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。     

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。     

微滴无保护剂玻璃化法冷冻附睾微量精子的研究

目的:探讨微滴无保护剂的玻璃化法冷冻经皮附睾穿刺抽吸术(PESA)所获微量精子的可行性及安全性。方法:通过比较微滴法冷冻(微滴组)和常规慢速冷冻(冷冻管组)这两种冷冻方法的复苏率、回收率及精子核DNA断裂发生率,评价两种冷冻方法的冷冻效果。结果:22例患者附睾精子标本经两种方法冷冻后,仅冷冻管组中有1例患者附睾精子标本中未见活动精子,余均有活动精子,冷冻管组与微滴组的复苏率均低,分别为(18.16±9.38)%和(21.99±10.95)%,两者差异无显著性(P>0.05)。冷冻管组与微滴组的回收率分别为(58.39±12.67)%和(70.82±14.94)%,两者差异有显著性(P<0.01)。22例患者附睾精子冷冻前都存在一定比例核DNA断裂,经单细胞凝胶电泳后出现典型的彗星精子,精子核DNA断裂发生率为(26.68±9.45)%。复苏后冷冻管组和微滴组精子核DNA断裂发生率分别为(28.68±12.54)%和(27.64±10.70)%,与冷冻前比较均未增加,差异均无显著性意义(P>0.05)。结论:微滴可作为微量精子冷冻的合适载体,并且微滴法无保护剂的玻璃化冷冻附睾微量精子可能是一种简单、安全而行之有效的冷冻方法。     

不同冷冻保护剂对人类精子冷冻复苏后活动能力的影响

目的:比较添加葡萄糖或蔗糖的冷冻保护剂对人类精子冷冻复苏后活动力的影响。方法:将50例志愿者的精液标本分别采用添加葡萄糖或蔗糖的保护剂冷冻,比较精子复苏后的活动力。结果:冷冻前,前向活动精子百分率为(58.4±5.7)%,活动率为(63.4±6.1)%,采用添加葡萄糖的冷冻保护剂冷冻复苏后的前向活动精子百分率为(43.8±7.6)%,活动率为(48.4±7.6)%;采用添加蔗糖的冷冻保护剂冷冻复苏后的前向活动精子百分率为(42.6±8.9)%,活动率为(48.0±8.5)%。冷冻前后精子的前向活动百分率和总活动率差异有统计学意义(P<0.01),采用2种不同的冷冻保护剂冷冻复苏后的精子前向活动百分率和总活动率没有统计学差异(P>0.05)。结论:冷冻对精子活动力损伤明显,添加蔗糖作为精子冷冻的保护剂是可行的。     

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。     

含甘油冷冻保护液和冷冻复苏过程对人精子运动状态的影响

目的: 观察添加含甘油冷冻保护剂和冷冻复苏过程对精子运动能力的影响。　方法: 采用计算机辅助的精液分析技术对 18份人精液的精子运动状态进行检测,对比分析加入含甘油冷冻保护剂前后与冷冻复苏后的结果。　结果: 加入高渗透压的冷冻保护剂后,精液中快速直线运动精子的比例增加(P<0. 05),同时精子运动曲线速度(VCL)、直线速度(VSL)和平均路径速度(VAP)值均增加(P均 <0. 005),但前向运动精子和精子活率并无改变;冷冻前和复苏后比较,精子的各项运动速度和各级运动精子的比例均明显下降 (P均≤0. 01),精子头侧摆幅度(ALH)减小,摆动性(WOB)、直线性(LIN)、以及前向性 (STR)差异均无显著性 (P>0. 05)。复苏后精子运动模式特征的差异仅表现在与处理前相比较时。　结论: 在添加甘油的高渗透压的冷冻保护剂后,精子的运动速度更快,a级精子增多。冻融过程的损伤削弱精子的运动能力甚至使部分精子丧失运动能力导致精子活率全面下降。不同的冷冻复苏环节对精子运动的影响有所不同。     

