In-depth study of the components of polymyxins is the key to controlling the quality of this class of antibiotics. Similarities and variations of components present significant analytical challenges. A two-dimensional (2D) liquid chromatography-mass spectrometr (LC-MS) method was established for screening and comprehensive profiling of compositions of the antibiotic colistimethate sodium (CMS). A high concentration of phosphate buffer mobile phase was used in the first-dimensional LC system to get the components well separated. For efficient and high-accuracy screening of CMS, a targeted method based on a self-constructed high resolution (HR) mass spectrum database of CMS components was established. The database was built based on the commercial MassHunter Personal Compound Database and Library (PCDL) software and its accuracy of the compound matching result was verified with six known components before being applied to genuine sample screening. On this basis, the unknown peaks in the CMS chromatograms were deduced and assigned. The molecular formula, group composition, and origins of a total of 99 compounds, of which the combined area percentage accounted for more than 95% of CMS components, were deduced by this 2D-LC-MS method combined with the MassHunter PCDL. This profiling method was highly efficient and could distinguish hundreds of components within 3 h, providing reliable results for quality control of this kind of complex drugs.

Objective:To analyze the clinical phenotype, sperm morphological characteristics and assisted reproductive therapy efficiency in patients with total globozoospermia.Methods:Four male patients with total globozoospermia were collected during November 2019 to May 2022 from Reproductive Medical and Genetic Center, the People's Hospital of Guangxi Zhuang Autonomous Region. Peripheral blood samples were collected for genetic detection and the whole exome sequencing to explore the pathogenic genes. Semen characteristics, sperm morphology and ultrastructure were analyzed. Four patients were treated with intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA). Conventional ICSI cycles ( n=9) were selected as control group, and the development dynamic parameters were monitored by Time-lapse. The fertilization and embryo development parameters, developmental dynamic parameters and clinical outcomes were analyzed between the two groups. Results:Four patients were complicated with low sperm motility and increased sperm DNA fragmentation. Sperm morphology analysis and acrosome fluorescence staining represented that all the spermatozoas were with a small round head lacked of acrosome. By the transmission electron microscope, it was observed that round-headed spermatozoas were lacked of acrosome completely, loose chromatin structure, vacuolation and other abnormal changes in the head, mitochondrial sheath in neck were reduced arranged in disorder, and the structure of "9+2" of the flagellar axial filament was incomplete. Of the 4 patients, 1 was homozygous deletion of DPY19L2 gene and 1 was homozygous frameshift mutation of DPY19L2 gene. There were no significance differences in fertilization rate, two pronuclei fertilization rate, day 3 high-quality embryo rate, day 6 blastocyst formation rate and day 6 high-quality blastocyst formation rate between total globozoospermia group and control group (all P>0.05). The developmental dynamic parameters as the time at which the second polar body is extruded, the time when both (or the last) PN disappear, two to six discrete cells in the total globozoospermia group were significantly earlier than those in control group, and the difference was statistically significant (all P<0.05). There was no significant difference in the embryo cleavage patterns between the two groups ( P>0.05). Among the 4 patients with total globozoospermia, 2 live births with signal healthy male baby were achieved by fresh embryo transfer, and 2 live births with one signal healthy male baby and one healthy female baby were achieved by frozen-thawed embryo transfer respectively. Conclusion:Abnormal morphology characteristics of spermatozoas from patients with total globozoospermia are obvious, patients with total globozoospermia could have favorable clinical outcomes following ICSI combined with AOA.

