精液保存和优化处理方法对精子DNA完整性的影响

目的:探讨不同精液保存和处理方法对精子DNA完整性的影响。方法:筛选2015年1~12月就诊的100例精液正常志愿者,将每例志愿者的精液混匀后分别对其进行不同的保存和优化处理;保存方法包括直接冷冻法、试剂冷冻法、室温保存4 h和24 h等,优化处理方法包括简单洗涤法、直接上游法和非连续密度梯度离心法。保存和处理后均采用精子染色体扩散实验(SCD)分析其精子DNA碎片指数(DFI);优化处理各组继续培养24 h,培养后再分析其精子活动率和DFI。结果:精液保存条件两两组间分析显示,直接冷冻法、试剂冷冻法和室温保存4 h的精子DFI分别为(27.3±6.4)%、(26.9±6.1)%和(24.7±6.8)%,结果无显著性差异(P0.05),而室温保存24 h则显著增加精子DFI[(35.6±9.0)%](P0.05)。与简单洗涤法的精子DFI[(13.7±2.0)%]相比,直接上游法的精子DFI[(9.1±1.3)%]和非连续密度梯度离心法的精子DFI[(8.0±2.5)%]均显著降低(P0.05);再培养24 h后,非连续密度梯度离心法处理的精子DFI[(11.5±4.2)%]最低(P0.05)。结论:保存条件和优化处理方法对精子DNA完整性有影响,临床工作中需选取最合适的精液处理方法。     

改良精子染色质扩散实验对精子DNA完整性检测的初步探讨

目的:通过对传统精子染色质扩散实验(SCD)方法进行改良,探索一种更简单、快速、准确的精子DNA完整性检测方法。同时对改良SCD法检测的正常参考值范围进行初步探讨。方法:对传统SCD法在实验过程、试剂配制及器具使用上进行了改良优化,同时对改良前后SCD法进行了比较。此外,利用改良SCD法对293例正常生育男性精液进行精子DNA完整性检测。结果:建立了改良SCD检测法,经统计分析改良前后测定结果无统计学差异,同时初步对改良SCD法检测的正常精子DNA百分比参考值范围进行了确定,其值为精子DNA异常发生率<32.58%。结论:改良SCD方法能更快速地对精子DNA的完整性进行检测,同时,由于降低了试剂成本,该法更容易在实际工作中得到普及。     

SCSA和SCD检测精子DNA完整性结果比较及与精子质量参数的相关性分析

目的:近年来精子DNA碎片预测男性生育受到广泛关注,其检测方法较多,但方法学的比较研究很少,本文旨在比较精子DNA完整性常用检测方法之间的差异,并分析DNA碎片与精子质量的相关性。方法:选择108例精液样本,采用精子染色质结构分析试验(SCSA)和精子染色体扩散实验(SCD)对样本分别进行精子DNA碎片检测分析,对两种方法的结果进行比较,并与精液常规、精子形态以及患者年龄进行相关性分析。结果:两种方法检测的精子DNA碎片化指数(DFI)存在显著的一致性(P0.01),DFI与精子活动率、前向运动精子百分率以及正常形态精子百分率呈显著负相关(P0.01),与畸形精子指数呈显著正相关(P_(SCSA)0.01,P_(SCD)0.05),SCSA得出的DFI与年龄呈显著正相关(P0.01),而SCD法的DFI并未发现类似的现象。结论:两种方法检测的结果对精子质量均有较好的预测价值,精子DFI作为一个检测指标,对男性生育力预测有一定的临床价值。但不同方法之间的检测结果差异较大,检测方法的标准化有待进一步研究。     

