不同获能时间对人类精子膜完整性的影响

本研究用人精子低渗肿胀试验（ＨＯＳ－ｔｅｓｔ）分析了１２例正常育龄男子的精液标本。精子分别用常规洗涤法、上游法和加入钙离子载体（Ａ２３１８７）诱导获能等３种不同方法处理后，分别在液化后、上游１小时后、获能３小时、６小时和２４小时后测定了精子的低渗肿胀率。结果显示经不同方法处理、在不同获能时间之后的精子，在２４小时之内其肿胀率保持在８７．８±１４．６％～７３．３±１１．９％之间。统计学分析表明各实验组肿胀率之间无明显差异。本研究提示在２４小时之内精子膜的完整性不受获能时间及不同处理方法的影响。     

Percoll、白蛋白处理前后精子低渗肿胀百分率的比较

本文报告了用低渗肿胀试验观察经Percoll梯度离心和白蛋白柱处理前后的精子低渗肿胀率，结果显示：Percoll、白蛋白柱处理前精子低渗肿胀率为73．8±8．0％。处理后的精子低渗肿胀率分别为84.6±72％、87．7±8.1％．与处理前比较差异均非常显著。提示精液经体外处理后再行人工授精是治疗不育症的一种手段。     

不育精液冷冻及解冻后上游处理对精子活率的影响

本文对男性不育患者４０份精液，根据精子的活动情况分为Ａ组（ｎ＝２４，精子活动率＞５０％），Ｂ组（ｎ＝１６，活动率＜５０％），精液均采用人工操作放置液氮超低温进行冷冻保存，一周以后进行解冻，精子复苏率分别为６８．９１％及６０．４９％；同时对３１例标本进行上游处理。Ａ组精子平均活率为（６１．３２±１３．０６）％。Ｂ组为（３２．９２±１１．６３）％，精子前向运动力分别为６．２６±１．３３级和４．０８±１．２５级；活动精子的回收率分别为５４．２６％和４７．５４％。通过分析发现，对精子质量低下，但有一定活动率的精子仍可作冷冻保存，并具有一定的可冻性及耐冻性。复苏后上游处理，不论其活率还是前向运动力均有明显提高（Ｐ＜０．０１），为做ＡＩＨ患者的精液冷冻保存及解冻后的精子作上游处理提供了一种实验方法及参考数据。     

碳酸氢盐在小鼠精子体外获能和顶体反应中的作用

检验了精子获能和透明带（ＺＰ）及孕酮激发顶体反应是否需要ＨＣＯ３／ＣＯ２。小鼠精子分别在改良的Ｔｙｒｏｄｅ（ｍＴ－Ｂ２５，含２５ｍｍｏｌ／ＬＨＣＯ３／ＣＯ２）或ｍＴ－Ｈ（无ＨＣＯ３Ｈ／ＣＯ２，含２０ｍｍｏｌ／ＬＨｅｐｅｓ）中预培养９０ｍｉｎ后，以２步７０％和３５％ｐｅｒｃｏｌｌ／ｍＴ－Ｈ洗涤精子，并将精子重新悬浮于ｍＴ－Ｂ２５，ｍＴ－Ｂ１５ｍＴ－ＢＨ和ｍＴ－Ｈ中，用ＩＺＰ／μｌ或１５μｍｏｌ／Ｌ孕酮激发精子顶体反应。在上述ｍＴ诸培养基中，“Ｂ”精子（以ＣＴＣ荧光染色法确定）发生率为６１％～６７％；顶体反应均可达到４１．０士１．４％～４８．０±１．４％。表示精子一旦在ＨＣＯ３／Ｃ０２环境中获能，顶体反应就可在无ＨＣＯ３／ＣＯ２环境中发生。然而，精子预先在ｍＴ－Ｈ中培养，与上述相同方法处理精子，“Ｂ”型精子（２２％～３３％）明显低于前者，对ＺＰ或孕酮的刺激不起反应（１４．Ｏ士３．６～２４．７士０．６％），甚至将精子重新悬浮于ｍＴ－Ｂ２５中也不发生反应（２２．０±９．５％），表示这些精子并未获能。上述结果表明小鼠精子获能依赖于ＨＣＯ３／ＣＯ２，但顶体反应则否。     

