Does Sperm DNA Fragmentation Index Continuously Decrease Over Time After Varicocelectomy in Varicocele-Induced Infertility? A Systematic Review and Meta-Analysis

Abstract

Varicocele (VC) is the most frequent and reversible cause of male infertility. One of the preferred management strategies to alleviate this problem is varicocelectomy. However, there are no researchers who have explored the relationship between better timing and postoperative sperm DNA fragmentation index (DFI) improvement in patients. We conducted this meta-analysis by enrolling published studies to find out the best waiting time after varicocelectomy to wait for better improvement of sperm DFI. A literature search was conducted using PubMed, Embase, Scopus, Web of Science, and Cochrane Library databases. The data from the pooled analysis were presented as mean difference (MD) along with a 95% confidence interval (CI). Heterogeneity was evaluated using I². Four studies were included after screening relevant literature. Statistical analysis revealed that after varicocelectomy, follow-up results within 3 months showed a significant improvement in sperm DFI compared with the preoperative period (MD: –3.66, 95% CI = [–5.17, –2.14], p < .00001), and follow-up results with 6 months showed a significant improvement in sperm DFI compared with the postoperative 3 months as well (MD: –1.51, 95% CI = [–2.73, –0.29], p = .02). Notably, no further improvement in sperm DFI was observed when the follow-up period reached 12 months (MD: –1.59, 95% CI = [–3.22, 0.05], p = .06). Six months after varicocelectomy may be the optimal time for sperm DFI compared with 12 months or even longer, which means it is also the preferable time for conception. However, more well-designed prospective studies are needed in the future to validate our conclusion.

Keywords

DNA fragmentation index varicocele varicocelectomy infertility

Introduction

Varicocele (VC) is a vascular condition marked by the abnormal twisting and dilation of the pampiniform plexus veins within the spermatic cord. It is commonly observed in young adults, predominantly on the left side, and can lead to male infertility, testicular hypofunction, pain, and discomfort. Approximately 15% of adult males in the general population suffer from VC. While some patients can conceive without intervention, primary infertile males have a 35% to 44% incidence of VC, with secondary infertile males rising to 45% to 81% (Jensen et al., 2017). The pathophysiology of varicocele-induced infertility is attributed to increased testicular temperature, hypoxia, oxidative stress, and hormonal changes. These factors impair spermatogenesis and cause sperm DNA breaks, known as sperm DNA fragmentation (SDF), reducing sperm quality (Paick & Choi, 2019).

Varicocelectomy is internationally accepted as an effective treatment for VC, particularly in patients with infertility (Will et al., 2011). Initially, patients with VC were more concerned about natural conception rates following surgery (Peng et al., 2015). Over time, the positive effects of varicocelectomy on semen quality have attracted increasing attention from clinicians and infertile couples. Although elevated SDF is mainly observed in men with abnormal semen parameters—count, motility, and morphology—it can coexist with normozoospermia (Jeremias et al., 2021). SDF, usually represented by the sperm DNA fragmentation index (DFI), can better evaluate sperm quality than conventional semen parameters.

It is essential for patients and clinicians to know the optimal time for semen quality improvement after VC surgery. Studies on different waiting times after varicocelectomy are well-designed and conducted. However, these studies have small sample sizes, and no meta-analyses compare improvements in sperm DFI across different follow-up times. Does a longer waiting time result in better semen quality, or does it negate the need for varicocelectomy? Therefore, we performed this meta-analysis to determine the optimal period for semen quality improvement after varicocelectomy and to provide statistically significant data to guide patients on the timing of conception.

Method

We conducted the present meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Protocols (Shamseer et al., 2015). Ethical approval and informed consent were not required due to the nature of the meta-analysis.

Search Strategy

An extensive search was conducted in PubMed, Embase, Scopus, Web of Science, and Cochrane Library from their inception dates to 30 June 2024, to identify studies on the effects of varicocelectomy on semen parameters over time after surgery. The keywords used for our search were (“varicocele” OR “varicocelectomy” OR “varicocele repair”) AND (“semen parameters” OR “semen quality” OR “semen” OR “sperm” OR “sperm parameters” OR “sperm quality”) AND (“DFI” OR “DNA fragmentation index”). The search strategy was adapted to the requirements of different electronic databases.

