Epigenetic effects of Qixiong Formula on sperm DNA methylation in a rat model of asthenozoospermia

Introduction

Male fertility is declining at an alarming rate due to changes in both environmental and lifestyle factors. Among the leading causes of male infertility is asthenozoospermia, a condition characterized by reduced sperm motility. According to the 6^th edition of World Health Organization (WHO) guidelines, asthenozoospermia is defined as a condition where the proportion of progressively motile sperm (PR) in semen is less than 32%, or less than 40% of the total of progressively motile sperm and non-progressive motile sperm (PR + NP) (1,2). The increasing incidence of this condition is closely associated with environmental pollution, unhealthy lifestyle habits, and delayed childbearing. Currently, approximately 8–12% of couples of reproductive age experience fertility issues (3), and male factors account for nearly half of these cases (4). In couples affected by asthenozoospermia, the likelihood of natural conception is significantly reduced, and if conception occurs, there is a heightened risk of complications such as abnormal embryonic development and miscarriage. Although the aetiology of asthenozoospermia is multifactorial, recent research has highlighted the involvement of epigenetic dysregulation, impaired energy metabolism, altered signal transduction, and structural abnormalities of sperm flagella (5-7). In particular, aberrant DNA methylation has emerged as a key area of interest in understanding the underlying mechanisms of reduced sperm motility.

DNA methylation is one of the most studied epigenetic modifications, involving the transfer of a methyl group from S-adenosyl methionine to cytosine residues in DNA, a reaction catalysed by DNA methyltransferases (DNMTs) (8-10). While classical genetic theories have long emphasised the central role of genes in regulating spermatogenesis and sperm maturation, emerging evidence indicates that epigenetic mechanisms, particularly DNA methylation, play a critical role in the regulation of gene expression during chromosomal recombination, segregation, and meiosis (11-13). Increasing evidence suggests that aberrant DNA methylation patterns are associated with impaired spermatogenesis and reduced semen quality. For instance, one study identified 696 CpG island DNA methylation levels that were associated with spermatogenesis, and found that the levels of DNA methylation in sperm of fertile men and male infertility patients were significantly different (14). Notably, abnormal methylation levels at imprinted genes such as MEST, H19, and IGF2 have been observed in patients with oligoasthenozoospermia (14). These findings support the hypothesis that defective sperm DNA methylation may contribute to reduced sperm motility.

Traditional Chinese medicine (TCM) has a long-standing history in the treatment of male infertility. Qixiong Formula (QXF; originally named Qixiong Zhongzi Decoction) is a well-established herbal prescription used at the Department of Andrology, Xiyuan Hospital, China Academy of Chinese Medical Sciences, specifically for the treatment of asthenozoospermia. In TCM theory, this formula is traditionally known to support reproductive health by “tonifying the Spleen and Kidney” (core TCM concepts linked to vital energy, nutrient metabolism, reproductive function), improving blood circulation, and thereby promoting spermatogenesis. A previous randomized controlled clinical trial has demonstrated that QXF can significantly improve PR and PR + NP in patients with asthenozoospermia with a favourable safety profile and no significant adverse effects (15). However, the specific mechanism by which QXF exerts its effects remains unknown. In this study, we aim to explore the potential mechanisms of QXF in the treatment of asthenozoospermia using a rat model, with a focus on changes in semen parameters, testicular histopathology, and sperm DNA methylation levels. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-462/rc).

Methods

Ethics approval

All animal experiments were performed under a project license (No. 2020XLC004-3) granted by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences, in compliance with China national and institutional guidelines for the care and use of animals.

Experimental materials

The research plan was formulated prior to the initiation of the experiment and is not subject to registration requirements. Clean-grade male Sprague-Dawley (SD) rats, weighing 180–220 g, were obtained from the Experimental Animal Center, Xiyuan Hospital, Chinese Academy of Chinese Medical Sciences {(Experimental Animal Production License: SCXK(Beijing)2019-0010}. Animals were housed under standard laboratory conditions with a 12-h light/12-h dark cycle, a controlled temperature of 18–23 ℃, and free access to food and water. Drinking water was replaced daily, and bedding was changed every other day to maintain hygienic conditions.

Ornidazole (ORN) capsules were obtained from Xi’an Wanlong Pharmaceutical Co., LTD (approval No. H20031257) and used to induce asthenozoospermia in rats. QXF was prepared by the Decoction Room of Xiyuan Hospital. The composition and dosage of QXF are: stir-fried Astragalus membranaceus (20 g), Rehmannia glutinosa (prepared rhizome, 15 g), Dioscorea opposita (Chinese yam, 10 g), Fructus corni (Cornelian cherry fruit, 10 g), Ligusticum chuanxiong (10 g), Cuscuta chinensis (10 g), Cructus lycii (goji berry, 15 g), etc., a total 129 g of crude drug. The clinical human equivalent dose was calculated based on a standard body weight of 70 kg, corresponding to 1.84 g/kg/day of crude drug. According to standard dose conversion for rats (6.3 times the human dose), the medium dose used in the experiment was 11.59 g/kg/day. Low and high doses were set at 5.79 and 23.18 g/kg/day, respectively.

Animal grouping, modeling and intervention

Sixty male SD rats, aged 6–8 weeks, were weighed using a standard electronic scale and individually numbered. They were randomly assigned to five groups (n=12 per group) using the random number table method: normal group, model group, QXF low-dose group (QXF-L), QXF medium-dose group (QXF-M) and QXF high-dose group (QXF-H).

