程序冷冻法、液氮蒸汽法冷冻人类精子的效果比较

目的:比较程序冷冻法和液氮蒸汽法冷冻保存人类精子的效果.方法:用同一批次GYC型冷冻保护剂,分别应用程序冷冻法和液氮蒸汽法对68份精液标本进行冷冻保存,并比较其冷冻复苏率.结果:应用程序冷冻法冷冻的精液标本,其复苏后前向运动精子(a b级)的冷冻复苏率为66.7%±8.1%,应用液氮蒸汽法冷冻的精液标本,其复苏后前向运动精子(a b级)的冷冻复苏率为63.9%±7.9%,两组相比无显著差异(p>0.05).结论:采用程序冷冻法和液氮蒸汽法冷冻人类精子,二者冷冻复苏率无显著差异,两种方法均可以用于人类精子库冷冻保存精液标本.     

含雌激素的冷冻保护剂对冻存正常精子质量的影响

目的:探讨含雌激素的冷冻保护剂对正常精液精子玻璃化冻存效果的影响。方法:将门诊收集的每例正常健康人精液平均分成5份,1份用于冷冻前指标测定,其余4份分别应用冷冻保护剂(CPM)甘油、甘油-卵黄-柠檬酸钠(GYC)、甘油+雌二醇(E2)、GYC+E2进行玻璃化冻存,检测冻存前、后精子活率、活力(a+b级)、正常精子形态百分率和精子尾部低渗肿胀率(HOSR)等精子功能的指标,评价4种CPM的冷冻保护效果。结果:4种CPM对精子的冷冻保护效果有区别,精子活率和活力、正常形态率和HOSR在冷冻后都有不同程度下降(P<0.01),甘油组、GYC组比甘油+E2组和GYC+E2组下降更明显(P<0.01)。GYC+E2组各参数高于甘油+E2组。结论:精子冷冻保护剂中加入雌激素可减少对正常精液冻融过程中精子的损伤,含雌激素的甘油复合型冷冻保护剂冷冻保护效果优于含雌激素的甘油型冷冻保护剂。     

含雌激素的冷冻保护剂对冻存正常精子质量的影响

目的:探讨含雌激素的冷冻保护剂对正常精液精子玻璃化冻存效果的影响。方法:将门诊收集的每例正常健康人精液平均分成5份,1份用于冷冻前指标测定,其余4份分别应用冷冻保护剂(CPM)甘油、甘油-卵黄-柠檬酸钠(GYC)、甘油+雌二醇(E2)、GYC+E2进行玻璃化冻存,检测冻存前、后精子活率、活力(a+b级)、正常精子形态百分率和精子尾部低渗肿胀率(HOSR)等精子功能的指标,评价4种CPM的冷冻保护效果。结果:4种CPM对精子的冷冻保护效果有区别,精子活率和活力、正常形态率和HOSR在冷冻后都有不同程度下降(P<0.01),甘油组、GYC组比甘油+E2组和GYC+E2组下降更明显(P<0.01)。GYC+E2组各参数高于甘油+E2组。结论:精子冷冻保护剂中加入雌激素可减少对正常精液冻融过程中精子的损伤,含雌激素的甘油复合型冷冻保护剂冷冻保护效果优于含雌激素的甘油型冷冻保护剂。     

冷冻保存精子受精机能的研究

精子的冷冻保存已应用于临床,但冻融后精子的受精能力尚未得到充分的评价。现比较冻融前后精子机能与受精情况,讨论冷冻对精子受精能力的影响。选择正常与不孕症患者214例,手淫法取精37℃放置30分钟,液化后取半量精液做常规检查及受精机能检查,余量冷冻保存。分组:A组<20×10~6/ml,B组:20～40×10~6/ml,C组:>40×10~6/ml。精子冷冻保存液为KS-Ⅱ液,采用自动程序冷冻法,在液氮中保存2～8周后,用37℃温水快速融冻,检查受精机能。(1)冻融后的精子复苏率:A组为46.6±45.3%,B组51.1±43.4％,C组63.5±29.4％平均59.3±35.4%,显示冻融后的复苏率普遍降低,AB组降低明显。(2)牛宫颈粘液精子穿透试验(BMP test):其冷冻前平均为41.1±17.2mm,冻融后为14.0±11.2mm,冻融后的精子穿透能力显著降低A组精子降低更为     

