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。     

精子形态对体外受精-胚胎移植妊娠结局的影响

目的:分析男性精液参数与卵裂率、优质胚胎率及妊娠率的相关性,探讨休外受精一胚胎移植(IVF-ET)周期中精子形态对精子功能的影响.方法:分析82个受精率100%的IVF-ET周期.以Diff-Quickstain方法染色,密度梯度离心法处理精液.WHO精子形态学标准评估精子形态,精子正常形态率≥14%为正常组,<14%...     

精液黏度增高对混合抗球蛋白反应(MAR)检测结果的影响及对策

目的:探讨精液黏度增高对混合抗球蛋白反应(mixed agglutination reaction,MAR)检测结果的非特异性影响及处理措施。方法:①连续监测高黏度组(n=23)及正常黏度组(n=22)精液标本黏度逐渐降低时MAR检测结果的伴随变化;②分别在31例黏度正常的活动精子中添加高黏度精浆以制备高黏度精液标本,同时应用设立空白测定对照法(A法)、洗涤精子法(B法)和加入纤维蛋白溶酶法(C法)3种方法同步处理制备的高黏度精液标本,比较处理后标本MAR检测结果与加入高黏度精浆前MAR测定值("真值")的差异性。结果:①高黏度组标本黏度与MAR检测结果呈明显相关性(r=0.912,P<0.001),正常组标本黏度变化与MAR检测结果无相关性(r=0.120,P>0.05);高黏度组较正常组MAR检测结果显著增高(P<0.001),但当高黏度组标本黏度降至正常后,其MAR检测结果与正常组无显著性差异(P>0.05);②采用A法或C法,其MAR检测结果与"真值"间均无显著性差异(P>0.05)。结论:精液黏稠度增高可导致MAR检测结果假阳性,在高黏度精液标本中添加纤维蛋白溶酶或设立空白测定对照可克服此影响。     

不育男性精子DNA碎片化指数与精液解脲脲原体及人型支原体感染关系的研究

目的:探讨解脲脲原体(Uu)和人型支原体(Mh)对不育男性精子浓度、活动力、正常形态率及精子DNA碎片化指数(DNA fragmentation index,DFI)的影响.方法:选取169份精液标本,其中129例特发不育者为不育组,40例正常生育者为对照组,计算机辅助精液分析(CASA)系统分析精子浓度和活动力.精子形态分析采用Shorr染色法,吖啶橙试验(acridine orange test,AOT)检测精子DFI.支原体检测采用培养法.结果:与对照组相比无Uu/Mh、Uu或Uu+Mh感染组精子DFI显著增高(P＜0.05),与无Uu/Mh感染组相比,Uu或Uu+Mh感染组DFI亦显著增高(P＜0.05).与正常生育对照组相比无Uu/Mh、Uu或Uu+Mh感染组正常形态率显著降低(P＜0.05),其余组间无统计学差异(P＞0.05).与正常对照组相比,无Uu/Mh、Uu、Mh和Uu+Mh感染组的精子浓度、a级、a+b级和a+b+c级精子百分比均显著降低(P＜0.05),而除无Uu/Mh感染组外,其余各组间无统计学差异(P＞0.05).结论:特发性不育男性的精子浓度、正常形态率、a级、a+b级和a+b+c级精子百分比较正常男性的均显著下降,Uu/Mh感染并非是最主要原因,可能是其他重要因素或综合因素影响的结果.特发性不育男性精子DFI明显增加,Uu是其中的主要因素之一.Uu可能主要通过对精子DNA,而不是常规参数的影响来造成男性生育力的下降.     

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。     

精子形态对体外受精胚胎移植妊娠结局的影响

目的：分析男性精液参数与卵裂率、优质胚胎率及妊娠率的相关性,探讨体外受精-胚胎移植（IVF-ET）周期中精子形态对精子功能的影响。方法：分析82个受精率100%的IVF-ET周期。以Diff-Quickstain方法染色,密度梯度离心法处理精液,WHO精子形态学标准评估精子形态,精子正常形态率≥14%为正常组,〈14%者为异常组。观察精液处理前后精子参数变化,比较妊娠组与未妊娠组的年龄、移植日子宫内膜厚度和精液各参数的情况。比较精液处理前后精子正常形态率与两组的正常受精率、卵裂率、优质胚胎率和妊娠率。结果：①密度梯度离心法处理后82例精液密度明显降低,活动力明显提高,差别有统计学意义。②精液处理前精子正常形态率：正常组11例（13.41%）,异常组71例（86.59%）;处理后正常组34例（41.46%）,异常组48例（58.53%）。异常组精液处理后正常精子形态的比例提高,中位数（P25,P75）分别为8.15%（6.38%,10.50%）vs.12.41%（9.98%,18.58%）,差别有统计学意义（P〈0.01）。③精液处理前精子正常形态率正常组和异常组的密度和活动力差别无统计学意义（P〉0.05）。④精液处理前后精子正常形态率正常组和异常组的妊娠率和优质胚胎率差异均无统计学意义（P〉0.05）;精子正常形态率正常组的卵裂率高于异常组（P〈0.05）。⑤妊娠组和未妊娠组除男女双方年龄差异有统计学意义外,移植日子宫内膜厚度和各精液参数差异均无统计学意义。结论：精子形态对妊娠结局无预测,密度梯度离心法能提高正常精子形态百分数,需根据精液处理后的结果选择受精方式。     

