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     

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。     

低温冷冻对人精子顶体超微结构的影响

目的评价低温冷冻及不同冷冻方法对人精子顶体超微结构的影响。方法将人的精子分别用速冻法及缓冻法冷冻1年：在冷冻前后分别用电镜观察精子顶体的超微结构，比较冷冻前后及不同冷冻方法间顶体超微结构的变化。结果顶体超微结构相对正常（异常）的精子在冷冻后显著减少（增加）；速冻法与缓冻法相比，顶体超微结构相对正常（异常）的精子减少（增加）更显著。结论低温冷冻将对人精子顶体的超微结构造成损害；缓冻法对人精子顶体超微结构的损害较轻。     

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。     

人精子膜功能完整性与精液参数的关系

目的探讨精子膜功能完整性与精液参数的关系。方法收集503例精液样本,其中对照组149例,不育组354例。根据WHO标准分析精子密度、活率、活力、精液白细胞浓度、精子形态和精子尾部低渗膨胀(HOS)率。结果不育组精子尾部低渗膨胀率显著低于对照组(P<0.05)。与对照组相比,HOS正常不育组和HOS异常不育组形态正常精子率、密度、活力、活率均显著降低(P<0.05),头部异常精子率、精液白细胞浓度显著增高(P<0.05);此外,HOS正常不育组的尾部异常精子率显著高于对照组,其精子活力、活率均显著高于HOS异常不育组(P<0.05)。精子HOS与精子密度、活力、活率呈显著正相关,与精液白细胞浓度呈显著负相关(P<0.05)。结论精子膜功能可能与精液参数密切相关,精子膜功能是评价男性生育力的重要指标。     

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。     

吸烟对男性不育患者精子DNA完整性及精子形态的影响分析

目的探讨吸烟对男性不育患者精子DNA完整性及精子形态的影响。方法 132例男性不育患者根据烟龄分为2组:A组烟龄1～5年;B组烟龄大于5年。正常对照组为100例非吸烟已生育健康男性。对其进行精子DNA完整性检测及精子形态学分析。结果 A组精子DNA碎片指数(DFI)和精子畸形率与正常对照组比较差异有显著性(P<0.05),而B组精子DNA碎片指数和精子畸形率与正常对照组比较差异有非常显著性(P<0.01)。结论长期吸烟可能会损伤男性精子DNA及影响精子形态,是引起男性不育的原因之一。     

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。     

精子DNA完整性与年龄及精液常规参数相关性分析

目的探讨精子DNA完整性对男性不育患者年龄及精液相关参数的关系。方法选取新疆医科大学第一附属医院生殖助孕中心就诊的808例接就诊的男性,进行精液常规分析,采用改进的精子染色质扩散法(sperm chromatin dispersion,SCD)检测精子DNA完整性(DFI),按精子DNA碎片率结果分为DFI15%(A组)、15%≤DFI30%(B组)、DFI≥30%(C组),同时按照WHO第五版标准进行精液常规分析,对DFI在各组间年龄、精子密度和精子活率差异及相关性进行分析。结果将B组和C组分别与A组比较,年龄差异具有统计学意义(P0.05),精子密度与精子活率差异无统计学意义(P0.05);精子密度与DFI无明显相关性,精子活率与DFI有显著负相关关系,精子活率随着DFI升高而显著降低。结论年龄是精子DNA碎片率的影响因素之一,精子DNA损伤影响精子活力,可作为判断精子质量的可靠指标。     

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.     