Objective:To analyze the clinical phenotype, sperm morphological characteristics and assisted reproductive therapy efficiency in patients with total globozoospermia.Methods:Four male patients with total globozoospermia were collected during November 2019 to May 2022 from Reproductive Medical and Genetic Center, the People's Hospital of Guangxi Zhuang Autonomous Region. Peripheral blood samples were collected for genetic detection and the whole exome sequencing to explore the pathogenic genes. Semen characteristics, sperm morphology and ultrastructure were analyzed. Four patients were treated with intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA). Conventional ICSI cycles ( n=9) were selected as control group, and the development dynamic parameters were monitored by Time-lapse. The fertilization and embryo development parameters, developmental dynamic parameters and clinical outcomes were analyzed between the two groups. Results:Four patients were complicated with low sperm motility and increased sperm DNA fragmentation. Sperm morphology analysis and acrosome fluorescence staining represented that all the spermatozoas were with a small round head lacked of acrosome. By the transmission electron microscope, it was observed that round-headed spermatozoas were lacked of acrosome completely, loose chromatin structure, vacuolation and other abnormal changes in the head, mitochondrial sheath in neck were reduced arranged in disorder, and the structure of "9+2" of the flagellar axial filament was incomplete. Of the 4 patients, 1 was homozygous deletion of DPY19L2 gene and 1 was homozygous frameshift mutation of DPY19L2 gene. There were no significance differences in fertilization rate, two pronuclei fertilization rate, day 3 high-quality embryo rate, day 6 blastocyst formation rate and day 6 high-quality blastocyst formation rate between total globozoospermia group and control group (all P>0.05). The developmental dynamic parameters as the time at which the second polar body is extruded, the time when both (or the last) PN disappear, two to six discrete cells in the total globozoospermia group were significantly earlier than those in control group, and the difference was statistically significant (all P<0.05). There was no significant difference in the embryo cleavage patterns between the two groups ( P>0.05). Among the 4 patients with total globozoospermia, 2 live births with signal healthy male baby were achieved by fresh embryo transfer, and 2 live births with one signal healthy male baby and one healthy female baby were achieved by frozen-thawed embryo transfer respectively. Conclusion:Abnormal morphology characteristics of spermatozoas from patients with total globozoospermia are obvious, patients with total globozoospermia could have favorable clinical outcomes following ICSI combined with AOA.

Objective:To develop the real-time radiotherapy monitoring system of three-dimensional (3D) point cloud by using depth camera and verify its feasibility.Methods:Taking the depth camera coordinate system as the world coordinate system, the conversion relationship between the simulation CT coordinate system and the world coordinate system was obtained from the calibration module. The patient's simulation CT point cloud was transformed into the world coordinate system through the above relationship, and registered with the patient's surface point cloud obtained in real-time manner by the depth camera to calculate the six-dimensional (6D) error, and complete the positioning verification and fractional internal position error monitoring in radiotherapy. Mean and standard deviation of 6D calculation error, Hausdorff distance of point cloud after registration and the running time of each part of the program were calculated to verify the feasibility of the system. Fifteen real patients were selected to calculate the 6D error between the system and cone beam CT (CBCT).Results:In the phantom experiment, the errors of the system in the x, y and z axes were (1.292±0.880)mm, (1.963±1.115)mm, (1.496±1.045)mm, respectively, and the errors in the rotation, pitch and roll directions were 0.201°±0.181°, 0.286°±0.326°, 0.181°±0.192°, respectively. For real patients, the translational error of the system was within 2.6 mm, the rotational error was approximately 1°, and the program run at 1-2 frames/s. The precision and speed met the radiotherapy requirement. Conclusion:The 3D point cloud radiotherapy real-time monitoring system based on depth camera can automatically complete the positioning verification before radiotherapy, real-time monitoring of body position during radiotherapy, and provide error visual feedback, which has potential clinical application value.

