冷冻保存对人精子膜蛋白PH-20和透明质酸酶活性的影响

目的:探讨冷冻保存对人精子膜蛋白PH-20和顶体内透明质酸酶活性的影响。方法:24例正常生育力精液标本行冷冻保存。免疫印迹检测PH-20蛋白在人精子中的表达,免疫荧光观察PH-20蛋白在人精子上的定位,采用改良透明质酸钠-明胶底物膜法检测精子顶体内透明质酸酶(HYD)活性(HYD阳性反应率、HYD活性强度)。结果:免疫印迹显示正常标本冷冻前、后精子均有PH-20蛋白的表达,其PH-20/β-actin平均光密度在正常组冷冻前为0.41±0.15;正常组解冻后为0.24±0.11,两者的平均光密度差异有统计学意义;间接免疫荧光染色后在荧光显微镜下观察冷冻前PH-20阳性率达72.88%±8.04%;解冻后PH-20阳性率降至46.97%±8.38%,差异有统计学意义;解冻后HYD活性与冷冻前比较均有显著性下降。结论:冷冻-解冻过程可引起精子PH-20蛋白表达减少和精子PH-20阳性率降低,以及受精过程中的关键酶-HYD活性减弱。     

人精子膜蛋白P34H表达与透明质酸酶活性的关系

目的探讨人精子膜蛋白P34H表达和顶体内透明质酸酶活性的关系。方法收集88例精液标本,其中正常生育组20例,不育男性68例。参照世界卫生组织(WHO)标准对标本进行精液常规分析,根据精液参数的不同,将68例不育标本分为正常精液参数不育组和精液参数异常不育组。Western blotting检测P34H蛋白在人精子中的表达,免疫荧光观察P34H蛋白在精子表面的阳性表达率,采用改良透明质酸钠-明胶底物膜法检测精子顶体内透明质酸酶(HYD)活性(HYD阳性反应率、HYD活性强度)。结果正常精液参数不育组和精液参数异常不育组的精子P34H/β-actin平均吸光度、精子P34H标记阳性率均明显低于正常生育组,经统计学分析差异均有显著性(P0.05);正常精液参数不育组和精液参数异常不育组的HYD活性(HYD阳性反应率、HYD活性强度)与正常生育组比较均有显著下降(P0.05)。相关性分析提示,精子P34H蛋白表达量与HYD活性(HYD阳性反应率、HYD活性强度)存在正相关性,相关系数r=0.449、0.431,P0.01;精子P34H阳性率与HYD活性(HYD阳性反应率、HYD活性强度)存在正相关性,相关系数r=0.727、0.691,P0.01。结论男性不育患者精子P34H蛋白表达减少,精子P34H阳性率降低以及HYD活性(HYD阳性反应率、HYD活性强度)减弱。     

冷冻保存对人类精子顶体完整性及超微结构的影响

目的探讨冷冻保存对人类精子顶体完整性及超微结构的影响.方法 2 0例正常生育力精液标本(A组)和27例不育症精液标本(B组)行冷冻保存.应用荧光标记豌豆凝集素法检测冷冻前、后精子顶体完整率.采用透射电镜观察冷冻精子头部超微结构的改变 (n=3).结果解冻后A和B组的顶体完整率与冷冻前的比较均有显著性下降(P<0.01) ,且B组的降低程度明显大于A组.透射电镜观察到冷冻精子头部超微结构发生不同程度的损伤,浆膜和顶体膜出现肿胀、破损,顶体结构异常改变显著增多,顶体内容物丢失,甚至顶体帽缺失.结论冷冻-解冻过程对精子顶体造成了损伤,引起顶体完整率降低和超微结构改变.     

两种冷冻保护剂对形态正常精子百分率影响

目的比较甘油和甘油-卵黄-柠檬酸钠(GYC)两种冷冻保护剂对精子形态影响.方法应用甘油和GYC两种冷冻保护剂(CPM)对精液标本进行速冻和缓慢冷冻保存,应用计算机精液分析仪进行精子形态分析.结果冷冻复温后形态正常精子百分率与冷冻前比较的形态正常精子百分率比较明显下降(P<0.001);速冻与缓慢冷冻法中两种保护剂间形态正常精子百分率比较均无显著性差异(P>0.05). 结论冷冻保存易造成形态正常精子百分率下降,两种保护剂对精子形态没有影响.     

