四种精液处理方法优选冷冻精子的比较

目的探讨洗涤法(SW)、密度梯度离心法(DGC)、上游法(SU)、磁性活性细胞分选法(MACS)对冷冻精液的优选效果,为供精人工授精(AID)和供精体外受精(IVF-D)提供可靠的数据。方法门诊收集健康男性精液,制备冷冻精液。冷冻精液复苏后,根据优选处理方法不同分为4组:SW组、DGC组、SU组和MACS组,每组样本各20例。比较分析各组优选前后精子浓度、前向运动精子活率、膜完整性、正常形态率及DNA碎片率等的差异。结果 SW组,处理前后前向运动精子活率、精子膜完整性、正常形态率及DNA完整率,均无显著性差异(P0.05);DGC和SU组中,优选后前向运动精子活率[分别为(78.19±12.13)vs.(34.17±8.61)、(82.20±7.17)vs.(42.86±16.38)]、精子膜完整率[分别为(84.62±5.84)vs.(68.17±6.74)、(81.44±4.47)vs.(65.19±6.15)]、正常形态率[(20.42±4.63)vs.(12.16±5.75)、(25.02±4.61)vs.(10.24±5.37)]及DNA完整率[(86.73±7.07)vs.(67.98±6.45)、(88.49±3.09)vs.(67.44±5.89)]均显著高于同组优选前(P0.05);MACS组中,优选后冷冻精子的膜完整性[(84.00±5.33)vs.(69.21±7.79)]、正常形态率[(13.72±4.58)vs.(7.90±3.80)]及DNA完整率[(91.95±2.82)vs.(69.79±3.90)]亦显著高于优选前(P0.05),但前向运动精子活率没有显著改善(P0.05)。结论四种方法在处理冷冻精子方面各有优缺点,临床可以根据精液标本的质量或者对于精液制备液的要求来选择不同的精子优化方法。MACS可以较好地优化DNA完整率,其在辅助生殖领域的应用具有一定前景。     

两种精子优选方法效果比较

目的　探讨精子经王氏管和密度梯度离心法处理后精子参数的变化 ,比较两者在宫内人工授精 (IUI)的临床效果。　方法　选择不育男性精液 15 7份 ,采用两种方法进行配对处理 ,比较两种分离方法前后精子活力、正常形态率、精子顶体形态和精子染色质等变化 ;分离后的精子用于IUI的临床妊娠率。　结果　两种方法处理后的精子活力 (a +b级 )、正常精子形态率、精子顶体完整率、正常染色质率与处理前比较有显著差异 (P <0 .0 1) ;但两种方法分离的精子用于IUI的临床妊娠率无显著差异 (P >0 .0 5 )。　结论　王氏管法和密度梯度离心法处理精子后均获得质量较好的精子 ,但用于IUI后临床妊娠率无明显差异     

鸡蛋白蛋白优选人精子

比较不同鸡蛋白蛋白（ Ｃ Ｅ Ａ）柱对 ３０ 份正常生育力精液进行精子优选的效果。分别以 ７５％单层 一步法（ Ｄ Ｃ）、７５％ 和１２５％ 双层一步法（ Ｓ Ｃ）及 ７５％ 预 选（ Ｆ Ｄ）再经 １０％ 、１５５％ 和 １７５％ 三层二步法（ Ｔ Ｃ） Ｃ Ｅ Ａ 柱进行优选。比较优选前后精子活动率、前向运动精子百分率和畸形率等常规指标及优选后各组精子回收率，并以盐酸喹啉染色比较优选前后精子 ｙ 小体阳性率。结果：优选后各组精子常规指标显著提高，以 Ｆ Ｄ 组精子回收率最高（３４７９％ ），高于上游法进行优选的回收率，与用人血清白蛋白进行优选的效果相当，可作为临床精子优选的有效手段。 Ｄ Ｃ、 Ｓ Ｃ 和 Ｔ Ｃ 各组回收率在 ２％ ～９％ 之间，显著低于 Ｆ Ｄ 组，但 ｙ 小体阳性 率均较优选前显著提 高， 在通过 Ｘ、 Ｙ 精子 分离以预防 Ｘ 连锁遗 传病上的可能用途有待进一步研究。     

