Redox Imbalance in Male Infertility: From Empirical Antioxidant Therapy to Precision Medicine and Epigenetic Insights

ABSTRACT

Background

Oxidative stress (OS) is a critical factor in male infertility, where excessive reactive oxygen species (ROS) impair sperm quality and DNA integrity. Although physiological ROS levels support sperm maturation, imbalance can trigger oxidative or reductive stress (RS), both harmful to fertility. Thus, empirical antioxidant therapy without confirmed OS may be unwarranted and risk overtreatment.

Methods

We searched PubMed for original and review articles on OS, RS, antioxidants, redox imbalance, and male infertility, emphasizing mechanisms, diagnostics, therapeutic outcomes, and epigenetic implications.

Main Findings

Although antioxidants may improve semen parameters, their effect on pregnancy and live birth rates remains inconclusive. Conventional diagnostics often overlook redox imbalance, leading to empirical antioxidant use. A precision redox approach—guided by oxidative profiling to assess redox balance—offers a more effective strategy. Although multiple tools have been proposed to assess redox status, identifying a clinically robust and reliable assay remains challenging. Emerging evidence shows that redox imbalance alters nuclear and mitochondrial epigenetics, potentially affecting embryo development and transgenerational health.

Conclusion

Managing male infertility should prioritize restoring redox balance rather than eliminating ROS. Future studies should prioritize standardized diagnostics, individualized antioxidant therapy, and a deeper understanding of how redox imbalance affects sperm epigenetics and offspring outcomes.

1. Introduction

Infertility is defined as the failure to achieve pregnancy after 1 year of unprotected intercourse, affecting 7%–10% of couples worldwide. Male factors contribute to nearly half of these cases, with causes ranging from genetic defects, varicocele, infections, hormonal disturbances, and lifestyle influences to idiopathic origins [1, 2, 3]. In many cases, male infertility remains idiopathic, with oxidative imbalance now recognized as a key contributing factor [1, 2, 3, 4]. Reactive oxygen species (ROS)—byproducts of cellular metabolism—can disrupt sperm structure and function when produced in excess, impairing semen quality and fertility potential [1, 2, 3, 4, 5]. Oxidative stress (OS), first conceptualized by Sies in 1985, is defined as a disturbance in the balance between pro‐oxidants and antioxidants in favor of the former, leading to potential cellular damage [6]. Agarwal et al. introduced the term “male oxidative stress infertility” to describe men with elevated ROS levels and poor semen parameters, previously lumped together as idiopathic infertility [7]. Contributing factors such as advanced paternal age, pollutants, poor diet, heat exposure, and obesity elevate ROS levels and adversely affect sperm through lipid peroxidation (LPO), sperm DNA fragmentation (SDF), and apoptosis [8].

To treat such conditions, numerous exogenous antioxidants have been employed; however, the clinical efficacy remains poorly substantiated by high‐quality evidence [1] [2, 3] Notably, recent findings warn against indiscriminate antioxidant use without evaluating OS, as this may induce reductive stress (RS)—a state equally harmful to sperm function [3]. Thus, infertility assessment should move beyond standard semen analysis to include redox evaluation. Maintaining redox homeostasis requires a tightly regulated balance between ROS and antioxidants, and identifying precise biomarkers via omics approaches is critical for improved outcomes [4, 5, 6, 7]. Although several biomarkers—such as gene mutations, epigenetic patterns, and mRNA profiles—have been linked to poor sperm quality or embryo development, the underlying mechanisms of male infertility remain unclear, underscoring the need for molecular‐level research [9, 10].

This review offers a comprehensive analysis of the dual role of ROS in male reproductive health, emphasizing redox balance and critically evaluating both the therapeutic benefits and risks of antioxidant therapy. We highlight the need for personalized, redox‐guided approaches in clinical practice. Additionally, we explore emerging links between redox imbalance and epigenetic alterations—factors that may impair fertility even in men with normal semen profiles, representing a significant gap in current knowledge. By examining these mechanisms, we aim to identify biomarkers and therapeutic targets for future applications.

