精子核成熟度与精液参数关系研究

目的:研究精子核成熟度与精液参数关系。方法:49例精液标本,其中生育组15例,不育组34例。应用精子质量自动检测系统(CASA)进行精子密度、活力分析,伊红染色进行活率分析,联苯胺染色评价精液白细胞,采用精子形态检测系统下人工修正方法分析精子形态,用苯胺蓝染色评价精子核成熟度。结果:不育组苯胺蓝染色阳性率显著高于生育组(P<0.05)。形态异常精子组中头部异常、颈部异常、尾部异常、无定型、其它畸形精子组苯胺蓝染色阳性率均显著高于形态正常精子组(P<0.05)。苯胺蓝染色阳性精子率与形态正常精子率、活力、活率均呈显著负相关(P<0.05);苯胺蓝染色阳性精子率与精子密度、精液白细胞浓度均无显著相关性。结论:精子核成熟度异常可导致男性生育力下降,精子核成熟度是评价男性生育力重要参考指标。     

封闭式薄片法对人类微量精子超快速玻璃化冷冻的研究

目的:通过应用封闭式薄片法对人类微量精子进行超快速玻璃化冷冻,旨在寻找一种微量精子冷冻的最佳方法。方法:选取于大连妇儿生殖中心就诊的正常男性精液标本20例,精液上游处理后用于实验。首先用含冷冻保护剂的不同微滴量(0.5μl,1.0μl,3.5μl)对冷冻效果进行比较,以期找到微量精子冷冻的最佳微滴量。同时用无冷冻保护剂法对微量精子进行冷冻,并与冷冻保护剂组进行比较。结果:3组冷冻微滴量解冻后精子的回收率、活动率及存活率比较,差异均无统计学意义(P0.05)。无冷冻保护剂组与冷冻保护剂组的解冻后精子回收率、活动率及存活率比较,差异均无统计学意义(P0.05)。结论:封闭式薄片法超快速冷冻人类微量精子,解冻后可获得较高的精子回收率、活动率与存活率。无冷冻保护剂法冷冻微量精子,解冻后与冷冻保护剂法的结果相似。     

少量人类精子以空卵透明带为冻贮载体的冷冻保存

目的:探讨空卵透明带作贮存载体冷冻保存少量人类精子。方法:将人或金黄地鼠卵内的细胞成份全部除去,制备成空卵透明带,分别显微注入人睾丸精子、附睾精子和射出精子后冷冻保存,并与正常供精者射出精子作对照。结果:解冻后卵透明带在溶液中容易识别寻找,卵透明带内的精子易于观察。附睾精子组(n=11)与供精者射出精子组(n=7)的冷冻精子活动率和存活率无显著性差异(P>0.05),但睾丸精子组(n=7)这两项参数值均显著低于附睾精子和射出精子组(P<0.01)。含6％、7.5％和9％不同甘油浓度的冷冻保护剂,对冷冻精子活动率无显著性影响(P>0.05);冻贮于人和金黄地鼠空卵透明带内的精子,两者的冷冻精子活动率也无显著性差异(P>0.05〕。结论:空卵透明带是冻贮少量人类精子的合适载体。     

复发性流产及胎儿发育异常与男性畸形精子的相关性探讨

目的:探讨男性畸形精子与不明原因复发性流产及胎儿发育异常发生的相关性。方法:筛选185例配偶不明原因复发性流产及22例配偶因胎儿发育异常终止妊娠的男性的精液,涂片染色后按照严格标准进行精子形态分析,与配偶有过正常妊娠史的继发不育男性精液进行精子形态学比较。结果:复发性流产及胎儿发育异常与对照组精子正常形态百分率间比较差异无统计学意义,且胎儿异常组和复发性流产组组间差异也无统计学意义。结论:发生不明原因复发性流产和胎儿异常的精子畸形率并不显著高于正常妊娠,畸形精子率高低是否作为一个影响自然妊娠和胚胎发育的独立因素,缺乏足够的临床依据。     