辅助生殖中的精子优选研究进展

精子优选是辅助生殖技术中的重要环节。精子活力和形态等常规参数是评估精子的重要内容,近年来精子超微结构、DNA完整性、凋亡和膜成熟度等特征逐渐受到关注,针对这些特征筛选精子成为精子优选技术发展的方向。本文介绍了近年来发展起来的电泳法、电动电位法、透明质酸结合法、膜联蛋白V选择法、形态选择性单精子注射技术(IMSI)和微流控芯片法的技术原理,除微流控芯片法外其它技术均已应用于IVF或ICSI治疗中,同时对各种技术的优缺点和应用效果等方面做一综述。     

微流控芯片在人类精子优选中的应用

目的 研究自制微流控芯片优选人类精子的效果.方法 自行构建微流控芯片装置.收集男性不育患者的精液样本:①分别置于无毒灭菌离心管与微流控芯片中1h、2h、3h、4h,比较各时间点两组精子活力的变化;②分别用微流控芯片优选精子40min、60min、80min,比较各时间点优选前后各项精子参数的变化;③分别用微流控芯片、上...     

优选技术对人精子染色体及超微结构的影响

目的 :了解优选技术对人精子染色体及超微结构的影响。　方法 :分别应用上游法、Percoll密度梯度离心法及双曲管优选法处理精液 ,观察优选前后精子染色体畸变率、精子性染色体比例及超微结构相对正常的精子百分率。　结果 :精子染色体畸变率、精子性染色体比例及超微结构相对正常的精子百分率在 3种优选方法优选前后均无显著改变 (P >0 .0 5 )。　结论 :3种优选方法不会增加精子超微结构的损害 ,对精子染色体没有产生影响     

卵胞浆内单精子注射技术精子优选方法的研究进展

卵胞浆内单精子注射(ICSI)技术是针对男性因素不育最为有效的方法之一,而男性因素不育主要涉及到少、弱、畸精子症等.不同质量的精子与ICSI受精、胚胎质量、着床以及临床妊娠均有着直接的关联,选择形态结构以及功能正常的精子是提高临床结局的有效方法.在精液分析结果的基础上,必须选择和应用正确的精子选择技术.本文就精子的优选...     

基于微流控芯片模拟生理状况下精子自然优选研究

目的:在微流控芯片上模拟生理状态下的精子与宫颈粘液相互作用过程,寻找一种自然的接近生理状态的精子优选方法。方法:按功能构建微流控芯片,基于精子与宫颈粘液相互作用在芯片通道中模拟生理状态下的精子自然优选过程,进而实现对精子的优选,同时在芯片上集成实时精子检测池,通过计算机辅助精子分析系统(CASA)实时对精子的各项参数进行测定。本研究用微流控芯片法和常规上游法同时分析30例标本,并与分选前比较。结果:分选前a+b级精子百分率为(29.78±11.24)%、正常形态精子百分率为(8.00±5.19)%、直线速度(VSL)为(18.89±4.90)μm/s、平均路径速度(VAP)为(26.84±5.13)μm/s、前向性(STR)为(70.15±7.61)%。常规上游法分选后各项参数相应为(71.65±11.18)%、(14.95±6.79)%、(24.14±5.95)μm/s、(32.61±6.36)μm/s、(73.87±9.34)%。微流控芯片分选后各项参数相应为(92.37±6.33)%、(23.33±7.67)%、(34.03±16.78)μm/s、(38.73±16.40)μm/s、(84.91±12.56)%。芯片法与分选前比较,各参数差异有统计学意义(P均<0.01);与上游法比较,各参数差异亦有统计学意义(P均<0.05)。结论:本研究在微流控芯片上模拟生理状态下精子与宫颈粘液的相互作用过程,实现了对精子的自然优选和实时检测。优选出的精子质量较分选前及上游法都有明显提高,为芯片上实现模拟生理状态下的受精过程奠定了基础。     

黄精赞育胶囊优选方处理前后精子的超微结构研究

目的 观察黄精赞育胶囊优选方作用前后精子的超微结构变化.方法 运用原子力显微镜(AFM)技术对比观察正常精子和病理性精子在黄精赞育胶囊优选方作用前后超微结构的动态变化.结果 获得精子头体、颈部和鞭毛等部位的实时超微结构图像,发现药物T预后活动力低精子超微结构的缺陷有逐步被修复的现象.结论 黄精赞育胶囊通过修复活动力低精子超微结构的病理形态学缺陷可能足优选方改善弱精子质量的机制之一.     

