Abstract
Male infertility accounts for a substantial proportion of global infertility, yet its molecular basis remains incompletely defined. Emerging evidence implicates epigenetic dysregulation in impaired spermatogenesis and abnormal sperm function. We synthesize genomic, transcriptomic, and single-cell studies to clarify how DNA methylation, histone modifications, and noncoding RNAs contribute to oligoasthenozoospermia. We also appraise single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin using sequencing, and integrative multi-omics for resolving cell-type and stage-specific regulation. These approaches resolve germ-cell lineages, map gene-regulatory networks, and link chromatin states with transcription across spermatogenesis. Finally, we discuss translational implications for diagnostics, biomarkers, and therapeutic strategies, including antioxidant regimens, varicocele management, and assisted reproductive technologies outcomes. In conclusion, integrating epigenetics with single-cell technologies has enhanced our understanding of male germ cell development and identified potential research directions to improve reproductive health.
Plain language summary
Male infertility affects nearly half of infertile couples worldwide, yet its causes remain complex and poorly understood. One of the most common forms, oligoasthenozoospermia (low sperm count and poor motility), often cannot be explained by genetic mutations alone. Recent studies show that epigenetic regulation—chemical changes that control gene activity without altering the DNA sequence—plays a major role in male infertility. This review summarizes the latest scientific progress on how three major epigenetic mechanisms—DNA methylation, histone modification, and non-coding RNAs—affect sperm production and function. Abnormal patterns of DNA methylation or histone modification can disrupt sperm development, while changes in non-coding RNAs can damage sperm DNA and reduce fertility potential. The article also highlights single-cell sequencing technologies such as scRNA-seq and scATAC-seq, which can analyze gene activity and chromatin accessibility in individual sperm cells. These tools help scientists uncover how epigenetic abnormalities arise and identify specific cell types or developmental stages affected in infertility. From a clinical perspective, the review discusses how oxidative stress damages sperm DNA and how antioxidant therapy and varicocele repair can improve sperm quality by restoring epigenetic balance. It emphasizes the importance of lifestyle factors—such as diet, smoking, and environmental exposures—in influencing sperm health through epigenetic mechanisms. Overall, integrating epigenetic research with advanced single-cell technologies offers new opportunities to improve diagnosis, develop targeted therapies, and enhance reproductive outcomes for men with infertility.
Keywords
Background
In recent years, the prevalence of infertility has been increasing yearly worldwide and has become a public health problem. 1 Studies have shown that nearly half of all infertile couples are due to male factors. 2 Male infertility commonly manifests as oligozoospermia, asthenozoospermia, azoospermia, teratozoospermia, or increased sperm DNA fragmentation (SDF). 3 Spermatogenesis is a highly ordered and complex biological process. 4 It involves the self-renewal and differentiation of spermatogonia, meiosis of spermatocytes, and maturation of spermatids into spermatozoa.4,5 This process requires the precise synergy of multiple regulatory mechanisms, including genetic, epigenetic, endocrine, and local microenvironmental regulation.5,6 A growing body of evidence indicates that aberrant epigenetic modifications can disrupt the normal processes of spermatogenesis, resulting in spermatogenic disorders and male infertility.7,8 To date, genetic factors have been implicated in only a minority of male infertility cases, 9 suggesting that non-genetic mechanisms, particularly epigenetic regulation, may exert a more significant influence on its pathogenesis.
Epigenetic studies include, for example, DNA methylation, histone modifications, non-coding regulatory RNAs, and a range of other regulatory mechanisms involved in the remodeling of chromatin. 10 Over 170 chemical modifications have been identified in RNA, several of which are implicated in spermatogenesis, embryonic development, and disease processes. 11 Meanwhile, single-cell sequencing technology offers a novel approach to investigating the role of epigenetic regulation in spermatogenesis and infertility, as it enables the resolution of genetic and epigenetic information at the single-cell level. 12 Single-cell genome sequencing is a technique that can detect DNA mutations and chromosomal abnormalities. Single-cell technologies such as DNA methylation sequencing and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) provide insight into the regulatory landscape of chromatin, whereas single-cell RNA sequencing (scRNA-seq) captures the corresponding gene expression programs.13,14 The integration of genetic and epigenetic information, enhanced by single-cell multi-omics and spatial omics technologies, provides unprecedented resolution to uncover regulatory programs of gene expression and their essential roles in spermatogenesis and reproductive biology.15,16
In conclusion, the rising global prevalence of male infertility, coupled with its complex and multifactorial etiology, has highlighted the significant role of epigenetic modifications in sperm as a crucial factor influencing reproductive health. This review aims to provide a comprehensive overview of current knowledge regarding sperm epigenetics, emphasizing key mechanisms such as DNA methylation, histone modifications, and non-coding RNAs (ncRNAs), along with their direct and indirect effects on male fertility. Furthermore, we examine the promising potential of single-cell sequencing technologies, which are poised to yield deeper insights into the molecular mechanisms underlying these epigenetic changes. These technological advancements could substantially improve the diagnosis and therapeutic strategies for male infertility.
