Objective To investigate the efficacy of discontinuous density gradient combined with swim-up method for bacterial removal in frozen semen,and to provide experimental evidence for the safe application of frozen semen in assisted reproductive technology.Methods A total of 208 frozen semen samples containing non-pathogenic bacteria,cryopreserved in our human sperm bank from January 2019 to December 2023,were selected.The thawed semen samples were processed using discontinuous density gradient combined with swim-up method.To simulate in vitro fertilization procedures,the processed sperm suspensions were subjected to 5-day in vitro culture for bacterial residue monitoring.Prior to cryopreservation,all semen samples underwent routine bacterial culture to exclude pathogenic microorganisms.Post-thaw semen samples,processed sperm suspensions,and 5-day culture media were sent to an independent third-party laboratory for comprehensive bacterial identification.Results Among the 208 frozen semen samples containing non-pathogenic bacteria,41 bacterial species were identified,with 1～4 species per sample.After processing with discontinuous density gradient combined with swim-up method,the sterility rate of the sperm suspension reached 89.42%(186/208),with residual bacteria including Enterococcus faecalis,Corynebacterium glucuronolyticum,Group B Streptococcus,and Corynebacterium glycinophilum.After 5-day in vitro culture,the sterility rate increased to 93.75%(195/208),with residual bacteria limited to Entero-coccus faecalis and Group B Streptococcus.Conclusions The combined method of discontinuous density gradient and swim-up technique effectively eliminates most bacteria in frozen semen.However,the clearance rate for Entero-coccus faecalis and Group B Streptococcus remains below 90%.Optimization of pre-freezing bacterial screening and post-thaw processing techniques is necessary to enhance the safety of frozen semen in assisted reproductive technology.

Objective To investigate the efficacy of discontinuous density gradient combined with swim-up method for bacterial removal in frozen semen,and to provide experimental evidence for the safe application of frozen semen in assisted reproductive technology.Methods A total of 208 frozen semen samples containing non-pathogenic bacteria,cryopreserved in our human sperm bank from January 2019 to December 2023,were selected.The thawed semen samples were processed using discontinuous density gradient combined with swim-up method.To simulate in vitro fertilization procedures,the processed sperm suspensions were subjected to 5-day in vitro culture for bacterial residue monitoring.Prior to cryopreservation,all semen samples underwent routine bacterial culture to exclude pathogenic microorganisms.Post-thaw semen samples,processed sperm suspensions,and 5-day culture media were sent to an independent third-party laboratory for comprehensive bacterial identification.Results Among the 208 frozen semen samples containing non-pathogenic bacteria,41 bacterial species were identified,with 1～4 species per sample.After processing with discontinuous density gradient combined with swim-up method,the sterility rate of the sperm suspension reached 89.42%(186/208),with residual bacteria including Enterococcus faecalis,Corynebacterium glucuronolyticum,Group B Streptococcus,and Corynebacterium glycinophilum.After 5-day in vitro culture,the sterility rate increased to 93.75%(195/208),with residual bacteria limited to Entero-coccus faecalis and Group B Streptococcus.Conclusions The combined method of discontinuous density gradient and swim-up technique effectively eliminates most bacteria in frozen semen.However,the clearance rate for Entero-coccus faecalis and Group B Streptococcus remains below 90%.Optimization of pre-freezing bacterial screening and post-thaw processing techniques is necessary to enhance the safety of frozen semen in assisted reproductive technology.

