (AMPK) plays an important role in the regulation of cell metabolism and energy balance and is associated with cell proliferation, survival and multiple signaling pathways. Recent reports found that AMPK is involved in tumor suppression and drug resistance. The aim of this study was to explore the effect of AMPK on the anti-tumor effect of adriamycin and underlying mechanism in breast cancer MCF-7/adr cells. Methods:The anti-proliferative effects of adriamycin was detected by methyl thiazolyl tetrazolium (MTT) assay in MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKαcells. The cell morphology in each group was stained with the lfuorescent dye Hoechst 33528, and the effects on apoptosis induction were examined by lfow cytometry (FCM). The intracellular concentration of adriamycin was detected by lfuorescence assay. The resis-tance-and apoptosis-related proteins were analyzed by Western blot. Results:The growth of breast cancer MCF-7/adr cells was inhibited by adriamycin in a dose-and time-dependent manner. The IC50 values at 24 and 48 h were (36.8±2.1) and (28.8±1.3) μg/mL, respectively. AMPKαover-expression enhanced the cytotoxic effect of adriamycin in MCF-7/adr-AMPKαcells in a dose-and time-dependent manner. Its IC50 values at 24 and 48 h were (16.0±0.7) and (4.2±0.2) μg/mL, respectively. Fluorescent morphological assay showed that AMPKαoverexpression contributed to adriamycin induced apoptosis in MCF-7/adr-AMPKαcells. After treatment with 1.0 μg/mL adriamycin for 48 h, the apoptosis rates of MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKα cells were (12.0±1.4)%, (12.7±1.6)% and (32.0±4.2)%, respectively, indicating that overexpression of AMPKα enhanced the adriamycin-induced apoptosis in MCF-7/adr cells. Fluorescence microplate assay showed that over expression of AMPKαsigniifcantly increased the intracellular accumulation of adriamycin, in a concentration dependent manner. Western blot analysis showed that, compared with MCF-7/adr and MCF-7/adr-vector cells, the expressions of Bax, caspase-3 and cleaved PARP proteins were increased. Meanwhile, Bcl-2 and P-gp protein expressions were decreased in MCF-7/adr-AMPKαcells. Furthermore, the release of cytochrome c from mitochondria into the cytosol was also observed in MCF-7/adr-AMPKαcells. Conclusion:AMP-Kαoverexpression can enhance the chemosensitivity of breast cancer MCF-7/adr cells to adriamycin through inhibiting the drug effux transporter and regulating the expression of apoptosis-related proteins.

OBJECTIVE:To establish a method for the content determination of amygdalin in Lianhua qingwen capsule. METH-ODS:HPLC was performed on the column of Phenomenex Kinetex XB-C18 with mobile phase of acetonitrile-0.2%Phosphoric acid so-lution(6∶94,V/V)at a flow rate of 1.0 ml/min,the detection wavelength was 207 nm,column temperature was 30℃,and the injec-tion volume was 10μl. RESULTS:The linear range of amygdalin was 43.16-215.80 μg/ml(r=0.999 7);the limit of detection was 0.431 6μg/ml,the limit of quantitation was 1.294 8μg/ml;RSDs of precision,stability and reproducibility tests no more than 0.69%;recovery was 95.16%-100.49%(RSD=1.67%,n=9). CONCLUSIONS:The method is simple and rapid with high accuracy and well reproducibility,and can be used for the content determination of amygdalin in Lianhua qingwen capsule.

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts (24.7%) were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts (66.3%) survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% (18/44) and 32.8% (20/61) respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.

Embryos with a poor morphological score at cleavage stage are usually discarded because they are considered unsuitable for transfer and cryopreservation. This study examined the in vitro blastocyst development after extended culture of these embryos and the clinical outcomes after transfer of these blastocysts in warming cycles. A total of 597 blastocysts (24.7%) were obtained from 2421 embryos with low morphological scores after extended culture. One hundred and sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from 30.8% to 32.6%. After warming, 61 out of 92 blastocysts (66.3%) survived and were transferred in 44 cycles. The clinical pregnancy rate and the implantation rate were 40.9% (18/44) and 32.8% (20/61) respectively. Thirteen healthy babies were born, and 5 pregnancies aborted spontaneously. Our study suggested that some blastocysts derived from embryos with a poor morphological score can be successfully vitrified and give rise to live births. Selection and vitrification of viable embryos after extended culture of embryos with a poor morphological score may constitute a proposal to avoid embryo wastage.
Adult ; Cryopreservation ; methods ; Embryo Culture Techniques ; methods ; Embryo Transfer ; methods ; statistics & numerical data ; Female ; Fertilization in Vitro ; methods ; statistics & numerical data ; Humans ; Infertility ; pathology ; therapy ; Middle Aged ; Pregnancy ; Pregnancy Outcome ; Vitrification ; Young Adult