To induce asthenozoospermia, ORN was freshly prepared each day by dissolving it in 0.5% carboxymethyl cellulose sodium (CMC-Na). Based on a previously established and validated rat model, the model group and all the intervention groups QXF-L, QXF-M and QXF-H) received ORN via oral gavage once daily for 28 consecutive days at a dose of 400 mg/kg (16). Rats in the QXF intervention groups were additionally treated with QXF at different doses: QXF-L (5.79 g/kg/day), QXF-M (11.59 g/kg/day), and QXF-H (23.18 g/kg/day). Based on the preliminary modeling and relevant research on TCM (17), the QXF solutions were administered by gavage once daily (1 mL/100 g body weight) for 28 days, concurrently with ORN. The normal control group received distilled water alone, while the model group was administered ORN with distilled water (no QXF).

Materials

Twenty-four hours after the last administration, all rats were weighed and anesthetized by intraperitoneal injection of 1% pentobarbital sodium at 0.04 g/kg. After fixation, the abdominal area was shaved and disinfected with 75% ethanol solution. A midline laparotomy was performed. and 3 mL of blood samples was collected from the abdominal aorta. The blood samples were allowed to clot at room temperature for 5 min, followed by centrifugation at 3,000 rpm for 15 min at 5 ℃. The resulting serum was collected and stored at −20 ℃. Subsequently, the scrotum was incised, and both testes and epididymis were carefully removed. Tissues were rinsed in cold saline, gently blotted dried with filter paper, placed on an ice tray. Surrounding connective tissues were removed, and weights of testes and epididymis recorded using an electronic balance.

Detection of semen parameters

Semen parameters were evaluated according to the method described by Alizadeh et al. (18). Briefly, the left epididymal tail was placed in normal saline containing 0.5% bovine serum albumin (BSA), and the suspension was filtered to remove tissue debris. The sperm suspension was placed in an incubator at 37 ℃ and incubated before further analysis. The detection of sperm parameters (including sperm concentration and motility) via the Computer-Aided Sperm Analysis (CASA) system serves as the primary indicator in this study.

Determination of organ coefficient and histopathological observation

The weight of both testes and epididymis was recorded, and the organ coefficient was calculated as follows: organ (mg/100 g) = bilateral organ weight (mg)/body weight (g) × 100%. For histopathological observation, testes and epididymal were fixed in Bouin’s solution for 24 h, then dehydrated, embedded in paraffin, and sectioned at a thickness of 4 µm. Sections were mounted and baked at 67 ℃ for 1 h, followed by standard hematoxylin and eosin (H&E) staining. The procedure included dewaxing with xylene, sequential hydration in 100%, 95%, 80% and 70% ethanol, hematoxylin staining, rapid differentiation in 1% hydrochloric acid ethanol, and bluing under running water. After counterstaining with 0.1% eosin, slides were dehydrated in gradient ethanol, cleared with xylene, and sealed with coverslips. Histological changes in testicular and epididymal tissue sections were observed under light microscope.

Detection of liver and kidney function

Serum levels of creatinine (Cr), blood urea nitrogen (BUN) and urea were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, the ELISA plates were coated with specific capture antibodies diluted in coating buffer solution and incubated overnight at 4 ℃. After washing, 200 µL of serum samples containing the target antigen were added to each well and incubated at 37 ℃ for 1–2 h. Following washing, 200 µL of enzyme-conjugated detection antibodies were added and incubated for an additional 1–2 h at 37 ℃. Substrate solution (200 µL) was then added and allowed to react at room temperature for 30 min. The reaction was stopped by adding stop solution, and optical density (OD) was measured at 450 nm using a microplate reader. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined by colorimetric assay, expressed as IU/L. A volume of 2.5 µL of serum was added to 12.5 µL of ALT or AST substrate and incubated at 37 ℃ for 1 h. Then, 25 µL of 0.2% 2,4-dinitrophenylhydrazine in 37% hydrochloric acid was added and incubated for 20 min under room temperature. Finally, 250 µL of 0.4 M NaOH was added, incubated for 30 min, and the absorbance was measured at 492 nm using a spectrophotometer.

Methylation status of sperm imprinted genes

The contralateral epididymis was collected and placed in 0.5% phosphate-buffered saline (PBS) solution. The tissue was allowed to precipitate at 4 ℃ for 4 h. The suspension was then centrifuged at 3,000 rpm for 15 min, after which the supernatant was discarded and the sperm pellet retained for further processing. Genomic DNA was extracted using a commercial DNA extraction kit, quantified and stored at −80 ℃ until further analysis. A total of 200 ng genomic DNA per sample was used for RRBS Library construction using the Acegen Rapid RRBS Library Prep Kit (Acegen, Suzhou, China, cat. No. AG0422). Genomic DNA was digested with MspI, end-repaired, adaptor-ligated, and subjected to bisulfite conversion, followed by polymerase chain reaction (PCR) amplification and purification. The qualified libraries were sequenced. The high-quality original sequencing data reads were used to compare the reference genome. The loci with the sequencing depth of more than 5× were selected to calculate the methylation level. Cluster analysis of genome-wide methylation levels was performed using principal component analysis (PCA), followed by analysis of differentially methylated regions (DMRs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were subsequently conducted to identify functional categories associated with the DMRs.