Objective To understand the changing distribution and antimicrobial resistance profiles of Klebsiella strains in 52 hospitals across China in the CHINET Antimicrobial Resistance Surveillance Program from 2015 to 2021.Methods Antimicrobial susceptibility testing was carried out according to the unified CHINET protocol.The susceptibility results were interpreted according to the breakpoints in the Clinical & Laboratory Standards Institute(CLSI)M100 document.Results A total of 241,549 nonduplicate Klebsiella strains were isolated from 2015 to 2021,including Klebsiella pneumoniae(88.0％),Klebsiella aerogenes(5.8％),Klebsiella oxytoca(5.7％),and other Klebsiella species(0.6％).Klebsiella strains were mainly isolated from respiratory tract(48.49±5.32)％.Internal medicine(22.79±3.28)％,surgery(17.98±3.10)％,and ICU(14.03±1.39)％were the top 3 departments where Klebsiella strains were most frequently isolated.K.pneumoniae isolates showed higher resistance rate to most antimicrobial agents compared to other Klebsiella species.Klebsiella isolates maintained low resistance rates to tigecycline and polymyxin B.ESBLs-producing K.pneumoniae and K.oxytoca strains showed higher resistance rates to all the antimicrobial agents tested compared to the corresponding ESBLs-nonproducing strains.The K.pneumoniae and carbapenem-resistant K.pneumoniae(CRKP)strains isolated from ICU patients demonstrated higher resistance rates to majority of the antimicrobial agents tested than the strains isolated from non-ICU patients.The CRKP strains isolated from adult patients had higher resistance rates to most of the antimicrobial agents tested than the corresponding CRKP strains isolated from paediatric patients.Conclusions The prevalence of carbapenem-resistant strains in Klebsiella isolates increased greatly from 2015 to 2021.However,the Klebsiella isolates remained highly susceptible to tigecycline and polymyxin B.Antimicrobial resistance surveillance should still be strengthened for Klebsiella strains.

Objective To establish an allogeneic rat model of endometriosis and to evaluate the effects of gonadotropin-releasing hormone (GnRH) agonist GenSci006 on experimental rat endometriosis. Methods Endometrium from SPF grade donor female SD rats were transplanted onto the abdominal wall of recipient female rats to construct an allogeneic endometriosis model. The rats undergoing sham surgery were divided into the sham group. Three weeks later, the length, width and height of the ectopic endometrium were measured, and the volume of the endometrium (V₁) was calculated before drug administration. The modeling rats were randomly divided into four groups: model group, triptorelin group (0.25 mg/kg), GenSci006-1 group (0.125 mg/kg) and GenSci006-2 group (0.25 mg/kg). Each group had 16 rats and received a single dose of the corresponding drug. The sham group and model group were administered an equal volume of solvent. Three weeks after administration, ectopic endometrium was measured to calculate the volume V₂ and inhibition rate. The effect of GenSci006 on rat uterus and ovarian tissues was assessed by comparing organ coefficients and changes in pathological sections. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of serum estradiol (E₂), progesterone (P₄), follicle stimulating hormone (FSH), and luteinizing hormone (LH). Real-time fluorescent quantitative PCR was used to detect the expression of GnRH receptor (GnRHR) mRNA in the hypothalamus and pituitary. Western blot was used to detect the expression of estradiol receptor alpha (ERα), beta (ERβ) and progesterone receptor (PR) in ectopic endometrium. Results Three weeks after administration, compared with the model group, the body weight of rats in the triptorelin and GenSci006-2 groups significantly increased (P < 0.05), while the volume of ectopic endometrium significantly decreased (P < 0.05). Compared with the sham group, the model group showed no significant changes in uterine and ovarian organ coefficients or endometrial thickness (P > 0.05). Compared with the model group, the uterine organ coefficients and endometrial thickness were significantly reduced in the triptorelin and GenSci006-2 groups (P < 0.05). Compared with the sham group, the serum levels of E₂, P₄, FSH and LH in the model group showed no significant changes (P > 0.05). Compared with the model group, the ovarian organ coefficient and serum P₄ levels of rats in the Triptorelin, GenSci006-1, and GenSci006-2 groups were significantly reduced (P < 0.05), while the serum LH levels of rats in the GenSci006-1 group were significantly increased (P < 0.05). However, there were no significant changes in serum E₂ and FSH levels in each group (P > 0.05). Compared with the model group, the expression levels of GnRHR mRNA in the pituitary tissue of rats in the triptorelin and GenSci006-2 groups were significantly downregulated (P < 0.05), with no significantly changes in the hypothalamus (P > 0.05). There were no significant changes in the expression level of GnRHR mRNA in the hypothalamus or the protein levels of ERα, ERβ and PR in the ectopic endometrial tissue in any group (P > 0.05). Conclusion The allogeneic endometriosis rat model is a suitable animal model for screening and evaluating drugs for treating endometriosis. The volume of ectopic endometrium, inhibition rate, uterine and ovarian organ coefficients, and serum E₂ levels may serve as indicators for detecting drug efficacy.