2. Reactive Oxygen Species in the Male Reproductive Tract

2.1. Sources and Physiological Roles of ROS in the Male Reproductive Tract

ROS, highly reactive byproducts of cellular respiration, are inevitably generated in all aerobic cells. Mitochondria serve as the primary source of ROS during oxidative phosphorylation, with additional contributions from cytosolic enzymatic reactions [3, 11, 12]. Since MacLeod's discovery of ROS in human sperm in 1943 [13], their dual role has been increasingly acknowledged. ROS in the male reproductive system arise from both systemic and local sources. Systemic OS is primarily driven by external factors such as smoking, alcohol, pollutants, and heavy metals, whereas local OS within the reproductive tract is more directly linked to impaired fertility [4, 5]. Although these contributors are associated with intense OS, their specific effects on semen quality have yet to be clearly determined [5]. Elevated ROS levels further impair spermatogenesis by inducing sperm dysfunction and disrupting the blood–testis barrier [1, 2, 3, 4, 5].

Regarding endogenous sources, residual cytoplasm in immature sperm contains ROS‐generating enzymes such as glucose‐6‐phosphate dehydrogenase and nicotinamide adenine dinucleotide phosphate oxidase. Sertoli cells, because of their high metabolic activity, contribute additional ROS. Pathological states, including diabetes, hypertension, chronic kidney disease, and hemoglobinopathies, are also associated with heightened OS and compromised male reproductive function [1, 2, 3, 4, 5]. Exogenous stressors further exacerbate OS and induce both thermal and nonthermal damage, impairing sperm motility, Leydig cell function, and DNA integrity [14]. Lifestyle factors such as tobacco and alcohol use increase leukocyte counts and toxic metabolite production, intensifying seminal OS. Environmental toxins (e.g., lead and mercury) and testicular insults amplify ROS generation and contribute to sperm dysfunction (Figure 1). Notably, increased OS has been documented after vasectomy reversal, suggesting that persistent obstruction may sustain oxidative damage [15].

FIGURE 1.

Origin and roles of ROS in male fertility. ROS generated from endogenous and exogenous sources play dual roles supporting fertility or causing sperm damage and infertility.

At physiological levels, ROS are essential for sperm fertilization processes such as capacitation, hyperactivation, acrosome reaction, and sperm–oocyte fusion [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. In terms of spermatogenesis, ROS contribute to germ cell proliferation and differentiation and regulate spermatogonial stem cell self‐renewal [12, 16, 17]. Capacitation—the final step enabling fertilization—depends on ROS‐mediated signaling and mitochondrial support [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. Hyperactivation, characterized by vigorous flagellar movement for zona pellucida penetration, is regulated by ROS via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway, tyrosine phosphorylation, and ionic fluxes [12, 16, 19]. The acrosome reaction, essential for oocyte penetration, is initiated by ROS‐induced Ca2+ influx from both intra‐acrosomal stores and extracellular sources. ROS also stimulate phospholipase A activity and membrane LPO, facilitating enzyme release [20, 21, 22, 23]. Low ROS levels enhance sperm–oocyte fusion by increasing membrane fluidity and promoting receptor‐mediated zona pellucida binding [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. ROS facilitate chemotaxis via progesterone signaling and trigger intracellular cascades (Ca2+ influx and cAMP/PKA activation) [16, 17, 18, 19, 20, 21, 22, 23, 24, 25]. However, when ROS levels deviate from physiological balance—either by excess or deficiency—they disrupt cellular function and impair fertility.