Percoll梯度离心前后精子功能指标分析

目的 :通过分析Percoll梯度离心法处理前后精子相关功能指标 ,评价该法在精子体外处理中的作用。　方法 :采用多次曝光摄影技术、瑞 姬氏染色法、低渗肿胀实验 ,观察处理前后精子运动速度、顶体完整率和低渗肿胀率的变化。　结果 :2 5例不育病人精液标本经Percoll梯度离心法处理前后 ,精子运动速度 (2 0 .5 6± 8.13μm/s和 2 5 .18± 8.30 μm/s ,P <0 .0 5 )、顶体完整率 (6 1.2 0 %± 2 1.11%和 70 .36 %± 17.70 % ,P <0 .0 1)和低渗肿胀率 (6 8.2 8%±17.33%和 76 .76 %± 17.10 % ,P <0 .0 5 )均有显著差异。　结论 :精液标本经Percoll梯度离心法处理后 ,各项精子功能指标均较处理前有明显改善 ,该法可广泛应用于辅助生殖医学中。     

不同浓度低渗肿胀试验结合伊红染色对精子质量的评估

本文应用不同浓度精子低渗肿胀试验结合伊红染色法，对８例生育男性和１０例不育男性的精子膜功能进行评估。结果表明：１．活精子尾部低渗肿胀率均达到９０％以上，生育组与不育组无显著性差异；２．在１５０ｍＯｓｍ低渗溶液的稀释倍数为３～７倍之间，精子头部膜的损害程度在生育组与不育组之间存在显著性差异；３．在不同浓度的低渗溶液中，精子头部膜与精子尾部膜对低渗溶液的顺应性并不一致。实验证明不同浓度低渗肿胀试验结合伊红染色可正确评价精子膜的整体功能。     

不同浓度低渗肿胀试验结合伊红染色对精子质量的评估

本文应用不同浓度精子低渗肿胀试验九红染色法，对８例生育男性和１０例不育男性的精子膜功能进行评估。结果表明：１、活精子尾部低渗肿胀率均达到９０％以上，生育组与不育组无显著性差异；２、在１５０ｍＯｓｍ低渗溶液的稀释倍数为３－７倍之间，精子头部膜的损害程度在生育组与不育组之间存在显著性差异；３、在不同浓度的低渗溶液中，精子头部膜与精子尾部膜对低渗溶液的顺应性并不一致。实验证明不同浓度低渗肿胀试验结合伊红     

体外获能过程中精子凋亡及其对体外受精影响的研究

目的 探讨精子体外获能进程与细胞凋亡之间的联系，以及获能过程中精子凋亡对体外受精的影响。方法 猪精子经钙离子载体A23187(IA)诱导处理后，在TALP以及添加10％胎牛血清(TALP FCS组)、25μmol／L，核酶抑制剂(ATA)、TALP ATA组3种培养液中孵育，以考马斯亮蓝和TUNEL检测顶体反应(AR)率和凋亡率；同时行体外受精，检查卵母细胞穿透率和卵裂率。结果 鲜精液的精子凋亡率为5．7％，经洗涤、上游处理后凋亡率下降为3％；获能培养过程中，尽管IA浓度影响精子寿命，胎牛血清推迟获能进程，但凋亡仅与AR直接相关。获能完成后4小时左右出现明显的凋亡增长峰，ATA可阻止获能后的凋亡效应；3种获能处理的精子体外受精后，TALP和TALP FCS组有超过30％以上的受精卵不发生卵裂，而TALP ATA组仅为20％，与阴性对照组相当。结论 精子在顶体反应发生的同时可能启动和(或)激活凋亡机制，4小时后出现凋亡效应；精子DNA的异常断裂影响受精卵的发育能力。     

解脲支原体感染对精子功能影响的实验研究

目的：研究解脲支原体感染对精子功能的影响。方法：正常精液一式2份,一份用解脲支原体Ⅳ（UUⅣ）型人工感染正常精子,一份作对照.于感染后4、8、16、24h观察精子的低渗肿胀试验、精子爬高试验、精子活动力及活率。结果：正常精子受UUⅣ型感染8h,爬高高度低于对照组（48.1±4.2mmvs69.5±1.6mm,P＜0.05）;感染16hg形精子百分率、活动力、活率明显下降;感染24h精子总肿胀率明显低于对照组（12.75±8.98％vs75.50±4.88％,P＜0.01）。结论：UUⅣ型对精子低渗肿胀率和毛细管中爬高力有明显影响、是引起男性不育的病因之一。     

水试验在评价人获能精子膜完整性的研究

水试验在评价人获能精子膜完整性的研究杨宁，文任乾，田佩玲，林德亮我们曾用低渗溶液对人精子在不同获能时间膜的完整性进行了探讨，结果表明获能２４小时内，用低渗膨胀试验未能发现精子膜完整性的变化。本实验用水试验进一步对不同获能时期的人精子膜完整性的情况进行...     

