刺五加水提物体外对弱精子症患者精子运动参数的影响

目的:研究不同浓度的刺五加水提物体外对弱精子症患者精子运动的影响,探讨其可能的作用机制。方法:将35例弱精子症患者经手淫法获得并通过上游优化处理的精子,与不同浓度刺五加水提物共同孵育30、60、120、180 m in后,应用计算机辅助精子分析系统(CASA)观察不同浓度(2.5、5、10、20 g/L)刺五加水提物对人精子各运动参数的作用。结果:不同浓度刺五加水提物能明显提高弱精子症患者精子的运动能力,在浓度为5 g/L和10 g/L时,精子活率(MOT)、前向运动精子百分率、曲线运动速度(VCL)、直线运动速度(VSL)和平均路径速度(VAP)与空白对照组相比,差异均有显著性(P<0.05)。结论:刺五加水提物在体外能明显改善弱精子症患者精子运动能力,其最佳浓度为10 g/L。     

睾酮体外对人精子运动参数的影响

目的通过研究睾酮体外对人精子运动参数的影响,探讨睾酮在男性不育症治疗中的作用。方法10例健康生育男性手淫获得精液,经上游优化处理后的精子与不同浓度的睾酮孵育10、30、60min,10例弱精子症患者手淫取精并与睾酮孵育10、60、120min后,采用计算机辅助的精液分析系统(CASA)检测精子的运动参数。结果50ng/dl(1.73nmol/L)睾酮在体外能显著增强正常人精子的直线速度(VSL)、曲线速度(VCL)和平均速度(VAP),而对活率、前向运动百分率无明显影响,而100ng/dl(3.47nmol/L)睾酮使精子活率和前向性运动百分率均有明显下降(P<0.01);1.04nmol/L～1.73nmol/L浓度睾酮能显著增强弱精子症患者精子的活率、前向运动百分率及VSL,并随着浓度的增加,睾酮作用显著增强,而对VCL和VAP无明显影响。结论低浓度(1.04nmol/L～1.73nmol/L)睾酮在体外显著增高弱精症患者精子的运动参数。     

稀少精子体外培养液对人精子活力的研究

目的:探讨稀少精子体外培养液(以下提到处简称激活剂)对人精子活力是否有改善作用,以帮助临床医生、实验室及患者更好的选择辅助生殖方式。方法:本研究选取门诊进行精液常规检查的标本178例,其中弱精子症组151例,正常活力精子组27例。每份标本共取200μl,分成均等的两份,分别加入等体积的激活剂(实验组)和F10(1×)(对照组),然后在37℃、5%的CO_2恒温箱中共孵育30 min,观察并记录两组孵育前后精子的浓度、活力(前向运动、非前向运动及不活动精子百分比)和存活率的变化。结果:激活剂作用于弱精子症组后,精子存活率与作用前及对照组相比,无统计学差异(P>0.05);正常活力精子组呈现出相同的结果。激活剂作用于弱精子症组后,精子前向活力、非前向活力提高的幅度分别是:14.02%和4.86%,而存活型不活动精子比例降低的幅度是19.01%,差异均具有显著性意义(P<0.01);正常活力精子组结果与弱精子症组相似。不论是弱精子症组还是正常活力精子组,存活型不活动精子减少的百分比与存活型不活动精子百分比呈正相关,其r值分别为0.260(P<0.01)(相关程度较弱)和0.679(P<0.01)(相关程度较强)。结论:1激活剂不影响精子存活率;2激活剂提高了弱精子症和正常活力精子的活动力;3精液中存活型不活动精子百分比越高,激活剂作用后,其转变为前向、非前向的百分比越大;且正常活力组的这种相关性强于弱精子症组。     

刺五加注射液与茶碱、咖啡因体外对精子运动功能影响的比较研究

目的:比较刺五加注射液与茶碱、咖啡因体外对精子运动功能的影响。方法:由12例弱精子症患者通过手淫获得并经上游优化处理的精子分别与一定浓度的刺五加注射液(10g/L)、咖啡因(7mmol/L)和茶碱(3mmol/L)一起孵化0h、1h、3h后,采用计算机辅助精液分析系统(CASA)检测精子的运动参数(精子活动率、前向性运动百分率、直线运动速度、曲线运动速度)。结果:刺五加注射液在体外能显著提高人精子活动率,前向性运动精子百分率,精子直线运动速度和曲线运动速度,其改善精子运动功能优于茶碱和咖啡因,差异均有显著性(P<0.05)。结论:中药刺五加注射液在体外能显著改善人精子的运动功能。     

