含雌激素的冷冻保护剂对少弱精子玻璃化冻存效果的研究

目的 比较添加雌激素的冷冻保护剂对少弱精子的冻存效果.方法 将40例少弱精子患者的精液标本分别采用甘油、甘油-卵黄-柠檬酸钠（GYC）、甘油＋雌激素（甘油＋ E2）、甘油-卵黄-柠檬酸钠＋雌激素（GYC＋ E2）四种冷冻保护剂（CPM）冷冻,比较冻融前后精子活率、活力（a＋b级）、正常精子形态百分率、精尾低渗肿胀率（HOSR）.结果 四种CPM对少弱精子的冷冻效果存在差别,精子活率、活力、正常形态百分率、精子尾部低渗肿胀率在冻后都有不同程度下降（P〈0.05）,但甘油＋E2、GYC＋ E2 能显著提高冻存后精子的活率及活力（a＋b级）.结论玻璃化冻存对精子存在损伤,含雌激素的冷冻保护剂可以提高低质量精液的冻存效果.     

人类精液冷冻保护剂应用研究概况

人类精液冷冻保护剂(cryoprotective medium,简称CPM)对人类精子在冷冻-解冻过程中减少或避免冷冻损伤起着关键性的作用。既往的研究表明,精子在冷冻时若不被CPM加以保护是很难存活的。自1949年Polge等发现了甘油对精子的冷冻保护特性后,几十年来各国科学家一直在发展和改良CPM,以求最大限度地保存精子冷冻前生理与功能的完整性。目前,人类精液CPM已经从使用单一甘油发展成为多种类型和配方的复合型CPM。本文就目前人类精液冷冻保存所使用的CPM及其应用概况作一综述。     

精子优选后冷冻保存对活性氧生成的影响

目的观察优选技术在精液冷冻保存中对常规精液参数、线粒体膜电位(△Ψm)和活性氧(ROS)的影响。方法分别用计算机辅助精子分析仪(CASA)、流式细胞仪(FCM)检测优选或未优选的精子在冷冻前后常规精液参数、△Ψm和精子细胞内ROS的变化。结果各冻融组精子活率、a级精子、(a+b)级精子、正常形态率均比冷冻前有不同程度降低,有显著性差异;而优选冻融组和优选加精浆冻融组间常规精液参数无显著性差异(P>0.05),但都明显高于原始冻融组(P<0.01)。原始精液冷冻后比冷冻前△Ψm下降(P<0.05),ROS显著上升(P<0.01)。优选冻融组和优选加精浆冻融组与冷冻前相比,以及组间无显著性差异(P>0.05);但与原始冻融组分别比较,△Ψm均显著提高(P<0.05),ROS均显著减少(P<0.01)。各冻融组△Ψm和ROS都有明显相关性,相关系数(r)依次为-0.528、-0.537、-0.606(P<0.001)。结论精液优选后冷冻可以在保持良好的△Ψm同时,减少ROS生成,因此建议临床可应用该技术冷冻保存精子。     

不同季节对北京地区捐精志愿者精子冷冻复苏率的影响

目的 比较不同季节之间精液冷冻前、复苏后参数以及精子冷冻复苏率是否有差异，探究季节因素是否是影响精液冷冻前后参数以及冷冻复苏率的因素。方法 应用回顾性研究方法，收集2017年1月到2022年4月来北京大学第三医院人类精子库捐精人群的精液冷冻前后参数以及精子冷冻复苏率数据。按照气象划分法对捐精时间进行不同季节分组。结果 一共分析了13 774份精液冷冻前后的常规数据。冷冻前精子浓度和冻前精子总活力，春季明显高于其他季节(P<0.05);复苏后精子浓度和复苏后精子总活力，春季高于其他季节(P<0.05)。精子冷冻复苏率，春、夏、秋、冬季分别为83.1(73.3～90.2)%、79.4 (68.7～87.3)%、81.8(72.5～88.2)%、82.7(74.4～88.9)%,春季高于其他季节(P<0.05)。多元线性回归分析发现季节是影响精子冷冻复苏率的主要因素。春季的精子冷冻复苏率比夏季约高3.7%(CI=3.0%～4.3%,P<0.001),春季的精子冷冻复苏率比秋季约高1.0%(CI=0.4%～1.8%,P=0.001)。结论 春季可作为精液冷冻保存的首选季...     

