Sperm DNA damage in men from infertile couples

Aim： To investigate the prevalence of high levels of sperm DNA damage among men from infertile couples with both normal and abnormal standard semen parameters. Methods： A total of 350 men from infertile couples were assessed. Standard semen analysis and sperm chromatin structure assay （SCSA） were carried out. Results： Ninety-seven men （28% of the whole study group） had a DNA fragmentation index （DFI） 〉 20%, and 43 men （12%） had a DFI 〉 30%. In the group of men with abnormal semen parameters （n = 224）, 35% had a DFI 〉 20%, and 16% had a DFI 〉 30%, whereas these numbers were 15% and 5%, respectively, in the group of men with normal semen parameters （n = 126）. Men with low sperm motility and abnormal morphology had significantly higher odds ratios （ORs） for having a DFI 〉 20% （4.0 for motility and 1.9 for morphology） and DFI 〉 30% （6.2 for motility and 2.8 for morphology） compared with men with normal sperm motility and morphology. Conclusion： In almost one-third of unselected men from infertile couples, the DFI exceeded the level of 20% above which, according to previous studies, the in vivo fertility is reduced. A significant proportion of men with otherwise normal semen parameters also had high sperm DNA damage levels. Thus, the SCSA test could add to explaining causes of infertility in cases where semen analysis has not shown any deviation from the norm. We also recommend running the SCSA test to choose the appropriate assisted reproductive technique （ART）.     

Sperm nuclear DNA fragmentation and its association with semen quality in Greek men

Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non‐normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non‐normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility.     

Blood and semen paraoxonase—arylesterase activities in normozoospermic and azoospermic men

Paraoxonase and arylesterase enzymes are corner stones of antioxidant defence. We aimed to compare azoospermic infertile men and normozoospermic individuals with respect to total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), paraoxonase and arylesterase levels in the blood and seminal plasma. Two‐hundred consecutive infertility patients and voluntarily participated were included. In the normozoospermic group, TAS, PON, arylesterase values were statistically significantly higher when compared with those in the azoospermic group, while lower TOS and OSI levels were observed in the blood and seminal plasma of azoospermic group. In the semen analyses of normozoospermic group, the correlation between semen volume, sperm concentration, sperm motility and morphology and TAS, TOS, OSI, PON and arylesterase values was examined. A negative correlation was determined between semen volume and OSI. Levels of serum oxidative parameters were higher in the azoospermic group relative to normozoospermic group, but antioxidant parameters were lower than those of the normozoospermic group. Oxidative stress performs an essential role in the aetiology of male infertility by negatively influencing sperm quality and function. Assessment of blood and seminal plasma oxidative profiles might be an important tool to better evaluation of sperm reproductive capacity and functional competence.     

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients’ samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.     

Clinical correlates of the biological variation of sperm DNA fragmentation in infertile men attending an andrology outpatient clinic

Determination of sperm DNA fragmentation, as assessed by the sperm chromatin structure assay (SCSA), has become an important tool for the evaluation of semen quality. The aim of the present study was to describe the biological variation of sperm DNA fragmentation in men attending an andrology clinic and to identify clinical correlates of the biological variation of sperm DNA fragmentation. For this study, two consecutive semen samples from 100 patients attending our andrology outpatient clinic were subjected to semen analysis, performed in parallel according to WHO guidelines and by SCSA. A good agreement between pairs of samples was found for SCSA-derived variables, as indicated by a significantly lower median coefficient of variation (CV) of the DNA Fragmentation Index (DFI) and the high DNA stainability (HDS) compared with WHO semen parameters. In half of the men attending our andrology clinic, however, the individual biological variation of DFI and HDS, expressed as CV of two samples, exceeded 10%. Dysregulation of spermatogenesis, as seen as testicular insufficiency or varicocele, was not associated with increased variability of DFI or HDS. A backward multiple linear regression analysis, however, indicated that the biological variation of DFI may be more profound in men with characteristics of normal spermatogenesis. In conclusion, we confirm previous reports that sperm DNA fragmentation has a lower biological variability than classical semen parameters. We hypothesize that the sperm chromatin structure may be more influenced in patients with normal spermatogenesis, whereas in men with disturbed spermatogenesis, the chromatin structure may be already so impaired that the effect of unidentified factors leading to variability of sperm DNA fragmentation in time may not be as profound.     

Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case–control study

Standard sperm parameters have a limited power for prediction of the chance of natural conception. Recent studies have indicated that the sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI), a measure for the fraction of sperms with DNA damage, is associated with fertility in vivo . The aim of this study was to evaluate the value of this parameter for prediction of infertility. One hundred and twenty-seven men from infertile couples with no known female factor and 137 men with proven fertility were included. Semen analysis was performed as recommended by the WHO. DFI was assessed using SCSA. Logistic binary regression was used to compute the odds ratios (OR) for infertility. As compared with men with a DFI <10%, men with a DFI between 10% and 20% had an increased risk for infertility (OR 2.5, 95% CI: 1.0–6.1). This was also true for men with a DFI >20% (OR 8.4; 95% CI: 3.0–23). In men with normal standard semen parameters (sperm concentration, motility and morphology) the OR for infertility was increased with DFI >20% (OR 5.1, 95% CI: 1.2–23), whereas if one of the standard semen parameters was abnormal, the OR for infertility was increased already at DFI above 10% (OR 16, 95% CI: 4.2–60). We conclude that SCSA DFI adds to the value of semen analysis in prediction of the chance of natural conception.     

Effect of antioxidant intake on sperm chromatin stability in healthy nonsmoking men

Oxidative stress is detrimental to sperm function and a significant factor in the etiology of male infertility. This report examines the association between dietary and supplementary intake of the antioxidants vitamin C, vitamin E, and beta-carotene and sperm chromatin integrity. Eighty-seven healthy male volunteers donated semen samples, completed food-frequency questionnaires, and provided information about their sociodemographic characteristics, medical and reproductive histories, and lifestyle habits. Sperm chromatin integrity was measured using the DNA fragmentation index (DFI) and related parameters, obtained from the sperm chromatin structure assay (SCSA). SCSA measures the susceptibility of sperm DNA to acid-induced denaturation in situ. After adjusting for age and duration of abstinence, there was no dose-response association between any DFI outcome and any antioxidant intake measure. Non-dose-related associations were found between beta-carotene intake and both the standard deviation of DFI (SD DFI) and the percent of immature sperm. Participants with moderate, but not high, beta-carotene intake had an increase in SD DFI compared with participants with low intake (adjusted means 206.7 and 180.5, respectively; P = .03), as well as an increase in the percentage of immature sperm (adjusted means 6.9% and 5.0%, respectively; P = .04). If antioxidant intake in the range studied is indeed beneficial for fertility in healthy men, it does not appear to be mediated through the integrity of sperm chromatin. The results of this study do not preclude possible beneficial effects of high antioxidant intake on sperm chromatin integrity for men with fertility problems.     

Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation

Male infertility has a complex etiology, and many times, the cause is unknown. While routine semen analysis provides an overview of basic semen parameters, such as sperm concentration, motility, viability and morphology, a significant overlap of these parameters has been reported in fertile and infertile men. Moreover, conventional semen parameters do not reveal the cellular or molecular mechanisms of sperm dysfunctions leading to infertility. Therefore, sperm functional parameters, including sperm chromatin integrity, are evaluated to provide information on subtle sperm defects that are not routinely identified. Incomplete or defective sperm chromatin condensation increases the susceptibility of the sperm DNA to oxidative damage or other factors. To evaluate sperm chromatin integrity, different methods with varying degrees of diagnostic and prognostic capabilities are available. Among these assays, SCSA, TUNEL and SCD assays are most commonly used. While these assays rather evaluate the DNA directly for damages, the aniline blue and chromomycin A3 stains test for the quality of chromatin condensation. Thus, this review discusses and compares different methods used to evaluate sperm chromatin integrity and condensation, and their inclusion in the routine evaluation of the male infertility.     

Sperm chromatin structure components are differentially repaired in cancer survivors