Objective:To investigate whether the early developmental dynamic phenotypes and kinetic parameters of embryos used for embryo selection are affected by the differences in the components of culture media utilized.Methods:The clinical data of patients undergoing in vitro fertilization (IVF) at Center for Reproductive Medicine and Genetics of People's Hospital of Guangxi Zhuang Autonomous Region from October 2016 to December 2018 were analyzed in a retrospective cohort study. According to the different culture media utilized, IVF cycles were divided into Cook group and Vitrolife group. After 1∶1 propensity-score matching (PSM), 59 IVF cycles were included in each group. Time-lapse imaging technology was used to analyze the early developmental dynamics of normal fertilized embryos between insemination and 68 h post insemination. Seven developmental dynamic phenotypes of embryos were annotated and the differences in the composition of dynamic phenotypes were compared between the two groups. Thirteen early developmental kinetic parameters were calculated, and the differences in the kinetic parameters of normal dynamic phenotypic embryos between the two groups were compared. According to the two published time-lapse embryo selection algorithms, the hierarchical distribution of normal dynamic phenotypic embryos of two groups was compared. Results:1) In Cook group, the composition of developmental dynamic phenotypes of embryos were 54.0% normal phenotype, 3.0% abnormal first cytokinesis (A1 cyt), 17.4% abnormal cleavage (AC), 5.2% reverse cleavage (RC), 3.2% chaotic cleavage (CC), 3.5% multinucleation (Mn) and 13.7% mixed phenotype, which were 49.3%, 4.0%, 19.1%, 7.5%, 2.1%, 6.4% and 11.6% in Vitrolife group, respectively. No statistically significant differences were observed between the two groups for the composition of dynamic developmental phenotypes ( P>0.05). 2) Compared with Vitrolife group, the 13 developmental kinetic parameters (tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, cc2, s2, t5_PNf and t8_PNf) of normal dynamic phenotype embryos in Cook group were slightly longer, and the average number of blastomeres in 68 h post insemination (EB68hpi) was less, but the differences were not statistically significant (all P>0.05). 3) No significant difference in hierarchical distribution of embryos was observed between Vitrolife group and Cook group according to algorithm A ( P>0.05). The difference of embryo hierarchical distribution between the two groups was statistically significant according to algorithm B ( P=0.040), the proportion of grade A + embryos in Vitrolife group was higher than that in Cook group [59.8% (125/209) vs. 43.3% (94/217)], and grade C proportion was lower [9.6% (20/209) vs. 20.3% (44/217)]. Conclusion:Although the early developmental dynamic phenotypes and kinetic parameters of embryos were not affected by the differences between Cook and Vitrolife sequential culture media, the applicability of different time-lapse embryo selection algorithms to the culture media is different, the embryo culture media utilized should be considered when selecting or constructing the algorithms.

Objective:To investigate whether the early developmental dynamic phenotypes and kinetic parameters of embryos used for embryo selection are affected by the differences in the components of culture media utilized.Methods:The clinical data of patients undergoing in vitro fertilization (IVF) at Center for Reproductive Medicine and Genetics of People's Hospital of Guangxi Zhuang Autonomous Region from October 2016 to December 2018 were analyzed in a retrospective cohort study. According to the different culture media utilized, IVF cycles were divided into Cook group and Vitrolife group. After 1∶1 propensity-score matching (PSM), 59 IVF cycles were included in each group. Time-lapse imaging technology was used to analyze the early developmental dynamics of normal fertilized embryos between insemination and 68 h post insemination. Seven developmental dynamic phenotypes of embryos were annotated and the differences in the composition of dynamic phenotypes were compared between the two groups. Thirteen early developmental kinetic parameters were calculated, and the differences in the kinetic parameters of normal dynamic