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。     

无渗透性冷冻保护剂的精子玻璃化冷冻研究进展

精子冷冻保存技术已经广泛应用于人类辅助生殖.近十余年来,国内外学者开始探索无冷冻保护剂的精子玻璃化冷冻,并获得了一定的成功[1].多项研究资料显示[2],使用无冷冻保护剂的玻璃化冷冻正常精液标本可取得与常规慢速冷冻相同的临床效果.但在这些报道的无冷冻保护剂的精子玻璃化冷冻研究中,实际上均存在一些非渗透性的冷冻保护剂,而...     

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。     

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。     

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。     

含雌激素的冷冻保护剂对少弱精子玻璃化冻存效果的研究

目的 比较添加雌激素的冷冻保护剂对少弱精子的冻存效果.方法 将40例少弱精子患者的精液标本分别采用甘油、甘油-卵黄-柠檬酸钠（GYC）、甘油＋雌激素（甘油＋ E2）、甘油-卵黄-柠檬酸钠＋雌激素（GYC＋ E2）四种冷冻保护剂（CPM）冷冻,比较冻融前后精子活率、活力（a＋b级）、正常精子形态百分率、精尾低渗肿胀率（HOSR）.结果 四种CPM对少弱精子的冷冻效果存在差别,精子活率、活力、正常形态百分率、精子尾部低渗肿胀率在冻后都有不同程度下降（P〈0.05）,但甘油＋E2、GYC＋ E2 能显著提高冻存后精子的活率及活力（a＋b级）.结论玻璃化冻存对精子存在损伤,含雌激素的冷冻保护剂可以提高低质量精液的冻存效果.     

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.     

睾丸精子冷冻的研究进展

全球约20％的育龄夫妇受不孕症影响,其中无精子症占男性不育病因的10％～15％,使用新鲜或冻融的睾丸精子行ICSI已成为无精子症患者实现生育梦想的主要方法.然而睾丸精子由于未在附睾中进一步成熟,其抗冷冻能力明显不如射精精子,使用常规冷冻方法冻融后精子活率差、回收率低.自首次使用冻融睾丸精子获得1例活产以来,睾丸精子冷冻...     

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。     

冷冻环玻璃化法冷冻小鼠卵母细胞的效果评价

目的评价ED15(15%ethylene glycol,EG+15%dimethylsulphoxide,DMSO)冷冻环玻璃化法冻存小鼠成熟卵母细胞的效果,以及对其纺锤体形态、染色体分布和发育潜能的影响。方法分别以小鼠新鲜成熟卵母细胞为对照组,玻璃化冷冻复苏卵母细胞为冷冻组。玻璃化冷冻以冷冻环为载体,以乙二醇(EG)及二甲亚砜(DMSO)联合作冷冻保护剂。解冻后观察细胞存活情况并对解冻后培养0、1、2h的卵母细胞行纺锤体及染色体免疫荧光染色,激光共聚焦扫描显微镜观察;其余卵母细胞行体外受精培养,计算受精率、囊胚形成率。结果冷冻组成熟卵母细胞存活率为98.2%,复苏后卵母细胞培养0、1、2h的纺锤体形态正常率及染色体分布正常率与对照组比较无显著性差异(分别为87.0%、90.9%、90.3%vs95%,P>0.05;91.3%、95.4%、93.5%vs90%,P>0.05);冷冻组受精率及囊胚形成率与对照组比较均无显著性差异(81.3%vs85.2%,P>0.05;76.1%vs75.4%,P>0.05)。结论ED15冷冻环玻璃化法冻存小鼠成熟卵母细胞复苏存活率高,对其纺锤体形态、染色体分布及发育潜能无明显影响。     

Male factors determining the outcome of intracytoplasmic sperm injection with epididymal and testicular spermatozoa

During a period of 8 years, 1,079 intracytoplasmic sperm injection (ICSI) procedures with aspirated epididymal or testicular spermatozoa were performed. Epididymal spermatozoa were used in 172 cycles and testicular spermatozoa or spermatids in 907 cycles. Multiple biopsies were obtained from at least two different locations in the testes. Retrieved spermatozoa were used after cryopreservation (frozen) or immediately after aspiration (fresh). Three hundred patients had obstructive azoospermia (OA) or ejaculation failure. In 414 cases, azoospermia was caused by impaired spermatogenesis resulting from maldescended testes, chemotherapy/radiotherapy, or by Sertoli-cell-only syndrome, genetic disorders or unknown aetiology. Transfer rates, pregnancy rates and birth rates per ICSI cycle showed no statistically significant differences between testicular and epididymal spermatozoa in men with OA (28% average birth rates in both cases). However, birth rates differed significantly with regard to the status of spermatogenesis. Treatment of men with nonobstructive azoospermia (NOA) resulted in a birth rate of 19% per cycle. In all patient groups, there was no difference in the birth rates achieved with fresh and cryopreserved spermatozoa. While testicular volume, follicle-stimulating hormone level and age of the male patient are no statistically significant prognostic factors, the underlying cause of azoospermia is the most important factor determining the outcome of ICSI with epididymal and testicular spermatozoa. The pregnancy rate is lower in NOA patients than in those with OA.     