自制金属冷冻板法冷冻人附睾精子的实验研究

目的:研究一种新型人精子冷冻方法对精子复苏率的影响,以探索人附睾精子、睾丸穿刺精子的最佳冷冻方法。方法:选取76例梗阻性无精子症患者的附睾穿刺(PESA)标本,按照自制金属冷冻板法和传统冷冻法分两组冷冻。采用计算机辅助精液分析系统检测冷冻前、后前向运动精子百分率,并对比两种方法对精子膜功能、DNA碎片指数(DFI)、顶体酶活性和精子畸形百分率的影响。结果:复苏后自制金属冷冻板法和传统冷冻法前向运动精子百分率[(12.0±7.5)%vs(8.0±5.1)%,P0.05]和低渗肿胀精子百分率[(22.0±17.5)%vs(18.0±20.5)%]比较有显著性差异(P0.05),均较冷冻前[(20.7±8.8)%和(30.0±13.5)%]显著下降(P0.05)。自制金属冷冻板法复苏后精子顶体酶活性显著高于传统冷冻法[(75.2±9.5)μIU/10~6精子vs(55.7±8.3)μIU/10~6精子,P0.05],均较冷冻前(120.0±10.5)μIU/10~6精子显著下降(P0.05)。两种方法复苏后畸形精子百分率和DFI无显著差异[(98.7±8.8)%vs(98.5±9.2)%,P0.05]和[(38.2±8.5)%vs(39.5±10.2)%,P0.05],并均较冷冻前[(97.2±9.5)%和(30.8±9.7)%]显著升高(P0.05)。自制金属冷冻板法和传统冷冻法冷冻复苏率[(65.2±12.0)%vs(52.3±18.0)%]有显著性差异(P0.05)。结论:自制金属冷冻板法是一种经济高效、操作简单的精子冷冻方法且能最大限度的节约精子;复苏后可以保证较好的精子复苏率、活动力和顶体酶活性。     

维生素E和B12对冷冻精子活力和DNA的保护作用

目的探讨维生素E(Vit E)和Vit B12对冷冻精子活力和DNA的保护作用。方法收集在我院生殖医学中心就诊的32例正常男性精液标本,按照添加维生素的不同分为3组:对照组:精液中只添加常规冷冻保护剂;Vit E组:添加常规冷冻保护剂和5mM的VitE;Vit B12组:添加常规冷冻保护剂和0.5%的Vit B12。比较3组精子复苏后的精子活动力、存活率、顶体酶活性、精子DNA碎片率(SDF)等指标。结果经液氮冷冻72h复苏后,Vit E组活动力(a+b)级的精子数、精子存活率均显著高于对照组(P0.01);顶体酶活性在各组间比较无显著性差异(P0.05);Vit E组和Vit B12组的SDF[分别为(12.16±0.50)%、(13.02±0.48)%]均显著低于对照组的(15.18±0.78)%(P0.01);Vit E组的精子DNA平均晕轮直径[(6.76±0.14)μm]显著高于对照组[(6.09±0.18)μm](P0.01),Vit B12组与对照组比较无显著性差异(P0.05)。结论精子冷冻保护剂中添加适当浓度的Vit E可以缓解冻融过程对精子造成的伤害,在一定程度上提高冻融后精子活动力、存活率及精子DNA完整性。     

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.     

经皮附睾穿刺取精精子冷冻复苏后卵细胞胞质内单精子注射治疗无精子症的初步研究

目的:回顾性分析27例无精子症患者经皮附睾穿刺取精术(PESA)所获精子冷冻复苏后行卵细胞胞质内单精子注射(ICSI)治疗后的效果及妊娠结局。方法:将诊断性附睾穿刺以及PESA治疗周期ICSI后所剩余活精子以常规方法加以冷冻,将复苏后找到了足量活精子并行ICSI的病例归为冻精组,而采用新鲜PESA活精子ICSI的病例则归为对照组。比较冻精组与对照组的受精率、种植率、临床妊娠率,同时分析两组间的妊娠并发症、新生儿出生及畸形等情况。结果:冻精组15个周期、对照组100个周期分别注射MⅡ期成熟卵子163、1 157个,受精率冻精组显著高于对照组(84.05%vs73.29%,P<0.05),种植率、临床妊娠率则两组间差异无显著性(23.07%vs15.73%;53.33%vs37.00%,P>0.05),新生儿出生体重差异亦无显著性(P>0.05)。冻精组共妊娠8例,已分娩5例,继续妊娠3例。对照组妊娠37例,已分娩30例,1例死胎;继续妊娠3例;流产4例。两组均未出现重大的妊娠并发症及新生儿畸形。结论:采用PESA冷冻精子ICSI是治疗男性无精子症的一种经济、有效、安全的方法;但PESA冻精复苏率有待于进一步提高。     

Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.     

Severe hypospermatogenesis in cases of nonobstructive azoospermia: should we use fresh or frozen testicular spermatozoa?

