Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

Aim: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. Methods: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10~6 vs. 17.6 x 10~6), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Conclusion: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART. i

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay （SCSA） parameters, that is, DNA fragmentation index （%DFI） and high DNA stainability （%HDS）, were assessed on the flesh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation （DGC） was followed by swimup treatment in comparison with DGC alone （P 〈 0.01）. Although the %DFI increased in a time-dependent manner with incubation both at room temperature （RT） and at 37℃ in air, the %DFI after 24 h at RT was significantly lower than that at 37℃ （P 〈 0.05）. Incubation with 5% CO2 was effective in maintaining sperm motility （P 〈 0.01）; however, it induced further elevation of %DFI （P 〈 0.001）. Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.

Comparison between the quality and function of sperm after semen processing with two different methods

Aim: To compare the recovery rate of morphologically normal and chromatin condensed spermatozoa from native se-men samples using the SpermPrep^(TM) filtration columns and Percoll gradient centrifugation and to determine the influenceof the two processing techniques on fertilization and pregnancy rates in an IVF-ET program. Methods: Sixteen se-men samples obtained from patient's husband were included in this study. Each was divided into two aliquots. The firstaliquot was processed with SpermPrep^(TM) filtration columns and the second, Percoll gradient centrifugation. Smears weremade before and after semen processing with both methods for the evaluation of chromatin condensation (chromomycineCMA3) as well as morphology (strict criteria) of spermatozoa. One hundred and seventy oocytes were retrieved fromthe patients and the oocytes from each patient were subdivided into two sets : one set was inseminated using spermatozoaprocessed with SpermPrep^(TM) and the other inseminated after semen processing with Percoll gradient centrifugation. Re-sults: The Percoll method yielded a significantly higher percentage of chromatin condensed (90.8 ±6.5% vs 82.3±8.8%, P = 0.017) and morphologically normal spermatozoa (12.9±7.4% vs 6.9±4.8%, P =0.001) in com-parison to SpermPrep^(TM). Whereas, sperm count recovery rate was significantly higher after the use of SpermPrep^(TM) thanafter the Percoll gradient centrifugation. The fertilization rate was similar between the two methods. Conclusion:Semen processing with Percoll should be recommended for intracytoplasmic sperm injection as the natural selection isbypassed and the SpermPrep^(TM) technique could be recommended for IVF and IUI programs as the sperm concentrationplays a more significant role in these procedures.

A laboratory modification to testicular sperm preparation technique improves spermatogenic cell yield

Testicular sperm extraction is a common procedure used to find spermatogenic cells in men with nonobstructive azoospermia. The laboratory processing of biopsied testicular tissues needs to be performed meticulously to acquire a high yield of cells. In this study, the effectiveness of mincing the tissues after testicular biopsy was assessed using histological evaluation, as was the possible adverse effect of residual tissue on the migration of spermatogenic cells during density gradient centrifugation. Our results indicate that testicular residual tissue, when laid on the density gradient medium along with the sperm wash, hinders the spermatogenic cells＇ forming a pellet during centrifugation, and therefore impairs the intracytoplasmic sperm injection procedure. Whereas the mean number of recovered cells from the sperm wash medium （SWM） with residual tissue is 39.435 ~ 24.849, it was notably higher （60.189 ~ 28.214 cells） in the SWM without minced tissues. The remaining tissue contained no functional seminiferous tubules or spermatogenic cells in histological sections. In conclusion, the remaining residual tissue after mincing biopsied testicular tissue does not add any functional or cellular contribution to spermatogenic cell retrieval; in fact, it may block the cellular elements in the accompanying cell suspension from migrating through the gradient layers to form a pellet during centrifugation and cause loss of spermatogenic cells.

The ability of sperm selection techniques to remove single- or double-strand DNA damage

A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are densitygradient centrifugation （DGC） and swim-up （SUP）. To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques （ART）, but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged （degraded） DNA, as characterized by the presence of both single- and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART.