Impact of sperm epigenetics on male infertility
Sperm DNA methylation and oligoasthenozoospermia
Methylation is a major form of epigenetic modification of genomic DNA. It serves as a crucial regulatory mechanism for genomic function and happens while adding methyl groups to DNA, catalyzed by DNA methyltransferases (DNMTs).17,18 In mammals, DNA methylation occurs predominantly on cytosine 5′-C at the 5′-CpG-3′ site, forming 5-methylcytosine (5mC). Two main types of these modifications are identified: de novo methylation, which targets unmethylated DNA strands, and maintenance methylation, which ensures methylation of the daughter strand when the parent strand is already methylated. 19 Recent research suggests that aberrant DNA methylation may adversely affect male semen quality, particularly by reducing sperm concentration (oligozoospermia) and impairing progressive motility (asthenozoospermia). 20
DNA methylation regulates gene expression, chromatin remodeling, and genome stability from spermatogonial cells to mature sperm.16,21,22 During the early stages of meiosis, when DNA replication and recombination occur, the genome shows a hypomethylated state. 23 In human sperm, approximately 70% of the DNA is methylated, and this widespread methylation is essential for spermatogenesis and offspring vitality. 24 However, limited information exists on how DNA methylation and its disruption affect sperm development and function during human spermatogenesis.23,25 Transposable elements (TEs) make up a significant portion of the mammalian genome, and during meiosis, the genome relies heavily on TE suppression.26,27 TE silencing is predominantly maintained through epigenetic mechanisms, 28 and variations in TE methylation or dysfunction in silencing pathways are often linked to male infertility.29,30
The methylation status of spermatogenesis-related genes directly regulates their expression during sperm production. 31 Both hyper- and hypomethylation can dysregulate gene expression, thereby compromising spermatogenesis and sperm quality. For example, significant hypomethylation of the DAZ3 promoter, especially at CpG sites −246/−247, has been reported in patients with idiopathic asthenozoospermia. 32 Conversely, VDAC2 promoter hypermethylation has been observed in men with impaired spermatogenesis and reduced sperm function. 33 Kumar et al. 34 reported pronounced hypermethylation of the SPATA promoters in patients with oligoasthenozoospermia (OAT), potentially contributing to impaired spermatogenesis. Moreover, aberrant methylation of the imprinted gene H19 directly impairs spermatogenic capacity, resulting in defective spermatogenesis and reduced sperm counts. 35 Aberrations in DNMTs, the enzymes responsible for establishing and maintaining genome-wide DNA methylation patterns, are closely associated with male infertility. In testes from infertile men, expression of DNMT1, DNMT3A, and DNMT3B is reduced relative to fertile controls and is accompanied by alterations in genome-wide DNA methylation, supporting a model in which methylation-induced DNMT inactivation is a key pathogenic driver of male reproductive dysfunction. 36 Taken together, these studies indicate that DNA methylation modulates gene expression during spermatogenesis by maintaining appropriate activation and repression of key spermatogenic genes.
Abnormal DNA methylation may also perturb chromatin compaction and the homeostasis of DNA repair, thereby compromising genome stability and increasing susceptibility to DNA breaks. 37 SDF is a key indicator of sperm quality and is widely used in the diagnosis of male infertility.38,39 The DNA fragmentation index (DFI) correlates closely with sperm concentration, motility, and morphology.40,41 Accordingly, assays of DNA integrity reflect overall sperm quality. In sperm with high DFI, methylation of genes such as CD14, CDKN1B, and SOX9 is reduced and inversely correlates with functional impairment. 42 Methylation of IGF2 is negatively associated with DFI, whereas methylation of KCNQ1 shows a positive association.43,44 In summary, aberrant DNA methylation is associated with DFI. Altered methylation may modulate gene expression and sperm function, thereby contributing to elevated DFI and oligoasthenozoospermia.
Varicocele is a common cause of male infertility, affecting approximately 15% of men. 45 It is identified in 19%–41% of primary and up to 80% of secondary infertility cases.46,47 In high-grade varicocele, increased DNA damage and reduced fertility have been linked to abnormal sperm DNA methylation. 48 Varicocele pathophysiology involves elevated oxidative stress, which increases genomic instability and compromises chromatin integrity in sperm. Bahreinian et al. 49 observed that sperm from men with varicocele display pronounced DNA hypomethylation, higher SDF, and greater oxidative stress. Under hypomethylated conditions, sperm are more susceptible to DNA damage. Santana et al. 50 observed lower genome-wide sperm DNA methylation in varicocele and a positive correlation between disease severity and the extent of hypomethylation. Further analyses identified multiple differentially methylated regions linked to gametogenesis, meiosis, and semen quality in patients with varicocele. 48 In addition, varicocele may, via oxidative stress, nutrient restriction, and elevated calcium, reduce DNMT3A/3B activity or enhance demethylation pathways, thereby promoting sperm DNA hypomethylation 51 ; this mechanism requires further validation. 45 Animal models of varicocele have shown testicular upregulation of TET2, increased 5-hmC, and consequent hypomethylation with altered gene regulation. 52 To date, only one study has evaluated the impact of varicocele repair on sperm DNA methylation. Tavalaee et al. 53 reported no overall change in sperm DNA methylation after varicocelectomy; however, methylation levels improved in oligozoospermic men who were more severely affected by varicocele. These findings suggest that varicocelectomy may influence sperm epigenetic marks and warrant further study.