微流控技术在精子优选及体外受精中的应用

由于传统辅助生殖技术(ART)成功率低,人们在不断追求探索新的技术。大量研究表明微流控技术具有革新传统体外受精(IVF)操作流程的潜能。与传统方法相比,微流控技术应用于精子优选及体外受精具有效率高、时间短、无离心损伤、实时观察筛选效果、微环境相似及自动化等显著优点,为ART提供了新平台。笔者综述了近年来微流控技术在精子活力评价与筛选、精子化学趋向性筛选、体外受精、精子浓度检测、精子分离富集等方面的应用。同时,本文简要讨论了微流控平台原理﹑结构设计及操作流程,聚焦各种方法的优缺点以及临床应用的可能性。可见微流控技术应用于ART仍有一些问题亟待解决,有理由相信其未来发展方向是通过制作高度集成化的平台即体外受精-芯片实验室(IVF-lab-on-a-chip)来实现。     

卵胞浆内单精子注射的精子优选技术的研究进展

随着辅助生殖技术的发展,卵胞浆内单精子注射(ICSI)技术为众多不孕不育夫妇带来了生育可能。精子的质量对于胚胎的生长发育和ICSI的临床结局有着至关重要的影响。先进的精子筛选技术可以提高具有高度DNA完整性的、结构完整的成熟精子用于受精的机会。本文简要综述了现有的ICSI精子优选技术及其对ICSI结局的影响,希望为ICSI技术的临床应用与进一步的发展提供新的思路。     

ART success and in vivo sperm cell selection depend on the ultramorphological status of spermatozoa

Management of male infertility has recently shifted from treatment of the subfertile man towards techniques of assisted reproduction (ART). This study aimed to evaluate the possible role of the ultramorphological status of the spermatozoon with respect to sperm selection in vivo and prediction of ART success. Ultramorphological sperm parameters were assessed retrospectively for 92 males with sufficient sperm density (10⁷ spermatozoa ejaculate⁻¹) whose wives conceived following a stepwise discarding of the female genital tract barriers, using intra-uterine insemination (IUI) ( n =26), in vitro fertilization (IVF) ( n =45) or intracytoplasmic sperm injection (ICSI) ( n =21). In parallel, sperm samples of 71 fertile males were examined. Normal ultramorphology of all head and tail subcellular organelles was found to be essential for the ability of spermatozoa to pass the lower female genital tract. The ultramorphological migration threshold for this barrier is apparently higher than that essential for oocyte fertilization. No specific indication associated with passage through the upper genital tract was found. A high prevalence of axonema defects was found to impair the ability of sperm cells to penetrate the oocyte investment. The natural fertility index, based on routine sperm parameters and the ultrastructural status of the spermatozoon's subcellular organelles was confirmed to be beneficial for directing patients to ART. A discriminative score based on axonema integrity was found to contribute additional information for the first choice decision between conventional ART and ICSI (75% prediction ability). Thus it may be helpful in finding the simplest and least expensive procedure with the greatest long-term chance for pregnancy.     

Artificial insemination with husband's sperm (AIH): techniques for sperm selection

Artificial insemination with husband's sperm (AIH) attempts to treat infertile couples by solving their particular infertility problems. This review is concerned with detailing and evaluating the experiences of different authors with AIH in infertile couples, types of insemination, and indications for insemination, with emphasis on techniques for sperm selection and separation: glass-wool filtration, separation on albumin columns, washed sperm, swim-up method, and spermatozoa selection in Percoll gradients.     

Sperm telomere length in motile sperm selection techniques: A qFISH approach

Several studies have associated telomere shortening with alterations in reproductive function. The objective of the present study was to determine telomere length (TL) in spermatozoa selected by either density‐gradient centrifugation (DGC) or swim‐up. The analysis of TL was performed using quantitative fluorescent in situ hybridisation (qFISH) using PNA probes in combination with a chromatin decompaction protocol in sperm cells. Results of TL were 24.64 ± 5.00 Kb and 24.95 ± 4.60 Kb before and after DGC, respectively, and 19.59 ± 8.02 Kb and 20.22 ± 5.18 Kb before and after swim‐up respectively. Sperm selected by DGC or swim‐up did not show any significant differences in TL as compared to nonselected sperm (p > .05). Negative correlations between TL and sperm motility (r = ?.308; p = .049) and concentration (r = ?.353; p = .028) were found. Furthermore, exposure of sperm to increasing concentrations of hydrogen peroxide during incubation resulted in a reduction in TL. These data indicate that oxidative stress may be one of the main factors involved in the reduction of TL in sperm. Preliminary clinical results from patients included in this study indicate that TL was shorter in spermatozoa from couples who never achieved a pregnancy compared to couples who did achieve at least one natural pregnancy (p < .05); however, the clinical utility of this biomarker still needs to be confirmed in further studies.     