Chemotherapy is often associated with male infertility. Our aim was to determine the effect of chemotherapy on sperm chromatin quality in cancer survivors. Sixteen men with advanced testicular cancer and 15 with Hodgkin lymphoma requiring chemotherapy were compared with 11 community volunteers. Eleven idiopathic infertile men with abnormal sperm chromatin were included as a positive control group. Semen analysis and sperm chromatin quality were determined prechemotherapy and at 6, 18, and 24 months posttreatment. DNA damage was determined by the sperm chromatin structure assay (SCSA). The level of DNA compaction was assayed by determining high DNA stainability (HDS, SCSA), the percentage of free thiols (monobromobimane-labeling assay), and the level of protamination (chromomycin A3-labeling assay). Sperm concentration and motility were dramatically decreased in cancer patients 6-18 months after chemotherapy compared with community volunteers but were not statistically different from community volunteers at 24 months posttreatment. High levels of DNA damage were observed prechemotherapy, with a tendency to remain high during the 24-month posttreatment period in testicular cancer patients; low DNA compaction (HDS, SCSA) persisted in testicular cancer patients 24 months postchemotherapy. Low levels of sperm DNA compaction were observed in cancer patients compared with community volunteers and infertile men. Sperm monobromobimane and chromomycin A3 labeling in cancer patients were similar to those from community volunteers by 18 months after treatment. Chemotherapy-induced damage to components of the sperm chromatin structure was repaired differentially over time. However, significant sperm DNA damage and low DNA compaction remained up to 24 months posttreatment. The assessment of complementary aspects of sperm chromatin quality is necessary to evaluate sperm samples in cancer survivors.     

Male infertility： risk factors in Mongolian men

Aim: To determine the most common risk factors of male infertility in Mongolian men attending an infertility clinic. Methods: A prospective, case-control study was conducted in which 430 men were enrolled. All the men had sought their first infertility evaluation between 1998-2002 in the State Research Center on Maternal Child Health, Ulaanbaatar, Mongolia. They were divided into two groups depending on the results of their semen analysis:191 with abnormal semen and 239 with normal semen profile. Univariate and multivariate analyses were performed to determine any association between risk factors and semen abnormality. Results: Logistic regression analysis demonstrated that the testicular volume, a history of sexually transmitted infections (STI), epididymitis and testicular damage all have statistically significant associations with semen abnormality, when controlled for multiple risk factors.Adjusted odds ratios of 3.4 for mumps orchitis, 2.3 for other orchitis and 3.9 for testicular injury were found.Gonorrhoea, the most commonly reported STIs in this study, gave an adjusted odds ratio of 1.0 for having one or more sperm abnormality. An adjusted odds ratio for subjects with a history of other STIs was 2.7. However, as a predictor of azoospermia, STIs had very high odds ratio, being 5.6 in patients with gonorrhoea and 7.6 in patients with other STIs. Conclusion: A history of pathology involving testicular damage appeared to have the strongest impact on male infertility in Mongolia. STIs have less impact on semen quality except when complicated by orchitis, epididymitis and vasal obstruction. (Asian J Androl 2004 Dec, 6: 305-311)     

乙肝病毒携带者不育男性精液质量分析

目的:分析携带乙肝病毒(HBV)的不育男性的精液质量,探讨HBV感染对男性精液质量的影响。方法:选择2018年门诊初诊的782例不育男性,年龄25~35岁,根据HBV感染情况分为小三阳组(血清学检查乙肝表面抗原、e抗体、核心抗体阳性,n=286)和大三阳组(血清学检查乙肝表面抗原、e抗原、核心抗体阳性,n=230),以未感染者作为对照组(n=266),对上述3组进行精液常规、精子顶体酶活性及精子染色质结构分析,比较3组结果是否有差异。结果:①小三阳组精子浓度[(71.49±60.03)×10^6/ml]、前向运动精子百分率[(30.70±14.79)%]、精子活率[(42.67±17.23)%]、精子存活率[(81.07±10.19)%]、正常形态精子百分率[(5.72±3.47)%]均低于大三阳组[(88.20±82.62)×10^6/ml、(34.88±15.60)%、(45.77±16.58)%、(82.55±7.55)%、(6.93±4.45)%]和对照组[(89.29±53.80)×10^6/ml、(37.82±13.63)%、(48.16±14.03)%、(85.26±6.39)%、(7.27±4.43)%],除精子存活率以外差异均有统计学意义(P<0.05);大三阳组精子浓度、前向运动精子百分率、精子活率、精子存活率、正常形态精子百分率均低于对照组,其中前向运动精子百分率、精子存活率的差异有统计学意义(P<0.05);②小三阳组的精子顶体酶活性[(57.07±26.38)μIU/10^6精子]显著低于大三阳组[(63.03±28.75)μIU/10^6精子,P<0.05]和对照组[(78.00±33.49)μIU/10^6精子,P<0.01];大三阳组的精子顶体酶活性显著低于对照组(P<0.01);③小三阳组精子DNA碎片指数[DFI,(14.79±9.46)%]和高可染性[HDS,(9.62±6.20)%]均高于大三阳组[(12.95±7.29)%、(8.43±4.72)%]和对照组[(11.60±5.98)%、(8.41±4.59)%],差异有统计学意义(P<0.05);大三阳组的DFI和HDS均高于对照组,仅DFI的差异有统计学意义(P<0.05)。结论:HBV携带者的男性精液质量显著低于未感染者,HBV感染可能是引起男性生育力降低的原因之一。     