Role of histone modifications in regulating spermatogenesis
Histone proteins are essential chromatin components, primarily classified into five types: H1, H2A, H2B, H3, and H4. Among them, H2A, H2B, H3, and H4 form a highly conserved histone octamer, each contributing two molecules.54,55 In eukaryotes, DNA wraps around the histone octamer to form nucleosomes; linker histone H1 binds the linker DNA to stabilize higher-order chromatin. 42 Histone modification is a post-translational process that primarily targets the lysine-rich regions of histones, particularly H3 and H4, inducing covalent modifications.56,57 The main modifications include acetylation, phosphorylation, methylation, ubiquitination, and glycosylation. Histone modifications play a crucial role in spermatogenesis, fertilization, and embryonic development, 58 particularly in sperm meiosis. After meiosis, most histones are first replaced by transition proteins (TP1/TP2) and subsequently by protamines (PRM1/PRM2), which compact the paternal genome and protect DNA integrity. 59 This histone-to-protamine exchange is facilitated by stage-specific H4K5/K8/K12/K16 acetylation. 60
Histone acetylation is catalyzed by histone acetyltransferases, which transfer an acetyl group from acetyl-CoA to the ε-amino group of lysine residues on the N-terminal tails of core histones H2A, H2B, H3, and H4. 61 During spermatogenesis, HATs and histone deacetylases jointly regulate histone acetylation in sperm. Proper acetylation relaxes nucleosomes and facilitates transcription. 62 Histone H4 acetylation (e.g., H4K5ac, H4K8ac, H4K12ac, H4K16ac) displays stage-specific patterns during spermatogenesis and is essential for the histone-to-protamine transition. 63 Sirtuin 1 (SIRT1), a class III histone deacetylase, is expressed throughout spermatogenesis. Germ cell-specific deletion of Sirt1 reduces sperm counts and causes aberrant histone modifications, defective protamine replacement, disrupted chromatin remodeling, and abnormal spermatozoa.64,65 Moreover, SIRT1 levels are significantly lower in men with oligoasthenozoospermia and even lower in those with varicocele, further supporting its role in male germ cells. 66
Histone phosphorylation involves the transfer of a phosphate group from ATP to specific amino acid residues on histone proteins, catalyzed by protein kinases. 67 This modification participates in diverse cellular processes, such as chromosomal folding, condensation, and segregation; transcriptional activation and repression; cell signaling; regulation of apoptosis; metabolism; and DNA damage repair. 68 In male germ cells, numerous studies have demonstrated that genotoxic stress, such as ionizing radiation or chemotherapeutic agents, which induce DNA double-strand breaks (DSBs), also triggers phosphorylation of the histone variant H2AX at Ser139, resulting in the formation of γH2AX. 69 Furthermore, flow cytometric analysis has revealed significantly higher γH2AX levels in sperm from infertile men compared with healthy controls. Therefore, γH2AX quantification by flow cytometry may serve as a sensitive biomarker for DSBs in human sperm. 70
Histone methylation can occur on all basic amino acid residues, including arginine, lysine, and histidine. 71 Lysine may be mono- (me1), di- (me2), or tri-methylated (me3); arginine may be mono-methylated (me1), symmetrically di-methylated (me2s), or asymmetrically di-methylated (me2a); whereas histidine methylation is relatively uncommon. 72 Histone methylation is tightly linked to chromatin structure and function, regulating gene expression by either promoting or repressing transcription through multiple mechanisms. Studies have demonstrated that, compared with fertile men, sperm from infertile men display similar localization patterns of H3K4me3 and H3K27me3 but reduced retention of these marks at loci associated with developmental transcription factors and specific imprinted genes. Moreover, changes in the methylation status of several candidate developmental promoters and imprinting loci have been detected in infertile men. 73 Schon et al. 74 analyzed the relative abundance of post-translational modifications on histones H3 and H4 in semen samples from 31 men with normal or abnormal semen parameters using nano-liquid chromatography–tandem mass spectrometry. The analysis revealed reduced H4 acetylation and altered H3K9 methylation in semen samples from men with asthenozoospermia, indicating that histone PTMs are closely associated with sperm motility.
