尿毒症患者精子形态特征的扫描电镜观察

目的:探讨尿毒症患者精子超微结构的形态特征。方法:应用扫描电镜观察10例尿毒症患者以及5名正常生育男性的精子。结果: 尿毒症患者精子异常有以下几种类型:①头部异常:包括精子顶体脱落、顶体缺失、核异常、尖头、小头、无头以及双头畸形精子;②颈部异常:包括精子颈部断裂、分离、颈部膨大;③尾部异常:包括线粒体膨大,线粒体缺失,无尾、双尾、短尾、卷尾等形态结构异常精子。正常生育男性精子头部、颈部、尾部形态结构正常。结论:尿毒症患者精子存在精子头部、颈部、尾部等多种形态结构异常。     

整合素亚基α_5、β_1在人精子的表达及其与受精的关系

目的 :研究精子表面整合素亚基 α5、β1的表达及其与精子功能状态和受精力的关系 ;整合素亚基 α5、β1的表达与不明原因不育症的关系。方法 :1 3例生育力正常男性的精液标本 ,9例临床诊断为不明原因不育症患者的精液标本。以精子穿透去透明带的金黄地鼠卵行体外受精试验 (SPA)检测精子受精力 ;对新鲜、获能和孕酮诱导顶体反应后精子行间接免疫荧光染色 ,流式细胞仪检测精子表面整合素亚基α5、β1表达阳性的精子百分率。以三色法染色 ,观察精子顶体反应的发生率。结果 :流式细胞仪检测显示正常组新鲜精子表面α5亚基的表达 (1 3 .3± 5 .4% )与对照组 (1 0 .7± 6 .4% )无显著差异 (P>0 .0 5 )。获能组精子表面 α5亚基的表达率 (6 5 .7± 1 7.6 % )比新鲜组和对照组显著增高 (P<0 .0 5 )。孕酮诱导顶体反应后 ,α5亚基的表达率 (6 5 .3± 1 8.9% )比新鲜组和对照组显著增高 (P<0 .0 5 ) ,但与获能精子组相比无显著差异 (P>0 .0 5 )。β1亚基的表达率分别为新鲜组1 2 .9± 7.4% ,获能组 1 5 .9± 7.9% ,诱导顶体反应组 1 6 .9± 6 .2 % ,与对照组 (分别为 :9.3± 3 .4% ,1 7.3± 7.8% ,1 2 .2± 8.7% )比较均无显著差异 (P>0 .0 5 )。获能精子表面α5亚基的阳性表达率与受精率有一定的线性相关 (r=0 .     

热水浸浴对不育患者精子参数的影响

目的:评价有热水浸浴习惯的不育患者精子参数的变化。方法:精液标本按照男子既往热水浸浴习惯与否进行分组,A组:26例,婚后多年未育,长期较热温度水(37℃)浴缸浸浴习惯;B组,26例,婚后多年未育,没有热水浸浴习惯。检测A、B组的精子活动率、前向运动精子率、精子浓度、精子畸形率和精子核空泡率。结果:与B组的精子参数比较,A组的精子活动率、前向运动精子率、精子浓度和正常形态精子率均有显著降低(P0.01),精子核空泡率显著升高(P0.01)。结论:较热温度水浸浴对睾丸精子发生过程有不良效应,导致精子参数显著降低。     

人类体外受精培养液内毒素水平的评价

目的:检测人类体外受精使用的商品性培养液的内毒素水平,评价人精子存活试验和小鼠2-细胞发育试验测试内毒素的差异。方法:36批次的商品性体外受精培养液,使用前(A组,n=36),及使用后的其中25份样品(B组,n=25)采用鲎试验法检测内毒素水平。另对比人精子存活试验和2-细胞胚胎发育试验的敏感性。结果:A组样品无内毒素阳性检出,B组有2份样品检出内毒素。A组和B组样品24h精子活动率改变与对照组比无显著性差异(P>0.05),但A组中有3份样品抑制小鼠2-细胞胚胎的发育。结论:体外培养环境和实验操作须防止内毒素污染。虽然商品性培养液未检出内毒素,仍须对其作严格的质量控制。人精子存活试验测试培养液质量和低内毒素水平的敏感性低于小鼠2-细胞发育试验。     

