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.     

圆头精子的冷冻保存

目的:探讨圆头精子的冷冻保存效果及其精子功能改变。方法:6例圆头精子症患者各提供1-3份精液标本,应用不含卵黄冷冻保护剂和二步降温方法冷冻保存圆头精子症标本,检测冷冻精子活动率、头-尾膜完整率、存活时间和顶体蛋白酶活性。结果:圆头精子标本冷冻保存后,精子活动率显著减少,膜完整率显著下降,头膜损伤-尾膜完整的精子率显著升高(n=13,P＜0.01)。体外存活时间缩短。精子冷冻前后无顶体蛋白酶活性。不含卵黄冷冻保护剂与含卵黄冷冻保护剂,以及两步冷冻与多步冷冻的效果比较未见显著差异(n=7,P＞0.05)。结论:圆头精子可经历冷冻保存后存活,但冷冻复苏率低。冷冻-解冻过程显著降低精子功能。不含卵黄冷冻保护剂和两步降温可应用于冷冻保存圆头精子症标本。     

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。     

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.     

应用改良巴氏染色法对前列腺炎合并不育症患者精液标本的分析

目的探讨改良巴氏染色法在前列腺炎合并不育症患者精液标本的应用. 方法前列腺炎合并不育症患者精液(n=40, A组)和男性不育症门诊初诊者精液(n=40,B组)作改良巴氏染色法分析,并比较2组的精子和非精子细胞染色效果.结果 A组的畸形精子率和白细胞数显著高于B组(P<0.01).A组高粘稠度精液标本染色后,背景易因涂片较厚而出现杂色,精子头、颈和尾部的染色效果与B组的没有差别.而且非精子细胞类型的着色效果也未见与B组的有差别.结论前列腺炎合并不育症患者精液的畸形精子率和白细胞数有较高的发生率.改良巴氏染色法可应用于前列腺炎高粘稠度精液标本的染色分析,实际操作应避免涂片较厚而染上背景色.     

冷冻保存对人精液影响的细胞遗传学研究

本文首次对冷冻保存人精液对其精子染色体畸变频率和类型的影响进行了研究。检测了13名正常人的精子在液氮中冷冻前和冷冻后的染色体畸变频率和类型。冷冻后精子总的染色体畸变率(17.8%)和冷冻前精子染色体总畸变率(13.4%)没有显著意义的差异。但分析特定的畸变类型时,发现仅亚单倍体频率在冷冻前(7。5%)和冷冻后(3.4%)有显著意义的差异。在冷冻前和     

精液体外优化对精子DNA以及IVF-ET妊娠结局的影响

目的探讨人工助孕前精液经体外优化制备技术处理后精子DNA的损伤以及对体外授精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)结局的影响。方法收集107例行IVF助孕患者的精液,按照WHO标准对处理前精液行精液常规分析,采用精子染色质扩散试验(sperm chromatin dispersion,SCD)检测处理前和处理后精子DNA碎片率,并收集IVF实验室及临床结局。根据处理前精子DNA碎片率将患者分为两组:精子DNA碎片率≤20%的患者为A组,精子DNA碎片率20%的患者为B组。结果优化处理后精子DNA碎片率显著低于处理前精子DNA碎片率(3.49±3.38%vs18.69±10.89%,P0.001);处理前和处理后精子DNA碎片率均与精子总活动率、前向运动率及受精率有显著负相关关系(Spearman相关系数:-0.508、-0.629、-0.283、-0.299、-0.399、-0.329,P0.05)与男方年龄、精液量、精子浓度、正常形态率及卵裂率无显著相关关系;B组处理后精子DNA碎片率高于A组,差别有统计学意义(P0.05);B组的精子总活动率、前向运动率和受精率均低于A组,差别有统计学意义(P0.05);两组男方年龄、女方年龄、不孕年限、获卵数、精液量、精子浓度、正常形态率、卵裂率、种植率和妊娠率之间的差异均无统计学意义(P0.05)。结论精液经过优化处理(密度梯度离心加上游法)可以显著降低精子DNA碎片率,精子DNA碎片率高与精子活力低下密切相关,并会降低受精率,但对种植率、妊娠率等临床结局没有显著影响。     