冷冻保存对人精子运动特征的影响

目的研究冷冻保存前后供精者精子运动特征的变化,了解冷冻保存对人精子受精潜能的影响。方法供精者精液标本68例,按照世界卫生组织推荐的方法和仪器行精液常规分析和计算机辅助精子分析(CASA)。入选精液加入甘油-卵黄-柠檬酸钠精子冷冻保护剂,行液氮蒸汽冷冻,1个月后复温,再次行CASA。结果冷冻后精子运动学参数中精子头侧摆幅度(ALH)和鞭打频率(BCF)升高,其它所有运动学参数均降低。精子动动学参数中除中速动动(Medium)、慢速运动(Slow)、平均路径速度(VAP)、直线运动速度(VSL)、ALH外,人精子冷冻前和解冻后各个参数都有显著的正相关(P<0.05)。除Medium、Slow、(曲线速度)VCL、ALH外,供精者精子各项参数冷冻前和解冻后比较,差异均有统计学意义(P<0.05)。冷冻复温后VCL>25μm/s、VSL>25μm/s。结论冷冻保存后人精子运动能力降低,但仍具有受精潜能。CASA用于冷冻精液的评估有一定的临床应用价值。     

冷冻复温后低温保护剂和精子激活剂对精子功能影响的研究

本文对冷冻复温后精子的功能进行了研究观察，发现精液保护剂（甘油－卵黄－枸橼酸钠）对冷冻复温后精子的存活有明显的保护作用，而单纯应用卵黄或甘油溶液却对精子功能存在不利的影响。体外处理精子时应用ＡＴＰ、肌苷对精子的功能无任何的改善。两种浓度的苯甲酸钠咖啡因（１２ｍＭ和４ｍＭ）对冷冻复温后精子在不同时间内的存活和ＣＭ穿透力均有明显的提高作用     

新型冷冻管承载设计对人类精子复苏率的影响

目的探讨精液冷冻管承载工具对人类精液活动力及复苏率的影响,优化精子冷冻保存的方案.方法取本库20例健康自愿供精的精液,同一例供精者的精液分成两份:实验组冷冻管用棉花包裹,对照组不做任何处理.通过相差显微镜镜检,利用计算机辅助精液分析(CASA)系统分析两组冷冻前后人类活动精子的运动参数,并作相关的统计学分析及比较.结果实验组冷冻复苏率为(66.71±6.51)%,对照组冷冻复苏率为(59.67±7.74)%(平均提高了7.04%)(P<0.01).结论冷冻管包裹棉花的冷冻前处理对精子有一定的保护作用,因而冷冻管承载工具的专门设计对精液冷冻有实质性意义.     

甘油-卵黄-柠檬酸钠型冷冻保护剂未经冷冻对人精子运动学参数的影响

目的 研究甘油-卵黄-柠檬酸钠(glycerol-egg yolk-sodium citrate,GYC)型冷冻保护剂对人精子运动学参数的影响.方法 供精者精液标本183例,按照世界卫生组织推荐的方法和仪器行精液常规分析和计算机辅助精子分析(computer-aided sperm analysis,CASA);加入GYC型冷冻保护液后再次行CASA获得精子运动学参数.结果 加入GYC型冷冻保护剂后,供精者精子各项运动学参数与加入冷冻保护剂前的运动学参数均有显著的正相关(P<0.05);加入冷冻保护剂后,除BCF外,供精者精子各项参数的改变均有统计学意义(P<0.05).结论 GYC型冷冻保护剂对人精子的运动学参数有影响;人精子冷冻保护剂的配方仍需改良.     

冷冻复温后低温保护剂和精子激活剂对精子功能影响的研究

本文对冷冻复温后精子的功能进行了研究观察，发现精液保护剂对冷冻复温后精子的存活有明显的保护作用，而单纯应用卵黄或甘油溶液却对精子功能存在不利的影响。体外处理精子时应用ＡＴＰ，肌苷对精子的功能无任何的改善。     

复温方法及保护剂对人冷冻精液复温后精子存活率的影响

作者对冷冻精液的四种复温方法及两种保护剂分别进行了202例和194例次的对比观察。结果表明:冷冻的复温效果受复温方法影响.快复温比慢复温效果好,37℃水浴和48℃水浴复温效果明显优于室温(22℃)空气中和孵箱(37℃)内放置(P<0.001),并结合观察结果,对快速复温及效果良好的机制进行了讨论。甘油和甘油-卵黄-柠檬酸钠复合剂两种冻精复温后,精子存活率十分接近(P>0.05).表明两种保护剂对精子的保护效能大致相同.     