精子染色质扩散实验检测精索静脉曲张及不明原因不育患者精子DNA碎片

目的了解精索静脉曲张(VC)及不明原因不育患者精子DNA碎片的发生比例。方法改进的精子染色质扩散(SCD)实验分析精子DNA碎片。检测VC不育患者39例,不明原因不育患者57例。以生育健康成年男性32例为对照组。结果VC不育患者SCD小光晕和无光晕精子(精子DNA碎片)比值平均为(36.6±18.9)%,VC不育组明显高于对照组(12.1±5.2)%(P<0.001),而大光晕和中光晕精子比值VC不育组明显低于对照组(P<0.01);不明原因不育患者精子DNA碎片比值平均为(26.8±10.2)%,与对照组[(12.1±5.2)%]比较有显著性差异(P<0.001)。结论SCD实验表明,VC及不明原因不育患者精子DNA碎片比值增高。     

高原环境对青年男性精子DNA损伤的影响

目的:探讨长期高原作业是否能造成精子DNA的损伤。方法:以53例驻守低海拔地区的部队官兵为对照组,51例高海拔地区的部队官兵为观察组进行调查和对照研究,通过单细胞凝胶电泳技术和染色质扩散实验,检测精子DNA的损伤情况。结果:观察组在进入高原前的各项指标与对照组之间均无明显差异(P>0.05)。观察组的总彗星细胞发生率、各级彗星细胞发生率(G1、G2、G3)和DNA异常精子百分率在进入高原地区前分别为:(5.56±3.98)%、(3.72±1.85)%、(1.57±1.07)%、(0.27±0.34)%和(16.59±12.07)%,进入高原地区半年后均有升高,分别为:(11.15±8.59)%、(5.97±3.26)%、(3.83±2.13)%、(1.35±1.53)%和(22.03±15.33)%,与半年前相比差异显著(P<0.05);二级彗星细胞发生率(G2)和DNA异常精子百分率在1年后有所下降,分别为(3.32±1.83)%和(20.54±15.52)%,但仍比进入高原地区前有明显升高(P<0.05)。结论:长期在高原地区作业可引起精子DNA损伤,但对生育潜能的影响有待进一步研究。加强高原地区作业人员的男性生殖健康预防保健工作仍然十分重要。     

精液体外处理对精子染色质完整性的影响

<正>精子染色质完整性损伤可以增加不育和遗传缺陷风险,精子染色质完整性损伤的因素及其保护机制已成为辅助生殖领域研究的热点之一。精液体外处理技术是辅助生殖技术(ART)常规操作,研究表明,经体外优选处理后,精子浓度、活力、形态等可很大程度改善[1],然而其对精子染色质完整性的影响目前尚存在争议。体外处理精液,去除精浆的同时     

精子DNA碎片对体外受精-胚胎移植妊娠结局的影响

目的比较不同精子DNA碎片指数(DFI)时胚胎发育和妊娠结局的差异,探讨精子DNA碎片对辅助生殖结局的影响。方法回顾性分析2016年10月至2018年12月在本院生殖医学中心接受体外受精-胚胎移植(IVF-ET)的年龄小于37岁且卵巢储备功能正常的1 041例患者的临床资料,根据男方入院时生育力评估检测的DFI值分为4组:DFI10%组(n=277)、10%≤DFI20%组(n=480)、20%≤DFI30%组(n=183)及DFI≥30%组(n=101),比较各组患者的年龄、获卵数、胚胎受精率、优质胚胎率及妊娠率和流产率等。结果 4组间获卵数、MⅡ卵率及卵裂率比较无显著性差异(P0.05)。10%≤DFI20%组的受精率(81.04%)显著低于DFI10%组(83.39%)和20%≤DFI30%组(83.59%)(P0.05)。优质胚胎率随着DFI升高呈现下降趋势,DFI10%组(44.87%)显著高于10%≤DFI20%组(41.21%)及DFI≥30%组(37.51%)(P0.05)。4组间的临床妊娠率及流产率比较均无显著性差异(P0.05)。结论对于年龄小于37岁、接受IVF的女性患者,DFI的升高会对优质胚胎率产生不良影响,但对临床妊娠结局未见明显影响。     