Inclusion and Exclusion Criteria

Studies included in our meta-analysis had to meet the following inclusion criteria: (a) conducted on human subjects aged 18 years or older; (b) compared sperm DFI in VC patients between pre-surgery and integer multiples of 3 months after surgery; and (c) provided sufficient data to pool results. Studies were excluded if they had: (a) insufficient or duplicated data; (b) patients younger than 18 years; (c) surgery not performed for infertility; (d) were reviews, meta-analyses, case reports, meeting abstracts, editorial comments, expert opinions, or animal studies; and (e) semen analysis unrelated to time after surgery. For studies published multiple times, those with the most informative and sufficient data were included in our meta-analysis.

Data Extraction and Quality Assessment

First, two writers independently conducted an initial screening based on titles and abstracts, excluding ineligible studies with precise reasons. Second, the eligibility of all potentially related research was reviewed in full text and finally confirmed during the data extraction process. Any disagreements were resolved by consensus or through consultation with a third reviewer. The quality of evidence (QoE) assessment of the articles was conducted using the Cambridge Quality Checklists (Murray et al., 2009). For each article, a screener made the initial assessment of QoE scores, a verifier checked the screener’s assessment, and a third writer affirmed the final agreed score. Funnel plots were used to assess publication bias. Sensitivity analyses were performed by excluding each study from the meta-analysis one at a time and assessing the pooled effect size to determine whether the meta-analysis outcomes were significantly influenced by any specific study.

Statistical Analysis

Statistical analyses were performed using Review Manager version 5.4. Results were expressed as mean differences (MDs) with 95% confidence interval (CI) because the extracted data were continuous variables. Heterogeneity was analyzed using Chi-square and I² tests. Heterogeneity was defined as p < .10 or I² > 50%. A fixed-effect model was used if p≥ .10 or I²≤ 50%. Otherwise, a random-effects model was used. p < .05 was considered statistically significant.

Results

We initially identified 277 records related to our topic. First, we excluded 209 duplicate records by comparing titles, authors, and publication years. Subsequently, 33 records were excluded due to irrelevance based on their titles and abstracts. This left 22 records for full-text eligibility assessment. After reviewing the full texts, only four studies were included in our meta-analysis. The detailed study selection process is illustrated in Figure 1. The included studies were published between 2015 and 2024 and involved a total of 505 VC patients. Table 1 provides detailed information on the first author’s name, year of publication, recruitment location, study design, sample size, population age, follow-up duration, and SDF assay. K. Ni’s study was categorized into three groups based on the degree of VC. The QoE assessment is presented in Table 2.

Figure 1.

Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Flow Chart for Inclusion/Exclusion of Studies

Table 1.

Characteristics of Studies Included in the Meta-Analysis

First author (year)	Country	Study design	Sample size	Age	Follow-up month after varicocelectomy	SDF assay
Abdelaziz et al. (2015)	Egypt	Prospective study	54	27.7±7.2	6 and 12 months	TUNEL
Ni, Steger, et al. (2016)	China	Prospective study	19	30.53±4.01	3 and 6 months	SCSA
Ni, Steger, et al. (2016)	China	Prospective study	18	30.22±3.56	3 and 6 months	SCSA
Ni, Steger, et al. (2016)	China	Prospective study	14	30.36±3.41	3 and 6 months	SCSA
Afsin et al. (2018)	Turkey	Prospective study	40	15-30	3, 6, and 12 months	TUNEL
Rochdi et al. (2024)	Morocco	Prospective study	360	38.5±1.6	6, 12, and 18 months	SCD

Note. SDF, sperm DNA fragmentation; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling; SCSA, sperm chromatin structure assay; SCD, sperm chromatin dispersion.

Table 2.

Quality of Evidence Assessment (Results of the Cambridge Quality Checklist)

First author	Year	Cambridge quality checklist			Total quality score (3–15)
First author	Year	Checklist for correlates (1–5)	Checklist for risk factors (1–3)	Checklist for causal risk factor (1–7)	Total quality score (3–15)
Abdelaziz et al. (2015)	2015	2	3	4	9
Ni, Steger, et al. (2016)	2016	3	3	4	10
Afsin et al. (2018)	2018	3	3	3	9
Rochdi et al. (2024)	2024	3	3	4	10

Sperm DFI in VC Patients Before and 3 Months After Surgery

Two studies compared sperm DFI between pre-surgery and 3 months after varicocelectomy. The fixed-effect model was used to pool the data due to low heterogeneity (I² = 0%, p = .60). After varicocelectomy, sperm DFI decreased by 3.66% from pre-surgery to 3 months post-surgery (MD: –3.66, 95% CI = [–5.17, –2.14]) (Figure 2).

Figure 2.