Changes in human sperm motility and DNA fragmentation index after incubation at different temperatures following density gradient centrifugation and swim-up procedures

Objective:The purpose of this study was to evaluate the sperm motility and DNA integrity at different temperatures to analyze whether the sperms are suitable on the second day for insemination of in vitro matured oocytes by intra-cytoplasmic sperm injection(ICSI)following density gradient centrifugation(DGC)and swim-up(SU)procedures.Methods:Semen samples were collected from 30 outpatients who visited the Center for Reproductive Medicine for semen analyses.Following sperm selection by DGC and SU procedures,the liquified semen samples were divided into three groups and incubated at 4,25,and 37°C,respectively.Following incubation for 24,48,and 72 hours,the sperm motility and sperm DNA fragmentation index(DFI)were analyzed.Results:Following the combination of DGC and SU procedures,the sperm motility(91.8%±8.6%vs.50.8%±13.1%)and DFI(5.1%±7.9%vs.13.0%±11.6%)were significantly improved(P<0.01)compared to those without any treatment.The sperm motility of the 3 groups significantly declined(P<0.05)post-incubation compared to that of the groups prior incubation.However,sperm motility significantly increased(76.9%±10.4%)(P<0.05)at 25°C compared to that of the other 2 groups(53.5%±11.0%and 47.6%±10.2%).Sperm DFI significantly increased(P<0.05)at 37°C following incubation for 24 and 72 hours in comparison to that of the other 2 groups.However,the sperm DFI did not significantly increase when the sperm samples were incubated at 4(5.7%±5.9%)and 25°C(6.8%±5.6%)for 24 hours compared to that before incubation(5.1%±7.9%).Conclusions:These results indicate that the sperm quality,in terms of motility and DFI,can be efficiently improved by DGC in combination with SU.Following which,the sperm samples can be incubated at 25°C and be used on the second day for insemination of in vitro matured oocytes by ICSI.

浮游板优化精液与密度梯度离心结合上游法的比较

目的应用浮游板和密度梯度离心结合上游法优化人类精液,明确优化效果并比较两种方法优化后精子参数的差异。方法收集正常精液样本85例和轻中度少弱精子症精液样本59例,每例样本分为原液组、密梯组和浮游组各1 ml进行后续处理。原液组在精液液化后未做任何处理;密梯组精液液化后用密度梯度离心法结合上游法进行处理,浮游组则在精液液化后应用精子浮游板进行处理。比较原液组、密梯组、浮游组3个亚组间的精子浓度、前向运动精子百分率、正常形态精子百分率;应用流式细胞仪检测精子线粒体膜电位和精子DNA碎片指数,使用单精子活性氧分析仪检测单精子活性氧水平,使用电子显微镜观察精子超微结构并进行组间比较。结果无论是正常精液还是轻中度少弱精子症精液,与原液组比较,密梯组与浮游组的前向运动精子百分率、正常形态精子百分率及精子线粒体膜电位均显著升高(P<0.05),精子DNA碎片指数显著降低(P<0.05);密梯组和浮游组各指标无显著性差异(P>0.05)。正常精液中,浮游组的精子浓度显著高于密梯组(P=0.029),单精子活性氧水平显著低于原液组(P=0.038)。轻中度少弱精子症精液中3亚组间的单精子活性氧水平均无显著性差异(P>0.05)。电子显微镜观察发现正常精液中3个亚组的精子均未见明显异常;轻中度少弱精子症精液中密梯组部分精子可观察到精子细胞膜断裂和线粒体肿胀等异常,而浮游组精子超微结构未见明显异常。结论密度梯度离心结合上游法和浮游板法用于精液的处理和优化,均可有效回收活力、形态和功能更好的精子。精子浮游板法的体外干预小,操作简便,可以获得活性氧更少、损伤更小的精子。

