五子衍宗丸对少弱精子症模型大鼠精子线粒体膜电位及超微结构影响

目的:研究五子衍宗丸对少弱精子症模型大鼠精子线粒体膜电位(MMP)水平及线粒体超微结构的影响。方法:取体重为200～220 g雄性SD大鼠60只,随机分成正常组,模型组,对照组(黄精赞育胶囊组),五子衍宗丸低、中、高剂量组,除正常组外,其他各组大鼠灌服雷公藤多苷[30 mg/(kg.d)],连续8周,制备少弱精子症模型。造模结束后,正常组、模型组给予等容量蒸馏水[10 ml/(kg.d)],对照组给予黄精赞育胶囊溶液[3.01 g/(kg.d)],五子衍宗丸各组分别给予水提液,低剂量组[2.30 g生药/(kg.d)],中剂量组[4.60 g生药/(kg.d)],高剂量组[9.20 g生药/(kg.d)],连续30 d。末次给药后30 min,采用荧光流式细胞技术(JC-1染色)测定精子MMP水平(JC-1+%),并通过透射电镜观察精子线粒体超微结构的改变。结果:①MMP:JC-1+%和强度分别为:正常组70.80±4.92、4 360±945;模型组33.77±6.19、1 4685±496;对照组56.34±10.35、3 277±895;五子衍宗丸低剂量组40.80±10.40、2 016±767;中剂量组59.40±6.51、3 897±643;高剂量组60.71±7.81、3 371±6467。造模各组大鼠精子线粒体JC-1+%及强度均明显下降,与正常组比较差异有显著性意义(P均<0.05);连续给药30 d后,给药各组均能明显提高精子MMP,增加JC-1+%,除低剂量组外,其他用药各组与模型组比较差异有显著性意义(P均<0.05)。②精子线粒体超微结构:雷公藤造模后,精子外膜松散、变性,线粒体肿胀、大小不一、线粒体膜不完整,轴丝结构不清或出现断裂。给予五子衍宗丸30 d后,精子外膜及线粒体膜结构完整,减少线粒体肿胀,轴丝及微管结构基本正常。结论:雷公藤多苷能降低精子MMP水平,破坏线粒体的结构。五子衍宗丸能明显提高少弱精子症模型大鼠精子MMP水平,减轻精子线粒体结构损伤。保护精子线粒体结构与功能的完整是五子衍宗丸治疗少弱精子症的机制之一。     

黄精赞育胶囊对弱精子症患者精子线粒体膜电位的影响

目的:观察黄精赞育胶囊对弱精子症患者精子线粒体膜电位的影响及临床疗效。方法:弱精子症患者70例分药物治疗组(n=39)和空白对照组(n=31)做队列观察研究。药物治疗组予黄精赞育胶囊口服3个月,1次4粒,每日3次。服用时可备孕,期间若配偶怀孕则停止用药。空白对照组不予药物干预。观察初次就诊和3个月后精液参数变化,及JC-1染色后流式细胞仪检测精子线粒体膜电位(MMP)值的变化。结果:两组各失随访1例。药物治疗组总有效率为71.05%,服药过程中3例患者女方自然妊娠。药物治疗组35例患者完成观察期结束后检查,MMP%、精子总活力、前向运动精子百分率(PR%)、精子形态均有改善,其变化值较空白对照组30例患者的测定值有显著差异(P0.01),分别为△MMP%:(20.28±14.21)%vs(1.19±10.36)%;△PR%:(17.58±12.73)%vs(2.26±8.29)%;△总活力:(20.68±14.12)%vs(3.46±8.67)%;△正常形态精子百分率:(3.37±3.99)%vs(0.23±3.48)%。MMP%与精子总活力(r=0.69)、PR%(r=0.75)、正常形态精子百分率(r=0.26)具有显著相关性(P0.01)。结论:黄精赞育胶囊能提高精子MMP值,改善线粒体功能,从而有效增加精子活力及PR%并改善精子形态,对治疗弱精子症安全有效。     

