Relationship between varicocele and sperm DNA damage and the effect of varicocele repair: a meta-analysis

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1 Reproductive BioMedicine Online (2012) 25, ARTICLE Relationship between varicocele and sperm DNA damage and the effect of varicocele repair: a meta-analysis Ying-Jun Wang, Rong-Qiu Zhang, Yan-Jun Lin, Rong-Gui Zhang, Wei-Li Zhang * Department of Urologic Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China * Corresponding author. address: 66zwl@sina.com (W-L Zhang). Dr Wang is currently reading for his PhD at Chongqing Medical University, China. His research topic is sperm function and sperm DNA damage. Abstract Varicocele, a cause of male infertility, occurs in nearly 40% of infertile males. It has been postulated that varicoceles may cause sperm DNA damage. Sperm DNA integrity has been recognized as one of the important determinants of normal fertilization and embryo growth in natural and assisted conception. Eighty-three human studies were identified after an extensive literature search involving the role of varicoceles in sperm DNA damage. Of the 83 studies, 12 were selected that measured similar types of reactive sperm DNA damage. Seven studies determined the damage of sperm DNA in varicocele-associated patients and six studies evaluated the efficacy of varicocelectomy. One study was a duplicate because both outcomes were included. Data were analysed using RevMan software. The overall estimate showed that patients with varicoceles have significantly higher sperm DNA damage than controls, with a mean difference of 9.84% (95% CI 9.19 to 10.49; P < ). A varicocelectomy can improve sperm DNA integrity, with a mean difference of 3.37% (95% CI 4.09 to 2.65; P < ). In conclusion, there is increased sperm DNA damage in patients with varicoceles and varicocelectomy may be a possible treatment; however, more studies with appropriate controls are needed to confirm this finding. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: meta-analysis, sperm DNA damage, varicocele, varicocelectomy, DNA fragmentation index, male infertility Introduction Varicoceles are defined as a vascular abnormality in the veins within the pampiniform plexus (Gat et al., 2004). Approximately 15% of adult males are believed to have clinical or subclinical varicoceles, although the prevalence in infertile males is as high as 40% (Schoor et al., 2001) /$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

2 308 Y-J Wang et al. Few topics in urology have been as controversial as the effect of varicocelectomy on male factor infertility (Baazeem et al., 2011; Cayan et al., 2000; Krause et al., 2002; Onozawa et al., 2002). Despite considerable research, the exact pathophysiological mechanism by which varicoceles can induce male factor infertility is not known with certainty, although several potential causes have been postulated (Saleh et al., 2003; Baccetti et al., 2006; Hendin et al., 1999). The Best Policies Practice Groups of the American Urological Association and the American Society for Reproductive Medicine (Jarow et al., 2002; Sharlip et al., 2002) have jointly stated that correction of varicoceles is indicated for infertile men with palpable lesions and one or more abnormal semen parameters. In contrast, the 2005 clinical guidelines from National Collaborating Centre for Women s and Children s Health (2004) stated Men should not be offered surgery for varicoceles as a form of fertility treatment because it does not improve pregnancy rates. There are also many conflicting opinions reported. Many studies report improvement after surgery (Cayan et al., 2000; Madgar et al., 2002; Onozawa et al., 2002; Segenreich et al., 1997), but other studies show no benefit (Breznik et al., 1993; Kamischke and Nieschlag, 2001; Krause et al., 2002). Clearly, there are differing opinions whether or not a varicocelectomy improves fertility. Sperm DNA integrity has been recognized as one of the vital determinants of normal fertilization and embryo growth in natural and assisted conception (Agarwal and Said, 2003; De Jonge, 2002). There is now clear evidence that infertile men possess substantially more sperm DNA damage than do fertile men (Evenson et al., 1980; Irvine et al., 2000; Shen and Ong, 2000; Spanò et al., 2000; Zini et al., 2001, 2002). Many observations have raised concerns regarding the safety of using DNA-damaged spermatozoa for assisted reproduction treatment and have asserted a negative correlation between DNA