微流控芯片优选人类精子研究

目的:探讨微流控芯片技术对精子的优选能力。方法:自行设计和制造微流控芯片,利用芯片对40例人精液标本进行精子分选实验,优化其分选条件,观察芯片处理前后精液各参数变化。同时对其中30例精液标本(A组:a+b级精子<20%组,n=15;B组:a+b级精子≥20%组,n=15)同时用芯片法和密度梯度离心法分选,比较2种方法分离前后精子活力、形态等参数的变化。结果:①优选后精子活力和精子正常形态率都可见显著提高(P<0.001;P<0.01)。②在精子活动力优选上A、B组芯片法均明显优于密度梯度离心法(P<0.01),尤其在A组这种优势更为明显(P<0.001)。而在精子形态优选上,2种方法无显著差异(P>0.05)。结论:微流控芯片技术在优选精子中具有较高的分选效率,且具有操作简单、分选时间短,对精子损伤小的特点,在辅助生殖技术中特别是体外受精中将有良好的应用前景。     

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。     

3106个不同来源精子ICSI周期临床妊娠结局分析

目的:分析精子的来源对卵胞质内单精子注射(ICSI)治疗结局的影响。方法:回顾性分析因男性不育行ICSI的3 106个新鲜周期,按精子来源分为:射精组(A组)、附睾穿刺取精(PESA)组(B组)、睾丸穿刺取精(TESA)组(C组)、冻融PESA精子组(D组)及冻融TESA精子组(E组),比较各组ICSI后胚胎发育及妊娠结局情况。结果:C组2PN受精率、卵裂率显著低于A组及B组;B组临床妊娠率、胚胎植入率显著高于A组及C组,A组、B组及C组间分娩率、异位妊娠率、流产率及新生儿畸形率无统计学差异(P>0.05);E组2PN受精率显著低于D组,但B组与D组之间、C组与E组间2PN受精率、优质胚胎率、多胎率、流产率及异位妊娠率均无统计学差异(P>0.05)。结论:PESA/TESA-ICSI、冻融PESA/TESA精子技术是治疗梗阻性无精子症安全有效的方法,建议首先选择附睾取精,并可将剩余PESA/TESA精子冻存。     

畸形精子症患者精子染色体非整倍体的研究

目的:研究畸形精子症患者精子染色体的非整倍体率。方法:应用18号、X和Y染色体着丝粒探针,采用荧光原位杂交(FISH)技术比较畸形精子症患者(畸精组,n=18)和生育力正常且精子正常形态率、浓度、活力等均正常男性(对照组,n=5)精子中18号、X和Y染色体的非整倍体率。结果:畸精组共计数精子58 178条,对照组共计数精子16 369条。畸精组和对照组杂交效率分别为97.5%和98.3%;染色体非整倍体类型主要有二体(XX18、YY18、XY18、Y1818和X1818)和二倍体(1818XX、1818YY、1818XY)。畸精组和对照组的18号染色体二体率分别为0.29±0.16%和0.03±0.02%,性染色体二体率分别为0.65±0.24%和0.05±0.02%,二倍体率分别为0.14±0.12%和0.04±0.03%。18号、X和Y染色体非整倍体率组间均有统计学差异(P<0.05)。结论:与生育力和精液各参数均正常男性相比,畸形精子症患者18号、X和Y染色体非整倍体率明显升高。     