解脲支原体感染对大鼠生精细胞线粒体核糖体蛋白S22的影响及知柏地黄汤的干预作用

目的:观察解脲支原体(UU)感染对大鼠睾丸生精细胞线粒体核糖体蛋白S22(MRPS22)表达的影响及知柏地黄汤的干预作用。方法:UU感染SD雄性大鼠模型45只,随机分成模型组、知柏地黄汤组、西药组,并以健康大鼠为对照组,每组15只。造模成功后第10天起知柏地黄汤组予以知柏地黄汤灌胃[1 g/(k·d)],西药组予以阿奇霉素灌胃[0.105 g/(k·d)],对照组、模型组予以同体积生理盐水灌胃,连续灌胃21 d。采用TUNEL法检测生精细胞凋亡,电镜观察生精细胞线粒体结构,流式细胞仪检测线粒体膜电位(MMP),RT-PCR检测大鼠睾丸生精细胞MRPS22 m RNA表达,Western印迹检测大鼠睾丸生精细胞MRPS22蛋白表达。观察各组大鼠睾丸生精细胞凋亡指数(AI)、生精细胞线粒体结构、MMP、生精细胞MRPS22 m RNA和蛋白表达情况。结果:模型组大鼠睾丸生精细胞AI[(11.23±1.65)%]显著高于知柏地黄汤组[(6.62±0.49)%]和西药组[(7.82±0.81)%](P 0. 01),知柏地黄汤组显著低于西药组(P 0.05)。知柏地黄汤组及西药组大鼠睾丸线粒体结构较模型组显著改善。模型组大鼠睾丸生精细胞MMP水平[(8. 77±1. 73)%]显著低于对照组[(22. 33±1. 66)%](P 0. 01),知柏地黄汤组[(18. 26±1. 32)%]显著高于模型组(P 0.01)和西药组[(15.91±1.69)%],西药组MMP水平显著高于模型组(P 0. 01)。模型组MRPS22 m RNA表达(8.02±3.21)与对照组(15.43±2.54)、知柏地黄汤组(11. 26±3. 82)相比均具有显著性差异(P 0. 01),知柏地黄汤组MRPS22 m RNA表达显著高于西药组(8. 79±2. 03)(P 0. 01)。模型组(22. 65±5. 31) MRPS22蛋白表达与对照组(33.31±7.09)、知柏地黄汤组(33.35±3.96)和西药组(28.11±4.13)相比均具有显著性差异(P 0. 01),知柏地黄汤组显著高于西药组(P 0.01)。大鼠睾丸生精细胞MRPS22蛋白表达量与MMP呈显著正相关(r=0.639,P 0.01)。结论:UU感染可能通过抑制大鼠睾丸生精细胞MRPS 22 m RNA及蛋白表达,降低MMP,引起生精细胞的凋亡。知柏地黄汤通过改善UU感染大鼠睾丸生精细胞MRPS22 m RNA及蛋白表达,提高MMP,减轻大鼠睾丸生精细胞凋亡。     

知柏地黄汤对解脲脲原体感染模型大鼠精子线粒体呼吸链复合物的影响

目的:观察知柏地黄汤对解脲脲原体(UU)感染模型大鼠精子线粒体呼吸链复合物的影响。方法:90只雄性SD大鼠,随机分成空白对照组、模型对照组、中药组(知柏地黄汤8.56 g生药/(kg·d))、西药组[强力霉素20 mg/(kg·d)]、中西药组(知柏地黄汤+强力霉素等效剂量之和),除空白对照组外,其他组均采用经大鼠膀胱注射解脲脲原体建立UU感染动物模型,然后,连续灌胃21 d(各组给予对应药物)后处死,取附睾标本检测精子质量,分光光度法检测精子线粒体呼吸链复合物(Ⅰ、Ⅱ、Ⅲ、Ⅳ)活性。结果:①模型对照组、空白对照组、中药组、西药组、中西药组建模10 d后UU培养阳性率分别为92.9%、0%、33.3%、26.7%、20.0%,模型对照组UU阳性率明显高于其他治疗组(P0.05)。②模型对照组、空白对照组、中药组、西药组、中西药组大鼠附睾精子浓度分别为(0.97±0.23)、(3.02±0.52)、(1.21±0.35)、(1.02±0.31)、(1.52±0.28)×10~6/ml,活动率分别为(58.62±15.36)%、(80.45±7.21)%、(75.52±8.78)%、(68.43±10.25)%、(78.25±7.67)%,a+b级精子百分率分别为(6.15±1.02)%、(10.32±1.14)%、(10.12±1.08)%、(9.01±1.27)%、(10.74±1.03)%;模型对照组大鼠附睾精子浓度、活动率及a+b级精子百分率明显低于空白对照组(P0.01);各治疗组大鼠附睾精子活动率及a+b级精子百分率明显高于模型对照组(P0.01)。③模型对照组、空白对照组、中药组、西药组、中西药组大鼠精子线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性分别为(31.54±16.25)、(136.86±6.34)、(100.68±14.41)、(81.68±6.78)、(124.06±5.54)μmol/(min·mg),(9.50±3.86)、(20.34±0.37)、(10.88±1.04)、(12.93±1.07)、(16.23±0.60)μmol/(min·mg),(5.58±1.79)、(19.60±0.61)、(11.34±1.35)、(13.87±1.23)、(15.96±0.69)μmol/(min·mg),(9.54±1.34)、(28.98±3.33)、(17.02±2.04)、(18.41±2.67)、(21.66±2.93)μmol/(min·mg)。模型对照组大鼠精子线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ活性明显低于空白对照组(P0.01);中药组、西药组、中西药组大鼠精子线粒体呼吸链复合物Ⅰ、Ⅲ、Ⅳ活性明显高于模型对照组(P0.01),西药组、中西药组大鼠精子线粒体呼吸链复合物Ⅱ活性高于模型对照组(P0.05)。结论:知柏地黄汤能够提高UU感染大鼠精子质量及线粒体呼吸链复合物活性。这可能是知柏地黄汤治疗UU感染所致不育症的机制之一。     