精子DNA碎片指数和精子畸形率对ICSI临床结局的影响

目的:通过评估ICSI治疗前的精子畸形率(SMR)和精子DNA碎片指数(DFI),探讨精子DFI和SMR对卵胞浆内单精子注射(ICSI)助孕结局的影响。方法:共入组79对因少弱精子症实施第一周期ICSI治疗的不孕夫妇,在进入治疗周期前3⁶个月,评价精子浓度、前向运动精子百分率、SMR及DFI。主要观察SMR和DFI与ICSI结局参数的关系。结果:79例少弱精子症患者DFI正常51例,异常28例,异常组的DFI值明显升高(14.18%vs 41.47%);巧合的是,SMR正常组同样为51例,异常组28例,异常组的SMR值亦明显升高(87.88%vs98.46%)。按DFI正常(DFI≤25%)与异常(DFI>25%)分组,或按SMR正常(≤96%)与异常(>96%)分组,组间的双方年龄、女方BMI、获卵数、移植胚胎数等基本情况差异无统计学意义。DFI正常和异常组间,SMR正常和异常组间的受精卵子数、可移植胚胎数、早期流产率无显著差异;异常组生化妊娠率(43.5%vs 61.5%)和临床妊娠率(39.1%vs 56.4%)降低,但差异无统计学意义(P=0.19及0.10)。精子DFI与SMR呈显著正相关(r=0.231,P<0.05)。结论:精子DFI增高(>25%),与按严格标准检测的SMR增高(>96%)男性行ICSI治疗,生化妊娠率和临床妊娠率降低,但与正常者比较未发现有统计学差异,可能与样本量小有关,有必要深入研究。     

精子DNA碎片指数对精子优化处理后IUI临床结局的影响

<正>近年来不孕不育发病率呈上升趋势,相关研究发现约有50%的不育因素由男性导致[1]。男性不育原因有很多,包括染色体异常、内分泌因素、精索静脉曲张、生殖系统感染或输精管梗阻等,但最终回归到精子的数量和质量方面。以往,男性生育力检查和评估主要依赖于精子浓度、精子活力、精子形态     

精子DNA完整性检测技术研究进展

氧化胁迫、精子染色质包装或分离异常、凋亡异常发生等因素可引起精子DNA损伤。精子DNA完整性异常,会影响受精和胚胎发育,最终导致不孕不育、流产、死产、子代智力低下和染色体疾病。传统的精液检查往往不能找到精子DNA完整性异常患者的不育原因,因此对这部分患者进行精子DNA完整性检查具有更重要的意义。目前已经建立了很多方法来检测精子DNA的完整性,现就常用的精子DNA完整性检测技术的原理、方法、步骤及其临床应用作一综述。     

Microencapsulation of human spermatozoa increases membrane stability and DNA longevity

The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation on human sperm membrane integrity (viability) and sperm DNA fragmentation (SDF) following storage for 24 hr at 37°C. The cumulative sperm viability (Log-rank, Mantel–Cox; Chi-square = 114.95, p = .000) and cumulative sperm DNA fragmentation (Log-rank, Mantel–Cox; Chi-square = 187.86, p = .000) of encapsulated spermatozoa were substantially improved when compared to control spermatozoa. Significant differences in the dynamic behaviour of different individuals were only apparent for sperm viability in microencapsulated samples (p = .021) while no significant differences were observed in control spermatozoa (p = .245); the equivalent comparison for SDF showed no differences (control p = .320; microencapsulated p = .432). We present potential scenarios for the use of microencapsulated human spermatozoa in reproductive medicine.     