2.2. Oxidative Stress–Induced Damage and Antioxidant Capacity

Spermatozoa are particularly vulnerable because of their limited cytoplasmic antioxidant reserves [16, 17, 18, 19]. OS disrupts membrane fluidity, triggers ATP leakage, impairs flagellar motion, and compromises the acrosome reaction. Immature sperm released during transit from the seminiferous tubules can generate ROS that damage adjacent mature sperm via apoptotic signaling [12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. Although mild oxidative damage may be reversible, severe or persistent OS leads to irreversible SDF and worse reproductive outcomes [27, 28]. ROS‐mediated mitochondrial dysfunction further impairs ATP production, worsening energy deficits and sperm vitality loss [29, 30]. Mateo‐Otero et al. demonstrated that ROS‐induced DNA damage impairs early embryogenesis and increases miscarriage risk [12]. Aziz et al. reported a significant correlation between ROS levels and abnormal sperm morphology, including amorphous heads, acrosomal defects, and tail deformities [16]. Lopes et al. found higher SDF and impaired antioxidant enzyme activity in patients with asthenozoospermia and OS [31]. Oxidative markers such as malondialdehyde (MDA) are elevated in men with poor semen profiles, as shown by Barik et al. and Oumaima Amma, who also noted decreased seminal antioxidant levels in teratozoospermia [32, 33]. A meta‐analysis of 65 studies involving > 5,800 patients by Chao Huang et al. revealed significantly elevated OS markers—such as MDA, nitric oxide (NO), and carbonyl proteins—and reduced levels of antioxidants—including glutathione, glutathione peroxidase (GPx), vitamins C and E, catalase and glutathione S‐transferase (GST)—in men with infertility [34]. Additionally, OS disrupts sperm motility by damaging mitochondrial membranes and impairing bioenergetic pathways. In fertile men, elevated total oxidant status was associated with reduced motility and lower total antioxidant capacity (TAC) [35].

Both enzymatic antioxidants, including superoxide dismutase, catalase, GPx, and peroxiredoxins (PRDX), and nonenzymatic antioxidants, such as proteins, vitamins, and trace minerals, work synergistically to maintain redox balance [18]. The TAC reflects the cumulative antioxidant potential of seminal plasma and can be measured via direct (e.g., Trolox equivalent antioxidant capacity) or indirect assays [36]. As OS arises from an imbalance between ROS and TAC, combining ROS and TAC measurements provides a more accurate index of redox status than either parameter alone [19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37]. Several studies have validated the clinical relevance of TAC in male fertility. Mahfouz et al. found significantly higher TAC levels in the fertile group than in the infertile group, with a diagnostic threshold of 1420 μM (76% sensitivity and 64% specificity) [37]. Similarly, Roychoudhury et al. identified a TAC cutoff of 1947 μM in a larger cohort, distinguishing fertile from infertile men [38]. Another study using the ferric‐reducing antioxidant power assay demonstrated lower TAC in men with abnormal semen profiles and found positive correlations with sperm concentration, motility, and morphology—highlighting the link between impaired antioxidant defenses and poor sperm quality [39]. At the molecular level, the Kelch‐like ECH‐associated protein 1–nuclear factor erythroid 2‐related factor 2 (KEAP1–NRF2) signaling pathway serves as a major regulator of cellular antioxidant responses, offering protective effects against testicular OS [40]. Darbandi et al. found that KEAP1 methylation in sperm remained unchanged despite elevated ROS levels, suggesting that antioxidant upregulation occurs via alternative pathways [41]. KEAP1 upregulation was associated with reduced sperm counts and elevated OS levels, whereas NRF2 inhibition promotes OS and testicular dysfunction [42].

3. Antioxidant Therapy: Use and Overuse for Redox Balance

3.1. Rationale and Current Applications of Antioxidant Therapy in Male Infertility

Multiple clinical trials have evaluated antioxidant therapy in male infertility with mixed results. For instance, a randomized controlled trial (RCT) by Jannatifar et al. found significant improvements in sperm quality after 3 months of N‐acetylcysteine (NAC, 600 mg/day) [43] and a systematic review by Syarif et al. echoed these findings across five RCTs [11]. Similarly, L‐carnitine and L‐acetylcarnitine therapy showed moderate effects on sperm motility and morphology in men with idiopathic infertility [44]. However, a Cochrane review of 34 trials (n = 2,876) and the large‐scale trial failed to demonstrate consistent improvements in semen parameters, DNA fragmentation, or live birth rates [45, 46]. Their further analyses found no correlation between antioxidant levels and fertility outcomes [47]. Furthermore, recent meta‐analyses, including a Cochrane review of 90 trials (n = 10,303), suggested potential benefits for clinical pregnancy, but not live birth, especially when high‐risk‐of‐bias studies were excluded [3, 48, 49]. Inconsistencies across studies—due to poor randomization, small sample sizes, and variable regimens—highlight the experimental nature of antioxidant use. Although generally safe, current evidence does not support their routine use in male infertility management [3, 44, 49].

