The assessment of oxidative stress in infertile patients with varicocele

OBJECTIVES

To assess oxidative stress markers, antioxidant capacity and cytokines in seminal plasma from infertile patients with varicocele, and to investigate seminal oxidative status and sperm DNA damage after varicocelectomy.

PATIENTS, SUBJECTS AND METHODS

The records were retrospectively evaluated for 28 azoospermic, 30 oligospermic (15 with varicocele and 15 without) and 30 patients with normal semen characteristics (15 with varicocele and 15 without). The mean (sd ) age of the men was 32.4 (5.6) years; all men with varicocele had a unilateral or bilateral microsurgical subinguinal varicocelectomy. The level of nitric oxide (NO), 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), hexanoyl‐lysine (HEL), superoxide dismutase (SOD) activity, interleukin (IL)‐6, IL‐8 and tumour necrosis factor‐α in seminal plasma were measured. In addition, sperm DNA fragmentation was analysed before and 6 months after varicocelectomy.

RESULTS

Azoospermic and oligospermic patients had a significantly higher HEL concentration and SOD activity in seminal plasma; those with varicocele had a significantly higher NO, HEL, and SOD activity in seminal plasma. There was a significant increase in sperm concentration and reduction in NO, HEL, 8‐OHdG level and SOD activity after varicocelectomy. Oligospermic patients with varicocele had a significantly higher IL‐6 level in seminal plasma, and there was a significant reduction after varicocelectomy. The percentage of apoptosis‐positive sperm decreased significantly after varicocelectomy.

CONCLUSIONS

This study indicates that the seminal plasma of patients with varicocele is under excessive oxidative stress, and partly even in patients with normospermia, and that varicocelectomy reduces oxidative stress in seminal plasma and ameliorates sperm DNA damage.     

Poor sperm quality and advancing age are associated with increased sperm DNA damage in infertile men

With increasing evidence for faulty paternal contribution to reproduction, there has been a steady increase in studies highlighting an association between sperm DNA damage, failed/delayed fertilisation and aberrant embryo development. Owing to prevailing ambiguity, the aims of the study were to analyse the genetic integrity of the male gamete and then to understand its association with age, standard semen parameters, lifestyle and occupational factors. The study included 504 subjects, attending university infertility clinic for fertility evaluation and treatment. Semen characteristics were analysed by standard criteria; terminal deoxynucelotidyl transferase-mediated nick end-labelling assay was employed for DNA damage assessment. The average incidence of sperm DNA damage in patients with normozoospermic semen parameters was <10%. Patients with oligozoospermia, severe oligozoospermia, oligoasthenoteratospermia, asthenoteratozoospermia and necrozoospermia had significantly higher level of sperm DNA damage (P < 0.001). Patients above 40 years of age had significantly high levels of DNA damage (P < 0.001) compared with their counterparts. Patients with varicocele and a history of alcohol consumption had higher incidence of spermatozoa with DNA damage (P < 0.01). Poor sperm characteristics in the ejaculate are associated with increased sperm DNA damage. Age-related increase in sperm DNA damage and association of the same with varicocele and alcohol consumption are also demonstrated.     

Evaluation of sperm DNA fragmentation index,Zinc concentration and seminal parameters from infertile men with varicocele

The aim of this study was to investigate the effect of varicocele on DNA fragmentation index (DFI), zinc concentration and seminal parameters in infertile patients. In this prospective study, 179 men with at least 1‐year history of infertility and varicocele were examined for semen quality at Hanoi Medical University Hospital (HMUH), Hanoi, Vietnam. In addition, an inverse correlation between zinc concentration and the degree of sperm DNA fragmentation in patients with clinical varicocele was found. The difference in mean values of sperm DNA fragmentation index in patients with various grades of varicoceles can be neglected, whereas most patients with varicocele of grades II and III had DFI >30%. Varicocele is associated with high levels of DNA damage in spermatozoa and reduced zinc levels that correlate with different grades of disease. Therefore, DNA fragmentation index and zinc concentration can be used as essential additional diagnostic test for patients with clinical varicocele. A study should be conducted to evaluate the benefits of zinc supplementation to improve seminal parameters in patients with varicocele.     