Statistical analysis

All experimental data in this study were analyzed and processed by SPSS 21.0 statistical software. Data were first assessed for normality and homogeneity of variance. For normally distributed data with equal variances, one-way analysis of variance (ANOVA) was used, followed by least significant difference (LSD) or Student-Newman-Keuls (SNK) post hoc tests for multiple comparisons. If variances were unequal, Welch’s ANOVA was applied, followed by the Games-Howell test. For non-normally distributed data, non-parametric tests were used. A P value <0.05 was considered statistically significant; P<0.01 was considered highly significant.

Results

Sperm concentration and motility

Compared with the normal group, the model group showed no significant change in sperm concentration (P>0.05, P=0.07), but exhibited a significant decrease in PR + NP levels (P<0.01, P=0.001), confirming the successful induction of asthenozoospermia model using ORN. Treatment with QXF at low, medium, and high doses did not significantly alter sperm concentration relative to the model group (P>0.05, P=0.73, P=0.82, P=0.82); compared with the model group, there was no significant difference in PR + NP levels between the low-dose group (P>0.05, P=0.52), while there was a significant difference in PR + NP levels between the medium dose and high-dose groups (P<0.05, P=0.042, P=0.04), but there was no dose-dependent effect (Figures 1,2).

Figure 1 Seminal concentrations of rats in each group. QXF-L: QXF low-dose group; QXF-M: QXF medium-dose group; QXF-H: QXF high-dose group. QXF, Qixiong Formula.

Figure 2 Comparison of sperm motility between rats in each group. QXF-L: QXF low-dose group; QXF-M: QXF medium-dose group; QXF-H: QXF high-dose group. *, P<0.05, compared with the normal group; **, P<0.01, compared with the normal group; ^#, P<0.05, compared with the model group; ^##, P<0.01, compared with the model group. NP, non-progressive motile sperm; PR, progressively motile sperm; QXF, Qixiong Formula.

Coefficient of testis and epididymis

There was no significant difference in body weight among the groups (Figure 3). In terms of testicular coefficient, there was no significant difference among all groups. However, epididymal coefficients were significantly reduced in the model group compared to the normal group (P<0.05, P=0.046). While no significant differences were observed between the QXF-L groups versus the model group (P>0.05, P=0.12), the QXF-M and QXF-H group showed a statistically significant increase in epididymal coefficient compared to the model group (P<0.05, P=0.045, P=0.04).

Figure 3 Comparison of epididymis and testicle coefficients in each group. QXF-L: QXF low-dose group; QXF-M: QXF medium-dose group; QXF-H: QXF high-dose group. *, P<0.05, compared with the normal group; ^#, P<0.05, compared with the model group. QXF, Qixiong Formula.

Histopathologic evaluations

The testicular tissue of rats in each group exhibited intact basic architecture. The seminiferous tubules were lined by a uniformly thick and plump epithelium, characterized by abundant cellular stratification and tightly ordered arrangement. The tubular lumina contained abundant, densely packed mature spermatozoa with normal morphology. The interstitial tissue showed no signs of edema or fibrosis, and the Leydig cells, distributed in clusters, displayed a plump morphology without significant abnormalities (Figure 4). In the normal group, the epididymal tubules were arranged in a regular and orderly manner with morphologically intact epithelial cells, and abundant sperm were observed within the lumen. In the model group, the number of sperm in the lumen of the epididymal tubules was reduced, accompanied by a noticeable increase in degenerated sperm. Compared to the model group, all QXF treatment groups showed a reduction in the number of degenerated sperm. Moreover, this improvement was dose-dependent, with higher doses of QXF leading to a further decrease in sperm degeneration (Figure 5).

Figure 4 Histopathological [photos of the testicular of each group of rats (HE staining, ×200). (A) Normal group. (B) Model group. (C) QXF low-dose group. (D) QXF medium-dose group. (E) QXF high-dose group. HE, hematoxylin-eosin; QXF, Qixiong Formula.

Figure 5 Histopathological photos of the epididymis of each group of rats (HE staining, ×200). (A) Normal group. (B) Model group. (C) QXF low-dose group. (D) QXF medium-dose group. (E) QXF high-dose group. HE, hematoxylin-eosin; QXF, Qixiong Formula.

The liver and kidney function

Compared with the normal group, in the model groups there were no significant differences in the serum levels of liver and kidney function parameters, such as urea, BUN, Cr, ALT and AST (P>0.05, P=0.88, P=0.62, P=0.81, P=0.45, P=0.39). Similarly, compared with the model group, there was no statistically significant difference in urea (P>0.05, P=0.91, P=0.67, P=0.89), BUN (P>0.05, P=0.78, P=0.83, P=0.92), Cr (P>0.05, P=0.61, P=0.57, P=0.49), ALT (P>0.05, P=0.72, P=0.68, P=0.85), and AST (P>0.05, P=0.58, P=0.79, P=0.64) in the intervention group (QXF-L, QXF-M, and QXF-H), as shown in Table 1.

Methylation

Global methylation patterns

Genome-wide methylation differences between groups

Comparative methylation analysis was performed to analyze the methylation levels between groups. This included methylation level distribution across the whole genome, within gene functional regions, and in the 2 kb upstream and downstream regions of genes. As shown in Figure 6, the number of hypermethylated genes in the model group was greater than the number of hypomethylated genes, suggesting overall hypermethylation in the ORN-induced asthenozoospermia model. Similarly, when comparing each QXF-treated group to the model group, hypermethylated genes outnumbered hypomethylated genes. Notably, the QXF high-dose group (QXF-H) showed fewer differentially methylated genes (DMGs) compared to the low- and medium-dose groups, suggesting a stabilising effect at higher doses.

