梗阻性无精子症患者精子顶体完整性与卵胞质单精子注射治疗结局之间的关系

目的:探讨梗阻性无精子症(OA)患者精子的顶体完整性及其与卵胞质单精子注射(ICSI)治疗临床结局之间的关系。方法:选取梗阻性无精子症患者共37例为试验组,同期进行体外受精治疗且精液常规参数正常的男性33例为对照组,应用荧光标记的豌豆凝集素法(PSA-FITC)检测精子顶体完整性,巴氏染色法分析精子形态,比较试验组与对照组的顶体完整率(AIR)、正常形态率(NFR)、受精率(FR)、卵裂率(CR)及优质胚胎率(OER),并将AIR与FR、NFR与FR进行相关性分析。结果:试验组的AIR、NFR、FR显著低于对照组(P<0.01),CR、OER试验组与对照组相比无统计学差异(P>0.05)。试验组AIR与FR呈显著正相关(r=0.595,P<0.01),NFR与FR显著正相关(r=0.463,P<0.01);对照组AIR与FR显著正相关(r=0.683,P<0.01),NFR与FR呈显著正相关(r=0.205,P<0.01)。结论:梗阻性无精子症患者的精子AIR较低。行皮下附睾抽吸术(PESA)-ICSI的梗阻性无精子症患者精子其AIR高则受精率也会高。     

精子DNA碎片与体外受精结局的关系

目的:探讨精子DNA碎片与体外受精(in vitro fertilization,IVF)结局的关系。方法:采用染色质扩散实验(sperm chromatin dispersion,SCD)对242例接受IVF的男方进行精子DNA碎片率(DNAfragmentation index,DFI)检测,按照WHO标准进行精液常规分析,将精子DFI、精液常规参数和IVF受精率、卵裂率、可移植胚胎率、优质胚胎率进行Spearman相关分析,将精子DFI、精液常规参数对生化妊娠、临床妊娠的影响进行Logistic回归分析。结果:精子DFI与精子前向活动率呈负相关(r=-0.355,P<0.001);密度梯度法处理前、后精子DFI均与IVF受精率呈负相关(r=-0.223,P<0.001)(r=-0.136,P<0.05);精子DFI、精液常规参数与卵裂率、可移植胚胎率、优质胚胎率无相关性;精子DFI与生化妊娠、临床妊娠结局无相关性。结论:精子DFI影响精子活力与IVF受精率,精子DFI检测对预测IVF受精率有一定的临床意义。     

两种精子优化处理方法在体外受精中的应用选择

目的:评价Isolate密度梯度离心法和上游法两种活动精子处理方法在体外受精-胚胎移植(IVF-ET)中的效果。方法:本中心2003.01-12完成的253个IVF-ET周期为研究对象,回顾性地比较了两种精子分离方法的受精率、卵裂率、优质胚胎率和临床妊娠率。结果:Isolate离心法和上游法两组比较,受精率分别为84.13%和94.06%,有显著差异(P<0.01);卵裂率93.81%和97.01%,有显著差异(P<0.01);优秀胚胎率62.80%和56.62%,差异显著(P<0.05);临床妊娠率53.5%和48.0%,差异无统计学意义(P>0.05)。结论:在IVF-ET治疗不孕不育中,Isolate离心法分离的活动精子在受精和卵裂上不如上游法,在优质胚胎率上优于上游法,但两者的临床妊娠率无显著差异,故两者同样适用于IVF精子处理。     

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。     

重组人睾丸精子结合蛋白对人精子获能及膜功能的影响

目的:探讨重组人睾丸精子结合蛋白(testis sperm binding protein,TSBP)对人精子获能及膜功能的影响。方法:采集22例健康生育男性新鲜精液样本,精子经Percoll密度梯度离心法优化处理后与不同浓度的重组人睾丸精子结合蛋白分别孵育培养1h或3h,取培养后精子进行孕酮诱导顶体反应与精子膜低渗肿胀功能检测。结果:0.1mg/mlTSBP处理组孕酮诱导精子顶体反应率及精子膜尾部低渗肿胀率均明显高于对照组(P<0.05);0.01mg/mlTSBP处理组各指标与对照组无显著差异。结论:重组人睾丸精子结合蛋白在体外可以促进人精子获能,对精子的膜功能具有保护作用。     