Generally, antioxidants are commonly used to counter OS by neutralizing ROS, largely because of their perceived “natural” health benefits derived from sources such as fruits, vegetables, and whole grains [50, 51]. However, limited regulatory oversight and aggressive marketing have led to their widespread and often unmonitored use. Ligny et al. found that 79% of over‐the‐counter male fertility supplements contained at least one ingredient exceeding the recommended daily allowance, with only 38% supported by clinical trials—most of which lacked methodological rigor [3]. This raises concerns about their clinical efficacy and highlights the potential risks associated with over‐supplementation [52, 53, 54]. Nevertheless, antioxidants remain widely prescribed in male infertility—even in the absence of confirmed OS. This is problematic, as ROS exert both beneficial and harmful effects depending on concentration. Through a global survey, Agarwal et al. (n = 1,327) reported that while 85.6% of reproductive specialists routinely prescribed antioxidants, only 34.3% performed OS‐related testing. Notably, 65.7% never assessed OS before treatment initiation, underscoring a significant disconnect between diagnosis and intervention [2].

According to the 2024 AUA/ASRM guidelines, clinicians should counsel patients on the uncertain efficacy of supplements, as current evidence does not support the routine use of any specific antioxidant (moderate recommendation; grade B) [55]. Similarly, the 2025 EAU guidelines advise against routine antioxidant therapy for idiopathic male infertility due to inconclusive benefits (level 1b, weak recommendation) [56]. The 2024 JUA guideline recognizes that antioxidant therapy has shown some improvements in semen quality and pregnancy outcomes, but deems the current evidence insufficient for routine recommendation in idiopathic male infertility (Grade C; Evidence Level II) [57]. The 2023 ESHRE consensus also concluded that, in the absence of strong data showing improved live birth outcomes, routine antioxidant use is not recommended for male or female infertility management [58].

3.2. Antioxidant Overuse and Its Potential Harm

Although antioxidants are intended to counteract OS, their excessive or unmonitored use may disrupt physiological redox signaling and contribute to adverse outcomes. A meta‐analysis by Bjelakovic et al. (68 RCTs, N = 232,606) reported an association between antioxidant supplementation and increased mortality, particularly in high‐quality trials [52]. Similarly, a Cochrane review (N = 296,707) found no benefit for primary or secondary mortality prevention and highlighted increased death risks linked to β‐carotene, vitamin E, and possibly high‐dose vitamin A [54]. A long‐term RCT involving 5220 healthy adults also found no protective effect against metabolic syndrome, reinforcing skepticism toward routine antioxidant use despite widespread marketing [59].

Because ROS play essential roles in apoptosis, immune defense, and detoxification, over suppression via antioxidants may impair these fundamental processes [53, 60]. β‐carotene supplementation was found to increase lung cancer risk among smokers, as reported in the CARET study [61]. Furthermore, high doses of vitamin E (≥ 400 IU/day) have been linked to elevated mortality rates [62]. In vitro studies by Peris et al. showed that NAC increased mitochondrial ROS in adipocytes, raising concerns about its role in inducing RS [63]. Similarly, high‐dose antioxidant use has been associated with SDF and reduced fertility. Animal studies have reported that excessive carnitine supplementation impaired sperm quality in healthy mice [64, 65]. Clinically, Rolf et al. found no improvement in semen quality or sperm survival after 8 weeks of high‐dose vitamins C and E in men with subfertility [66]. Verma et al. further demonstrated a dose‐dependent effect of vitamin C, where low concentrations were beneficial; however, higher levels induced motility loss and LPO [67]. Redox homeostasis is especially critical during embryogenesis, where antioxidant excess may lead to developmental abnormalities [60]. For example, high vitamin A intake in early pregnancy has been linked to congenital defects [68]. Overall, these findings underscore that antioxidant therapy, while potentially beneficial, carries real risks when misused or overdosed.

3.3. Reductive Stress and Its Effect on Male Reproductive Health

RS refers to an overly reduced redox state caused by excessive endogenous or exogenous antioxidants and reducing agents, disrupting key cellular functions [3]. Initially proposed by Gregory J. Gores in 1989 through studies on rat hepatocytes, RS was found to trigger mitochondrial dysfunction and promote toxic oxygen species production under respiratory inhibition [41, 60]. This pathological redox imbalance impairs mitochondrial homeostasis, cellular metabolism, and critical growth signaling pathways (Figure 2). In this context, reductive conditions may paradoxically generate ROS through abnormal electron transfers, contributing to cellular damage [64]. RS has been increasingly implicated not only in male infertility but also in broader pathologies such as cancer, heart failure, and neurodegenerative diseases [60, 69, 70].