rhGM-CSF体外对人精子运动参数的影响

目的观察体外添加不同浓度的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)对人精子运动参数的影响,探讨其在精子运动中的作用机制。方法健康生育男性和弱精子症患者各10例手淫取精,经简易上游优化处理后的精子与不同浓度rhGM-CSF溶液孵育10min、30min、60min后,采用计算机辅助的精液分析系统检测精子各项运动参数的变化。结果对精液运动参数正常的标本,1ng/ml和10ng/ml的rhGM-CSF溶液能显著提高精子的活率、前向性运动百分率,在10ng/ml时平均直线运动速度也有显著提高。对弱精子症患者精液标本,1ng/ml和10ng/ml的rhGM-CSF溶液能显著提高精子的前向运动百分率和直线运动速度,10ng/ml的rhGM-CSF对精子活率和平均路径速度也有显著性提高。两类精子的曲线运动速度均无显著性变化。结论1ng/ml～10ng/ml浓度范围的rhGM-CSF体外能显著改善精子的运动功能。     

双丁酰环磷酸腺苷对人精子运动功能的影响

目的 :通过研究双丁酰环磷酸腺苷 (dbcAMP)对体外人精子的运动功能有无影响 ,了解环磷酸腺苷 /蛋白激酶A(cAMP/PKA)信号传导通道是否参与人精子运动功能的调节。　方法 :10例健康生育男性手淫获得并经上游优化处理的精子与不同浓度的dbcAMP一起孵育 2 0、30、6 0min后 ,采用计算机辅助的精液分析系统 (CASA)检测精子的运动参数。　结果 :dbcAMP在体外能显著提高人精子的活率及前向性运动百分率 ,并且随浓度的增加 ,这种作用显著增强 ,而对精子的形态及直线速度 (VSL)和曲线速度 (VCL)无明显影响。　结论 :dbcAMP在体外能提高人精子的运动功能     

钙通道拮抗剂体外对人精子运动参数的影响

目的　探讨钙通道拮抗剂在体外对人精子运动参数的影响。方法　将不同类型的钙通道拮抗剂在体外与生育男性经上游优化处理的精子分别共同孵育 10、2 0、3 0、60min ,与正常组进行对照研究。采用计算机辅助精子分析系统检测人精子运动参数 (精子活率、精子前向运动百分率、畸形率、平均路径速度、精子侧摆幅度和摆动幅度 )。结果　硝苯地平 (10 μmol/L)在体外对精子活率 (P <0 .0 1)、精子前向运动百分率 (P <0 .0 1)、精子平均路径速度 (P <0 .0 0 1)、精子侧摆幅度 (P <0 .0 1)和摆动幅度 (P <0 .0 1)等指标的差异有非常显著性。结论　人精子细胞中存在L 型电压依赖性钙通道 ,且L 型钙通道拮抗剂可导致男性不育。     

弗司扣林对人精子运动功能的影响

目的 通过研究弗司扣林(forskolin)对体外人精子的运动功能有无影响，了解环磷酸腺苷／蛋白激酶A(cAMP／PKA)信号传导途径是否参与人精子运动功能的调节。方法 10例健康成年男性手淫获得新鲜精液，经上游优化处理后与不同浓度的forskolin一起孵育20、30、60min后，采用计算机辅助的精子分析系统(CASA)检测精子的各项运动参数，并进行对比分析。结果 forskolin在体外能显著提高人精于的活率及前向性运动百分率，而对精子的形态及直线速度(VSL)和曲线速度(VCL)无明显影响。结论 forskolin在体外能提高人精子的运动功能，此结果为证实cAMP/PKA信号传导通道参与调节人精子运动功能提供了一定的实验依据。     

阴道毛滴虫代谢产物体外对精子活力的影响

目的 :研究阴道毛滴虫代谢产物体外对人精子活力的影响。　方法 :阴道毛滴虫体外培养 ,除去虫体 ,留取培养液 ,稀释成三个浓度 ;将 10例正常生育男性的优化精子分为等体积的 4份 ,组成以下 4组 :A组加入未培养滴虫的空白培养液 ,B ,C ,D组依次加入前述浓度梯度的培养液 (1.2× 10 9/L、6× 10 8/L、1.2× 10 8/L) ,分别于 30s、1、2、4和 6h时采用计算机辅助精子分析系统 (CASA)分析精子运动参数。　结果 :阴道毛滴虫浓度在 6× 10 8/L以上时 ,精子的活率和活力与对照组相比都显著降低 (P <0 .0 5 ) ,并呈浓度依赖性和时间依赖性。　结论 :阴道毛滴虫代谢产物体外能抑制精子活力 ,可能是造成不孕不育的原因之一。     