微量精子冷冻复苏行卵胞浆内单精子注射的临床结局

目的:探讨经皮睾丸穿刺取精术(TESA)获得的微量精子经冷冻复苏后行卵胞浆内单精子注射(ICSI)治疗非梗阻性无精子症患者的临床效果。方法:回顾性分析2015年10月至2017年8月在我院生殖中心因少、弱、畸形精子症行射出精子常规ICSI及非梗阻性无精子患者TESA获得的微量精子行新鲜或冷冻后ICSI治疗,共238个周期的临床资料,132个周期为常规ICSI精子组,63个周期为冷冻TESA精子组,43个周期为新鲜TESA精子组,比较3组的实验室指标和临床妊娠结局。结果:常规ICSI精子组获卵数(10.58±5.37枚)与冷冻TESA精子组(10.73±4.19枚)和新鲜TESA精子组(10.88±4.67枚)相比差异无统计学意义(P0.05)。3组患者卵子成熟率、正常受精率、优质胚胎率相比差异无统计学意义(P0.05)。冷冻TESA精子组的临床妊娠率、多胎率、流产率(47.62%、26.67%、6.67%)与常规ICSI精子组(48.48%、25.00%、6.25%)及新鲜TESA精子组(51.16%、22.73%、4.55%)相比差异无统计学意义(P0.05)。结论:经皮睾丸穿刺取精术后对有活动精子的睾丸组织进行冷冻复苏行ICSI可以获得较好的治疗效果,也是治疗非梗阻性无精子症不育患者的有效方法。     

供精精液参数与受精妇女妊娠结局的Logistic回归分析

目的:探讨供精精液参数与受精妇女妊娠结局的关系。方法:回顾性分析本院生殖中心实施供精人工授精(AID)治疗的1903个周期,对女方年龄、授精精液采集后冷冻保存前和冻融复苏后各项参数和同一周期授精次数与术后妊娠结局进行单因素和多因素Logistic回归分析。结果:精子采集后冷冻保存前活动率(OR=1.982,P=0.042)、精子冻融复苏后前向运动精子总数(OR=1.699,P=0.031)和同一周期授精次数(OR=2.178,P=0.010)显著影响受精妇女的妊娠几率。结论:精子采集后冷冻保存前活动率>70%、精子冻融复苏后前向运动精子总数>30×106、同一周期授精2次能提高受精妇女的妊娠几率。     

精子线粒体膜电位与男性精液参数和体质量指数的相关性研究

目的探讨精子线粒体膜电位(MMP)与精子常规参数及体质量指数(BMI)的相关性。方法在本院辅助生殖科行精液分析的男性患者,禁欲3~7 d后,以手淫方式获取精液样本。通过计算机辅助精液分析仪检测精液常规,改良巴氏染色检查精子形态,JC-1染色后经流式细胞仪评估MMP。结果与对照组(56.68%±11.13%)相比,弱精子组MMP(41.24%±9.71%)显著降低。MMP与BMI呈显著负相关(r=-0.25,P0.01),与精子总活力(r=0.63,P0.01)、前向运动力(r=0.64,P0.01)及正常精子形态率(r=0.37,P0.01)呈显著正相关,而与年龄、精液量、精子浓度和精子数量其余参数的相关性均无统计学意义。结论精液中精子MMP是评估精子功能的重要指标,对临床综合分析男性不育症因素具有一定的参考意义。     

附睾降解性不活动精子症的研究

目的 :探讨精子不活动是否由附睾降解引起。方法 :本文选择 5例精液中无活动精子的不育患者 ,应用睾丸精子活力检测、精子头 -尾膜完整性结合试验和透射电镜观察 ,探讨其精子不活动的原因。结果 :5例患者睾丸活检组织所分离的睾丸精子 ,经孵育后的活动率为 2 %～ 1 1 %。睾丸精子组中头膜 -尾膜均完整的精子率显著高于射出精子组 ( P<0 .0 1 )。睾丸精子未见明显的降解 ,但透射电镜显示射出精子的浆膜、核等结构表现显著的降解变化。结论 :本组患者的精子可能经历了病理性附睾降解 ,引致精子丧失活动性 ,对这类患者采用活动的睾丸精子作辅助生育治疗有可能改善成功率。     

人精子2种扫描电镜样品制备方法的比较

目的:比较人精子2种扫描电镜样品制备方法的效果。方法:10例精液标本,同一例标本分别采用扫描电镜一般样品制备程序(方法A,A组)和本实验室方法(方法B,B组)处理。方法:B组减少了固定后洗涤精子、脱水和醋酸异戊脂置换的时间,增加了多级乙醇浓度梯度。观察2组开裂精子率的差别。结果:应用2种标本制备程序处理的各例标本,均可观察到开裂或断裂的精子。裂开部位可发生在精子头部、颈部或尾部,以发生在头部为多见,其次为颈部。A组的开裂精子率为28.9±16.2%,B组的为14.1±7.7%,B组的显著少于A组(P<0.01)。结论:方法B的程序更适合于人精子标本的制备,可减少精子表面的开裂或结构断裂,有利于人精子超微形态学观察。     

