Detection of DNA fragmentation in human spermatozoa: correlation with semen parameters

To determine the prevalence of high levels of sperm DNA damage among infertile men with normal and abnormal semen parameters, 90 patients were subdivided into the following three groups. Group A ( n  = 30): men with normal semen parameters who acted as the controls. Group B ( n  = 30): asthenozoospermic men and group C ( n  = 30): teratozoospermic men, suffering from male infertility. DNA damage was evaluated by the rate of DNA fragmentation index (DFI) as assessed by the terminal desoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. It was found that the difference was not significant between the percentage of DFI in patients with asthenozoospermia and the normospermic men (9.46% ± 8.68 and 8.19 ± 6.84 respectively, P- value not significant). The patients with teratozoospermia showed a significantly higher percentage of DNA fragmentation compared with the controls (respectively 21.37 ± 17.26% and 8.19 ± 6.84%, P  < 0.001). There was a positive correlation between abnormal sperm morphology and the DFI ( r  = 0.44, P  < 0.01) in group C. It is concluded that the impairments of sperm parameters were associated with an increase of DNA fragmentation; this association was strictly related to atypical forms.     

Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

Aim： To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality. Methods： Two early apoptotic changes in the semen of 56 men were assessed using Annexin V （AN）/propidium iodide （PI） staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential （MMP）. The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. Results： The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN^-/PI^- spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL （＋） spermatozoa. As for the AN^-/PI^＋ fraction, we found an opposite result in comparison to AN^-/PI^- spermatozoa. The level of early apoptotic AN^＋/PI^- spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN^＋/PI^＋ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL （＋） spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL （＋） spermatozoa. Conclusion： Although early apoptotic AN^＋/PI^- spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa （especially, the percentage of AN^-/PI^- spermatozoa）, and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization. （Asian J Androl 2008 Mar; 10： 227-235）     

Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa

Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r = -0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA.     

Influence of semen processing technique on human sperm DNA integrity

Objectives. To compare the effects of density-gradient centrifugation and swim-up technique on sperm DNA integrity.Methods. Semen samples (n = 22) were obtained from consecutive nonazoospermic men presenting for infertility evaluation. Individual samples were divided into three aliquots (whole semen, density-gradient centrifugation, and swim-up) for subsequent analysis of sperm motility and DNA integrity. Sperm DNA integrity was evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA.Results. Mean sperm motility (±SEM) improved significantly after processing with two-layer density-gradient and swim-up compared with whole semen (65.6% ± 4.0% and 73.0% ± 3.0% versus 52.0% ± 3.6%, respectively, P <0.005), with no significant difference in motility between Percoll-treated and swim-up-treated spermatozoa. In contrast, the percentage of spermatozoa with denatured DNA was reduced significantly in swim-up-treated but not in Percoll-treated spermatozoa compared with whole semen (4.8% ± 1.2% and 13.6% ± 3.6% versus 10.1% ± 2.3%, respectively, P <0.0001).Conclusions. Although density-gradient centrifugation is comparable to swim-up technique in recovering spermatozoa with enhanced motility, spermatozoa recovered after swim-up possess higher DNA integrity. These data urge us to reexamine our current sperm processing techniques in order to minimize sperm DNA damage.     

精浆弹性蛋白酶与精液常规参数及精子DNA完整性的相关性研究

目的:观察男性不育患者精浆弹性蛋白酶水平与精液质量及精子DNA完整性之间的关系,探讨生殖道炎症隐性感染对男性不育的影响。方法:选择202例男性不育患者依照精浆弹性蛋白酶实验结果并结合其他生殖道感染炎症标志物将男性不育分为确认感染组和隐性感染组,选择同期因女方因素不孕前来检查的生育力评估正常,排除生殖道炎症及其他影响生育的致病因素的健康男性32例作为对照组。统计分析不育组与正常对照组、不育组中确认感染组与隐性感染组、不育组中弹性蛋白酶水平正常者与升高者之间精浆弹性蛋白酶水平与精液质量及精子DNA完整性之间的差异。精子浓度及活力用计算机辅助精子分析系统检测,精子形态检查采用Diff-Quik染色法,精子DNA完整性分析用精子染色质扩散法(SCD),精浆弹性蛋白酶用酶联免疫吸附法。结果:正常对照组和不育组的正常形态精子百分率分别为(11.9±9.2)%和(4.1±3.3)%、精子DNA碎片指数(DFI)分别为(10.9±8.7)%和(21.3±12.7)%,精浆弹性蛋白酶分别为(220.9±73.1)ng/ml和(1687.3±1621.2)ng/ml,前向运动精子百分率(PR)分别为(49.7±10.8)%和(19.1±10.3)%,两组相互比较各指标均具有统计学差异(P均0.05)。不育组中弹性蛋白酶升高者较弹性蛋白酶正常者,PR显著降低,DFI显著升高,差异均具有统计学意义(P0.05)。隐性感染和确证感染患者间精液量、精子浓度、PR、正常形态精子百分率和DFI的差异均无统计学意义(P均0.05)。结论:生殖道炎症感染无论是显性或者是隐性有可能导致DNA损伤程度增加,从而影响男性生育。     