虾青素在奥硝唑致少弱精子症大鼠氧化损伤中的保护作用研究

目的:探讨虾青素(AST)对奥硝唑(ORN)致少弱精子症大鼠精子质量的改善作用及其机制。方法:40只成年雄性SD大鼠,随机分成5组,每组8只。分别为A组(溶剂对照组):0.5%羧甲基纤维素钠溶剂+1 ml玉米油、B组(ORN低剂量造模组):ORN 400 mg/(kg·d)、C组(ORN高剂量造模组):ORN 800 mg/(kg·d)、D组(ORN低剂量造模+AST治疗组):ORN 400 mg/(kg·d)+AST 20 mg/(kg·d)、E组(ORN高剂量造模+AST治疗组):ORN 800 mg/(kg·d)+AST 20 mg/(kg·d),灌胃3周后水合氯醛腹腔麻醉大鼠;取1侧附睾尾部检测精子浓度和活力,另1侧附睾组织匀浆,检测谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GR)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量。结果:与A组相比,B组附睾尾精子活力,附睾GSH-Px、SOD活性显著降低,MDA含量显著上升(P﹤0.05);C组附睾尾精子活力、浓度、睾丸系数,附睾组织GSH-Px、GR、CAT、SOD活性均显著降低,而MDA含量显著上升(P﹤0.05)。AST治疗组:与B组相比,D组附睾尾精子活力和附睾组织SOD活性显著增加[(45.3±8.7)%vs(66.3±8.9)%;(116.7±25.3)U/mg prot vs(146.1±23.8)U/mg prot,P﹤0.05],而MDA含量显著下降[(1.68±0.45)nmol/mg prot vs(1.19±0.42)nmol/mg prot,P﹤0.05];与C组相比,E组实验后体重增量、精子活力显著增加,而MDA含量显著下降[(89.0±9.5)%vs(99.9±4.1)%;(17.9±3.5)%vs(27.3±5.3)%;(2.03±0.30)nmol/mg prot vs(1.52±0.41)nmol/mg prot,P﹤0.05]。结论:AST可提高ORN致大鼠少弱精子症模型的精子质量,其机制可能是提高了大鼠附睾的抗氧化能力。     