人类卵子冷冻技术的进展

人类卵子冷冻作为生育力保存的一种方式,在辅助生殖技术（ART）中起着关键的作用。由于伦理及研究条件的限制,卵子冷冻技术一直发展缓慢。近年,随着冻融技术及胞浆内单精子注射技术（ICSI）的发展,卵子冷冻已广泛应用于临床。玻璃化冷冻因其简单、细胞损伤小、复苏存活率高正在逐步取代慢速冷冻法,被大多数生殖医学中心所采纳。但卵子冷冻保存过程中仍存在交叉感染的风险及卵子冷冻损伤,液氮紫外线消毒、蒸气氮存储系统或封闭载杆可以防止交叉感染,相对于慢速冷冻法,玻璃化冷冻在减少卵子冷冻损伤方面更有优势。     

用精子染色质结构检测未治疗肿瘤患者的DNA损伤

睾丸癌、Hodgkin氏病和白血病属于育龄男性最常见的肿瘤。由于肿瘤治疗可继发不育,在放、化疗或手术之前,常让这些患者冷冻保存精液。但是,相当比例的这类患者在治疗前精液质量已有明显的下降,冷冻前精液质量低下伴有复苏效果低下。睾丸癌的精液质量低下,可能与生精细胞本身的缺陷有关。其它肿瘤如何影响精液质量,目前尚不清楚。研究治疗前肿瘤患者冷冻前后精液的特性及精子染色质缺陷间的相关性。     

人精子冷藏库成功的必要条件:精子质量与冷冻前放置时间

在不孕门诊中提供冷冻保存精液十分必要,尤其获得性免疫缺陷综合征出现后,日益主张使用供者冷冻精液以替代新鲜精液。研究对供者精液建立有效的精液冷冻保存方法,以保证融冻后有高质量的精子。在1987年～1989年间对52名符合条件的供者精液进行分析。精液是在禁欲3天后手淫法取得,作全面精液分析,献精液标准为。容积2～6ml;精子浓度大于80×10~6/ml;活动力大于50％,绝大多数前向运动;正常形态精子大于45％。冷冻保存液每100ml中含15％甘油,20％卵黄,1.15g枸橼酸钠,1.8g葡萄糖,1g甘氨酸及青霉素10万IU,链霉素50mg,pH为7.3。每一容积的精液用32℃等量的冷冻保存液稀释后按规定方法在液氮蒸汽中冷却。精液的融冻在32℃进行,使用前复查精子活     

精子氧化损伤及抗氧化剂的研究

随着对男性生殖健康的关注,人们开展了许多关于精子氧化损伤和抗氧化剂在男性生殖系统中的应用研究.在正常的生理条件下,精子产生微量的活性氧(reactive oxygen species,ROS),这对于受精、顶体反应和获能是必需的.然而,如果产生的ROS增加超过自身清除能力的增加,就会导致精子质膜的过氧化损伤和DNA完整性的丢失,最终导致精子细胞死亡、降低受精率.目前,实验室和临床已经有许多研究关注抗氧化剂在氧化损伤引起的男性生殖问题中的作用,主要通过抑制ROS的产生或者消除过多的ROS来保护精子免受氧化损伤的影响,特别是在缺乏精浆的体外处理中尤为重要.     