FIGURE 2.

The redox balance spectrum” and male infertility. Oxidative and reductive stress represent opposing redox imbalances, both disrupting sperm function, whereas oxidative balance supports normal male reproductive processes.

In reproductive health, excessive antioxidant intake may disrupt the physiological roles of ROS, which are essential for normal sperm function. A double‐blind RCT by Stenqvist et al. in men with elevated sperm DNA fragmentation index (DFI ≥ 25%) showed that a 6 month antioxidant supplementation improved sperm concentration but did not reduce DFI [71]. Similarly, Ménézo et al. observed that while antioxidant therapy reduced SDF, it also significantly increased sperm nuclear decondensation, potentially affecting paternal epigenetic regulation during embryogenesis [72]. Intense sperm decondensation may disrupt chromosome condensation and lead to cytoplasmic fragments in the embryo, potentially altering fertility outcomes [60]. These findings emphasize the need for antioxidant therapy to restore, rather than overshoot, redox homeostasis—balancing the benefits of ROS regulation with the risks of inducing RS [51, 60, 73]. Redox imbalance—whether due to excessive ROS production or uncontrolled antioxidant use—can disrupt cellular homeostasis and impair fertility. This duality highlights the need for a more suitable approach: antioxidant therapy can be beneficial only when precisely targeted.

4. Redox‐Based Precision Antioxidant Therapy: The Need for Appropriate Tools

Personalized antioxidant therapy represents a critical evolution in the management of male infertility. Nonselective antioxidant use, especially without evaluating redox status, may trigger compensatory stress responses or worsen underlying dysfunctions [18, 64, 74]. In a prospective study, Vessey et al. found that L‐carnitine improved semen parameters only in men with elevated ROS, emphasizing the need for individualized therapy [75]. In this context, Meng et al. proposed the “precision redox” framework based on the 5R principles: delivering the right antioxidant species to the right place, at the right time, in the right amount, and to the right target [74].

A cornerstone of precision medicine is the ability to assess redox status accurately (Figure 3). Although semen analysis is fundamental in male fertility evaluation, it does not capture redox complexity [76]. Direct ROS measurements, such as luminol‐based chemiluminescence and MDA assays, offer diagnostic value. For instance, Desai et al. proposed an ROS cutoff of > 0.0185 × 10⁶ cpm/20 × 10⁶ sperm with 78% sensitivity and 82% specificity [77], whereas other studies have confirmed high ROS levels in men with infertility but with modest specificity [78] [79] TAC, which reflects total antioxidant defense, correlates with semen quality in some studies but lacks standardization and cost‐effectiveness for routine use [4, 38, 76, 80, 81]. DNA oxidation biomarkers such as 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) show promise but remain costly and technically demanding [82, 83]. Beccati et al. reported a correlation between blood leukocyte ROS production and seminal OS in men with idiopathic infertility, implying potential diagnostic value; however, Guz et al. found no consistent systemic‐to‐seminal redox relationship [84, 85]. These results underscore the complexity and current limitations in using systemic ROS markers to reliably assess OS in the male reproductive tract.

FIGURE 3.

Suggesting a framework for precision redox medicine in male infertility. A precision medicine approach tailors redox‐based interventions in male infertility according to measured redox status.

Emerging tools such as the MiOXSYS system measure oxidation–reduction potential (ORP), a real‐time index of redox balance in semen [18, 86, 87]. Agarwal et al. suggested a cutoff of 1.34 mV/10⁶ sperm/mL with 98.1% sensitivity; however, specificity remained low (40.6%) [88]. Selvam et al. defined a physiological ORP range, deviations from which may indicate OS or RS, each needing tailored management [18]. A pilot study showed that antioxidant therapy improved sperm concentration only in men with high baseline ORP, indicating potential for guiding treatment selection [86]. ORP is now listed in the WHO 6th edition as a research methodology [76] and a meta‐analysis confirmed its inverse association with semen quality [89]. However, ORP has limitations, as Tomita et al. reported it to be a poor predictor of fertilization and pregnancy outcomes [87]. Castleton et al. reported that MiOXSYS and OxiSperm II failed to accurately reflect OS or sperm function due to confounding by sperm concentration [90]. Similarly, Joao et al. noted poor sensitivity/specificity of the ORP index, reinforcing the need for independent biomarker validation [91]. ORP reflects the dynamic electron flow during redox reactions and may be confounded by the reductive nature of the seminal environment, whereas TAC measures overall antioxidant levels without capturing real‐time redox imbalances [91, 92]. Furthermore, the assay remains investigational due to the lack of strong published evidence linking ORP with reproductive outcomes. Technical issues—such as sample viscosity, poor liquefaction, and the need for standardized timing postejaculation—further limit its reliability [76]. Moreover, the exclusive availability of MiOXSYS also hampers independent validation and cross‐platform comparison [90, 92].