In vitro comparison of soybean lecithin-based extenders for cryopreservation of buffalo (Bubalus bubalis) semen

The objective of the present study was to compare the quality of frozen buffalo semen processed in tris-citric egg yolk extender with two different commercially available soybean lecithin-based extenders. Split pooled ejaculates, possessing more than 70 % visual sperm motility were divided in three aliquots and diluted in AndroMed®, Bioxcell®, or tris-citric egg yolk (TCE) extenders. Post-thaw motion characteristics, viability, plasma membrane integrity, acrosome morphology, and DNA integrity of buffalo sperm were studied after thawing and incubation for 4 h. Results demonstrate that total motility in frozen–thawed semen processed in TCE extender were similar when compared with AndroMed® or Bioxcell® extenders (P?>?0.05). Kinematic parameters, such as average path velocity and linearity index in the soybean lecithin-based extenders were comparatively superior to TCE extender. After 4 h of incubation, proportions of overall and progressive motility decreased in all extenders but were comparatively superior in soybean lecithin-based extenders than TCE extender (P?<?0.05). The proportion of post-thaw sperm viability, plasma membrane integrity and normal apical ridge remained similar in all extenders (P?>?0.05). The post-thaw sperm viability was comparatively superior in soybean lecithin-based extenders than TCE after incubation at 37 °C for 4 h (P?<?0.05). The type of extender had not been the significant effect on the percentage of spermatozoa with DNA damage after thawing and incubation for 4 h. In addition, our results suggest that soybean lecithin base extenders can be used as a substitute for tris-citric egg yolk extender in cryopreservation of buffalo semen.     

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.      

Evaluation of in vivo reproductive toxicity of potassium chromate in male mice

To evaluate the effects of potassium chromate on mice sperm cells after a short-term exposure, male ICR-CD1 mice were administered with 5 or 10 mg K₂CrO₄/bw for 4 consecutive days. One group of mice was sacrificed at day 5, starting from the beginning of the experiment and another group was sacrificed at day 35. Testis and epididymis histology was evaluated by light microscopy and testicular cells populations were evaluated by flow cytometry (FCM). Spermatozoa were collected from the epididymis and their morphology and several functional parameters (density, motility, viability, mitochondrial function, acrosome integrity) were evaluated. Furthermore, DNA fragmentation and chromatin status of sperm cells were assessed at both experimental periods.Besides a reduction in seminiferous tubules diameter, exposure to potassium chromate did not induce further histopathological changes in mice testis or epididymis. These results were supported by the analysis of testicular cellular subpopulations by FCM. Concerning spermatozoa morphology, an increase in the percentage of multiple abnormalities and a decrease in the percentage of normal spermatozoa were found at days 5 and 35, respectively. Although spermatozoa mitochondrial function or viability was not affected, its motility was significantly reduced by potassium chromate exposure at both experimental periods. A decrease in acrosome integrity was found in mice injected with 10 mg K₂CrO₄/bw after 35 days. Exposure to potassium chromate did not affect either DNA fragmentation or chromatin susceptibility to acid denaturation of sperm cells. In this work, we were able to show the effects of potassium chromate on spermatozoa physiological parameters such as motility, morphology and acrosome status and also demonstrate that the doses tested did not induce DNA damage to sperm cells after one spermatogenic cycle.     

Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.     

Effect of short-term exposure to radio frequency emitted by base transceiver station (BTS) antenna on epididymal sperms

This study was conducted to assess the effect of short-term exposure to RF waves generated by BTS antenna on the viability and motility of stored sperm in different parts of the epididymis. One hundred testes from slaughtered bulls were collected and used for this study. The testes were divided into two groups, test and sham-exposed, each group, according to time of exposure to RF (1 to 5?h), was divided into five subgroups, ten in each group. After a defined time, the motility (tail of the epididymis) and viability of sperms (in three parts of the epididymis) were evaluated in both groups. In the head of the epididymis, the reduction of sperm viability observed in the test group was 13% (after 3?h), 18% (after 4?h) and 21% (after 5?h) compared to the sham-exposed group, which was statistically significant (P?<?0.05). In the test group, sperm viability was significantly reduced in the body and tail sections after 4 and 5?h compared to the sham-exposed group (P?<?0.05). After 3?h of exposure to RF, the percentage of progressive motility of sperm decreased (8.7%), while the percentage of slow progressive motility increased significantly (6.4%) after 4?h compared to the sham-exposed group (P?<?0.05). These results suggested that short-term exposure to RF generated by mobile BTS has a deleterious effect on the quality of epididymal sperm (viability and motility) and that this effect is more severe in the head section and increases with increased exposure time.     

Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa

BACKGROUND: Oxidative stress induced by reactive oxygen species (ROS) isassociated with an impaired fertilization ability of spermatozoa.We investigated the effects of adding antioxidants to a spermpreparation medium on the functional parameters of the spermatozoa. METHODS: Spermatozoa were washed with Ham's F-10 media containing theantioxidants, ethylenediaminetetraacetic acid (EDTA) and catalase,at various concentrations, and then the ROS levels in spermsuspensions, and the forward motility, acrosome reaction, DNAintegrity and lipid peroxidation of the spermatozoa were assessed. RESULTS: The ROS levels were significantly lower in sperm suspensionswashed with the antioxidants (196312 rlu; relative light units)than in control sperm (604 rlu, P < 0.05). The addition of10 µM EDTA to the sperm preparation medium significantlyimproved the motility of the spermatozoa compared with the controlgroup, the groups containing EDTA at other concentrations andthe groups containing catalase. Catalase significantly increasedthe acrosome reaction rate of the spermatozoa. Both EDTA andcatalase significantly decreased the DNA fragmentation rateof the spermatozoa. However, the antioxidants did not reducelipid peroxidation. CONCLUSIONS: Supplementing sperm preparation medium with EDTA or catalasesignificantly improved the overall functional parameters ofthe spermatozoa by reducing the ROS levels.     

过氧化氢对生育男性精子凋亡及核DNA完整性的影响

目的研究体外过氧化氢对生育男性精子凋亡及精子DNA完整性的影响。方法58例正常生育男性均来自吉林大学第二临床医院泌尿男科,精子Percoll优选后制作精子悬液,用伊红Y染色分析精子活率,用瑞-姬染色分析精子凋亡率,用精子核吖啶橙荧光染色检测精子DNA完整性。结果外加过氧化氢浓度为0.02mmol/l时,精子活率显著低于对照组（P〈0.05）。当浓度为0.20mmol/l时,精子活率显著低于对照组（P〈0.05）,精子凋亡率显著高于对照组（P〈0.05）。外加过氧化氢浓度为0.02mmol/l、0.2mmol/l时,精子DNA完整性均显著低于对照组（P〈0.05）,且精子DNA完整性随实验浓度增加而下降。结论体外过氧化氢对精子活率、凋亡率与DNA完整性有影响,高浓度的过氧化氢可促使精子凋亡,损伤精子DNA完整性,导致男性不育。     

An important role of actin polymerization in the human zona pellucida-induced acrosome reaction.

The effects of inhibitors of actin polymerization and depolymerization, cytochalasins and phalloidin, on the human zona pellucida (ZP)-induced acrosome reaction (AR) were investigated. Motile spermatozoa, selected by swim-up technique from normozoospermic men, were incubated in medium with or without the actin modulators. Oocytes (four per test) which had failed to fertilize in vitro were added and incubation continued for 2 h. The spermatozoa bound to the ZP were dislodged by repeatedly aspirating the oocytes with a small-bore pipette and the status of the acrosomes was determined by fluorescein-labelled Pisum sativum agglutinin (PSA). Double immunofluorescent staining with PSA and an anti-actin monoclonal antibody illuminated the acrosomal region of acrosome-intact spermatozoa. In calcium ionophore-induced AR spermatozoa, actin staining was confined to the equatorial segment, post-acrosomal region and tail. Cytochalasins B and D significantly inhibited ZP-induced AR in a dose-dependent manner (P < 0.001). Both inhibitors had no effect on the acrosome of spermatozoa in the insemination medium. Cytochalasin B or D (10-40 micromol/l) had no effect on total percentage motile spermatozoa but decreased sperm velocity and hyperactivation. Phalloidin had no effect on the ZP-induced AR or sperm motility. In conclusion, actin polymerization plays an important role in human ZP-induced AR.