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.     

冷冻保存对人类精子膜完整性的影响

本文建立“低渗肿胀－伊红”（ＨＯＳ－ＥＹ）结合试验探讨冷冻保存人类精子头部和毛部膜完整性的影响，ＨＯＳ－ＥＹ试验按照精子尾部肿胀与否和头部着色与否区分为４种膜类型。冷冻保存后，头膜－尾膜均完整的Ⅳ型精了率和头膜－尾膜的均损伤的Ｉ型精子率分别显著减少和增多（ｎ＝５０，Ｐ〈０．００１），表明冷冻－解冻过程对精子膜完整性有严重影响，头膜损伤－尾膜完整的Ⅲ型精子率在冷冻组显著增多，提示精子头部或尾部的冷东     

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.     

应用人精子头-尾膜完整性试验对生育力组及不育组精液的评价

目的应用人精子头-尾膜完整性试验,比较生育力组与不育组男性精液4种精子膜完整性类型的差异.方法精液标本分为生育力组(n=32)和不育组(n=50),采用"低渗肿胀-伊红拒染"结合试验,检测人精子头-尾膜完整性.结果头膜尾膜均损伤的Ⅰ型精子和头膜损伤-尾膜完整的Ⅲ型精子,生育组和不育组存在明显差异(P<0.01);头膜完整-尾膜损伤的Ⅱ型精子,生育组和不育组无明显差异(P>0.05);头膜-尾膜均完整的Ⅳ型精子,生育组明显高于不育组(P<0.01);Ⅳ型精子率与生育组精子活动率(n=32, r=0.82,P<0.01)和不育组精子活动率(n=50,r=0.80,P<0.01)均呈显著相关性.结论生育组具有较多头膜-尾膜均完整的精子."低渗肿胀-伊红拒染"结合试验能够清晰显示包括过渡型膜损伤的4种膜完整性类型的精子,精子头-尾膜完整性可以作为评价精子生理学的一项检测指标.     

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。     

冷冻保存对正常和不育症精子PH-20表达和凋亡的影响

目的探讨冷冻保存对人精子膜蛋白PH-20表达和精子凋亡的影响。方法 14例正常生育力精液标本(A组)和20例不育症精液标本(B组)行冷冻保存。Western blotting检测PH-20蛋白在人精子中的表达,免疫荧光用来观察PH-20蛋白在人精子上的定位,应用末端脱氧核苷酸转移酶(Td T)介导的原位末端标记(TUNEL)法检测精子凋亡情况。结果解冻后正常生育组和不育组的PH-20/β-actin平均吸光度与冷冻前比较均有显著性下降(P0.05);解冻后正常生育组和不育组的PH-20阳性率与冷冻前比较均有显著性下降(P0.05)。解冻后正常生育组的精子凋亡率与冷冻前的比较差异无显著性意义(P0.05)。而解冻后不育组的精子凋亡率与冷冻前的比较均显著性下降(P0.05),且不育组的降低程度大于正常生育组。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,但冷冻保存对正常生育者的精子凋亡率无显著影响。     

小鼠精子3种活体染色法的比较分析

目的 比较小鼠精子伊红、锥兰和苯胺黑-伊红3种活体染色法的存活率,结合活动率,低渗肿胀-伊红结合(HOS-EY)试验评价小鼠精子存活率的方法.方法 取昆明系小鼠(n=15)附睾尾精子制成精子悬液,分别应用0.15%伊红、0.25%锥兰和10%苯胺黑-1.0%伊红3种活体染色法检测精子存活率,同时采用精子活动率和HOS-EY试验作对照测试.结果 小鼠精子用3种活体染色法测得的存活率没有显著差异(P＞0.05),存活率与活动率有直线回归关系(P＜0.05),HOS-EY试验中头-尾膜完整的精子率低子活动率和存活率(P＜0.05).结论 伊红、锥兰和苯胺黑-伊红3种活体染色法均可应用于小鼠精子存活率检测,结合检测精子活动率和HOS-EY试验可以进一步反映小鼠精子的存活状态.     

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.     

玻璃化冷冻技术在卵母细胞冻存上的应用

玻璃化冷冻保存技术由于具有操作简便、冻融损伤小并且不需要仪器精密控温的特点，已经成为最具应用前景的冷冻保存技术。玻璃化冷冻受到冷冻保护剂成分、浓度、作用时间、作用温度以及降温速率的影响。卵母细胞由于其特殊的细胞学特性，冷冻保存相对困难。玻璃化冻存技术应用于卵母细胞的冷冻保存能获得较好的存活率、受精率以及卵裂率。但是，经过玻璃化冻融的卵母细胞受精后囊胚形成率还不理想，高浓度的冷冻保护剂是否具有细胞毒性，是否会影响胚胎发育等问题尚待解答。因此，还需要更深入的研究来改善卵母细胞的玻璃化冷冻保存技术。     