Histone ubiquitination refers to the covalent attachment of a small 76–amino acid protein, ubiquitin, to the ε-amino group of lysine residues on histones, mainly via an isopeptide bond. 75 During spermatid elongation, histones H2A, H2B, H3, and their variant TH2B undergo different levels of mono- and poly-ubiquitination. This modification loosens chromatin structure, promoting histone removal and subsequent degradation. 63 Studies have demonstrated that the RING finger E3 ligase RNF8 mediates histone ubiquitination in an enzyme activity–dependent manner, which subsequently promotes histone acetylation and facilitates histone eviction during spermatogenesis and the DNA damage response. 76 In RNF8-knockout mice, the absence of RNF8 leads to failed sperm maturation during spermatogenesis and male infertility, accompanied by a dramatic decrease in testicular histone ubiquitination. 77 Plant homeodomain finger protein 7 (PHF7), a newly identified RING-type E3 ligase, specifically catalyzes ubiquitination of histones H2AK119 and H3K14. 78 Disruption of PHF results in male infertility in mice, characterized by markedly reduced sperm motility and progressive movement. 79
A hallmark of spermiogenesis is the gradual transformation of the spherical sperm head nucleus into an ovoid or falciform shape. 80 Sperm-head shaping is central to spermatid differentiation, with histone–protamine replacement as a key step. 81 In this widespread chromatin-remodeling program, most histones are first replaced by testis-specific variants. TPs are then incorporated into the spermatid nucleus. Subsequently, PRMs replace TPs in late spermatids, packaging the genome into a highly condensed nucleus. 82 This process compacts chromatin, thereby protecting paternal DNA from environmental insults. TP1 and TP2 are the predominant transition proteins in spermatids. TP1 is a 6.2-kDa protein composed of approximately 20% arginine and 20% lysine, whereas TP2 is a 13-kDa protein containing approximately 10% arginine, 10% lysine, and 5% cysteine. 83 Mice lacking Tnp1 and Tnp2 exhibit severe spermatogenic defects, widespread reductions in sperm motility, and abnormal sperm morphology. The study further showed that single Tnp mutations produce phenotypes distinct from double-heterozygous mutations, underscoring both functional redundancy and unique roles of transition proteins in sperm development. 84
Most mammals express two protamine variants, Prm1 and Prm2. 85 These proteins bind tightly to DNA via a central, arginine-rich DNA-binding domain. 86 Maintenance of a species-specific Prm1:Prm2 ratio is essential for normal fertility.87,88 Alterations in the protamine ratio in mice and humans are linked to increased SDF, lower fertilization rates, and abnormalities in sperm morphology and motility.89,90 Recent biochemical and mass spectrometry analyses revealed that Prm1 and Prm2 from mature sperm carry multiple PTMs, including phosphorylation, acetylation, and methylation. 91 These modifications may confer lineage-specific functions. For instance, loss of lysine (K)49 acetylation on Prm1 markedly alters sperm chromatin composition and results in subfertility in mice. 90 Whether PTMs on human PRMs exert similar effects remains to be determined. 85 Numerous genes regulating histone–protamine replacement have been identified. Deletions or mutations in these genes can disrupt this process, leading to abnormal spermatogenesis and reduced male fertility or even infertility. For example, disruption of Fam170a enhances ubiquitination of H2A and H2B, accelerates removal of core histones, disturbs the timing of transition-protein expression and degradation, and decreases protamine incorporation during spermatogenesis. Collectively, these alterations impair histone–protamine exchange and cause defects in spermatogenesis. 92
During the histone-to-protamine transition, about 10%–15% of histones are retained in the human sperm genome. 93 These histones are mainly located at gene promoters and regulatory elements enriched with unmethylated CpG regions, indicating a role in transcriptional regulation and genome organization after fertilization. 93 The mechanisms governing histone retention during spermatogenesis remain unclear. 94 However, evidence supports a model in which histone variants H3.1, H3.2, and H3.3 are stably incorporated into CpG island nucleosomes. Epigenetic modifications on these histones, such as H3K4me3 and H3K27me3, together with the cessation of transcription and histone turnover during spermatogenesis, promote the retention of specific histone variants. 95 H4 tail acetylation and butyrylation dynamically compete at active promoters and are associated with histone eviction dynamics during spermatogenesis. 60 Furthermore, the transcription factors CTCF and BORIS may regulate histone retention through bipartite occupancy in testis-specific genomic regions, particularly those linked to spermatogenesis and transcriptional regulation. 96 These retained histones play a key role in mediating the paternal genome’s contribution to embryonic development. Paternal DNA packaging in sperm may transmit epigenetic information to the zygote during early embryogenesis.73,97 For example, elevated levels of histone H3K27me3 and H4K20me3 in sperm chromatin correlate with increased sperm DNA damage, reduced fertilization rates, and lower embryo quality. 98 Abnormal histone retention in sperm from men with recurrent pregnancy loss may profoundly affect sperm epigenetic imprinting patterns. Such disruptions may interfere with the finely tuned developmental processes of the embryo, ultimately resulting in pregnancy loss. 99
Non-coding RNAs and oligoasthenozoospermia
During transcription in the nucleus, RNA polymerase reads the DNA sequence to synthesize complementary messenger RNA (mRNA), which then passes through the nuclear pore into the cytoplasm and binds to ribosomes for protein synthesis. During translation, the ribosome sequentially scans codons along the mRNA, utilizing transfer RNA (tRNA) to deliver amino acids in the correct order for functional protein synthesis, ultimately leading to the expression of genetic information. 100 The complexity of higher organisms cannot be explained by protein-coding genes alone. 101 Recent high-throughput transcriptomic analyses have revealed that while up to 90% of the eukaryotic genome is transcribed, protein-coding sequences constitute only 1%–2% of the total genomic DNA, with the vast majority being ncRNA.102,103 Recent research has shown that ncRNAs, especially short-stranded ncRNAs, are essential for male fertility and actively participate in every stage of spermatogenesis.104,105