硫辛酸对奥硝唑所致少弱精子症大鼠精子发生的保护作用研究

目的:研究硫辛酸(LA)对奥硝唑(ORN)所致少弱精子症模型雄性大鼠生精功能的保护作用。方法:取70只雄性SD大鼠,随机分成7组,每组10只,分别为:A组(溶剂对照组):1 ml 0.5%羧甲基纤维素钠(CMC-Na)+1 ml橄榄油;B组(低剂量ORN造模组):400 mg/kg ORN+1 ml橄榄油;C组(低剂量ORN+低剂量LA治疗组):400 mg/kg ORN+50 mg/kg LA;D组(低剂量ORN+高剂量LA治疗组):400 mg/kg ORN+100 mg/kg LA;E组(高剂量ORN造模组):800 mg/kg ORN+1 ml橄榄油;F组(高剂量ORN+低剂量LA治疗组):800 mg/kg ORN+50 mg/kg LA;G组(高剂量ORN+高剂量LA治疗组):800 mg/kg ORN+100 mg/kg LA。连续给药20 d,处死大鼠,称量大鼠体重、睾丸、附睾、精囊重量,计算脏器指数,检测附睾精子计数及活力,睾丸、附睾做HE染色观察组织形态学改变。结果:与A组相比,E组大鼠体重增量、睾丸、附睾脏器指数明显减少[(117.67±11.53)g vs(88.11±12.65)g;(1.06±0.12)%vs(0.65±0.13)%;(0.21±0.03)%vs(0.17±0.01)%,P均0.01];与E组相比,F组大鼠附睾脏器指数明显增加[(0.17±0.01)%vs(0.20±0.02)%,P0.01],G组大鼠体重增量、睾丸、附睾脏器指数明显增加[(88.11±12.65)g vs(102.70±16.10)g,P0.05;(0.65±0.13)%vs(0.95±0.06)%,P0.01;(0.17±0.01)%vs(0.19±0.02)%,P0.05],精囊脏器指数各组之间并无明显差异。与A组相比,B组大鼠精子活力明显降低[(74.12±8.73)%vs(40.25±6.08)%,P0.01],E组大鼠精子计数与活力明显降低[(38.59±6.40)×105/100 mg vs(18.67±4.59)×105/100 mg;(74.12±8.73)%vs(27.58±8.43)%,P均0.01];与B组相比,C、D组大鼠精子活力明显增加[(40.25±6.08)%vs(58.13±7.62)%;(40.25±6.08)%vs(76.04±8.44)%,P均0.01];与E组相比,F、G组大鼠精子计数及活力明显增加[(18.67±4.59)×10~5/100 mg vs(25.63±9.66)×10~5/100 mg,P0.05;(18.67±4.59)×10~5/100 mg vs(29.92±4.15)×10~5/100 mg,P0.01;(27.58±8.43)%vs(36.56±11.08)%,P0.05;(27.58±8.43)%vs(45.05±9.59)%,P0.01]。A、B、C、D组大鼠睾丸、附睾组织形态学无明显改变。E组大鼠睾丸与A组相比,生精小管腔内可见坏死脱落的生精细胞,生精细胞层次不清,排列紊乱;附睾管腔中精子数目明显减少,并伴有较多非细胞成分存在。F、G组大鼠睾丸生精小管内精子数目增加,但仍可见到生精小管内有生精细胞脱落,生精细胞层次不清晰,排列紊乱,但较E组有明显改善;F、G组大鼠附睾管腔中精子数目明显减少,可见散在、脱落的生精细胞,但较E组有明显改善。结论:LA能够改善ORN对大鼠造成的生殖系统损伤,提高精子质量,对生殖系统有较好的保护作用。     

RHO/ROCK信号通路在精子抗冷冻损伤中的作用

目的:探讨RHO/ROCK信号通路在人精子抗冷冻损伤中的作用,为高效精液冷冻保护剂的研制提供理论依据。方法:选取健康精液25份,每份精液分为新鲜组、对照组与RHO通路抑制剂组(抑制剂组)。检测冷冻前后各组精液精子活力、精子存活率、精子膜完整率、正常形态精子百分率、精子DNA碎片指数(DFI)、精子顶体酶活性及精子线粒体膜电位变化;免疫荧光染色检测RHOa/ROCK蛋白在精子中的表达。结果:抑制剂组精液冷冻复苏后精子活力[(57.50±6.83)%vs(51.20±7.70)%,P=0.002]、精子存活率[(60.24±5.53)%vs(52.87±5.07)%,P=0.001]、精子膜完整率[(67.10±4.43)%vs(59.78±5.56)%,P=0.001]、正常形态精子百分率[(7.46±1.28)%vs(4.83±1.11)%,P=0.001]、精子DFI[(18.87±4.07)%vs(27.64±6.64)%,P=0.001]、精子线粒体膜电位(63.11±2.97 vs 56.30±4.28,P=0.001)指标均明显优于对照组;抑制剂组冷冻后精子顶体酶活性与对照组差异无统计学意义(98.30±11.33 vs 97.65±9.31,P0.05)。免疫荧光染色显示RHOa/ROCK蛋白在精子头、颈部广泛表达。结论:RHO/ROCK信号通路在精子冷冻损伤中具有一定作用,抑制其通路活性可明显提高精子抗冷冻损伤的能力。     

