不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏效果的比较

目的比较不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏的效果。方法将15份上游后的精液标本分别采用常规精子冷冻和不使用冷冻保护剂的冷冻环玻璃化法冷冻,比较精子复苏后的活力、形态及精子膜的完整性三项指标。结果冷冻前、前向活动精子百分率、正常精子形态百分率及精子膜完整率分别为(79±6.42)%、(34±9.36)%和(91±5.18)%;不采用冷冻保护剂的玻璃化法冷冻复苏后,三者分别为(42.20±8.35)%、(31.00±7.63)%和(50.00±9.34)%。常规冷冻法冷冻复苏后,三者分别为(38.00±15.80)%、(30.00±5.24)%和(47.00±13.67)%。冷冻前后前向活动精子百分率和精子膜完整率的差别有统计学意义,但两种冷冻方法相比差异无统计学意义。结论使用不加入冷冻保护剂的玻璃化方法冷冻人的精子是可行的,可取得与常规冷冻相同的效果。     

甘油-卵黄-柠檬酸钠型冷冻保护剂未经冷冻对人精子运动学参数的影响

目的 研究甘油-卵黄-柠檬酸钠(glycerol-egg yolk-sodium citrate,GYC)型冷冻保护剂对人精子运动学参数的影响.方法 供精者精液标本183例,按照世界卫生组织推荐的方法和仪器行精液常规分析和计算机辅助精子分析(computer-aided sperm analysis,CASA);加入GYC型冷冻保护液后再次行CASA获得精子运动学参数.结果 加入GYC型冷冻保护剂后,供精者精子各项运动学参数与加入冷冻保护剂前的运动学参数均有显著的正相关(P<0.05);加入冷冻保护剂后,除BCF外,供精者精子各项参数的改变均有统计学意义(P<0.05).结论 GYC型冷冻保护剂对人精子的运动学参数有影响;人精子冷冻保护剂的配方仍需改良.     

精子冻融技术及其研究进展

精子冻融是辅助生殖技术的基础,冻融可能造成精子结构损伤和活力功能丧失,因此优化冻融方法十分必要。提高冷冻前精液质量、进行精子优选、添加合适的冷冻保护剂、选择适宜的冷冻复苏方法等均可有效地减少冻融损伤,有利于保存男性生育力。本文对精子冻融的基本原理、方法改良、针对特殊质量精液的冷冻方法及冷冻后精子质量评估进行了综述。     

蜂蜜作为人精子冷冻保护剂的初步探讨

本文对蜂蜜作为人精子冷冻保护剂的可能性进行了初步探讨,结果表明,用“甘油-蜂蜜”配方可取得与现行的“甘油-卵黄-葡萄糖-柠檬酸钠”配方相同的冷冻保护效果(P>0.05).蜂蜜的使用浓度以6％较佳。实验结果还提示保护剂的渗透压对精子存活率有一定影响。初步考虑蜂蜜的作用机制与提供能源、调节代谢、维持渗透压有关。它的应用简化了配制保护剂的操作程序、避免了使用卵黄带来的弊端,且价廉易得,因而有推广价值。     

精子冷冻环无保护剂玻璃化冷冻方法的初步研究

目的探讨精子冷冻环无保护剂玻璃化冷冻方法的可行性。方法正常精液标本上游处理后,进行常规冷冻和冷冻环无保护剂的玻璃化冷冻,复苏后分别从活力参数及电镜下超微结构等指标比较两种冷冻方法的效果。结果两种方法冷冻后精子存活率、活动率之间差异无显著性(48%∶48%;44.5%∶43.5%,P>0.05),但均较未冷冻的89%和88.5%明显下降(P<0.001)。超微结构亦较未冷冻时发生了一定的改变,但核结构基本保持完整。结论精子冷冻环无保护剂的玻璃化冷冻是一种简单、方便而行之有效的冷冻方法。     

不同冷冻方法对人睾丸精子冷冻保存效果的研究

目的比较不同冷冻保护剂和不同冷冻载体对人睾丸精子的冷冻保存效果。方法选取在兰州大学第一医院生殖医学专科医院进行诊断性睾丸穿刺获得成熟活动精子的患者,以及行睾丸精子ICSI术后的患者,在知情同意下将剩余睾丸组织冷冻保存,共46例。将每例睾丸组织分为4份,随机分成4组进行冷冻:1.8ml冷冻管+甘油复合冷冻剂(A组,46例);1.8ml冷冻管+Fasrun冷冻保护剂(B组,46例);0.25 ml麦管+Fasrun冷冻保护剂(C组,46例);超微量冷冻麦管+Fasrun冷冻保护剂(D组,46例)。冷冻标本经液氮熏蒸后投入液氮,比较各组复苏后精子活力和复苏率。结果冷冻前各组的活动睾丸精子能够满足ICSI操作所用精子量;冷冻复苏后,C组和D组能够容易找到正常活动精子(≥0.1/200倍视野)的例数显著高于A组、B组(P0.05);D组解冻后运动精子复苏率显著高于A组、B组(P0.05),C组解冻后运动精子复苏率显著高于A组(P0.05)。结论应用改良的超微量冷冻麦管+Fasrun冷冻保护剂能够提高睾丸精子的冷冻复苏率。     