The aim of this comparative clinical study was to examine whether the fertilizing potential of frozen-thawed testicular sperm in the most severe cases of hypospermatogenesis is reduced compared with fresh testicular sperm. The results could determine the necessity of using fresh testicular sperm cells, which mandates involving the spouse by performing simultaneous in vitro fertilization intracytoplasmic sperm injection (IVF-ICSI) treatment in this subgroup of nonobstructive azoospermia (NOA) patients. We studied 13 couples in which the husband was diagnosed as having NOA and few motile testicular sperm cells or only immotile testicular sperm cells were isolated by testicular sperm extraction (TESE). Each couple underwent both an ICSI cycle, in which fresh testicular sperm that were retrieved shortly beforehand were injected, and a consecutive cycle, which used frozen-thawed sperm that were retrieved in the original TESE procedure but were cryopreserved and stored until use. We found that motility was lost during the freezing and thawing process in some cases, which resulted in significantly more cycles with only immotile sperm cells for injection in the frozen-thawed sperm group (38.5%) than in the fresh sperm group (7.7%; P < .05). Availability of only immotile sperm cells significantly reduced fertilization rates in both fresh and frozen-thawed groups, but the respective overall fertilization rate (44.9% vs 41.1%) and quality of embryos and pregnancy rate (18.2% vs 15.4%) were not significantly different between groups. Implantation rates were more favorable in the fresh sperm group (10.5% vs 5.9%), but not significantly so. We conclude that, although cryopreservation does impair motility, which results in significantly more cycles with only immotile sperm cells for ICSI in the most severe forms of hypospermatogenesis, fertilization and pregnancy rates are not significantly compromised.     

Epididymal epithelial cells cultured in vitro prolong the motility of bovine sperm

It is well known that the epididymis is an excellent environment to maintain sperm viability. Therefore, we used different sections of bovine epididymis (caput, corpus, and cauda) to develop epithelial cell culture monolayers to identify factors that will increase sperm survival in the freezing-thawing process. Each epididymal section was dissected and treated with collagenase to obtain epithelial cell clusters. The cells were cultured in RPMI-1640 medium with 10% serum at 38.5 degrees C. A confluent monolayer was obtained after 5-7 days in culture and preliminary characterization using cytokeratin antibody indicated that the cell culture contained 85%-95% of epithelial cells. These cellular cultures were tested for their ability to maintain motility of epididymal and frozen-thawed spermatozoa. Washed spermatozoa were added to obtain a final dilution of 1 x 10(6) spermatozoa/mL. The motility of frozen-thawed spermatozoa was also recorded after incubation in conditioned media. Our results show that cocultures of spermatozoa and epididymal cell monolayers for 24 and 48 hours were beneficial for maintaining epididymal and frozen-thawed sperm motility (36.0% and 20.4%) compared with spermatozoa cultured with fibroblast cells or in the absence of a cell monolayer (0%; P < .01). The conditioned medium provides favorable conditions for sperm motility. Results with conditioned medium on maintenance of frozen-thawed sperm motility suggest that epididymal cells in vitro secrete beneficial factors that prolong the sperm survival.     

Sole use of sucrose in human sperm cryopreservation

Glycerol alone or in combination with other additives is one of the most widely used and successful cryoprotectants for human sperm. The glycerol method requires rigorous post thaw sample washing for use in ART and this may lead to low sperm yield from oligospermic samples. In this study the feasibility of the use of sucrose in sperm cryopreservation was explored. Sucrose as cryoprotectant was combined with direct plunging of sample into liquid nitrogen (vitrification) as a freezing method. Sucrose treated sperm from normozoospermic and severly oligozoospermic samples underwent rapid freeze and thaw. Motility and viability were evaluated before freezing (after sucrose equilibration) as well as post freezing (after thaw). The 100 mM concentration of sucrose showed better cryoprotectant features compared to that of higher concentrations (200-1000 mM). Sucrose (100 mM)treated sperm maintained low but acceptable motility (30%) and satisfactory viability (60%) after freezing and thawing. The cryoprotectant capacity of sucrose for normozoospermic and oligozoospermic samples were identical. The sucrose method utilizes rapid freezing of a micro volume of sample and thus quickly freezes, thaws, and maximizes recovery of the sperm from the sample.     

Cryopreservation of rhesus monkey (Macaca mulatta) epididymal spermatozoa before and after refrigerated storage

Recently, there has been an increased interest in preservation of epididymal sperm as a potential source of material for genetic resource banking; however, cryopreservation of epididymal sperm from the rhesus monkey has not been explored. This study evaluated the effect of prolonged refrigerated storage of the intact cauda epididymides at various conditions on the postthaw motility of rhesus monkey epididymal spermatozoa, and also tested whether altering cryoprotectants and cooling methods could improve post-thaw motility for epididymal sperm after refrigerated storage. Motility before freezing decreased significantly after refrigerated storage (0 degrees C) for a period of 24 or 48 hours. Although postthaw motility was not significantly different after 24 hours of refrigerated storage, epididymides stored at a higher temperature (4 degrees C-10 degrees C) yielded better results, but postthaw motility still decreased significantly after 48 hours of refrigerated storage at 4 degrees C. Comparisons of glycerol and ethylene glycol at 3% and 6% revealed similar postthaw motility. However, consistently high postthaw motility was obtained with 3% glycerol throughout all freezing trials regardless of whether samples were collected fresh or after refrigerated storage for 24 or 48 hours. Cooling at a higher rate of 220 degrees C/min was found to yield better postthaw motility than the slower rate of 29 degrees C/min. Thawing time duration was evaluated, and a minimum of 30 seconds was required for thawing 0.25-mL straws containing 50-microL semen samples. An overall average of 42% postthaw motility was obtained for rhesus monkey epididymal sperm packed in 3% glycerol and cooled after 24 or 48 hours refrigerated storage. These postthaw motility results for epididymal sperm indicate that this method should be practical for use in preserving epididymal sperm, even if tissue must be shipped from sites remote from the cryopreservation laboratory.     