精子核DNA完整性测定方案的优化及其在辅助生殖技术中的应用价值

目的:对现有精子核DNA完整性测定方案进行优化,并探讨其在辅助生殖技术(ART)中的应用价值。方法:选择2021年1月1日至2022年12月8日于江西中医药大学附属生殖医院就诊的拟接受体外受精-胚胎移植(IVF-ET)助孕的194对夫妇作为研究对象。采集男方的精液作为对照组(n=194),同一精液经双层密度梯度离心法优化处理后精子混合液作为观察组(n=194)。根据精子核DNA碎片率(DFI)测定结果将对照组及观察组各分为3个亚组,对照A组和观察A组：DFI<15%,对照B组和观察B组：DFI 15%～30%,对照C组和观察C组：DFI≥30%。对观察组与对照组的DFI值及各亚组间的助孕及妊娠情况进行比较。结果:(1)观察组DFI明显低于对照组,差异有统计学意义[(13.55±10.17)%vs.(18.56±11.54)%,P<0.05]。(2)6个亚组间的受精率、卵裂率及优胚率两两比较,差异无统计学意义(P>0.05)。(3)6个亚组的妊娠率和着床率比较,差异有统计学意义(P<0.05);其中,对照A组、对照B组、观察A组、观察B组4组的临床妊娠率(均在65...     

解脲脲原体感染与精子DNA完整性的相关性的研究

目的:探讨不育男性解脲脲原体(Uu)感染与精子核DNA完整性及精液各参数的相关性。方法:95例不育症患者的精液用Uu培养液进行解脲脲原体的培养,按WHO的标准,用计算机辅助精液分析系统(CASA)进行精液各参数的分析,然后通过精子低渗肿胀实验,吖啶橙(AO)荧光染色检测精子膜的完整性和精子核DNA的完整性。结果:Uu阳性40例,Uu阴性55例,Uu阳性组与阴性组比较,精子核DNA完整性与精子的密度、a+b级精子百分率以及精子的肿胀率都有显著性差异(P<0.01,P<0.05)。同时Uu阳性组与阴性组的精子核DNA完整性与精子的肿胀率存在显著的正相关性(r=0.438,P=0.014;r=0.438,P=0.01)。结论:Uu感染能够降低精子膜的完整性以及精子核DNA的完整性,这可能是Uu致男性不育的又一因素。     

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。     

常规IVF-ET中完全体外受精失败发生的相关因素的探讨

目的:探讨IVF-ET完全体外受精失败发生的相关危险因素。方法:回顾性分析行常规体外受精-胚胎移植(IVF-ET)的2 429个周期。应用单因素及多因素Logistic回归分析夫妇之间受孕史、精子正常形态率、精液量、精液浓度、精液活力、男方生育史、女方年龄、女方原发/继发不孕、是否有输卵管性因素、不孕年限、获卵数、月经第3日的FSH、T、PRL、LH、E2、hCG注射前LH、E2、P对完全受精失败发生的影响。结果:IVF完全不受精发生率为5.7%。完全受精失败组的精子正常形态率(11.1±5.8%)、a+b级百分比(47.4±10.5%)显著低于受精组(13.4±5.3%、50.1±8.6%)(P<0.05);完全受精失败组的女方原发不孕构成比(69.1%)、男方原发不孕构成比(74.8%)、无管性因素构成比(30.2%)、夫妇间无受孕史构成比(79.1%)显著高于受精组(36.3%、41.7%、13.6%、44.3%)(P<0.05)。此外,畸精组的完全不受精发生率(15.0%)显著高于精子形态正常组(5.2%)。Logistic回归分析亦显示精子正常形态率、精液浓度、夫妇间受孕史、不孕年限与完全受精失败有显著相关性(P<0.05)。结论:对治疗周期中畸形精子症患者、精液浓度、活力偏低、男方原发不育、女方原发不孕、无输卵管性因素、不孕年限长、夫妇之间无孕史的患者应纳入受精失败的高危人群,考虑行部分ICSI,以保障受精,对防止完全不受精的发生具有积极意义。     