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD)of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further development capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%)(P＞0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P＜0.05). There was no significant difference in cytoplasm remains between methanol/acetic acid method and Tween/HCL method (P＞0.05). The hybridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P＞0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chromosomal diseases.

To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID50, 10 TCID50 and 1 TCID50). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID50 of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.

Objective To observe echocardiographic characteristics of bicuspid aortic valve (BAV) with Sievers classification.Methods Clinical and echocardiography data of 121 patients with BAV were retrospectively analyzed.According to Sievers classification,the patients were divided into type 0 (no raphe),type 1 (one raphe) or type 2 (two raphes).BAV of type 1 was further divided into three subtypes,including 1(L-R) subtype (the raphe positioned between the left and the right coronary sinuses),1(R-N) subtype (the raphe positioned between the right coronary sinus and non-coronary sinus) and 1 (N-L) subtype (the raphe positioned between the left coronary sinus and non-coronary sinus).The diameters of aorta in BAV patients with Sievers classification obtained with echocardiography were compared.Results There were 3 patients (3/121,2.48％) of type 0 and 118 patients (118/121,97.52％) of type 1 BAV.In patients of type 1 BAV,there were 80 cases (80/121,66.12％) of subtype 1 (L-R),33 (33/121,27.27％) of subtype 1 (R-N) and 5 (5/121,4.13％) of subtype 1 (N-L).There was no type 2 patient among these 121 cases.The diameters of the annulus and sinus of Valsalva in subtype 1 (R-N) BAV patients were less than those in subtype 1 (L-R) BAV (P=0.01,0.02).There was no significant difference of the diameters of the annulus,sinus of Valsalva,sinotubular junction nor the proximal ascending aorta among different subtypes of BAV (all P＞0.05).Conclusion Subtype 1(L-R) is more common in BAV patients,which often associated with larger diameters of annulus and sinuses of Valsalva than subtype 1 (R-N) on echocardiography.

Objective:To explore the technical advantages of MR cholangiopancreatography (MRCP) with single breath holding high parallel acquisition factor 3-D variable flip angle fast spin echo (3D-SPACE) sequence.Methods:From November 2018 to March 2019, 75 patients who underwent MRCP examination in our hospital were prospectively enrolled, with single breath holding high parallel acquisition factor 3D-SPACE sequence and free breathing navigation gated 3D-SPACE sequence. Three experienced radiologists scored the overall image quality, artifacts, CBD visibility, left and right hepatic ducts, right anterior and posterior branches, second and third branches, main pancreatic duct and gallbladder duct with four scales. Paired t test was used for statistical analysis. Results:The scanning time of single breath holding method (18 s) was significantly shorter than that of free breathing diaphragm navigation method［264（226，313）s］, and the difference between the two methods was statistically significant ( Z=-7.520， P<0.001). The SNR, CR and CNR (8.31±4.23, 0.92±0.30, 11.46±5.77) of single breath holding method were lower than those of free breathing diaphragm navigation method (11.23±5.70, 0.93±0.38, 15.06±7.37), and the differences between the two methods were also statistically significant ( t=4.378, 3.429, 4.063, P<0.05). The overall image quality, artifact, the CBD, left and right hepatic duct, right anterior and posterior branchs, the second and third branches, main pancreatic duct and cystic duct of single breath holding method were higher than those of free breathing diaphragm navigation method, and the differences between the two methods were statistically significant ( P<0.001). Conclusions:Compared with the free breathing diaphragm navigation gated 3D-SPACE MRCP imaging method, the single breath holding high parallel acquisition factor 3D-SPACE MRCP imaging method has less artifacts and examination time, but higher visibility to pancreaticobiliary tree and work efficiency, which is worthy of further promotion.