Figure 6 Comparison of methylation levels of each component. QXF-L: QXF low-dose group. QXF-M: QXF medium-dose group; QXF-H, QXF high-dose group. DMR, differentially methylated region; QXF, Qixiong Formula.

Average methylation level in DMRs

To further visualise methylation alterations, average methylation levels within DMRs were calculated and used to generate cluster heat maps (Figure 7). The model group exhibited markedly higher methylation levels compared to the normal group. In contrast, methylation levels in the QXF-L, QXF-M, and QXF-H groups were significantly reduced relative to the model group, indicating a demethylating effect of QXF across all treatment doses (Figure 7).

Figure 7 Clustering heat maps of DMR methylation levels among groups. (A) Model vs. normal. (B) Model vs. QXF-L. (C) Model vs. QXF-M. (D) Model vs. QXF-H. The abscissa represents the comparative combination group, the ordinate represents the clustering effect of methylation level value, and the blue to red represents the low to high methylation level. QXF-L: QXF low-dose group. QXF-M: QXF medium-dose group; QXF-H, QXF high-dose group. DMR, differentially methylated region; QXF, Qixiong Formula.

Functional enrichment of DMGs

GO enrichment analysis

GO enrichment analysis was performed on DMR-associated genes located in genebody regions and within ±2 kb of the transcription start and end sites. GO terms were categorised into biological processes, cellular components, and molecular functions. Results were filtered based on criteria (observed >2, fold change >2, adjusted P<0.05) and ranked by significance. The top GO terms included: “Model vs. Normal”: ATP binding, perikaryon, positive regulation of transcription by RNA polymerase II; “QXF-L vs. Model”: PDZ domain binding, receptor complex, nervous system development; “QXF-M vs. Model”: ATP binding, perikaryon, memory; “QXF-H vs. Model”: sequence-specific DNA binding, receptor complex (Figure 8).

Figure 8 Bar graph of GO enrichment analysis of DMR genes in each group. (A) Model vs. normal. (B) Model vs. QXF-L. (C) Model vs. QXF-M. (D) Model vs. QXF-H. The ordinate is the enriched GO term, and the x-coordinate is the −log₁₀ (adjP). The longer the column is, the more significant the difference is in the statistical test of the GO term. The numbers on the right of the column indicate the number of genes enriched and the enrichment multiple. QXF-L: QXF low-dose group. QXF-M: QXF medium-dose group; QXF-H, QXF high-dose group. BP, biological process; CC, cell component; DMR, differentially methylated region; GO, Gene Ontology; MF, molecular function; QXF, Qixiong Formula.

KEGG pathway enrichment analysis

KEGG enrichment analysis was performed using metrics including enrichment fold change, Q value, and the number of DMR-associated genes involved in each pathway. Twenty key biological pathways were identified across group comparisons. Pathways most relevant to sperm function and showing the highest enrichment significance (highlighted in red in Figure 9) included: “Model vs. Normal”: cAMP signalling pathway, focal adhesion, ECM-receptor interaction, calcium signalling pathway; “QXF-treated groups”: cAMP, Rap1, PI3K-Akt, and calcium signalling pathways.

Figure 9 Bubble plots of KEGG metabolic pathways enriched by DMR genes in each group. (A) Model vs. normal. (B) Model vs. QXF-L. (C) Model vs. QXF-M. (D) Model vs. QXF-H. The vertical axis represents pathway name, and the horizontal axis represents fold change. The size of the dots represents the number of DMR-related genes in this pathway, and the color of the dots corresponds to different adjP ranges. QXF-L: QXF low-dose group. QXF-M: QXF medium-dose group; QXF-H, QXF high-dose group. DMR, differentially methylated region; KEGG, Kyoto Encyclopedia of Genes and Genomes; QXF, Qixiong Formula.

Discussion

Infertility affects over 15% of couples worldwide, with male factors contributing to more than 50% of cases. Despite ongoing research, the aetiology of male infertility remains elusive in approximately 30–40% of patients, leaving a significant proportion without a clearly defined cause (11). In particular, asthenozoospermia, defined by reduced sperm motility with normal or near-normal sperm concentration, is one of the leading causes of male subfertility. While assisted reproductive technologies (ARTs) such as intracytoplasmic sperm injection (ICSI) offer effective solutions in many cases, pharmacological treatments that directly target sperm motility remain limited, particularly for idiopathic forms. Moreover, conventional antioxidant therapies have yielded inconsistent results in clinical practice, highlighting the need for alternative or complementary approaches with better mechanistic rationale (19). TCM, which has been used for centuries in this context, offers promising alternatives with reported clinical efficacy and favourable safety profiles (20,21). In our previous RCT, clinical study, QXF was shown to significantly improve sperm motility in patients with asthenozoospermia, without inducing notable side effects (13). Building on these findings, the present study evaluated the effects of QXF in a validated rat model of asthenozoospermia. Treatment with QXF significantly enhanced PR, non-progressive motility (NP), and total motility (PR + NP), confirming its therapeutic potential. Importantly, no significant alterations in liver or kidney function were observed in QXF-treated rats, further supporting its safety. Numerous agents have been used to establish animal models of male infertility, including tripterygium glycosides, cyclophosphamide, gossypol, bisphenol A, and ORN. Among these, ORN has demonstrated particular relevance for asthenozoospermia modelling. While tripterygium glycosides induce widespread testicular toxicity—including increased germ cell apoptosis and sperm malformations (22), and other agents such as cyclophosphamide, gossypol, and bisphenol A also impair spermatogenesis (23-25), these models may not precisely mimic the clinical presentation of asthenozoospermia without affecting sperm concentration. The use of ORN as a model compound dates back to 1988, when McClain et al. demonstrated that oral administration of 400 mg/kg/day for 61 days reduced fertility in male rats, an effect that was reversible upon discontinuation (26). Similar results were reported by Oberländer et al. (27). A previous study suggested that even 200 mg/kg may impair sperm motility (28), however, our preliminary experiments found that only the 400 mg/kg dose consistently induced asthenozoospermia without affecting sperm concentration, while 200 mg/kg had no significant effect and 800 mg/kg produced inconsistent results (16). Accordingly, we selected 400 mg/kg as the optimal dose for model induction in this study. Our findings confirm the feasibility and reproducibility of this dosage in generating a stable asthenozoospermia model.