人精子血管内皮生长因子及其受体2的表达和顶体酶活性的关系

目的:探讨人精子血管内皮生长因子(VEGF)及其受体2(VEGFR2)表达和精子顶体酶活性的关系。方法:收集92例精液标本,按照世界卫生组织(WHO)颁布的标准对精液进行常规分析检测。免疫印迹法检测VEGF蛋白在人精子中的表达,免疫荧光法观察VEGFR2在人精子上的定位,采用改良明胶底物膜法检测精子顶体内顶体酶活性(顶体酶阳性反应率、顶体酶活性强度)。结果:免疫印迹检测结果显示,不育各组精子VEGF/GAPDH平均光密度均明显低于正常生育组;免疫荧光染色检测结果显示,不育各组精子VEGFR2阳性率均明显低于正常生育组;不育各组的顶体酶活性(顶体酶阳性反应率、顶体酶活性强度)与正常生育组比较均有显著下降。相关性分析提示精子VEGF蛋白表达量与顶体酶活性存在正相关性;精子VEGFR2阳性率与顶体酶活性存在正相关性。结论:男性不育患者精子VEGF蛋白及其受体2表达降低并伴随顶体酶活性减弱。     

人精液一氧化氮含量与男性不育的相关性

目的:研究精液中一氧化氮(NO)含量对精子凋亡的影响及其与男性不育之间的相关性.方法:采用精子质量检测系统对精液标本进行常规检查;应用硝酸还原酶法测定精液中NO的含量;应用瑞-姬染色和TdT介导的原位末端标记(TUNEL)法检测凋亡精子;观察凋亡精子的形态结构改变.结果:不育组精液中NO(58 37±14 14)μmol/l比正常生育组精液中NO(35 20±8 23)μmol/l含量高;正常生育组精子凋亡率9 67%±2 54%比不育组精子凋亡率33 98%±10 54%低.结论:不育组精液中精子的凋亡率随着NO含量的增高而增加.高浓度的NO导致精子凋亡率增加而致使男性生育力下降.     

男性泌尿生殖道支原体和衣原体感染对精液质量影响与不育关系分析

目的探讨男性泌尿生殖道解脲支原体(UU)和沙眼衣原体(CT)感染对精液质量的影响与不育的关系。方法选择2014年1月至2015年5月在我院生殖中心就诊的500例不育男性患者(不育组)和60例已生育健康男性(已育组)作为研究对象,取尿道分泌物采用PCR基因扩增法作沙眼衣原体(CT)和解脲支原体(UU)检测,并取其精液用计算机辅助精液分析系统(CASA)结合人工精液分析方法对精液液化时间、精子浓度、精子存活率、前向活动力和精子形态等参数进行分析检测。结果不育组单独感染CT(22%)、UU(46.6%)及混合感染CT+UU(8%)阳性率均明显高于已育对照组,有显著性差异,差异有统计学意义(P0.05);CT感染组和UU感染组之间精液分析的主要参数无显著性差异(P0.05),但和已育正常对照组比较,有显著性差异,差异有统计学意义(P0.05)。结论 CT和UU在不育男性中感染率较高,可影响精液各项参数异常,会导致精液质量下降,引起男性不育。     

男性不育精液中尿酸含量与生殖细胞凋亡的探讨

目的探讨人精液中尿酸含量与生殖细胞凋亡的关系.方法参照WHO标准方法,进行精液常规分析,按精子密度、活动率不同分为(正常、＜20、20～40、＞40)4个组.采用尿酸酶-过氧化物酶偶联法检测精液尿酸含量.用脱氧核苷酸末端转移酶(TdT)介导的缺口末端标记(TUNEL)和瑞-姬染色法,分别检测和观察生殖细胞的凋亡.结果75例不育者精液尿酸含量和生殖细胞的凋亡率分别为(263.87±57.15)μmol/L和(16.38±1.25)%与正常生育组(397.60±52.1)μmol/L、(4.61±1.23)%比较呈显著性差异(P＜0.01).精子密度和活动率随精液尿酸含量减少而降低,生殖细胞调亡率随之上升(P＜0.01).不育组精液尿酸含量与生殖细胞的凋亡地显著性负相关(r=-0.93,P＜0.05).凋亡的生殖细胞体积缩小,核染色质致密,凝聚在核周形成新月形,或核裂解形成凋亡小体.结论精液尿酸含量与生殖细胞的凋有着密切关系.低精液尿酸含量可使睾丸生殖细胞凋亡率增加,使精子密度和活率下降而致男性不育.     