Forest Plot of Sperm DFI Before and 3 Months After Varicocelectomy

Sperm DFI in VC Patients at 3 and 6 Months Post-Surgery

Two studies compared sperm DFI at 3 and 6 months after varicocelectomy. The fixed-effect model was used to pool the data due to low heterogeneity (I² = 0%, p = .93). After varicocelectomy, sperm DFI decreased by 1.51% from 3 to 6 months post-surgery (MD: –1.51, 95% CI = [–2.73, –0.29]) (Figure 3).

Figure 3.

Forest Plot of Sperm DFI Between 3 and 6 Months After Varicocelectomy

Sperm DFI in VC Patients at 6 and 12 Months Post-Surgery

Three of the four studies evaluated sperm DFI at 6 and 12 months after surgery. The pooled results showed no significant improvement in sperm DFI, with an MD of −1.59 (95% CI = [−3.22, 0.55]). Heterogeneity was significant (I² = 73%, p = .03) (Figure 4).

Figure 4.

Forest Plot of Sperm DFI Between 6 and 12 Months After Varicocelectomy

Sensitivity Analysis and Publication Bias

Sensitivity analysis was performed on sperm DFI at 6 and 12 months after surgery. This involved omitting one study at a time to assess the stability of the pooled results and evaluate the influence of each study. In addition, no significant publication bias was detected using the funnel plots, which were symmetrical (Figure 5).

Figure 5.

Funnel Plots for Publication Bias. (A) Funnel Plot for Publication Bias of Sperm DFI Before and 3 Months After Varicocelectomy. (B) Funnel Plot for Publication Bias of Sperm DFI Between 3 and 6 Months After Varicocelectomy. (C) Funnel Plot for Publication Bias of Sperm DFI Between 6 and 12 Months After Varicocelectomy

Discussion

Currently, the prevailing view links clinical VC closely to infertility, based on epidemiological findings showing a high prevalence of VC in infertile populations (Kupis et al., 2015). Studies conducted over the past century have concluded that VC is associated with poorer semen quality, impaired sperm function, and altered testicular histology. No single theory can fully explain the relationship between infertility and VC. VC can cause male infertility through several mechanisms. Impairment of venous drainage can increase oxidative stress, elevate scrotal temperature, induce testicular hypoxia, and cause abnormal metabolite reflux (Su et al., 2021). VC can raise scrotal temperature by up to 2.6°C (Garolla et al., 2015). Elevated scrotal temperature adversely affects spermatogenesis, leading to increased SDF and reduced sperm fertilization capability (Agarwal et al., 2009).

Although semen analysis is a crucial tool for evaluating male infertility, it remains sub-optimal for assessing and managing male infertility, raising concerns about its effectiveness in distinguishing between fertile and infertile men (Barbărosie et al., 2021; Snow-Lisy & Sabanegh, 2013). SDF has emerged as a more useful tool than conventional semen parameters for providing a functional assessment of male fertility (Lewis et al., 2008; Santi et al., 2018). SDF is a marker of chromatin damage in spermatozoa. Chromatin damage may include a range of DNA defects, such as single or double-strand breaks, base deletions or modifications, inter- or intra-strand DNA cross-linkages, or protamine deficiency (Esteves et al., 2014). Assessing DNA integrity is important due to the role of chromatin compaction and structural stability in producing fully functional sperm cells (García-Peiró et al., 2011; Ni, Spiess, et al., 2016; Rogenhofer et al., 2013; Spanò et al., 2000). The recent sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen emphasizes the importance of SDF assessment in managing male infertility (World Health Organization [WHO], 2021).

Varicocelectomy remains the gold standard for treating VC. Like the debate surrounding infertility and VC, the impact of varicocelectomy on semen quality remains contentious. Nonetheless, most published literature, including original research (Kibar et al., 2002) and meta-analysis (Agarwal et al., 2007; Qiu et al., 2021; Schauer et al., 2012), acknowledges the positive impact of varicocelectomy on semen quality. Baazeem et al. (2011) conducted a comprehensive meta-analysis of 22 studies, confirming the positive impact of varicocelectomy on semen quality. They concluded that varicocelectomy improves sperm concentration (by 12.32 × 10⁶/ml, p < .0001), total motility (by 10.86%, p < .0001), and progressive motility (by 9.69%, p = .003). Several studies have also investigated whether varicocelectomy improves pregnancy outcomes, which is a crucial question (J. H. Evers et al., 2008, 2009; J. L. Evers et al., 2001). About 50% of men with clinically palpable VC have elevated DFI. Varicocelectomy is associated with a reduction in DFI, particularly in higher-grade VC (Smit et al., 2010). A reduction in DFI following varicocelectomy may lead to higher clinical pregnancy rates (Ni et al., 2014). Overall, the evidence supports the positive impact of varicocelectomy on pregnancy outcomes.