精液优化处理后DNA碎片指数与IVF-ET胚胎质量及妊娠结局的关系

目的:评估经密度梯度离心联合上游法优化处理后的精子DNA碎片指数(DFI)与辅助生殖体外受精-胚胎移植(IVF-ET)胚胎质量及妊娠结局的关系。方法:回顾性分析2022年4月至12月在郑州大学第一附属医院生殖医学中心因单纯输卵管因素行IVF-ET的257个周期,比较男方精液优化前后精液参数和精子DFI;按优化后精子DFI将其分为高DFI组(DFI>5)与低DFI组(DFI≤5),比较两组的胚胎发育及妊娠结局;根据临床妊娠情况分为妊娠组与非妊娠组、持续妊娠组及早期流产组,比较精子DFI情况。结果:与处理前相比,精液优化处理后前向运动精子及正常形态精子百分比提高,精子DFI下降(P<0.001)。精子优化处理后高DFI组的早期流产率高于低DFI组(P<0.05)。105例临床妊娠周期中早期流产组精液优化处理前、后DFI均高于持续妊娠组(P<0.05)。结论:密度梯度离心联合上游优化处理是一种有效的精液制备方法,可提高精子前向运动能力和正常形态精子比例,降低精子DFI;优化后精子高DFI可能增加临床妊娠后早期流产的风险。

优化精液处理中的密度梯度离心法以提高受精结局的探讨

【目的】本研究旨在改进生殖男科领域现有的精液处理方法,特别是针对双层密度梯度法中的300×g 20 min处理条件,以提高受精结局。【方法】收集2020年7月和9月以及2022年3月和5月在中山大学附属第六医院生殖医学中心进行辅助生殖助孕的1623例患者的精液标本进行实验。预实验中,比较四种不同的双层密度梯度方法(200×g 10 min、200×g 20 min、300×g 10 min和300×g 20 min)处理后标本的精子DNA碎片率和回收率。然后,筛选出一种最优的方法作为新方法,并与目前在用的旧方法(300×g 20 min双层梯度法)进行对比,检验受精率是否有统计学差异。在新方法的基础上,进一步优化为单层密度梯度法,并与双层密度梯度法进行对比,检验是否存在统计学差异。实验过程中严格控制温度、离心速度和离心时间,同时记录每组样本的数量和处理条件。【结果】在保证足够的精子回收率的基础上,发现四种双层密度梯度法中300×g 10 min的精子DNA碎片率低于300×g 20 min。因此,选择300×g 10 min作为新方法进行试验。结果表明,新方法300×g 10 min的总受精率、二核体(2pn)受精率均高于300×g 20 min,且差异有统计学意义(P<0.05);300×g 10 min的卵裂率也略高于300×g 20 min,但差异没有统计学意义(P>0.05)。单层密度梯度法的总受精率、2pn受精率均高于双层密度梯度法,但差异没有统计学意义(P>0.05);单层密度梯度法的卵裂率高于双层密度梯度法,囊胚形成率低于双层密度梯度法,且差异均有统计学意义(P<0.05)。【结论】相对于现有的300×g 20 min双层梯度法,300×g 10 min双层梯度法成功提高了总受精率、2pn受精率、卵裂率,缩短了精液优化处理的时间;而单层密度梯度法虽提高了卵裂率、节约了试剂成本和操作时间,但其囊胚形成率却出现了下降的情况。这些发现为生殖男科领域的精液处理方法提供了有益的指导和启示。

Percoll梯度离心前后精子功能指标分析

目的 :通过分析Percoll梯度离心法处理前后精子相关功能指标 ,评价该法在精子体外处理中的作用。 方法 :采用多次曝光摄影技术、瑞 姬氏染色法、低渗肿胀实验 ,观察处理前后精子运动速度、顶体完整率和低渗肿胀率的变化。 结果 :2 5例不育病人精液标本经Percoll梯度离心法处理前后 ,精子运动速度 (2 0 .5 6± 8.13μm/s和 2 5 .18± 8.30 μm/s ,P <0 .0 5 )、顶体完整率 (6 1.2 0 %± 2 1.11%和 70 .36 %± 17.70 % ,P <0 .0 1)和低渗肿胀率 (6 8.2 8%±17.33%和 76 .76 %± 17.10 % ,P <0 .0 5 )均有显著差异。 结论 :精液标本经Percoll梯度离心法处理后 ,各项精子功能指标均较处理前有明显改善 ,该法可广泛应用于辅助生殖医学中。