miRNA is found in mature sperm and male reproductive organs, including the testes, epididymis, and prostate. 106 Certain miRNAs are associated with sperm concentration, motility, and morphology. In comparison to semen from normal fertile men, semen from oligospermic patients shows higher levels of miR-22, miR-21, miR-375, miR-148a, miR-423-5p, miR-320a, miR-30a, miR-423-3p, and miR-221, while levels of miR-25, miR-122, miR-152, miR-34b, miR-192, and miR-335 are reduced. 107 Moreover, miR-26b-5p, miR-27a-3p, and miR-23b-3p are strongly positively correlated with sperm count, motility, and morphology. 108 The expression of hsa-miR-196a-2 (rs11614913) is altered in men with oligozoospermia. 109 A separate study shows that miR-525-3p directly regulates SEMG1 expression by binding to its 3′-UTR and is expressed at lower levels in asthenozoospermic patients. 110
SDF, characterized by single- or double-strand breaks, is one of the most common forms of nuclear damage. SDF and sperm miRNAs are key molecular regulators of male reproduction. 111 In the male reproductive system, altered expression of the miR-34/449 family can cause defective ciliary development in the ductal epithelium, leading to testicular dysfunction and sperm aggregation, and consequently reducing sperm concentration. In addition, abnormal ciliary development impairs flagellar formation, increasing morphological abnormalities and reducing sperm motility. 112 Interestingly, miR-449b-5p levels in sperm show a significant positive correlation with SDF. 111 Li et al. 113 reported that miR-374b and miR-26b are closely linked to apoptosis and DNA damage, and their altered expression may directly or indirectly contribute to SDF. One study revealed that miR-322 is downregulated in mouse seminal plasma, which may contribute to sperm DNA damage. 114 Further research demonstrated that miR-322 promotes GC-2 cell apoptosis by directly regulating Ddx3x expression. 115 Moreover, miR-424 is downregulated in infertile men and may induce spermatogenic cell apoptosis and DNA damage by directly targeting Ddx3x, contributing to male infertility. 115 Research on DNA fragmentation and ncRNA remains limited, and the underlying mechanisms are still unclear.
Varicocele-related male infertility is linked to the expression of several miRNAs. Neslihan et al. 116 found that miR-145 expression in sperm is reduced in varicocele cases. Studies have shown that in men with varicocele-related infertility, the expression of miRNA-122, miRNA-181a, and miRNA-34c5 in seminal plasma is significantly decreased. These miRNAs correlate positively with sperm concentration, motility, and normal morphology. 117 Moreover, miR-15a expression is significantly reduced in varicocele patients. miR-15a inhibits HSPA1B expression by directly binding to its 3′-UTR. HSPA1B is a stress-induced chaperone protein. Thus, it is inferred that miRNAs regulate sperm cell stress responses. 118 Interestingly, miR-210-3p expression in the seminal plasma of varicocele patients increases significantly with varicocele severity. This could serve as a useful clinical biomarker for screening sperm abnormalities caused by varicocele. 119
RNA molecules longer than 200 nucleotides are referred to as long non-coding RNAs (lncRNAs). 120 lncRNAs play a vital role in sperm biology, including sperm formation, maturation, and function. 121 Systematic studies of lncRNA expression profiles in human mature sperm reveal a correlation between lncRNA expression and sperm motility. For instance, lnc32058, lnc09522, and lnc98487 were upregulated in the same oligospermic sperm sample, and their expression levels correlated with progressive sperm motility. 122 Lu et al. 123 found that Linc00893 is downregulated in sperm samples from asthenozoospermic patients, and its expression positively correlates with sperm motility. NEAT1, a nucleus-enriched lncRNA that scaffolds paraspeckles, is highly conserved throughout evolution and widely expressed in mammalian cells, where it plays a role in spermatogenesis. 57 Studies show that NEAT1 is significantly downregulated in severely oligospermic individuals compared to fertile males. 124 Mahmoud et al. found that serum TUG1 levels in asthenozoospermic patients are abnormally correlated with total testosterone levels and sperm motility. 124 Studies have also shown that the average telomere length in oligospermic patients is shorter, and the level of long non-coding telomeric repeat-containing RNA correlates positively with progressive sperm motility in normal sperm donors. 125 These lncRNAs could serve as potential biomarkers for diagnosing various types of male infertility, including azoospermia, oligozoospermia, asthenozoospermia, and teratozoospermia.
PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that form piRNA-induced silencing complexes (piRISC) in the germlines of various animal species. 126 piRISC protects genomic integrity by silencing TEs. 126 Recent studies show that hsa-piR-002528 and hsa-piR-017183 are upregulated across all infertility subtypes (oligozoospermia, asthenozoospermia, and azoospermia), while hsa-piR-023244 and hsa-piR-023338 exhibit subtype-specific expression patterns. In the asthenozoospermia group, steroid 5α-reductase type 2 (SRD5A2) mRNA is upregulated and negatively correlates with hsa-piR-002528 and hsa-piR-023338, suggesting that its regulatory effect may influence sperm motility and count. 127 In addition to piRNAs, small RNAs derived from tRNA (tsRNAs) are also associated with oligozoospermia and asthenozoospermia. Neslihan et al. 128 found that the expression of 5′tRF-Glu-CTC in seminal plasma is significantly elevated in oligospermic individuals compared to those with normal sperm. 5′tRF-Glu-CTC is involved in ribosomal function and translation control. 128 Its expression varies under different pathophysiological conditions, making it a potential new marker of sperm quality and male fertility 128 (Figure 1).