左卡尼汀对冷冻精子运动参数和精子线粒体功能的影响

目的:探讨左卡尼汀(LC)对人冷冻精子的影响。方法:将每份供精志愿者精液分为6组:新鲜精液组(FE组);常规冷冻组(Non-LC组,冷冻保护剂中未添加LC); LC 1～4组,冷冻保护剂中LC浓度分别为:1、2. 5、5、10 mmol/L。通过观察冷冻复苏后精子活力和运动参数改变,筛选最佳LC工作浓度。伊红-苯胺黑染色法评估精子质膜完整性(PMI),JC-1法评估精子线粒体膜电位(MMP),DCFH-DA法评估活性氧(ROS),探索LC对精子冷冻损伤的影响机制。结果:与FE组相比,冷冻复苏后精子(Non-LC组和LC各组),前向运动精子百分率(PR%)和运动参数(VAP、VSL、VCL)均明显下降(P 0. 05)。与Non-LC组相比,LC 3组PR%[(47. 0±4. 3)%vs(41. 9±4. 6)%,P=0. 0261)]和VAP [(38. 9±4. 2)μm/s vs (34. 9±2. 6)μm/s,P=0. 0152)]改善明显,确定5 mmol/L为实验最佳LC工作浓度。与FE组比较,Non-LC组冷冻复苏后精子PMI[(52. 7±5. 7)%vs (75. 5±5. 4)%]、MMP[(44. 5±3. 5)%vs(57. 3±4. 4)%]明显降低(P 0. 01),ROS[(12. 5±3. 9)%vs(6. 8±2. 4)%]明显升高(P 0. 01);与Non-LC组比较,LC组精子PMI[(70. 1±8. 2)%]和MMP[(50. 3±3. 4)%]明显升高(P 0. 01、0. 05),ROS[(8. 4±5. 3)%]明显降低(P 0. 05)。结论:LC可能通过降低精子ROS,提高精子MMP,保护精子质膜,改善冷冻后精子活力和运动参数。     

LonP1表达与线粒体稳态以及精子活力的关联性研究

目的探究线粒体lon蛋白水解酶(LonP1)对精子线粒体稳态的维持调节作用及其可能存在的调节机制。方法收集2016年6月至2017年6月于温州医科大学附属第一医院生殖中心进行精液常规检查的120名男性精液标本,根据精子活力分为正常活力精子(NS)和弱精子症精子(AS)两组,采用计算机辅助精液分析系统(CASA)检测各组精子运动参数,通过免疫荧光法和免疫蛋白印迹法检测精子中LonP1的蛋白表达水平,流式细胞术检测各组精子的线粒体膜电位强度(MMP,以绿色荧光强度/红色荧光强度比值表示,比值越小代表膜电位越高)和线粒体内活性氧(mROS)水平,比较两组各指标参数差异,并分析LonP1表达水平和精子活力及精子线粒体功能相关指标(MMP,mROS)的关联性。另外,NS组精液经密度梯度离心法处理后,沉淀精子加入培养液重悬,以LonP1功能抑制剂CDDO-ME 2.5μmoL/L和5μmol/L为终浓度进行体外培养,同时设置空白对照组(0μmol/L),比较各组精子运动参数、LonP1表达水平以及精子线粒体功能相关指标。结果 (1)人类成熟精子中存在LonP1的表达,且稳定表达于精子中段的线粒体鞘部;(2)NS组和AS组精子LonP1表达水平(0.52vs.0.22)与精子活力(69.89%vs.26.10%)、MMP(4.72%vs.12.73%)呈正相关(P0.05),与mROS含量呈负相关(418.85vs.460.52)(P0.05);(3)经LonP1功能抑制剂CDDO-ME处理后,与空白对照组比较,CDDO-ME药物处理组的LonP1蛋白表达水平随CDDO-ME作用浓度增加而显著降低(P0.05),5μmol/L浓度组的MMP水平显著下降(78.81%vs.7.34%)、mROS含量增加(640.68vs.462.26),差异均有统计学意义(P0.05);(4)CDDO-ME处理后,除精子活率(PR+NP)外,精子运动参数平均路径(VAP)和曲线速率(VSL)均随CDDO-ME作用浓度增加而下降(P0.05),与空白对照组相比,5μmol/L浓度组的精子活力(PR)(52.08%vs.65.15%)、曲线速率(VCL)(48.28μm/s vs.78.78μm/s)、侧摆幅度(ALH)(1.28μm vs.1.93μm)、直线性(LIN)(38.83%vs.56.05%)和前向性(STR)(45.53%vs.81.25%)均显著下降,差异有统计学意义(P0.05)。结论精子线粒体鞘部存在LonP1的稳定表达,LonP1能够通过精子线粒体稳态维持改善精子活力。     