IVF周期精子处理前、后凋亡率与受精率和胚胎质量的相关性分析

目的:探讨体外受精-胚胎移植(IVF-ET)中精子处理前、后凋亡率的变化以及处理前、后精子凋亡率与受精率和胚胎质量的相关性。方法:收集行IVF者的精液55例,密度梯度离心结合上游法处理精液,IVF当日通过流式细胞术检测处理前、后精子凋亡指标——caspase-3(CP3)活化率,于受精后16 h、48 h、72 h分别观察受精率、卵裂率、优质胚胎率。结果:共获卵690枚,平均受精率、卵裂率、优质胚胎率分别为:84.49±14.92%、94.62±11.09%、47.11±27.17%;处理后精子CP3活化率较处理前有明显降低(22.84±6.05%vs 34.80±6.56%,P<0.05);处理前精子CP3活化率与受精率、卵裂率、优质胚胎率均无显著相关性(Spearman相关系数:0.056,0137,-0.060,P>0.05),处理后精子CP3活化率与受精率及优质胚胎率呈显著负相关(Spearman相关系数:-0.375,-0.416,P<0.01),与卵裂率无显著相关性(Spearman相关系数:-0.156,P>0.05)。结论:密度梯度离心结合上游法处理精子后可以显著降低精子凋亡率,处理后精子凋亡率可能影响受精率和优质胚胎率,可为临床预测受精结局及胚胎发育情况提供参考指标。     

两种活动精子分离法的体外受精结局比较

目的:评价Percoll密度梯度法和上游法两种活动精子分离方法在体外受精-胚胎移植(IVF-ET)中的效果。对象和方法:以本中心1999年6月～2001年1月完成的362个IVF-ET治疗周期为研究对象,回顾性地比较了两种活动精子分离方法的受精率、卵裂率、胚胎发育状况和临床妊娠结局。结果:Percoll法和上游法两组比较,受精率分别为71.8％和71.2％,卵裂率88.4％和89.2％,差异均不显著(P>0.05)。取卵后d2,两组胚胎的细胞数分别为2.7±1.3个和3.0±1.0个,差异不显著(P>0.05);两组<20％碎片的优良胚胎率分别为77.6％和65.9％,差异具有统计学意义(P<0.01)。两组临床妊娠率分别为43.7％和36.6％,妊娠周期的活婴出生率分别为70.3％和60.7％,均具有显著性差异(P<0.01)。结论:在IVF-ET治疗不育与不孕中,Percoll法分离的活动精子临床妊娠结局优于上游法。     

固相透明质酸法优选精子的效果观察

目的：对比研究高通量的固相透明质酸（HA）分离新技术较传统密度梯度法在精子优化分离效果方面的优势性。方法：收集新鲜精液标本31份,每份标本液化后被均分成2部分,一部分精液以固相HA法分离精子,即将精液加入到底部表面包被有HA的培养皿中,洗脱掉未结合的精子,分离、收集与HA结合的精子：另外一部分精液以传统的密度梯度法优化分离精子,观察2种方法提取后精子各项研究指标的差异性,以及相对变化幅度。结果：与密度梯度法组相比,固相HA法组处理后精子的正常形态率、获能2h酪氨酸磷酸4~（TP）、ATP（精子获能2hTP率一未获能精子TP率）、诱发顶体反应（AR）和△AR（诱发AR率一自发AR率）等指标明显增高CP〈0．001）,增长幅度依次为：1．55倍（95％CI=1．04,~2．81）、1．81-fg-~（95％CI=0．89-6．11）、3．35-9}（95％CI=1．04-10．32）、1．37倍（95％CI=0．96～2．71）和1．88倍（95％CI=1．09,-4．71）;而固相HA法组自发AR、核蛋白不成熟度和DNA碎片率显著降低（P〈O．001）,分别是密度梯度法组的68％（95％CI=19％-102％）、47％（95％CI=2~／~103％）和21％（95％CI=0％～70％）;2种方法处理后精子的前向运动率和活动率无统计学差异（P〉0．05）。结论：基于成熟精子头部具有HA受体可与外源性HA特异性结合原理的精子。固相HA分离技术,与传统的密度梯度法相比,分离后的精子具有更优的受精潜能和成熟度,可显著提高分离精子的整体质量,有望进一步改善辅助生殖结局。     