Currently, no single assay has emerged as a definitive tool for evaluating redox imbalance, prompting ongoing efforts to apply diverse techniques. Riley et al. demonstrated that among three fluorescent probes tested on native semen, only MitoSOX Red (via flow cytometry) reliably detected spontaneous ROS production in viable sperm and showed a strong correlation with DNA fragmentation [93]. Expanding on this, Calamai et al. used a similar approach in a larger subfertile cohort, identifying elevated ROS levels in 29% of patients—findings that were independent of conventional semen parameters [94]. The development of simple, rapid, and cost‐effective point‐of‐care tests is essential to implement precision redox medicine effectively and avoid overtreatment or RS risks.

Although no gold standard currently exists for assessing redox imbalance in semen, several tools can provide clinically useful evidence of OS and may help guide treatment decisions. Each assay has distinct advantages and limitations, as well as varying levels of complexity, meaning their adoption depends largely on the resources and expertise of individual centers [92, 95]. Chemiluminescence remains the most established research method with high sensitivity and reproducibility, though it requires large semen volumes and lacks standardized cutoffs [95, 96, 97]. MiOXSYS offers rapid, cost‐effective ORP measurement with minimal manipulation, yet its accuracy is influenced by semen viscosity, and it lacks robust validation for reproductive outcomes [76, 86, 92]. OxiSperm II is inexpensive and simple to use but measures only a single ROS species with limited precision, while TAC provides an overall antioxidant profile but suffers from methodological variability and the absence of clinical thresholds [37, 38, 97]. Flow cytometry enables multiparametric ROS detection in viable sperm, though it remains technically demanding and poorly standardized [93, 95].

Despite their limitations, the use of these assays can provide valuable evidence of redox imbalance, thereby complementing semen analysis, which fails to capture redox dynamics [76]. Among these assays, TAC, MiOXSYS, and chemiluminescence can be regarded as practical options, and the implementation of any available method is preferable to the current practice in which redox status is rarely assessed. Such evidence supports a more rational and targeted use of antioxidant therapy to restore redox balance, moving beyond purely empirical treatment strategies [2]. Specific assessment of redox status offers more direct and biologically meaningful information compared with SDF, which remains an indirect marker and may be influenced by multiple confounding factors [98]. The wider implementation of these diagnostic tools will contribute to generating the robust evidence base required to refine methodologies, achieve validation, and ultimately establish their clinical utility.

5. Redox Imbalance and New Findings on Genomic and Epigenomic Alterations

While OS is a well‐recognized factor in male infertility, its precise effects on the genomic and epigenomic stability of sperm remain underexplored. Elevated ROS levels and increased SDF are hallmark indicators of redox‐induced damage associated with male infertility [99, 100]. Redox imbalance in these areas of sperm DNA may contribute to diverse offspring outcomes, depending on the extent and location of the damage, as well as the effectiveness of DNA repair mechanisms [101, 102, 103, 104]. Certain regions of the genome are particularly vulnerable to redox stressors, making them more susceptible to oxidative damage. These genomic “hot spots” are also known sites for copy number variations and microdeletions [101, 105]. Recurrent OS targeting these vulnerable sites may compromise chromosomal stability and increase the risk of heritable defects. This suggests that paternal redox imbalance could have long‐term consequences not only for fertility but also for the health of future generations.