However, fewer studies have investigated the optimal waiting period after VC repair for improved sperm DFI and conventional semen parameters. Al Bakri et al. (2012) conducted the first prospective study comparing semen parameters at different time points after varicocelectomy. They found that semen quality improved after varicocelectomy at 3, 6, and 9 months. However, no additional improvements in semen parameters were observed beyond 6 months. Another prospective study compared semen parameters, including volume, sperm concentration, morphology, and progressive motile sperm count, between 3 and 6 months after surgery. No significant differences were found in these parameters between the two time points after surgery (Ghaed et al., 2020). Regarding sperm DFI, Hamidi Madani et al. (2024) compared DFI levels before surgery and at 4 and 12 months post-surgery. The findings showed a consistent decline in DFI post-surgery, with a less pronounced decrease from 4 to 12 months compared with the decline from pre-surgery to 4 months post-surgery. Our meta-analysis, which included a larger VC population, showed that sperm DFI before surgery was higher compared with 3, 6, and 12 months, and longer post-surgery. This supports the positive impact of varicocelectomy on sperm DFI in VC patients. However, no further improvements in sperm DFI were observed between 6 and 12 months post-surgery.

Several studies have shown an association between elevated SDF and failed natural pregnancy (Evenson et al., 1999; Siddhartha et al., 2019; Simon et al., 2017; Spanò et al., 2000). A systematic review and meta-analysis of men from couples with recurrent idiopathic miscarriage found higher SDF levels compared with fertile controls (MD: 11.98%, 95% CI = [6.64–17.32], p < .001) (Tan et al., 2019). With advances in assisted reproductive technology (ART), methods such as in vitro fertilization (IVF), intrauterine insemination (IUI), and intracytoplasmic sperm injection (ICSI) are now available for men with clinical VC and infertility (Vahidi et al., 2018). These methods have been shown to improve the chances of conception for infertile couples (Tournaye, 2012). For older couples, ART may be more effective than varicocelectomy. Sperm DFI is a valuable parameter for assessing fertility. DFI reflects sperm DNA integrity and damage (Yang et al., 2019). Studies suggest that DNA fragmentation levels can predict ART outcomes. The clinical threshold for DFI is currently set at 25%. Men with a DFI greater than 25% are at a higher statistical risk of reproductive problems (Birowo et al., 2020). In ART, SDF is recognized as a factor associated with failed IUI (Bungum et al., 2007; Duran et al., 2002; Rilcheva et al., 2016). Couples opting for ART may miss out on the potential benefits of varicocelectomy. Therefore, it is crucial for couples to determine the optimal timing to achieve the best improvement in sperm DFI.

Our results identify the optimal waiting time after VC surgery for both patients and clinicians. For couples experiencing VC-related infertility, varicocelectomy should be offered to men. Couples should consider ART if no significant improvements in sperm DFI are observed 6 months after surgery. To the best of our knowledge, this is the first meta-analysis to examine the effect of timing on sperm DFI following varicocelectomy. Previous meta-analyses primarily focused on improvements in conventional semen parameters and sperm DFI. Less attention was given to the timing after varicocelectomy. Our results emphasize the importance for couples and physicians to consider the optimal timing for sperm DFI improvement, which is a more reliable indicator of sperm quality, and to manage infertility more effectively after varicocelectomy. However, there are several limitations to consider when interpreting our results. First, the small number of studies included in our meta-analysis limited the statistical power. Second, given that the normal spermatogenic cycle is approximately 3 months, and due to limited data, no further analyses were conducted to assess improvements between 6 and 12 months after surgery, such as at 9 months or longer time.

Conclusion

Our meta-analysis found that the decrease in sperm DFI following varicocelectomy is sustained at 3, 6, and 12 months. However, sperm DFI does not show further improvement beyond 12 months post-varicocelectomy compared with the results observed at 6 months. Consequently, 6 months after varicocelectomy may be the optimal time for conception in managing VC-related infertility. This also highlights the importance of timing for physicians managing VC patients after varicocelectomy. Notably, further well-designed prospective studies are needed to validate our conclusions.

Footnotes

Acknowledgements

All authors have no acknowledgments to disclose.

Author Contributions

TJ conceived of the study and drafted the manuscript. LHW and LZ participated in the procedure. LHW carried out data curation. LZ performed the statistical analysis. TJ and HJ participated in its design and coordination. All authors read and approved the final manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science and Technology Innovation Joint Fund Project of Fujian Province (No. 2021Y9117).

ORCID iD

Lihong Wang

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