复方玄驹胶囊联合维生素E对不育患者精子染色质损伤的影响

目的:初步探讨复方玄驹胶囊联合维生素E治疗方案对轻度少精子症和/或弱精子症患者精子染色质损伤的临床疗效。方法:50例精液异常的男性不育患者随机分为实验组(n=24)和对照组(n=26),分别予以复方玄驹胶囊+维生素E和单纯维生素E治疗3个月,运用计算机辅助精液分析系统(CASA)及精子染色质结构分析(SCSA)方法分析两组患者治疗前后精液常规参数和精子DNA损伤指数(DFI),比较治疗前后精液常规参数及精子DFI的变化。结果:实验组治疗后前向运动精子率为(21.55±8.68),对照组为(21.47±11.53),两组相比差异没有统计学意义(P>0.05)。在实验组中治疗前DFI为34.09±10.32,治疗后DFI为29.57±12.19,与治疗前相比显著下降(P<0.05)。结论:复方玄驹胶囊联合维生素E治疗可有效改善不育患者精液质量,对精子染色质损伤有一定的改善作用。     

不育男性精子染色质结构与精液常规参数的关系

目的 研究不育男性精子染色质结构参数与精液常规参数的关系.方法 采集36例男性不育患者和18例健康对照者的精液标本,用TUNEL法检测精子DNA碎片化,CMA3染色法检测精子染色质包装质量,按照世界卫生组织标准(1999)检测精液常规参数.结果 不育组的精子TUNEL阳性率和CMA3阳性率均显著高于健康对照组,差异具有统计学意义(P＜0.05),精液常规参数正常的不育男性精子TUNEL阳性率和CMA3阳性率也显著高于健康对照组,差异具有统计学意义(P＜0.05),精子TUNEL阳性率和CMA3阳性率与精液常规参数之间具有显著的相关性(P＜0.05).结论 不育男性的精子染色质结构检测结果与健康男性存在显著差异,精子染色质结构检测可提供反映男性生育能力的信息.     

Sperm antibodies in men with oligozoospermia and azoospermia

Spermatozoal antibodies found in the sera of oligozoospermic and azoospermic men are often related to infertility or subfertility. In this study the sera of 36 such men were examined for the presence of sperm antibodies. During the routine analysis of the semen, note was taken of the type and degree of agglutination in the ejaculates. The semen and serum of 15 normozoospermic men were used as controls. The gelatin agglutination, micro-agglutination and tube-slide agglutination tests were compared. A correlation between the state of fertility and the presence of sperm antibodies was found. The presence of sperm antibodies is more likely to be a corollary than a cause of oligozoospermia or azoospermia.     

精子染色质结构分析的临床意义

目的探讨女方不明原因流产和不明原因不孕与其配偶精子染色质性状之间的关系;方法通过精子染色质结构分析(SCSA)对22例不明原因流产和36例不明原因不孕妇女配偶精子,以及20例正常对照者精子进行DNA完整性检测,并比较它们之间的差异;结果对照组与不明原因流产组以及不孕组之间精子DNA完整性存在差异,且差异具有统计学意义(P<0．05),而不明原因流产组与不孕组之间精子DNA完整性比较无明显差异(P>0．05);结论精子染色质结构分析有可能作为不明原因流产和不孕的辅助诊断方法在临床应用。     

High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men

Single nucleotide polymorphisms (SNPs) in 7000 bp of the mitochondrial genome, encompassing 15 coding regions from COI to ND5, were characterized by single strand polymorphism analysis and confirmed by DNA sequencing. About 2.4% of normozoospermic men and 8.4% of men with poor semen quality had at least one nucleotide substitution. Most of the substitutions occurred in the third codon and did not change the amino acid. Hydrophobicity plots of the proteins with changes in an amino acid as a result of a nucleotide substitution suggested that they did not affect the function of the protein. The two most common substitutions at nucleotide (nt) 9055 and 11719 had significantly higher frequencies in men with reduced sperm motility. Eleven percent of the men with poor semen parameters and 1.3% of normozoospermic men had a 9055 substitution, 12% of the men with poor semen parameters had a substitution at nt 11719, but none of the normozoospermic men had this substitution. All the patients with these substitutions had reduced sperm motility and/or low sperm count. These SNPs in the mitochondrial genome were in a homoplasmic state. Thus, we propose that possessing these mitochondrial mutations compromises the semen quality of these men.     