Objective:To explore the effects of consecutively repeated application of emergency contraception pills (ECPs) containing levonorgestrel (LNG) on the female fertility and the health outcomes of F1 generation rats.Methods:Female SPF rats were intragastric administered with LNG-ECPs consecutively for 3 (P-3), 6 (P-6) and 12 (P-12) estrous cycles (three times in each estrous cycle), respectively. Under each administration schedule, rats were randomly divided into 2 groups according to body weight stratification using random numbers generated in Excel, i.e. LNG-ECPs group and solvent control group, administered with 0.12 mg/kg LNG-ECPs and corresponding volumes of 0.5% CMC-Na, respectively. Four hours after the last dosing, half of the animals (12-18) in each group were allotted randomly for dissection (6-9) and mating (6-9), respectively. The remaining half (12-18) were recovered for 3 estrous cycles, and then were randomly allocated for dissection (6-9) and mating (6-9). Organ coefficients were calculated. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, testosterone, anti-Müllerian hormone (AMH) and free thyroid hormone 3 (fT3) were examined by enzyme linked immunosorbent assay (ELISA). Ovarian tissues were sectioned and stained with hematoxylin and eosin (HE) for follicle counting. In addition, the pregnancy rate and litter size of the female rats were recorded, and the growth indexes and behavioral parameters of the cubs were measured. Moreover, RNA sequencing (RNA-seq) of the ovarian tissues was performed to establish the differential expression gene profile of ovarian injury induced by LNG-ECPs. Then gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed.Results:1) After consecutive administration for 3 and 6 estrous cycles, LNG-ECPs showed no significant impact on the serum hormone levels and female fertility (all P>0.05), and the growth indexes and behavioral parameters of the F1 generation (all P>0.05). 2) After consecutive administration for 12 estrous cycles, the serum levels of FSH [(0.21±0.17) U/L], LH [(0.27±0.08) U/L] and progesterone [(0.68±0.23) μg/L] in LNG-ECPs group decreased significantly compared with those in solvent control group [(1.00±0.82) U/L, P=0.043; (1.00±0.50) U/L, P=0.006; (1.00±0.20) μg/L, P=0.027], while the level of estradiol [(2.24±1.03) μg/L] and testosterone [(1.25±0.25) μg/L] increased noticeably compared with those in solvent control group [(1.00±0.35) μg/L, P=0.019; (1.00±0.07) μg/L, P=0.044]. The number of primordial follicles (4.88±2.36) lost distinctly, while the number of atretic follicles (24.38±5.01) increased markedly in LNG-ECPs group compared with those in solvent control group (16.13±9.36, P=0.005; 19.13±2.30, P=0.018). In addition, the weight-loaded swimming (WLS) time of the F1 generation rats from the LNG-ECPs group [(157.13±32.29) s] reduced obviously compared with those from the solvent control group [(198.06±40.01) s, P=0.003]. Moreover, after recovering for 3 estrous cycles, LNG-ECPs significantly increased the levels of FSH [(2.48±1.18) U/L], LH [(1.60±0.41) U/L], testosterone [(1.37±0.23) μg/L] and the ratio of FSH/LH (1.61±0.41) compared with those in solvent control group [(1.00±0.67) U/L, P=0.024; (1.00±0.27) U/L, P=0.014; (1.00±0.18) μg/L, P=0.011; 1.00±0.49, P=0.042], respectively. Additionally, the serum levels of estradiol [(0.49±0.15) μg/L] and AMH [(0.79±0.15) μg/L] were significantly lower than those in solvent control group [(1.00±0.37) μg/L, P=0.011; (1.00±0.10) μg/L, P=0.016]. In addition, the number of primordial follicles in rats of LNG-ECPs group (6.25±5.06) were obviously less than that in solvent control group (12.00±5.56, P=0.048). Furthermore, the total distance in open field [(89.85±36.98) m] and the swimming time in WLS [(112.00±29.52) s] in rats treated with LNG-ECPs both decreased distinctly compared with those in solvent control group [(147.55±23.13) m, P<0.001; (137.69±25.85) s, P=0.014]. 3) According to transcriptomic analysis, Cd5, Cxcr1, Lexm, Fga, Mybphl and Gstm5 were the significant differential expressed genes (DEGs) in the ovarian tissues of rats. These DEGs were involved in pathways related to steroid hormone biosynthesis, including terpenoid backbone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion. Additionally, these genes were involved in metabolic processes, such as carbon metabolism, butanoate metabolism, cysteine and methionine metabolism. And the genes were also involved in immunoregulatory processes including cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptors. Conclusion:Consecutively repeated administering LNG-ECPs to the female rats in a short-term period (<12 cycles) did not demonstrate significant adverse effects on female fertility and the growth and development and the behaviors of their F1 generation cubs. However, long-term repeated treatment with LNG-ECPs (12 cycles) caused ovarian injury on female rats and showed negative impacts on the health of the F1 generation cubs, and no significant improvement was observed after recovering for 3 estrous cycles.

