[Application value of frozen-thawed mouse round spermatids in in vitro fertilization].

OBJECTIVE: To investigate the application value of the frozen-thawed round spermatids of the mouse in in vitro fertilization (IVF). METHODS: Haploid spermatids of the mouse obtained in vitro were divided into a frozen-thawed and a fresh group and oocytes were collected from 6－8 weeks old female mice. After diamidino-phenyl-indole (DAPI) staining, the oocytes were subjected to intracytoplasmic round spermatid injection (ROSI), 259 in the frozen-thawed and 238 in the fresh group. Comparisons were made between the two groups in the capacities of fertilization and embryonic development. RESULTS: The survival rate of the frozen-thawed haploid spermatids was (75.9 ± 2.3) %. No statistically significant differences were observed between the frozen-thawed and fresh groups after ROSI in the rates of fertilization (51.9 vs 55.7%, P >0.05), 2-cell embryos (51.0 vs 62.2%, P >0.05), 4－8-cell embryos (41.8 vs 42.9%, P >0.05), or morula-blastocysts (12.2 vs 21.4%, P >0.05). CONCLUSIONS: Frozen-thawed round spermatids of the mouse are similar to fresh ones in their capacities of fertilization and embryonic development.

[An optimal method for cryopreservation of microamount round spermatids of the mouse].

OBJECTIVE: To search for an optimal protocol and freezing conditions for the cryopreservation of microamount round spermatids of the mouse. METHODS: We compared the survival rates of frozen-thawed microamount round spermatids of the mouse achieved by vitrification or standard slow freezing with different concentrations of glycerol (5, 7, or 9%) and different lengths of equilibrium time (0, 15, 30, 45, or 60 min). RESULTS: Under the conditions of 7% glycerol and 30 min equilibrium, both vitrification and standard slow freezing achieved high survival rates of spermatids, and the former obtained an even higher rate than the latter ([72.9 ± 15.4]% vs [58.2 ± 17.7]%, P < 0.05). CONCLUSION: A high rate of frozen-thawed microamount round spermatids of the mouse can be achieved by vitrification under the conditions of 7% glycerol and 30 min equilibrium.

[EGF and SCF promote the proliferation and differentiation of mouse spermatogenic cells in vitro].

OBJECTIVE: To study the promoting effects of the epidermal growth factor (EGF) and stem cell factor (SCF) on the proliferation and differentiation of spermatogenic cells in mice. METHODS: We cocultured in vitro spermatogenic cells of male mice aged 7 - 8 days in the medium with EGF and/or SCF at the concentrations of 5, 10, 20, 40 and 100 ng/ml, respectively. Then we observed the survival rate and morphological changes of the spermatogenic cells, detected the expressions of the pachytene-specific phosphoprotein gene (P19) and haploid sperm cell-specific transition protein gene (TP1), and analyzed the ploidy of the cells. RESULTS: After cocultured with EGF or SCF for 2 - 4 days, the spermatogenic cells began to proliferate in masses or chains in all concentration groups, most obviously in the 20 ng/ml EGF and 40 ng/ml SCF groups. At 7 days, both the number and survival rate of spermatogenic cells were significantly higher in the 20 ng/ml EGF and 40 ng/ml SCF groups than in the others (P < 0.05), and meanwhile, the P19/TP1 ratio was obviously decreased and the rate of haploid sperm markedly increased in the 40 ng/ml SCF group (P < 0.05). Combination of EGF and SCF remarkably promoted the proliferation of the spermatogenic cells (P < 0.05). CONCLUSION: Both EGF and SCF could increase the number and survival rate of spermatogenic cells. SCF could promote the formation of haploid sperm, and the combination of the two cytokines could enhance the effect on the proliferation of spermatogenic cells.

[Application of chemical activation to in vitro fertilization by round spermatid injection in mice].