The influence of cryoprotective media and processing procedures on motility and migration of frozen—thawed human sperm

AIM: The study was designed to examine the effects of cryoprotective media, and glycerolating and thawing procedures on human sperm motility and gel penetrating ability. METHOD: Fifteen unselected donors provided semen varying in quality that was distributed in a factorial design across three cryoprotectants (glycerol, egg yolk-citrate-glucose-glycerol and egg yolk-tris-glucose-glycerol). Also, glycerol was added at room temperature versus at 4 degrees C. Two thaw temperatures were tested (laboratory air temperature for 10 min versus a 65 degrees C waterbath for 4 seconds). The proportion of total and progressively motile sperm was estimated immediately after thawing and following incubation at 35 degrees C for 2 h. Migration of sperm for 30 min at 37 degrees C through polyacrylamide gel was tested.RESULTS: Donors differed greatly, with post-thaw total motility of sperm ranging from 9 to 44% (P<0.05). Egg yolk-citrate-glucose-glycerol and egg yolk-tris-glucose-glycerol were superior to glycerol alone (post-thaw values of 35, 37 and 21%, respectively, P<0.05). This was due primarily to poor sperm survival when semen was cooled to 4 degrees C without glycerol or egg yolk. The two thaw temperatures gave similar results. Sperm migration tests paralleled the motility results, but were more sensitive in detecting differences. CONCLUSION: Egg yolk, particularly in a tris-based medium that is widely used in domestic animals, improved the cryopreservation of both good and poor quality human semen.     

A new instant cryoprotectant for human sperm

A new instant cryoprotectant, mainly containing culture medium, human serum albumin, and kallikrein, and its laboratory handling in the cryoprotection of semen are described. A comparative study distributed to three cryobiological centers using either egg yolk citrate medium or instant cryoprotectant showed that neither had a significantly better or worse mean percentage recovery rate. The advantage of the new instant cryoprotectant presented consists clinically in the absence of egg yolk and technically in its easy handling. Therefore, ICP is judged as an alternative to ECM in the cryopreservation of human sperm.     

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.     

低密度脂蛋白和高密度脂蛋白相对全蛋黄溶液在猕猴精子低温冷冻中的作用

最近,住各种动物精子低温冷冻的实验中,提取于鸡蛋蛋黄中的低密度脂蛋白被视为优于全蛋黄溶液。与此同时,有研究认为蛋黄中的高密度脂蛋白可能对解冻后精子的生存产生负面影响。关于低密度脂蛋白和高密度脂蛋白在猕猴精子低温冷冻作用的研究还未有过报道。本研究在加和不加渗透型抗冻剂（甘油）两种情况下,通过与全蛋黄溶液对比的手段评价了两种脂蛋白在猕猴精子低温冷冻中的作用。此外,本研究也尝试多种能够改造精于膜与低密度脂蛋白结合体成分的添加剂对保存解冻精子活力的作用。我们的实验结果表明,低密度脂蛋白是伞蛋黄溶液在猕猴精子低温冷冻中发挥保护作用的主要成分。不管有无添加甘油,低密度脂蛋白与令蛋黄溶液在保存解冻精子活性方面作用相近,并未显现出任何优越性。通过加入胆同醇、胆固醇环糊精复合物或卵磷脂来改变精子膜与低密度脂蛋白结合体成分的方法并未改善解冻精子的存活状况;但加入甲基-β-环糊精则明显降低解冻精于的活力。高密度脂蛋白对猕猴精子低温冷冻保存不存在负面影响。对猕猴精子低温冷冻保存而言,本研究表明全蛋黄溶液相比较于稀释液中的有效成分低密度脂蛋白仍然具有优越性。     

精子形态与精子运动参数关系研究

目的:探讨精子形态与精子运动参数关系。方法:783例不育患者,采用精子自动分析系统分析精子运动参数,采用精子形态检测系统下人工修正方法进行精子形态分析。精子形态正常组241例,异常组542例。结果:精子形态异常组精子曲线速度(VCL)、摆动性(WOB)、平均路径速度(VAP)均显著高于精子形态正常组(P<0.05,P<0.001),而精子形态异常组精子平均移动角度(MAD)、直线性(LIN)、前向性(STR)均显著低于精子形态正常组(P<0.05,P<0.001);形态正常精子百分率与MAD、LIN、WOB、STR有显著正相关性,而与侧摆幅度(ALH)有显著负相关。结论:精子形态异常和运动能力弱相伴产生,形态异常对精子运动的影响可能只是这一现象产生的次要原因,而一些共同作用于精子形态和运动能力的因素也许是导致这一现象产生的主要原因。     

Cryopreservation alters the levels of the bull sperm surface protein P25b

Fertility of frozen-thawed bull sperm is reduced by cryopreservation. Freezing-thawing procedures can result in as much as a sevenfold fertility decrease. Sperm mortality and loss of motility do not fully explain the reduced fertility of cryopreserved semen; they may be partially explained by the loss of sperm surface proteins, which are necessary for fertilization. We have previously identified P25b, a sperm surface protein, which is associated with the fertility index of bulls used for artificial insemination. Using Western blotting techniques, we have evaluated P25b levels before and after cryopreservation of bull spermatozoa in extenders based on either egg yolk or milk. Long storage periods (28 days) in liquid nitrogen results in a threefold decrease of P25b levels associated with cryopreserved versus fresh spermatozoa. Over a short storage period (3-7 days), a stable P25b level was observed on spermatozoa cryopreserved in extender containing either egg yolk or milk. A decrease in P25b levels associated with spermatozoa was observed after 5 days of storage in egg yolk extender, whereas a significant decrease was observed after 14 days of sperm storage in milk extender (P < .05). Therefore, the loss of P25b may be responsible, at least in part, for the decrease in fertility following the freezing-thawing procedure of bull semen. Moreover, the cryopreservation extender used may have different effects on the loss of sperm surface proteins after even brief storage periods in liquid nitrogen. Considering that a sperm protein similar to P25b exists in humans (P34H), these results may have significant clinical applications in which frozen semen is used.     