Relationship between sperm cell ubiquinone and seminal parameters in subjects with and without varicocele

Summary. In a previous paper it was demonstrated that Coenzyme Q10, a lipidic molecule with important antioxidant properties, is present at remarkable levels in human seminal fluid, and shows a direct correlation with seminal parameters (sperm count and motility). In patients with varicocele, on the contrary, correlation with sperm motility was lacking and a higher proportion of Coenzyme Q10 was found in seminal plasma. In the present study, the levels of Coenzyme Q10 in the cell pellet of spermatozoa, obtained after centrifugation of semen, were evaluated. In nonvaricocele subjects it was observed that a higher concentration of Coenzyme Q 10 (expressed as ng of the molecule per million of cells) was present in the spermatozoa of oligospermic and asthenospermic patients (sperm count <20*10⁶ spermatozoa ml⁻¹, sperm motility <40%). This relationship was not observed in varicocele subjects, who also showed slightly lower intracellular absolute values of the coenzyme.
Since Coenzyme Q10 is an antioxidant molecule involved in the defence of the cell from free radical damage, higher intracellular concentrations may represent a mechanism of protection of the spermatozoa. In varicocele patients, this mechanism could be deficient, leading to higher sensitivity to oxidative damage.     

腹腔镜术与常规手术治疗精索静脉曲张不育症351例疗效观察

本文对３５１例精索静脉曲张不育症患者采用腹腔镜精索静脉结扎术（２４４例,其中１３例连同精索内动脉一并结扎）和经腹股沟精索静脉结扎术（常规手术,１０７例）,术后随访３～３２个月。在术后８周,两组精子计数、活动率、活动力均较术前有显著改善（Ｐ〈０．０１）。腹腔镜组精索内动脉结扎与否,其精子改善情况无显著差异（Ｐ〉０．０５）。两组相较,腹腔镜组术后精子计数、活动力的改善更为显著（Ｐ〈０．０１）。在术后１４周时,常规手术组精子计数仍明显低于腹腔镜组（Ｐ〈０．０５）。随访期间腹腔镜组妊娠者１４９例,妊娠率６１．０７％（其中动脉结扎者７例,妊娠率５３．８５％）,常规手术组妊娠者６２例,妊娠率５８．９３％,两组妊娠率无明显差异（Ｐ〉０．０５）。     

4977‐bp mitochondrial DNA deletion in infertile patients with varicocele

Varicocele is the abnormal inflexion and distension of veins of the pampiniform plexus within spermatic cord and is one of the amendable causes of male infertility. It can increase reactive oxygen species (ROS) production in semen and cause oxidative stress. The purpose of this study was to analyse spermatozoa mtDNA 4977‐bp deletion in infertile men with varicocele. To detect 4977‐bp deletion in spermatozoa mtDNA, semen samples of 60 infertile patients with clinical varicocele and 90 normal men from northern Iran were prepared. After extraction of spermatozoa total DNA, Gap polymerase chain reaction (Gap PCR) was performed. 4977‐bp deletion was observed in 81.66% of patients with varicocele, while approximately 15.55% of controls had this deletion. As spermatozoa from patients with varicocele had a high frequency of occurrence of 4977‐bp deletion in mtDNA [OR = 24.18, 95% confidence interval (CI) = 10.15–57.57, P < 0.0001], varicocele may induce mtDNA deletion in spermatozoa and cause infertility in north Iranian men. However, to determine the relation between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility, larger population‐based studies are needed. It is concluded that there is an association between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility in the population studied.     

精索静脉曲张不育患者精子核成熟性研究

利用苯胺兰细胞化学染色法和ＳＤＳ、ＳＤＳ一ＥＤＴＡ处理精子研究精子核的成熟性。结果发现，２１例精索静脉曲张不育患者精子核蛋白存在明显的转型缺陷（不育组２７．８２±９．７３，对照组４０．６２±１１．７３，Ｐ＜０．０１）；其精子核抗解聚能力明显下降，Ｐ＜０．０１（ＳＤＳ）和＜０．０５（ＳＤＳ一ＥＤＴＡ）；α一糖苷酶活性越低，核蓝染精子百分数越高（ｒ＝－０．８１，Ｐ＜０．０１），而核稳定精子百分数越低（ＳＤＳ组ｒ＝０．７５；ＳＤＳ一ＥＤＴＡ组ｒ＝０．７２，Ｐ＜０．０１）．结果说明，精子核的成熟性异常与精索静脉曲张不育有密切的联系。附睾功能的异常可能是精索静脉曲张导致不育的机制之一。     