The epididymal coefficient is a key macroscopic indicator for evaluating epididymal physiological function, and its changes are directly associated with the microscopic pathological features of the epididymis (29). The significantly decreased coefficient in the model group is a manifestation of organic damage, caused by the reduction and atrophy of epididymal duct epithelial cells, impaired secretory function, and decreased intraluminal sperm count. In contrast, the coefficient in the high-dose QXF group (QXF-H) showed a statistically significant increase, which suggested an improvement in cellular structure, a restoration of secretory function, and an increase in sperm density, confirming the protective effect of the formula on the epididymis. There was no significant difference in the coefficient between the low/medium-dose QXF groups (QXF-L/M) and the model group; however, sperm motility in the former two groups was significantly improved, which further supported the effectiveness of QXF.

Endocrine assessment and semen analysis remain central to the evaluation of male infertility, alongside medical history and physical examination. However, these standard investigations often fail to identify the underlying causes of male infertility, especially in idiopathic cases. In recent years, epigenetic modifications have emerged as crucial regulators of sperm development and function. In particular, abnormal DNA methylation has been increasingly associated with poor sperm parameters and various forms of male infertility, including asthenozoospermia, oligozoospermia, and teratozoospermia (30-32). As research in male infertility advances, DNA methylation profiling has become a key focus (33-35). The advent of high-throughput technologies such as reduced representation bisulfite sequencing (RRBS) has significantly improved the resolution and cost-efficiency of genome-wide methylation analysis (36-39). For example, He et al. used RRBS to analyse sperm samples from patients with asthenozoospermia and oligoasthenozoospermia and reported hypermethylation of the MEG8 DMR region in asthenozoospermia, and of MEG8, GNAS, and SNRPN in oligozoospermia (40). Similarly, Aarabi et al. observed global hypomethylation in sperm DNA from men with idiopathic infertility (41), while Schrott et al. demonstrated widespread methylation alterations in sperm associated with cannabis use (42).

In the present study, after confirming the efficacy of QXF in improving sperm motility in the rat model of asthenozoospermia, we explored DNA methylation changes in five groups using RRBS. As expected, the model group showed aberrant hypermethylation at multiple loci compared to healthy controls. Interestingly, QXF-treated groups—especially QXF-H—displayed a shift toward hypomethylation, suggesting that QXF may exert its effects in part by restoring a healthy methylation profile. While the QXF-L group showed some hypermethylated genes compared to normal rats, the dominant trend in all QXF-treated groups relative to the model group was demethylation, which is consistent with the reactivation of gene expression. This observation aligns with the well-established principle that hypomethylation of gene promoters is generally associated with increased transcription, while hypermethylation can lead to gene silencing (43). These methylation adjustments may play a key role in improving sperm motility following QXF administration.

Functional enrichment analysis is a fundamental bioinformatics approach designed to identify biologically relevant patterns within a predefined set of genes. It statistically evaluates whether certain biological functions, pathways, or processes are over-represented in the gene set of interest compared to a background expectation, thereby suggesting their potential collaborative roles in specific cellular mechanisms or disease-related pathways (44). In the present study, GO and KEGG enrichment analyses of DMGs revealed multiple signalling pathways relevant to sperm function. Among them, the cAMP and cGMP-PKG pathways are particularly notable. cGMP is involved in spermatogenesis, sperm capacitation, and testosterone synthesis (45,46), while cAMP is known to regulate the initiation and maintenance of sperm motility (47,48). These findings support the view that QXF may exert multi-pathway regulatory effects on sperm motility at the epigenetic level.

One particularly interesting finding is the recurrent enrichment of the PI3K/Akt signalling pathway across all QXF-treated groups, but not in the normal vs. QXF-L comparison. This suggests that PI3K/Akt signalling may be specifically involved in the therapeutic effect of QXF in asthenozoospermia, rather than being a baseline feature of QXF exposure. Numerous components in QXF can exert regulatory effects on the PI3K/AKT pathway, such as Astragalus polysaccharides, Cornus officinalis polysaccharides and ligustrazine (49-51). The corresponding mechanisms may be associated with antioxidant activity, autophagy regulation, and anti-inflammatory effects. This is consistent with our previous predictions based on network pharmacology analyses (52) and they provide a clear direction for future research aimed at identifying the key TCM components and elucidating their molecular mechanisms. The PI3K/Akt pathway, involving phosphatidylinositol 3-kinase and the downstream serine/threonine kinase Akt (PKB), regulates a wide range of cellular processes including proliferation, differentiation, apoptosis, and cytoskeletal remodelling (53-55). Recent studies have shown that this pathway also plays a key role in sperm motility, potentially through the regulation of autophagy (56-59).