Objective:To explore the effects of consecutively repeated application of emergency contraception pills (ECPs) containing levonorgestrel (LNG) on the female fertility and the health outcomes of F1 generation rats.Methods:Female SPF rats were intragastric administered with LNG-ECPs consecutively for 3 (P-3), 6 (P-6) and 12 (P-12) estrous cycles (three times in each estrous cycle), respectively. Under each administration schedule, rats were randomly divided into 2 groups according to body weight stratification using random numbers generated in Excel, i.e. LNG-ECPs group and solvent control group, administered with 0.12 mg/kg LNG-ECPs and corresponding volumes of 0.5% CMC-Na, respectively. Four hours after the last dosing, half of the animals (12-18) in each group were allotted randomly for dissection (6-9) and mating (6-9), respectively. The remaining half (12-18) were recovered for 3 estrous cycles, and then were randomly allocated for dissection (6-9) and mating (6-9). Organ coefficients were calculated. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, testosterone, anti-Müllerian hormone (AMH) and free thyroid hormone 3 (fT3) were examined by enzyme linked immunosorbent assay (ELISA). Ovarian tissues were sectioned and stained with hematoxylin and eosin (HE) for follicle counting. In addition, the pregnancy rate and litter size of the female rats were recorded, and the growth indexes and behavioral parameters of the cubs were measured. Moreover, RNA sequencing (RNA-seq) of the ovarian tissues was performed to establish the differential expression gene profile of ovarian injury induced by LNG-ECPs. Then gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed.Results:1) After consecutive administration for 3 and 6 estrous cycles, LNG-ECPs showed no significant impact on the serum hormone levels and female fertility (all P>0.05), and the growth indexes and behavioral parameters of the F1 generation (all P>0.05). 2) After consecutive administration for 12 estrous cycles, the serum levels of FSH [(0.21±0.17) U/L], LH [(0.27±0.08) U/L] and progesterone [(0.68±0.23) μg/L] in LNG-ECPs group decreased significantly compared with those in solvent control group [(1.00±0.82) U/L, P=0.043; (1.00±0.50) U/L, P=0.006; (1.00±0.20) μg/L, P=0.027], while the level of estradiol [(2.24±1.03) μg/L] and testosterone [(1.25±0.25) μg/L] increased noticeably compared with those in solvent control group [(1.00±0.35) μg/L, P=0.019; (1.00±0.07) μg/L, P=0.044]. The number of primordial follicles (4.88±2.36) lost distinctly, while the number of atretic follicles (24.38±5.01) increased markedly in LNG-ECPs group compared with those in solvent control group (16.13±9.36, P=0.005; 19.13±2.30, P=0.018). In addition, the weight-loaded swimming (WLS) time of the F1 generation rats from the LNG-ECPs group [(157.13±32.29) s] reduced obviously compared with those from the solvent control group [(198.06±40.01) s, P=0.003]. Moreover, after recovering for 3 estrous cycles, LNG-ECPs significantly increased the levels of FSH [(2.48±1.18) U/L], LH [(1.60±0.41) U/L], testosterone [(1.37±0.23) μg/L] and the ratio of FSH/LH (1.61±0.41) compared with those in solvent control group [(1.00±0.67) U/L, P=0.024; (1.00±0.27) U/L, P=0.014; (1.00±0.18) μg/L, P=0.011; 1.00±0.49, P=0.042], respectively. Additionally, the serum levels of estradiol [(0.49±0.15) μg/L] and AMH [(0.79±0.15) μg/L] were significantly lower than those in solvent control group [(1.00±0.37) μg/L, P=0.011; (1.00±0.10) μg/L, P=0.016]. In addition, the number of primordial follicles in rats of LNG-ECPs group (6.25±5.06) were obviously less than that in solvent control group (12.00±5.56, P=0.048). Furthermore, the total distance in open field [(89.85±36.98) m] and the swimming time in WLS [(112.00±29.52) s] in rats treated with LNG-ECPs both decreased distinctly compared with those in solvent control group [(147.55±23.13) m, P<0.001; (137.69±25.85) s, P=0.014]. 3) According to transcriptomic analysis, Cd5, Cxcr1, Lexm, Fga, Mybphl and Gstm5 were the significant differential expressed genes (DEGs) in the ovarian tissues of rats. These DEGs were involved in pathways related to steroid hormone biosynthesis, including terpenoid backbone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion. Additionally, these genes were involved in metabolic processes, such as carbon metabolism, butanoate metabolism, cysteine and methionine metabolism. And the genes were also involved in immunoregulatory processes including cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptors. Conclusion:Consecutively repeated administering LNG-ECPs to the female rats in a short-term period (<12 cycles) did not demonstrate significant adverse effects on female fertility and the growth and development and the behaviors of their F1 generation cubs. However, long-term repeated treatment with LNG-ECPs (12 cycles) caused ovarian injury on female rats and showed negative impacts on the health of the F1 generation cubs, and no significant improvement was observed after recovering for 3 estrous cycles.