冷冻保护剂与精液比例对冷冻复苏后精子活动力的影响

目的:比较冷冻保护剂与精浆的不同比例对人类精子冷冻复苏后活动力的影响。方法:将57例志愿者的精液标本分别采用冷冻保护剂∶精液=1∶1和冷冻保护剂∶精液=1∶3的体积比进行冷冻,比较精子复苏后的前向活动率和总活动率。结果:冷冻前的前向运动精子百分率和总活动率分别为(58.60±5.57)%和(66.17±5.24)%。采用冷冻保护剂∶精液=1∶1比例进行冷冻复苏后的前向运动精子百分率和总活动率分别为(40.53±8.97)%和(51.23±9.30)%;采用冷冻保护剂∶精液=1∶3比例进行冷冻复苏后的前向运动精子百分率和总活动率分别为(44.70±8.67)%和(51.50±7.40)%。冷冻前后精子的前向运动百分率和总活动率差异有显著性(P<0.05)。两种不同比例的保护剂相比冷冻后前向运动精子百分率差异有显著性(P<0.05),而总活动率差异没有统计学意义。结论:冷冻对精子活动力损伤明显,冷冻保护剂∶精液=1∶3比例较冷冻保护剂∶精液=1∶1比例可提高冷冻后前向运动精子百分率。     

采用冷冻精子与新鲜精子进行卵胞浆内单精子注射的结果比较

目的　比较冷冻精子与新鲜精子进行卵胞浆内单精子注射 ( ICSI)助孕技术的治疗效果。　方法　对 1 6 1对不育夫妇进行 1 6 3个辅助生殖技术治疗周期 ,其中采用冷冻精子 47个周期 ,比较了冷冻精子组与新鲜精子组 1 1 6个周期的受精率、卵裂率、A级胚胎率与临床妊娠率。　结果　冷冻精子组 (组 I)的受精率为 77.6 %、卵裂率为 92 .9%、A级胚胎率为 6 5.4%、临床妊娠率为 45.5% ;新鲜精液严重异常组 (组 II)分别为 54 .4%、94.0 %、45.7%及 2 5.0 % ;新鲜精液轻、中度异常组 (组 III)分别为 73 .5%、92 .5%、46 .8%及 2 9.3 %。组 I的受精率、A级胚胎率和临床妊娠率明显高于组 II( P<0 .0 5) ;与组 III比较无显著性差异 ( P>0 .0 5)。　结论　患者本身的精子质量直接影响 ICSI的受精率 ,精子冷冻复苏处理不影响 ICSI的受精率、卵裂率和优质胚胎率     

不同类型无精子症患者新鲜与冷冻精子妊娠结局比较

<正>ICSI的成功应用是男性不育治疗史上的一项重大突破,在治疗男性不育具有重要意义~([1])。ICSI技术现在已广泛应用于严重少、弱、畸精子症和无精子症患者,常规体外受精失败史以及行植入前遗传学检查等患者,这些患者可通过ICSI技术成功孕育自己的生物学后代。无精子症在普通人群中发病率为1%,不育患者中发病率为10%～15%~([2-3])。外科取精技术的发展,尤其是附睾或睾丸精子成功应用于辅助生殖技术,更是无精子症治疗史上的里程碑式     

冷冻复温后低温保护剂和精子激活剂对精子功能影响的研究

本文对冷冻复温后精子的功能进行了研究观察，发现精液保护剂对冷冻复温后精子的存活有明显的保护作用，而单纯应用卵黄或甘油溶液却对精子功能存在不利的影响。体外处理精子时应用ＡＴＰ，肌苷对精子的功能无任何的改善。     

速冻和缓慢冷冻法对精子运动特征的影响

目的了解冷冻方法对人精子运动特征的影响。方法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运动特征分析。结果冷冻复温后精子运动能力与冷冻前精子运动能力比较明显下降(P<0.001,P<0.05);速冻与缓慢冷冻方法保存的精子运动参数相比较差异均无显著性(P>0.05)。结论冷冻保存易导致精子运动能力下降,速冻与缓慢冷冻方法对精子运动参数影响无明显差异。     

Canine sperm vitrification with sucrose: effect on sperm function

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m ) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.     