fragmentation index (DFI) and fertilization rate (Host et al., 2000; Huang et al., 2005; Lopes et al., 1998a,b; Sun et al., 1997). Over the past decade, there has been a growing body of research investigating the role of sperm nuclear DNA integrity as a factor in male infertility. Sperm DNA damage (percentage DFI >30% by sperm chromatin structure assay; SCSA) has been associated with reduced potential for natural and intrauterine insemination-assisted pregnancy (Bungum et al., 2007; Evenson et al., 1999; Spanò et al., 2000). Sperm DNA damage has also been associated with a modest reduction in IVF pregnancy rate and, more importantly, with a significant increase in the risk of pregnancy loss after IVF and intracytoplasmic sperm injection (ICSI) (Collins et al., 2008; Zini et al., 2008a,b). Although the influence of sperm DNA damage on late reproductive (fetal and post-natal) outcomes in humans remain unknown, sperm DNA damage has been shown experimentally to impact negatively on intracytoplasmic sperm injection embryo development, pregnancy rates and offspring health and genomic stability (Adiga et al., 2010; Fernández-Gonzalez et al., 2008). Recently, several excellent reviews have given a descriptive summary of the literature regarding the relationship between sperm DNA damage and varicocele (García-Peiró et al., 2011; Bertolla et al., 2006; Blumer et al., 2008, 2011; Chen et al., 2004; Ishikawa et al., 2007; Saleh et al., 2003; Smith et al., 2006; Talebi et al., 2008; Wu et al., 2009). Almost all of these reviews conclude that varicoceles may increase sperm DNA damage. There are also many opinions in the literature that surgical varicocelectomy significantly reduces sperm DNA damage in infertile men with palpable varicoceles (Acar et al., 2008; Azadi et al., 2010; Chen et al., 2008; Dada et al., 2010; Hurtado de Catalfo et al., 2007; La Vignera et al., 2011; Lacerda et al., 2011; Moskovtsev et al., 2009, 2010; Smit et al., 2010; Werthman et al., 2008; Zini et al., 2005, 2009), but disagreements in the efficacy still exist because a variety of studies have used different methods. In addition, several factors that might be responsible for these controversies remain to be clarified. First, the types and mechanisms of sperm DNA damage vary among different studies. Second, multiple techniques have been used to measure DNA defects in human spermatozoa (Agarwal and Allamaneni, 2005; Perreault et al., 2003). Finally, the sample sizes in some original studies are too small to generate a significant result. Therefore, this study conducted a meta-analysis on the published reviews. The objective was to evaluate the alterations in the concentrations of sperm DNA damage in varicocele patients and to determine the efficacy of varicocele repair by combining several publications from the literature. Materials and methods The Medline database from 1963 to August 2011 was searched using the following search terms: human sperm DNA, human sperm DNA damage and human sperm chromatin in combination with varicocele, varicocelectomy, microsurgery and high ligation. Additional studies were identified from the study reference lists. Only full articles published in English were searched. Two investigators (WYJ and ZRQ) independently reviewed the papers for eligibility and discrepancies were resolved by group discussion. All the published studies analysing the relationship between varicocele and sperm DNA damage and the effect of varicocele repair were considered for inclusion in the meta-analysis if they satisfied the following criteria: (i) clinical study in human; and (ii) sperm DNA damage detected by SCSA, TdT (terminal deoxynucleotidyl transferase)-mediated dudp nick-end labelling (TUNEL) and Comet analysis. Studies were excluded if they had no original data available for retrieval and duplicate publications were also excluded. The studies included in the final analysis utilized one of three tests of sperm DNA damage: SCSA, TUNEL and the neutral Comet assay. The SCSA is an objective, flow cytometry-based assay that measures the susceptibility of DNA to denaturation (under acid conditions) and provides a quantitative but indirect assessment of sperm DNA damage. The TUNEL assay is a semi-quantitative (microscopy-based) assay that provides a direct assessment of sperm DNA fragmentation by labelling DNA breaks (Zini et al., 2008a). The neutral Comet assay is technically simpler to use, is more cost efficient and has a comparably higher sensitivity for detecting DNA damage. This study included only studies that employed SCSA, TUNEL or Comet analysis to detect sperm DNA damage, because these methods are most widely used and thought to have a higher value than others (Li et al.,