梯度离心分离家兔精子试验

用Percoll梯度离心分离家兔精子,收取梯度管上、中、下各层精子给母兔人工授精。上层精子得到10窝共62只仔兔,其中雌性29只(46.8％),雄性33只(53.2％),中层精子得到5窝共31只仔兔,其中雌性16只(51.6％),雄性15只(48.4％);下层精子得到12窝共61只仔兔,其中雌性39只(64.0％),雄性22只(36.0％)。

Percoll梯度离心与精子运动轨迹图像分析

目的：研究Percoll梯度离心后精子运动轨迹的变化。方法：Percoll梯度离心法处理精子运动状况较差的精液标本，分析精子运动轨迹的变化。结果：在24例精液标本中，Percoll处理前后的精子平均运动速度分别为18.4±4.6μm／s和25.7±5.4μm／s.二者之间具有显著差异（P＜0.01）。结论：用Percoll梯度离心法处理精子，可提高精子运动速度，改善精子质量。

人睾丸组织中成熟精子的分离方法初探

探索简单、适用的体外分离睾丸组织中成熟精子的方法,以用于男性无精子症病人行卵母细胞内单精子注射受精-胚胎移植为目的。用含不同浓度透明质酸酶的10 %人血白蛋白人类输卵管液培养人睾丸曲细精管组织匀浆2 4 h,再用Percoll梯度离心法洗涤后计算所获各级精子的总数,并设置前后对照和空白对照进行比较。用不同浓度透明质酸酶处理后的4个实验组分别与2个对照组比较,实验组所获得的各级精子的总数高于对照组,有显著差异(P<0 .0 1)。实验组中透明质酸酶浓度分别为5 0、10 0、15 0 u/ ml的10 %人血白蛋白人类输卵管液处理组获得的各级精子总数组间比较,处理前对照和处理后空白对照之间比较,无显著差异(P>0 .0 5 )。而用浓度为2 0 0 u/ ml的10 %人血白蛋白人类输卵管液处理组获得的各级精子的总数与其它浓度处理组之间比较存在统计学差异(P<0 .0 1) ,其所获得的各级精子总数相似,但活动能力较其它浓度处理组低。透明质酸酶结合Percoll梯度离心可从人睾丸曲细精管组织匀浆中分离出较多更纯的成熟精子,但过高浓度的透明质酸酶可能会影响分离出的精子的活动能力。

两种精子优选方法效果比较

目的 探讨精子经王氏管和密度梯度离心法处理后精子参数的变化 ,比较两者在宫内人工授精 (IUI)的临床效果。 方法 选择不育男性精液 15 7份 ,采用两种方法进行配对处理 ,比较两种分离方法前后精子活力、正常形态率、精子顶体形态和精子染色质等变化 ;分离后的精子用于IUI的临床妊娠率。 结果 两种方法处理后的精子活力 (a +b级 )、正常精子形态率、精子顶体完整率、正常染色质率与处理前比较有显著差异 (P <0 .0 1) ;但两种方法分离的精子用于IUI的临床妊娠率无显著差异 (P >0 .0 5 )。 结论 王氏管法和密度梯度离心法处理精子后均获得质量较好的精子 。

体外操作对人精子运动参数及细胞内活性氧簇的影响

目的:研究体外物理操作(离心以及微量加样器抽吸)对人精子运动参数以及细胞内活性氧簇(ROS)的影响,旨在优化人精子体外处理方法。方法:7例精液常规参数正常的标本,采用不同离心力(200 g,600 g)及离心时间(5 min,15 min)以及不同微量加样器抽吸次数(2,6,10次)进行处理,测试精子运动参数和细胞内ROS的变化。结果:1活动精子百分比以及活精子细胞内ROS水平主要受到离心时间的影响(P<0.05);前向运动精子百分比(PR)以及平均运动速率(VAP)均不受离心力及离心时间的影响(P>0.05)。单因素方差分析多重比较结果提示,当离心时间为5 min以上时精子活动力显著下降,而ROS水平显著增加(P<0.05)。2加样器抽吸2次以上可致人精子活动力显著下降(P<0.05),抽吸6次以上可导致人精子PR及VAP显著降低(P<0.05);但抽吸操作并未显著影响精子细胞内ROS的水平。结论:在人精子体外操作时应尽量缩短离心时间并控制加样器抽吸次数从而保证正常的精子运动参数,也应尽量缩短离心时间从而减少体外操作所致的细胞内ROS升高。