Dalbergiae Odoriferae Lignum has the effects of removing stasis, stanching bleeding, regulating Qi and relieving pain, and has a good therapeutic effect on coronary heart disease, angina pectoris, myocardial infarction and other cardiovascular diseases. Through the previous literature research, our group found that there were differences in the original plants of Dalbergiae Odoriferae Lignum described in ancient works on materia medica, and the corresponding efficacy was also significantly different. Therefore, this paper made an in-depth study and summarized various botanical sources of Dalbergiae Odoriferae Lignum involved in literature. The results showed that Dalbergiae Odoriferae Lignum had been divided into imported varieties(Fanjiang) and domestic varieties(Guangjiang or Tujiang). Among them, the former original plants mainly concerned with Dalbergia parviflora Roxb. and Dalbergia sissoo Roxb., while the latter concerned with Acronychia pedunculata (L.) Miq., Dalbergia benthamii Prain, Dalbergia pinnata (Lour.) Prain, Dalbergia hancei Benth., Dalbergia tsoi Merr. et Chun and Dalbergia odorifera T. Chen. The above research results help to enrich the understanding of Dalbergiae Odoriferae Lignum and have positive practical significance for the protection and rational utilization of its original plant resources.

Taking "count down your weight—start from 'diet'" as an example, this article discusses the design and practice of SPOC (small private online course) mixed teaching based on MOOC (massive open online course) in general medical courses. By designing teaching methods and teaching content, and using formative evaluation methods, the SPOC mixed teaching was implemented for 201 students from Sichuan University in the spring of 2020. According to the teaching evaluation and preliminary teaching effect, students generally believed that teaching resources were relatively abundant and the communication effects were generally recognized, as well as, it could significantly improve students' interest in and effect of general medical courses.

10.3969/j.issn.1007-3969.2013.04.004

Objective To establish the synthetic method of the substrate for angiotensin-converting enzyme(ACE),and investigate the assay method of sernm ACE. Methods The substrate was prepared by the use of N-hydroxysuccinimide ester in peptide synthesis,and serum ACE was determined by spectrophotometric assay using suhstrate. Results The substrate of furanacryloyl-phenylalanyl-glycyl-glycine with purity of 99.5% was obtained.The K_m value for the substrate was 0.25 mmol/L,the normal reference interval for serum ACE activity was 46.7 U/L to 113.3 U/L,and the within-run and between-run coefficient of variation(CVs)ranged from 3.9 to 4.9%.The serum ACE activity in patients with squamous carcinoma and adenocarcinoma of lung were significantly decreased(P<0.05),while that in patients with lung small cell carcinoma was not significantly decreased(P>0.05). Conclusion The synthesized substrate could be reliably used as a marker for diagnosis of different lung cancers in clinics.