多疣壁虎精子超微结构

目的:研究多疣壁虎精子超微结构,探讨爬行类精子进化规律.方法:用光镜及透射电镜观察多疣壁虎的精子.结果:多疣壁虎精子总长度约65～75 μm,头部弯曲长约27～30μm,尾部约 40～47 μm.用透射电镜观察多疣壁虎精于超微结构,可见其精子具有以下特点:顶体复合体由顶体帽、顶体下问隙、穿孔器、中央管、顶体下核帽5个部分组成;中段的轴丝复合体与线粒体鞘之间具有微管鞘(microtubule sheath)结构,线粒体鞘向后延伸包围主段,形成终环后隐窝结构;主段具有厚的圆筒状纤维鞘.结论:多疣壁虎精子超微结构比较研究显示爬行类精子具有种的特异性,提示了爬行类可能为多源起源.     

Does ICSI require acrosomal disruption? An ultrastructural study

BACKGROUND: Aggressive immobilization of sperm prior to ICSI significantly improves fertilization rates, but the mechanism of this effect is not yet clear. This study was performed in order to assess the characteristics of mechanically immobilized human sperm by transmission electron microscope (TEM). METHODS: Sperm obtained from ejaculated semen samples from three different donors were immobilized in a standard manner for ICSI. They were then injected into the perivitelline space of mouse oocytes in order to be able to locate them by TEM. Intact motile sperm injected subzonally served as controls (n=160). Finally, the 'carrier' oocytes were fixed and processed for TEM. RESULTS: A total of 300 sperm were mechanically immobilized and inserted into the perivitelline space of mouse oocytes. Ultrathin sections revealed consistent alterations in the acrosomal region including disruption of the plasma membrane, and disruption, vesiculation or even loss of the acrosome. Thus, all of the sperm assessed had undergone some disorganization of the head, in contrast to a majority of control sperm. CONCLUSIONS: Immobilization of sperm for ICSI by compressing and rolling the sperm tails induces a variable disruption and sometimes loss of the acrosome. This could well be a reason for the higher success rates when ICSI is performed using immobilized sperm.     

Antisperm antibodies modify plasma membrane functional integrity and inhibit osmosensitive calcium influx in human sperm

BACKGROUND: The hypo-osmotic swelling (HOS) test evaluates the ability of the functional sperm plasma membrane to stretch following cell swelling when exposed to hypo-osmotic solutions. Sperm samples with low HOS scores show low fertilization and pregnancy rates during assisted reproductive techniques, though data are controversial. The aim of this study was to compare the results of the HOS test in a group of normozoospermic men with those in a group of subjects affected by autoimmune infertility due to the presence of antisperm antibodies (ASA) bound to the sperm surface. METHODS: Sperm from normozoospermic and from infertile subjects affected by autoimmune infertility were exposed to hypo-osmolar conditions to verify the effects on intracellular calcium concentrations and acrosome reaction. RESULTS: Sperm samples from infertile men with ASA showed HOS test scores that were significantly lower than those of normozoospermic subjects despite similar sperm viability percentages. Sperm with ASA bound to their plasma membrane showed a reduced rise in intracellular calcium concentrations and acrosome reaction after hypo-osmotic challenge with respect to sperm from normozoospermic subjects without ASA. CONCLUSIONS: Infertile subjects with ASA have a reduced sperm plasma membrane functional integrity that could explain, at least in part, the low fertilization and pregnancy rates observed in these subjects during assisted reproductive procedures. Evaluation for the presence of ASA in all sperm samples showing low HOS test scores in the presence of normal sperm viability percentages is suggested.     