Influence of motility and vitality in intracytoplasmic sperm injection with ejaculated and testicular sperm

The vitality of spermatozoa used for intracytoplasmic sperm injection (ICSI) is a crucial factor for fertilization, establishment and outcome of a pregnancy in assisted reproductive technique cycles. The sperm origin may also be a limiting factor, although little is known about this issue. It is known that the motility of injected spermatozoa and their origin from ejaculate or testicular biopsies are important predictors in terms of fertilization, pregnancy and birth rates. Oocytes of patients in 2593 cycles were retrieved in our in vitro fertilization programme and inseminated via ICSI. We used motile (group 1, n = 2317) or immotile ejaculated spermatozoa (group 2, n = 79), motile sperm retrieved from testicular biopsies (group 3, n = 62) and immotile spermatozoa from testicular biopsies (group 4, n = 135). Female age and number of oocytes retrieved did not differ significantly among the groups. The fertilization rates were as follows: 67.1% in group 1, 49.8% in group 2, 68.3% in group 3 and 47.8% in group 4. The pregnancy rates in cases where three embryos had been transferred amounted to 35.7% in group 1, 17.3% in group 2, 38.3% in group 3 and 20.5% in group 4. The embryo quality showed no differences between groups 1 and 3 (14.5), and between groups 2 (11.8) and 4 (10.8). The abortion rate was similar in groups 1-3, but increased in group 4 (26.6%, 27.3%, 31.6% and 55.5%). Irrespective of their origin, the fertilization potential of injected spermatozoa was found to be influenced by motility. The resulting pregnancy and birth rates, i.e. the potential of the resulting embryos to implant and to achieve viable pregnancies, seem to be additionally dependent on the sperm origin. This was well shown by declining rates when spermatozoa in a relatively early stage of maturity had been used. We see increasing evidence that the degree of sperm maturity has an important impact on the outcome of ICSI. In obstructive azoospermia, spermatozoa retrieved from the epididymis should be used rather than testicular biopsy spermatozoa, or testicular sperm should be preincubated in culture medium before ICSI.     

Need for sperm retrieval and cryopreservation at vasectomy reversal

PURPOSE: Controversy exists on whether to obtain sperm for cryopreservation routinely at vasectomy reversal. With recent improvements in in vitro fertilization with intracytoplasmic sperm injection, it is now possible to obtain a small amount of testicular tissue for cryopreservation in the event of reversal failure. However, to our knowledge no studies exist of who is most likely to benefit from this procedure. MATERIALS AND METHODS: We reviewed 84 consecutive vasectomy reversals performed by 1 surgeon (J. I. S.) between July 1996 and March 2000 with followup available for 77. We grouped cases by procedure as vasovasostomy, vasoepididymostomy and vasovasostomy with vasoepididymostomy as well as bilateral or unilateral. Sperm was retrieved at reversal in 15 of 46 vasovasostomy (none used), 11 of 18 vasoepididymostomy (3 used) and 13 of 20 vasovasostomy with vasoepididymostomy (none used) cases. RESULTS: The overall anastomotic patency rate after unilateral or bilateral vasovasostomy, unilateral vasovasostomy with contralateral vasoepididymostomy and unilateral or bilateral vasoepididymostomy was 96%, 83% and 57%, respectively. The natural pregnancy rate without in vitro fertilization was 57%, 50% and 14%, respectively. The most recent vasoepididymal anastomoses were performed by the Berger triangulation technique with a 78% patency and 25% pregnancy rate. Only 8% of men with banked sperm eventually used it for assisted reproductive techniques, in whom unilateral or bilateral vasoepididymostomy failed in all. CONCLUSIONS: We currently do not recommend routine sperm retrieval for cryopreservation in men who undergoing vasovasostomy. We encourage men who require bilateral vasoepididymostomy to bank sperm at reversal. In men who undergo vasovasostomy with vasoepididymostomy we base the decision on preoperative counseling and intraoperative findings.     

Reliable single sperm cryopreservation in Cell Sleepers for azoospermia management

Conventional sperm freezing methods perform best when freezing sperm samples containing at least hundreds of spermatozoa. In this severe male factor infertility case series, we examined the reproductive outcomes in 12 intracytoplasmic sperm injection cases where spermatozoa used were frozen in Cell Sleepers. Cell Sleepers are novel devices in which individual spermatozoa can be frozen in microdroplets. The case series included five men with obstructive azoospermia, six with nonobstructive azoospermia and one with cryptozoospermia, in whom microscopic sperm retrievals from testicular sperm extraction (TESE), micro‐TESE extracts and a centrifugation procedure resulted in less than 50 spermatozoa. A total of 304 microscopically retrieved spermatozoa were frozen in 20 Cell Sleepers using a rapid manual cryopreservation method. A total of 179 mature oocytes were injected with recovered thawed spermatozoa, resulting in a fertilisation rate of 65.9% (118 of 179), with no total fertilisation failures. In 10 cases, an embryo transfer was performed, three on day 3 and seven on day 5, resulting in a per cycle pregnancy rate of 58.3% (seven of 12). Four of the pregnancies have progressed past 20 gestation weeks. The recovery and use of spermatozoa that were frozen in Cell Sleepers was uncomplicated and effective and eliminated the need to perform any microscopic sperm retrieval procedures on the day of oocyte collection. Modification of the routine sperm cryopreservation methodology to include the use of Cell Sleepers increases the range of sperm samples that can be effectively cryopreserved, to include men with severe male factor fertility.     