磷脂酰肌醇3激酶抑制剂体外对弱精子症患者精子运动参数的影响

目的:研究磷脂酰肌醇3激酶(PI3K)的特异性抑制剂LY294002在体外对弱精子症患者精子运动参数的影响,并探讨其可能的作用机制。方法:弱精子症患者和精液参数正常男性各10例,手淫法留取精液,经上游优化处理后的精子与不同浓度的LY294002孵育10、30、60 m in后,采用计算机辅助精液分析系统检测精子的运动参数。结果:LY294002在体外能显著提高弱精子症患者精子的活动率、前向性运动精子百分率和直线运动速度、平均路径运动速度。结论:LY294002在体外显著增强弱精子症患者精子的运动功能。     

Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa

The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation-thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 degrees C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 degrees C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline.     

己酮可可碱和2—脱氧腺苷提高冷冻复温后精液质量的研究

在光学显微镜下观察和测定了己酮可可碱（ＰＦ）和２－脱氧腺苷（２－ＤＡ）在体外对冷冻复温后精液精子的活率、活力及其毛细管穿透值的影响。结果显示ＰＦ和２－ＤＡ均能提高冷冻复温后精子的活率、活力及其毛细管穿透值，其最适浓度分别为４．０ｍｍｏｌ／Ｌ和２．０ｍｍｏｌ／Ｌ，与对照组比较均有显著性差异（Ｐ〈０．０１）。两组间比较，２－ＤＡ优于ＰＦ（Ｐ〈０．０１）。提示ＰＦ与２－ＤＡ合用不具有协同作用，反而有相互     

The application of pentoxifylline in the stimulation of sperm motion in men undergoing electroejaculation

The ability of pentoxifylline to stimulate the motion characteristics of antegrade and retrograde sperm collected at the time of electroejaculation with a rectal probe was assessed in six neurologically impaired men. Before electroejaculation, the bladder was rinsed and instilled with 20 to 30 ml Ham's F-10 medium. Washed sperm were incubated with various doses (0, 0.1, 1, and 3 mmol/L) of pentoxifylline. Video sequences were recorded at intervals from 0 to 4.5 hours and analyzed for sperm motion parameters using manual and computer-assisted semen analysis. The results were compared with equimolar concentrations of caffeine. Both pentoxifylline and caffeine demonstrated a dose-dependent stimulation of percent motility and other motion parameters. A maximal stimulation of two-fold to three-fold for both percent motility and curvilinear velocity, and 30% to 60% for straight line velocity was observed after incubation under these conditions. A significant increase in mean linearity was observed in samples incubated with 0.1 mmol/L pentoxifylline at 1.5 hours. Significant lateral head displacement was not observed at any time point. Two couples underwent gamete intrafallopian transfer (GIFT) in conjunction with this electroejaculation sperm stimulation procedure, and one has since delivered a normal child. These studies show that pentoxifylline stimulation can improve the movement characteristics of asthenospermic sperm from neurologically impaired men. Such sperm stimulation techniques do not affect the fertilization process and may improve the chances for conception in some cases of male-factor infertility.     

体外应用己酮可可碱和2—脱氧腺苷提高冷冻复温后精液质量的研究

本文在光学显微镜下观察和测定了己酮可可碱和２脱氧腺苷在体外对冷冻复温后精液精子的活率、活力及其毛细管穿透值的影响。结果：两者均能提高上述三项参数。其最适浓度分别为４．０ｍｍｏｌ／Ｌ、２．０ｍｍｏｌ／Ｌ，与对照组比较均有显著性差异（Ｐ＜０．０１）。两组间比较，２脱氧腺苷优于己酮可可碱（Ｐ＜０．０１）。     

Pentoxifylline acts synergistically with A23187 to increase the penetration of zona-free hamster oocytes by cryopreserved human spermatozoa

