The literature included in this study was sourced from the Web of Science Core Collection (WoSCC) database, as it is one of the most systematic and authoritative databases, which is widely recognized for its effectiveness in bibliometric research (25, 26). The search was conducted on June 22, 2023, using the following search query: (TS= “Spermatozoa” OR “Sperm” OR “male fertility” OR “male sterility” OR “male reproduction”) AND (TS= “Reactive Oxygen Species” OR “Oxidative stress”). The following search limitations were applied:

After screening, a total of 5,319 articles were retrieved. For articles meeting these inclusion criteria, all records, including titles, authors, abstracts, keywords, and references, were exported as plain text files and saved as download_txt files. Then, they were imported into CiteSpace 5.7.R5, and after deduplication, 5,301 articles remained for further analysis.

The researchers used CiteSpace 5.7.R5 to analyze countries, institutions, authors, and citations. CiteSpace allows for data visualization by constructing citation networks and co-occurrence networks, presenting the structure, patterns, and distribution of scientific knowledge in a specific discipline or field (27). In this study, the nodes in the network were represented using a yearly ring method, where the overall size of nodes reflects their citation or occurrence frequency, the width of the yearly ring represents the number of papers published in a given year, and the connections between nodes indicate shared citations or co-occurrence frequency with other nodes. The specific parameters used in this study were: Time slicing: Jan. 2014 to Dec. 2023, Years per slice: 1, Term source: Title, Abstract, Author Keywords, and Keyword Plus, Node type: Author, Institution, Country, Keyword, Reference, Cited Author, and Cited Journal, Link strength: Cosine, Selection Criteria: g-index, k = 20, Pruning: Pathfinder, Pruning the merged network, Visualization: Cluster View-Statistics, Show Merged Network. Additionally, keyword clustering analysis was conducted using VOSviewer 1.6.19, with parameters set to default values. The colors of nodes represent their membership in specific clusters, with different colors indicating different clusters, allowing for the discovery of structural distributions of research hotspots.

A total of 5,301 articles were included in the analysis. The annual number of publications increased from 352 in 2014 to 751 in 2022, as shown in Figure 2 . Through statistical analysis, it was found that in the last decade, the author with the highest number of publications was Ashok Agarwal, who published 66 papers. The country/region with the most publications was Peoples R China, where 898 papers were published, and the institution with the most publications was Univ Sao Paulo, publishing 115 papers. The centrality of authors, institutions, and countries/regions was generally low, indicating a need for increased collaboration in the field. The top 5 authors, countries/regions, and institutions in terms of publications in the last decade are detailed in Table 1 .

After conducting co-citation analysis on all journals, the researchers observed that the number of nodes increased from 401 in 2014 to 690 in 2022. Additionally, the number of connections grew from 1,203 to 2,070 during this period. These findings indicate that research in this field has garnered widespread attention in recent years, with increased mutual referencing and a trend of further development. From Figure 3A , it is evident that the journal with the highest number of citations and occupying a central position in this field is “Fertil Steril” with 2,894 citations and a centrality score of 0.62. Detailed information on the top 10 most frequently cited journals is provided in Table 2 .

Furthermore, this study conducted burst detection analysis on the cited journals. When a journal experiences a sudden increase in citation frequency within a specific period, it is marked in red. As shown in Figure 3B , starting from 2021, many journals have exhibited a sudden increase in co-citation frequency, and this trend has continued. Among them, “Antioxidants-Basel” stands out with a burst intensity of 103.18.

The researchers utilized CiteSpace to conduct co-citation analysis on the top 10% most frequently cited references within each time period. Co-citation relationships are established when two references are both cited by a third reference. References with a high co-citation frequency indicate their significant influence within their respective field of research, often referred to as core references. The co-citation network comprised a total of 1,015 nodes and 4,456 connections. Figure 4 displays the authors and publication years of articles cited more than 50 times. The top 10 most frequently cited references are listed in Table 3 (17, 18, 28–35).

Upon conducting cluster analysis on references cited more than 5 times, this study identified a total of 64 clusters, with clustering metrics showing Modularity Q = 0.7964 and Weighted Means Silhouette = 0.9141, indicating significant modularity and reasonable clustering results. Detailed clustering results are presented in Figure 5A . The fish-eye view ( Figure 5B ) illustrates the temporal distribution of cited references in different categories, with nodes positioned further to the right representing references cited more recently. Additionally, citation burst detection ( Figure 5C ) revealed bursts in citations each year from 2014 to 2023, indicating a sustained trend of development in this field of research.

It is noteworthy that over the past decade, the most frequently cited reference with the highest burst intensity was a review article by Agarwal A et al. (28), titled “Effect of Oxidative Stress on Male Reproduction,” published in World J Mens Health in 2014, cited 187 times with a burst intensity of 52.23.

Based on a co-occurrence analysis of keywords using CiteSpace, the researchers found a total of 678 nodes and 6,781 connections in the keyword co-occurrence network. The most frequently occurring keywords were “Oxidative stress” (3766), “spermatozoa” (1068), “sperm” (914), “apoptosis” (876), “antioxidant” (863), “lipid peroxidation” (847), “cryopreservation” (678), “DNA damage” (655), and so on. These keywords reflect the main research directions and content in this field. In Figure 6A , keywords that appeared more than 500 times are highlighted.