孕酮对人类精子透明带结合能力影响的研究

目的:探讨不同浓度孕酮对精子透明带结合能力的影响。方法:梯度离心洗涤精子后,将精子悬液分成5组,分别加入不同浓度的孕酮溶液,孕酮终浓度为0μmol/L、0.1μmol/L、0.5μmol/L、5μmol/L、10μmol/L,体外获能3.5 h。将取卵后常规IVF或ICSI授精7d内未受精的废弃卵子,共收集空白组57只卵子,实验组55只卵子,与精子体外共培养;2 h后观察卵子透明带结合精子数。结果:10μmol/L、5μmol/L孕酮组透明带精子结合数(66.6±5.3,64.5±5.1)显著高于空白组(61.5±4.1,59.8±5.0),差异有统计学意义(P<0.05)。低浓度孕酮(0.1μmol/L、0.5μmol/L)与空白对照组比较,无统计学差异(P>0.05)。结论:10μmol/L、5μmol/L孕酮可提高精子与卵子透明带结合力。低浓度孕酮(0.1μmol/L、0.5μmol/L)对精子透明带结合没有显著影响。     

卵母细胞滑面内质网聚集对早期胚胎及妊娠结局的影响

目的:研究卵胞浆内精子注射(ICSI)周期卵母细胞滑面内质网(sER)聚集对早期胚胎体外发育及妊娠结局的影响并分析其原因。方法:99例患者ICSI治疗107个周期,于HCG日测定血清激素E2、P、LH浓度以及子宫内膜厚度。根据所获MⅡ期卵母细胞是否出现sER聚集分为A、B两组,A组为所获MⅡ期卵母细胞均未出现sER聚集周期共90例;B组为有至少一枚MⅡ期卵母细胞出现sER聚集周期,共17例。比较A、B两组HCG日E2、P、LH水平、子宫内膜厚度,及治疗周期数、年龄、病程、Gn总量、Gn天数、获卵数、受精率、可用胚胎率、优质胚胎率、移植胚胎数、周期临床妊娠率、种植率、流产率和原发、继发不孕患者比例的差异。并且对比B组sER聚集阳性[sER(+)]与sER聚集阴性[sER(-)]卵母细胞的受精率、可用胚胎率和优质胚胎率。结果:B组HCG日E2水平明显高于A组(P<0.05,3141.18±604.47 vs 2635.12±825.46),而两组间P和LH水平以及子宫内膜厚度均无显著差异(P>0.05)。B组优质胚胎率显著低于A组(P<0.05,47.83%vs 57.67%),而B组Gn天数(P<0.01,13.35±1.66 vs 11.83±2.4)和流产率(P<0.05,100%vs 17.86%)均明显高于A组,两组在年龄、治疗周期数、病程、Gn总量、获卵数、受精率、可用胚胎率、移植胚胎数、临床妊娠率、种植率以及原发、继发不孕患者比例等各方面均无显著差异(P>0.05)。B组sER(+)卵母细胞的优质胚胎率显著低于sER-卵母细胞(P<0.01,20.69%vs 52.9%),而受精率和可用胚胎率无显著性差异(P>0.05)。结论:ICSI周期卵母细胞sER聚集对早期胚胎体外发育及妊娠结局均有不良影响,sER聚集可能与HCG日高E2水平及长时间Gn刺激有关。     

The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation

Purpose: To study the effect of different sperm preparation methods and incubation times post preparation on sperm DNA fragmentation. Methods: Sperm DNA fragmentation was assessed by sperm chromatin dispersion (SCD) method on motile sperm prepared by gradient centrifugation or swim-up and incubated in IVF medium for up to 24 hours. Data were analyzed to discover the effect of preparation methods and incubation times on sperm DNA Fragmentation Index (DFI). Results: There were no differences in DFI in sperm samples prepared by gradient centrifugation method or swim-up (3.87?±?2.14 vs. 3.45?±?1.83, p?=?0.544). However, an increase was observed in DFI in samples prepared by swim-up after 6–8 hours compared with by gradient centrifugation (34° vs. 13°, p?=?0.04). In the swim-up group, the DFI level at 4 hours was already significantly higher than it was initially. However, following gradient centrifugation, the DFI at 8 hours was significantly higher than the initial DFI level. Conclusion: Sperm samples prepared by gradient centrifugation may be more stable compared to samples prepared by swim-up in terms of DNA Fragmentation.     