While our findings offer strong mechanistic insight, they remain exploratory. The present study assessed global DNA methylation profiles, but did not evaluate methylation status at specific candidate genes, nor did it directly link methylation changes to alterations in gene or protein expression. In addition, the small sample size, the absence of positive controls, and incomplete exploration of the underlying molecular mechanisms constitute further limitations to our study. Future studies will address these shortcomings by expanding the cohort size and conducting in-depth mechanistic investigations. Additionally, the long-term therapeutic effects of QXF require further evaluation. Specifically, this can be achieved by detecting semen parameters several days after drug withdrawal; meanwhile, measuring serum sex hormone levels (including testosterone) should help rule out indirect hormonal effects.

Conclusions

In this study, an asthenozoospermia rat model was successfully established using ORN, and genome-wide methylation profiling of sperm DNA was performed using RRBS. The results demonstrated that QXF exerts dual regulatory effects on aberrant DNA methylation patterns in asthenozoospermia, specifically by reducing abnormal hypermethylation and enhancing hypomethylation at key genomic loci. Albeit the study is not devoid of limitations, such as the lack of analysis of the methylation status at specific candidate genes and the absence of a long-term follow-up, these findings contribute to a deeper understanding of the epigenetic mechanisms underlying asthenozoospermia and suggest that the therapeutic effects of QXF involve a dominant demethylating action, supplemented by targeted methylation induction. Furthermore, functional enrichment analyses revealed that cGMP/PKG, cAMP, and PI3K/Akt signalling pathways may play critical roles in mediating the effects of QXF on sperm motility and function.

Altogether, this study provides novel experimental evidence supporting the efficacy of QXF in treating asthenozoospermia and offers a scientific basis for its mechanism of action, paving the way for future translational and clinical research.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-462/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-462/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-462/prf