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。     

精子活动力对卵胞浆内单精子注射结局的影响

目的:探讨精子活动力与ICSI结局的关系。方法:对2001.12-2005.04在本中心完成ICSI治疗的139个周期进行分析。根据卵母细胞形态特征对928个MⅡ期卵进行分级(Ⅰ级、Ⅱ级和Ⅲ级),并分别用可获得的精子行显微穿刺。结果:用A级、B级和C级精子穿刺各级别的MⅡ期卵母细胞后,受精率在三者间无显著性差异,但高于D级精子行ICSI(P<0.05)。用A、B、C或D级精子行穿刺后一旦使卵受精,卵裂率无明显差异。卵母细胞为Ⅰ级或Ⅱ级时,用A级、B级和C级精子穿刺后,优质胚胎率在三者间无显著性差异,但高于D级精子行ICSI;卵母细胞为Ⅲ级时,用A、B级精子行ICSI后,优质胚胎率高于C、D级精子行ICSI(P<0.05)。结论:精子活动力不影响ICSI后卵裂率;不活动精子行ICSI后受精率和优质胚胎率,总体上较活动精子低。卵母细胞质量较差时,活动精子的活动力等级对优质率胚胎有一定影响。     

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.     

Annexin V magnetic-activated cell sorting versus swim-up for the selection of human sperm in ART: is the new approach better then the traditional one?

Purpose

To investigate whether the sperm fertilizing potential can be improved by selecting a non-apoptotic fraction using magnetic activated cell sorting (MACS), and to compare the results with the conventional swim-up method.

Methods

Twenty five male patients attending the andrology laboratory for sperm DNA fragmentation analysis. The sperm were prepared by density gradient centrifugation (DGC) and subsequently divided into three aliquots. The first was further separated into Annexin V-negative (non-apoptotic) fraction using MACS, the second was further processed by swim-up, while the third was left unseparated as a control. The impact of the combination of DGC with the two sperm preparation techniques on sperm quality was evaluated by comparing ‘rapid progressive’ motility, normal morphology according to Tygerberg’s strict criteria and DNA integrity (by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling [TUNEL]) for each aliquot.

Results

Sperm preparation that combines DGC with conventional swim-up method can provide sperm of higher quality in terms of motility, morphology and extent of DNA fragmentation compared to the Annexin V-negative (non-apoptotic) fraction derived from the combination of DGC with MACS.

Conclusions

Integrating MACS as a part of sperm preparation technique will not improve sperm fertilizing potential to the same extent as the traditional swim-up separation procedure.     

Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique

OBJECTIVE: To compare the effects of density-gradient centrifugation and swim-up on sperm apoptosis by using a multiparameter flow cytometric method. DESIGN: Autocontrolled split-sample study. SETTING: Tertiary infertility center. PATIENT(S): Sixty-two male partners of couples undergoing infertility investigations. INTERVENTION(S): Each sample was analyzed both before and after semen preparation by optical microscopy and by flow cytometry. MAIN OUTCOME MEASURE(S): Percentage of viable, apoptotic, and necrotic sperm and recovery rate of total motile, progressive motile, and viable sperm before and after the two sperm preparation methods. RESULT(S): Compared with the original semen, the mean percentages of apoptotic and necrotic sperm were significantly lower after both sperm preparation methods. The mean percentage of viable sperm was significantly higher after swim-up compared with gradient centrifugation. The recovery rates of total motile, progressive motile, and viable sperm were significantly higher using gradient centrifugation compared with swim-up. The viable sperm percentage and the progressive sperm motility were significant predictors for negative difference between the two methods in terms of viable sperm percentage after preparation. CONCLUSION(S): Both sperm preparation methods allow obtaining a sperm population with a low percentage of apoptotic sperm. Therefore, the risk of using apoptotic sperm for clinical treatment seems to be rather low. The choice of method will depend on whether IVF/ICSI or intrauterine insemination is to be performed.     

Sperm preparation before freezing improves sperm motility and reduces apoptosis in post-freezing-thawing sperm compared with post-thawing sperm preparation

Purpose

To evaluate whether sperm preparation (swim-up technique) before freezing improves the percentages of sperm motility, sperm viability, and non-apoptotic spermatozoa after freezing-thawing process compared with preparation after cryopreservation.

Methods

Semen samples from 65 infertile males were equally divided into two aliquots one of which was processed for swim-up prior to cryopreservation and one of which was processed following cryopreservation. Sperm count, motility, and apoptosis index were measured in each group.

Result (s)

The total sperm count and the total motile sperm count decreased after thawing in both the pre-preparation and non-preparation groups compared with neat semen group (P < 0.001). Moreover, the percentage of apoptotic sperm in the pre-preparation group after cryopreservation was lower than that in the non-preparation group (P < 0.05), whereas the percentage of vital sperm with progressive motility was higher than that in the pre-preparation group (P < 0.001).

Conclusion (s)

Semen preparation by swim-up before freezing resulted in better sperm quality and fewer apoptotic sperm than sperm preparation after thawing. Therefore, sperm preparation before cryopreservation should be considered in routine sperm cryopreservation.     

Preparation of oligozoospermic and/or asthenozoospermic semen for intrauterine insemination using the SpermPrep semen filtration column.

OBJECTIVE: To improve the quality of oligozoospermic and/or asthenozoospermic semen by the SpermPrep (Fertility Technologies Inc., Natick, MA) semen filtration column. DESIGN: The SpermPrep column was applied for semen manipulation in oligozoospermia and/or asthenozoospermia (sperm count less than 20 x 10(6)/mL, sperm motility less than 40%). After concentration of motile sperm using a 40% Percoll density gradient centrifugation, the sperm suspension was filtered through the SpermPrep column. The percentage yield of motile sperm by the SpermPrep method was compared with those by a two-layer Percoll density gradient (Pharmacia, Uppsala, Sweden) centrifugation and a swim-up method. Infertile couples with poor quality semen were treated with intrauterine insemination (IUI) with motile sperm by the three preparations through three cycles. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital. PATIENTS: Twenty-one couples with long-standing infertility because of poor quality semen. MAIN OUTCOME MEASURE: Recovery of motile sperm, sperm motility, and outcome of IUI were evaluated among three semen preparations. RESULTS: Motility was improved by the SpermPrep method in 32 of 33 cases of oligozoospermia and/or asthenozoospermia. Percentage yield of motile sperm by the SpermPrep method was significantly greater than those by the two-layer Percoll density gradient and swim-up methods (42.7 +/- 4.6 versus 22.1 +/- 3.1 and 13.8 +/- 3.5), but there is no significant difference in the sperm motility among three semen preparations. After one treatment cycle for each preparation, 2 of 21 women conceived after IUI with motile sperm separated in the SpermPrep method. CONCLUSIONS: The SpermPrep method is an improved semen manipulation method for oligozoospermia and/or asthenozoospermia.     