益精方对小鼠精子线粒体功能的影响

目的观察益精方对环磷酰胺所致少弱精子症模型小鼠精子线粒体功能的影响。方法将40只雄性昆明小鼠随机分为对照组(CG)、模型组(MG)、小剂量中药治疗组(SG)和大剂量中药治疗组(LG)。按60mg/kg体质量给CG小鼠腹腔注射生理盐水(NS),给MG、SG、LG小鼠腹腔注射环磷酰胺,每天1次,连续5d,第6天开始按体质量分别给SG和LG小鼠灌服益精方,剂量分别为人类常规用量(以60 kg为标准)的1倍和5倍,MG小鼠给予等体积的NS灌胃,每天1次,连续34 d,CG常规饲养,然后对小鼠进行精液常规分析,流式细胞术检测精子线粒体功能。结果 CG、MG、SG和LG组小鼠附睾精子密度分别为(5.20±1.34)、(1.73±0.03)、(2.08±0.01)、(3.31±0.56)×106/m L;精子活力[(a+b)级精子百分率]分别为(14.49±0.30)%、(6.64±1.88)%、(11.99±1.01)%、(19.40±3.13)%;精子活率[(a+b+c)级精子百分率]分别为(68.39±15.13)%、(39.96±4.89)%、(62.28±4.43)%、(73.61±5.05)%。MG组精子密度、精子活力、精子活率显著低于CG组(P0.05);经治疗后LG组精子密度、精子活力和精子活率明显增加,与MG组比较有显著性差异(P0.05),而与CG组在精子活力和精子活率上没有明显区别。在CG、MG、SG和LG组,线粒体功能良好精子的百分率分别为(1105.93±0.33)%、(848.55±6.15)%、(888.77±22.46)%、(1000.885±72)%,线粒体功能丧失精子的百分率分别为(27.94±0.32)%、(42.43±1.00)%、(35.11±1.23)%、(30.91±0.44)%,MG小鼠精子线粒体功能良好的表达量明显下降,而线粒体功能丧失的表达量明显增多,与CG小鼠比较均有明显差异(P0.05);经治疗后,LG小鼠精子线粒体功能良好的表达量显著性增加(P0.05),同时线粒体功能丧失的表达量明显减少,与MG小鼠比较有明显差异(P0.05)。结论腹腔注射环磷酰胺可使小鼠精子密度、精子活力、精子活率降低,大剂量益精方可以增加小鼠精子密度,并增加精子线粒体功能,提高精子活力和精子活率,从而达到治疗少弱精子症的目的。     

Accurate sperm morphology assessment predicts sperm function

Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.     

附睾、睾丸精子卵浆内单精子注射治疗阻塞性无精症引起不育

为评价附睾或睾丸精子卵浆内单精子注射（ＩＣＳＩ）治疗阻塞性无精症引起不育的疗效，对３１例无精子男性不育患者配偶进行超排卵治疗３７个周期，获卵当日从患者附睾取精，其中２４例获得活动精子，７例失败，改用钳取睾丸曲细精管从中分离精子以供ＩＣＳＩ。结果：共获卵４５３个，附睾、睾丸精子ＩＣＳＩ受精率分别为５５７％、６１７％，平均每周期移植胚胎３７个，总妊娠率每周期２９７％。结论：只要获得活动精子ＩＣＳＩ，阻塞性无精症患者也有机会生育     