Funding: This work was supported by National Natural Science Foundation of China (Nos. 82174217 and 81703929), and High-Level Key Discipline Construction Project of Traditional Chinese Medicine by the National Administration of Traditional Chinese Medicine (No. zyyzdxk-2023238).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-462/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. 2020XLC004-3) granted by the Animal Ethics Committee of Xiyuan Hospital, China Academy of Chinese Medical Sciences, in compliance with China national and institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 6th edition. Geneva: WHO Press; 2021.
2. Agarwal A, Baskaran S, Parekh N, et al. Male infertility. Lancet 2021;397:319-33. [Crossref] [PubMed]
3. Vander Borght M, Wyns C. Fertility and infertility: Definition and epidemiology. Clin Biochem 2018;62:2-10. [Crossref] [PubMed]
4. Tournaye H, Krausz C, Oates RD. Concepts in diagnosis and therapy for male reproductive impairment. Lancet Diabetes Endocrinol 2017;5:554-64. [Crossref] [PubMed]
5. Esteves SC, Zini A, Coward RM, et al. Sperm DNA fragmentation testing: Summary evidence and clinical practice recommendations. Andrologia 2021;53:e13874. [Crossref] [PubMed]
6. Krzastek SC, Smith RP, Kovac JR. Future diagnostics in male infertility: genomics, epigenetics, metabolomics and proteomics. Transl Androl Urol 2020;9:S195-205. [Crossref] [PubMed]
7. Santi D, Corona G, Salonia A, et al. Current drawbacks and future perspectives in the diagnosis and treatment of male factor infertility, with a focus on FSH treatment: an expert opinion. J Endocrinol Invest 2025;48:1085-100. [Crossref] [PubMed]
8. Gunes S, Esteves SC. Role of genetics and epigenetics in male infertility. Andrologia 2021;53:e13586. [Crossref] [PubMed]
9. Shen FL, Zhao YN, Yu XL, et al. Chinese Medicine Regulates DNA Methylation to Treat Haematological Malignancies: A New Paradigm of "State-Target Medicine". Chin J Integr Med 2022;28:560-6. [Crossref] [PubMed]
10. Zhang M, Zhang JY, Sun MQ, et al. Realgar (α-As(4)S(4)) Treats Myelodysplastic Syndromes through Reducing DNA Hypermethylation. Chin J Integr Med 2022;28:281-8. [Crossref] [PubMed]
11. Wang F, Yu WX, Yan B, et al. Current status of sperm DNA methylation studies. Chin J Fam Plann 2019;27:973-5.
12. Denomme MM, McCallie BR, Parks JC, et al. Inheritance of epigenetic dysregulation from male factor infertility has a direct impact on reproductive potential. Fertil Steril 2018;110:419-428.e1. [Crossref] [PubMed]
13. Jenkins TG, Aston KI, James ER, et al. Sperm epigenetics in the study of male fertility, offspring health, and potential clinical applications. Syst Biol Reprod Med 2017;63:69-76. [Crossref] [PubMed]
14. Camprubí C, Salas-Huetos A, Aiese-Cigliano R, et al. Spermatozoa from infertile patients exhibit differences of DNA methylation associated with spermatogenesis-related processes: an array-based analysis. Reprod Biomed Online 2016;33:709-19. [Crossref] [PubMed]
15. Wang F, Gao QH, Geng Q, et al. Effectiveness and Safety Evaluation of Qixiong Zhongzi Decoction () in Idiopathic Asthenozoospermia Treatment: A Randomized Controlled Trial. Chin J Integr Med 2020;26:146-51. [Crossref] [PubMed]
16. Yan B, Wang F, Gao QH, et al. Linggui Fang protects the reproductive system of asthenospermia rats: An experimental study based on the L-carnitine pathway. Zhonghua Nan Ke Xue 2019;25:1113-7.
17. Liu S, Feng Q, Gong Z, et al. Linggui formula improves asthenozoospermia by modulating seminal plasma exosomes Wnt signaling via the LRP6/GSK3/Septin4 pathway. J Ethnopharmacol 2026;354:120495. [Crossref] [PubMed]
18. Alizadeh A, Taleb Z, Ebrahimi B, et al. Dietary Vitamin E Is More Effective than Omega-3 and Omega-6 Fatty Acid for Improving The Kinematic Characteristics of Rat Sperm. Cell J 2016;18:262-70. [Crossref] [PubMed]
19. Li KP, Yang XS, Wu T. The Effect of Antioxidants on Sperm Quality Parameters and Pregnancy Rates for Idiopathic Male Infertility: A Network Meta-Analysis of Randomized Controlled Trials. Front Endocrinol (Lausanne) 2022;13:810242. [Crossref] [PubMed]
20. Bai X, Liu Z, Tang T, et al. An integrative approach to uncover the components, mechanisms, and functions of traditional Chinese medicine prescriptions on male infertility. Front Pharmacol 2022;13:794448. [Crossref] [PubMed]
21. Wen Q, Xu H, Zou H, et al. The effect of Chinese herbal medicine on male factor infertility: study protocol for a randomized controlled trial. Front Endocrinol (Lausanne) 2024;15:1418936. [Crossref] [PubMed]
22. Xu Y, Fan YF, Zhao Y, et al. Overview of reproductive toxicity studies on Tripterygium wilfordii in recent 40 years. Zhongguo Zhong Yao Za Zhi 2019;44:3406-14. [Crossref] [PubMed]
23. Delgarm N, Morovati-Sharifabad M, Salehi E, et al. Exploring the main effects of phoenix dactylifera on destructive changes caused by cyclophosphamide in male reproductive system in mice. Vet Res Forum 2022;13:249-55. [Crossref] [PubMed]
24. Santana AT, Guelfi M, Medeiros HC, et al. Mechanisms involved in reproductive damage caused by gossypol in rats and protective effects of vitamin E. Biol Res 2015;48:43. [Crossref] [PubMed]
25. Lü L, Liu Y, Yang Y, et al. Bisphenol A Exposure Interferes with Reproductive Hormones and Decreases Sperm Counts: A Systematic Review and Meta-Analysis of Epidemiological Studies. Toxics 2024;12:294. [Crossref] [PubMed]
26. McClain RM, Downing JC. The effect of ornidazole on fertility and epididymal sperm function in rats. Toxicol Appl Pharmacol 1988;92:488-96. [Crossref] [PubMed]
27. Oberländer G, Yeung CH, Cooper TG. Induction of reversible infertility in male rats by oral ornidazole and its effects on sperm motility and epididymal secretions. J Reprod Fertil 1994;100:551-9. [Crossref] [PubMed]
28. Mo PJ, Pang XJ, Peng Y. Effect of ornidazole on epididymis carnitine in male rats. Acta Med Univ Sci Technol Huazhong 2009;38:1672-0741.