Impact of sperm epigenetics on male infertility.
Single-cell sequencing technology
Single-cell sequencing is an emerging technology that resolves gene-expression and epigenetic states at single-cell resolution. It provides a powerful approach to elucidate the molecular mechanisms of spermatogenesis and oligoasthenozoospermia. 129 By integrating genomics, transcriptomics, and epigenomics, it enables dynamic mapping of regulatory networks across spermatogenic stages and identification of key determinants of male fertility. Among these methods, scRNA-seq and its derivatives are widely used for transcriptomic profiling. They delineate gene-expression patterns in germ cells across developmental stages and serve as core tools for studies of male reproductive biology. 130 Epigenetic profiling methods, such as the assay for transposase-accessible chromatin using sequencing (ATAC-seq), reveal chromatin accessibility and nucleosome positioning at single-cell resolution. 131 At present, single-cell studies in male fertility largely rely on scRNA-seq, whereas ATAC-seq is used less frequently. Nevertheless, because it requires small input, is experimentally simple and efficient, and offers high sensitivity, ATAC-seq shows considerable promise for probing chromatin accessibility and epigenetic abnormalities during spermatogenesis. 132 Looking ahead, integrative multi-omics analyses combining scRNA-seq and scATAC-seq will enable systems-level understanding of the links between epigenetic dysregulation and oligoasthenozoospermia.
Single-cell RNA sequencing
In the steady-state adult testis, spermatogenesis is highly ordered but shows pronounced temporal and spatial heterogeneity. 4 Conventional bulk sequencing cannot resolve intra- and intercellular variation and may miss rare populations, such as late-lineage SSCs or transitional cell types, because signal averaging obscures their signals. 133 scRNA-seq overcomes these limitations by quantifying mRNA across spermatogenic cells, thereby detecting lineage variation and stage-specific heterogeneity. 134 scRNA-seq also facilitates pathological analysis, particularly in human biopsy specimens. Wang et al. performed scRNA-seq on 174 testicular cells from non-obstructive azoospermia (NOA) donors and, using a normal spermatogenesis dataset as reference, identified missing cell types and differentially expressed genes in the remaining cell types. 135 Zhao et al. 136 analyzed >80,000 single-cell transcriptomes from 10 healthy donors and 7 NOA patients. In congenital cases (Klinefelter syndrome and Y-chromosome microdeletions), Sertoli cells were mature but showed aberrant immune responses, whereas cells in idiopathic NOA (iNOA) were physiologically immature. They further showed that inhibiting Wnt signaling promotes maturation of iNOA Sertoli cells, restoring their ability to support germ-cell survival. 136 Additional studies reported that the WNT activator RSPO2 and the receptor LGR4 are expressed in human testicular germ cells and peritubular myoid cells, respectively, suggesting that Wnt signaling may regulate spermatogenesis via peritubular myoid cells. However, its specific role in germ-cell proliferation and differentiation remains unclear. 137 Using scRNA-seq, Chen et al. 138 found that, relative to healthy testes, spermatocytes in obstructive azoospermia testes show attenuated meiosis and increased apoptosis, ultimately leading to loss of spermatids. In OAT, scRNA-seq datasets remain scarce. This scarcity is expected because scRNA-seq typically requires testicular tissue from sperm-retrieval procedures such as TESE or microdissection TESE (mTESE), whereas many OAT patients undergo in vitro fertilization (IVF) using ejaculated sperm. Only severe OAT cases generally require testicular sperm extraction.138,139 Thus, single-cell transcriptomics helps elucidate disease mechanisms and therapeutic targets across male infertility, offering new avenues for diagnosis and treatment.
