目的 建立医疗过程中血液样本可控性采集模式,保证样本的采集、处理过程可回溯,为肿瘤、自身免疫性疾病、心脑血管疾病及代谢性疾病的研究提供高质量的血液样本.方法 通过建立网络化样本采集管理流程,将样本采集电子申请、项目管理以及样本处理中涉及血液样本的各项前处理因素整合到电子工作程序中,保证标准流程的执行情况处于可控状态,形成规范化的血液采集、前处理、保存和管理办法.结果 仁济医院血液样本库有临床疾病20余种,含有完整的临床及随访资料.血液样本与相应的组织样本、临床信息相匹配;系统记录每份样本采集时间、到库时间、入库时间等;可通过组合查询筛选相应质量要求的样本.共采集血液样本40000余份,抽样检查90%的样本分子生物学指标稳定.结论 仁济医院可控性血液采集、处理模式和管理模式为综合性医院血液生物样本库建设提供管理经验,为血液样本的规范化、高质量采集、前处理、保存提供详细操作经验参考,为肿瘤、自身免疫性疾病、心脑血管疾病及代谢性疾病的研究提供高质量的血液样本及信息资源.

Genome-wide association studies (GWAS) have identified several common loci contributing to non-obstructive azoospermia (NOA). However, a substantial fraction of NOA heritability remains undefined, especially those low-frequency [defined here as having a minor allele frequency (MAF) between 0.5 and 5%] and rare (MAF below 0.5%) variants. Here, we performed a 3-stage exome-wide association study in Han Chinese men to evaluate the role of low-frequency or rare germline variants in NOA development. The discovery stage included 962 NOA cases and 1348 healthy male controls genotyped by exome chips and was followed by a 2-stage replication with an additional 2168 cases and 5248 controls. We identified three low-frequency variants located at 6p22.2 (rs2298090 in HIST1H1E encoding p.Lys152Arg: OR = 0.30, P = 2.40 × 10(-16)) and 6p21.33 (rs200847762 in FKBPL encoding p.Pro137Leu: OR = 0.11, P = 3.77 × 10(-16); rs11754464 in MSH5: OR = 1.78, P = 3.71 × 10(-7)) associated with NOA risk after Bonferroni correction. In summary, we report an instance of newly identified signals for NOA risk in genes previously undetected through GWAS on 6p22.2-6p21.33 in a Chinese population and highlight the role of low-frequency variants with a large effect in the process of spermatogenesis.

目的 初步研究β-catenin蛋白在不同类型睾丸肿瘤中的表达特性,并探讨β-catenin分子的表达与睾丸肿瘤的关系,为临床诊断或治疗睾丸肿瘤提供一定依据.方法 随机收集我院于2012年1月至2012年12月期间确诊的21例睾丸肿瘤组织和1例梗阻性无精子症(OA)患者的睾丸活检组织(作为正常对照,其睾丸生精功能正常);同时,选取5只9周龄的ICR小鼠作为实验补充.采用了免疫组化、Western blots和RT-qPCR等方法来检测β-catenin的表达情况.结果 在梗阻性患者中,β-catenin在精原细胞的胞膜和胞浆中呈现低表达;在小鼠中,β-catenin也仅表达在精原细胞的胞浆和胞膜上.在睾丸肿瘤组织中,睾丸孤立性纤维肿瘤中可检测到β-catenin的mRNA和蛋白的高表达,分别为OA患者的9.4倍和17倍,且其β-catenin主要异位表达在细胞核中,可检测到磷酸化的β-catenin的表达.然而,β-catenin在睾丸精原细胞肿瘤和间质细胞肿瘤中不表达或低表达.结论 本研究初步表明β-catenin与正常的睾丸生精过程及病理的睾丸孤立性纤维肿瘤发生发展相关,而与睾丸精原细胞肿瘤和间质细胞肿瘤之间无明显相关性.但仍需扩大样本数量进行研究,或采用动物实验深入研究.

目的:拟通过比较梗阻(OA)和非梗阻性无精子(NOA)症患者精液细胞DNA倍体分析与细胞学检查结果的特点,以期建立一种无创的诊疗方案以鉴别OA与NOA.方法:NOA患者20例和OA患者10例,按照WHO方法进行精液分析诊断,获取其精液标本,液化后离心取沉淀物,1份采用体积分数70％冰乙醇重悬精液沉淀,4℃固定后用PI染色,采用流式细胞仪进行DNA倍体分析.同时,取另1份精液沉淀涂片,进行Diff-Quick染色和免疫细胞化学染色.结果:DNA倍体分析结果表明,20例NOA患者精液中均存在单倍体(1N)、二倍体(2N)和四倍体(4N)细胞,其中2N细胞含量最高,约占71.25±8.73％.1N细胞的百分比含量为5.46±2.93％,4N细胞含量为3.28±2.54％.10例OA患者精液中有8例只存在2N细胞,含量为71.67±13.09％,未检测到1N和4N,2例未检测到各种倍体细胞.精液细胞学检查结果表明,20例NOA患者中有15例患者精液中可检测出生精细胞,5例患者精液中未检出生精细胞.10例OA患者精液中均未检出生精细胞,其中2例没有任何细胞.结论:精液细胞DNA倍体分析与细胞学检查作为2种无创的检查方法,均可作为评估患者生精功能的辅助诊疗手段,两者结合可作为鉴别OA和NOA的辅助诊断指标.

Objective: To investigate the influence of culture temperature and concentration of fluorescence dye Hoechst 33258 (H258) on vital labeling of human sperm. Methods: Fourteen semen samples were randomly selected, and were cultured with 0 mg/mL (control), 0.01 mg/mL and 0.1 mg/mL H258 for 1 h under different temperatures (0°C, room temperature and 37°C) after washing. Progressive motility of human sperm was assessed, and H258 staining rates of the sperm were recorded. Semen samples without treatment were served as blank controls. Results: Under the conditions of this research, H258 concentrations and culture temperatures had significant impact on sperm motility. The percents of progressive motility of sperm gradually decreased with the increase of H258 concentrations and decrease of culture temperatures (P<0.01). The staining rates of the sperm gradually increased with the culture temperatures and mass concentrations of H258, and there were significant differences among staining rates of the sperm cultured with different temperatures at the mass concentration of 0.1 mg/mL (P<0.01). Conclusion: H258 can be used in vital labeling of human sperm. The optimal staining condition was 37°C+0.1 mg/mL H258 in this study. Culture condition should be determined by specific purpose.

Male factor infertility affects one-sixth of couples worldwide, and non-obstructive azoospermia (NOA) is one of the most severe forms. Our previous genome-wide association study (GWAS) identified three susceptibility loci for NOA in Han Chinese men. Here we test promising associations in an extended three-stage validation using 3,608 NOA cases and 5,909 controls to identify additional risk loci. We find strong evidence of three NOA susceptibility loci (P<5.0 × 10(-8)) at 6p21.32 (rs7194, P=3.76 × 10(-19)), 10q25.3 (rs7099208, P=6.41 × 10(-14)) and 6p12.2 (rs13206743, P=3.69 × 10(-8)), as well as one locus approaching genome-wide significance at 1q42.13 (rs3000811, P=7.26 × 10(-8)). In addition, we investigate the phenotypic effect of the related gene (gek, orthologous to CDC42BPA) at 1q42.13 on male fertility using a Drosophila model. These results advance our understanding of the genetic susceptibility to NOA and provide insights into its pathogenic mechanism.