3 Varicocele, sperm DNA damage and varicocele repair ). Besides, other methods do not have enough original data available or not frequently been reported. This study was divided into two parts. The first part was to evaluate the alterations in the concentrations of sperm DNA damage in patients with varicoceles. The diagnosis of varicoceles was assessed in these patients clinically by genital examination and confirmed by scrotal colour Doppler ultrasound. The control group was a group of healthy fertile volunteers. The second part was to determine the efficacy of varicocele repair. Studies of infertile men with a diagnosis of unilateral or bilateral palpable varicoceles and at least one abnormal semen parameter were included. Surgical varicocelectomies (high ligation, inguinal or micro-surgery) were reviewed. The effects of embolization or selective catheterization of the internal spermatic vein on semen parameters were not considered. The patients served as their own controls and the effect change was compared over time. The studies did not include a control group in the sense of a no treatment control, and the nature of the comparison groups varied across the studies. Statistical analysis The raw data obtained from individual studies were examined for completeness of observations and to avoid duplication of data. These data were entered into the RevMan software used by the Cochrane Collaboration (RevMan, version 4.2.8; Cochrane IMS). A random-effects model was applied for calculating the weighted mean difference. This measure was chosen to enable clinically relevant interpretation of the results. The data were examined for heterogeneity using the chi-squared Total articles retrieved by MEDLINE search (n = 83) Articles excluded after screening abstracts and/or titles (n = 53) Full articles reviewed (n = 30) Articles excluded with reasons: Sperm DNA not measured by SCSA, TUNEL or Comet assay (n = 9) Combination treatment (n = 1) Duplicate publication (n = 3) Have other comparison groups (n = 1) No original data available (n = 4) Included articles (n = 12) SCSA (n = 5) TUNEL (n = 2) One study included both SCSA and TUNEL Cometassay (n = 6) Figure 1 Article selection process for meta-analysis. SCSA = sperm chromatin structure assay; TUNEL = TdT (terminal deoxynucleotidyl transferase)-mediated dudp nick-end labelling.

4 310 Y-J Wang et al. statistic. A significance level of 0.05 was chosen for all statistical tests. Results Eighty-three potentially relevant studies were identified from the MEDLINE database. Among the 83 studies, 12 met the predefined selection criteria, of which two used the TUNEL assay (La Vignera et al., 2011, Smith et al., 2006), six used the SCSA (Moskovtsev et al., 2009; Smit et al., 2010; Smith et al., 2006; Talebi et al., 2008; Zini et al., 2005, 2009) and five used the Comet assay (Bertolla et al., 2006; Blumer et al., 2008, 2011; Lacerda et al., 2011; Wu et al., 2009) to evaluate sperm DNA damage. A typical flow schematic for the selection process is shown in Figure 1. Seven studies fulfilled the inclusion criteria for conducting the meta-analysis to evaluate the alterations of sperm DNA damage in patients with varicoeles and the main characteristics of the included studies are presented in Table 1. Six studies were included to determine the efficacy of varicocele repair, and the main characteristics of the included studies are presented in Table 2. One study was a duplicate because it included both outcomes. Six studies were excluded because they did not satisfy the criteria. Two studies sperm DNA were not measured by SCSA, TUNEL or Comet assay (Azadi et al., 2010; Chen et al., 2008). Four studies did not have the original data available (Dada et al., 2010; Nasr-Esfahani et al., 2009; Sakamoto et al., 2008; Werthman et al., 2008). A total of 240 patients and 176 normal healthy donors (controls) were included in the meta-analysis to evaluate sperm DNA damage in patients with varicoceles. The overall estimate indicated that patients with varicoceles have significantly higher sperm DNA damage than controls, with a mean difference of 9.84% (95% CI 9.19 to 10.49; P < ; Figure 2). A total of 177 patients with varicoceles were treated by surgery. The outcomes were collected to determine the efficacy of varicocele repair. The result