Computerized sperm motility and application of sperm cryopreservation

An objective method for measuring sperm motion characteristics was developed on an Intellect 100 Quantel Image Analysis System suitable for various image cytometric applications. It provided overall analysis of percent motility (% MS) as well as individual and mean measurements of motion characteristics, including vigor characteristics such as curvilinear velocity (Vc), straight line velocity (Vsl), and trajectory pattern characteristics, that is, progressiveness ratio (PR) and amplitude of lateral head displacement (Alh). Evaluation of the method for reproducibility and accuracy showed reliable measurements of these parameters measured on a minimum of 70 motile sperm, sufficient to describe adequately the sperm population. A study was performed comparing motion characteristics of 30 semen samples falling in a normal range before and after cryopreservation in cryoprotector medium (CM). A mean motility rate of recovery (MRR) of 45% was obtained. Only sperm count and concentration in motile forms among initial semen variables correlated weakly with MRR. Velocity recovery rate (VRR) approached 1 with a marked variability among ejaculates. Distribution profile of Vc was highly modified by freezing in CM: spermatozoa that were initially fast and progressive were the most resistant to cryoaggression. PR and Alh values were little affected by freezing in CM. The tolerance of various samples from a given patient was highly variable for % MS and Alh and less variable for Vc and PR. This illustrates the difficulty in predicting the effect of freezing on motility characteristics and, therefore, of extrapolating from semen variables the ability of frozen-thawed samples to fertilize.     

Temperature-induced sperm nuclear vacuolisation is dependent on sperm preparation

The influence of temperature during incubation on the degree of sperm nuclear vacuolisation was assessed by two different experiments. In a first experiment, motile spermatozoa from 24 patients were prepared by the swim-up technique and incubated either at room temperature or at 37 °C for up to 4 h. The presence of sperm nuclear vacuoles was determined by contrast-enhanced high magnification microscopy. No statistically significant difference was found in the degree of sperm nuclear vacuoles in both groups (RT: 45.6 ± 17.6%; 37 °C: 48.4 ± 17.0%) following 4 h of incubation. In a second experiment, spermatozoa from six patients were either prepared by swim-up or washed and incubated at 37 °C. After 4 h of incubation, a significant increase in sperm nuclear vacuolisation was found in washed sperm (from 51.5 ± 15.4% to 68.6 ± 9.0%; P < 0.05) but not in swim-up sperm (from 51.5 ± 15.4% to 48.2 ± 17.1%; n.s.). Our data show that the mode of sperm preparation does influence sperm nuclear vacuolisation at 37 °C (Experiment II). However, sperm nuclear vacuolisation is unaffected by temperature in motile sperm after preparation and isolation by swim-up.     

Efficacy of sperm wash media in improving sperm motility

Spermatozoa from 15 fertile men were washed with Ham's F10 and incubated with two commercially available sperm nutrient media for 2, 4, 6, and 24 h. Both sperm capacitation medium (Irvine Scientific Co., Santa Ana, CA) and Pro-ception (Milex Products, Inc., Chicago, IL) proved to be capable of improving sperm motion characteristics. These media may be used for incubating sperm for intrauterine insemination or for in vitro fertilization.     

Effects of altered epididymal sperm transit time on sperm quality

The epididymal sperm transit time seems to have an important role in the process of sperm maturation, and it seems that alterations to the transit can harm the process. The aim of the present work was to evaluate the influence of altered sperm transit time through the epididymis on sperm parameters and fertility of rats, as well as the role of testosterone in the alterations. Sprague–Dawley adult male rats were randomly assigned to four different groups and were treated for 12 days: (i) 10 μg/rat/day DES, to accelerate the transit; (ii) 6.25 mg/kg/day guanethidine sulphate, to delay the transit; (iii) same treatment as group 1, plus androgen supplementation; (iv) control animals received the vehicles. Guanethidine treatment delayed the sperm transit time through the epididymal cauda, provoking increased sperm reserves in this region. Animals exposed to DES showed an acceleration of sperm transit time in the epididymis, and consequently decreased sperm density in both epididymal regions, the caput-corpus and cauda, and diminished sperm motility. In both cases sperm production was not altered. Testosterone supplementation was able to restore the transit time to values close to normality, as they were higher than in the control rats. The same occurred in relation to sperm motility. Rats exposed to DES presented lower fertility after in utero artificial insemination using sperm collected from the proximal cauda epididymis. Therefore, it was concluded that the acceleration of rat sperm transit time appeared to harm normal sperm maturation, thus decreasing sperm quality and fertility capacity, in an androgen-dependent way.     

Assessing sperm function

This article reviews basic semen tests and new fertility tests that are providing great insights to the rapidly developing understanding of male infertility. Finally, promising new tests under development are mentioned with their potential clinical applications.     

Canine sperm vitrification with sucrose: effect on sperm function

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m ) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.