While DNA serves as the genetic foundation for reproduction and inheritance, epigenetic mechanisms also play a key role in regulating how genetic information is expressed [103]. Recent evidence has shown that redox imbalance can alter the epigenetic landscape of sperm by increasing DNA damage, modifying histone and DNA methylation patterns, and dysregulating the expression of small noncoding RNAs, potentially leading to mutations [106, 107, 108, 109, 110]. Several studies have shown that increased SDF—commonly associated with OS—is linked to distinct epigenetic alterations. Khezri et al. found that boar sperm with higher fragmentation exhibited site‐specific DNA methylation changes affecting pathways such as antioxidant defense [111]. In humans, Song et al. reported differential methylation in both imprinted and nonimprinted genes in sperm with poor DNA integrity, whereas Liu et al. observed widespread promoter hypermethylation in high SDF samples [106, 112].

A common OS‐related condition, such as varicocele, also exerts significant epigenetic effects on male fertility. Shojaei et al. found altered m6A RNA methylation enzymes alongside OS and sperm dysfunction [113], whereas Moshari et al. reported disrupted DNA methylation/demethylation processes and impaired embryo development [114]. Darbandi et al. demonstrated that elevated ROS were significantly associated with altered methylation of the H19–IGF2 imprinting region, reduced concentration and motility, and increased SDF in normozoospermic men [115]. Hug et al. reported that OS increases DNA hydroxy methylation, indicating disruption of the sperm epigenome. Notably, antioxidant supplementation caused mild but unexpected epigenetic changes, raising concerns about potential transgenerational effects [9]. Jiang et al. also found that L‐carnitine supplementation decreased H3K9me3 methylation, suggesting its potential role in modulating sperm epigenetic integrity [116]. Redox imbalance in sperm affects DNA integrity and disrupts key epigenetic reprogramming events. These alterations may compromise the paternal genetic and epigenetic contribution to the embryo, potentially impairing early embryonic development and reducing embryo quality. Appropriate epigenetic reprogramming during spermatogenesis is essential for ensuring healthy embryo development and long‐term offspring well‐being [117, 118]. Transgenerational epigenetic alterations, as shown by Lee et al., can impair male fertility, with prenatal PM2.5 exposure causing persistent DNA methylation changes, sperm defects, and elevated SDF across three generations [119].

Altered expression of specific miRNAs may disrupt germ and somatic cell regulation, contributing to impaired spermatogenesis and male infertility [120]. During spermatogenesis, OS may disrupt protamine replacement, potentially affecting epigenetic regulation. Although semen parameters may appear normal, epigenetic disruptions—such as altered protamine expression and increased SDF—should be considered as critical, yet often overlooked, contributors to male infertility. Because protamines are essential for chromatin condensation, redox‐induced alterations in their expression may compromise sperm nuclear integrity and disrupt early developmental programming, thereby affecting fertility, implantation success, and embryonic viability [9, 121, 122]. In patients with elevated SDF, distinct changes in the expression profiles of microRNAs were observed, suggesting their potential involvement in the molecular mechanisms underlying DNA damage [108, 112, 123]. For instance, miRNA‐122, miRNA‐181a, and miRNA‐34c5 are downregulated in oligoasthenoteratozoospermia men with varicocele, correlating with varicocele grade and bilaterality [109]. miRNA‐424/322 is associated with elevated SDF, and miR‐1255a, miR‐921, and miR‐3156‐5p may contribute to DNA damage via the phosphatidylinositol‐3′‐kinase‐AKT pathway [108, 123]. Similarly, Roach et al. showed that miR‐196a, a miRNA linked to the Nrf2‐mediated antioxidant response, remained ~100‐fold elevated, suggesting its potential as a biomarker of epigenetic disruption in sperm from chronic paternal alcohol exposure [124].

Emerging evidence demonstrates that redox imbalance can affect not only nuclear gene expression but also mitochondrial‐derived small RNAs (mt‐tsRNAs), highlighting its dual effect on both nuclear and mitochondrial regulation [10, 125]. Notably, single‐embryo transcriptomic analyses have demonstrated that sperm‐derived mt‐tsRNAs are transferred to the oocyte at fertilization, potentially influencing early embryonic gene expression—highlighting a novel mechanism of paternal non‐nuclear inheritance that may shape offspring metabolic health [10]. Thus, redox imbalance represents a critical but underrecognized factor in shaping both reproductive potential and transgenerational health outcomes. Together, these insights underscore that redox imbalance intersects with epigenetic machinery at multiple levels—nuclear and mitochondrial—mediating not only subfertility but also programming of long‐term offspring health. A better understanding of redox dynamics is essential for unraveling their epigenetic footprints and their role in reproductive medicine.