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。     

Sperm DNA damage and cytokines in varicocele: A case-control study

Varicocele is a vascular disease characterised by the abnormal enlargement of the pampiniform plexus veins and a well-known cause of male infertility. The aim of this study was to investigate the relationship between sperm DNA fragmentation (SDF) and inflammation in the pathogenesis of varicocele. We included 84 varicocele patients and 85 normozoospermic healthy controls, further analysed according to the body mass index, the smoking habit (smokers/non-smokers) and the varicocele severity (low/high grade). Semen parameters, SDF (by TUNEL) and inflammatory cytokines (by Luminex xMAP analysis) were evaluated. Varicocele patients showed significantly reduced semen parameters (volume, total sperm number, progressive motility, normal morphology) and increased SDF. Moreover, we observed a significant reduction of IFN-γ, IL-6, TNF-α and an increase of IL-10. No difference was reported according to the smoking habit, body mass index and varicocele severity. The observed cytokines pathway suggests the establishment of a chronic inflammatory condition, which may contribute to the alteration of semen quality. A thorough knowledge of the cytokine network might contribute to better understanding the link between inflammation and semen quality in varicocele and its impact on reproductive health.     

Randomised clinical trial of comparing effects of acupuncture and varicocelectomy on sperm parameters in infertile varicocele patients

The aim of the study was to evaluate the effect of the acupuncture treatment on sperm parameters and pregnancy rates in patients with primary infertility. Between January 2008 and May 2010, 30 men with the primary infertility (one year of unprotected intercourse, healthy wife) and varicocele with normal hormone levels and abnormal semen analysis were randomised into two groups. Group 1 underwent subinguinal microscopic varicocelectomy, and Group 2 underwent acupuncture treatment twice a week for 2 months. Both groups were evaluated with semen analysis at 6 months after the treatment. Patients in both groups evaluated with telephone calls and e‐mail in terms of pregnancy. The mean age of the patients was 27.2, and groups were comparable regarding the age (P = 0.542). The pre‐treatment sperm concentration, motility and morphological characteristics were similar in both groups. Sperm concentration and motility improved significantly in both groups after the treatment. Increase in sperm concentration was higher in the acupuncture group compared to the varicocelectomy group (P = 0.039). The average follow‐up was 42 months, and pregnancy rates were emphasised 33% in both groups. Acupuncture treatment in primary infertile varicocele patients with semen abnormalities seems to be effective and has comparable results with the varicocelectomy treatment.     

精索静脉曲张不育患者的精液常规、精子动态参数和精浆生化指标分析

目的观察精索静脉曲张（VC）伴不育患者的精液质量和精浆生化水平的变化。方法利用计算机辅助精液分析系统（CASA）及酶免法分别检测52例VC不育患者、85例非精索静脉曲张不育患者和21例正常生育男性的精液常规、精子动态参数以及精浆酸性磷酸酶、精浆锌（Zn）、果糖、α-糖苷酶的含量。结果与对照组相比,VC组患者A级精子率、（A＋B）级精子率、精子直线速度（VSL）、直线性（LIN）、侧摆幅度（ALH）、精浆锌含量、α-糖苷酶的含量与对照组相比显著下降（P〈0.05）。与非VC不育组相比,VC不育组（A＋B）级精子率、精子直线速度（VSL）、侧摆幅度（ALH）、精浆锌含量、α-糖苷酶的含量也明显降低（P〈0.05）。并且随着精索静脉曲张程度的增加,VC患者A级精子率、（A＋B）级精子率、精子曲线速度VCL、直线速度VSL、直线性LIN、平均路径速度VAP、前向性STR、侧摆幅度ALH、精浆中锌含量、α-糖苷酶的含量有下降趋势。结论 VC能够降低精子活动率、精子动态活动能力以及改变了精浆微环境,从而导致男性生育力下降。     

Novel association between sperm deformity index and oxidative stress-induced DNA damage in infertile male patients