Furthermore, this study conducted cluster analysis of keywords using VOSviewer for keywords that co-occurred more than 30 times. We identified four clusters: Cluster 1 in red (124 keywords), Cluster 2 in green (94 keywords), Cluster 3 in blue (67 keywords), and Cluster 4 in yellow (24 keywords). Details of the keywords within each cluster are shown in Figure 6B .

It is evident that Cluster 1 primarily includes keywords related to oxidative stress, sperm development, and apoptosis. Cluster 2 contains keywords related to sperm cryopreservation and cryoprotectants. Cluster 3 focuses on keywords related to male infertility, such as “male infertility,” “smoking,” “varicocele,” and “sperm DNA fragmentation.” Cluster 4 includes fewer keywords with lower occurrence frequencies, primarily related to such terms as “metabolism” and “involvement.”

Furthermore, burst detection analysis in Figure 6C revealed the existence of burst keywords nearly every year from 2014 to the present. These keywords have evolved from terms like “human spermatozoa,” “mitochondrial membrane potential,” and “glutathione peroxidase” in 2014 to terms like “inflammation,” “P53,” “health,” and “polyunsaturated fatty acid” after 2020.

Cellular oxidative stress can result from an increase in the production of reactive oxygen species (ROS), such as hydroxyl radicals (OH), superoxide anions (O2−), and hydrogen peroxide (H2O2) (43). It can also occur due to a decrease in antioxidant defenses. The antioxidant system includes enzymatic factors like superoxide dismutase, catalase, and glutathione peroxidase, as well as non-enzymatic factors like glutathione, N-acetylcysteine, vitamin E, coenzyme Q10, carnitines, myo-inositol, and lycopene, along with trace elements such as selenium, zinc, and copper (44–46).

Sperm cells are among the first to exhibit susceptibility to oxidative damage. This susceptibility arises from several factors. Sperm cells possess abundant mitochondria to provide energy for their motility, and mitochondria are considered a primary source of ROS. ROS, by affecting electron transfer in the respiratory chain, activate intracellular signaling cascades, leading to increased ROS production within mitochondria (47). Sperm cells also have plasma membranes rich in polyunsaturated fatty acids (PUFAs), making them vulnerable to oxidative stress. When the sperm plasma membrane undergoes lipid peroxidation (LPO), it disrupts membrane permeability, resulting in ATP efflux and impairing flagellar motility, ultimately affecting sperm motility and fertilization potential (15).

The impact of oxidative stress on male fertility is further evidenced by its role in exacerbating sperm DNA damage (48). Existing research indicates that an increase in sperm DNA damage can affect the developmental potential of embryos post-fertilization, leading to an elevated risk of miscarriage and an increased likelihood of birth defects in offspring (49, 50). Studies have shown that sperm obtained through testicular sperm aspiration (TESA) exhibit significantly lower DNA fragmentation than ejaculated sperm and have better outcomes in intracytoplasmic sperm injection (ICSI) treatments (51). Currently known sperm DNA breaks include single strand DNA breaks (SSBs) and double strand DNA breaks (DSBs), which cause different clinical reproductive effects (52). For one thing, SSBs is linked to oxidative stress and is widespread in all regions of the genome in the form of multiple breakpoints that can lead to prolonged conception or infertility (53). For another, DSBs are mainly confined to the sperm nuclear stroma and may be associated with inadequate DNA repair during meiosis, resulting in an increased risk of miscarriage, decreased embryo quality, and increased risk of implantation failure during ICSI cycles (54). Studies have shown that oocytes can repair DNA damage of sperm, mostly through base excision repair (BER). But this repair ability decreases with oocyte aging and associated decent in female gamete’s quality. A previous study in mice showed that this might be due to the significant decrease in the expression of some key genes of DSB repair pathway, such as Brca1 and Brca2, in MII oocytes with age (55).

With advancements in technology, assessing sperm quality solely based on traditional seminal parameters evidently falls short of meeting contemporary clinical demands. Oxidative stress levels are now regarded as a novel indicator for evaluating sperm quality. In 2019, literature introduced the concept of “Male Oxidative Stress Infertility” (56). This concept utilizes the Male Infertility Oxidative System (MiOXSYS) to measure the oxidation-reduction potential (ORP) of the semen as a means to assess oxidative stress levels. The advantage of this method is that it can detect oxidative stress levels in real time. The sample may be either semen or seminal plasma. This method needs short time and is easy to operate, but its disadvantage is that the detection result is affected by the viscosity of the sample. Henkel et al. conducted a prospective observational study demonstrating that seminal ORP values detected by MiOXSYS have significant predictive power for fertilization rate, blastocyst development rate, implantation/clinical pregnancy, and live birth rate during ICSI cycles. Based on the assisted reproduction results, the relevant calculated cut-off values for seminal ORP predicting good fertilization (>80%), blastocyst formation (>60%), implantation/clinical pregnancy and live birth were ≤0.709, ≤0.530, ≤0.465 and ≤0.393 mV/10⁶ sperm/ml, respectively, with an average value of 0.51 mV/10⁶ sperm/ml (57). Interestingly, these figures were significantly lower than the threshold ORP of 1.34 mV/10⁶ sperm/ml for men with abnormal and normal semen parameters in previous studies (58).