6. Research Gaps and Future Perspectives

Despite growing interest in redox‐based therapies, significant knowledge gaps limit the clinical utility of antioxidants in male infertility. Key uncertainties include the optimal type, dosage, and duration of supplementation, as well as the lack of standardized thresholds for redox imbalance in serum and seminal plasma. Consequently, many interventions remain empirical and inconsistently applied [55, 56, 126]. Although several large‐scale studies have demonstrated improvements in semen parameters following antioxidant therapy, evidence supporting their effect on pregnancy or live birth rates remains inconclusive. This discrepancy likely arises from heterogeneous study designs, insufficient assessment of OS, and inadequate patient stratification. A key limitation of previous trials is the absence of OS‐based patient selection, with antioxidants often prescribed for general sperm abnormalities, elevated SDF, or idiopathic subfertility without confirming redox imbalance [92]. Importantly, OS should be recognized as a contributing—but not exclusive—factor in male infertility, highlighting the need for more refined and individualized diagnostic approaches [3, 43, 45].

Emerging biomarkers, such as sperm DNA oxidation markers and ORP, show promise for better‐targeted interventions while requiring validation in large, well‐characterized cohorts. The absence of a robust and clinically validated assay for seminal OS highlights a critical knowledge gap in the diagnostic approach to male infertility [92]. Similarly, individualized proteomic and metabolomic profiling could help tailor therapy by identifying specific redox imbalances [83, 127]. RS detection remains a particular challenge due to the absence of reliable biomarkers and the compartmentalized nature of redox states across organelles [128, 129]. Future research should prioritize developing sensitive, organelle‐specific redox assays and exploring their implications on gamete quality and embryogenesis. This will enable antioxidant pharmacology to align with the “5R” principle, shifting from generalized treatments to personalized, safe, and effective strategies for managing male infertility [74, 130]. Current evidence also highlights the need to explore how redox imbalance affects genomic “hotspots” vulnerable to oxidative damage as well as epigenetic alterations, including DNA methylation and histone modification [101, 102, 103], and sperm‐specific microRNAs [123]. Recent advances in genetics and epigenetics provide valuable insights that may help elucidate previously unexplained cases of male infertility, particularly idiopathic and unexplained forms, which remain significant knowledge gaps in reproductive medicine.

7. Conclusions

ROS exerts a dual influence on male reproductive health—supporting essential sperm functions at physiological levels, yet contributing to impaired sperm quality, DNA fragmentation, and infertility when present in excess. Disruptions in redox homeostasis, whether oxidative or reductive, can compromise fertilization potential and may have downstream effects on embryonic development and offspring health. Although antioxidant therapy has gained popularity in clinical practice, its efficacy remains inconsistent. This discrepancy highlights the “antioxidant paradox,” where therapeutic benefit is only observed when antioxidant interventions are aligned with the individual's specific redox imbalance.

Progress in the field is hindered by methodological variability, a lack of validated and standardized redox biomarkers, and insufficient patient stratification based on oxidative status. Consequently, empirical antioxidant use carries the risk of overtreatment and may overlook opportunities for more precise and effective interventions. Future advancements will require the integration of redox profiling and adherence to the “5R” principle—administering the right antioxidant species, at the right place, time, level, and target. Furthermore, emerging evidence links redox imbalance to epigenetic alterations in the sperm genome, suggesting that its effects may extend beyond fertilization to affect transgenerational health. To address these challenges, well‐designed, large‐scale randomized controlled trials should evaluate fertility endpoints and incorporate molecular analyses to elucidate redox‐related genomic and epigenomic changes, ultimately guiding the implementation of precision redox medicine in male infertility management.

Disclosure

The authors have nothing to report.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Nguyen H. D. K., Kuroda S., Nguyen N. T., Yumura Y., and Takeshima T., “Redox Imbalance in Male Infertility: From Empirical Antioxidant Therapy to Precision Medicine and Epigenetic Insights,” Reproductive Medicine and Biology 24, no. 1 (2025): e12693, 10.1002/rmb2.12693.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.