Aim: To investigate the impact of abnormal sperm morphology using the sperm deformity index (SDI) on reactive oxygen species (ROS) production and its correlation with sperm DNA damage. Methods: Semen samples were collected from men undergoing infertility screening (n=7) and healthy donors (n=6). Mature spermatozoa were isolated and incubated with 5mmol/L β-nicotinamide adenine dinucleotide phosphate (NADPH) for up to 24h to induce ROS. Sperm morphology was evaluated using strict Tygerberg‘s criteria and the SDI. ROS levels and DNA damage were assessed using chemiluminescence and terminal deoxynucleotidyl transferase-mediated fluoresceindUTP nick end labeling (TUNEL) assays, respectively. Results: SDI values (median [interquartiles]) were higher in patients than donors (2 [1.8,2.1] vs. 1.53 [1.52, 1.58], P=0.008). Aliquots treated with NADPH showed higher ROS levels (1.22 [0.30, 1.87] vs. 0.39 [0.10, 0.57], P=0.03) and higher incidence of DNA damage than those not treated (10 [4.69, 24.85] vs. 3.85 [2.58, 5.10], P=0.008). Higher DNA damage was also seen following 24h of incubation in patients compared to donors. SDI correlated with the percentage increase in sperm DNA damage following incubation for 24h in samples treated with NADPH (r=0.7, P=0.008) and controls (r=0.58, P=0.04).Conclusion: SDI may be a useful tool in identifying potential infertile males with abnormal prevalence of oxidative stress (OS)-induced DNA damage. NADPH plays a role in ROS-mediated sperm DNA damage, which appears to be more evident in infertile patients with semen samples containing a high incidence of morphologically abnormalspermatozoa.     

Obesity leads to higher risk of sperm DNA damage in infertile patients

There has been a growing interest over the past few years in the impact of male nutrition on fertility. Infertility has been linked to male overweight or obesity, and conventional semen parameter values seem to be altered in case of high body mass index （BMI）. A few studies assessing the impact of BMI on sperm DNA integrity have been published, but they did not lead to a strong consensus. Our objective was to explore further the relationship between sperm DNA integrity and BMI, through a 3-year multicentre study. Three hundred and thirty male partners in subfertile couples were included. Using the terminal uridine nick-end labelling （TUNEL） assay, we observed an increased rate of sDerm DNA damage in obese men （odds ratio （95% confidence interval）： 2.5 （1.2-5.1））.     

Assessment of sperm functions in infertile patients with varicoceles

Acrosin activity, acrosome reaction and nuclear chromatin condensation were studied in 24 infertile patients with varicoceles and 26 fertile men with or without varicocele. Chromatin condensation, assessed by aniline blue staining, and acrosin activity, evaluated by gelatinolysis technique, were significantly affected in the group of infertile patients. Defective chromatin condensation and defective acrosin activity were detected in 67% and 50% of the infertile patients, respectively. No significant difference was found between the two groups regarding the acrosome reaction, which was assessed by the triple staining technique after exposure of spermatozoa to low temperature (4 degrees C). This study identified a subgroup of infertile patients with normal standard semen parameters but impaired sperm functions. Results of the sperm function tests and standard semen parameters were not correlated. Therefore, it is concluded from this study that important sperm functions are impaired in patients with varicocele and that the gelatinolysis technique and aniline blue staining are effective tools for assessment of the fertilization potential of varicocele patients.     

alpha-1,4-Glucosidase activity in infertile oligoasthenozoospermic men with and without varicocele

This work aimed to assess seminal alpha-1,4-glucosidase activity in infertile oligoasthenozoospermic men associated with and without scrotal varicocele. Eighty men were investigated. They were divided into three groups: group 1 (n = 20), fertile normozoospermic men; group 2 (n = 30), oligoasthenozoospermia with varicocele; and group 3 (n = 30), oligoasthenozoospermia without varicocele. The patients underwent medical history, clinical examination, conventional semen analysis and estimation of seminal plasma alpha-1,4-glucosidase activity by double-beam spectrophotometer method and serum testosterone by radioimmunoassay method. There was a significant decrease in the mean seminal alpha-1,4-glucosidase activity levels in infertile men versus controls (mean +/- SD; 7.66 +/- 0.433, 2.088 +/- 0.565, 5.384 +/- 0.85 mU ml(-1) respectively). Mean serum testosterone levels demonstrated nonsignificant differences between studied groups. Seminal alpha-1,4-glucosidase activity levels demonstrated significant correlation with sperm count, sperm motility percentage and serum testosterone in oligoasthenozoospermia with varicocele group and demonstrated nonsignificant correlation in other groups. It is concluded that varicocele-induced hypoxia is the adverse effect that causes both oligoasthenozoospermia and decreased seminal alpha-1,4-glucosidase levels.     