雷公藤单体(TW_(19))雄性抗生育活性的研究

雷公藤单体(TW_(19))系从雷公藤中分离、提纯出的一个二萜类化合物,本实验观察其对雄性大鼠抗生育作用及可能机理.TW_(19)(400μg/kg·d)给药三周后无抗生育作用,五周后生育力丧失,附睾尾部精子活力和密度明显降低,畸形率升高,附睾重量减轻,但对附睾上皮的形态变化无明显影响,管腔内可见畸形精子及脱落生精细胞.附睾尾部液中肉毒碱含量,给药组较对照组显著降低.TW_(19)对附睾头部α-糖苷酶和酸性磷酸酶含量没有明显影响;对生精上皮影响轻微,各期曲细精管基本正常,少数表现出精子细胞轻度损伤;TW_(19)可使睾丸重量减轻,并可显著降低睾丸透明质酸酶含量,但对睾丸乳酸脱氢酶-C_4没有明显抑制作用;对前列腺、储精囊等附属性腺器官重量没有影响.结论:TW_(19)对雄性大鼠具有确定的抗生育效果,作用环节可能在精子细胞及附睾.     

Flow cytometric comparison between swim-up and Percoll gradient techniques for the separation of frozen-thawed human spermatozoa.

Viable human spermatozoa were recovered from cryopreservation by either swim-up or Percoll density gradient techniques and their cell-surface oligosaccharide components were compared to those of fresh sperm. Sperm were labeled with FITC-Con A and analyzed by fluorescence microscopy and flow cytometry. By fluorescence microscopy, fresh sperm were uniformly labeled in the neck region alone. Frozen-thawed sperm, obtained by either swim-up or Percoll density gradients, showed two patterns of staining: one sperm population was stained in the neck region only, whilst another showed staining in both the neck and acrosome regions. No difference between sperm recovered by the two techniques was apparent. Flow cytometry profiles of fresh sperm revealed a single peak of fluorescence, whereas cryopreserved sperm gave two peaks of fluorescence. Samples recovered by Percoll density gradient contained significantly more sperm with the same fluorescence pattern as that observed for fresh sperm than did those recovered by swim-up. Cryopreservation alters the cell-surface oligosaccharide components of spermatozoa. Recovery of cryopreserved sperm by Percoll density gradient yields a greater proportion of sperm which resemble fresh sperm than does swim-up.     

Evaluation of acrosome reaction and viability of human sperm with two fluorescent dyes

Objective: To evaluate the usefulness of the Hoechst 33258/FITC-Pisum sativum agglutinin (FITC-PSA) staining for simultaneous assessment of viability and acrosome reaction rate (%AR) of human sperm. Material and Methods: Fresh sperm was collected 13 fertile donors provided fresh semen. We used motile sperm selected by the ”swim-up” procedure using modified HTF. Hoechst 33258 was added and co-incubated with sperm for 10 min. Samples were washed free of unbound stain and the sperm were mounted as smears on glass slides. After drying, sperms were incubated with FITC-PSA for 30 min. Sperm were examined by fluorescence microscopy. Also, FITC-Concanavalin A (FITC-ConA) staining and vital staining with yellowish eosin were performed simultaneously. The correlation of viability and %AR were analyzed. Results: Four different staining patterns were observed and clearly distinguished as follows: a) Viable acrosome-reacted sperm, b) Viable acrosome-intact sperm, c) dead acrosome-reacted sperm, d) dead acrosome-intact sperm. There was significant correlation between the results obtained by Hoechst 33258 and vital staining methods in viability of human sperm (r=0.927, P<0.001). There was significant correlation between the two methods (FITC-PSA and FITC-ConA) in %AR of human sperm (r=0.92, P<0.001). Conclusion: Viability and acrosome status of a human sperm can be easily assessed simultaneously by Hoechst 33258/FITC-PSA staining method. This combination method is considered useful to evaluate sperm function. Received: 8 May 2000 / Accepted: 31 July 2000     

Comparison of protective media and freezing techniques for cryopreservation of human semen

OBJECTIVE: To evaluate the influence of cryopreservation medium and freezing-thawing techniques on human sperm motility and morphology. STUDY DESIGN: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST-yolk ?zwitterion-citrate-egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]? and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST-yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40-50 and >50%) in fresh semen. RESULTS: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST-yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean+/-standard deviation) was 49+/-15.7, 43+/-15.2 and 52+/-16.8% when TEST-yolk was used and 34+/-17.8, 32+/-18.2 and 50+/-13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST-yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57+/-26.4, 38+/-8.6 and 38+/-17.3% in groups with >50, 40-50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using SAS/STAT software. CONCLUSIONS: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST-yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.     