Effect of pentoxifylline on immotile testicular spermatozoa

Purpose: A model for differentiating live and dead sperm cells during intracytoplasmic sperm injection (ICSI) is proposed. Methods: We used pentoxifylline, a phosphodiesterase inhibitor known to enhance sperm motility, to initiate motility in testicular spermatozoa. Ten immotile testicular sperm samples were divided into two parts for examination of sperm motility with and without pentoxifylline treatment at 30, 60, and 90 min. Results: The samples without pentoxifylline remained immotile even after 90 min of incubation: the addition of pentoxifylline initiated sperm motility in all samples: 51.8±10.2, 64.4±9.4, and 70.8±8.9% (mean±SD) at 30, 60, and 90 min, respectively. Conclusions: That pentoxifylline may be used to differentiate live testicular spermatozoa during ICSI, which may improve fertilization and pregnancy rates, is suggested.     

己酮可可碱和水溶性维生素E在精子优选过程中对精子质量的影响

目的:探索在精子优化分离过程中,己酮可可碱(pentoxifylline,PF)和水溶性维生素E(water-soluble vitamin E,WsVitE)对改善精子质量的价值。方法:分别以密度梯度法、上泳法常用精子分离液作为对照组,在同批号对照组分离液中添加PF和/或WsVitE后作为试验组;将32份新鲜精液标本每份分为4份,分别以密度梯度法、上泳法2种方式优化分离,每种分离方式均设对照组和试验组;比较分离后精子的质量差异。结果:2种分离方法处理后的新鲜精子,其前向运动率、获能后酪氨酸磷酸化水平、Ca2+载体诱发精子顶体反应率,试验组均优于对照组(P<0.01);25℃孵育24 h后,试验组较对照组的精子前向运动率、活动指数、正常形态率更高,而DNA碎片、脂质过氧化水平更低(P<0.01);上泳法试验组分离率精子各项指标总体优于密度梯度法试验组(P<0.05)。结论:在精子分离液中加入抗氧化剂VitE和磷酸二酯酶抑制剂PF可明显改善分离后精子的质量;上泳法分离后的精子质量更高、损伤更轻。     

Comparison of the in vitro fertilization rate by human sperm capacitated by multiple-tube swim-up and Percoll gradient centrifugation

Two sperm preparation methods, a multiple-tube swim-up and Percoll-gradient centrifugation, were employed in our human in vitro fertilization program. The fertilization rate of these two sperm preparation methods was compared when they were employed in semen samples of <60 million motile sperm/ml. The results described here suggest that both of these methods gave a similar fertilization rate in these semen samples, i.e., 72±8% for the Percoll-gradient centrifugation method and 66±8% for the multiple-tube swim-up method.     

Comparison of cryopreservation outcome following intracytoplasmic sperm injection and conventional in vitro fertilization

Purpose Our purpose was to compare the success rate of transferring frozen-thawed embryos generated from either intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Methods A retrospective review of all frozen—thawed embryo transfer (ET) cycles between January 1995 and April 1997 was performed. There were 83 and 204 transfer cycles of frozen — thawed multicellular embryos generated from conventional IVF (group A) and ICSI (group B), respectively. The survival rate of frozen — thawed embryos and the outcome following ET in both groups were assessed. Results The groups did not differ in age (31.7±4.6 and 30.6±6.0; mean±SD) or number of embryos transferred (3.5±1.1 and 3.8±1.3 for groups A and B, respectively). An acceptable pregnancy rate per ET was achieved in both groups, but the rate was significantly higher (P = 0.04) for group A than group B, 32.5 and 20%, respectively. Group A included frozen embryos of a higher quality than those of group B, but the proportion of embryos surviving after thawing was significantly higher for group B than group A (92.5 and 85.6%, respectively; P = 0.0004). The abortion rate did not differ between the two groups: 22 and 26.8% for groups A and B, respectively. Conclusions Although an overall high pregnancy rate was achieved following frozen-thawed ET, it was lower for cycles in which embryos had been generated from ICSI. This difference may be attributed to a lower prefreezing embryo quality in the ICSI group. Embryos originating from ICSI were not vulnerable to cryopreservation and, when implanted, resulted in a comparable abortion rate to thawed embryos of conventional IVF.     