小鼠精于3种活体染色法的比较分析

目的 比较小鼠精子伊红、锥兰和苯胺黑-伊红3种活体染色法的存活率，结合活动率，低渗肿胀-伊红结合(HOS-EY)试验评价小鼠精子存活率的方法。方法 取昆明系小鼠(n＝15)附睾尾精子制成精子悬液，分别应用0．15％伊红、0.25％锥兰和10％苯胺黑-1．0％伊红3种活体染色法检测精于存活率，同时采用精子活动率和HOS-EY试验作对照测试。结果 小鼠精子用3种活体染色法测得的存活率没有显著差异(P＞0.05)，存活率与活动率有直线回归关系(P＜0．05)，HOS-EY试验中头-尾膜完整的精子率低于活动率和存活率(P＜0．05)。结论 伊红、锥兰和苯胺黑—伊红3种活体染色法均可应用于小鼠精子存活率检测，结合检测精子活动率和HOS-EY试验可以进一步反映小鼠精子的存活状态。     

Andrology: Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol

Use of a cryoprotective agent is indispensable to prevent injuryto human spermatozoa during the cryopreservation process. However,addition of cryoprotective agents to spermatozoa before coolingand their removal after warming may create severe osmotic stressfor the cells, resulting in injury. The objective of this studywas to test the hypothesis that the degree (or magnitude) ofhuman sperm volume excursion can be used as an independent indicatorto evaluate and predict possible osmotic injury to spermatozoaduring the addition and removal of cryoprotective agents. Glycerolwas used as a model cryoprotective agent in the present study.To test this hypothesis, first the tolerance limits of spermatozoato swelling in hypoosmotic solutions (iso-osmotic medium dilutedwith water) and to shrinkage in hyperosmotic solutions (iso-osmoticmedium with sucrose) were determined. Sperm plasma membraneintegrity was measured by fluorescent staining, and sperm motilitywas assessed by computer-assisted semen analysis before, duringand after the anisosmotic exposure. The results indicate firstlythat motility was much more sensitive to anisosmotic conditionsthan membrane integrity, and secondly that motility was substantiallymore sensitive to hypotonic than to hypertonic conditions. Basedon the experimental data, osmotic injury as a function of spermvolume excursion (swelling or shrinking) was determined. Thesecond step, using these sperm volume excursion limits and previouslymeasured glycerol and water permeability coefficients of humanspermatozoa, was to predict, by computer simulation, the cellosmotic injury caused by different procedures for the additionand removal of glycerol. The predicted sperm injury was confirmedby experiment. Based on this study, an analytical methodologyhas been developed for predicting optimal protocols to reduceosmotic injury associated with the addition and removal of hypertonicconcentrations of glycerol in human spermatozoa.     

Cryopreservation of spermatozoa: a 1996 review

Both the ability to freeze human spermatozoa and the possibilityof pregnancy following intrauterine insemination have existedfor >40 years. There have been a number of improvements duringthat time concerning the methods of freezing and thawing humanspermatozoa. Initially, the use of the cryoprotective propertiesof glycerol allowed a major improvement; subsequently, changeswere mainly empirical. It was a long time before specific cryobiologicalstudies were undertaken. However, the necessity for these becameapparent with the partial recovery or sometimes loss of motilityafter freezing either subfertile semen before chemotherapy orradiotherapy, or spermatozoa collected from non-physiologicalsituations (epididymal or testicular spermatozoa). The maintrends in improvement have defined end-points other than thepercentage of motility recovery or the assessment of ultrastructuraldamage. More sensitive criteria of the objective assessmentof motility, energy status, damage to the plasma membrane orto subcellular elements, chromatin stability and chromosomaldamage have been proposed as complementary end-points to betterassess sperm cryopreservation. A different approach was relatedto the biochemical environment and physical conditions imposedon spermatozoa during the freezing and thawing process. Biochemicalchanges were assessed following different combinations of variousextenders which attempted either to better preserve some parameteror to avoid the tendency towards drastic increase in osmoticpressure. Analysis of physical conditions was linked to therate of cooling, freezing ad warming, and was based on cryobiologicalstudies. Finally, even though such improvements are not negligible,many questions remained unanswered. The extensive use of frozenspermatozoa during assisted reproductive techniques, togetherwith the development of assisted fertilization using surgicallycollected spermatozoa, creates the need for additional studiesto improve the cryopreservation of human spermatozoa. Keywords: cryopreservation/review/spermatozoa     

Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing

The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.     

Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage

When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0+/-1.2%) to spermatozoa cryopreserved by TYB (26.6+/-2.2%) via cryopreservation by 10% (v/v) glycerol (19.9+/-1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6+/-3.4% by glycerol; 19.6+/-3.7% by MM and 22.6+/-3.9% by TYB; P = 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa.     

Changes in carnitine and acetylcarnitine in human semen during cryopreservation.

L-Carnitine and acetylcarnitine concentrations were determined in spermatozoa and seminal plasma from 15 men, in both fresh ejaculate and frozen-thawed semen with cryoprotective medium. Sperm motility was also evaluated. In fresh samples, the levels of carnitine and acetylcarnitine in seminal plasma were comparable whereas in spermatozoa, acetylcarnitine predominated. Cryopreservation did not change the carnitine and acetylcarnitine levels in seminal plasma nor the carnitine concentration in spermatozoa; by contrast, the acetylcarnitine level in spermatozoa was decreased in 14 cases (110 +/- 8 versus 210 +/- 20 nmol/10(8) cells). This decrease in acetylcarnitine content was greater during semen dilution in cryoprotectant than after the freezing/thawing process. Motility was also decreased in all cases after the freezing/thawing process. These results suggest that acetylcarnitine recovery in spermatozoa is further evidence of the deleterious effect of the cryoprotective medium in the cryopreservation of semen.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification

BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000 degrees K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.     

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.