精子质量分析仪检测男性不育精液状况的研究

应用精子质量分析仪对１３１例不育及正常人精液进行精子活力指数（ＳＭＩ）检测，并与常规精液分析进行比较，发现ＳＭＩ与常规方法测得的精液质量参数有很好的相关性。不育组的ＳＭＩ值显著低于正常对照组（Ｐ〈０．０１）。对ＳＭＩ值〈８０．８０￣１６０和〉１６０的精液质量参数进行比较，发现各组间均有显著性差异（Ｐ〈０．０５）。提示用精子质量分析仪测得的ＳＭＩ能客观地反映精液质量和男性生育力状况，使用简单易掌握，     

Association of TNFα, TNFR1, and TNFR2 polymorphisms with sperm concentration and motility

Tumor necrosis factor α (TNFα) is a multifunctional cytokine that regulates various cellular processes related to spermatogenesis. Two types of cell receptors, TNFR1 and TNFR2, mediate TNFα activity. In the present study, we sought to explore the association of TNFα -857C→T, TNFR1 36A→G, and TNFR2 676T→G polymorphisms with sperm concentration and motility. Two hundred ninety men were examined during infertility investigation; of those, 170 men were normozoospermic and 120 were oligospermic. Polymerase chain reaction analysis revealed significant differences in genotype distribution of the TNFR1 36A→G polymorphism between normozoospermic and oligospermic men. Men with oligozoospermia presented TNFR1 36A/A genotypes less frequently than normozoospermic men (P < .001). The presence of the TNFR1 36G allele was significantly increased in oligospermic men (P < .001). Furthermore, the presence of the TNFR1 36G allele was associated with lower sperm concentration in normozoospermic men (P < .03) and in the total study population (P < .001), and with lower sperm motility in normozoospermic men (P < .007) and in the total study population (P < .001). No significant associations were found between TNFα -857C→T and TNFR2 676T→G polymorphisms and semen quality. The TNFR1 36A allele is associated with increased sperm concentration and motility in our series, supporting the significance of TNFR1 gene in semen quality.     

Relationships between serum hormone levels and semen quality among men from an infertility clinic

Participation rates in epidemiologic studies on semen quality are generally very low, raising concerns as to the potential for selection bias. Since hormones both initiate and maintain spermatogenesis, they may serve as surrogates of semen quality in epidemiologic studies. For this reason, in the present study, we explored the influence and predictive ability of reproductive and thyroid hormones on semen quality among men who were partners in an infertile couple. Between 1999 and 2003, 388 men were recruited from Massachusetts General Hospital Andrology Laboratory for clinical evaluation of fertility status. Fresh semen samples were assessed for quality (concentration, motility and morphology) and the serum levels of hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin B, sex hormone-binding globulin (SHBG), testosterone, free androgen index, free T4, total T3, and thyroid-stimulating hormone (TSH), were measured. Multiple logistic regression revealed increased odds for below-reference sperm concentration and morphology in men with increased FSH, and decreased odds for below-reference sperm concentration and motility in men with increased inhibin B. When FSH and inhibin B were divided into quintiles, the relationships with sperm concentration showed evidence of a threshold value. However, the ability of specific FSH (10 IU/L) and/or inhibin B (80 pg/mL) cutoff values to predict semen quality was lower than in previous reports. In multiple linear regression analysis, FSH and LH were inversely associated with sperm concentration, motility, and morphology. Inhibin B and free T4 were positively associated with sperm concentration, while there was a suggestive positive association between testosterone and sperm motility. In conclusion, we have found that FSH, LH, inhibin B, testosterone and free T4 levels are associated with human semen parameters. Additional consideration should be given to the utility of serum hormone levels as a surrogate for semen quality in epidemiologic studies in which the collection of semen is difficult due to logistical and/or volunteer rate constraints.     

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.