Improved techniques for collecting motile spermatozoa from human semen

A simple apparatus to collect moving sperm by non-traumatic means which can be used for artificial insemination is described. The technique is based on enhancing the process of migration from the seminal fluid into a top-layered artificial medium in an ordinary test tube. This has been achieved by controlling 3 main variables: 1) The dilution of migrated sperm was minimize by using only 0.5 ml of the medium layered onto 1 ml semen; 2) increasing the surface area between these media by turning the test tube from a vertical to almost a horizontal position; 3) stimulating sperm activity by incubation at 37 degrees C under air: 5% CO2 for 30 min. When restored to a vertical position approximately 0.3 ml medium, sufficient for most AIH or IVF procedures, was gently aspirated. The effects of these variables on the rate of sperm migration was tested one at a time, and increments that ranged from 20% to as much as 10-fold were detected. When these 3 variables were optimized and 58 semen specimens analyzed, it was found that motility increased from 42 to 87%, velocity from 24.5 to 27.3 micron per sec, whilst abnormal forms dropped from 37 to 15%. The final concentration of motile sperm was 23 X 10(6)/ml compared to an original mean concentration of 34 X 10(6)/ml, indicating a relative recovery of 68%. Oligoasthenospermic specimens revealed similar changes in sperm motility, velocity, morphology and recovery. However, due to the low initial content of moving sperm (4.8 X 10(6)/ml), their final concentration was also low (2.7 X 10(6)/ml). Such specimens required additional preliminary preparation to increase the sperm concentration prior to the migratory procedure.     

Improved techniques for separating motile spermatozoa from human semen

A simple and atraumatic method for concentrating washed, motile spermatozoa from normal and subnormal semen specimens is described. It incorporates a modified technique of centrifugation in which sperm are spun onto a soft, fluid cushion, thereby minimizing mechanical damage. Following initial semen dilution to 5-10 ml in an artificial medium, the mixture is transferred to a test tube and layered onto 1 ml of oily contrast medium (Lipiodol). After centrifugation at 300 g for 10 min all but 0.5 ml of the supernatant is discarded, and the unshaken test tube is incubated at 37 degrees C for 15-20 min, during which time the motile sperm migrate into the upper 0.5 ml. After this incubation, 0.3 ml of the upper layer is removed which is sufficient for most IVF and AIH purposes. It contains concentrated, washed, motile spermatozoa that are free of debris and most abnormal forms. No change in pH or osmolarity and no diffusion of any iodine from the oil base into the top layer were detected.     

Increased fructolysis of kallikrein-stimulated human spermatozoa

Significant stimulation of human sperm motility by kallikrein, a kinin-releasing proteinase, was observed to occur in semen specimens with reduced sperm motility during an incubation period of 24 hours at 22 degrees C. Parallel to an increase of the total sperm motility an increase of the fructose utilization was measured after addition of a single dose of kallikrein (1 KU/ml) within the same incubation period.     

Primary male infertility in Kuwait: a cytogenetic and molecular study of 289 infertile Kuwaiti patients

Infertility is one of the major public health problems, affecting 15% of couples who attempt pregnancy; in 50% of these, the male partner is responsible. Chromosomal abnormalities and Y microdeletions in the azoospermia factor (AZF) region are known to be associated with spermatogenetic failure. In the present study, 289 patients with primary male infertility because of spermatogenetic failure were studied in order to highlight the molecular background of male infertility in Kuwait, and to avoid the possibility of transmission of any microdeletions/chromosomal aberrations to offspring via intracytoplasmic sperm injection (ICSI). Of the 289 infertile men, 23 patients (8%) had chromosomal aberration in the form of Klinefelter syndrome/variant (16/23; 69.6%), XYY syndrome (3/23; 13%), XX male syndrome (2/23; 8.7%), 45,X/46X, i(Yp)(1/23; 4.4%) and 45,XY, t(9;22) (1/23;4.4%). Y-chromosome microdeletion in the AZFb and AZFc regions were detected in 7/266 cases (2.6%). Testicular biopsy was carried out in 31 azoospermic patients, of whom five men had Sertoli-cell only syndrome, while 26 patients had spermatogenic arrest. In conclusion, this study showed that the frequency of both chromosomal anomalies and Y microdeletions were found in 10.4% of the infertile men. The potential risk of transmitting these genetic disorders to offspring provides a rationale for screening infertile men prior to ICSI.     

睾丸切开显微取精辅助非阻塞性无精子症患者生育

目的:探讨睾丸切开显微取精术在辅助男性非阻塞性无精子症患者生育的效果。方法:采用睾丸切开显微取精术获取精子,结合卵浆内单精子显微注射技术,辅助1例非阻塞性无精子症不育患者人工受精。结果:精子获取成功,结合卵浆内单精子显微注射技术使患者妻子获得妊娠,并成功分娩1健康女婴。结论:睾丸切开显微取精术为非阻塞性无精子症患者生育,提供了一种新的方法。     

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。     

冻融兔卵母细胞培养不同时间再激活效应的研究

目的探讨冻融兔卵母细胞行卵胞浆内单精子注射(ICSI)后,不同培养时间、不同激活方法,卵母细胞受精及胚胎发育能力。方法共收集兔卵母细胞470枚,玻璃化冷冻,解冻行ICSI后培养半小时,分为3组激活:组1(n=46):钙离子激活;组2(n=40):氯化锶(SrCl_2)激活;组3(n=33):无水酒精激活;再同样方法解冻一部分卵母细胞,ICSI后培养1 h,分为2组激活:组4(n=30):钙离子激活;组5(n=26):氯化锶激活;对照组(n=39):ICSI后直接培养,不激活。各组得到的囊胚玻璃化冷冻,受体兔HCG准备后,胚胎解冻后移植。结果卵母细胞解冻行ICSI后,组5的卵母细胞受精率、分裂率和囊胚形成率最高,分别为53.8%,26.9%和7.7%,所获囊胚的母兔可能怀孕。结论冷冻卵母细胞解冻行ICSI后,适当延长培养时间,使用合适的激活方法可能会提高卵母细胞的受精以及早期胚胎发育能力。