司机职业对男性精液质量的影响

目的探讨司机职业与男性精液质量的相关性.方法422例不育男性(司机63例、非司机359例)和61例生育男性精液进行计算机辅助分析(CASA)和精子形态学分析.结果(1)不育组和生育组比较,不育组精子密度、活动率、前项运动级别显著性降低,畸形率显著性升高(P＜0.01).(2)不育司机组和不育非司机组比较,不育司机组精子密度、活动率和前项运动级别显著性降低,畸形率显著性升高(P＜0.05).不育司机组精子密度、活动率和前项运动级别与驾龄之间呈显著性负相关,畸形率与驾龄之间呈显著性正相关(P＜0.01).结论司机职业可能对男性精液质量有不良影响.     

不育男性精子膜蛋白P34H的表达与中性α-糖苷酶活性的关系

目的:探讨男性不育患者精子膜蛋白P34H的表达与精浆中性α-糖苷酶(NAG)活性的关系。方法:收集精液标本,分为正常生育组和不育组。参照WHO标准方法对标本进行精液常规分析,免疫印迹检测P34H蛋白在人精子中的表达,免疫荧光观察P34H蛋白在人精子上的定位,采用底物酶法检测精浆NAG活性。结果:免疫印迹结果显示,正常生育组的精子P34H蛋白表达和精子P34H阳性率均显著高于不育各组;精子P34H蛋白表达及阳性率在NAG活性正常组与NAG活性异常组差异无统计学意义。相关性分析提示精子P34H蛋白表达及P34H阳性率与精浆NAG活性呈正相关性。结论:精子P34H蛋白表达减少、精子P34H阳性率降低以及NAG活性减弱均会导致男性生育力低下,附睾精子蛋白P34H量和NAG活性可用于评价男性生育能力。     

乙酰左旋肉碱对人类精子冷冻前后顶体完整性及超微结构的保护作用探讨

目的 研究乙酰左旋肉碱对人精子冷冻前后顶体完整性及超微结构的保护作用.方法 将18例患者的精液标本液化及PureSperm梯度离心处理后分为2组,分别为A组(对照)和B组(7.5 mmol/L乙酰左旋肉碱),液氮中冷冻2周,比较冻融前后各组精子存活率、 活力、 头部和尾部超微结构损伤比例和顶体完整性.结果 精子冷冻后存活率和活力均较冷冻前明显下降(P<0.05),B组精子解冻后存活率和活力明显高于组A(P<0.05).冷冻后精子头部和尾部损伤比例均较冷冻前明显增加(P<0.05),而B组精子解冻后头部和尾部损伤比例均较A组明显下降(P<0.05).精子解冻后顶体完整性较冷冻前明显下降(P<0.05),B组精子解冻后精子顶体完整性较A组明显提高(P<0.05).结论 冷冻过程对人精子产生损伤,冷冻保护剂中添加7.5 mmol/L乙酰左旋肉碱可以提高精子解冻后顶体完整性,减轻冷冻损伤对于精子头尾超微结构的损伤,起到冷冻保护作用.     