冻存睾丸精子用于男性生殖力储备的探讨

目的:通过研究对无精子症患者实施睾丸活检或其他手术时冷冻睾丸精子经复苏后行卵细胞胞质内单精子注射(ICSI)助孕的临床效果,探讨冻存睾丸精子作为男性生殖力储备的有效性。方法:回顾性分析了在本院实施睾丸活检或其他手术时冷冻睾丸精子的患者96例,其中的55例已在本中心复苏冷冻精子行ICSI助孕共60个周期,评估其冷冻精子复苏、卵子受精、卵裂、可移植胚胎、优质胚胎、临床妊娠及其分娩情况。结果:复苏冻存睾丸精子60个周期均获成功,复苏后行ICSI技术助孕,受精率77.6%(513/661),2PN受精率69.4%(459/661),卵裂率99.4%(510/513),可利用胚胎率84.5%(431/510),优质胚胎率40.8%(208/510);所有周期均有可移植胚胎;新鲜胚胎移植52个周期,临床妊娠30例(临床妊娠率57.7%),双胎妊娠11例(其中1例双胎自然减为单胎),单胎妊娠19例,种植率为38.7%(41/106),流产率为3.33%(1/30)。目前,已经出生了20例健康婴儿(12个男婴,8个女婴),未发现先天缺陷儿;另外13例(7例单胎和6例双胎)继续妊娠中。结论:睾丸精子冷冻复苏后行ICSI助孕可以得到较好的临床结局。冻存睾丸精子是无精子症男性生殖力储备的有效方式。     

捐精者精子冷冻复苏率影响因素分析

目的:探讨季节、血型及精液参数等对捐精者精子冷冻复苏率的影响。方法:回顾性分析陕西省人类精子库捐精者4 088份精液标本,研究季节、血型、禁欲时间、精液量、精子形态、冷冻前精子活力及浓度对精子冷冻复苏率的影响。结果:捐精者精子冷冻复苏率随着精子浓度增高而增加,相关性分析提示精子浓度与冷冻复苏率呈正相关(r=0.247,P0.01)。而精子冷冻前活力和精子冷冻复苏率呈负相关(r=-0.262,P0.01)。禁欲第6天组的精子冷冻复苏率[(70.2±5.4)%]明显高于其他禁欲时间组(P0.01)。精子正常形态率20%组的精子冷冻复苏率[(71.4±5.1)%]要高于其他各组(P0.01)。A型血精子冷冻复苏率明显高于B型血[(69.1±4.8)%vs(69.8±4.7)%,P0.01];季节、精液量与精子冷冻复苏率之间无明显相关性(P0.05)。结论:捐精者的精子浓度、活力、形态及禁欲时间对于预测精子冷冻复苏率有一定的价值,而季节、血型、精液量与捐精者精子冷冻复苏率无明显相关性。     

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4 hr after warming with concentrations up to 100 μM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.     

Protective effect of butylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique

Butylhydroxytoluene (BHT), a synthetic analogue of vitamin E, shows antioxidant and antiviral properties and has been successfully used for mammalian sperm cryopreservation. In this study, BHT was included in a vitrification solution to determine its cryoprotective effect on human spermatozoa. Spermatozoa were selected by swim‐up and vitrified in close sealed straw using either a combination of human tubal fluid (HTF), sucrose and BHT 1 mm (VMBHT), or only HTF and sucrose (VM). The optimal concentration of BHT was determined by the observation of preserved progressive sperm motility (PSM) after warming and detection of plasma membrane (PMI), membrane mitochondrial potential (ΔΨm) and DNA integrity. The presence of reactive oxygen species (ROS) was also detected. The PSM was significantly higher in the VMBHT group (80.86 ± 5.41%) compared with the VM group (68.9 ± 3.67%) (P < 0.05). Butylhydroxytoluene significantly preserved DNA integrity (4.0 ± 0.1% versus 6.1 ± 1.6%; P < 0.05) and reduced ROS production (5.5 ± 2.2 versus 8.6 ± 1.8%; P < 0.05). Plasma membrane and ΔΨm showed no statistical differences. One millimolar BHT effectively maintained cell function and due to its antioxidant and antiviral properties could be used in semen cryopreservation of patients with viral infections transmitted by seminal plasma.