Morphometrical comparison of human spermatozoa obtained from semen and swim-up methodology

Because morphology is regularly established in semen smears, but not in swim-up spermatozoa, we were interested in comparing some morphological parameters of semen and swim-up spermatozoa to establish if the cells selected by the swim-up method were morphologically similar to those considered normal in semen. Normal human semen samples were divided into two aliquots. One of these aliquots was washed by centrifugation with B₂ medium and sperm smears were prepared with the resulting pellet as a control. The other aliquot was used to perform swim-up separation and the spermatozoa from the supernatant were used as experimental smears. Both groups were stained according to the triple stain technique and spontaneous acrosome reaction and viability were determined. Video microscopy and computer-assisted image processing of live and non-reacted sperm cells were used to establish morphometrical parameters of the sperm head in both populations. The following set of morphometrical parameters were considered: width, length, width/length ratio, acrosome area, head area, and acrosome area/head area ratio. An increase in head width, a decrease in head length and a subsequent increase of width/length ratio were found in swim-up cells compared with the control group. A slight increase in acrosome area/head area ratio was also observed in swim-up spermatozoa. Through the swim-up methodology we were able to select a subpopulation of oval shaped heads with spermatozoa having a bigger acrosome area in comparison to semen.     

ICSI outcome in patients with high DNA fragmentation: Testicular versus ejaculated spermatozoa

Sperm DNA fragmentation (SDF) has emerged as an important biomarker in the assessment of male fertility potential with contradictory results regarding its effect on ICSI. The aim of this study was to evaluate intracytoplasmic sperm injection (ICSI) outcomes in male patients with high SDF using testicular versus ejaculated spermatozoa. This is a prospective study on 36 men with high‐SDF levels who had a previous ICSI cycle from their ejaculates. A subsequent ICSI cycle was performed using spermatozoa retrieved through testicular sperm aspiration. Results of the prior ejaculate ICSI were compared with those of the TESA‐ICSI. The mean (SD) SDF level was 56.36% (15.3%). Overall, there was no difference in the fertilization rate and embryo grading using ejaculate and testicular spermatozoa (46.4% vs. 47.8%, 50.2% vs. 53.4% respectively). However, clinical pregnancy was significantly higher in TESA group compared to ejaculated group (38.89% [14 of 36] vs. 13.8% [five of 36]). Moreover, 17 live births were documented in TESA group, and only three live births were documented in ejaculate group (p < .0001). We concluded that the use of testicular spermatozoa for ICSI significantly increases clinical pregnancy rate as well as live‐birth rate in patients with high SDF.     

Analysis of sperm chromosomal aneuploidy and DNA integrity in infertile couples with unexplained recurrent miscarriage

Objective: To evaluate sperm chromosome aneuploidy and DNA fragmentation in infertile couples with unexplained recurrent miscarriage (URM).Methods: Multi-color fluorescence in situ hybridization (FISH) protocol was performed with the probes specific for the chromosomes 13, 18, 21, X and Y in the control group (n= 5) and the infertile patients with URM (n=12). The level of DNA fragmentation was determined by Sperm Chromatin Dispersion (SCD) test in the patients with URM (n=48) and the fertile men (control group, n=32).Results: The patients with URM had total numerical abnormality rate of 3.91% (n=12) for the chromosomes 13, 18 and 21 and 1.98% for the chromosomes X and Y respectively, which significantly higher than that of the control group (1.29% and 0.61%, n = 5, P<0.01). A proportion of total sperm DNA fragmentation was detected in infertile patients with URM (2.1±10.3%) (n = 48), which significantly higher than the control group(2.1±5.2%, P<0.01).Conclusions: Spermatozoa from couples with URM contained high rates of DNA fragmentation and chromosomal aneuploidy. Screening for sperm chromosomal aneuploidy and sperm DNA fragmentation may provide the useful information for investigating male unexplained infertility.     