29. Zhang GW, Wan XX, Wan CC, et al. Lipoic acid protects spermatogenesis in male rats with ornidazole-induced oligoasthenozoospermia. Zhonghua Nan Ke Xue 2018;24:297-303.
30. Geng Q, Gao R, Sun Y, et al. Mitochondrial DNA content and methylation in sperm of patients with asthenozoospermia. J Assist Reprod Genet 2024;41:2795-805. [Crossref] [PubMed]
31. Song B, Chen Y, Wang C, et al. Poor semen parameters are associated with abnormal methylation of imprinted genes in sperm DNA. Reprod Biol Endocrinol 2022;20:155. [Crossref] [PubMed]
32. Zhang J, Li X, Wang R, et al. DNA methylation patterns in patients with asthenospermia and oligoasthenospermia. BMC Genomics 2024;25:602. [Crossref] [PubMed]
33. Shacfe G, Turko R, Syed HH, et al. A DNA Methylation Perspective on Infertility. Genes (Basel) 2023;14:2132. [Crossref] [PubMed]
34. Rotondo JC, Lanzillotti C, Mazziotta C, et al. Epigenetics of Male Infertility: The Role of DNA Methylation. Front Cell Dev Biol 2021;9:689624. [Crossref] [PubMed]
35. Oluwayiose OA, Wu H, Saddiki H, et al. Sperm DNA methylation mediates the association of male age on reproductive outcomes among couples undergoing infertility treatment. Sci Rep 2021;11:3216. [Crossref] [PubMed]
36. Hattori N, Liu YY, Ushijima T. DNA Methylation Analysis. Methods Mol Biol 2023;2691:165-83. [Crossref] [PubMed]
37. Ashapkin VV, Kutueva LI, Vanyushin BF. Quantitative Analysis of DNA Methylation by Bisulfite Sequencing. Methods Mol Biol 2020;2138:297-312. [Crossref] [PubMed]
38. Beck D, Ben Maamar M, Skinner MK. Genome-wide CpG density and DNA methylation analysis method (MeDIP, RRBS, and WGBS) comparisons. Epigenetics 2022;17:518-30. [Crossref] [PubMed]
39. Nakabayashi K, Yamamura M, Haseagawa K, et al. Reduced Representation Bisulfite Sequencing (RRBS). Methods Mol Biol 2023;2577:39-51. [Crossref] [PubMed]
40. He W, Sun T, Zhang S, et al. Profiling the DNA methylation patterns of imprinted genes in abnormal semen samples by next-generation bisulfite sequencing. J Assist Reprod Genet 2020;37:2211-21. [Crossref] [PubMed]
41. Aarabi M, San Gabriel MC, Chan D, et al. High-dose folic acid supplementation alters the human sperm methylome and is influenced by the MTHFR C677T polymorphism. Hum Mol Genet 2015;24:6301-13. [Crossref] [PubMed]
42. Schrott R, Acharya K, Itchon-Ramos N, et al. Cannabis use is associated with potentially heritable widespread changes in autism candidate gene DLGAP2 DNA methylation in sperm. Epigenetics 2020;15:161-73. [Crossref] [PubMed]
43. Maurano MT, Wang H, John S, et al. Role of DNA Methylation in Modulating Transcription Factor Occupancy. Cell Rep 2015;12:1184-95. [Crossref] [PubMed]
44. Chen L, Zhang YH, Wang S, et al. Prediction and analysis of essential genes using the enrichments of gene ontology and KEGG pathways. PLoS One 2017;12:e0184129. [Crossref] [PubMed]
45. Wu K, Mei C, Chen Y, et al. C-type natriuretic peptide regulates sperm capacitation by the cGMP/PKG signalling pathway via Ca(2+) influx and tyrosine phosphorylation. Reprod Biomed Online 2019;38:289-99. [Crossref] [PubMed]
46. Cisneros-Mejorado A, Hernández-Soberanis L, Islas-Carbajal MC, et al. Capacitation and Ca(2+) influx in spermatozoa: role of CNG channels and protein kinase G. Andrology 2014;2:145-54. [Crossref] [PubMed]
47. Piroozmanesh H, Jannatifar R, Ebrahimi SM, et al. Cyclic adenosine monophosphate (cAMP) analog and phosphodiesterase inhibitor (IBMX) ameliorate human sperm capacitation and motility. Rev Int Androl 2022;20:S24-30. [Crossref] [PubMed]
48. Tourzani DA, Battistone MA, Salicioni AM, et al. Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation. Int J Mol Sci 2021;22:10241. [Crossref] [PubMed]
49. Bakeer MR, Soliman SS, Ahmed O, et al. Astragalus polysaccharides protect against Di-n-butyl phthalate-induced testicular damage by modulating oxidative stress, apoptosis, and the PI3K/Akt/mTOR pathway in rats. Front Vet Sci 2025;12:1616186. [Crossref] [PubMed]
50. Liu Y, Wang C, Hui T, et al. Cornus officinalis loganin attenuates acute lung injury in mice via regulating the PI3K/AKT/NLRP3 axis. J Ethnopharmacol 2025;351:120104. [Crossref] [PubMed]
51. Du HY, Wang R, Li JL, et al. Ligustrazine protects against chronic hypertensive glaucoma in rats by inhibiting autophagy via the PI3K-Akt/mTOR pathway. Mol Vis 2021;27:725-33.
52. Zhao F, Liu SJ, Gao QH, et al. Network pharmacology-based study of Chinese herbal Qixiong formula in treating oligoasthenospermia. World Journal of Traditional Chinese Medicine 2020;6:481-9.
53. Yue TT, Cao YJ, Cao YX, et al. Shuxuening Injection Inhibits Apoptosis and Reduces Myocardial Ischemia-Reperfusion Injury in Rats through PI3K/AKT Pathway. Chin J Integr Med 2024;30:421-32. [Crossref] [PubMed]
54. Wang JS, Gong XF, Feng JL, et al. Explore the effects of pulmonary fibrosis on sperm quality and the role of the PI3K/Akt pathway based on rat model. Andrologia 2022;54:e14348. [Crossref] [PubMed]
55. Chen KQ, Wei BH, Hao SL, et al. The PI3K/AKT signaling pathway: How does it regulate development of Sertoli cells and spermatogenic cells? Histol Histopathol 2022;37:621-36. [Crossref] [PubMed]
56. Fernandez MC, Yu A, Moawad AR, et al. Peroxiredoxin 6 regulates the phosphoinositide 3-kinase/AKT pathway to maintain human sperm viability. Mol Hum Reprod 2019;25:787-96. [Crossref] [PubMed]
57. Liao HY, O'Flaherty C. Lysophosphatidic Acid Signalling Regulates Human Sperm Viability via the Phosphoinositide 3-Kinase/AKT Pathway. Cells 2023;12:2196. [Crossref] [PubMed]
58. Deng CY, Lv M, Luo BH, et al. The Role of the PI3K/AKT/mTOR Signalling Pathway in Male Reproduction. Curr Mol Med 2021;21:539-48. [Crossref] [PubMed]
59. Zhu Y, Yin Q, Wei D, et al. Autophagy in male reproduction. Syst Biol Reprod Med 2019;65:265-72. [Crossref] [PubMed]