The relationship between sperm parameters and fertilizing capacity in vitro: A predictive role for swim-up migration

The relationship between sperm parameters and fertilizing capacity in vitro was examined retrospectively, with the aim of finding predictive criteria for successful in vitro fertilization. Three hundred thirty semen samples were used to inseminate 1462 oocytes. Conventional parameters of sperm concentration and percentage motility in the ejaculate as well as swim-up migration were analyzed in relation to fertilization rate. It was shown that the probability of fertilizing human oocytes in vitro decreases significantly when (a) the sperm concentration is below 20×10⁶ spermatozoa/ml ejaculate (P=0.006), (b) motility is lower than 80% (P=0.002), or (c) less than 4×10⁶ motile spermatozoa/ml are concentrated in the swim-up fraction (P<0.0001). It was also demonstrated that nonfertilizing sperm could not be distinguished from fertilizing sperm by the conventional criteria but rather by the average concentration of motile spermatozoa in the swim-up fraction [12.5±1.5 and 22.3±2.3×10⁶/ml for the 0 and the 100% fertilization groups, respectively (mean±SE;P<0.01)]. Thus, the swim-up migration technique can serve as a predictive test for the in vitro fertilizing capacity of sperm.     

Increased recovery of swim-up spermatozoa by application of "antigravitational" centrifugation.

OBJECTIVE: To compare the quantity and quality of sperm recovered following the 10-minute application of an antigravitational force during the swim-up procedure with the standard 60-minute swim-up (SSU) procedure. DESIGN: Prospectively controlled in vitro study. SETTING: Private andrology laboratory and hospital-based infertility practice. INTERVENTION(S): Equal aliquots of semen were evaluated following various intervals of antigravitational centrifugation swim-up (ACSU). ACSU and SSU were then compared. PATIENT(S): Thirty-eight men undergoing therapeutic testing. MAIN OUTCOME MEASURES: Sperm concentration, sperm motility, and progressive sperm motility. RESULTS: The number of sperm recovered from the ACSU procedure was significantly higher than from the SSU procedure. No significant differences in percent motile sperm and progressive motile sperm recovery between the two procedures were observed. CONCLUSION: The ACSU procedure yields a higher number of motile spermatozoa in a much shorter time.     

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.     

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。     

Sperm-preparation techniques for men with normal and abnormal semen analysis. A comparison

OBJECTIVE: To compare two commonly used sperm-preparation techniques, density gradient centrifugation and swim-up procedures, with respect to their effects on acrosome reaction (AR), hypoosmotic swelling (HOS) and nuclear maturity in men with abnormal and normal semen analyses. STUDY DESIGN: In accordance with World Health Organization criteria, 23 men with abnormal (group I) and 20 men with normal (group II) semen analyses were included in a prospective, controlled study. Each semen specimen was divided into aliquots in order to assess AR, HOS and nuclear maturity, determined with acridine orange staining, in both raw and processed semen samples using the density gradient centrifugation and swim-up techniques. RESULTS: Initial semen samples in group I revealed diminished AR, HOS and nuclear maturity rates in comparison to those in group II. In group I, density gradient centrifugation improved AR, HOS and nuclear maturity rates more than did swim-up. However, in group II it improved only the AR; HOS rates were better than with swim-up. There was a significant positive correlation between sperm concentration and HOS rate in raw semen samples from group I. In the same group, motility and morphology correlated with the nuclear maturity rate but not with AR and HOS rates. Semen samples with better motility (> 20%) or morphology (> 25%) showed better nuclear maturity rates (> 50%) in men with abnormal semen analyses. Motility had a sensitivity of 77% and specificity of 90% in predicting nuclear maturity. Morphology had similar sensitivity but lower specificity (70%). CONCLUSION: Density gradient centrifugation is superior to the swim-up technique in improving AR, HOS and nuclear maturity rates in men with abnormal semen analyses. However, when only nuclear maturity rate is taken into account, the swim-up technique seems to be sufficient for selecting spermatozoa in men with normal semen analyses. The nuclear maturity rate also correlates with sperm morphology and motility.