The effect of sperm DNA fragmentation on intracytoplasmic sperm injection outcome

Our study objective was to assess the effect of various sperm DNA fragmentation levels on clinical intracytoplasmic sperm injection outcome. This retrospective study included 392 patients who underwent ICSI and performed sperm DNA fragmentation testing before the procedure. Based on sperm DNA fragmentation cut-off values, the patients were differentiated into 3 groups as <20%, 20%–30% and >30%. According to the female status, patients were differentiated into favourable group (n = 259) with female age <35 years and anti-Mullerian hormone level ≥7.1 pmol/L; and unfavourable group (n = 133) with female age ≥35 years and anti-Mullerian hormone level ≤7.1 pmol/L. The patient's medical records were reviewed, and patient's demographic, laboratory data including semen analysis, sperm DNA fragmentation determined by means of sperm chromatin dispersion, hormonal profile and data regarding intracytoplasmic sperm injection cycle were collected. This cohort reported that the clinical reproductive outcomes of intracytoplasmic sperm injection showed no statistical significance with increase sperm DNA fragmentation levels. In sperm DNA fragmentation above 30%, favourable females had significantly higher clinical pregnancy rate and live birth rate than unfavourable females, while fertilisation rate and miscarriage rate showed no significance between the subgroups. High sperm DNA fragmentation is linked to poor semen parameters.     

In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation

The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham′s F10 medium plus bovine albumin at 37° and 5% CO₂ for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen‐free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.     

Study of aneuploidy rate and sperm DNA fragmentation in large-headed, multiple-tailed spermatozoa

The aim of this study was to analyse the meiotic segregation and DNA fragmentation rates in ejaculated spermatozoa of Tunisian men who presented the macrocephalic sperm head syndrome and to compare the results with those from 20 fertile men with normal semen profiles. Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay. Fluorescence in situ hybridisation for chromosomes X, Y and 18 was performed for the study of meiotic segregation. Despite a normal blood karyotype, patients with large-headed spermatozoa showed a significantly higher incidence of sperm chromosomal abnormalities compared with the control group. For all the patients, tetraploidy, triploidy and diploidy were the most observed abnormalities. A very high level of DNA fragmentation was shown for these patients. In conclusion, our results demonstrated that patients with large-headed, multiple-tailed spermatozoa had significantly higher incidence of sperm chromosomal abnormalities and very high level of DNA fragmentation. So intracytoplasmic sperm injection should not be recommended to these patients, not only because of its low chances of success rate but also because of its high genetic risk.     

The presence of human papillomavirus in semen does not affect the integrity of sperm DNA

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO‐LiPA test PCR‐based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses.     

Selection of viable human spermatozoa with low levels of DNA fragmentation from an immotile population using density gradient centrifugation and magnetic‐activated cell sorting

We aimed to determine whether density gradient centrifugation and magnetic‐activated cell sorting (DGC‐MACS) could select viable spermatozoa, with lower levels of DNA fragmentation, from an immotile population. Analysis involved sixteen patients, each with a sperm count ≥10⁷/mL. All samples were immotile despite exhibiting a live population >40%. Spermatozoa were prepared using DGC‐MACS and selected spermatozoa evaluated for membrane and DNA integrity using the hypo‐osmotic swelling (HOS) test, vital staining and the TUNEL test. The mean proportion of spermatozoa with an intact membrane in control, DGC and DGC‐MACS populations, was 52.5 ± 12.21%, 69.38 ± 7.87% and 81.81 ± 5.29%. The mean proportion of live spermatozoa in control, DGC and DGC‐MACS populations, was 65.88 ± 12.77%, 77.25 ± 7.39% and 85.81 ± 5.2%. DGC‐MACS reduced the within‐sample discrepancy between HOS test and vital stain results from 13.18% to 4.12%. The mean proportion of spermatozoa exhibiting DNA damage in control, DGC and DGC‐MACS populations, was 9.56 ± 3.39%, 5.25 ± 1.61% and 2.75 ± 1.13%. Finally, analysis showed that 71.23% of the DNA‐fragmented spermatozoa in unprocessed samples were removed following DGC‐MACS and that the addition of MACS to an existing DGC protocol reduced fragmented spermatozoa by a further 26.15% compared to DGC alone. Consequently, DGC‐MACS is a clinically viable method able to select viable spermatozoa with lower levels of DNA fragmentation from an immotile population.