精子DNA损伤与辅助生殖技术结局

精子DNA损伤发生的机制包括染色质包装异常、氧化应激和细胞凋亡等。此外,冷冻保存和体外制备技术均能影响精子DNA的完整性。精子DNA损伤与精液参数、精子的受精能力和受精后的胚胎发育潜能、辅助生殖技术（ART）结局及后代遗传风险密切相关。对近年有关精子DNA损伤的机制、冷冻保存和精子制备、精子DNA损伤与精液参数及其与ART结局关系的相关文献进行综述。     

体外操作对人精子运动参数及细胞内活性氧簇的影响

目的：研究体外物理操作（离心以及微量加样器抽吸）对人精子运动参数以及细胞内活性氧簇（ROS）的影响,旨在优化人精子体外处理方法。方法：7例精液常规参数正常的标本,采用不同离心力（200 g,600 g）及离心时间（5 min,15 min）以及不同微量加样器抽吸次数（2,6,10次）进行处理,测试精子运动参数和细胞内ROS的变化。结果：①活动精子百分比以及活精子细胞内ROS水平主要受到离心时间的影响（P＜0.05）;前向运动精子百分比（PR）以及平均运动速率（VAP）均不受离心力及离心时间的影响（P＞0.05）。单因素方差分析多重比较结果提示,当离心时间为5 min以上时精子活动力显著下降,而ROS水平显著增加（P＜0.05）。②加样器抽吸2次以上可致人精子活动力显著下降（P＜0.05）,抽吸6次以上可导致人精子PR及VAP显著降低（P＜0.05）;但抽吸操作并未显著影响精子细胞内ROS的水平。结论：在人精子体外操作时应尽量缩短离心时间并控制加样器抽吸次数从而保证正常的精子运动参数,也应尽量缩短离心时间从而减少体外操作所致的细胞内ROS升高。     

Addition of Tempol in semen cryopreservation medium improves the post-thaw sperm function

Abstract

Despite extensive research carried out for optimization and commercialization of sperm cryopreservation media, percentage of motility and viability remain low following cryopreservation. These adverse effects have been partially ascribed to reactive oxygen species (ROS) production during cryopreservation. Therefore, we proposed that addition of a cell permeable antioxidant like Tempol, with superoxide dismutase (SOD) mimetic action, may overcome these effects in an optimized commercially available cryo-protective medium. Therefore, semen samples were cryopreserved in the presence or absence of Tempol. A concentration of 5?μM Tempol was defined as optimal since it significantly improved motility and viability post thawing and reduced DNA fragmented sperm. In addition, percentage of ROS positive sperm was reduced. These effects of Tempol can be attributed to cell permeability characteristic and ability to reduce superoxide production both at intra- and extra-cellular levels. Tempol may hold the potential for clinical applications.     

供精精液冻存时间对人工受精结局及子代出生缺陷的影响

目的:探讨精液冻存时间对供精人工受精(AID)结局及子代出生缺陷的影响。方法:回顾性分析2007年4月~2013年4月在本生殖中心实施AID助孕治疗的6038例妇女共14 290个AID周期的临床资料,分析供精精液冻存0.5~1年、1~3年、3~5年、5~7年、7~10年对AID妊娠率、流产率、子代出生缺陷率的影响。结果:供精精液不同冻存时间的妊娠率、流产率未见统计学差异(P0.05);子代出生缺陷发生率为1.33%,不同冻存时间子代出生缺陷率未见统计学差异(P0.05)。结论:供精精液冻存0.5~10年,并不影响AID的临床妊娠率、流产率和子代出生缺陷率,用于人工受精安全有效,能获得类似自然妊娠的效果。     

Effect of tubal explants and their secretions on bovine spermatozoa: modulation of ROS production and DNA damage

Although low levels of reactive oxygen species (ROS) play a physiological role in maintaining sperm function, an increase in ROS generation above these levels may result in the induction of sperm membrane and DNA damage. The main objective of this study was to determine whether bovine oviducal explants (TU) and their conditioned media (CM) have a modulatory effect on the production of ROS, and consequently, on sperm DNA integrity. Thawed sperm were exposed to bovine TU and to CM obtained from the ampullar and isthmal regions after 4 and 12h, and DNA damage and intracellular ROS production was assessed by TUNEL and DHE and SYTOX Green, respectively. Co-incubation of spermatozoa with oviducal explants from the ampullar region (TUa) for 4h resulted in a statistically significant increase in the percentage of spermatozoa with DNA damage compared with controls (P=0.0106), and this increase was positively correlated with ROS levels. Conversely, although the incubation of spermatozoa with explants and conditioned media from the isthmal region (TUi and CMi, respectively) for 12h resulted in an increase of spermatozoa with DNA damage compared with controls (P<0.0001), this increase was not correlated with ROS levels. In conclusion, significant oxidative stress may take place in the oviduct, particularly during short-term incubation, and this may be related to changes in the antioxidant factors present in the oviducal cells and secretions. A redox imbalance in pro-oxidants and antioxidants in the oviduct may lead to oxidative stress and sperm DNA damage.     