弱精子症患者精子A激酶锚定蛋白AKAP82异常表达的研究

目的:通过测定弱精子症患者精子A激酶锚定蛋白AKAP82,了解其AKAP82表达是否异常。方法:41例弱精子症患者(实验组)和27例健康成年男性(对照组)精液,采用计算机辅助精液分析系统检测精子的运动参数。分别对两组精子进行W estern印迹和免疫荧光检测。结果:实验组中有11例在W estern印迹中出现异常条带或缺少正常条带,其中2例W estern印迹和免疫荧光检测结果均异常,9例患者W estern印迹检测结果异常;对照组仅2例W estern印迹检测结果异常。实验组与对照组之间差异有显著性(P<0.05)。结论:AKAP82的表达异常可能是精子活力低下的原因之一。     

腹腔镜直肠癌术中保留Denonvilliers筋膜对中青年男性排尿及性功能影响

目的探讨腹腔镜直肠癌术中保留Denonvilliers筋膜对中青年男性排尿及性功能影响。方法选择2012年12月至2015年12月收治的中青年直肠癌患者80例均实施腹腔镜直肠癌根治术,按照随机数字法分为两组,各40例,筋膜保护组术中注意对Denonvilliers筋膜加以保护,常规组常规操作。应用SPSS13.0软件分析,术后尿动力学、手术前后IIEF-5评分、术后精液常规及DFI指数等计量资料以均数±标准差(±s)表示,使用t检验;术后6个月性功能相关指标比较采用χ2检验,P0.05差异有统计学意义。结果筋膜保护组最大尿流量、膀胱顺应性、最大逼尿肌压力及最大尿道压力均显著高于常规组(P0.05);两组术后筋膜保护组IIEF-5评分显著优于常规组(P0.05);筋膜保护组精子密度、精子活动率及精子活力均显著高于常规组(P0.05),且精子DNA碎片显著少于常规组(P0.05),性欲增强及勃起功能增强的比例显著高于常规组(P0.05),出现逆行射精的比例显著低于常规组(P0.05)。结论腹腔镜直肠癌根治术,术中保护Denonvilliers筋膜,能减少对排尿功能的影响,减少术后勃起功能障碍及对精子活性的影响。     

Free radical scavenger effect of rebamipide in sperm processing and cryopreservation

Aim: To study the effect of rebamipide added to semen samples and cryoprotectant on reactive oxygen species (ROS) production. Methods: Semen samples from 30 fertile and healthy volunteers were collected by masturbation after 2 days - 3 days of abstinence. After liquefaction, the specimens were diluted with sperm wash media to a uniform density of 20×106/mL. Rebamipide was added to semen samples and cryoprotectant to a final concentration of 10 μmol/L, 30 μmol/L, 100 μmol/L or 300 μmol/L. Specimens were incubated at 37 ℃ in a 0.5 % CO2 incubator for 1 h or cryopreserved at -196 ℃ LN2 for 3 days. The sperm motility and viability and the levels of ROS and lipid peroxidation of sperm membrance were assessed before and after incubation and cryopreservation by means of computer assisted semen analyzer, eosin-nigrosin stain, chemiluminescence and thiobarbituric acid assay, respectively. Results: The sperm motility was significantly increased after incubation with 100 μmol/L and 300 μmol/L rebamipide (P     

鸡抗人精子卵黄抗体IgY的制备

目的 :探索一种快速和经济的从鸡卵黄制备抗人精子抗体IgY的方法。　方法 :用洗涤过的人精子免疫开产前的实验母鸡 ,间隔 7d加强免疫 ,4次加强注射后收集免疫鸡蛋 ,分离蛋黄和蛋清 ,去除蛋黄中的脂质后经 4次硫酸铵沉淀去除其它杂蛋白 ,再经SephadexG 2 5层析柱脱盐后获得卵黄抗体IgY。　结果 :鸡抗人类精子卵黄抗体IgY能使人类精子凝集 ,对照组用未免疫的鸡血清和卵黄抗体均为阴性。　结论 :产蛋母鸡可用来低成本和高效地生产抗人类精子抗体 ,精子凝集试验结果提示抗精子卵黄抗体可成为未来避孕药具的极有潜力的候选方法