showed varicocele repair could improve sperm DNA integrity significantly, with a mean difference of 3.37% (95% CI 4.09 to 2.65; P < ; Figure 3). Discussion In this systematic review of 12 studies, seven were identified in which varicocele-associated patients had damage of sperm DNA integrity and six involved the efficacy of varicocele repair. One article was a duplicate because it included both outcomes. In the seven studies, a total of 240 patients and 176 normal healthy donors (controls) were included. The overall estimate indicated that patients with varicoceles have significantly higher sperm DNA damage than controls, with a mean difference of 9.84% (95% CI 9.19 to 10.49; P < ). In the six studies, 177 patients with varicoceles were treated by surgery. The outcomes were collected to determine the efficacy of varicocele repair. The result showed that varicocele repair could improve sperm DNA integrity significantly, with a mean difference of 3.37% (95% CI 4.09 to 2.65; P < ). One strength of a systematic review is the improved precision of the summary odds ratio estimates compared with the individual studies. The combined estimate in the seven studies was significantly different from unity, Table 1 Study Characteristics of included studies addressing effects of varicocele on sperm DNA damage outcomes. Design Subjects Assay Varicocele patients (n) Controls (n) Smith et al. (2006) Unspecified Randomly selected SCSA/TUNEL La Vignera et al. (2011) Unspecified Randomly selected TUNEL Talebi et al. (2008) Prospective study Randomly selected SCSA Blumer et al. (2011) Unspecified Randomly selected Comet assay Wu et al. (2009) Retrospective study Randomly selected Comet assay 15 5 Blumer et al. (2008) Prospective study Randomly selected Comet assay Bertolla et al. (2006) Controlled prospective study Randomly selected Comet assay SCSA = sperm chromatin structure assay; TUNEL = TdT (terminal deoxynucleotidyl transferase)-mediated dudp nick-end labelling. Table 2 Study profiles Characteristics of included studies addressing effects of varicocelectomy on sperm DNA damage outcomes. Study design Subjects Assay Varicocele patients (n) Type of surgery Zini et al. (2009) Prospective study Consecutive selection SCSA 25 Microsurgical Smit et al. (2010) Unspecified Consecutive selection SCSA 49 Unspecified Zini et al. (2005) Retrospective study Consecutive selection SCSA 37 Microsurgical Lacerda et al. (2011) Prospective study Consecutive selection Comet 27 Microsurgical Moskovtsev et al. (2009) Prospective study Consecutive selection SCSA 9 Unspecified La Vignera et al. (2011) Unspecified Consecutive selection TUNEL 30 Microsurgical SCSA = sperm chromatin structure assay; TUNEL = TdT (terminal deoxynucleotidyl transferase)-mediated dudp nick-end labelling.

5 Varicocele, sperm DNA damage and varicocele repair 311 Study or Subgroup Bertolla et al 2006 Blumer et al 2008 Blumer et al 2011 La Vignera et al 2011 Smith et al 2006 Smith et al. (2006) Talebi et al 2008 Wu et al 2009 varicocele group Control group Mean Difference Mean Difference Mean SD Total Mean SD Total Weight 1.8% 6.8% 7.7% 32.7% 7.2% 30.0% 0.7% 13.1% IV, Fixed, 95% CI IV, Fixed, 95% CI [9.07, 18.65] 3.40 [0.91, 5.89] 0.60 [-1.72, 2.92] 3.00 [1.87, 4.13] [26.00, 30.80] [17.02, 19.38] [35.70, 50.80] 3.86 [2.07, 5.65] Total (95% CI) % 9.84 [9.19, 10.49] Heterogeneity: Chi² = , df = 7 (P < ); I² = 99% Test for overall effect: Z = (P < ) Favours experimental Favours control Figure 2 Forrest plot of meta-analysis on the concentrations of sperm DNA damage in varicocele patients compared with donors. Study or Subgroup After treatment Before treatment Mean Difference Mean Difference Mean SD Total Mean SD Total Weight IV, Fixed, 95% CI IV, Fixed, 95% CI Lacerda et al % [-5.73, -2.37] Moskovtsevet al % [-26.71, -5.89] La Vignera et al % [-3.98, -1.82] Smit et al % [-10.51, 0.51] Zini et al % [-4.38, -1.82] Zini et al % [-11.91, -2.09] Total (95% CI) % [-4.09, -2.65] Heterogeneity: Chi² = 9.88, df = 5 (P = 0.08); I² = 49% Test for overall effect: Z = 9.12 (P < ) Favours experimental Favours control Figure 3 Forrest plot of meta-analysis on the effect of varicocele repair on DNA damage in patients with varicoceles compared with pretreatment. indicating that varicoceles have an effect on sperm DNA damage. Although the number per study was not large, the odds ratios of the individual studies were all greater than unity. In contrast, a weakness of this meta-analysis was the highly variable study characteristics, specifically