精索静脉曲张患者精液质量及其不育患者手术前后精液变化的观察

目的观察精索静脉曲张（VC）患者的精液质量和精子形态学改变，以及VC不育患者手术前后精液的变化。方法121例VC患者精液按WHO标准常规分析并对精子形态学进行评价，23例健康男性精液检查结果作为对照。并对21例VC不育患者术前及术后的精液进行检测分析。结果121例VC患者的精子密度、（A＋B）级活动力精子（％）、活率、有效精子数、活动精子数、活力指数以及正常形态精子比例较对照组明显降低（P〈0．01）；畸形精子中小头、锥形头和无定形头精子数较对照组增多（P〈0．01）。21例VC不育患者手术后精子质量和精子形态学较术前明显改善。结论VC可以引起精液质量下降导致不育，精子形态学分析是判定VC患者精子受损的一个敏感指标，手术能有效地改善精液质量。     

Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation

Varicocele has been associated with reduced male reproductive potential. With the advances in biomolecular techniques, it has been possible to better understand the mechanisms involved in testicular damage provoked by varicocele. Current evidence suggests the central role of reactive oxygen species (ROS) and the resultant oxidative stress (OS) in the pathogenesis of varicocele-associated male subfertility although the mechanisms have not yet been fully described and it is likely to be multifactorial. Excessive ROS is associated with sperm DNA fragmentation, which may mediate the clinical manifestation of poor sperm function and fertilization outcome related to varicocele. Testing of ROS/OS and DNA fragmentation has the potential to provide additional diagnostic and prognostic information compared to conventional semen analysis and may guide therapeutic management strategies in individual patient.     

一氧化氮合酶对精索静脉曲张患者精子功能的影响

为探讨一氧化氮、一氧化氮合酶对精索静脉曲张（VC）患者精子功能的影响,本文测定15例VC不育患者外周静脉血清、精索静脉血清和精浆中一氧化氮（NO）及一氧化氮合酶（NOS）,并与12例有生育能力的对照组进行了对比研究。结果显示VC组精索静脉血清和精浆中NO含量及NOS活性,不仅高于其外周静脉血清（P＜0．01）,而且高于对照组（P＜0．01）,但两组外周静脉血清中两项指标无显著性差异（P＞0．05）。提示VC时可能是由于曲张精索静脉血和精浆中NOS的产生增加导致精子生成障碍或（和）精子活力下降,从而导致不育症。     

Hypoxia pathway has more impact than inflammation pathway on etiology of infertile men with varicocele

This study aimed to compare main molecular markers of hypoxia (HIF1‐α and P53) and inflammation (TLR‐2, TLR‐4 and TNF‐α) pathways between infertile men with varicocele and fertile individuals. Sperm parameters such as sperm concentration, motility and morphology were assessed according to World Health Organization (Laboratory manual for the examination and processing of human semen. Geneva, Switzerland, 2010) guideline in 20 infertile men with grade II or III varicocele, and 20 fertile men candidate of family balancing. In addition, sperm DNA fragmentation and molecular markers involved in hypoxia and inflammation pathways were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and real‐time PCR respectively. Mean of sperm parameters (concentration, motility and morphology) and DNA integrity were significantly lower in infertile men with varicocele compared to fertile individuals. Unlike markers involved in inflammation pathway, mean expression of markers of hypoxia pathway (HIF1‐α and P53) was significantly higher in infertile men with varicocele compared to fertile individuals (p < 0.05), and also a significant correlation was observed between expression of HIF1‐α and P53 (r = 0.461; p = 0.003). Overall, the result of this study suggests higher likelihood of involvement of hypoxia pathway, in comparison with inflammation pathway, in pathogenesis varicocele associated with male infertility.     

Comparative study of gene expression in patients with varicocele by microarray technology

The aim of the present study was to evaluate gene expression profile by microarray technology and validation by real-time PCR in paired samples of testicular biopsies (pre-surgery and post-surgery) in two patients with varicocele. The microarray analysis showed increased expression levels after surgery in 215 and 52 genes in patient 1 and 2, respectively, as well as decreased expression levels in 65 genes in patient 1 and 358 genes in patient 2. Real-time PCR confirmed the differential expression of the five selected genes: MT1M, PHLDA1 and INSL3 had decreased expression levels and CCIN and PRM2 increased expression levels compared with pre-surgery biopsies. In conclusion, both techniques showed decreased expression levels of genes involved in stress situations associated with varicocele and increased expression levels of genes involved in normal function of spermatogenesis.