OBJECTIVE: To search for an optimal activation protocol by comparing the chemical activation effects of single-activator and combined activation protocols on mouse oocytes following injection of round spermatids (ROSI) from spermatogenic cells cultured in vitro. METHODS: Using different concentrations of ethanol, ionomycin (Ion), calcium ionophore A23187 (CIA), strontium chloride (SrCl2), cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP) , we activated post-ROSI oocytes for different times, and activated them by combined protocols at optimal concentrations and action times according to different activation channels. We compared the activation effects of single-activator and combined activation protocols by comparing the rates of fertilization, cleavages, and morulas and blastocysts. RESULTS: With a single activator, the optimal protocols of different activators were as follows: 7% ethanol for 6 min, 5 micromol/L CIA for 5 min, 5 micromol/L Ion for 5 min, 2 mmol/L 6-DMAP for 2 h, 10 mmol/L SrCl2 for 1.5 h, and 10 microg/ml CHX for 1.5 h, among which 10 mmol/L SrCl2 for 1.5 h achieved the highest rate of morulas and blastocysts, significantly better than CHX (P < 0.05) but with no remarkable difference from other activators. The ethanol + 6-DMAP group showed a significantly higher rate of morulas and blastocysts (29.63%) than all other combined activation groups and single-activator groups except SrCl2 (P < 0.05), and it also exhibited higher rates of normal fertilization, cleavages and morula than the SrCl2 group, but with no significant difference. CONCLUSION: The single-activator 10 mmol/L SrCl2 for 1.5 h and the combined activation of 7% ethanol for 6 min + 2 mmol/L 6-DMAP for 2 h are the optimal protocols for chemical activation of mouse oocytes following ROSI, and the combined activation of ethanol + 6-DMAP is even superior to the single-activator protocol.

CT-guided microcoil localization of pulmonary nodules: the effect of the position of microcoil proximal end on the incidence of microcoil dislocation.

OBJECTIVES: To evaluate the effect of the position of microcoil proximal end on the incidence of microcoil dislocation during CT-guided microcoil localization of pulmonary nodules (PNs). METHODS: This retrospective study included all patients with PNs who received CT-guided microcoil localization before video-assisted thoracoscopic urgery (VATS) resection from June 2016 to December 2019 in our institution. The microcoil distal end was less than 1 cm away from the nodule, and the microcoil proximal end was in the pleural cavity (the pleural cavity group) or chest wall (the chest wall group). The length of microcoil outside the pleura was measured and divided into less than 0.5 cm (group A), 0.5 to 2 cm (group B) and more than 2 cm (group C). Microcoil dislocation was defined as complete retraction into the lung (type I) or complete withdrawal from the lung (type II). The rate of microcoil dislocation between different groups was compared. RESULTS: A total of 519 consecutive patients with 571 PNs were included in this study. According to the position of microcoils proximal end on post-marking CT, there were 95 microcoils in the pleural cavity group and 476 in the chest wall group. The number of microcoils in group A, B, and C were 67, 448 and 56, respectively. VATS showed dislocation of 42 microcoils, of which 30 were type II and 12 were type I. There was no statistical difference in the rate of microcoil dislocation between the pleural cavity group and the chest wall group (6.3% vs 7.6%, x2 = 0.18, p = 0.433). The difference in the rate of microcoil dislocation among group A, B, and C was statistically significant (11.9%, 5.8%, and 14.3% for group A, B, and C, respectively, x2 = 7.60, p = 0.008). In group A, 75% (6/8) of dislocations were type I, while all eight dislocations were type II in group C. CONCLUSIONS: During CT-guided microcoil localization of PNs, placing the microcoil proximal end in the pleura cavity or chest wall had no significant effect on the incidence of microcoil dislocation. The length of microcoil outside the pleura should be 0.5 to 2 cm to reduce the rate of microcoil dislocation. ADVANCES IN KNOWLEDGE:: CT-guided microcoil localization can effectively guide VATS to resect invisible and impalpable PNs. Microcoil dislocation is the main cause of localization failure. The length of microcoil outside the pleura is significantly correlated with the rate and type of microcoil dislocation. Placing the microcoil proximal end in the pleura cavity or chest wall has no significant effect on the rate of microcoil dislocation.

[ERK and P38MAPK expressions and human sperm motility].

OBJECTIVE: To compare the phosphorylation and protein expression of extracellular-signal regulated protein kinase (ERK) and P38 mitogen activated protein kinase (P38 MAPK) in the ejaculated spermatozoa of healthy volunteers and asthenospermia males, and to explore the correlation of ERK and P38 MAPK with human sperm motility. METHODS: Semen samples were collected from 20 healthy volunteers (sperm concentration > or = 20 x 10(6)/ml, grade a sperm > or = 25% or grade a + b sperm > or = 50%) and 20 infertile males with asthenospermia (sperm concentration > or = 20 x 10(6)/ml, grade a sperm < 25% and grade a + b sperm < or = 40%) and classified as a control and an asthenospermia group. Total protein in spermatozoa was extracted from all the subjects, and Western blotting was used to detect phosphorylation and protein expression levels of ERK and P38 MAPK. RESULTS: Compared with the control group, the protein expression levels of ERK and P38 MAPK and the phosphorylation level of P38 MAPK were significantly increased in the asthenospermia group (P < 0.05), but there were no statistically significant differences in the phosphorylation level of ERK between the two groups (P > 0.05). CONCLUSION: The up-regulated protein expressions of ERK and P38MAPK and increased phosphorylation level of P38 MAPK in human sperm may be involved in the pathogenesis of asthenospermia.