精浆弹性硬蛋白酶与精子 DNA 完整性及精液参数的影响分析

目的：探讨男性不育患者精浆弹性硬蛋白酶水平与精子 DNA完整性及精液质量之间的关系。方法选择148例男性不育患者根据精浆弹性硬蛋白酶的检测结果将所有患者分为 A、B、C3组。 A组为精浆弹性硬蛋白酶含量＜290 ng／mL;B组为精浆弹性硬蛋白酶含量290～1000 ng／mL;C 组为精浆弹性硬蛋白酶含量＞1000ng／mL。用酶联免疫吸附试验（ ELISA）检测精浆中弹性硬蛋白酶,精子 DNA完整性检测用精子染色质扩散法（ SCD）,精液参数用计算机辅助精子分析系统检测。结果 A组与 C 组相比精子存活率与前向运动精子百分率（PR）均升高、精子DNA碎片指数（DFI）明显降低,差异均具有统计学意义（ P＜0．05）。 B组的上述指标与A组相比,差异均无统计学意义（ P ＞0．05）。结论精浆弹性硬蛋白酶与精子DNA完整性及精液质量密切相关,生殖道感染是影响男性精液质量的一个重要原因。     

Sperm DNA integrity: correlation with sperm plasma membrane integrity in semen evaluated for male infertility

Previous independent studies have indicated that abnormally low parameters of sperm DNA integrity and sperm membrane integrity correlate to reduced fertility due in part to implantation disorders. The purpose of our study was to evaluate the relationship between sperm plasma membrane functional integrity assessed by the hypo-osmotic swelling test (HOST) and sperm DNA integriy test assesed by DNA fragmentation index (DFI). Semen samples from 102 random patients were evaluated in terms of standard semen parameters and assessed by DFI and HOST. Both tests showed a significant correlation to standard semen parameters (p < .05). In addition, patients with abnormal HOST results had a higher likehood of a subnormal or abnormal DFI result (p < .001). Our results suggest that a common sublethal insult may manifest as abnormalities in both the nucleus and the plasma membrane that act at the implantation and/or subsequent levels of development rather than at the fertilization stage.     

Catalase improves motility,vitality and DNA integrity of cryopreserved human spermatozoa

Cryopreservation of human spermatozoa offers a pre‐therapeutic possibility of preserving progenity in patients with testicular tumours. We aimed to investigate effects of cryopreservation and addition of catalase on sperm motility, vitality and DNA integrity in fresh and swim‐up spermatozoa. Semen samples were collected from 50 fertile men. Each sample was divided into two parts. First part was subdivided into two equal aliquots: both cryopreserved with and without catalase. The second part was subdivided into two equal aliquots: both processed by swim up and then cryopreserved with or without catalase. Semen analyses, sperm vitality and sperm DNA integrity were performed. Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and % of DNA damage showed significant increase in processed and cryopreserved processed samples when compared with fresh and cryopreserved fresh samples. There was no significant difference in sperm concentration while there was significant increase in % of progressive motility and sperm vitality and % of DNA damage showed significant decrease in samples with catalase when compared with samples without catalase (either fresh or processed). Catalase supplementation (fresh and processed) during cryopreservation results in better post‐thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity.     

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.     