Isolation of motile spermatozoa from semen samples using microfluidics

A microfluidic device was designed with two parallel laminar flow channels where non-motile spermatozoa and debris would flow along their initial streamlines and exit one outlet, whereas motile spermatozoa had an opportunity to swim into a parallel stream and exit a separate outlet. Motile sperm samples were prepared with density gradient separation (n = 5). Sperm motility was assessed the following day after exposing aliquots to polydimethylsiloxane (PDMS) used to construct the device. There was no difference in sperm motility when compared with unexposed aliquots (P > 0.05). Unprocessed semen samples (n = 10) were placed in wider channels and sperm motility and strict morphology were assessed from sorted outlets. Sperm motility increased from 44 +/- 4.5% to 98 +/- 0.4% (P < 0.05) and morphology increased from 10 +/- 1.05% to 22 +/- 3.3% (P < 0.05) following processing. Finally, density gradient prepared samples (n = 6) containing 5 x 10(6) motile spermatozoa/ml and 50 x 10(6) round immature germ cells/ml were sorted and assessed in a similar fashion. The ratio of motile spermatozoa to round immature germ cells in the wide inlet (1:10) was significantly improved in the thin outlet (33:1) (P < 0.05). This microfluidic device provides a novel method for isolating motile, morphologically normal spermatozoa from semen samples without centrifugation. This technology may prove useful in isolating motile spermatozoa from oligozoospermic samples, even with high amounts of non-motile gamete and/or non-gamete cell contamination. A movie sequence showing streaming and sorting of spermatozoa may be purchased for viewing on the internet at www.rbmonline.com/Article/847 (free to web subscribers).     

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.     

Semen manipulation: improved sperm recovery and function with a two-layer Percoll gradient

The authors compared a simple, two-layer Percoll density gradient technique with the swim-up technique for semen preparation in 128 men. In samples from normospermic (n = 55), oligospermic (n = 26), and asthenospermic (n = 29) men, the Percoll technique significantly improved yield, percent motility, and absolute number of motile sperm recovered, but in samples from oligoasthenospermic men (n = 18), only percent motility was improved. The Percoll density gradient also selected sperm with markedly improved function as assessed by both the sperm penetration assay and the fertility index. In 37 samples negative on the sperm penetration assay when processed with the swim-up technique, 19 (51%) became positive when processed with the Percoll technique. The Percoll density gradient is an improved method for semen manipulation as it allows greater recovery of sperm with higher motility and improved sperm function.     

Advantage of combining magnetic cell separation with sperm preparation techniques

The selection of vital, non-apoptotic spermatozoa is a prerequisite for achieving optimal conception rates in assisted reproductive techniques. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa. The objective of the present study was to optimize the integration of magnetic cell sorting in standard sperm preparations and to correlate the effect of different sperm preparation procedures on apoptotic markers. Semen specimens collected from 15 healthy donors were prepared by either density gradient centrifugation or by one-step sperm wash technique separately and in combination with magnetic cell sorting. The preparation methods were evaluated by assessment of semen parameters (motility, viability and morphology) as well as markers of apoptosis (levels of active caspase-3, integrity of membrane mitochondrial potential and externalization of phosphatidylserine). The apoptotic markers were measured using fluorochrome dyes coupled with flow cytometry. The results showed that the combination of density gradient centrifugation and annexin-V magnetic cell sorting was superior to all other sperm preparation methods in terms of providing motile, viable and non-apoptotic spermatozoa. This study clearly shows the advantage of integrating magnetic cell sorting as a part of sperm preparation, which in turn may positively affect the success rates of assisted reproductive techniques.     

Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation

Objective: To compare the Enhance (Percoll; Conception Technologies, San Diego, CA) and PureSperm (Gen X International, Madison, CT) sperm preparation methods with respect to recovery (percentage of motile sperm), motility (%), path and progressive velocities (μm/s), and hyperactivation (%).

Design: Comparison of sperm processing methods.

Setting: University medical center–based clinical andrology laboratory and infertility program.

Patient(s): Twenty-five men who presented for semen analysis.

Intervention(s): Each of 25 semen specimens were divided and each aliquot was prepared using two different density gradient centrifugation methods.

Main Outcome Measure(s): The motile sperm recovery, percent motility, motion parameters, and percent hyperactivation were measured for each semen specimen (n = 25) before and after separation with the use of the two methods.