External quality control in the andrology laboratory: an experimental multicenter trial

An external quality control study for semen analysis was performed involving 10 andrology laboratories in geographically separate locations. For the evaluation of sperm concentration, eight samples with different concentrations, and for the assessment of sperm morphology three slides prepared from different semen samples were distributed. Sperm motility was evaluated in five samples delivered cryopreserved to the participants. The coefficients of variation (CVs) for sperm counts varied with the sperm concentrations showing highest variability for low and lowest for high concentrations (range 23% to 73%). The CV for sperm morphology ranged from 25% for normal heads to 87% for abnormal midpieces. The CV for motility of sperm was 21%. For comparison the mean CVs for internal quality control were as follows: 10% for concentration, 8% for morphology (normal heads), and 8% for motility.     

Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates

Patients (155) were selected at random for fresh or cryopreserved semen and inseminated on the predicted day of ovulation. Semen analysis was performed using a microcomputerized multiple-exposure photography system. Frozen semen was used with either glycerol or TEST-yolk (TEST-buffered 20% egg yolk with 10% glycerol) as the cryoprotectant. Cryopreservation resulted in significant decreases in all semen parameters measured. Of these, velocity appeared to be the least effected. TEST-yolk provided significantly more protection against a reduction in velocity compared with glycerol. A total of 18, 17, and 27 patients conceived using fresh, glycerol, or TEST-yolk-preserved semen, respectively. For these same groups, a cumulative pregnancy rate of 52.9%, 27.1%, and 68.5%, respectively, was observed (not significant). The total number of motile sperm per insemination used for fresh artificial inseminations resulting in conception (132.4 X 10(6] was significantly greater than the number used for successful glycerol- and TEST-yolk-preserved semen (approximately 24 X 10(6]. These results demonstrate that although the number of motile sperm of cryopreserved ejaculates are dramatically reduced compared with the fresh counterparts, if a minimum criteria for ejaculate quality is established, the use of cryopreserved semen can offer a viable, effective, and relatively safe alternative to artificial insemination by donor with fresh semen.     

Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants

OBJECTIVE: To evaluate the ability of two cryopreservation methods and three cryoprotectants to preserve sperm quality. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): Twenty infertile men and 10 healthy donors. INTERVENTION(S): In the first experiment, semen was cryopreserved by either the Irvine Scientific method (IS) or the Cleveland Clinic Foundation (CCF) method. In the second experiment, semen was cryopreserved by the IS method and one of three cryoprotectants: TES and Tris yolk buffer, Sperm Freezing Medium, or Enhance Sperm Freeze. MAIN OUTCOME MEASURE(S): Postthaw sperm motility, cryosurvival, and kinematics. RESULT(S): Percentages of postthaw sperm motility and cryosurvival were higher in the IS cryopreservation method compared with in the CCF method (15.94 +/- 9.19 vs. 12.07 +/- 7.31 and 47.42 +/- 17.44 vs. 35.76 +/- 17.56). However, the CCF method resulted in significantly better sperm kinematics. Postthaw motility in the donors and patients was highest in the samples frozen in TES and Tris yolk buffer medium. CONCLUSION(S): The IS method was associated with more flash freezing compared with the CCF method and resulted in better preservation of sperm motility and a higher cryosurvival rate. TES and Tris yolk buffer was most effective at protecting sperm from the negative effects of the cryopreservation process. This may be due to the presence of egg yolk along with glycerol.     

Spermatozoal characteristics from fresh and frozen donor semen and their correlation with fertility outcome after intrauterine insemination.