乙肝病毒对人精液参数和精子DNA完整性的影响

目的:探讨精液中乙肝病毒存在对精液参数和精子DNA完整性的影响。方法:采用实时荧光定量PCR技术对153例血清乙肝表面抗原阳性患者精液进行乙肝DNA定量检测,采用精子染色质扩散法检测精子DNA损伤指数(DFI),对比分析精液HBV DNA阳性组(A组,n=43)与阴性组(B组,n=110)精液参数包括精液体积、精子浓度、存活率、前向运动精子百分率、平均直线速率(VSL)、平均路径速率(WAP)结果,以及HBV DNA拷贝数与精子DNA损伤指数的关系。结果:乙肝感染者精液HBV DNA检出率为28.1%(43/153);两组年龄、精液体积、精子浓度无统计学差异(P>0.05),A组精子存活率显著低于B组[(51.4±17.1)%vs(58.0±18.8)%,P>0.05],前向运动精子率显著低于B组[(24.5±10.1)%vs(29.6±13.3)%,P>0.05],VSL显著低于B组[(19.9±4.5)μm/s vs(23.7±4.0)μm/s,P<0.01],WAP显著低于B组[(23.4±5.3)μm/s vs(26.5±7.0)μm/s,P<0.01];A组精子DNA碎片指数显著高于B组[(24.2±9.4)%vs(19.3±8.0)%,P<0.01];精液中HBV DNA拷贝数与精子DNA碎片指数呈正相关(r=0.819,P<0.01)。结论:精液中HBV DNA存在对精子数量尚未构成显著影响,但可能改变一些精液参数,包括精子活力和运动速度以及精子DNA完整性,精液中乙肝病毒载量和精子DNA损伤之间呈现病毒载量-效应关系。     

精子DNA完整性与精液参数的相关性研究

目的 探讨精子DNA完整性与精液参数之间的相关性.方法 收集2009年3月至2009年7月在解放军105医院生殖中心就诊的220例男性患者的精液标本,采用吖啶橙荧光染色法(AOT)检测精子核DNA碎片指数(DFI).按DFI值分为DFI≤30%、30%50%3组,对DFI与各项精液参数的相关性进行分析.结果 精子活率、活力力、正常形态率随DFI值的升高逐渐降低,各组间均有显著性差异(P<0.05);DFI与精子活率、活动力、正常形态率均呈显著负相关(P<0.05),与精液量及密度无显著相关性.结论 精子DNA损伤可导致精子形态学异常和活力下降,精子DFI是评估男性生育力的一个较好的参考指标.     

Quality of human spermatozoa: relationship between high‐magnification sperm morphology and DNA integrity

The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)‐selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI‐morphological classes (Class 1 versus Class 3, respectively) (P > 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI‐selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei.     

吸烟对男性精液质量、精子DNA完整性及Chk1基因表达的影响

目的探讨吸烟对男性精液质量、精子DNA完整性Chk1基因表达的影响。方法将1 128例研究对象分为不吸烟组及吸烟组,且依据每日吸烟支数将吸烟组划分为轻度吸烟组、中度吸烟组、重度吸烟组,依据吸烟年限将吸烟组划分为:短烟龄组、中烟龄组、长烟龄组。对各组患者行精液常规参数、精子形态、精子DNA完整性及精子Chk1基因mRNA相对表达量检测。同时就精子DNA完整性及精子Chk1基因mRNA相对表达量与精液常规参数的相关性进行分析。结果(1)吸烟组与不吸烟组总体比较:吸烟组前向运动精子率及DNA双链精子百分率较不吸烟组显著下降(P0.05)。(2)不同烟量的影响:轻度、中度及重度吸烟组与不吸烟组相比,前向运动精子均显著下降(P0.05),且重度吸烟组头部精子缺陷率较不吸烟组显著增高(P0.05)。(3)不同烟龄的影响:长烟龄组精液量、浓度、总数及前向运动精子率较不吸烟组均下降显著(P0.05),中烟龄组较不吸烟组,仅前向运动精子率显著下降(P0.05)。长烟龄组头部精子缺陷率较不吸烟组显著增高(P0.05)。(4)相关性分析:精子DNA损伤程度与男性精液浓度及前向运动精子率间存在相关性(P0.05)。吸烟组患者较不吸烟组患者Chk1基因mRNA相对表达量降低(P0.05),且精子Chk1基因mRNA表达水平与男性精液浓度及前向运动精子率间存在相关性(P0.05)。结论吸烟可引起男性精液质量下降,且随着吸烟时间的延长及日吸烟量的增加,精液质量下降更为显著。而且,吸烟引发的精液质量下降不仅与精子DNA完整性存在一定的相关性,同时与DNA损伤修复相关基因Chk1下调相关。     

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.     

Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men

Normal chromatin condensation is important for sperm fertilising ability. However, routine semen analysis does not identify defects in sperm chromatin structure. This study aimed to investigate the condensation of chromatin and DNA integrity in spermatozoa of infertile men and deduce the relationship with sperm quality, as measured by conventional semen parameters. Semen analysis was carried out to assess sperm quality according to World Health Organization criteria. The remaining aliquot of each sample was processed for transmission electron microscopy, chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays. The ultrastructural analysis of spermatozoa from infertile men showed heterogeneity in sperm nuclear morphology. Some spermatozoa displayed a round nucleus with incomplete chromatin condensation. Immunoreactivity with antitransitional protein and antiprotamine antibodies indicated nuclear maturation defects in the spermatozoa of infertile men. Spearman's correlation analysis indicated a positive correlation between the percentages of CMA3- and TUNEL-positive spermatozoa. In addition, these two parameters were negatively correlated with sperm concentration, motility and normal morphology. This study demonstrated that men with morphologically normal spermatozoa of <30% have greater degree of protamine deficiency and DNA damage than men with morphologically normal spermatozoa of >30%. Evaluation of chromatin integrity appears to be a useful tool for assessing male fertility.     

Interaction between prostasomes and spermatozoa from human semen

Prostasomes are prostate-derived organelles that occur freely in human seminal plasma. They promote forward motility of spermatozoa probably by closely interacting with them in an unknown manner. We have studied the interaction between human prostasomes and spermatozoa by applying them as two separate samples in free-zone electrophoresis. During the run these samples approached each other and finally fused into one single peak that was not further dissociated. Both the spermatozoa and prostasomes displayed a net-negative surface charge, the latter being less negative. This discrepancy in charge was even more pronounced by pretreatment of prostasomes with neuraminidase, which, however, did not affect the interaction. This implies a strong interaction of a probable hydrophobic character between cells and organelles. The presence of prostasomes and spermatozoa in the fused, single peak was confirmed by transmission electron microscopy. Evidence for interaction was apparent in transmission electron microscopy after embedding in a hydrophilic, but not in a hydrophobic, resin. This observation supports the view that the bonds between prostasomes and spermatozoa are of hydrophobic character. This type of interaction enables the prostasomes to act in close vicinity to spermatozoa and may create the prerequisites for a proper microenvironment of the spermatozoa favoring their forward motility.     

精子DNA完整性与精液常规指标相关性分析

目的 探讨精子DNA完整性与精液常规指标的关系.方法 按照WHO<人类精液及精子-宫颈粘液相互作用实验室检验手册>要求对370例男性患者进行精液常规检测及精子DNA完整性分析.以上述手册设定的各精液常规检测指标参考值为依据分组,比较各组间精子DNA完整性是否存在差异,进而分析精子DNA完整性与各精液常规指标的相关性.结果 370例患者中,以不同年龄(岁)(≤30和＞30)、精子活率(%)(≥60和＜60)、前向运动精子活力(%)(≥50和＜50)分组,比较各组间精子DNA完整性无统计学差异(P＞0.05);而以不同精子密度(106/ml)(＜20和≥20＝、精液粘稠度(适中和粘稠)分组,各组间精子DNA完整性比较存在统计学差异(P＜0.05),其中不同密度精子DNA完整性差异显著.结论 与精子数量相关的精子发生过程是影响精子DNA完整性的主要原因;年龄因素及精子运动能力相关的附性腺功能与精子DNA完整性无关;选择合适的精液处理方式,尽量不破坏精子DNA完整性对于男科实验室精液检测及辅助生育技术过程中受精所需的精子悬液的制备等至关重要.     

Supplementation of quercetin for advanced DNA integrity in bull semen cryopreservation

The aim of this study was to identify the effects of adding quercetin (Q) to Tris extender in order to identify levels of oxidative stress in bull sperm after freeze thawing. Ejaculates were collected via artificial vagina from Holstein bulls. Semen was divided into five tools and diluted to a final concentration of 15 × 10⁶ spermatozoa/ml with the Tris extender containing Q (25, 50, 100 and 200 μg/ml) and no additive (control; C). All examples were equilibrated at 4°C during 4 hr then were loaded into 0.25‐ml straws and frozen using a controlled rate. Sperm motility and motility characteristics were determined using the computer‐assisted semen analyser. Sperm membrane integrity was assessed using the hypoosmotic swelling test. Sperm chromatin integrity was investigated using the single cell gel electrophoresis. Total antioxidant capacities were performed colorimetrically. Q supplementation used as an antioxidant did not produce better results in the proportion of sperm progressive and total motility, plasma membrane integrity and sperm abnormalities. Q supplementation exhibited the favourable tail length, tail DNA and tail moment. In conclusion, when whole parameters are considered, Q25 can be added to the Tris extender due to its positive effect on sperm DNA integrity and no adverse effect on the progressive and total motilities of sperm.