phenotypic embryos between the two groups were compared. According to the two published time-lapse embryo selection algorithms, the hierarchical distribution of normal dynamic phenotypic embryos of two groups was compared. Results:1) In Cook group, the composition of developmental dynamic phenotypes of embryos were 54.0% normal phenotype, 3.0% abnormal first cytokinesis (A1 cyt), 17.4% abnormal cleavage (AC), 5.2% reverse cleavage (RC), 3.2% chaotic cleavage (CC), 3.5% multinucleation (Mn) and 13.7% mixed phenotype, which were 49.3%, 4.0%, 19.1%, 7.5%, 2.1%, 6.4% and 11.6% in Vitrolife group, respectively. No statistically significant differences were observed between the two groups for the composition of dynamic developmental phenotypes ( P>0.05). 2) Compared with Vitrolife group, the 13 developmental kinetic parameters (tPNa, tPNf, t2, t3, t4, t5, t6, t7, t8, cc2, s2, t5_PNf and t8_PNf) of normal dynamic phenotype embryos in Cook group were slightly longer, and the average number of blastomeres in 68 h post insemination (EB68hpi) was less, but the differences were not statistically significant (all P>0.05). 3) No significant difference in hierarchical distribution of embryos was observed between Vitrolife group and Cook group according to algorithm A ( P>0.05). The difference of embryo hierarchical distribution between the two groups was statistically significant according to algorithm B ( P=0.040), the proportion of grade A + embryos in Vitrolife group was higher than that in Cook group [59.8% (125/209) vs. 43.3% (94/217)], and grade C proportion was lower [9.6% (20/209) vs. 20.3% (44/217)]. Conclusion:Although the early developmental dynamic phenotypes and kinetic parameters of embryos were not affected by the differences between Cook and Vitrolife sequential culture media, the applicability of different time-lapse embryo selection algorithms to the culture media is different, the embryo culture media utilized should be considered when selecting or constructing the algorithms.

Objective:To develop a 3D visualization technology-assisted patient positioning system for radiotherapy and compare it with traditional patient positioning method for breast and pelvic radiotherapy.Methods:A total of 40 patients receiving radiotherapy in Changzhou No.2 People′s Hospital from June 2020 to April 2021 were selected for this study, including 20 patients with breast cancer and 20 patients with pelvic cancer.3D visualization reconstruction was carried out using the CT data of the patients for positioning. Then the 3D visualization models were integrated with the real treatment environment and were then shifted to the isocentral positions of accelerators through interactive operations. Based on this, the patients were actually positioned. Every week, all of the patients were firstly treated with traditional positioning, followed by 3D visualization-guided positioning. As a result, 240 times of positioning data of all patients were collected in three weeks. They were compared with the data of cone-beam CT(CBCT)-guided positioning, which served as the gold standard.Results:The absolute positioning errors of 3D visualization-guided positioning along x, y and z axes were (1.92±1.23), (2.04±1.16), and (1.77±1.37)mm, respectively for patients with breast cancer and were (2.07±1.08), (1.33±0.88), and (1.99±1.25)mm, respectively for patients with pelvic cancer. Compared with traditional positioning method , the accuracy of 3D visualization-guided positioning along x、 y, and z axes was increased by 38.83%, 52.40% and 33%, respectively for patients with breast cancer and was improved by 36.84%, 54.04% and 52.58% for patients with pelvic cancer, with all differences being statistically significant along y and z axes ( t=2.956-5.734, P< 0.05). Meanwhile, the error distribution of the two positioning method was statistically significant along in y axis for patients with breast cancer( χ2=7.481, P<0.05) and was statistically significant along each axis for patients with pelvic cancer( χ2=5.900, 6.415, 7.200, P<0.05). Conclusions:The positioning method guided by 3D visualization technology can effectively improve the positioning accuracy of patients with breast cancer and patients with pelvic cancer and is of value in potential clinical application.