Hypoosmotic swelling test in normo-, oligo-, astheno- and oligoasthenozoospermic men before and after swim-up separation of spermatozoa

Semen samples from a total 58 men were examined by routine semen analyses and the hypoosmotic swelling test. Samples were classified as normal, oligo-, astheno- or oligoasthenozoospermic on the basis of spermatogram findings. The latter three groups showed a significant decrease in the percentage of HOS positive forms in comparison to normal spermograms. All these samples were treated with the swim up technique to select motile spermatozoa, using a procedure similar to that routinely employed in clinical settings for homologous intrauterine insemination (IUI). Following swim-up, the ejaculate supernatant and residual precipitate were subjected to the hypoosmotic swelling test (HOS test), and the percentage of normal forms was determined in the three groups. The results showed greater percentages of HOS positive, normal and HOS positive-normal forms in the group of normal individuals than in any of the other three groups. The supernatant used in IUI showed a significant increase in percentage HOS positive spermatozoa, normal forms and spermatozoa which were both normal and HOS positive in comparison with the other two groups in normal and oligozoospermic samples, but not in samples which presented suboptimal motility (astheno- and oligozoospermia). In conclusion, the swim-up technique is effective in separating high-quality spermatozoa in normo- and oligozoospermic samples, although its effectiveness with astheno- and oligoasthenozoospermic samples should be questioned.     

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep™ II filtration, swim-up and Percoll density gradient methods

Summary. The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim-up method, Percoll density gradient method, and SpermPrep™ II filtration method. Thirty-three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim-up method, whereas aliquot 2 was filtered via a SpermPrep™ II column. The Percoll density gradient method was compared with the SpermPrep™ II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep™ II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim-up method. Furthermore, significant differences were found in most of sperm morpho-metric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep™ II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep™ II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swim-up method.     

蒸馏水在精子计数和低渗肿胀试验中的应用

目的:探讨蒸馏水在精子计数和低渗肿胀试验中的应用。方法:收集37份精液标本,每份标本分别用蒸馏水和碳酸氢钠-甲醛稀释液作1∶20稀释,然后用血细胞计数池进行计数并比较,同时记录蒸馏水稀释时精子肿胀百分率。另收集26份精液标本用于低渗肿胀试验结果的比较。结果:蒸馏水作为计数稀释液与碳酸氢钠-甲醛液相比,精子计数结果差异无显著性(P>0.05)。蒸馏水稀释时所得的精子肿胀百分率与WHO推荐的低渗肿胀试验结果亦无差异(P>0.05)。结论:蒸馏水不仅可取代碳酸氢钠-甲醛稀释液作为精子计数稀释液,亦可取代低渗肿胀液用于低渗肿胀试验分析。     

Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization

Human ejaculates (n = 83) were analyzed for standard sperm parameters (concentration, motility, and morphology), as well as for the ability of the spermatozoa to react (swell) in a hypoosmotic medium (Jeyendran et al, 1984). Subsequently, the fertilizing capacity of the spermatozoa was tested by their ability to fertilize human oocytes in vitro. Although the sperm concentration was adjusted for in vitro fertilization, no adjustments were made for sperm motility and morphology. Correlation of the in vitro fertilizing capacity of the spermatozoa with the hypoosmotic swelling test (r = 0.56) was much higher than with standard sperm parameters (r varied from -0.04 to 0.25). Complete overlap was noted with standard semen parameters whether the ejaculate did or did not fertilize oocytes and ranged from very low to very high values in both cases. By contrast, all the semen samples that fertilized oocytes showed a 60% or higher reaction in the hypoosmotic swelling test, whereas the majority of the "infertile" semen samples showed less than 60% swelling. It therefore appears that, under the conditions of our studies, the hypoosmotic swelling test is a more accurate predictor of successful in vitro fertilization outcome than the conventional semen parameters. A combination of all parameters, however, is likely to be most useful. The hypoosmotic swelling test is simple and economical, and it is recommended that this test be further scrutinized for its value as an additional tool in the assessment of the in vivo fertilizing capacity of ejaculated spermatozoa.     