OBJECTIVE: To determine if conventional sperm parameters, specific characteristics of sperm motion determined by computer-aided semen analysis (CASA), sperm penetration assay (SPA), and/or spontaneous acrosome reaction assay could best predict fertility outcome after intrauterine insemination (IUI) from frozen donor sperm. DESIGN: A retrospective analysis of 2,245 cycles of therapeutic donor IUIs were initially studied; 1,147 cycles that met selection criteria were used in this report. SETTING: A university-based assisted reproductive technology center. PATIENTS, PARTICIPANTS: All IUIs were performed on women with documented patency of at least one fallopian tube, ovulatory cycles, and who did not receive human menopausal gonadotropins. Sperm donors had to be used for at least four different recipients (mean of 15) and at least 14 different cycles of insemination (mean of 41). INTERVENTIONS: None. MAIN OUTCOME MEASURE: Pregnancy. RESULTS: Statistical comparisons were made between donors of different relative fertility by using the Mann-Whitney test, Spearman's rank correlation, and multiple regression analysis. These analyses demonstrated that the most significant predictors of the fertility of frozen-thawed donor sperm were curvilinear velocity, straight line velocity, and the total number of motile sperm inseminated. The number of sperm with spontaneous acrosome reactions negatively correlated with fertility outcome, and SPA provided no predictive value. CONCLUSIONS: Our study supports the hypothesis that the study of sperm motion characteristics using CASA after thawing and washing of cryopreserved sperm is a better predictor of fertile outcome after IUI than analysis of fresh semen.     

Prospective,randomized, blinded evaluation of donor semen quality provided by seven commercial sperm banks

OBJECTIVE: To evaluate variability in donor semen quality between seven commercial donor sperm banks, within sperm banks, and between intracervical insemination and intrauterine insemination. DESIGN: Prospective, randomized, blind evaluation of commercially available donor semen samples. SETTING: An academic andrology laboratory. PATIENT(S): Seventy-five cryopreserved donor semen samples were evaluated. INTERVENTION(S): Samples were coded, then blindly evaluated for semen quality. MAIN OUTCOME MEASURE(S): Standard semen quality parameters, including concentration, motility parameters, World Health Organization criteria morphology, and strict criteria morphology. RESULT(S): Significant differences were observed between donor semen banks for most semen quality parameters analyzed in intracervical insemination samples. In general, the greatest variability observed between banks was in percentage progressive sperm motility (range, 8.8 +/- 5.8 to 42.4 +/- 5.5) and normal sperm morphology (strict criteria; range, 10.1 +/- 3.3 to 26.6 +/- 4.7). Coefficients of variation within sperm banks were generally high. CONCLUSION(S): These data demonstrate the variability of donor semen quality provided by commercial sperm banks, both between banks and within a given bank. No relationship was observed between the size or type of sperm bank and the degree of variability. The data demonstrate the lack of uniformity in the criteria used to screen potential semen donors and emphasize the need for more stringent screening criteria and strict quality control in processing samples.     

Cryopreservation of Low Number of Human Spermatozoa; Which is Better: Vapor Phase or Direct Submerging in Liquid Nitrogen?

Cryopreservation and subsequent survival of semen samples with low numbers of human spermatozoa in assisted reproductive technology (ART) facilities can be challenging. The aim of this study was to compare the quality of warmed human spermatozoa following vitrification using direct submerging (DS) in liquid nitrogen (LN₂) or with LN₂ vapour (V). Normozoospermic ejaculates were prepared by the swim-up technique and the motile sperm fraction was divided into three groups: (i) fresh (control); (ii) DS methods in LN₂ (DS Group); or (iii) cryopreserved in V (Group V). Cryopreservation was performed in a small volume using a Cryotech device. After warming, sperm parameters (motility, viability, acrosome, DNA fragmentation and chromatin integrity) were assessed using cytochemical assays. Progressive motility, viability, chromatin integrity and DNA fragmentation differed significantly after warming when compared with the control. Sperm progressive motility and total motility rates were significantly higher in the DS Group compared to Group V. However, normal morphology, acrosome integrity and DNA damage were not significantly different between two groups. Also, sperm chromatin condensation using chromomycin-A3 (CMA3) and Aniline Blue (AB) staining showed fewer alterations in the DS Group compared to Group V. The rates of spermatozoa with an intact acrosome decreased significantly after thawing from 81.30?±?6.76% to 68.84?±?14.70% in the DS Group and to 60.92?±?8.06% in Group V. Cryopreservation of a small number of spermatozoa with the DS method showed superior outcomes with regard to motility, viability and chromatin configuration.