CT-guided transthoracic needle biopsy of pulmonary lesions: comparison between the cutting needle and aspiration needle.

OBJECTIVES: To compare CT-guided transthoracic cutting needle biopsy (TCNB) with transthoracic aspiration needle biopsy (TANB) for pulmonary lesions with respect to the diagnostic accuracy and complication rate. METHODS: Of the 859 cases that underwent consecutive CT-guided biopsy of pulmonary lesions, 713 cases confirmed by surgical pathology or clinical follow-up were enrolled. Of these, the first consecutive 275 cases underwent TANB, and the remaining 438 received TCNB. The final diagnosis determined the accuracy of biopsy. Based on the post-biopsy CT and clinical medical records, the presence or absence of biopsy-related complications was determined. The χ2 test was used to compare the differences between TCNB and TANB in terms of diagnostic accuracy and complication rate. RESULTS: Among the 713 biopsy lesions, the final diagnosis was malignant in 411 cases and benign in 302 cases. As compared to TANB, the diagnostic accuracy of TCNB (98.9% vs 93.8%, χ2 = 14.35, p < 0.01), sensitivity to malignant lesions (97.8% vs 90.6%, χ2 = 10.58, p < 0.01), negative predictive value (97.6% vs 84.8%, χ2 = 19.03, p < 0.01), and specific diagnostic rate for benign lesions (73.4% vs 57.9%, χ2 = 7.29, p < 0.01) were improved. On the other hand, a statistical difference was detected between TCNB and TANB with respect to the incidence of pneumothorax (20.6% vs 13.1%, χ2 = 6.46, p = 0.01), hemorrhage (32.2% vs 13.1%, χ2 = 33.03, p < 0.01), and hemoptysis (8.2% vs 3.3%, χ2 = 6.87, p < 0.01). One patient died just several minutes after TCNB due to severe hemorrhage with hemoptysis. CONCLUSIONS: Compared to TANB, CT-guided TCNB improves the diagnostic accuracy of pulmonary lesions, but complication rate increases significantly. ADVANCES IN KNOWLEDGE: In general, TCNB should be recommended, especially for highly suspicious benign lesions. For patients with small lesions adjacent to vessels or vessels within the lesion, TANB should be considered.

CT-Guided Microcoil Localization of Small Peripheral Pulmonary Nodules to Direct Video-Assisted Thoracoscopic Resection without the Aid of Intraoperative Fluoroscopy.

OBJECTIVE: To evaluate the feasibility, safety, and effectiveness of CT-guided microcoil localization of solitary pulmonary nodules (SPNs) for guiding video-assisted thoracoscopic surgery (VATS). MATERIALS AND METHODS: Between June 2016 and October 2019, 454 consecutive patients with 501 SPNs who received CT-guided microcoil localization before VATS in our institution were enrolled. The diameter of the nodules was 0.93 ± 0.49 cm, and the shortest distance from the nodules to the pleura was 1.41 ± 0.95 cm. The distal end of the microcoil was placed less than 1 cm away from the nodule, and the proximal end was placed outside the visceral pleura. VATS was performed under the guidance of implanted microcoils without the aid of intraoperative fluoroscopy. RESULTS: All 501 nodules were marked with microcoils. The time required for microcoil localization was 12.8 ± 5.2 minutes. Microcoil localization-related complications occurred in 179 cases (39.4%). None of the complications required treatment. A total of 463 nodules were successfully resected under the guidance of implanted microcoils. VATS revealed 38 patients with dislocated microcoils, of which 28 underwent wedge resection (21 cases under the guidance of the bleeding points of pleural puncture, 7 cases through palpation), 5 underwent direct lobectomy, and the remaining 5 underwent a conversion to thoracotomy. In 4 cases, a portion of the microcoil remained in the lung parenchyma. CONCLUSION: CT-guided microcoil localization of SPNs is safe and reliable. Marking the nodule and pleura simultaneously with microcoils can effectively guide the resection of SPNs using VATS without the aid of intraoperative fluoroscopy.