In addition to seminal ORP measurement, there are alternative methods for oxidative stress assessment, including total antioxidant capacity (TAC), superoxide dismutase (SOD), and malondialdehyde (MDA) assays. However, it remains challenging to incorporate these tests into routine use due to their cost, complexity, time sensitivity, and the potential requirement for sophisticated equipment, substantial and well-handled sample volumes, as well as extensive technical training (59). Apart from the mentioned indicators, the sperm DNA fragment index (DFI) is also considered an indirect marker that can provide insights into oxidative stress (60). There are a variety of methods to test sperm DNA fragments, such as sperm chromatin structure assay (SCSA), sperm chromatin dispersion (SCD) assay, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate notch end labeling (TUNEL) assay, and single cell gel electrophoresis (Comet) assay (61). The advantage of TUNEL, SCSA, and SCD lies in their highly standardized protocol, but TUNEL and SCSA require flow cytometer. Comet assay is able to differentially detect MAR-region double-strand breaks, but technique and analysis are not standardized between laboratories. In earlier studies, the cut-off value for DFI was usually set to DFI = 27% or 30% (62, 63), but some studies in recent years have used a lower cut-off value, such as DFI = 20% (64–66). The author believes that it is difficult to determine the optimal ORP or DFI cut-off value due to different clinical scenarios and study objects. In clinical work, doctors still need to take this as a reference to propose a more personalized treatment plan. In general, while oxidative stress assessment is considered an emerging marker for evaluating male fertility, there remains a lack of scientifically practical detection methods and diagnostic standards. Further research is needed to establish these measures for the diagnosis and management of high oxidative stress levels.

Currently, sperm cryopreservation has become one of the essential techniques in assisted reproduction and fertility preservation. Research reports have indicated the adverse effects of freezing technology on sperm vitality, fertilization capacity, and DNA integrity, with these effects being particularly noticeable in infertile men or low-quality sperm samples (67). Oxidative stress (OS) is considered one of the primary reasons for the decreased sperm survival rate and capabilities following ultra-low-temperature storage. Studies have confirmed that during the process of freezing and thawing, changes occur in the fluidity of the sperm mitochondrial membrane, leading to mitochondrial dysfunction and an increased release of reactive oxygen species (ROS), ultimately causing single-strand and double-strand DNA breaks (68).

In recent years, researchers have conducted detailed studies on the mechanisms of cryopreservation-induced damage and the effects of antioxidant additives using animal sperm. For example, studies involving the cryopreservation of ram semen have revealed that freezing leads to the opening of the mitochondrial permeability transition pore (mPTP), resulting in increased permeability of the inner mitochondrial membrane, which leads to an elevation in calcium (Ca2+) and ROS release (69). Compounds such as MitoQ and L-carnitine have been found to mitigate cryopreservation-induced sperm damage by specifically targeting mitochondrial oxidative stress (70, 71). Supplementation of melatonin in the cryopreservation medium for ram sperm has been shown to inhibit the opening of mPTP, thereby improving crucial aspects of mitochondrial oxidative phosphorylation kinase (OXPHOS kinase), oxygen consumption, ATP synthesis, as well as sperm vitality, motility, and kinetics. Interestingly, melatonin-treated cryopreserved sperm have demonstrated higher rates of blastocyst formation and pregnancy following in vitro fertilization and artificial insemination (72).

Antioxidant therapy is considered one of the crucial strategies for improving sperm quality (73). Maintaining a healthy diet and lifestyle is the primary approach to reducing oxidative stress within the body (74). Additionally, treatments such as surgery for varicocele (75) and anti-inflammatory therapies (76) have been shown through research to effectively enhance semen ROS (Reactive Oxygen Species) levels. Furthermore, supplementation with various antioxidants is beneficial for neutralizing ROS within the body. These include enzymes like superoxide dismutase (SOD), glutathione (GSH), and N-acetylcysteine (NAC), as well as non-enzymatic antioxidants like vitamin A, vitamin E, coenzyme Q10, and essential trace elements (77, 78). Researchers have also been exploring the use of plant extracts to improve oxidative stress treatment (79). Although the efficacy of many antioxidants has been demonstrated through studies, no consensus has been reached regarding the standardized dosages and durations of antioxidant intake. In addition, some studies have shown that exogenous antioxidant intake is not appropriate for all infertile men. Antioxidant paradox and reductive stress have been carefully elaborated, suggesting that excessive antioxidant intake may have adverse effects on male fertility (80, 81). Therefore, it is necessary to test the REDOX status of patients before proceeding with antioxidant therapy. The management of patients with such conditions remains a significant challenge for both researchers and clinical practitioners.