Single-cell assay for transposase-accessible chromatin using sequencing
The scATAC-seq uses the Tn5 transposase to insert adaptors into accessible chromatin regions, enabling genome-wide capture and sequencing of open chromatin. 140 This method profiles open chromatin at the single-cell level and evaluates chromatin accessibility across the genome. By analyzing data from hundreds to thousands of cells, it reveals cell-to-cell variability in chromatin organization and maps regulatory landscapes at single-cell resolution. 141 In spermatogenesis research, scATAC-seq has elucidated the relationship between chromatin architecture and transcriptional activity. Maezawa et al. employed ATAC-seq to map chromatin-accessibility dynamics from spermatogonia to spermatids, revealing stage-specific chromatin reprogramming during spermatogenesis. 132 At the onset of meiosis, somatic-type accessible regions in early spermatogonia that maintain stemness and proliferation gradually close, whereas new meiotic-accessible regions emerge, activating transcription factors such as A-MYB and RFX2. In late spermatids, chromatin becomes highly compacted, retaining only limited open regions that support genes essential for sperm maturation. Dynamic remodeling of open chromatin is closely associated with redistribution of active histone marks (e.g., H3K4me3 and H3K27ac), suggesting that chromatin accessibility reprogramming drives transcriptional diversity during spermatogenesis. 132 Using scATAC-seq, Wu et al. analyzed 5300 testicular cells from three healthy men and demonstrated pronounced chromatin-accessibility dynamics during human spermatogenesis. As spermatogonial stem cells progress to the pachytene stage, the number of accessible regions first increases and then gradually decreases. Genome-wide analysis revealed significantly higher chromatin accessibility on chromosomes 19 and 17. Across developmental stages, binding sites for transcription factors such as CTCF, GLI3, DMRT6, NFY, and ISL1 were enriched in accessible chromatin. 142 In summary, scATAC-seq has revealed dynamic chromatin remodeling during human spermatogenesis, including alterations in both chromatin accessibility and gene expression. ATAC-seq enables genome-wide mapping of open chromatin regions in specific temporal and spatial contexts, facilitating the identification of transcriptional genes and their cis-regulatory elements. 143
scRNA-seq and scATAC-seq
Combined scRNA-seq and scATAC-seq offer unprecedented resolution for elucidating the complex epigenetic mechanisms underlying spermatogenesis. 14 Their integration enables joint analysis of active regulatory sequences and transcripts at the single-cell level, facilitating the identification of upstream regulatory elements of target genes. In human adult hSSCs, ATAC-seq and DNA methylation analyses on SSEA4+ cells, combined with scRNA-seq and bulk RNA-seq, systematically mapped the relationship between chromatin accessibility and transcriptional activity across developmental stages, revealing pronounced spatiotemporal specificity. 144 In mice, ATAC-seq analysis of spermatocytes and spermatids revealed that GCN5 deficiency markedly reduces genome-wide chromatin accessibility, especially at promoters and enhancers of spermatogenesis-related genes. Integration with RNA-seq further showed that reduced chromatin accessibility strongly correlates with downregulation of spermatogenic genes such as Prm1, Tnp1, and Odf2. These findings suggest that GCN5 regulates nucleosome dynamics through H3K9/K14 acetylation, maintaining chromatin accessibility and transcriptional activity as a central epigenetic regulator in spermatogenesis. 145 In human single-cell datasets, Wu et al. applied scATAC-seq to characterize chromatin organization and accessibility in normal males. They found that TLE3 was specifically upregulated in differentiating spermatogonia, maintaining the balance between spermatogonial stem cells and their differentiating counterparts. PFN4 exhibited high chromatin accessibility during the pachytene/diplotene and round spermatid stages, participating in actin cytoskeleton remodeling and meiotic regulation. Integrative analysis of scATAC-seq and scRNA-seq data revealed a strong positive correlation between chromatin accessibility and gene expression. In differentiating spermatogonia and pachytene/diplotene stages, genes with increased accessibility—such as ZNF207, CBX5, TLE3, and PFN4—were synchronously upregulated, indicating that chromatin opening directly promotes activation of spermatogenesis-related genes. 141
In summary, single-cell multi-omics approaches provide a comprehensive framework for elucidating molecular mechanisms underlying disease and reproductive development. scRNA-seq has uncovered previously unrecognized testicular cell populations involved in spermatogenesis. In parallel, scATAC-seq maps chromatin accessibility at single-cell resolution, enabling identification of transcription factors active during meiosis. 146 Spatial transcriptomics integrates gene expression with tissue architecture, compensating for the inherent absence of spatial context in single-cell methods. 146 Together, these technologies enable detailed exploration of male infertility mechanisms at single-cell resolution. With continued advancements in sequencing and analytical technologies, their applications in reproductive epigenetics are expected to expand further.
Epigenetic perspective on treating oligoasthenozoospermia
Oligoasthenozoospermia arises from multilayered molecular abnormalities, including gene-expression imbalance, epigenetic dysregulation, and disruption of the spermatogenic microenvironment. Epigenetic stability of sperm is indispensable for maturation, fertilization competence, and early embryonic development. Accordingly, enhancing this stability may improve clinical outcomes.