Comparison of costs of different anaesthetic techniques

The costs of anaesthetic drugs, intravenous agents as well as gases, were studied for different anaesthetic techniques in a medium-sized operative procedure, cholecystectomy. Three anaesthetic breathing systems were used: a non-rebreathing system, a circle absorber system with medium fresh gas flows of 3-6 l/min, and a low-flow circle system. Anaesthesia without volatile inhalation agents used with a low-flow technique was the least expensive, and anaesthesia with isoflurane in a non-rebreathing system was the most expensive. The costs of anaesthesia without volatile inhalation agents in a non-rebreathing system, enflurane anaesthesia in a circle system with medium fresh gas flows, and isoflurane anaesthesia with low-flow technique were similar.     

The ability of sperm selection techniques to remove single- or double-strand DNA damage

A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are density-gradient centrifugation (DGC) and swim-up (SUP). To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques (ART), but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged (degraded) DNA, as characterized by the presence of both single- and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART.     

Comparison of two techniques used to collect normal and motile sperm

To improve the quality of sperm for intra-uterine insemination (IUI) or in-vitro fertilization (IVF) swim-up (SU) and migration-sedimentation (MS) methods were compared, for the selection of morphologically normal and motile sperm. In 74 patients consulting for couple infertility, it was shown that MS gave a better yield of motile sperm than did SU. An improvement in morphology and motility was achieved, especially in asthenospermia (motility less than 40%) and/or teratospermia (normal shape less than 40%). The percentage of midpiece and tail abnormalities was lowered by the MS technique. It is proposed that the MS method be used for IUI and IVF, adapting the number of tubes to the initial sperm concentration.     

Choosing among different technical variations of percoll centrifugation for sperm selection

Summary.  We compared the efficiency of different methodological variations of the centrifugation through discontinuous Percoll gradients (PC) to improve semen samples in the laboratory. Five different combinations of the number, volume and density of PC layers were assayed in 14 semen samples presenting various qualities. Each specimen was divided into five aliquots and processed simultaneously. The percentage of spermatozoa showing optimal movement (VAP > 30 μm s^-1 and STR > 80%) after PC selection when the number of gradients was reduced to three or two (PC-3: 43.3%, PC-2: 41.3%) and when the volume of layers was diminished to 0.5 ml (mPC-3: 44.2%, and mPC-2: 48.1%), was higher than in classical columns with four gradients of 1 ml (PC-4: 26.3%). The absolute recovery of optimal sperm was better with PC-2, mPC-2 and mPC-3. In samples showing low concentration or motility of spermatozoa, mPC-2 was the most effective technique. PC-2 and mPC-2 showed a tendency to eliminate more red blood cells contaminating the samples. Straight line velocity and straightness were similarly improved by all the methods. We conclude that the technique of PC centrifugation with only two gradients is simpler and more effective for sperm selection and in cases of poor samples can be used with low-volume layers.     

Effect of 655-nm diode laser on dog sperm motility

Sperm motility depends on energy consumption. Low-level laser irradiation increases adenosin triphosphate (ATP) production and energy supply to the cell. The aim of this study is to analyse whether the irradiation affects the parameters that characterise dog sperm motility. Fresh dog sperm samples were divided into four groups and irradiated with a 655-nm continuous-wave diode laser with varying doses: 0 (control), 4, 6 and 10 J/cm². At 0, 15 and 45 min following irradiation, pictures were taken of all the groups in order to study motility with computer-aided sperm analysis (CASA). Functional tests were also performed. Average path velocity (VAP), linear coefficient (Lin) and beat cross frequency (BCF) were statistically and significantly different when compared to the control. The functional tests also showed a significant difference. At these parameters, the 655-nm continuous-wave diode laser improves the speed and linear coefficient of the sperm.     

Comparison of two sperm preparation techniques using automated sperm motion analysis: migration sedimentation versus swim-up

Migration sedimentation and spermatozoa swim-up techniques were used for obtaining spermatozoa from the semen samples of 39 infertile men. Concentration, percentage of motile sperm, velocity, linearity, and motility index of the sperm preparations obtained by both methods were compared using the CellSoft automated sperm motion analyzer. The mean velocity of the spermatozoa obtained after the migration sedimentation technique was significantly higher than that with swim-up technique. Since it is not necessary to centrifuge spermatozoa with the migration sedimentation technique, this method may be more desirable than other techniques using centrifugation.