不同冷冻保护剂在羊卵巢整体冻存中的效果比较

目的 应用自制梯度浓度混合-程控降温器官灌注仪,灌注羊完整卵巢,并行程序化冷冻,探讨冻融羊完整卵巢的效果并筛选最佳冷冻保护剂组合。 方法 将收集的28个羊完整卵巢,随机分配到新鲜对照组（A组）、海藻糖组（B组）、甘油组（C组）,二甲基亚砜（DMSO）组（D组）。冻融后组织切片行HE染色及原位缺口末端转移酶标记技术（TUNEL）检测,并通过RT-PCR技术检测组织Bcl-2相关X蛋白(Bax)及冷诱导RNA结合蛋白（CIRP）的mRNA表达。 结果 B组正常卵泡形态比率与A组差异无显著性(P>0.05),C组及D组正常形态卵泡比率显著低于A组,差异有统计学意义(P<0.05)。 B、C、D 3组冻存组基质细胞密度均低于A组,其中D组最低,差异具有统计学意义(P<0.05)。冻存组中,B组TUNEL反应阳性的细胞数目显著少于C组及D组,差异具有统计学意义(P<0.05),且均显著多于新鲜组。A组及B组bax基因 mRNA的表达量明显低于C组及D组（P<0.05）;B组CIRP基因 mRNA的表达量明显高于C组及D组,差异均具有统计学意义(P<0.05)。 结论 羊卵巢整体冻存后可较好保存组织学形态,海藻糖组的冷冻保护剂在组织结构保存及抑制细胞凋亡方面优于甘油组及DMSO组。     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

Fertilization in vitro of human oocytes by spermatozoa collected in different stressful situations

It has been shown that semen quality is impaired in couplesundergoing in-vitro fertilization (IVF), probably due to stress.A possible effect of stress on the ability of spermatozoa tofertilize human oocytes in vitro was analysed in the presentstudy composed of 26 couples with normozoospermic men undergoingIVF. A semen sample was obtained during the infertility work-upand was cryopreserved (sample 1). A second sample (sample 2)was provided after oocyte retrieval during the IVF cycle. Sample1 was thawed and both samples were washed and preincubated foroocyte insemination. One-hundred-and-five oocytes were inseminatedusing thawed sample 1, and 120 with sample 2.Semen parameterssuch as density, progressive motility and percentage of abnormalforms were compared between sample 1, before and after freezing,and sample 2. Only motility was significantly (P<0.01) decreasedby cryopreservation in sample 1, but no parameter was significantlydifferent when fresh sample 1 was compared to sample 2. Thefertilization rate was 78.6% using sample 1 in comparison to87.5% when sample 2 was employed (not significant, NS). Cleavagerates were 77.7 and 89.7%, respectively (NS). A group of fivepatients undergoing IVF who needed donor semen served as a controlfor the effect of sperm cryopreservation on IVF. In these cases,the donor was asked to provide a fresh sample. Half of thissample was frozen and thawed. Subsequently, fresh and thawedsamples were prepared for insemination and oocytes inseminatedeither with the fresh preparation (n=24) or the frozen and thawedspermatozoa (n=22). There was a significant (P<0.05) decreasein motility in the thawed sample, but fertilization and cleavagerates were not different. These data suggest that the stressfulsituation induced by IVF treatment in normozoospermic men doesnot affect the ability of spermatozoa to fertilize human oocytesin vitro. Cryopreservation of human spermatozoa before IVF maybe a good policy in couples especially suspected of being understress during this procedure.     

The outcome of intracytoplasmic injection of fresh and cryopreserved ejaculated spermatozoa--a prospective randomized study

BACKGROUND: The overall aim of this prospective, randomized study was to compare the reproductive potential of fresh and frozen-thawed ejaculated spermatozoa from oligoasthenoteratozoospermic patients in an intracytoplasmic sperm injection (ICSI) procedure. METHODS: All patients consenting to participate in this study had a sperm sample frozen prior to the start of a cycle. Patients were randomized using a random number table to undergo ICSI with either fresh (group A, n = 118) or frozen-thawed (group B, n = 122) spermatozoa. All prognostic variables were equally distributed among the two groups. RESULTS: The pregnancy rate per started cycle was 29.7% in group A and 38.5% in group B, P > 0.05. A significant difference was observed in the rate of ongoing pregnancies between group A (23.7%) and group B (35.2%), P < 0.05. CONCLUSION: From our data we can conclude that cryopreservation of spermatozoa from men with poor sperm quality does not negatively affect fertilization and pregnancy rates after ICSI. A larger study will be needed to investigate whether the use of cryopreserved spermatozoa can be helpful in selecting the most vital spermatozoa for ICSI.     

Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa

Semen cryopreservation is an important procedure in the treatment of human infertility. However, the ability of spermatozoa to survive freeze/thaw processes varies between patients. Cryopreservation-induced stress may result in membrane injury with consequent loss of sperm motility and viability. We investigated the relationship between the physico-chemical state of the human sperm membranes and their tolerance to cryopreservation. Conventional characteristics of 20 semen samples were analysed before and after cryopreservation as well as their membrane fluidity assessed by measuring the fluorescence polarization anisotropy, which is inversely proportional to the fluidity. Correlation between fluidity and post-thaw recoveries of motile and viable spermatozoa were examined. Results showed that membrane anisotropy markedly varies between patients. In cryopreserved spermatozoa, anisotropy values were significantly higher than in fresh spermatozoa. Furthermore, recovery of motile and viable spermatozoa after freeze/thaw was strongly correlated with anisotropy of fresh spermatozoa (P < 0.05). The higher the membrane fluidity was before freezing, the better was the response of spermatozoa to cryopreservation. The results indicate that the freeze/thaw process results in a rigidifying effect on the sperm membrane and suggest that sperm adaptability to freeze/thaw-induced stress could be dependent on their initial membrane fluidity. The latter finding has practical implications for predicting the response of spermatozoa following freezing and thawing and for improving the recovery of viable spermatozoa.     

两种冷冻方法冻融人卵巢组织异种移植后血管生成素-2表达变化的比较

目的 通过检测血管生成素-2（Ang-2）的表达,探讨两种冷冻方法对人卵巢组织的冻融及异种移植后的效果。 方法 共收集16例人卵巢组织标本,每例分成2份,其中1份应用程序化冷冻方法进行冷冻,另1份应用玻璃化冷冻方法进行冷冻。解冻后分别移植到去卵巢的雌性裸鼠颈部皮下。32只SCID裸鼠随机分为两组：A组16只,移植以程序化方法冷冻复苏的人卵巢组织;B组16只,移植以玻璃化方法冷冻复苏的人卵巢组织。解冻后及移植6周后观察两组卵巢组织中始基卵泡的存活及颗粒细胞Ang-2的表达情况。 结果 解冻后,A组Ang-2的阳性率明显高于B组（P<0.05）,始基卵泡正常率两组相比差别不明显（P＞0.05）。移植后,始基卵泡正常率及Ang-2的阳性率两组相比均无明显差别（P＞0.05）。解冻后和移植后相比,两组的Ang-2阳性率及A组的始基卵泡正常率差别均不明显（P＞0.05）,B组移植后始基卵泡正常率明显低于解冻后（P<0.05）。 结论 与玻璃化冷冻相比,程序化冷冻对人卵巢组织始基卵泡颗粒细胞Ang-2的表达影响更小。将解冻后的人卵巢组织进行异种移植不影响Ang-2的表达。     

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.     

Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa

The present study evaluated the effects of cryopreservation on progesterone-induced variations of calcium ion concentration [Ca(2+)](i), plasma membrane potential and acrosome reaction in human spermatozoa. Spermatozoa from 10 fertile donors were divided in two equivalent aliquots, one used as control (fresh spermatozoa) and the other used after freezing-thawing. Measurement of spermatozoa [Ca(2+)](i) before and after freezing-thawing showed a significant reduction of basal [Ca(2+)](i) in thawed spermatozoa (P < 0.01). Progesterone induced a rise of [Ca(2+)](i) both in fresh and thawed spermatozoa with a significant reduction after freezing-thawing (P < 0.01). The monitoring of sperm plasma membrane potential demonstrated that progesterone induced plasma membrane depolarization in fresh spermatozoa that was absent in thawed spermatozoa. The inhibitory effects of freezing-thawing on progesterone induced [Ca(2+)](i) and plasma membrane potential variations in human spermatozoa were closely related to the inhibition of the acrosome reaction. In conclusion the present study demonstrates that freezing-thawing procedures reduce the responsiveness of human spermatozoa to progesterone in terms of [Ca(2+)](i) rise and completely inhibit its effects on plasma membrane potential variations, thus supporting the hypothesis that freezing-thawing procedures may differently modify the plasma membrane receptors for progesterone in human spermatozoa which are known to express at least two receptors for this steroid in their plasma membrane.