Surgical sperm retrieval after previous vasectomy and failed reversal: clinical implications for in vitro fertilization

OBJECTIVE: To investigate the effect of the interval between previous vasectomy reversal on retrieval rates of epididymal and testicular spermatozoa using percutaneous epididymal sperm aspiration (PESA), or testicular sperm extraction (TESE), and the subsequent reproductive potential of these gametes in intracytoplasmic sperm injection (ICSI) cycles. PATIENTS AND METHODS: Sixty-six consecutive sperm retrievals were considered in patients who were azoospermic after previous vasectomy, of whom 54 had had a previous failed reversal, the remainder deciding against a reversal. PESA and TESE retrieval rates were noted, as were the time since vasectomy and the interval between vasectomy and unsuccessful reversal. The presence of palpable epididymal cysts was noted, with their effect on sperm retrieval rates. Fertilization and pregnancy rates were analysed in subsequent ICSI cycles using freshly retrieved spermatozoa or frozen-thawed cryopreserved spermatozoa. RESULTS: All 66 patients had sperm retrieved successfully; the success rates for PESA were not significantly affected by previous failed reversal when compared with patients who had not had a reversal, at 14 of 54 (26%) vs five of 12 (P=0.3). The interval since vasectomy did not affect PESA retrieval rates but there was a significantly poorer retrieval rate for PESA in the presence of palpable epididymal cysts, at seven of 35 (20%) vs 12 of 23 (52%) (P=0.012). Fertilization rates were significantly lower using cryopreserved spermatozoa retrieved from either the epididymis or testis (50% vs 70%, P=0.007), although subsequent implantation and pregnancy rates were not significantly different. CONCLUSION: Surgical sperm retrieval is successful in all cases of azoospermia secondary to vasectomy, either by PESA or TESE. There are no clinical markers to indicate which patients will have successful PESA after vasectomy, although the presence of epididymal cysts is associated with significantly lower retrieval rates. The reduction in fertilising ability of cryopreserved spermatozoa does not affect clinical pregnancy rates in ICSI cycles.     

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre‐freezing equilibration times (2–3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm ). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60–85°C min^?1), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm ) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified–warmed sperm variables were at their best when the spermatozoa was diluted in TCG–6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.     

Functional significance of the sperm head morphometric size and shape for determining freezability in iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples

In the present study, computer-automated sperm head morphometry of epididymal samples was used to determine if sperm head area and shape are useful measurements for separating "good" and "bad" Iberian red deer freezers. A microscope slide was prepared from single diluted sperm fresh samples collected from 38 mature stags. Slides were air-dried and stained with Hemacolor. The sperm head area and shape (length/width) for a minimum of 145 sperm heads were determined for each male by means of the Sperm-Class Analyser. The remainder of each sample was frozen. After thawing, sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. All sperm parameters evaluated at thawing were placed in a statistical database and a multivariate cluster analysis performed. Mean sperm parameters of the 2 clusters generated ("bad" and "good" freezers) were compared by ANOVA. Our results show that sperm quality at thawing for all sperm parameters evaluated was significantly higher (P < .01) for "good" freezers than for the "bad" ones (sperm motility index: 67.4 +/- 2.0 vs 57.1 +/- 2.8; NAR: 67.1 +/- 2.5 vs 54.5 +/- 3.5; viability: 68.8 +/- 2.0 vs 60.1 +/- 2.8; HOST: 71.3 +/- 2.2 vs 63.1 +/- 3.1). Additionally, differences (P < .01) in epididymal sperm head area and shape were found between "good" and "bad" freezers before freezing, with the smallest overall sperm head dimensions found in the "good" freezers group (area: 32.04 microm2 vs 34.42 microm2). Thus, the lower the sperm head area in the fresh samples, the greater the sperm cryoresistance. Our results show that the 2 groups of males also differ in sperm head shape in fresh samples (good: 1.96 vs poor: 1.72; P < .01). It is possible that sperm head area and shape influence total sperm volume, thus causing differences in heat exchange as well as in movements of water, ions, and cryoprotectants and, in turn, on sperm freezability.