The number of cryopreserved human spermatozoa which penetrated zona-free hamster oocytes afier stimulation with 2μmol A23187 per litre was increased by the hrther addition of 0.6 or 3.6 mmol pentoxifjrlline per litre. With spermatozoa prepared by washing by repeated centrifugation, the median numbers of sperm headd/egg were 1.9, 7.9 and 10.8 in the presence of 0, 0.6 or 3.6 mmol pentoxifylline per litre, respectively. A similar effect was observed with spermatozoa prepared on a Percoll gradient. As A23187 inhibited sperm motility, and this was exacerbated by pentoxifylline, the increased penetration rate of hamster oocytes cannot be explained by improved sperm motility. The number of spermatozoa stimulated to acrosome react by 2 μmol A23187 per litre was increased 3-fold by 3.6 mmol pentoxifylline per litre and 4-fold by 5 mmol caffeine per litre. These data suggest that CAMP may act synergistically with Ca²⁺ to stimulate the acrosome reaction. Pentoxifjrlline may improve the fertility of poor-quality human spermatozoa by enhancing their ability to respond to the Ca²⁺ signal produced by binding to the zona pellucida.     

Effect of pentoxifylline on the intrinsic swimming forces of human sperm assessed by optical tweezers

It is still controversial whether in vitro exposure of sperm to pentoxifylline increases sperm motility and force, which is defined as the product of velocity by beat frequency of the tail. Laser optical tweezers have been successfully used in the past to evaluate sperm force in basal conditions. The aim of this prospective study was to determine whether exposure of human sperm to pentoxifylline has any effect on sperm intrinsic forces. Twelve healthy subjects undergoing routine semen analysis were enrolled in the study. Ten exhibited normal semen parameters, 2 exhibited asthenozoospermia. Each semen specimen was washed and, after swim-up, resuspended in human tubal fluid (HTF) and divided into 2 aliquots. One aliquot was incubated with pentoxifylline for 30 minutes (final concentration = 3.6 mM); the second aliquot, without pentoxifylline, served as a control. After 30 minutes the pentoxifylline-treated aliquot was divided into 2 portions, 1 of which was washed to remove the pentoxifylline, the other was left in prolonged coincubation with the chemical. The main outcome was the measurement of sperm intrinsic force in milliwatts (mW), which was assessed by means of a noninvasive infrared laser optical trap created by a continuous wave, 1064-nm Nd:YAG laser beam directed in an inverted microscope. Exposure of sperm to pentoxifylline consistently increased sperm relative escape force from the laser optical trap. The increase ranged from 33% to 154% over baseline force compared with controls. The average absolute increase in sperm force rose from 37 mW to 79 mW (P < .05). Specimens with sperm having an initial low relative escape force gained the highest relative increase. The effect of pentoxifylline on sperm force, already apparent after 5 minutes, reached a peak at 30 minutes and persisted for up to 3 hours in sperm that were left in coincubation and in those on which the pentoxifylline had been washed off. In conclusion, pentoxifylline significantly increases sperm intrinsic relative force in normozoospermic and asthenozoospermic samples. This experiment confirms that optical tweezers can provide an accurate determination of sperm force in in vitro conditions. Clinical data must now establish whether a documented increase in sperm force is an important parameter for assessing sperm fertilizing capacity.     

The role of glucose in supporting motility and capacitation in human spermatozoa.

Glucose has been reported to be beneficial to human sperm for optimal capacitation and fertilization, although it is unclear whether glucose is required for providing extra metabolic energy through glycolysis, or for generating some other metabolic product. In this study, the effects of sugars on human sperm capacitation, motility, and energy production were investigated. The glucose concentration that supported the greatest number of acrosome reactions was 5.56 mmol L(-1). Compared with incubations with no added sugar, this concentration of glucose, fructose, mannose, or galactose appeared to slightly increase the number of acrosome reactions occurring after 18 hours of capacitation, or following induction by 2 micromol A23187 + 3.6 mmol pentoxifylline L 1, but only glucose had a statistically significant effect. Glucose supported increased penetration of zona-free hamster oocytes, but its advantage was not statistically significant. The addition of 5.56 mmol glucose or fructose L(-1) to sugar-free medium immediately increased the adenosine triphosphate (ATP) concentration and motility of sperm. These parameters were then stable for 3 hours, but declined markedly after 18 hours. In the absence of a glycolysable sugar, motility began to decline in the first hour and only 2% or 3% of sperm remained motile after 18 hours. Glucose or fructose was required to support hyperactivated motility. 2-Deoxyglucose was detrimental to the ATP concentration and motility of sperm, and supported fewer spontaneous or progesterone-stimulated acrosome reactions than were observed in the absence of a sugar. We conclude that glycolytic ATP production is required for vigorous motility and hyperactivation in human sperm. Other products of glucose metabolism are not essential to support capacitation, but they may have a small, enhancing effect.     