The direct effect of Hochuekki-To on human sperm fertilizability using the hypoosmotic swelling test.

The concentration and motility of human sperm increase is examined following the oral administration of a traditional herbal medicine, Hochuekki-To using the hypoosmotic swelling (HOS) test. After 1 h incubation in Hochuekki-To, the motility of sperm and the results of the HOS test improved, particularly for severely abnormal sperm. For both the original and incubated sperm, the results of the HOS test improved when Hochuekki-To was used in the swim-up medium for the swim-up washing method rather than when Whittingham's T6 solution was used. Hochuekki-To had a favorable, direct effect on human sperm functions, suggesting an effect on the physiological integrity of sperm membrane.     

不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏效果的比较

目的比较不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏的效果。方法将15份上游后的精液标本分别采用常规精子冷冻和不使用冷冻保护剂的冷冻环玻璃化法冷冻,比较精子复苏后的活力、形态及精子膜的完整性三项指标。结果冷冻前、前向活动精子百分率、正常精子形态百分率及精子膜完整率分别为(79±6.42)%、(34±9.36)%和(91±5.18)%;不采用冷冻保护剂的玻璃化法冷冻复苏后,三者分别为(42.20±8.35)%、(31.00±7.63)%和(50.00±9.34)%。常规冷冻法冷冻复苏后,三者分别为(38.00±15.80)%、(30.00±5.24)%和(47.00±13.67)%。冷冻前后前向活动精子百分率和精子膜完整率的差别有统计学意义,但两种冷冻方法相比差异无统计学意义。结论使用不加入冷冻保护剂的玻璃化方法冷冻人的精子是可行的,可取得与常规冷冻相同的效果。     

Does the hypoosmotic swelling test predict human sperm viability?

Human sperm viability is essential for successful fertilization. Eosin Y is the usually accepted method for sperm viability assessment, though the hypoosmotic swelling test has been proposed for the selection of viable spermatozoa in procedures such as intracytoplasmic sperm injection. The present study was designed to determine the value of hypoosmotic swelling test in the prediction of sperm viability. For this purpose, hypoosmotic swelling and eosin Y were performed in parallel and in combination, on both fresh and freeze-thawed semen. Rates for eosin Y were significantly higher than for the hypoosmotic swelling test in fresh semen, with a weak, though significant correlation between the two tests (r = 0.47, p < 0.05). When both tests were performed in succession (hypoosmotic swelling test followed by eosin Y), 14.6% of swollen sperm incorporated the dye. Following exposure to hypoosmotic conditions, sperm viability decreased by 35%. When sperm were killed by freezing, hypoosmotic swelling test rates were higher than eosin Y. Results indicate that these two tests cannot be used interchangeably, since 15% of the swollen sperm apparently died, suggesting that plasma membrane integrity is lost before the capacity to maintain osmotic equilibrium.     

Evaluation of human sperm hyperactivated motility and its relationship with the zona-free hamster oocyte sperm penetration assay

The authors studied hyperactivated motility of human spermatozoa as a method of evaluating capacitation by examining its relationship to results of zona-free hamster oocyte sperm penetration assays (SPA) of semen samples from 50 men attending the infertility clinic. Hyperactivated motility was assessed in the seminal plasma and after swim-up preparation of spermatozoa at 1, 3, and 24 hours of incubation in capacitation media using a computer-assisted semen analysis system equipped with a hyperactivation module. Hyperactivated motility reached a peak at 1 hour and plateaued at 3 hours. The percentage of spermatozoa in seminal plasma with star-spin hyperactivated motility was significantly lower in the group showing no penetration in the SPA. The hyperactivated motility characteristics did not differ in the groups with positive or negative penetration. Correlation analysis failed to show any significant relationship between the hyperactivated motility parameters and SPA score. When the hyperactivated motility characteristics were compared in samples with normal and abnormal semen analyses, the total percentage of spermatozoa with hyperactivated motility and the percentage with star-spin at 3 hours were significantly lower in the group with abnormal semen analysis. The data indicate that lower hyperactivated motility of spermatozoa was found in patients with a score of zero for SPA and in patients with abnormal semen analysis. It was concluded that although no direct correlations were found between the results of SPA and hyperactivated motility, evaluating hyperactivated motility may still be useful as an early indicator of capacitation abnormalities of human spermatozoa not measured by SPA.