兔骨髓间充质干细胞的分离、培养与鉴定

目的优化获取兔骨髓间充质干细胞并对其进行纯化、鉴定,同时对其生物学性状进行研究。方法取新生新西兰大耳白兔骨髓5.0 mL,采用密度梯度离心法、贴壁筛选法并结合传代对其进行纯化,观察细胞形态、超微结构和表面标志物的表达,从而对其进行鉴定。观察第1、3、5、8、10代细胞的增殖情况,并绘制出生长曲线。结果经密度梯度离心、贴壁筛选并结合传代所得到的间充质干细胞很纯,第3代和第5代细胞形态单一,细胞排列具有典型的漩涡状结构。第1、3、5代细胞增殖能力强,生长旺盛;而第8、10代细胞增殖明显减弱。所分离培养的细胞表达CD44、CD90,不表达CD34,电镜观察为低分化细胞。结论分离培养的细胞为间充质干细胞且成分单一,具有干细胞特性,第3、5代细胞很纯且其增殖能力强,适用于做以后的研究。

优选后精子DNA碎片指数及与体外受精的关系

目的探讨优选前和优选后精子DNA碎片指数(DFI)的变化及其对体外受精(IVF)的影响。方法检测105例IVF助孕患者男方精液密度梯度离心优选前及优选后的精子DFI,分析优选前后精子DFI与IVF受精率、卵裂率、优质胚胎率、妊娠结局等的关系。结果优选处理后精子DFI较处理前明显降低,差异有统计学意义[(13.88±8.63),(29.68±9.94),P<0.05];优选后的精子DFI与IVF受精率呈负相关关系(P<0.05)。结论优选后精子DFI与IVF受精率密切相关,其对IVF的受精率具有预测价值。

Percoll梯度离心前后精子功能参数的分析

作者通过Percoll梯度离心技术观察处理前后精子相关功能的指标 ,以评价该技术在精子体外处理中的效果 .方法 采用精子前向运动试验 (PMS)、精子尾部低渗肿胀试验 (HOS)和DNA荧光染色有效精子计数试验 (ESC) ,观察精子处理前后PMS ,HOS和ESC的变化 .结果 32例不育病人精液标本经Percoll梯度离心法处理后 ,精子的PMS ,HOS及ESC等指标均明显高于处理前(P <0 .0 1) .该结果表明 ,体外精液标本经Percoll梯度离心法处理后 ,各项精子功能指标均较处理前有明显提高 .

梯度离心在体外处理精子结合抗体中的应用

目的:比较单纯PureSperm方法和PureSperm联合上游法处理精子结合抗体阳性精子的效果。方法:采用单纯梯度PureSperm及PureSperm联合上游法处理精子结合抗体阳性的精子,比较2种处理方法处理后精子结合抗体消退情况。结果:2种方法处理后,精子结合抗体滴度较处理前降低(P<0.01);梯度PureSperm联合上游法处理后,精子结合抗体滴度的下降较单纯PureSperm方法更显著(P<0.05)。结论:采用梯度PureSperm离心或梯度PureSperm离心联合上游法处理精液,可以提高精子质量,再行宫腔内人工授精可能提高免疫性不育的治疗效果。

密度梯度离心法收集逆行射精患者尿液中精子用于人工授精

目的:探讨密度梯度离心法收集逆行射精患者尿液中的精子用于人工授精的可行性。方法:碱化患者尿液,应用两层密度梯度离心法收集尿液中的精子,在配偶排卵期内行宫腔内人工授精。结果:5例逆行射精患者,在经过共18次收集尿液中精子及对配偶实施人工授精术后,有3例成功妊娠并有2例已足月分娩。结论:密度梯度离心法收集逆行射精患者尿液中的精子,方法简单、快捷有效,用于人工授精具有可行性。