梗阻性无精子症患者睾丸精子左旋肉碱培养后关键基因表达量的变化研究

目的:探讨肉碱对人精子活力的影响及其对男性不育的治疗作用。方法:以梗阻性无精子症患者睾丸穿刺取得的精子为研究对象,通过普通培养液、培养液中添加100mmol/L及250mmol/L肉碱进行培养,比较培养前后精子活动率的变化,以及用RT-PCR检测男性生殖细胞特异性基因Vasa、Dazl、Acr、Prm1及ATPase6.0表达量的变化,探讨左旋肉碱与有关精子发生和成熟过程的重要功能基因表达的关系。结果:培养24～72h,添加100mmol/L的左旋肉碱培养液运动精子数明显较未添加和添加250mmol/L组高(P<0.01);100mmol/L左旋肉碱培养液组中典型的快速前向运动精子数明显较多,巴氏染色法观察其具有正常的精子结构,RT-PCR检测表明100mmol/L的肉碱可提高精子中Acr、Prm1、Dazl及ATPase6.0基因的表达量,而250mmol/L的肉碱组Dazl、Acr、Prm1基因的表达减少。结论:合适浓度的左旋肉碱可通过上调一些生殖相关基因的表达使得睾丸精子培养后活力增强,有利于对睾丸穿刺患者行单精子注射时精子的选择。如培养液中肉碱浓度过高,可能由于肉碱的毒性作用,反而使得这些基因的表达量减少。     

Effect of dilauroylphosphatidylcholine on human spermatozoa.

To achieve higher rates of acrosomal loss for in vitro studies of human sperm function, the effect of liposomes prepared from the phospholipid, dilauroylphosphatidylcholine (PC12), on human spermatozoa was investigated. In general, acrosome loss was induced with PC12 with an associated decline in the percentage of motile spermatozoa. Reduction of bovine serum albumin concentration in the incubation medium from 12 mg/mL to 3 mg/mL resulted in a more pronounced effect of PC12, with a significant reduction (P less than 0.001) in the percentage of motile spermatozoa within 1 hour of incubation with PC12. The percentage of spermatozoa observed to be acrosome-free under staining with fluorescein-conjugated Concanavalin A lectin was significantly reduced (P less than 0.05) when spermatozoa were incubated with PC12 (260 mumol/L) in the absence of calcium. The percentage of motile spermatozoa was not different during PC12 incubations when Ca2+ concentration (0, 2.5, and 5 mmol/L) was altered. An active motility pattern was restored in PC12-treated human spermatozoa by subsequent incubation in a defined medium containing 7.5 mmol/L adenosine 5'-triphosphate and 20 mumol/L cyclic adenosine 3', 5'-monophosphate. It was demonstrated that PC12-treated human spermatozoa were capable of binding to the zona pellucida of salt-stored human oocytes once an active motility pattern had been restored.     

Human sperm immobilization effect of Carica papaya seed extracts： an in vitro study

AIM: To examine if the seed extracts of Carica papaya, which showed antispermatogenic/sperm immobilization properties in animal models, could cause human sperm immobilization in vitro. METHODS: Chloroform extract, benzene chromatographic fraction of the chloroform extract, its methanol and ethyl acetate sub-fractions and the isolated compounds from the sub-fractions i.e., ECP 1 & 2 and MCP 1 &2, of the seeds of Carica papaya were used at concentrations of 0.1%, 0.5%, 1% and 2%. Sperm motility was assessed immediately after addition of extracts and every 5 minutes thereafter for 30 minutes. RESULTS: There were dose-dependent spermicidal effects showing an instant fall in the sperm motility to less than 20% at 2% concentration. Isolated compounds ECP 1 & 2 were more effective inducing a motility of less than 10%. Many of the spermatozoa became vibratory on the spot. Total inhibition of motility was observed within 20-25 min at all concentrations of all products. Scanning and transmission electron microscopy revealed deleterious changes in the plasma membrane of the head and mid-piece of spermatozoa. Sperm viability test and the number of abnormal spermatozoa after completion of incubation suggested that the spermatozoa were infertile. The effects were spermicidal but not spermiostatic as revealed by the sperm revival test. CONCLUSION: The results reveal spermicidal activity in vitro of the seed extracts of Carica papaya.