Cytosolic and mitochondrial ROS: which one is associated with poor chromatin remodeling?

The aim of this study was to determine if there are any associations between impaired chromatin packaging and the origin of reactive oxygen species (ROS) production. Cytosolic ROS, mitochondrial ROS, DNA protamination, and apoptosis were evaluated with dichlorofluoresceindiacetate (DCFH-DA), dihydrorhodamine 123 (DHR123), chromomycin A3 (CMA3), and YO-Pro-1 (Y1)/propidium iodide (PI), respectively, by flow cytometry (FCM) in 40 infertile individuals. Percentages DCF+ and R123+ sperm were positively associated with percentage CMA3+ sperm and negatively associated with percentage apoptotic sperm. No correlation was observed between CMA3+ sperm and the percentage of apoptotic sperm. Under protamination of sperm is not associated with the origin of ROS production, but their relationship may suggest an association with general physiological dysfunction of sperm. Furthermore, under protamination does make sperm prone to apoptosis. Rather, it is likely that apoptosis is induced by ROS production. Considering that these conclusions are derived from correlative analyses, additional studies including an interventive approach are required.     

Effect of density gradient centrifugation on reactive oxygen species in human semen

Density gradient centrifugation can separate motile sperm from immotile sperm and other cells for assisted reproduction, but may also remove antioxidants from seminal plasma, resulting in oxidative stress. Therefore, we investigated reactive oxygen species (ROS) concentrations and distribution in semen before and after density gradient centrifugation. We assessed semen volume, sperm concentration, sperm motility, and ROS levels before and after density gradient centrifugation (300 x g for 20 minutes) in 143 semen samples from 118 patients. The ROS removal rate was evaluated in ROS-positive samples and ROS formation rate in ROS-negative samples. Thirty-eight of 143 untreated samples (26.6%) were ROS-positive; sperm motility was significantly lower in these samples than in ROS-negative samples (p < 0.05). After density gradient centrifugation, only seven of the 38 ROS-positive samples (18.42%) exhibited a ROS-positive lower layer (containing motile sperm) with a ROS removal rate of 81.58%, whereas the upper layer was ROS-positive in 24 samples (63.16%). In the ROS-negative group (n = 105), ROS was detected in 19 samples after centrifugation (18.10%, ROS generation rate), of which 18 were ROS-positive only in the upper layer or interface and the other was ROS-positive in both layers. Density gradient centrifugation can separate motile sperm from immotile sperm as well as remove ROS (including newly generated ROS). This data supports the view that density gradient centrifugation can select motile spermatozoa without enhancing oxidative stress.

Abbreviations: ROS: reactive oxygen species; SOD: superoxide dismutase; GPx: glutathione peroxidase; DNA: deoxyribonucleic acid; DGC: density gradient centrifugation; IUI: intrauterine insemination; IVF: in vitro fertilization; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA: ethylenediaminetetraacetic acid; HTF: HEPES-buffered human tubal fluid; IMSI: intracytoplasmic morphologically selected sperm injection; SMAS: sperm motility analyzing system; CASA: computer-assisted semen analyzer; WHO: World Health Organization     