Result(s): There was no difference in the percent motility and motile count between specimens prepared with Enhance (Percoll) and PureSperm and fresh specimens. Statistically significant differences were found (fresh versus test) in the velocities and in hyperactivation (PureSperm only), and no differences were found between the processing methods.

Conclusion(s): PureSperm appears to be as effective as Percoll (Enhance) for the recovery of good, progressively motile sperm for use in IUI or other assisted reproductive techniques.     

Comparison of three sperm preparation media.

OBJECTIVE: In assisted reproduction, spermatozoa must be effectively separated from seminal plasma to undergo capacitation, a prerequisite for fertilization. Percoll was recently withdrawn from the American market because of safety concerns. The purpose of this study was to evaluate the quality of sperm separated by two different sperm washing media, and compare these results to sperm quality after separation on a Percoll (Perwash) gradient. MATERIALS AND METHODS: Semen samples from 10 normozoospermic men were compared after separation on three media: Perwash, a Percoll-type medium (Conception Technologies, La Jolla, CA), ISolate (Irvine Scientific, Santa Ana, CA), and SpermFertil (Embryotech Laboratories, Wilmington, MA). Semen characteristics examined were: sperm count, percentage motility, curvilinear velocity, lateral head displacement, percentage recovery of motile sperm, viability, hypo-osmotic swelling, and penetration in bovine cervical mucus. Sperm morphology was scored using World Health Organization and Kruger's strict criteria. The motility of the sperm was examined at one-hour intervals for three hours to determine which method produced samples with the longest period of motility. RESULTS:Total motile sperm count, motility, curvilinear velocity, and percentage of normal morphological forms as determined by the WHO method were significantly lower in specimens prepared by SpermFertil than in ISolate or Perwash specimens (P < .05). Tail abnormalities were significantly more frequent in specimens prepared by SpermFertil than in ISolate and Perwash specimens (P < .001). Percentage recovery of motile sperm was significantly higher in ISolate and Perwash specimens than in SpermFertil specimens (P < .05). Semen characteristics were similar in specimens prepared with ISolate and with Perwash. At all time intervals, sperm motility was higher in ISolate and Perwash specimens than in SpermFertil specimens (P < .001). CONCLUSION: Sperm samples separated with a SpermFertil column have poorer sperm quality in several respects than samples prepared with ISolate or Perwash. This finding and the similarity between ISolate and Percoll procedures suggests that ISolate is a good alternative to Percoll-type media to prepare sperm for assisted reproduction.     

Screening of sperm velocity by fluid mechanical characteristics of a cyclo-olefin polymer microfluidic sperm-sorting device

The microfluidic sperm-sorting (MFSS) device is a promising advancement for assisted reproductive technology. Previously, poly(dimethylsiloxiane) and quartz MFSS devices were developed and used for intracytoplasmic sperm injection. However, these disposable devices were not clinically suitable for assisted reproduction, so a cyclo-olefin polymer MFSS (COP-MFSS) device was developed. By micromachining, two microfluidic channels with different heights and widths (chip A: 0.3 × 0.5 mm; chip B: 0.1 × 0.6 mm) were prepared. Sorted sperm concentrations were similar in both microfluidic channels. Linear-velocity distribution using the microfluidic channel of chip B was higher than that of chip A. Using confocal fluorescence microscopy, it was found that the highest number of motile spermatozoa swam across the laminar flow at the bottom of the microfluidic channel. The time required to swim across the laminar flow was longer at the bottom and top of the microfluidic channels than in the middle because of the low fluid velocity. These results experimentally demonstrated that the width of microfluidic channels should be increased in the region of laminar flow from the semen inlet to the outlet for unsorted spermatozoa to selectively recover spermatozoa with high linear velocity.     

Improved motile sperm recovery by a hyperosmotic percoll gradient

Purpose: Our purpose was to investigate whether a new, relatively hyperosmotic Percoll gradient, Enhance-S, can improve total motile sperm recovery rates compared with the commonly used Percoll gradient Perception. Methods: Semen specimens from each of 17 donors were divided into two equal aliquots. One part was washed using Percoll Perception, while the other was prepared using Percoll Enhance-S. Results: Compared to the unwashed specimen, sperm motion characteristics (motility and velocity) improved significantly after Percoll separation using either the Perception or the Enhance-S gradient. There was no difference in motility or velocity in spermatozoa recovered after wash with either of the two preparations. However, the total motile sperm recovery was significantly higher using the Percoll Enhance-S gradient than with the Percoll Perception gradient (P < 0.0024). Conclusion: The new Percoll Enhance-S gradient provides significantly more total motile sperm than the Percoll Perception gradient.     

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。