输精管结扎术后附睾精子及其吻合术后再现精子超微结构的观察

目的观察输精管结扎术后附睾精子和吻合术后再现精子超微结构的演变情况及其相关关系。方法15例要求作输精管吻合术的对象,手术时取出附睾精子（A组）并追踪其吻合术后精液再现精子（B组）,同时应用光镜和电子显微镜分别观察A组、B组的精子形态和结构的改变,并进行统计学分析。结果光镜分析显示：B组与A组比较,精子密度和尾畸比例下降有显著性;而精子活动率和头畸比例升高有显著性;正常形态的比例有增高的趋势。电镜分析显示：B组与A组比较,正常形态精子显著升高,而尾部畸形精子明显降低,两者差异均有统计学的显著性;头畸形和颈畸形的比例在两组间差异没有显著性;而两组间尾部畸形比例呈正相关。透射电镜显示内部结构在头、颈、尾均有多细胞器、多种形式的改变。结论本文用电镜观察进一步证实了输精管结扎术后附睾精子形态向输精管吻合术后精液再现精子变化的规律。不论电镜还是光镜分析,精子尾部异常都是由高比例（A组）向低比例（B组）转化,正常形态的精子均由低比例（A组）向高比例（B组）转化;精子头部的异常光镜分析是一个由低比例（A组）向高比例（B组）转化的过程,而电镜分析没有改变。本文在A组和B组未有发现与输精管结扎术有关的精子形态学上特征性的改变。     

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.     

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.     

前列腺炎合并不育症患者的抗精子抗体评价

目的评价前列腺炎合并不育症患者的抗精子抗体关系.方法应用混合球蛋白试验(MAR试验)、浅盘精子凝集试验(TAT试验)和浅盘精子制动试验(SIT),对35例前列腺炎合并不育症患者(A组)的血清和精子表面抗精子抗体进行检测,随机选择35例男性不育门诊初诊者作对照(B组).结果 A组采用TAT检出血清抗精子抗体阳性5例,滴度水平在1:8～16,SIT未测出阳性,采用MAR试验检出精子表面抗体阳性8例.B组采用TAT检出血清抗体阳性4例,SIT阳性1例,采用MAR试验检出精子表面抗体阳性2例.经t检验,A组精子表面抗精子抗体阳性率显著高于B组(P＜0.01).结论前列腺炎合并不育症患者存在着精子免疫因素,且表现出精子表面抗体发生率升高,临床对这类不育患者治疗要重视前列腺炎的抗炎处理.     

Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate

Cryopreservation causes extensive damage to spermatozoa, thereby impairing their fertilizing ability. The purpose of this study was to determine if the direct addition of pentoxifylline to the seminal plasma before cryopreservation improved sperm motility and acrosome reaction. Semen specimens from 15 healthy volunteers were divided into two aliquots. One aliquot was treated by adding 5 mM pentoxifylline directly to the seminal plasma (treatment group) and the other aliquot received no treatment (control group). Both aliquots were then cryopreserved by using the liquid nitrogen freezing method. The percentage of motile spermatozoa and various motion characteristics were then evaluated by performing computer-assisted semen analysis. The sperm viability was determined with a supra-vital dye, Hoechst-33258, and the acrosome reaction (spontaneous and calcium ionophore-induced) was monitored using fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA) binding assays. Pentoxifylline treatment significantly increased the sperm motility, the amplitude of lateral head displacement, the hyperactivation status, and the frequency of spontaneous acrosome reactions before freezing (P < 0.05). After post- thaw, no difference in motion characteristics (except percentage motility) between treated and control groups were observed. Acrosome loss due to the freeze-thaw process was less in the pentoxifylline- treated group (P = 0.0003). In addition, the percentage of cryopreserved acrosome-intact spermatozoa that underwent further acrosome reactions in response to calcium-ionophore challenge was significantly higher in the treated group (P = 0.03). Pentoxifylline treatment before freezing improved the acrosome reaction to ionophore challenge in cryopreserved spermatozoa. Treatment with pentoxifylline appears to minimize sperm damage during the freeze-thaw process and may improve fertilization rates with assisted reproductive procedures such as intrauterine insemination or in-vitro fertilization.