data collection (prospective or retrospective), definition of a varicocele (unilateral or bilateral, grade I, II and III varicoceles), population characteristics (unselected, age and semen parameter), surgical varicocelectomy (high ligation, inguinal or microsurgery), sperm DNA test type and sperm DNA test cut off. The finding of an association between sperm DNA damage and varicoceles was consistent with the results reported in studies using methods other than SCSA, TUNEL or Comet assay for the measurement of DNA damage. García-Peiró et al. (2011) reported a significant increase in DNA stainability and in DNA-degraded spermatozoa, associated with the presence of a varicocele. Enciso et al. (2006) concluded that individuals with varicoceles exhibit a higher yield of sperm cells with the greatest nuclear DNA damage in a population with fragmented DNA by the sperm chromatin dispersion test. Ishikawa et al. (2007) evaluated the extent of oxidative DNA damage in testes of men with varicoceles using an immunohistochemical assessment of 8-hydroxydeoxyguanosine. Oxidative DNA damage caused by oxidative stress (OS) was related to impaired spermatogenesis in patients with varicoceles. The finding of high seminal OS in patients with varicoceles may indicate that OS plays a role in the pathogenesis of sperm DNA damage in these patients. Varicoceles are strongly associated with OS. This association might be attributed to an increase in nitric oxide (NO) and the release of NO synthase and xanthine oxidase in the dilated spermatic veins of men affected with varicoceles (Mitropoulos et al., 1996; Romeo et al., 2003). Another potential explanation for DNA damage in patients with varicoceles might be related to the elevated intratesticular temperature that is associated with varicoceles, thus affecting testicular function (Turner, 2001). This hypothesis might be explained by the direct thermal damage to nuclear DNA at the level of seminiferous tubules (Fujisawa et al., 1988). However, the exact mechanism(s) of increased sperm DNA damage in patients with varicoceles warrants further research. In a recent study, varicocelectomies were associated with a significant increase in pregnancy and live-birth rates for couples who underwent intrauterine insemination, although standard semen parameters were not improved in all patients (Daitch et al., 2001). Therefore, the improvement in pregnancy rates after varicocelectomy may be due to a factor not tested during routine semen analysis, such as sperm DNA damage (Saleh et al., 2003). The current study showed that varicocele repair could significantly improve sperm DNA integrity (mean difference 3.37%, 95% CI 4.09 to 2.65; P < ). In other studies, there was no

6 312 Y-J Wang et al. improvement in semen parameters after varicocelectomy among men with Y-chromosome microdeletions or abnormal karyotypes (Cayan et al., 2001) and there were reduced pregnancy outcomes after varicocelectomies among men with increased testis tissue cadmium and microdeletions in the sequence of the L-type voltage-dependent calcium channel (Marmar and Benoff, 2005). On the basis of the data from the extant literature, this meta-analysis concludes that there is an increase in sperm DNA damage in patients diagnosed with varicoceles. A surgical varicocelectomy may be a possible treatment for decreasing the damage. In the future, studies should be conducted with appropriate controls to determine the usefulness of varicocelectomies. A substantial weakness of the study is the second part, where patients served as their own controls in the evaluation of the effect of varicocelectomy. It is well-recognized in biological studies that patients selected for an abnormality in a specific highly variable biological factor (in this case, sperm DNA fragmentation) will have regression towards the mean improvement in that variable after an intervention or with observation alone. That is why a control group is critical for evaluation of the effect of such an intervention. In the future, studies should be conducted with appropriate controls to determine the usefulness of varicocelectomies. Large-scale randomized controlled trials are still needed in support of the results. Studies should determine the aetiological role of sperm DNA damage in varicocele. Acknowledgements The authors thank the Department of Urology of The Second Affiliated Hospital, Chongqing Medical University for their support of this study. 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