Objective:To explore the volume resolution of prostate motion target by four-dimensional (4D) ultrasound.Methods:The prostate ultrasound model was selected, and the group comparison study was conducted using 4D ultrasound to outline the prostate target under different motion amplitudes (A) and motion period (T). The simulated A value was set as 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, and 5 mm, respectively. The T value was set as 1 s, 2 s, 3 s, and 4 s, respectively. The volume of the target of the model prostate was calculated, and the static ultrasound image of the target was used as the control group to analyze the difference between two groups.Results:When the model was still, the size of the target of ultrasound was consistent with that of CT scan ( P>0.05). When the A values were 0.5 mm and 1 mm, there was no statistical difference between the volume in period 1-4 s and the volume in the target at rest (all P>0.05). When the A values were 2 mm and 3 mm, and the T values were 1 s, 2 s and 3 s there was statistical difference between the volume of target and that of of static ultrasonic target (all P<0.05). When the A value was 2 mm and the T value was 4 s, there was no statistical difference between the target volume and the static target volume ( P=0.710). The range within the group was 6.7 cm 3, and the standard deviation was 1.15 cm 3. When the A value was 3 mm and the T value was 4 s, the volume repeatability of the target was poor, and the range within the group was 14.4 cm 3; when the A values were 4 mm and 5 mm, and the T values were 1-4 s, the range within the group was 3.27-17.63 cm 3 and 6.51-21.02 cm 3, respectively. The volume repeatability of the target under each period was extremely poor, which could not meet the clinical requirements. Conclusion:4D ultrasound can provide reliable reference data for patients′ target delineation within 1-4 s of motion cycle and within 1 mm of motion amplitude, which exerts on effect upon the original position of probe.

Objective:To establish a technical system for early judgment and rescue artificial oocyte activation of oocytes after fertilization failed, intracytoplasmic sperm injection (ICSI) and to explore the activation efficiency and application value.Methods:Firstly, a retrospective analysis was performed in 150 ICSI cycles cultured by Time-lapse system during January 2017 to March 2018 from Reproductive Medical and Genetic Center, the People's Hospital of Guangxi Zhuang Autonomous Region, the time distribution of the second polar body (Pb2) exclusion and its relationship with fertilization and embryo development outcomes, the feasibility of Pb2 exclusion as an early indicator of fertilization failure in ICSI was discussed. Secondly, 225 fertilization failed oocytes from 93 ICSI cycles during March 2018 to June 2019 were collected for randomized controlled trials according to different artificial activation modes. They were divided into three groups including non-activation group (NAOA group), rescue activation group (RAOA group) and late activation group (LAOA group). At the same time, the rest of injected oocytes from ICSI cycles include in this study were used as control group. Fertilization and embryo development parameters were used to explore the efficiency and application value of rescue artificial activation for after fertilization failure oocytes ICSI.Results:Time-lapse monitoring showed that the fertilization rate and 2PN fertilization rate were 99.91% and 97.76% in the Pb2-exclusion group, 0.03% and 0% in the without Pb2-exclusion group after ICSI, with significant differences between the two groups ( P<0.001); the exclusion time of Pb2 after ICSI was (3.04±1.45) h, and the distribution and proportion of Pb2 exclusion time were 0-3 h (58.00%), 3-5 h (36.29%), 5-8 h (3.92%) and >8 h (1.21%). The Pb2 exclusion rate, the fertilization rate and the 2PN fertilization rate in NAOA group were all 0%, and the 2PN fertilization rate (36.00%), day 3 (D3) high-quality embryo rate (8.00%), day 5 (D5) blastocyst formation rate (0%) and D5 high-quality blastocyst formation rate (0%) in LAOA group were significantly lower than those in RAOA group (60.00%, 44.19%, 51.16%, 25.58%) ( P=0.005, P=0.002, P<0.001, P=0.005), and also in control group (97.63%, 48.62%, 63.23%, 37.94%) ( P<0.001); the Pb2 exclusion rate (84.00%), the fertilization rate (81.33%), the 2PN fertilization rate (60.00%) and the 2PN cleavage rate (95.56%) in RAOA group were significantly lower than those in control group (100.00%, 99.68%, 97.63%, 99.51%) ( P<0.001, P<0.001, P<0.001, P=0.04). However, there was no significant difference in D3 high-quality embryo rate, D5 blastocyst formation rate and D5 high-quality blastocyst formation rate between the two groups ( P>0.05). Conclusion:Time-lapse monitoring of the Pb2 exclusion can be used as an early judgment indicator of fertilization failure in ICSI cycles. Rescue artificial activation of fertilization failed oocytes in 5 h after ICSI can achieve normal fertilization and acceptable embryo development outcomes.