不同冻贮载体在人类微量精子冷冻保存技术中的应用效果

目的:评估不同冻贮载体在人类微量精子的冷冻保存技术中的应用效果。方法:在Pubmed、ScienceDirect、SpringerLink、Willey Online Library、CNKI、维普数据库、万方数据库检索2016年12月之前发表的、有关人类微量精子冷冻保存技术的文献,筛选纳入的文献均为非随机试验性研究。根据纽卡斯尔-渥太华量表(NOS)对所纳入的研究文献进行方法学质量评估和数据提取。结果:纳入了31个研究,包含了13种不同的冻贮载体,各研究的精子回收率59%~100%,平均存活率73.7%,有9个研究将解冻精子用于卵胞浆内单精子注射(ICSI)受精,受精率18%~71%,已报道5例采用冻融微量精子ICSI受精后行胚胎移植并获得妊娠,其中3例获得健康活产婴儿。结论:目前仍没有一种较为理想的冻贮载体用于微量精子冷冻保存,还需要多中心临床试验去评估这些冻贮载体的有效性。     

Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca²⁺ concentration in boar spermatozoa during cryopreservation

Protein tyrosine phosphorylation in sperm is associated with capacitation in several mammalian species. Although tyrosine phosphorylated proteins have been demonstrated in cryopreserved sperm, indicating capacitation-like changes during cryopreservation, these changes have not yet been quantified objectively. We monitored tyrosine phosphorylation, intracellular calcium and sperm kinematics throughout the cryopreservation process, and studied the relationships among them in boar spermatozoa. Sperm kinetics changed significantly during cryopreservation: curvilinear velocity, average path velocity and straight line velocity all decreased significantly (P < 0.05). While the percentage of sperm with high intracellular calcium declined (P < 0.05), global phosphorylation increased significantly (P < 0.01). Specifically, cooling to 5 °C induced phosphorylation in the spermatozoa. After cooling, a 32-kDa protein not observed in fresh semen appeared and was consistently present throughout the cryopreservation process. While the level of expression of this phosphoprotein decreased after addition of the second extender, frozen-thawed spermatozoa showed an increased expression. The proportion of sperm cells with phosphorylation in the acrosomal area also increased significantly (P < 0.05) during cryopreservation, indicating that phosphorylation might be associated with capacitation-like changes. These results provide the first quantitative evidence of dynamic changes in the subpopulation of boar spermatozoa undergoing tyrosine phosphorylation during cryopreservation.     

低温冷冻和孵育对人精子氧化应激水平的影响

目的：探讨低温冷冻和孵育对人精子氧化应激水平的影响。方法：60份精液，每份分成5份分别列入5组（处理前对照组、低温冷冻空白实验组、低温冷冻样本组、孵育空白实验组和孵育样本组）。以晚期氧化蛋白产物（AOPP）为蛋白氧化指标，丙二醛（MDA）为脂质过氧化指标，用分光光度法分别检测AOPP和MDA的水平。结果：低温冷冻样本组MDA水平明显高于处理前对照组和低温冷冻空白实验组（P〈0．05），但AOPP水平无明显差异（P〉0．05）。孵育样本组AOPP和MDA水平均高于处理前对照组、孵育空白实验组及低温冷冻样本组（P〈0．05）。结论：精子的低温冷冻和孵育，均存在氧化应激损伤。低温冷冻主要造成精子的脂质过氧化损伤，孵育可致精子的脂质过氧化损伤和蛋白氧化损伤。     

Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.     

EFFECTS OF CRYOPRESERVATION ON PENETRATION ABILITY OF HUMAN SPERMATOZOA INTO BOVINE CERVICAL MUCUS

Cryopreserved sperm exhibit lower fertilizing capacity in comparison to fresh sperm, partly due to effects of glycerol as the common cryoprotectant medium. Since standard semen analysis is not a good predictive method to assess sperm fertilizing capacity, functional tests like cervical mucus penetration may provide more useful information. A total of 24 semen samples were examined before and after cryopreservation for sperm parameters as well as number and motility of penetrated sperm into bovine cervical mucus (BCM) as an alternative for human cervical mucus. Freezing and thawing procedures have negative effects on sperm penetration into cervical mucus. No significant relation was noticed between sperm motility percentage or its penetration into BCM before and after cryopreservation, which denotes the variability in resistance of sperm to damaging effects of freezing.