Currently, antioxidants are widely used as empirical therapy for oligoasthenozoospermia. Common agents include vitamins C and E, selenium, and coenzyme Q10. Growing evidence indicates that oxidative stress is a central driver of genomic damage and epigenetic alterations in sperm. 147 Heightened oxidative injury disrupts chromatin architecture and topology and interferes with the binding of epigenetic regulators. It also perturbs DNA-methylation patterns and the balance between 5mC and 5hmC, and damages up to 18,000 sperm-delivered mRNAs and sncRNAs, thereby increasing DNA fragmentation, lowering fertilization rates, and compromising embryo quality. 147 Targeted antioxidant therapy can reduce the accumulation of oxidative DNA damage in sperm. 148 By attenuating oxidative damage, antioxidant regimens can protect the paternal genome, preserve critical epigenetic marks, and improve reproductive outcomes in natural conception and assisted reproductive technologies (ART). However, in control mice with normal redox status, high doses of redox-active agents markedly disrupted systemic redox balance and impaired sperm DNA integrity. 149 These findings underscore the bidirectional risk of oxidative versus reductive stress, with potential perturbations to 5mC/5hmC balance and epigenetic stability. Therefore, using reliable biomarkers to directly assess DNA damage or oxidative-stress levels, and to guide the selection and dosing of antioxidant therapy, may improve sperm quality and help ensure the genetic and epigenetic health of offspring. 147
Varicocele is a common etiologic factor in oligoasthenozoospermia. 150 Oxidative stress is a central mediator of its pathophysiology. 151 Infertile men with or without varicocele exhibit elevated reactive oxygen species (ROS), and this pattern indicates that varicocele increases ROS production and exacerbates oxidative damage. 152 The presence of varicocele often correlates with greater oxidative-stress severity. 152 Substantial evidence shows that varicocelectomy reduces oxidative stress, and oral antioxidants, which have favorable safety profiles, can serve as adjuncts to surgery. 153
For severe oligoasthenozoospermia, ART offers a viable path to conception. Pharmacologic and surgical interventions can improve sperm quality; however, in some patients, genetic and epigenetic sperm abnormalities are difficult to fully reverse. Consequently, these abnormalities may persist despite ART and impair post-fertilization genome reprogramming and early embryonic development, underscoring the critical role of paternal epigenetic status in determining ART outcomes. 97 Studies have documented aberrant methylation of imprinting genes (e.g., H19, IGF2) in oligoasthenozoospermia; these changes may be transmitted through IVF and disrupt early embryogenesis. 154 Additional reports describe altered imprint methylation and differential methylation at Alu repeats in sperm from men with abnormal semen parameters, findings potentially associated with ART outcomes. 155 Beyond DNA methylation, sperm small noncoding RNAs (sncRNAs) also influence ART outcomes. Hua et al. showed that differentially expressed tsRNAs, rsRNAs, and miRNAs correlate with embryo quality, and that tsRNAs and rsRNAs may serve as clinical biomarkers for assessing sperm quality in IVF. 156 In sum, epigenetic regulation is indispensable for maintaining appropriate gene expression under physiological conditions. Although ARTs, including intracytoplasmic sperm injection (ICSI) and IVF, expand reproductive options for many infertile couples, the transmission of aberrant epigenetic marks can increase the risk of subfertility and other adverse health outcomes in offspring.
Lifestyle interventions are the cornerstone of oligoasthenozoospermia management, intended to optimize reproductive health and increase natural conception rates. The epigenetic basis of male infertility is partly driven by extratesticular factors. External factors, such as environmental toxins, poor dietary habits, tobacco use, alcohol intake, and psychosocial stress, can trigger epigenetic alterations that disrupt normal spermatogenesis. Evidence demonstrates that exposure to endocrine-disrupting chemicals, including phthalates, modifies sperm DNA methylation patterns, leading to decreased motility and diminished fertility potential. 157 Similarly, oxidative stress associated with lifestyle factors alters histone acetylation and DNA methylation, thereby impairing chromatin remodeling during spermatogenesis. 6 Smoking is associated with hypomethylation of the H19 imprinting control region (H19-ICR) in oligozoospermia and hypermethylation of the SNRPN imprinting control region (SNRPN-ICR) in asthenozoospermia, alterations linked to male infertility. 158
Conclusion and future perspectives
This review synthesizes evidence on how diverse sperm epigenetic factors regulate genes critical to male fertility and highlights the central roles of epigenetic alterations in male germ-cell development. Current studies show that aberrant epigenetic processes, particularly DNA methylation errors and abnormal histone modifications, are linked to impaired spermatogenesis, reduced fertilization competence, and compromised early embryonic development. Nevertheless, the molecular architecture underlying male infertility and defective spermatogenesis remains incompletely defined. Identifying the principal molecular drivers and modifying factors of male infertility remains a major challenge in reproductive medicine. 159
When studying epigenetic changes in human sperm, investigators must account for methodological limitations. A comprehensive accounting of genetic and epigenetic contributions is essential for improving diagnosis. Single-cell sequencing is likely to be particularly transformative. Single-cell multi-omics provides high-resolution views of disease mechanisms and reproductive development. scRNA-seq has uncovered previously unrecognized testicular cell populations involved in spermatogenesis. In parallel, scATAC-seq maps chromatin accessibility at single-cell resolution, enabling identification of transcription factors active during meiosis. Integrating these data with spatial transcriptomics will restore the spatial context lacking in single-cell assays. Together, these technologies deepen understanding of human spermatogenesis and enable identification of molecules implicated in male germ-cell development. They also provide powerful tools to investigate reproductive disorders and open avenues to elucidate disease etiology and to develop preventive and therapeutic strategies.
The etiology of oligoasthenozoospermia is multifactorial, and preserving sperm epigenetic stability is crucial for maturation, fertilization competence, and early embryonic development. As a regulator of normal cellular development, epigenetics also offers opportunities for therapeutic innovation. With deeper insights into spermatogenesis, restoring this process via in vivo or ex vivo gene therapy and pharmacogenomic approaches is becoming plausible. Overall, epigenetic alterations are frequent in male infertility but are unlikely to be the sole determinants. Leveraging emerging technologies such as single-cell sequencing will be essential to define more precisely the role of epigenetics in male infertility. Clarifying these relationships will refine current therapeutic strategies and catalyze the development of new interventions.