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.     

Using two different thawing temperatures and their effect on the motility recovery of human cryopreserved sperms in cancer patients

Human sperm cryopreservation is a widely used technique which helps the male partner for the fertility insurance by preserving their sperms for a long time. Cancer patients suffer from low semen quality especially who undergo chemotherapy or radiation treatments may face complete loss of their sperm production. This study aimed to investigate the effects of different thawing temperature (37?°C & 40?°C) on sperm motility recovery after cryopreservation-involving four types of cancer tumors at variable ages between 16 and 42?years- to help cancer patient for storage their sperms. A detailed semen analysis was collected under guidelines for 30 samples. The samples were then analysed and frozen in liquid nitrogen. The semen samples were subsequently thawed at 37?°C & 40°Cfor 3?min. This was then followed by statistical analysis of the comparative motilities. Results: thawing of cryopreserved human sperm at 40?°C results in a statistically significant increase in motility recovery compared with thawing at 37?°C.     

A simple,rapid and economic manual method for human sperm DNA extraction in genetic and epigenetic studies

Over the recent years, isolation of high quantities of pure, intact, double-stranded, highly concentrated and not contaminated genomic DNA is prerequisite for successful and reliable large scale genetic and epigenetic analysis and also high quantities of pure DNA are required for these types of studies. The DNA extraction methods developed for human somatic cells are not effective for sperm because of the high rating of nuclear compaction and DNA integrity in sperm cells. In the present study, an economic, reliable and simple method of DNA extraction procedures, were examined to ascertain their relative effectiveness for extracting DNA from human sperm. The quality and quantity of the extracted DNA were assessed by spectrophotometric measurements, methylation-specific PCR (MSP) amplification, and gel electrophoresis. This method was applied successfully from sperm samples (N?=?50, age?=?33.9?±?7.8?years) with DNAs absorbency ratios of A260/A280 (1.82?±?0.03) and A260/230 (1.95?±?0.16). Gel electrophoresis revealed non-degraded and suitable quality genomic DNA for epigenetic and genetic analysis. The purity and quality of this protocol was closer to the optimum value sand no contamination during manual extraction was observed. It concluded that this described method might have a sufficient quality for subsequent molecular analysis and general genetic and epigenetic methods.     

ANDROLOGY: Influence of Swim-Up Method on the Recovery of Spermatozoa From Different Types of Semen Samples

Purpose: To compare the effectiveness of swim-up method, using different types of semen samples.Methods: In this retrospective study undertaken in university medical college infertility centre, subfertile couples undergoing Intra Uterine Insemination were selected. A total of 600 semen samples used for the preparation of sperm samples using swim-up method were analyzed. Relative Yield was calculated from the sperm count and motility before and after swim up from each semen sample in six different groups.Result(s): Statistically significant increase in relative yield was found in oligospermic samples (20.41) followed by teratospermia (16.98). However, relative yield was low in asthenospermic (11.97) and normal (>60 million/ml) samples (11.66).Conclusion(s): Semen samples with good sperm count resulted in poor recovery after swim up. Swim-up method appears to be effective for oligospermic samples. Modifications like multiple tube swim up, using appropriate incubation time based on the initial semen parameter, will enhance the sperm yield after swim up.